key: cord- -acbo zs authors: thomas, l.h.; howard, c.j.; parsons, k.r.; anger, h.s. title: growth of mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with mycoplasma dispar date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: acbo zs inoculation of tracheal organ cultures from bovine foetuses with mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed – days later by lifting and detachment of overlying epithelium. the effect was associated with large numbers of m. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. ciliary activity was undiminished for up to days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. in contrast, m. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. the cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma. studies of microorganisms isolated from the respiratory tract have been greatly advanced by the use of the organ culture technique first described by i-ioorn and tyrrell ( ) . since then the technique has been used by collier et al. ( collier et al. ( , to investigate the effects of m. pneumoniae in organ cultures of hamster trachea, by cherry and taylor-robinson ( ) to study m. mycoides var capri in chicken tracheal organ cultures, by pijoan et al. ( ) to investigate four porcine mycoplasmas in pig tracheal organ cultures and by thomas and howard ( ) to study four bovine mycoplasmas in foetal calf tracheal organ cultures. m. bovis is a significant pathogen for the bovine respiratory tract thomas et al., ) ; it was thought therefore that a useful insight into its pathogenicity might be obtained by its inoculation into tracheal organ cultures and by comparison with another pathogen of the bovine respiratory tract, m. dispar (thomas and howard, ; howard et al., ) . this paper describes the results of this investigation. m. bovis strains were grown in broth (gourlay and leach, ) or solid media (howard et al., ) , except that ampicillin ( mg m - ) was substituted for penicillin, and thallium acetate was omitted from broth used to grow inocula. numbers of organisms were measured as colony forming units (cfu). the ab/ strain has been shown to be pathogenic for the calf respiratory tract thomas et al., ) , strain ab/ was isolated from an outbreak of mastitis and strains sm /c and minr were isolated from two separate outbreaks of calf pneumonia. m. dispar strain gri was grown in broth as described above. numbers of organisms were measured as colour change units (ccu) (gourlay and leach, ) as this species frequently fails to produce colonies on agar and ccu give a more accurate assessment of the number of viable organisms present. this strain has also been shown to be pathogenic for the calf respiratory tract gourlay et al., ) . tracheal organ cultures were prepared from four bovine foetuses at - months gestation (thomas and howard, ) . they were maintained as rings and rolled singly (experiments and ) in -oz bottles with ml of medium , except that mycostatin was omitted from the medium and % heated, foetal calf serum was added. cultures were inoculated with . ml or . ml of mycoplasmas and the maintenance medium was sampled -- h later (day sample). thereafter medium was sampled and changed on days , , and post-inoculation (experiment ) and on days , , , , , , , , , , , . rings were removed for sampling on days , and (experiment ) and days , , , , and (experiment ) . for experiment six tracheal rings from one of two foetuses were placed in a bottle in ml of medium. duplicate cultures from each foetus were inoculated with . ml of one of the four strains of m. bovis. the maintenance medium was sampled on day following inoculation and thereafter medium and tissue were sampled at - day intervals until the termination of the experiment on day . the maintenance medium was also changed on the day of sampling. sampled rings were divided into segments and pieces taken for estimation of mycoplasma numbers, histology, including immunoperoxidase labelling, and electron microscopy (experiment only). uninoculated control cultures were maintained in parallel with inoculated cultures in all three experiments and sampled at the same time for comparison. all rings were examined for ciliary activity at -- day intervals. pieces of tracheal tissue were fixed in formol-sublimate (mercuric formalin) for up to h and then transferred to % alcohol before embedding in paraffin wax and sectioning. serial sections were then stained by haematoxylin/eosin, giemsa or by the unlabelled antibody immunoperoxidase method. paraffin sections of formol-sublimate fixed tissue were stained by the peroxidase-antiperoxidase method [sternberger et al., , adapted by parsons et al., ( ] using primary antiserum to m. bovis and m. dispar prepared in rabbits (thomas et al., ) . samples of tracheal tissue were diced and fixed in fresh % phosphate buffered glutaraldehyde for h followed by h fixation in % buffered osmium tetroxide (millonig, ) . dehydration was performed in ascending grades of methanol and completed in propylene oxide. the tissues were embedded in araldite and polymerized overnight at °c. sections, -- nm thick, were cut on a cambridge huxley ultra microtome, using glass knives, and stained with uranylacetate and lead citrate (venable and coggeshall, ) for examination in a philips electron microscope using an accelerating voltage of kv. in this preliminary experiment lasting days, three organ cultures were inoculated. titres in the maintenance medium rose from "s cfu ml- at day to a maximum of l s cfu m - at day and fell to . cfu m - on day . no effect on ciliary activity was detected. microscopically, little or no cytopathic effect was detected in the epithelial layer, apart from a slight lifting and detachment by day . however in the lamina propria loss of cell nuclei was associated with large numbers of m. bovis located by ipx labelling. by day m. bovis was progressively infiltrating between cells of the epithelium and accumulating in the lamina propria. organisms were also detected in large numbers in the peritracheal connective tissue surrounding the convex margin of the ring but with little or no apparent cytopathic effect. mean titres obtained from maintenance medium and in tissue for the duplicate cultures are shown in table i . titres in medium at day reflected the relative titre of the four inocula and ranged between < " and s cfu m - , but following the first change of medium at day numbers of mycoplasmas in both medium and tissue varied by < cfu ml -~ for all four strains of m. bovis for the duration of the experiment. it should be noted that numbers in tissue were approximately -fold higher than those shown in table i due to the initial % dilution involved in the trituration of the tissue. ab/ . medium < . . . . , . tissue nd , . . , . ab/ . medium . . . . , . tissue nd . . . . . sm /c . medium . . . . , . tissue nd . . . , . minr . medium . . . . , . tissue nd . . . , . anumber of organisms cfu m - ( n) at time zero. bnumbers in tissue estimated as a % suspension in mycoplasma broth. cmean titre of duplicate cultures, one from each foetus, each containing six tracheal rings at start of experiment. nd, not done. microscopically, changes closely resembled those seen in experiment : little or no cytopathic effect was detected in the epithelial layer but a loss of cellular structure in the lamina propria was associated with the presence of large numbers of m. bovis. some difference was seen in the distribution of the strains of m. bovis as detected by immunoperoxidase labelling, in that strain minr showed less propensity to colonise the lamina propria. this same observation was made on all samples from day through to conclusion of the experiment at day . were first re-isolated on or after the seventh day (medium or tissue). on day , ~' ccu m - were isolated from the medium of one culture and on day , s' and . ccu m - were isolated from the medium and tissue respectively of the same culture. a second culture sampled on day contained . and . ccu ml -t in medium and tissue, respectively. no mycoplasmas had been isolated from this culture previously. the remaining three cultures were concluded on days, , and , and no mycoplasmas were isolated. titres were essentially similar to those obtained in the first two experiments. mycoplasmas were isolated from the medium of two of the five organ cultures on the second day after inoculation with m. bovis. by day mycoplasmas were isolated from the medium of all four remaining cultures at titres of s'°-- "s cfum - . titres remained at this level until the last culture was sampled on day . numbers of mycoplasmas in the tissue were similar to those in the maintenance medium sampled on the same days ( ":-- . cfu ml- ). activity was normal in cultures inoculated with m. dispar compared to control cultures up to day but declined sharply to very faint activity by day . by this time the ciliated margin of the cultures was ragged and uneven due to the presence of extravasated cells. no reduction in ciliary activity could be detected in cultures inoculated with m. bovis up to days after inoculation. by days, however, ciliary activity had declined to one third of that of uninoculated control cultures. in contrast, m. bovis had virtually no cytopathic effect on the ciliated epithelium for days following inoculation in spite of large numbers of organisms infiltrating between the columnar epithelium, accumulating in the lamina propria and amassing in the region of the basement membrane (fig. ) . infiltration could be detected by day following inoculation and, although pleomorphic organisms were seen by electron microscopy in spaces between the columnar epithelial cells, the cell membranes of the adjacent cells were apparently normal (fig. ) . by day , some parts of the epithelial layer were still intact but in other areas there was lifting and detachment of the epithelium. the effect of m. bovis on the connective tissue cells of the lamina propria was striking: by day a significant loss of cell nuclei was apparent and by day little or no cellular structure, excepting a few epithelial cells lining the secretory glands, was discernible (fig. ) . no comparable effect was seen in control cultures (fig. ) or in cultures inoculated with m. dispar. as described in experiments and , the cytopathic effect was associated with large numbers of m. bovis and organisms were also present in large numbers in the peritracheal connective tissue investing the outer, convex margin of the culture but had no apparent effect. findings from the three experiments with m. bovis in organ cultures of bovine trachea are in close agreement. the capacity of m. bovis to penetrate between the cells of the respiratory epithelium without causing damage to those cells is remarkable and is in sharp contrast to the action of m. dispar, which, as described in an earlier paper (thomas and howard, ) and confirmed in the present study acts, entirely at the ciliated margin of the epithelial layer to produce its cytopathic effect. the ability of all four strains of m. bovis used to penetrate the respiratory epithelium and enter the intercellular spaces resembles to some extent the reported action of m. pneumoniae in organ cultures of hamster trachea (collier et al., ) . however, m. pneumoniae attaches, forms clumps and is restricted to the ciliated margin of the epithelium; furthermore this organism appears to possess a special modification for attachment at this site, none of which was observed for m. bovis. subsequent studies have also shown that m. bovis, in contrast to m. pneumoniae, is non-motile (w. bredt and l.h. thomas, unpublished observations) . the eventual and relatively insignificant ciliastatic effect of m. bovis is probably attributable to the extensive necrosis in the underlying tissues, rather than to a direct effect as seen in the cultures inoculated with m. dispar. the relatively slow onset of the cytopathic effect due to m. dispar compared with that seen earlier (thomas and howard, ) may be attributed to the low titre of inoculum used. this was designed to ensure that the onset of the cytopathic effect for each mycoplasma might coincide; a high titre inoculum for m dispar ( -- . ccu ml -~) produced a marked cytopathic effect within h (thomas and howard, ) . compatible with their behaviour in vivo. m. dispar causes an exudative bronchiolitis with peribronchiolar and alveolar round cell infiltration whereas m. bovis appears to be a more invasive pathogen, causing extensive coagulative necrosis in lung parenchyma with apparently little effect on the respiratory epithelium (thomas et al., ) . some caution should be used, however, in interpreting in vivo effects for these mycoplasma from their behaviour in organ culture; although a progressive penetration of m. bovis from the epithelial layer to the lamina propria was apparently demonstrated, the cut, transverse surface of the tracheal ring does expose the lamina propria for direct penetration by the mycoplasmas. the colonisation of the peritracheal connective tissue by m. bovis is probably an artefact but nevertheless the lack of cytopathic effect in this location is surprising. the work described here supports the view that m. bovis is second only to m. mycoides subsp, rnycoides in its pathogenicity for bovine tissue. the mechanism whereby m. bovis produces its cytopathic effect remains to be elucidated. a toxin has been described for m. bovis (geary et al., ) and the relevance of such a toxin to its pathogenicity in lung tissue discussed (thomas et al., ). however we have, to date, been unable to demonstrate any toxic effect for organ cultures using a cell free filtrate of medium from cultures infected with m. bovis (thomas and howard, unpublished observations) . the apparent similarity of action in vitro and in vivo suggests that organ cultures may be a convenient way of investigating mechanisms of pathogenicity in m. bovis and m. dispar. large quantity production of chicken embryo tracheal organ cultures and use in virus and mycoplasma studies biologic effects of mycoplasma pneumoniae and other mycoplasmas from man on hamster tracheal organ culture mycoplasma pneumoniae in hamster tracheal organ culture: immunofluorescent and electron microscopic studies inflammatory toxin from mycoplasma bovis: isolation and characterization a new mycoplasma species isolated from pneumonic lungs of calves (mycoplasma dispar sp. nov.) pneumonia and arthritis in gnotobiotic calves following inoculation with mycoplasma agalactiae subsp, bovis pathogenicity of some mycoplasma and acholeplasma species in the lungs of gnotobiotic calves on the growth of certain "newer" respiratory viruses in organ cultures induction of pneumonia in gnotobiotic calves following inoculation of mycoplasma dispar and ureaplasmas (t-mycoplasmas) induction of immunity in calves to mycoplasma bovis infection of the respiratory tract advantages of a phosphate buffer for osmium tetroxide solution in fixation localisation of enteropathogens in paraffin-embedded tissue by immunoperoxidase the effect of porcine mycoplasmas on pig tracheal organ cultures the unlabelled antibody-enzyme method of immunohistochemistry. preparation and properties of soluble antigen-antibody complex --horseradish peroxidase --and its use in the identification of spirochaetes replication of a bovine coronavirus in organ cultures of foetal trachea effect of mycoplasma dispar, m. bouirhinis, acholeplasma laidlawii and t-mycoplasmas on explant cultures of bovine trachea the pathology and microbiology of mycoplasma bouis infection in gnotobiotic calves, including combination with respiratory syncytial virus a simplified lead citrate stain for use in electron microscopy we acknowledge the technical assistance of miss n. rolley and mr. paul sopp with the isolation of mycoplasmas. mr. p.f. dennis and mr. brian turfrey prepared the histological sections. mr. h. kay, environmental health officer at the fmc abattoir, salisbury, gave assistance in obtaining the organ culture tissues. key: cord- -ydrnanug authors: brock, k.v.; potgieter, l.n.d. title: detection of bovine viral diarrhea virus in serum from cattle by dot blot hybridization assay date: - - journal: vet microbiol doi: . / - ( ) -y sha: doc_id: cord_uid: ydrnanug a dot blot hybridization assay was developed for use as a rapid screening test to detect bovine viral diarrhea virus (bvdv) in serum from infected cattle. a . . kilobase cdna, prepared from the bvdv genome, was molecularly cloned and used in this study. insert cdna was removed from the puc plasmid vector by pst-i restriction endonuclease digestion and purified from plasmid dna by agarose electrophoresis and electroelution. the hybridization probe was prepared by nick translation in the presence of gamma dct( )p and labelled to a specific activity of × ( ) cpm/μg of dna. specificity was determined by dot blot hybridization of infected cell culture supernate from nine different bvdv strains. the probe hybridized equally with all strains of bvdv tested, which included four cytopathic and five noncytopathic strains of bvdv. serum was collected from veal calves with respiratory tract disease, unthriftiness, anorexia, and/or poor conditions. serum samples were treated with nonidet p detergent and denatured with formaldehyde and heat prior to application on . micron nylon membrane filters using a vacuum dot blot apparatus. hybridization was done under relatively stringent conditions ( % formamide at °c). a total of serum samples from different calves were tested and of these samples, ( %) were positive by dot blot hybridization for bvdv rna. eight calves ( %) out of , tested to weeks later, remained positive for bvdv rna. . transplacental infection is a common sequel in persistently-infected cattle or after bvdv infections in susceptible, pregnant heifers and cows (orban et al., ; liess et al., ) . the virus is fetopathic, primarily early in pregnancy; however, fetal infections may result in persistent immunotolerant infections in postnatal life (duffel and harkness, ; harkness, ) . such animals may appear to be healthy but some die pre-maturely, often after chronic illness, and all have the potential of developing mucosal disease (orban et al., ; liess et al., ; duffel and harkness, ) . evidence indicates that mucosal disease is precipitated by superinfection of persistently-infected, seronegative animals with a different strain of bvdv and thus represents the final outcome of in utero infection (lies et al., ) . another significant repercussion of in utero infection is that persistently-infected cattle may be the primary source of bvdv in nature (harkness, ) . novel approaches for identifying persistently-infected animals are required since methods currently employed are inefficient, expensive and of questionable accuracy (coria et al., ; howard et al., ) . since the virus is endemic in the u.s.a., attempts to maintain a herd free of infection to the virus likely would invite disastrous economic losses (harkness, ) . the aim of rational control measures should be to break the cycle of transmission by identifying (and removing) persistently-infected animals and by preventing transplacental infections (duffel and harkness, ; harkness, ) . another important objective must be prevention of bvdv infection of stressed animals (harkness, ) . effective bvdv control, therefore, would require screening of herds for persistently-infected cattle and use of effective vaccines. measures for the control and prevention of bvdv are based on the detection and identification of persistently-infected individuals within a herd. at present, virus isolation is the only method available to detect persistentlyinfected animals (duffel and harkness, ; harkness, ; bezek et al., ) . a rapid, specific, and sensitive detection assay would facilitate herd screening for bvdv infection. the purpose of this study was to evaluate the use of a dot blot hybridization assay to detect bvdv in serum from infected cattle. bvdv strains used in this study included five cytopathic strains ( , auburn, nadl, singer, oregon c v) and five non-cytopathic strains ( , , new york- , tgan, and neb) . the origin of strains and has been described (potgieter et al., ) ; the other bvdv strains were obtained from dr. s.r. bolin, national animal disease center, ames, ia. dulbecco's minimum essential medium supplemented with % fetal bovine serum that had been treated with beta-propiolactone ( . % final concentration) to remove adventitious viruses was used for cell growth (brock et al., ) . primary bovine turbinate (btu) cell cultures, passaged to times and negative for bvdv by indirect immunofluorescence and dot blot hybridization assay, were used for propagating bvdv. complete monolayers of btu cell cultures ( cm ) were inoculated with . to . ml of test serum for h. the inoculum was removed after h, replaced with medium, and incubated at °c in a . % co incubator for days. cell cultures were examined daily for development of cytopathic effect. cells were removed by scraping and a drop of cell suspension was placed onto a glass slide and fixed in acetone for min. bvdv antigen was detected by indirect fluorescence assay using an anti-bvdv antiserum (nadl and new york- strain) prepared in a gnotobiotic calf. the indirect fluorescence assay also was done to determine serum antibody titers to bvdv using btu cell cultures infected with the nadl strain of bvdv. a . ml aliquot of the cell suspension from the first passage was inoculated into btu cell cultures for a second passage as described above and tested by indirect fluorescence assay for bvdv antigen. cdna was obtained from purified bvdv (strain ) genomic rna and cloned into the puc plasmid vector (brock et al., ) . a . kilobase (kb) cdna clone was identified from the cdna clones and used to prepare hybridization probes (brock et al., ) . the insert cdna fragment was removed from the puc plasmid by restriction endonuclease digestion with pst-i and purified from plasmid dna by agarose electrophoresis and electroelution (maniatis et al., ) . purified cloned cdna sequences were labelled with dct p to a specific activity of - × cpm/g by nick translation as described by rigby et al. ( ) . labelled dna was separated from unincorporated nucleotides by gel filtration through sephadex g- (maniatis et al., ) . prior to hybridization, probe was heated for min at °c and chilled on ice. whole blood was collected from veal calves with respiratory tract disease, unthriftiness, anorexia, and/or poor condition. serum was removed from clotted blood samples following centrifugation at g for min. serum samples ( . ml) were immediately denatured as described below and applied to membrane filters. the remaining portion was frozen at - °c for further use. negative control samples from uninfected btu cell culture supernate and positive control samples from bvdv-infected cell cultures (strain , ccidso/ml) also were denatured and applied to each membrane filter to be hybridized. hybridization blots were prepared, hybridized, and washed following hybridization as described by maniatis et al. ( ) . hybridization of cdna probes with bvdv rna was done by the dot blot method as described by shockley et al. ( ) . samples tested included various strains of cell culturegrown bvdv, uninfected cell culture supernatant fluids, and serum. specimens were clarified by centrifugation at g for min. samples were denatured by adding nonidet p- to a concentration of . % and the mixture was incubated on ice for min (white and bancroft, ) . then an equal volume of % formaldehyde and % × ssc ( × ssc is . m nac , . m trisodium citrate, ph . ) was added and the mixture was heated at °c for rain. a /tl volume of the treated sample was applied to a . micron nylon membrane filter (biodyne membranes, icn biomedicals, irvine, ca) using a dot blot apparatus. the nylon membrane filters were airdried and baked at °c for min before they were placed into plastic bags with prehybridization solution at °c for - h with gentle agitation. the cdna probes ( cpm/ml) were denatured by heating at °c for min followed by rapid cooling in ice and added to the hybridization solution ( % formamide, ×ssc, . % sds and /tl/ml denatured salmon sperm dna). hybridization was allowed to proceed at °c for - h. the hybridized filters were washed ( x ssc and . % sds, four times for min at o c and then . × ssc and . % sds, twice for min at ° c ) and dried at room temperature on filter paper before their placement into radiographic film cassettes containing radiographic film and an intensifying screen for - h. the cdna hybridization probe reacted equally with infected cell culture supernates of all cytopathic strains ( , auburn, nadl, singer, oregon c v) and non-cytopathic strains ( , , new york-l, tgan, and neb) tested. previously, as little as - pg of purified bvdv rna resulted in detectable hybridization signals with the hybridization probe used in this study (brock et al., ) . to determine the minimum detection level of bvdv in serum, serial tenfold dilutions of bvdv infected cell culture supernatant ( ccidso/ml) were made in bvdv negative, fetal bovine serum. virus was detected by dot blot hybridization assay to minimum level at a - dilution in the serum. a total of serum samples from different calves were tested and ( %) of these samples were positive by dot blot hybridization for bvdv rna. an autoradiograph of a hybridized dot blot membrane filter containing test samples is shown in fig. . virus isolation was attempted on the first samples collected in addition to dot blot hybridization assay. bvdv was present in of the samples determined positive by dot blot hybridization assay ( table ) . virus was isolated from three of the samples. one of the serum samples from which virus was isolated was negative for bvdv by dot blot hybridization assay. serum antibody levels of the eight dot blot hybridization positive samples ranged from < : to : (table ) . serum from eight calves ( %) out of , tested to weeks later, remained positive for bvdv rna. results from the dot blot hybridization assay were available within to h. the sensitivity and specificity of hybridization probes for the presence of viruses is equal or greater than other laboratory methods such as immunoassays, virus isolation, and electron microscopy (richman et al., ; teramoto et al., ) . the sensitivity of hybridization of probes with target sequences is dependent on the methods of labelling, hybridization conditions, detection system, and hybridization system (richman et al., ) . from the results of this study, it is concluded that a dot blot hybridization assay can detect bvdv in serum from naturally-infected cattle and may be applicable as a diagnostic assay. however, the hybridization results do not correlate well with standard diagnostic methods such as virus isolation since virus was isolated from only two out of eight dot blot positive samples (table ) . when detection of virus passaged in btu cell cultures by hybridization is compared with virus isolation, the results highly correlate. however, dissimilar results were obtained from individual animals samples. comparing hybridization results of virus-laden serum with virus isolation, virus was detected in a dilution of cell culture supernate ( ccidso) in fetal bovine serum. in this study, bvdv was isolated from only % ( / ) of samples positive by hybridization assay. the presence of anti-bvdv serum antibody in some samples may account for the difficulty in the isolation of virus from serum (table ) . following the acute phase of postnatal bvdv infections, virus can not be isolated from serum. virus may remain in serum in antibody complexes following the development of antibody. therefore, samples taken to days after infection may be negative for virus by isolation in cell culture but positive by hybridization. the preferred sample for isolation of bvdv from infected animals is white blood cells (buffy coat) from heparinized whole blood instead of serum (bezek et al., ) . also, virus isolation may not have correlated with hybridization results due to insufficient detection of some antigenically-heterologous field strains of bvdv by the anti-bvdv antibody used in the indirect fluorescence antibody assay (castrucci et al., ; coria et al., ) . also it may be possible that antibody present in fbs used to supplement cell culture interfered with the isolation of bvdv. the disparity between the hybridization results and virus isolation would suggest the hybridization probe is reacting nonspecifically. however, several factors support the specificity of the hybridization probe for bvdv. the hybridization probe consisted only of insert dna removed from plasmid dna by restriction endonuclease digestion and electroelution to prevent nonspecific reactions by plasmid dna. control puc plasmid dna did not hybridize with the bvdv hybridization probe and probe prepared from puc plasmid dna alone did not hybridize with samples identified as positive with the bvdv hybridization probe. the hybridization probe has previously been used against several bovine rna and dna viruses (bovine respiratory syncytial virus, bluetongue virus, bovine coronavirus, and bovine herpesvirus i ) without any cross-hybridization (brock et al., ) . the possibility that the probe reacts nonspecifically with a factor in bovine serum samples is not probable. hybridization results of serum samples taken from a different population of steers and bulls identified only five animals that were positive by hybridization using identical assay conditions (unpublished data). assuming comparable results, if random nonspecific reactions occur, more positive animals would have been expected. also in this study, initial samples collected from some animals were positive and following the second sampling to weeks later, the samples taken from the same animals were negative. one animal was negative from the first sample taken and positive on the second sample by hybridization. another important factor in the evaluation of the assay results was the definition of the positive endpoint of the dot blot hybridization assay. in this study, samples that had an equal intensity of hybridization signal compared with the positive control dots of bvdv-infected cell cultures were considered positive for bvdv rna by hybridization (fig. ) . the variability in the hybridization signals was likely due to the varying amounts of bvdv rna present in the samples. hybridization of some samples resulted in low level signals below the intensity of the controls and were considered negative although virus may have been detectable by virus isolation (fig. ) . although the hybridization probe can detect a minimum quantity of to pg of bvdv rna (brock et al., ) , the evaluation of hybridization results from clinical samples at this low threshold level is difficult and impractical as a diagnostic assay. the use of polymerase chain reaction technology to amplify the template in the samples may improve definition between positive and negative signals when a minimum amount of bvdv rna is present. it was thought that serum was the best sample for testing for bvdv infection. serum samples would be easier to obtain from a field situation than white blood cells from buffy coats. control and prevention of bvdv infection centers around the ability to detect and remove persistently-infected animals which may have levels of to ccidso/ml of serum (harkness, ) . white blood cells may remain infected with bvdv following the clearance of virus from the serum in acutely-infected animals (malmquist, ) . therefore, the detection of bvdv in buffy coat cells would not differentiate acute and persistent infections of bvdv. although the presence of virus in serum does not indicate a persistent infection, a continual presence of virus in the serum of persistently-infected animals would be expected. the high percentage ( %) of calves infected with bvdv demonstrates that bvdv is very prevalent especially in stressful environments. the calf sample population did not represent a normal population in this study, therefore the prevalence of bvdv infection may actually be lower in a given herd. the eight out of calves that remained positive by hybridization to weeks after initial testing were not characterized as persistently-infected animals which indicates that acute bvdv infections may not be resolved following the development of antibody. it is probable that the animals identified as positive by hybridization represented postnatal infections and were not persistently infected since virus was not isolated from serial samples and most animals had anti-bvdv serum antibody. reoccurring, postnatal infections with antigenically-heterologous bvdv strains may be common in mixed and stressed populations of animals such as veal calves. the results from this study are not considered conclusive. the disagreement between virus isolation and hybridization requires further investigation. however, the results are important in demonstrating the difficulty encountered in comparing two assays; the ability to detect infectivity of bvdv and the detection of bvdv nucleic acids. due to the potential problems that may be encountered in attempting to isolate bvdv it is difficult to ignore the potential detection of bvdv by hybridization assay. however, bvdv hybridization assay results must be examined closely along with virus isolation until further controlled studies can be done to support results obtained by hybridization assay. although current theories on the pathogenesis of mucosal disease, acute bvdv infections, and persistent infections from in utero infection are accepted by many researchers, many aspects of the complex pathogenesis of bvdv infection remain unknown (orban et al., ; liess et al., ; duffel and harkness, ; harkness, ; horzinek and van berlo, ) . the development of new technologies such as hybridization probes to detect viral genomes may provide new or different information than is currently accepted on the pathogenesis of bvdv infection. hybridization probes are very sensitive and specific for detecting bvdv rna, and although correlating results with standard tests such as virus isolation is difficult, there are important diagnostic applications of these methods. conclusion dot blot hybridization can be used to detect bvdv in serum from cattle naturally-infected with bvdv. the hybridization assay is extremely sensitive and specific for detection of bvdv rna. however, the results of the hybridization assay in detecting natural infections did not correlate with virus isolation as a diagnostic assay. immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves molecular cloning of complementary dna from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application a study of immunologic relationships among serologically heterologous strains of bovine viral diarrhea virus by cross immunity tests differentiation of cytopathic and noncytopathic isolates of bovine viral diarrhea virus neutralization bovine virus diarrhoea-mucosal disease infection in cattle the control of bovine viral diarrhoea virus infection the pestiviruses: where do they belong? an enzyme-linked immunosorbent assay (elisa) for the detection of antibodies to bovine viral diarrhoea virus (bvdv) in cattle sera studies on transplacental transmissibility of a bovine virus diarrhoea (bvd) vaccine virus in cattle: ii. inoculation of pregnant cows without detectable neutralizing antibodies to bvd virus to days before parturition ( st to th day of gestation) bovine viral diarhea-mucosal disease: etiology, pathogenesis, and applied immunity molecular cloning a laboratory manual studies on transplacental transmissibility of a bovine virus diarrhoea (bvd) vaccine virus: i. inoculation of pregnant cows to days before parturition ( th to th day of gestation) experimental production of bovine respiratory tract disease with bovine viral diarrhea virus role of bvd virus in shipping fever of feedlot cattle: case studies and diagnostic considerations rapid viral diagnosis labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with dna polymerase i diagnosis of porcine and bovine enteric coronavirus infections using cloned cdna probes comparison of enzyme-linked immunosorbent assay, dna hybridization, hemagglutination, and electron microscopy for detection of canine parvovirus infections cytoplasmic dot hybridization salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work (article no. ) was supported in part by cooperative research agreement - - from the u.s. department of agricultural science and education administration. key: cord- -v tff gk authors: nguyen, t.d.; bottreau, e.; aynaud, j.m. title: transmissible gastroenteritis (tge) of swine: in vitro virus attachment and effects of polyanions and polycations date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: v tff gk four transmissible gastroenteritis virus (tgev) strains (purdue- , d- , -sg and gep-ii) and two cell lines (swine testis-st and pig kidney-rpd) were used to study virus attachment and cell susceptibility. virus attachment was partially thermodependent and the rate varied, depending on the strain. identical tgev inocula produced a higher plaque number by plaque assay in the swine testis cell line (st) than in the pig kidney cell line (rpd) but [( )h]uridine-labèlled virus was found associated equally well with both cell lines. a field tgev strain (gep-ii), which was unable to multiply in cell cultures, appeared able to inhibit the attachment of radiolabelled cell-passaged virus. therefore, the susceptibility to tgev infection was apparently not determined at the virus-to-cell attachment stage. the attachment sites on the cell surface were specific, however, differences in tgev attachment determinant between strains were not observed. attachment of all the virus strains tested was enhanced by deae-dextran and inhibited by dextran sulfate, poly-l-lysine (pll), poly-l-α-ornithine (plo) and protamine sulfate. attachment of virus to the host cell plasma membrane is the first step of the virus replication cycle and has been suggested to be one of the major determinants of cell susceptibility to virus infection. in most cases, this process occurs when viral protein (s) bind specifically to cell surface receptors (tardieu et al., ) . it was also demonstrated that the degree of virus virulence can be determined at this level ( mak et al., ; mims and white, ) . transmissible gastroenteritis virus (tgev) is a member of the coronaviridae. in the natural host, it infects intestinal epithelial cells, causing severe diarrhea in newborn piglets, but only mild diarrhea in adult swine (bohl, ) . the virus replicates in vitro in several porcine cell culture types (bohl, ) . however, viral isolation in cell cultures from field outbreaks is not always successful (dulac et al., ) . in contrast to virulent infection, peroral vaccination of sows, using attenuated virus, resulted in poor immunity ( saif and bohl, ; bohl, ) . in the latter cases, it was not clear whether or not virus attached to target cells. in this paper, we report the results of experiments on tgev attachment in vitro using four virus strains and two cell culture types. the effect of polycations and polyanions on virus attachment was also examined. the four tgev strains: purdue- , d- , -sg and gep-ii and two cell lines: swine testis (st) and pig kidney (rpd) have been described elsewhere ( laude et al., ; aynaud et al., ) . all but the gep-ii strain can multiply in these two cell lines. strain -sg is a tgev derived from d- by passages in cell cultures and stomach juice of adult swine alternatively (aynaud et al., ) . minimal essential medium (mem) containing iu ml- of penicillin and pg ml-x of streptomycin sulfate was used. for cell growth, the medium was supplemented with % fetal calf serum. virus stock was produced by infecting confluent monolayers of rpd cells in -cm plastic tissue-culture flasks (falcon) with the cell-passaged strains and by inoculation of non-immune newborn piglets for the gep-ii strain. a plaque assay (aynaud et al., ) was used with the following modifications. a volume of . ml of virus suspension containing - plaque forming units (p.f.u.), was inoculated onto the cell monolayers ( ~ × ~ cells per well) in six-well plastic plates (costar). virus attachment was allowed to occur at ° c. at the times indicated in fig. , the inoculum was removed from the cell sheets by washing twice with mem and replaced with mem containing % agarose (indubiose). #ci ml- of [ - h] uridine (amersham, sp. act. ci tool-l). the virus harvested was then partially purified by pelleting in an r- rotor centrifuge (beckman) at ×g for h at ° c. the pellet was resuspended in mem by vortexing and diluted to give a titer of ~ s p.f.u. ml- , corresponding to c.p.m, ml- . the attachment of labelled virus was carried out at ° c. confluent monolayer cells ( ~ × cells per well) grown in -well plastic plates (costar) were incubated with the radiolabelled virus suspension ( . ml per well) for min with gentle rocking. following three washings with mem to remove the inoculum, the cells were solubilized with n naoh then neutralized with . n hc . the whole content of a well was transferred to a scintillation vial. the radioactivity was measured using acs scintillation liquid (amersham) in an lkb liquid scintillation counter. the unlabelled tge virus (strains purdue- , d- , -sg and gep-ii) was first inoculated onto the cell monolayers, grown in -well plastic plates, following incubation for h at °c, the virus was removed by extensively washing the cells with cold mem. the radiolabelled virus was then inoculated and further steps in the experiment were performed as described above. results are expressed as a/ao x , a being the radioactivity associated with the cells which were first inoculated with different amounts of unlabelled virus and ao the radioactivity bound to the control cells. the following products were used. polycations: deae-dextran (pharmacia, mol. wt. -- ), protamine sulfate ( sigma, salmonine, grade x), poly-l-lysine (sigma, mol. wt.= ), poly-l-~-ornithine (sigma, mol. wt. -- ). polyanions: heparin (choay-france), iu ml- and dextran sulfate ( sigma, mol. wt. = ) and an amorphous product: dextran t (pharmacia, mol. wt.= ). these were prepared immediately before use by dilution in mem. the minimum toxic concentration for the cells had been determined previously. the effect of these chemicals on virus attach- ment was tested by using the plaque assay as described above, except that the inoculum containing - p.f.u. was prepared in the presence of the chemical at appropriate concentrations and incubated with confluent monolayer cells for min at ° c. the inoculum was then removed by extensive washing and replaced with mem containing % agarose. the chemicals were omitted from control cultures. to test whether the chemicals had an effect on viral replication step (s) following attachment, after incubation with virus alone and removal of the inoculum, the cells were incubated with the chemicals for min at ° c. the cells were then washed free of the chemicals and mem containing % agarose was added. plaques were formed by infectious virus particles which were attached to the cells and not removed by washing. the percentage of infectious virus particles in the inoculum which attached to the cells, as a function of incubation time, is indicated in fig. . it can be seen that the attachment rate of tgev strain purdue- to the cells was much greater than that of other strains (d- and -sg). after h of incubation with the cells, most of the infectious virus particles ( > % ) of the purdue-ll strain were found attached to the cells, whereas attachment was only % in the case of the d- and -sg strains. results of attachment of [ h ] uridine-labelled virus to the st and rpd cells showed that for any strain tested, the radioactivity was found equally associated with both cell types (table i) . differences in susceptibility to tgev infection, indicated by plaque formation, between the st and rpd cells and effects of temperature on virus attachment were studied by incubating virus inoculum with the cells for min at either or °c. it was found (table ii) that at °c, the infectious virus particles attached to the cells better than at °c (p < . ). the plaque numbers observed also showed that the st cells were significantly (p < . ) more susceptible than the rpd cells. this difference was also observed in infected cells to which agar-overlay medium was added without removal of the inoculum (table iii) . the attachment of the labelled virus was reduced by pre-incubation of the cells with unlabelled virus. the reduction was inversely proportional to the m.o.i. (for purdue- , d- and -sg strains) and dilutions (for the gep-ii strain ) of the unlablled virus previously inoculated. figure shows a typical experiment using the labelled virus strain purdue-ll and the st cells. experiments using the rpd cells and other labelled virus strains (d- and -sg ) gave similar results (not shown). these. results suggested a specificity of tgev attachment sites on the cell surface. moreover, the strain gep-ii, which does not multiply in either st or rpd cells, was found to be able to attach to the cells and to inhibit the subsequent attachment of labelled virus. deae-dextran enhanced virus attachment (fig. ) , when tgev partially purified by pelleting was used. the curves reach a plateau beginning at a concentration of /zg ml- of deae-dextran. an inhibitory effect on virus attachment was obtained when poly-l-lysine, protamine sulfate and dextran sulfate were used (figs. , and ) . the effect of poly-l-~x-ornithine was similar to that of poly-l-lysine (data not shown). similar plaque numbers were observed in the control and in the pre-infected cells which were incubated with polyanions or polycations (for min at °c ) after the removal of inoculum. no effect of heparin and dextran was observed. the effect of all chemicals was consistent for all tgev strains tested. our determination of an incubation period ( min) that preceded maximal virus attachment allowed the effects of temperature, polyanions and polycations on virus attachment to be studied. the results shown in table i suggest that the attachment of tgev is partially thermodependent. differences in attachment rate between strains were also observed. the tgev strain purdue-ll attached to the cells much faster than the others. although both the tgev strains purdue- and -sg were established as high passage strains ( and passages, respectively), the attachment rates of these strains were different (fig. ) . the attachment rates of d- and -sg were found to be similar, however. differences betweeen these two strains have been described (aynaud et al., ) , -sg having higher resistance to acidity and digestive enzymes and smaller plaques in st cells. it is evident that these two characteristics are not related to attachment rate. no explanation is available for the difference in virus attachment rate between strains. interference with infectious virus attachment by varying numbers of defective particles which are present in the different virus inocula is unlikely. although the infectious virus titre of the -sg strain is lower (by log, aynaud et al., ) , the intensity of virus-antibody reaction in elisa is similar for the three virus strains (unpublished data), suggesting a -fold greater number of defective particles for the -sg strain compared to the others. the incubation of ~ - p.f.u. with x cells in a well ( for plaque formation ) would have rendered such an interference insignificant. all three virus strains produced a higher number of plaques in st cells than in rpd cells (table ii) , but the radioactively labelled virus was found to be similarly associated to both cell types. this strongly suggests that the difference in plaque number observed between the two cell lines is not determined at the level of virus attachment. also, the field tgev strain gep-ii, which was unable to multiply in cell cultures even in the presence of deae-dextran, was able to attach to the cells, thereby inhibiting the subsequent attachment of labelled virus. a higher synthesis of viral rna was observed for the three virus strains in st cells than in rpd cells (nguyen, ) . a defective multiplication of tgev may be an explanation for the low susceptibility of pig kidney cell lines which has been described by garwes et al. ( ) . according to these authors, the multiplication of tgev in llc-pk cells is stopped at the maturation step. their observations and our present findings tend to reinforce the statement that genetic susceptibility or resistance to coronavirus is not determined at the level of virus receptors ( shif and bang, ) . the results shown in fig. , on the other hand, demonstrate a specificity of the attachment sites of tgev on the cell surface. it is interesting to note that the difference in attachment site between virus strains which has been dem-onstrated for other viruses, e.g., reovirus (weiner et al., ) , was not observed in the case of tgev. moreover, until now only one antigenic determinant of tgev has been reported (bohl, ) and the present study suggests that this also applies to the attachment determinant. studies of foot and mouth disease virus showed that attachment and antigenicity determinants are different (meloen et al., ) . it is likely that tgev possesses the same characteristic as neutralizing polyclonal antibodies from infected swine were unable to inhibit virus attachment . since the absolute ratio of tgev particles to p.f.u. has not been established, we could not determine the number of virus attachment sites per cell. at an m.o.i, of ~ - p.f.u. per cell, the unlabelled virus strain purdue- inhibited attachment of nearly % of the same radiolabelled virus strain at a m.o.i, of ~ p.f.u. per cell. in agreement with sturman and holmes ( ) , who demonstrated that the coronavirus receptors/cell ratio was low ( ~ ), our results suggest that the ratio of tgev attachment sites per cell could not be high, in contrast to many other viruses, for which the host cell possesses as many as × attachment sites (armstrong et al., ) . effects of polyanions and polycations on the virus cycle as well as on cellular uptake have been studied. these substances are believed to change the electric charges of ligands or cell receptors by combining with them and in that way enhancing or inhibiting the macromolecule (virus)-cell interactions (ryser, ; toyoshima and vogt, ) . the infectivity of other coronaviruses has been reported to be enhanced by deae-dextran (bradburne and tyrrell, ; takayama and kirm, ; hirano et al., ; sato et al., ) . our results on tgev have identified another coronavirus member with this characteristic. the effects of deae-dextran (polycation, enhancer), dextran sulfate (polyanion, inhibitory) and dextran ( amorphous, no effect) are compatible with the explanation for the mechanism of action by electric charges of these molecules, but if the activity of polycations and polyanions relies only on their electric charges, contradictions can be seen in our results. among polycations used, deae-dextran had an enhancing effect whereas others (pll, plo, protamine sulfate) were inhibitory. such contradictions might be explained by the size of molecules used, since the effect of these molecules on cell uptake depends upon size as well as charge (ryser, ) . differences between strains in the level of enhancement or inhibition of virus infectivity by polyanions and polycations (figs. , and ) might be explained by differences in their rate of attachment to the cells. the most important observation was the one-way effect of these molecules on virus attachment with all the strains studied. this again leads to the conclusion that there is no difference in the nature of attachment sites between tgev strains. studies on reovirus receptor of l cells and comparison with reovirus receptor of erythrocytes transmissible gastroenteritis (tge) of swine: survivor selection of tge virus mutants in stomach juice of adult pigs transmissible gastroenteritis the propagation of coronavirus in tissue cultures isolement du virus de la gastroenterite transmissible du porc sur cultures cellulaires et comparaison antig~nique avec deux souches americaines defective replication of porcine tgev in a continuous cell line effect of diethyl aminoethane dextran on plaque formation of mouse hepatitis virus in vitro properties of low and high passaged strains of transmissible gastroenteritis coronavirus of swine studies of the early events of the replication cycle of three variants of mengo encephalomyelitis virus in mouse fibroblast cells the main antigenic determinant detected by neutralizing monoclonal antibodies on the intact foot and mouth disease virus particle is absent from isolated vp viral pathogenesis and immunology.v. determinant of viral virulence and host resistance caractgrisation in vitro du coronavirus de la gastroent~rite transmissible (get) et immunog~nicit~ d'un mutant att~nu~ ( -sg) neutralizing iga and igg do not inhibit attachment of transmissible gastroenteritis (tge) virus a membrane effect of basic polymers dependent on molecular size passive immunity in tge of swine: ig classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus inducement of cytopathic changes and plaque formation by porcine encephalomyelitis virus in vitro interaction of mouse hepatitis virus and macrophages from genetically resistant mice i. adsorption of virus and growth curves molecular biology of coronaviruses improved method for titration of mouse hepatitis virus type in a mouse cell culture interaction of viruses with cell receptors enhancement and inhibition of avian sarcoma viruses by polyanions and polycations interaction of reovirus with cell surface receptor. i. murine and human lymphocytes have a receptor for haemagglutinin of reovirus type key: cord- -wu tuvy authors: katz, jonathan b.; shafer, amy l.; eernisse, kenneth a.; landgraf, john g.; nelson, eric a. title: antigenic differences between european and american isolates of porcine reproductive and respiratory syndrome virus (prrsv) are encoded by the carboxyterminal portion of viral open reading frame date: - - journal: vet microbiol doi: . / - ( ) -b sha: doc_id: cord_uid: wu tuvy antigenic differences between european and american isolates of porcine reproductive and respiratory syndrome virus (prrsv) were revealed by serologic analysis of a recombinant protein derived from prrsv open reading frame (orf ). the hydrophilic carboxyterminal amino acids encoded by the orf of a european (lelystad) isolate of prrsv were expressed as a recombinant fusion protein (bp -p) in a baculovirus gene expression system. sera from gnotobiotic swine exposed to prototypic reference european and american isolates of prrsv and sera from conventionally reared european and american swine convalescing from naturally acquired prrsv infections were used to characterize the bp -p protein. sera from gnotobiotic and conventionally reared swine exposed to european isolates of prrsv were significantly more reactive (p < . ) with bp -p than were the corresponding american prrsv antisera using the indirect immunoperoxidase monolayer assay (ipma). prototypic european, but not american, prrsv antisera also recognized bp -p using western immunoblotting and radioimmunoprecipitation assay (ripa) procedures. however, gnotobiotically derived antiserum to an atypical american-origin prrsv was reactive with bp -p by both ipma and western immunoblot. despite a predicted potential for n-linked glycosylation, studies with tunicamycin and peptide-n-glycosidase f (pngase f) indicated that bp -p was not n-glycosylated in either insect cell cultures or trichoplusia ni larvae infected with the recombinant baculovirus. sera from rabbits inoculated with bp -p failed to neutralize both the european (lelystad) and american (atcc vr- ) reference isolates of prrsv and did not react by ipma with prrsv-infected cell cultures. taken together, the data suggest that the carboxyterminal portion of prrsv orf encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between european and most american isolates of prrsv. f ( pngase f) indicated that bpo -p was not n-glycosylated in either insect cell cultures or trichoplusia ni larvae infected with the recombinant baculovirus. sera from rabbits inoculated with bpo -p failed to neutralize both the european (lelystad) and american ( atcc vr- ) reference isolates of prrsv and did not react by ipma with prrsv-infected cell cultures. taken together, the data suggest that the carbox- in the few years since it was initially described (keffaber, ; lindhaus and lindhaus, ; paton et al., ) , the disease complex now known as the porcine reproductive and respiratory syndrome (prrs) has become an economically significant swine health problem throughout europe and north america (goyal, ) . the viral etiology of prrs was confirmed both in the netherlands (meulenberg et al., ; terpstra et al., ) and in the united states with the isolation of two previously unknown prrs viruses (prrsv), a prototypic european prrsv (lelystad isolate) and a prototypic american prrsv (vr- isolate). these to nm enveloped rna viruses wensvoort et al., ) share common antigenic determinants but may be differentiated using both polyclonal antisera and monoclonal antibodies (nelson et al., ) . serologic analysis of european and american swine naturally exposed to prrsv has confirmed that geographically based antigenic differences do exist between most north american and most european isolates of prrsv; these differences are consistent with those first noted between the lelystad and the vr- isolates bautista et al., ) . there is some evidence, however, that antigenically intermediate prrsv or possibly both prototypic antigenic types may exist within the north american swine population (bautista et al., ) . prrsv has been tentatively classified as an arterivirus, along with equine arteritis virus (eav) and two other proposed members of that genus: lactate dehydrogenase elevating virus (ldv) and simian hemorrhagic fever virus (shfv) (plagemann and moennig, ) . prrsv contains a to kb single strand positive polarity polyadenylated genome (plagemann and moennig, ) and utilizes a nested ' coterminal mr.na gene expression strategy (meulenberg et al., ; conzelmann et al., ) . the prrsv genome contains open reading frames (orfs), including the large ' terminal orfs la and lb encoding the putative viral polymerase and the ' terminal orf encoding the nucleocapsid protein (meulenberg et al., ; conzelmann et al., ) . prrsv virions are believed to contain two other structural, presumably envelope, proteins (nelson et al., ) . it has not been determined which of the orfs through encode these proteins or the identities and functions of the other proteins encoded by these orfs. recombinant dna technology permits the expression of individual gene segments in order to better understand the antigenicity of their protein products and their roles in the host-virus relationship. we report here the expression and serologic analysis of a markedly hydrophilic portion of prrsv orf (lelystad isolate). this segment was of additional interest because comparison of ldv, eav, and prrsv orfs through revealed that the orf protein products of these viruses would be the most divergent in size but potentially the most universally highly n-glycosylated of all the proteins encoded by those orfs (den boon et al., ; conzelmann et al., ; godeny et al., ) . these features suggested to us that prrsv orf might encode a glycoprotein mediating virus-host cell interactions; therefore, it might be a target of host defense mechanisms subjected to selective evolutionary pressure. we hypothesized that it might account in part for the geographically oriented antigenic differences between the majority of european and north american prrs viruses, and that was the basis for this study. the lelystad and vr- isolates of prrsv were provided by c. terpstra, central diergeneeskundic institut, the netherlands, and by d. chladek, boehringer ingelheim inc., usa, respectively. a british reference prrsv isolate, humberside , was a gift from s. edwards, central veterinary laboratory, united kingdom. prrsv isolates and were recovered in this laboratory from midwestem american swine tissues. antisera to the isolate had been found uniformly cross-reactive with numerous north american prrs virus isolates; isolate was of interest because it was completely fastidious for replication in swine pulmonary alveolar macrophages. all viruses except were propagated in cultures of marc- cells, which are a prrsv permissive cell line subcloned from ma- cells (kim et al., ) . the identity of all viruses was confirmed using prrsv-specific monoclonal antibodies in an indirect immunoperoxidase monolayer assay (ipma) format. gnotobiotic swine were used to prepare antisera to each prrsv isolate. each lo-dayold piglet was intranasally inoculated with lo to lo tcid,, of one virus isolate, and the resultant convalescent antiserum was collected between to days post-inoculation. twenty field origin sera derived from clinical cases of prrs in swine from northern germany were generously provided by t. blaha, tierartzliche hochschule, hanover, germany. fifteen american field origin prrsv seropositive swine sera were also obtained from d. kinker, iowa state university, ames, ia, usa. each serum originated from a separate swine herd experiencing prrs, and these sera represented samples from herds located in states in the eastern, southern, and midwestem united states. the ipma ( holm-jensen, ) was used to evaluate serial -fold serum dilutions for reactivity against both the lelystad and the prrsv isolates. these viruses had been inoculated h previously onto separate marc- cell monolayers. immunodetection was accomplished using protein g-horseradish peroxidase (hrp) conjugate and aminoethylcarbazole substrate (zymed, so. san francisco, ca, usa). a prrsv serum neutralization (sn) procedure was used to evaluate selected sera as recently described (yoon et al., ) . recombinant orf antigen, cesium chloride purified prssv (nelson et al., ) , and ultrafiltered concentrates of cell cultures infected with the lelystad isolate were used as antigens in western immunoblotting following denaturing polyacrylamide gel electrophoresis in % to % or % to % gradient gels (laemmli, ; burnett, ) . immunodetection was again accomplished with protein g-hrp conjugate and an insoluble tetramethylbenzidine (tmb) substrate (kirkegaard and perry inc., gaithersburg, md, usa). a radioimmunoprecipitation assay (ripa) recently described for prrsv (nelson et al., ) was used with the same antigens to evaluate sera from the gnotobiotic swine inoculated with prrsv isolates. a hydrophobicity/hydrophilicity plot (kyte and doolittle, ) of the putative orp protein revealed a markedly hydrophobic amino terminus followed by the remaining predominantly hydrophilic % ( of amino acids) of the protein (fig. ) . we postulated that this latter portion might be externalized and, therefore, available to interact with host cell receptors or antibodies and that the amino terminus perhaps represented a biosynthetically cleaved leader peptide or otherwise cryptic segment not directly interactive with the host. to focus our examination on the hydrophilic body of the protein, we excluded the hydrophobic segment through a subunit cloning strategy. total rna was extracted from a cm* cell monolayer infected with the lelystad isolate of prrsv. a -mer oligonucleotide complementary to the viral rna sequence (meulenberg et al., ) nucleotides downstream from the ore termination codon (positions , to , inclusive) was used to prime viral cdna synthesis in a ~ reaction (maniatis et al., ) . a base pair (bp) cdna segment was then amplified through cycles of the polymerase chain reaction (pcr) (saiki et al., ) using a ' primer located between positions , to , and a ' primer ( , to , ) to which a pst i recognition sequence had been appended (fig. ) . following xho i and pst i digestion and gel purification, the pcr product was directionally ligated into a baculovirus polyhedrin gene transfer plasmid, pacsg-his-nt (pharmingen, inc., san diego, ca, usa). the resulting plasmid contained the carboxyterminal amino acid-encoding portion of orf fused in frame to a ' vector sequence encoding amino acids including a polyhistidine domain. the resulting fusion polypeptide had a calculated mass of , daltons ( kda) . following dna sequence verification, recombinant plasmid dna ( ug) was cotransfected with bsu -digested acnpv baculoviral dna ( . ug) (pharmingen, inc.) into a spodopteru frugiperda sf- cell culture using a liposomal transfection reagent (dotap, boehringer marmheim, indianapolis, in, usa). clonally purified baculoviruses expressing prrs virus-immunoreactive material were identified by ipma. separate pcrs of cell culture fluids from monolayers infected by these cloned viruses were used to confirm the presence of the orf gene sequence and the absence of the nonrecombinant baculovirus polyhedrin gene sequence. the latter pcr employed primers spanning the polyhedrin gene insertion site (invitrogen, inc., san diego, ca, usa). one rdna baculovirus, bp , was used for recombinant protein production. recombinant protein (bpo -p) was produced by infecting both sf- cell cultures and -day-old trichoplusia ni larvae according to standard procedures ( o'reilly et al., ) . ] asparagine amidase (boehringer mannheim, inc.) in a further effort to evaluate the n-glycosylation status of the protein. the polyhistidine sequence within bp -p enabled efficient affinity purification of both cell culture and larval origin bpo -p using immobilized nickel ion chromatography as previously described (janknecht et al., ) . purified larval and cell culture origin bpo -p was adjuvanted (fatunmbi et al., ) with avridine (pfizer, inc., groton, ct, usa) and used to immunize four rabbits each on four biweekly occasions. sera from these animals were then evaluated by ipma, sn, western blotting, and ripa for anti-prrsv reactivity. bf'o -p expression was detected in the cytoplasm of infected sf- cells using gnotobiotically-derived lelystad isolate antiserum in an ipma format (fig. ) . bpo -p expression was noted within h post-infection (hpi) and persisted at least to hpi. comparison of bp infected and uninfected cell culture proteins by sds-page revealed the presence of a new protein in the infected cell cultures (fig. a) . the size of this protein was consistent with the kda mass predicted from the amino acid sequence of the fusion protein encoded by bp . the prrsv-specific identity of this protein was confirmed by western immunoblotting (fig. c ). bpo -infected insect larvae contained a similarly sized immunoreactive protein within hpi (figs. b, c). protracted incubation of affinity purified larval bp -p with pngase f did not result in a detectable reduction in apparent bpo -p molecular mass (fig. c ). there was also no detectable decrease in the molecular mass of bp -p from tunicamycin treated cell cultures relative to untreated controls (fig. c ). the ipma, sn, ripa, and western immunoblot procedures were used to evaluate rabbit antisera developed against both larval and cell culture origin bpo -p. none of these sera exhibited respectively. lane : molecular mass markers (kda) panel c: immunoblot of affinity purified larval bpo -p protein with and without prior digestion with pngase f (lanes and , respectively). immunoblot of sf- cell culture origin bpo -p protein produced in the presence or absence of tunicamycin (lanes and , respectively). lane : cell culture control infected with recombinant baculovirus containing vector-only transfer plasmid sequences. prrsv sn activity at a : dilution. all sera were ipma-reactive at : dilution using bp -infected sf- cells but failed to react at a : dilution with isolate-infected marc- cells. these sera also did not react at the : dilution with lelystad isolateinfected cell monolayers fixed between and hpi. two of the rabbit antipeptide sera were reproducibly reactive by western immunoblot with a diffuse ( to kda) band of antigen found in homogenates of marc- cells infected h previously with the lelystad isolate (fig. ) . repeated ripa and western blot attempts using these sera were unsuccessful in identifying a virus-specific protein in purified virion preparations. antisera to gnotobiotic lelystad, humberside , and virus isolates reacted specifically at : dilutions with nitrocellulose blotted bpo -p, while antisera to vr- and virus isolates were totally nonreactive at the same or lower dilutions (fig. b) . ripa results were consistent with these findings. these sera were also evaluated by ipma against lelystad and infected marc- cells and bpo -infected sf- cells (table ) . antisera to the lelystad and humberside isolates were differentially reactive with cells fig. . western immunoblot analysis of sera from two rabbits inoculated with bpo -p. lane : molecular mass markers &da). lanes and : blots of rabbit anti-(bp -p) sera, showing reactivity against to kda proteins found in cells infected with prrsv (lelystad isolate) hours previously. lane : blot of rabbit serum (same as used in lane ) against uninfected cell antigens. lanes and : blots of hyperimmune anti-bpgf-p rabbit serum and gnotobiotic anti-prrsv (lelystad) swine serum, respectively, against the bpo -p immunogen. hyperimmune rabbit serum also detects small ( - kda) breakdown products of bp -p resulting from its catabolism in infected insect cells. 'german sera significantly more reactive (p < . ) with lelystad and bp viral antigens than the corresponding american sera and significantly (p<o.ol) less reactive with antigen than with the other viral antigens. damerican sera significantly more reactive (p < . ) with viral antigen than the corresponding german sera and significantly (p < . ) more reactive with antigen than with the lelystad and bp viral antigens. knot determined. infected with the lelystad isolate and also recognized bpo -p in infected sf- cells. antisera to the vr- and isolates were differentially reactive with isolate infected cells. antiserum to the isolate was nearly equally reactive with cell cultures infected with either of the prrsv and with the bp virus (table ) . convalescent sera from field origin prrsv-infected german and american swine were differentially and antithetically reactive by ipma. a two-way analysis of variance was used to assess the serologic differences. the geometric mean titer (gmt) of the german sera was significantly greater (p < . ) against lelystad and bp viral antigens than against the viral antigen. conversely, american sera were significantly (p < . ) more reactive with viral antigens than either of the other two antigens (table ) . taken together, the data demonstrate that immunoreactive epitopes within the carboxyterminal portion of the protein encoded by lelystad prrsv orf are expressed during the course of european prrsv infections, and that this polypeptide sequence is at least partially responsible for the serologic differences observed between european and american prrsv isolates bautista et al., ) . antiserum to the macrophage-fastidious and antigenically intermediate isolate was reactive with bpo -p, further suggesting the importance of the orf protein in the serologic differentiation of prrsv isolates. the similarity between the ripa and western immunoblot reactivity patterns of bp -p using european and american prssv antigens and gnotobiotically derived convalescent swine antisera suggests that perhaps both linear and conformational orf epitopes may be involved in the antigenic differentiation of european and american prrs viruses. our data support the hypothesis (bautista et al., ) that coexisting high lelystad and atcc vr- prrs virus antibody responses found in some american swine may result from infections with a virus of mixed antigenicity as opposed to dual infections with prototypic american and european-like prrsv isolates. like the homologous predicted ldv and eav orf proteins, the prrsv orf protein contains the greatest number of sites for potential n-glycosylation of all the proteins encoded by orfs through (den boon et al., ; conzelmann et al., ; godeny et al., ) . n-linked glycosidic moieties have frequently been found important in mediating viral infectivity, in directing soluble and cell-associated host immune responses, in protecting critical viral protein epitopes from immune attack, and in interacting with viral polypeptide components to ensure correct viral glycoprotein conformational structure (rademacher et al., ; li et al., ; grigera et al., ) . attempts to produce observably deglycosylated and nonglycosylated forms of bpo -p using both tunicamycin inhibition and enzymatic deglycosylation approaches indicated that the recombinant protein did not contain n-linked glycosidic modifications. these findings must be interpreted cautiously. despite the theoretical potential for a high degree of n-linked glycosylation, the carboxyterminal portion of the native orf protein may in fact not be glycosylated. perhaps more likely however, the aminotetminal peptide sequence omitted during bp construction may have represented a signal sequence required to direct nascent orf into the er-golgi complex for glycosylation during insect larval and sf- cell infections. although insect cells and tissues are generally assumed to be glycosylation-competent, differences between mammalian and insect glycosylation patterns have been observed (rademacher et al., ; shimokawa and smith, ; o'reilly et al., ) and could also account for the data. the addition or removal of very small n-linked glycosides to the bp -p protein might have been undetectable given the molecular size resolution of the western immunoblotting, but this is unlikely because viral n-linked glycosidic moieties are usually substantial in apparent molecular mass (rademacher et al., ) . regardless of the foregoing considerations, the orp of the lelystad isolate clearly encodes epitopes recognized immunologically by the swine host during the course of both experimental and natural infections. monospecific bpo -p rabbit antipeptide antisera reacted by immunoblot but not by ripa with a diffuse band of antigen ( - kda) found repeatedly in virus-infected cell cultures. this material was specific to virus infected cells, but was apparently not of virus structural protein origin, because it was not detected by either ripa or immunoblot using purified virus preparations. the difference between the ripa and the western immunoblot performance of the rabbit antipeptide antisera using unpurified infected cell culture material may be due to the presentation of the relevant reactive linearized nonstructural virus protein epitopes under the conditions of the denaturing immunoblot but not under the milder conditions of the ripa. monospecific bp -p antisera were not neutralizing, but the functional significance of this finding is unclear because other workers have suggested that antibody responses to prrsv may not necessarily be protective and might even participate in antibody-dependent enhancement of infection (choi et al., ) . the failure of monospecific rabbit bpo -p anti-peptide antisera to react with prrsv-infected cells is consistent with the ideas that the natural orf translation product may be conformationally (epitopically ) different from the recombinant bp -p fusion protein, or that the native protein may be a nonstructural, perhaps soluble, virus gene product. lactate dehydrogenase elevating virus, eav, shiv, and apparently prrsv all possess three structural proteins including a nucleocapsid protein encoded by the ' terminal orp (nelson et al., ) . the other two presumed prrsv structural proteins are kda and kda in apparent molecular mass (nelson et al., ) and are thus far removed from the - kda band identified here. although the precise identity and functional role of the prrsv orf gene product thus remain enigmatic, a differential virus serotype-specific serologic response is clearly directed against it by swine exposed to prrsv. serologic survey for lelystad and vr- strains of porcine reproductive and respiratory syndrome (prrs) virus in u.s. swine herds characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) electrophoretic transfer of protein from sds-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein a antibody-dependent enhancement of sirs virus replication isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome (prrs) virus, a member of the arterivirus group enhancement of antibody response of turkeys to trivalent avian influenza vaccine by positively charged liposomal avridine adjuvant complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase elevating virus ( ldv) virology porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus i. detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. a comparative examination using cf, pla, and npla assays rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus reproductive failure of unknown etiology enhanced replication of porcine reproductive and respiratory syndrome ( prrs) virus in a homogenous subpopulation of ma- cell line a simple method for displaying the hydropathic character of a protein cleavage of structural proteins during assembly of the head of bacteriophage t glycosylation is necessary for the correct folding of human immunodeficiency virus gp in cd binding ratselhafte schweinkrankenheit molecular cloning: a laboratory manual differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome (pius) virus by monoclonal antibodies baculovirus expression vectors: a laboratory manual lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses primer-directed enzymatic amplification of dna with a thermostable dna polymerase expression of prostaglandin endoperoxidase synthase- in a baculovirus system experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with lelystad virus: koch's postulates fulfilled antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus a modified semm neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome (prrs) virus in swine sera we thank tom glasson, gene hedberg, wayne romp, melanie harris, and nancy platter for illustrative services and careful manuscript preparation. key: cord- -ckqghr b authors: johnson, michael e.; paul, prem s.; gorziglia, mario; rosenbusch, ricardo title: development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: ckqghr b a dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. recombinant complementary dna (cdna) representing gene (the gene encoding the neutralization antigens in vp glycoprotein) from osu (porcine rotavirus serotype ) and gottfried (porcine rotavirus serotype ) strains were used to determine the optimal hybridization conditions which allow specific detection of group a porcine rotaviruses. probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cdna with ( )p by the random primer extension method. probes were hybridized at various stringencies with viral rna from different rotavirus serotypes bound to nylon membranes. hybridization at low stringency ( % base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. hybridization at high stringency ( % base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. subgenomic gene fragments were then tested to provide more specific probes. a bp fragment from osu gene between nucleotides and and a bp fragment from gottfried gene between nucleotides and were found to be specific as hybridization probes. these studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene to detect and differentiate serotypes of porcine rotavirus. additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype. rotaviruses, members of the family reoviridae, are a major cause of viral diarrhea in many animals and man (woode et al., ; flewett and woode, ; estes et al., ) . rotaviruses have been grouped into five antigenically distinct groups (a to e) with group a probably being the most prevalent (pedley et al., ) . group a rotaviruses share a common group antigen (woode et al., ) on protein vp , an inner capsid protein (greenberg et al., b) . group a rotaviruses have been further classified into two subgroups (i and ii) based on the subgroup antigen also located on vp (greenberg et al., b; estes et al., ) . the majority of animal rotaviruses possess the subgroup i antigen while the majority of human rotaviruses have the subgroup ii antigen (hoshino et al., ) . porcine group a rotavirus strain osu has a subgroup i antigen and the gottfried strain of group a rotavirus has a subgroup ii antigen (hoshino et al., ) . neutralization antigens vp , formerly vp (liu et al., ) , and vp are associated with the outer capsid of rotavirions (greenberg et al., c) . the viral genome of group a rotaviruses consists of segments of double stranded rna that migrate in a characteristic electrophoretic pattern in polyacrylamide gels (kalica et al., ) . the group a rotavirus common and subgroup antigens on vp are coded for in gene segment (estes et al., ; greenberg et al., a) . neutralization antigens of vp and vp are encoded by gene segments and either or , respectively (kalica et al., ; greenberg et al., a) . at least seven serotypes of group a rotaviruses have been reported in accordance with the unified scheme of serotyping rotaviruses from animals and man (hoshino et al., ) . in swine, two serotypes of group a rotaviruses have been recognized (bohl et al., ) . osu and gottfried strains have been designated as the prototype strains for porcine rotavirus serotypes and (bohl et al., ) . according to the unified serotyping scheme established for rotaviruses occurring in animals and man, osu has been classified as serotype and gottfried as serotype . recently, two new strains of porcine rotavirus were isolated in iowa (paul et al., a) . these new strains, designed isu- and isu- , were shown by neutralization tests to be new porcine rotavirus serotypes. two additional rotavirus serotypes have been reported in mexico (ruiz et al., ) and australia (nagesha and holmes, ) . the most common methods for detection of rotavirus are: electron microscopy, enzyme-linked immunosorbent assay (elisa) and polyacrylamide gel electrophoresis (page). serotyping of rotaviruses has been traditionally done by tissue culture isolation followed by plaque reduction neutralization (hoshino et al., ) or fluorescent focus inhibition assays that use hyperimmune antisera to each of the rotavirus serotypes (woode et al., ) . recently, several groups of investigators have developed serotype-specific monoclonal antibodies for use in elisa for serotyping of rotavirus field isolates (shaw et al., ; coulsen et al., ; taniguchi et al., ) . while this approach appears promising, studies have shown that monoclonal antibody based elisa may not detect all rotavirus isolates belonging to the same serotype (coulson et al., ; taniguchi et al., ) . as an alternative, nucleic acid hybridization procedures for detection ofrotavirus have been used (pedley and mcgrae, ; lin et al., ) . similar techniques for differentiating rotavirus serotypes in humans using recombinant cdna hybridization probes specific for gene segment were demonstrated by dimitrov et al. ( ) and lin et al. ( ) . in the present study, a dot blot hybridization assay is described for the detection and serotypic differentiation of porcine rotavirus utilizing hybridization probes prepared from recombinant cdna representing gene from osu and gottfried strains (porcine rotavirus serotypes and , respectively). rhesus monkey kidney cells, ma- (national veterinary services laboratory, ames, ia), were used to propagate rotaviruses. cells were maintained in dulbecco's modified eagle's medium (dmem) (gibco laboratories, grand island, ny) supplemented with % serum plus (hazelton research products, inc., lenexa, ks). the cell monolayers were washed twice with dmem prior to inoculation with virus. the virus inoculum was pretreated with #g of trypsin (type ix; sigma chemical co., st. louis, mo) per ml for h at ° c. the inoculum was absorbed onto cultures of ma- cells for . h in roller bottles and the culture media was replaced with dmem. swine testicular (st) cells (american type culture collection, rockville, md) were used to propagate transmissible gastroentitis (tge) virus and porcine parvovirus (ppv). cells were maintained in eagle's minimum essential medium (hazelton research products, inc., lenexa, ks) supplemented with lactalbumin hydrolysate ( . %) (sigma chemical co., st. louis, mo) and % serum plus. virus inoculum was absorbed onto monolayers of st cells for h prior to addition of fresh medium. rotavirus strains were cultivated in ma- cells. the strains used were as follows: rhesus rotavirus (rrv), representing serotype ; bovine rotavirus strain ncdv, representing serotype and genetic reassortant rotaviruses rrv × d and rrv × ds- containing genes from rrv and the vp gene from human rotavirus serotypes and , respectively (obtained from harry greenberg, veterans administration hospital, palo alto, ca); porcine osu strain (serotype , porcine rotavirus serotype ) originally isolated by bohl et al. ( ) (obtained from gerald woode, texas a & m university, college station, tx); porcine gottfried strain (serotype , porcine rotavirus serotype ) originally isolated by bohl et al. ( ) (obtained from linda saif, oardc, wooster, oh ); porcine rotavirus isolates isu- and isu- (paul et al., a) ; porcine rotavirus serotype field isolates , and from iowa; porcine rotavirus serotype isolates r, , and (janke et al., ) from infected pigs in missouri (provided by robert solorzano, university of missouri, columbia, mo). additional non-rotavirus samples included tge virus miller strain (atcc, rockville, md) pseudorabies vi-rus (prv) strain k (paul et al., ) and porcine parvovirus (ppv) strain nadl- (paul and mengeling, ) . fecal specimens were collected from -day-old gnotobiotic piglets day post-infection with rotaviruses. pigs were infected with osu-infected cell lysates, group c rotavirus cowden strain (atcc, rockville, md) or group b rotavirus (obtained from ken thiel, oardc, wooster, oh). after extensive cytopathic effect, virus infected cell cultures were frozen and thawed twice and cells and supernates were concentrated by centrifugation at x g for h. the pellets were resuspended in pbs and extracted with trifluorotrichloroethane (genetron; e.i. du pont de nemours and co., wilmington, de) and clarified by centrifugation at x g for min. each viral suspension was pelleted through % sucrose at xg for h. the virus pellet was resuspended in pbs and virus particles were further purified by isopycnic centrifugation in cesium chloride density gradients (mccrae, ) . the virus band was collected, virions pelleted at xg for h and the virus pellet resuspended in ml of te buffer ( mm tris-hc , ph . , mm edta). rotavirus double-stranded rnas (dsrnas) were obtained by extractions with an equal volume of phenol-chloroform ( : ) followed by a second extraction of the supernatant with an equal volume of chloroform. rna in the supernatant was precipitated with ethanol by overnight incubation at - °c followed by centrifugation at ×g in a microcentrifuge. the rna pellet was resuspended in te buffer and stored at ° c. confirmation of viral rna was achieved by electrophoresis in laemmli . mm-thick slab gels (laemmli, ) with % resolving and . % stacking gels. electrophoresis was performed at a constant current of ma for h at ° c, and gels were stained with silver (herring et al., ) . viral rna was extracted from fecal samples as previously described (paul et al., b ) . briefly, . g fecal samples were diluted with . ml of sodium acetate buffer, sonicated and clarified by centrifugation at xg for min. the supernatants were harvested, extracted with . ml of phenol-chloroform and clarified by centrifugation at xg in a micro-centrifuge for min. the presence of viral rna was confirmed by polyacrylamide gel electrophoresis. viral rna was obtained from tgev infected cell cultures by extraction with acid guanidium thiocyanate and phenol-chloroform as described previously (chomczynski and sacchi, ) . porcine parvovirus dna was isolated from st cells infected with ppv by phenol-chloroform extraction (davis et al., ) . pseudorabies virus dna was isolated from porcine kidney cell line, mvpk, infected with prv and purified on sodium iodine gradients (paul et al., ) . cellular dna from uninfected ma- cells was obtained by previously described procedures (davis et al., ) . rotavirus dsrna extracted from cesium chloride purified virions was quantitated spectrophotometrically at a wavelength of nm. assuming dsrna has an optical density (od) similar to double stranded dna, an od of corresponds to approximately /~g/ml (maniatis et al., ) . rotavirus dsrna concentration was additionally confirmed by visual comparison of various dilutions of the virus dsrna with dilutions oflambda dna (brl, bethesda, md) standards in % agarose gels stained with ethidium bromide. tgev rna, ppv dna, prv dna, and ma- cellular dna were quantitated spectrophotometrically. various concentrations of rotavirus dsrnas were heat denatured ( ° c, min) in # mm tris-hc (ph . ), mm edta (lin et al., ) , quenched on ice, and further diluted with an equal volume of cold × ssc ( x ssc = . m nac , . m sodium citrate). the denatured samples were filtered onto zeta-probe blotting membranes (bio-rad laboratories, richmond, ca) that had been presoaked with × ssc by using a -well filtration manifold (schleicher and schuell, inc., keene, nh). after airdrying, the membranes were baked in a vacuum oven for h at °c. in addition to rotavirus dsrnas, negative control nucleic acids were spotted onto the membranes. these osu gene cdna cloned in pbr plasmid (gorziglia et al., ) was excised from the plasmid by psti cleavage. gottfried gene cdna cloned in plasmid ptz r was excised from the plasmid by digestion with restriction enzymes hindiii and ecori. the cleavage products were separated on . % agarose gels, the insert excised from the gel, separated from the gel matrix by phenol-chloroform extraction and concentrated by ethanol precipitation (maniatis et al., ) . purified gene cdna inserts were resuspended in te in appropriate volumes and stored at °c. five subfragments were excised from gene from both osu and gottfried cdna inserts ( fig. and table ). the five subfragments (a-e) from osu gene were obtained by digestion of the bp gene cdna insert with sau ai, taqi, and bali restriction enzymes. the five subfragments (f-j) from gottfried gene cdna were obtained by digestion with sau ai, and double as probes. osu gene cdna (lane ) was digested with sau ai, taqi and ball (lanes , and respectively). gottfried gene cdna (lane ) was digested with sau ai (lane ) and with alul and bamhi (lane ). kb dna ladder was used as a marker for molecular size (lanes and ). cleaved cdna was electrophoresed in a . % agarose gel and stained with ethidium bromide. subfragment f was not readily apparent in this photograph. digestion with alui and bamhi. the bp subfragment (e) lying between nucleotides and of osu gene cdna was excised with restriction enzyme bali. the bp subfragment lying between nucleotides and was excised with restriction enzymes alui and bamhi. the subfragments were isolated in agarose gels and purified as previously described. the whole gene cdna inserts and the cdna subfragments were labeled by the random primer extension method (amersham, arlington heights, il) using p-dctp ( ci/mmol) according to the manufacturer's protocol. specific activities of the radiolabelled probes ranged between x and x cpm//zg of dna. several different parameters were chosen to vary the stringency conditions for hybridization. the stringency was adjusted by changing the temperature of hybridization, varying the salt concentration, and by the addition of different amounts of formamide. increasing the temperature of the hybridization minimizes interaction of the probe with partially homologous sequences. as the salt concentration increases, the rate of hybridization increases. conversely, the rate of hybridization decreases as formamide concentration increases. formamide acts to lower melting temperature (tm), the temperature at which nucleic acids are % denatured. therefore, low temperatures ( - ° c) are usually used with high salt concentration ( × to × ssc) with the inclusion of % formamide. hybridization performed at high temperatures ( to o c ) require low salt concentration ( . x to . × ssc ) and may include formamide. initial studies utilized the conditions reported by dimitrov et al. ( ) . membranes with the immobilized nucleic acids were prehybridized in heat sealed plastic bags for h at either ° c or ° c. for some membranes, prehybridization was carried out at °c with % formamide. for all membranes, the prehybridization solution consisted of xssc, xdenhardt's solution ( ×denhardt's= . % each of bovine serum albumin, polyvinylpyrrolidone, and ficoll), mm tris (ph . ), mm edta, . % sodium dodecyl sulfate (sds), and ~tg/ml of sonicated, denatured salmon testes dna (sigma chemical co., st. louis, mo). hybridization was performed for - h in a solution ( - / /cm of membrane) of x ssc, x denhardt's solution, and tris, edta, sds and salmon testes dna as in prehybridization solution, % deionized formamide when required and p-labeled, denatured probes. additional studies were performed utilizing a different set of stringency conditions. the prehybridization solution contained . x ssc or × ssc, mm tris (ph . ), % sds, . % bovine serum albumin, . % polyvinyl-pyrrolidone, . % ficoll, mm edta, /zg/ml of salmon testes dna/ ml, and % deionized formamide. prehybridization was carried out for h at either °c in xssc ( . m na +) or at °c in . xssc ( . m na +) (flores et al., ) . hybridization was conducted for - h with the p-labeled probes using the same reagents as in the prehybridization. following hybridization, membranes were washed by one of two procedures. initial studies utilized the method described by dimitrov et al. ( ) as follows: three times in x ssc- . % sds, twice in . x ssc- . % sds and twice in x ssc. these washes were performed at the same temperature as the hybridization. membranes hybridized in % formamide were washed according to the method reported by flores et al. ( ) . membranes were washed four times at room temperature in x ssc- . % sds, twice in x ssc- . % sds, and twice in x ssc. the last two washes were done at the temperature of hybridization. the air-dried membranes were exposed to kodak xar film (rochester, ny) with intensifying screens at - ° c. the exposure time of the autoradiographs varied between and h. the exposure time was considered correct when ng of homologous dsrna and ng of heterologous dsrna were both detected. homology between pbr plasmid dna sequences and e. coli dna sequences was examined by hybridization of a p-labeled pbr dna probe to phenol-chloroform extracted porcine fecal samples (data not shown ). the probe reacted with a number of the fecal samples whether positive or negative for the presence of rotavirus. these results strongly indicated the presence of homologous sequences in the genomes pf pbr and e.coli. because the strong signal sometimes created by this apparent homology could diminish its specificity to discriminate rotavirus serotypes, all future hybridizations were performed with the gene cdna inserts or selected regions from these inserts without the plasmid. rotavirus cdna probes corresponding to gene from osu strain were tested for reactivity with heterologous rna representing rotaviral serotypes through ( fig. a) . under conditions of low stringency ( ° c, x ssc ) with % base pair (bp) mismatch, reactions of each probe with homologous rna from cell culture propagated virus was visibly stronger than reactions with heterologous rna. however, under these conditions, some reactivity with rna from heterologous rotavirus serotypes did occur. this reactivity was especially evident at the ng level and most notable between osu (porcine serotype ) and gottfried (porcine serotype ). increasing the temperature of hybridization to °c ( % bp mismatch) and °c with % optimization of stringency conditions for the dot blot hybridization assay. rotaviral rna representing serotype (rrv × d), serotype (rrv × ds- ), serotype (rrv), serotype (gottfried), serotype (osu), and serotype (ncdv) were spotted at two concentrations ( ng and ng) and hybridized to osu gene (a) and gottfried gene (b) probes under various conditions of stringency ranging from low ( °c, × ssc, % formamide) to high ( °c, . ×ssc, % formamide). formamide ( % bp mismatch) the reactivity with heterologous rna was slightly decreased. these results led to the imposition of additional changes in the stringency conditions. under conditions of low stringency ( °c, × ssc, % formamide) the allowable bp mismatch is % and the results were similar to those observed at ° c, % formamide ( % bp mismatch ). the osu gene probe did react with ng of gottfried rna as well as with rna from serotype (rrv×d), serotype (rrv), and serotype (ncdv) rotavirus. however, the reactions of the gottfried gene probe ( fig. b ) with heterologous rna decreased appreciably with only minimal reaction at the ng level with osu and serotypes (rrv×d) and (rrv). a slight increase in reactivity with the gottfried gene probe and ncdv was observed under these conditions. increasing the conditions of stringency ( °c, . xssc, % formamide) the allowable bp mismatch was decreased to %. under these conditions, reactivity of both the osu and gottfried gene probes with rna from heterologous serotypes was eliminated. however, under these conditions, there was also a substantial decrease in the signal generated by hybridization of the probes with homologous rna. the strong signal generated at the ng level at low stringency ( % bp mismatch ) was diminished substantially and barely apparent with the osu gene probe and homologous rna at high stringency. based on these observations, additional studies for differentiating rotavirus serotypes were performed at stringencies permitting % bp mismatch ( °c, ×ssc, % formamide) and % mismatch ( ° c, . x ssc, % formamide). the specificity of the dot blot hybridization assays was further evaluated with the inclusion of rotavirus rna from porcine field isolates used as positive controls, and nucleic acid from several other sources used as negative controls. under conditions of low stringency ( % bp mismatch), the osu gene probe reacted very intensely which homologous rna, as well as with rna from porcine field isolates , , and rna from a fecal sample taken from a gnotobiotic pig that had been infected with osu (fig. a) . moderate reactivity was also observed with rna from porcine field isolates , r, , and new porcine rotavirus serotype isu- . the diminished signal with these samples indicates a reduction in the homology of the nucleotide sequence of gene from that with osu. these differences were further amplified under conditions of high stringency ( % bp mismatch). rotaviruses , , , r and have been serotyped as porcine rotavirus serotype . hybridization of the osu gene at high stringency was only indicated in those samples with the strongest signal at low stringency (fig. b) . these included field isolates , , the gnotobiotic fecal sample, and, of course, homologous osu rna. in addition, reaction with rna from heterologous serotype (rrv x d), serotype (rrv), serotype (ncdv) and isolate isu- seen at low stringency (fig. a) were absent at high stringency (fig. b) . field isolates , r, and did not react with the osu probe under these conditions. however, as indicated previously, the signal generated with homologous osu rna was decreased from the ng level to the ng level. specificity of the osu gene probe was further confirmed by the lack of hybridization with ng of negative control nucleic acids from uninfected ma- cell dna, e. coli dna, yeast trna, &[. fig. . specificity and sensitivity of gottfried gene probe under conditions of low stringency. viral rna from osu and gottfried were spotted in -fold dilutions (starting with ng at the top left) on replicate membranes and hybridized to the gottfried gene probe. negative control nucleic acid included uninfected ma- cell dna, e. coli dna, yeast trna, tgev rna, prv dna, and ppv dna. rna from group b and group c rotavirus were spotted at three volume of /a, /a and /d from phenol-chloroform extracts. in addition, rna from rotavirus serotypes i, , , and were spotted along with isu- . tgev rna, prv dna, ppv dna, and rna from fecal samples taken from gnotobiotic pigs infected with group b or group c rotaviruses (fig. b ). similar results were observed with the gottfried gene probe. under conditions of low stringency, the gottfried gene probe reacted strongly with homologous gottfried rna and moderately with isu- , ncdv, and rnas. reaction with osu and other heterologous rnas was observed only at the ng level (fig. ) . under conditions of high stringency, hybridization of the gottfried gene probe was observed only with homologous gottfried rna indicating a strong lack of nucleotide homology between the vp genes of prototype gottfried and heterologous rna (data not shown). the adverse effect of decreased sensitivity was observed again with the gottfried gene probe under conditions of high stringency. the level of detection was decreased from ng to ng. however, sensitivity could be increased by longer exposure (data not shown). as with the osu gene probe, the gottfried gene probe did not react with the negative control nucleic acids. selected regions of gene as serotype specific hybridization probes results using complete gene probes demonstrated the improved sensitivity ( ng level) of the assay under conditions of low stringency. however, and gottfried (b) gene cdna. regions ofgene used as probes are depicted in schematic as subfragments a-j. rotavirus rna from infected cell cultures representing serotype (rrv×d), serotype (rrv×ds ), serotype (rrv), serotype (gottfired), serorype (osu), serotype (ncdv), and isolates isu- and isu- were spotted on replicate membranes at two concentrations ( ng and l ng) and hybridized to the selected probes under low stringency hybridization conditions ( °c, × sscd, % formamide). osu and gott-,"~;~a c,,hf~.rn,~ntc under conditions of low stringency, the probes reacted with rna from heterologous rna, especially at higher concentrations. therefore, we hypothesized that serotype specific probes prepared from selected regions of gene and the utilization of low stringency conditions may improve the specificity while maintaining sensitivity. five subfragments of gene from osu and gottfried (table ) were p-labeled and hybridized to heterologous rotavirus rna under both high and low stringency conditions. significant background was observed with subfragments a-d from osu gene and subfragments f-i from gottfried gene (fig. ) . the bp subfragment from osu gene cdna (subfragment e) and the bp subfragment from gottfried gene cdna (subfragment j) were hybridized to rotavirus rna from serotype (rrv x d), serotype (rrv x ds ), serotype (rrv), serotype (gottfried), serotype (osu), serotype (ncdv) and isolates isu- and i su- (fig. ) . under conditions of low stringency, the base pair mismatch is %. subfragment probe e reacted primarily with osu rna but also reacted slightly with rna from isu- (fig. a ). the gottfried subfragment probe j reacted only with gottfried rna (fig. b ). epidemiological studies to determine the prevalence and distribution of porcine rotaviruses have been hampered by the lack of suitable assays capable of both detection and differentiation of porcine rotavirus serotypes. due to the involvement of two viral proteins, vp , formerly vp (liu et al., ) , and vp in stimulating neutralizing antibodies, results from traditional methods can be ambiguous . recently, a number of groups have developed vpt-serotype specific monoclonal antibodies (mab) to be used with an enzyme-linked immunosorbent assay (elisa) for the determination of human rotavirus serotypes (coulson et al., ; taniguchi et al., ) . although sensitive and efficient, these methods do have their limitations. coulson et al. ( ) reported that rotavirus from some human stool specimens did not react with their specific mab but did react with other mabs to the same serotype. this non-reactivity of serotype-specific mabs may have been caused by small base changes in the nucleotide sequence that codes for the neutralization epitopes within the vp gene (dyall-smith et al., ; green et al., ) or possibly by changes in the proximity of the neutralization epitopes present in the protein's native form versus that of the linear molecule (green et al., ; morita et al., ) . in another study, taniguchi et al., ( ) reported the inability of a serotype-specific mab based elisa to determine the rotavirus serotype of % of infected human stool samples, possibly due to intraserotype antigenic variation. dot blot hybridization assays have been utilized by a number of groups both for the detection of human rotavirus in clinical samples (flores et al., ; pedley and mc-crae, ) and for the detection and characterization of enteric coronavirus infection of baby pigs (shockley et al., ) . additional studies have been reported in which improved cdna probes of gene-specific segments of the rotavirus genome were used to characterize human rotavirus by subgroup and serotype (dimitrov et al., ; lin et al., ; flores et al., ) . our initial studies used stringency conditions reported by dimitrov et al. ( ) in which serotyping of rotaviruses was achieved with the use of cloned cdna of gene (which codes for the subgroup antigen) and gene (which codes for the neutralization antigen). they reported that rotavirus serotypes and could be differentiated with the gene cdna probe by using conditions of low stringency ( ° c), and comparison with the results obtained with hybridization at °c, % formamide. in addition, a further increase in the stringency conditions to ° c, % formamide allowed differentiation of serotypes and from serotype . based on the equation tm (dna-rna)= . +i . log (na+)+ . (%g+c)+ll. (%g+c) - / #bp in duplex- . (% formamide) (wahl et al., ) where (g+c) was given a value of . (flores et al., ) , the base pair mismatch allowed for the formation of stable hybrids was % at low stringency and % at high stringency. however, green et al. ( ) reported - % divergence in the amino acid sequences of the vp proteins of several different human rotavirus serotypes and to % homology among rotaviruses of the same serotype. in addition, % nucleotide identity between osu and gottfried strains has been reported indicating that the conditions of stringency would have to be increased to differentiate porcine rotavirus serotypes and . the conditions of stringency used by flores et al. ( ) were found to be suitable for our hybridization assay. under low stringency ( ° c, x ssc, % formamide) the calculated base pair mismatch was %. as expected, under these conditions some reactivity was observed with the cdna probes and heterologous rna. under these conditions, however, a sensitivity level of at least ng was achieved with rna from homologous rotavirus. in addition, rna from isolates and from iowa and r, , and from missouri, all typed as porcine rotavirus serotype , could be identified with the osu probe (fig. a) . reactivity was also observed with rna from isu- , recently reported to be a new serotype of porcine rotavirus. similar reactivity of isu- with the gottfried probe was observed (fig. ) . these data indicate that isu- gene may have higher homology with osu and gottfried than that observed with osu or gottfried with other rotavirus serotypes. use of this new porcine serotype is suggested for the assessment of the specificity of osu and gottfried probes in the detection of porcine rotavirus serotypes. under conditions of high stringency ( °c, . x ssc, % formamide) the allowable base pair mismatch for the formation of stable hybrids was reduced to %. under these conditions, a high level of specificity was achieved as indicated by the elimination of reactivity with heterologous rna. however, these conditions decreased the level of sensitivity for homologous rna from ng at low stringency to only ng (fig. b ). in addition, the reactivity of isolates and was greatly reduced and the reactivity with isolates , r, and was eliminated (fig. b) . these results indicate sufficient diversity may exist within the vp gene among rotaviruses of the same serotype that, at conditions of high stringency, they may not be identified. results obtained with different stringencies indicated sensitivity was higher at low stringency but specificity was lower. based on these observations, we concluded that probes prepared from selected regions ofgene that are serotype specific and hybridized to rotavirus rna under low stringency conditions may provide the specificity and sensitivity achieved at two different stringencies. recently, several groups have determined the nucleotide sequence of the vp gene of more than rotavirus strains. comparison of their deduced amino acid sequences have revealed six discrete divergent regions (a to f) among different serotypes (green et al., ; green et al., ) : amino acids - (region a), - (region b), - (region c), - (region d), - (region e), and - (region f). a comparison of these same regions among rotaviruses belonging to the same serotype revealed - % homology (green et al., ) . furthermore, dyall-smith et al. ( ) identified three regions corresponding to regions b, d, and e as the antigenic sites involved in serotype-specific neutralization. based on these reports, serotype-specific probes were prepared from nucleotide sequences corresponding to some of the previously described regions. the bp subfragment from osu gene (fragment e) lies between bases and (fig. a ) and encompasses coding regions corresponding to region c-f. the bp subfragment from gottfried gene (fragment j) lies between bases and (fig. b ) and encompasses coding regions corresponding to regions b-d. under low strongency conditions ( ° c, x ssc, % formamide ) the probes were highly specific for rna from homologous serotypes. these results showed that subfragments from the middle ofgene from both osu (subfragment e) and gottfried (subfragments i and j ) were most specific for homologous rotavirus rna. sequences within these subfragments contained more divergent regions among rotavirus serotypes than the other subfragments. surprisingly, subfragment g of gottfried gene was less specific than subfragment j although both subfragments were nearly identical in size and location within gene . possible contamination of subfragment g with subfragment h could have occurred with excission of subfragment g from the agarose gel. these two subfragments are nearly identical in size (subfragment g was bp and subfragment h was bp) and subfragment h may have a large percentage of conserved sequences among rotavirus serotypes. however, this problem can be avoided in future studies by amplification of serotype-specific regions by polymerase chain reaction technology. sensitivity studies and further evaluation of the specificity of these probes are currently under investigation. practical application of the dot blot hybridization assay may require some modification. these include densitometry to minimize subjective comparisons of the reactions, the use of probes representing gene that encodes vp , and the construction of cdna probes from selected regions in which highly conserved bases at the ' end have been removed (flores et al., ) . specific regions of dna can now be amplified using synthetic oligonucleotides by polymerase chain reaction (pcr) and used as probes. additionally, the use of nonradioactive labeled probes would eliminate the costly, cumbersome, and time consuming method of radioactive labeling. recently, an enzyme-labeled oligonucleotide probe was developed to detect human rotavirus rna in clinical samples using a dot blot hybridization assay (olive and sethi, ) . the authors reported the assay to be highly sensitive and specific when compared with polyacrylamide gel analysis and enzyme-linked immunoassay. results of this study, combined with the availability of amplification techniques and alternative methods of labeling probes, offer the potential for nucleic acid probes to be used as versatile diagnostic tools for detection and serotyping of porcine rotaviruses, especially for the detection of noncultivable rotaviruses. hybridization assays could also facilitate the collection of epidemiological information such as the distribution and prevalence of porcine rotavirus serotypes. such information would be useful in formulating strategies for future development of efficacious vaccines for rotavirus-induced diarrhea in swine. isolation and serotyping of protein rotaviruses and antigenic comparison with other rotaviruses single-step method of rna isolation by acid guanidium thiocyanate-phenol-chloroform extraction simple and specific immunoassay using monoclonal antibodies for serotyping human rotaviruses basic methods in molecular biology detection of rotaviruses by nucleic acid hybridization with cloned dna of simian rotavirus sa genes location of the major antigenic sites involved in rotavirus serotype-specific neutralization rotaviruses: a review the rotaviruses. brief review a dot hybridization assay for detection ofrotavirus conservation of the fourth gene among rotaviruses recovered from asymptomatic newborn infants and its possible role in attenuation dot hybridization assay for distinction ofrotavirus serotypes molecular cloning of human rotavirus genome gene sequence of the vp serotype specific glycoprotein of gottfried porcine rotavirus comparison of the amino acid sequences of the major neutralization protein of four human rotavirus serotypes prediction of human rotavirns serotype by nucleotide sequence analysis of the vp protein gene gene coding assignments for growth restriction, neutralization and subgroup specificities of the wa and ds- strains of human rotavirus serological analysis of the subgroup protein of rotavirus using monoclonal antibodies production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaquereduction neutralization analysis by plaque reduction neutralization assay ofintertypic rotaviruses suggests that gene reassortment occurs in vivo single and mixed infections of neonatal pigs with rotaviruses and enteroviruses: virological studies detection of differences among human and animal rotaviruses, using analysis of viral rna genes of human (strain wa) and bovine (strain uk) rotaviruses that code for neutralization and subgroup antigens cleavage of structural proteins during the assembly of the head of bacteriophage t cdna probes of individual genes of human rotavirus distinguish viral subgroups and serotypes identification of simian rotavirus sa genome segment product molecular cloning: a laboratory manual double-stranded rna viruses analysis of serotype-specific neutralization epitopes of vp of human rotavirus by the use of neutralizing monoclonal antibodies and antigenic variants new porcine rotavirus serotype antigenically related to human rotavirus serotype detection of human rotavirus by using an alkaline phosphatase-conjugated synthetic dna probe in comparison with enzyme-linked immunoassay and polyacrylamide gel analysis evaluation of a modified live-virus vaccine for the prevention of porcine parvovirus-induced reproductive disease in swine differentiation ofpseudorabies (aujeszky's disease) virus strains by restriction endonuclease analysis isolation of two new serotypes of porcine rotavirus from pigs with diarrhea isolation of a bovine rotavirus with a "super short" rna electrophoretic pattern from a calf with diarrhea a rapid screening assay for detecting individual rna species in field isolates of rotaviruses definition of two new groups of atypical rotaviruses molecular and antigenic characterization of porcine rotavirus ym, a possible new rotavirus serotype specific enzyme-linked immunoassay for rotavirus serotypes and diagnosis of porcine and bovine enteric coronavirus infection using cloned cdna probes direct serotyping of human rotavirus in stools by an enzyme-linked immunosorbent assay using serotype -, -, -, and -specific monoclonal antibodies to vp molecular hybridization of immobilized nucleic acids: theoretical concepts and practical considerations the isolation of reovirus-like agent associated with diarrhoea in colostrum-deprived calves in great britain morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice, and foals antigenic relationships among some bovine rotaviruses: serum neutralization and cross-protection in gnotobiotic calves the authors thank ms. mary brooks, ms. marti morgan, ms. laura pusateri, mr. mark dennis, and ms. deb deyoe for technical assistance. this study was supported in part by grants from the iowa biotechnology council, iowa livestock health advisory council, and usda formula funds ( section of the farm bill). key: cord- - g zklzy authors: pocock, d.h. title: characterisation of rotavirus isolates from sub-clinically infected calves by genome profile analysis date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: g zklzy rotaviruses isolated from sub-clinically infected calves from a single farm were analysed by genome profile analysis. the isolates showed genomic variation and eight different profiles were observed, including one which was atypical for group a rotaviruses. the ′ terminal labelling method for the analysis of genome profiles used in this study required only ng of viral rna, an increase of -fold in sensitivity over ethidium bromide staining for detecting all rotavirus genome segments. however, dual infections involving two rotaviruses with distinct profiles could not be detected if the concentrations of the viruses differed by > -fold. although rotaviruses are frequently associated with acute diarrhoea in man and animals, sub-clinical infections are common (chrystie et al., ; de leeuw et al., ) . in a recent longitudinal survey mcnulty and logan ( ) reported the detection of rotavirus in % of calves during the first weeks of life and of these % showed no clinical signs. a similar survey carried out on children found that rotavirus shedding was detected to the same degree in children with or without diarrhoea (barron-romero et al., ) . the reasons for sub-clinical reactions to infection have not been fully elucidated, but there is experimental evidence that oral administration of antibody during virulent rotavirus infection eliminates any clinical signs of the infection (snodgrass and wells, ; bridger and brown, ) . differences in virulence among bovine rotaviruses have also been reported (carpio et al., ) but naturally occurring avirulent strains have not yet been isolated and characterised. since genomic diversity among rotaviruses was found to be extensive (kalica et al., ; rodger and holmes, ) , genome profile analysis has been used to characterise virus isolates. to improve the sensitivity of this technique clarke and mccrae ( ) described a method in which the viral rna was ' terminal labelled prior to electrophoresis, so that the rna could then be detected in the polyacrylamide gels by autoradiography instead of ethidium bromide staining. this report investigates the use of this technique for the characterisation of rotaviruses isolated from subclinically infected calves, as the first stage in a study of asymptomatic rotavirus infections in calves. the uk strain of bovine rotavirus (bridger and woode, ) was grown in ma cells with serum-free eagles mem containing . pg m - trypsin (sigma chemical co. ltd., type ). isolates of bovine rotavirus were obtained from sub-clinically infected calves on a farm where such infections were common (reynolds, ) . rotavirus was detected in the faecal samples of these animals by enzymelinked immunosorbent assay (elisa) (reynolds et al., ) . ten ml of a % faecal suspension were extracted twice with ml fluorocarbon (arklone p, ici ltd.) and the virus pelleted from the aqueous phase by centrifugation at x g for h. cell culture grown virus was similarly pelleted from supernatant fluids after clarification at x g for rain. virus pellets were resuspended in buffer ( mm naci; mm cac ; . % sds; mm tris/hc ph . ) and proteinase k (sigma chemical co. ltd., protease type xi) was added to give a final concentration of gg m - . the mixture was incubated at °c for min and extracted twice with vol phenol:chloroform ( : v/v). sodium acetate (ph . ) was added to the aqueous phase to give a final concentration of . m and the rna was precipitated with % ethanol. after centrifugation at x g for rain, the rna pellet was dissolved in . ml distilled water and stored at - °c. rna concentrations were determined by spectrophotometry: d: nm was assumed equivalent to pg mr' (galuard and joklik, ) . the method of rna labelling was essentially that described by clarke and mccrae ( ) , except that pg rnase-free bovine serum albumin (miles laboratories ltd.) was added to the reaction mixture to prevent adsorption of the t rna ligase to the reaction vessel. unincorporated radioactivity was removed by chromatography on whatman cfll cellulose columns. electrophoresis was carried out on % polyacrylamide gels with a % stacking gel using the discontinuous buffer system described by laemmli ( ) , at either ma constant current for -- h or overnight at ma. the gels were either stained with a p g m - aqueous solution of ethidium bromide (eb) for h to detect unlabelled rna or dried onto filter paper and autoradiographed when p-labelled rna was used. rna extracted from the uk strain and from an isolate with a c profile was adjusted to give equal concentrations as estimated by spectrophotometry. mixtures containing a constant amount of c rna and varying amounts of uk strain rna were prepared and ' terminally labelled as above. samples of the labelled ds-rna mixtures containing c.p.m. were electrophoresed on a % gel. by ' terminal labelling of ng of viral rna, all rotavirus segments were detected after autoradiographic exposure for h (fig. la) . the amount of rna could be reduced to ng if the exposure time was extended to -- days (data not shown). a comparable gel stained with ethidium bromide (fig. lb) showed that the smallest amount of rna which gave a complete pattern was ~g, i.e. there was a -fold difference in sensitivity between the two methods. genome profiles were obtained from the rotavirus-positive faecal samples of all calves, and the patterns fell into eight types, c --c (fig. ) . there was clear evidence of genomic variation and this variation was not confined to any segment or group of segments. the profile type c differed from the normal pattern produced from group a rotaviruses as no band was observed in the normal segment region of the gel but a band was evident between segments and . viruses with these profiles were detected in the faecal samples of eight calves by an elisa specific for group a rotaviruses indicating that they must belong to this group. profiles containing the expected segments of ds-rna were found from samples. one profile, however, had an extra four bands suggesting that this calf had been infected with two viruses having c and c type profiles. in this study only one sample showed evidence of mixed infection with two rotaviruses. it was therefore important to determine whether mixed infections were rare or whether the technique simply could not detect them. to investigate the latter, mixtures containing known amounts of ds-rna from two rotavirus isolates with disparate genome profiles (uk strain and a c type) were used to simulate the type of samples that might be recovered from calves concurrently infected with these two viruses. after ' terminal labelling the rna segments were analysed by electrophoresis (fig. ) profiles, and meant that in a faecal sample containing two rotaviruses if one virus was present at a concentration -fold less than the other, i.e. log dilution, it would not be detected. rotaviruses isolated from sub-clinically infected calves were characterised by genome profile analysis. the ' terminal labelling of rotavirus ds-rna used in this study was found to require only ng of viral rna to give a genome profile after electrophoresis. comparison of this technique with ethidium bromide staining for the detection of rotavirus ds-rna segments showed it to be -fold more sensitive, allowing the successful analysis of all the elisa positive samples used. herring et al. ( ) used silver staining to visualise the genome segments and reported an increase of -to -fold over ethidium bromide. however this method suffers the same problem as ethidium bromide staining in that low molecular weight segments are difficult to detect, because they both depend on the total weight of rna present in each band whereas the ' terminal labelling method relies on the number of molecules of each segment. genomic variation was observed between isolates, and this was not confined to any particular genome segment or group of segments, a finding similar to that reported by rodger and holmes ( ) for rotaviruses from calves with enteric disease. eight different profiles were observed and all were typical for group a rotaviruses (pedley et al., ) , except for the c type which was typified by the lack of a band in the segment region and the appearance of a band between segments and . variation in the position of segment has been reported for subgroups and of human group a rotaviruses (kalica et al., ) , although not to the extent seen with the c isolates. rotaviruses with unusual genome profiles have been detected in man and animals (pedley et al., ; snodgrass et al., ) , but these isolates have distinct group antigens and their genome profiles lack a triplet of segments , and , having similar electrophoretic mobilities, characteristic of group a viruses. therefore, the detection of c profile viruses by a group a specific elisa and the presence of the , and segment triplet in their genome profiles both confirm their assignment to group a. the detection of these viruses highlights the caution needed if genome profile analysis alone is used to assign isolates to a particular rotavirus group. the detection of calves dually infected with rotaviruses by the appearance of extra bands in genome profiles was found to depend on the relative rates of excretion of the two virus strains. it is therefore possible that other rotavirus variants were present in the samples but remained undetected, and underlines the requirement of cloning these isolates before further experimental work, e.g. animal inoculation, is undertaken. work is now in progress to adapt these genetic variants of rotavirus for growth in cell culture so that further antigenic and virulence characterisation can be carried out. one isolate, with a c profile, has been isolated and cloned in cell culture and shown by inoculation into gnotobiotic calves to be avirulent (bridger and pocock, ) . asymptomatic rotavirus infections in day care centers development of immunity to porcine rotavirus in piglets protected from disease by bovine colostrum variation in virulence of bovine rotaviruses neonatal calf diarrhoea: identification of a reoviruslike (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy comparative virulence of different bovine rotavirus isolates asymptomatic endemic rotavirus infections in the newborn a rapid and sensitive method for analysing the genome profiles of field isolates of rotavirus rotavirus infections in calves in dairy herds quantitation of the relatedness of reovirus serotypes , and at the gene level rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels comparison of human and animal rotaviruses by gel electropboresis of viral rna distinctive ribonueleic acid patterns of human rotavirus subgroups and cleavage of structural proteins during the assembly of the head of bacteriophage t longitudinal survey of rotavirus infection in calves molecular characterization of rotaviruses with distinct group antigens the epidemiology of enteric virus infections of calves evaluation of elisa and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces comparison of the genomes of simian, bovine and human rotaviruses by gel electrophoresis and detection of genomic variation among bovine isolates rotavirus infection in lambs: studies on passive protection comparison of atypical rotaviruses from calves, piglets, lambs and man key: cord- - z crx authors: lu, wei; duhamel, gerald e.; benfield, david a.; grotelueschen, dale m. title: serological and genotypic characterization of group a rotavirus reassortants from diarrheic calves born to dams vaccinated against rotavirus() date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: z crx two strains of bovine rotavirus (brv), designated strain nebraska scottsbluff- (ns- ) and ns- , were isolated from neighboring cow-calf beef cattle ranches where dams had been vaccinated with a commercial vaccine containing group a brv strain neonatal calf diarrhea virus (ncdv)-lincoln (p :g ). nothern blot by hybridizations using whole genomic rna probes indicated that strains ns- and ns- had identical group a rna electrophoretic patterns and were homologous at all gene segments. strain ns- was compared with reference group a brv strains using serological and genotypic methods. in vitro virus neutralization assays indicated that strain ns- was neutralized by a g -specific neutralizing monoclonal antibody (mab) and guinea pig hyperimmune serum (gphs) raised against brv strain b (p :g ), but not by g -specific neutralizing mab or gphs raised against brv strain brv strain b (p :g ). nucleic acid hybridization experiments using whole-genomic rna probes revealed that gene segment of strain ns- differed from brv strains ncdv-lincoln and b , but hybridized with strain b . conversely, gene segment of strain ns- hybridized with brv strain b , but not with brv strains ncdv-lincoln and b . a g-specific cdna probe produced by reverse transcription polymerase chain reaction (rt-pcr) amplification of strain ns- hybridized specifically only with g strains ncdv-lincoln and b , but not with g strain b . co-electrophoresis experiments using strains ns- , b , and b further confirmed these results, suggesting that strain ns- was a naturally-occurring reassortant brv between strains b and b . taken together these results indicated that a naturally-occurring group a brv reassortant with a p gene different from the vaccine virus was responsible for the diarrheal syndrome observed on both ranches. results from this study also indicate the existence of at least different gene segments among group a brv infecting cattle. strains b and b . taken together these results indicated that a naturally-occurring group a brv reassortant with a p gene different from the vaccine virus was responsible for the diarrheal syndrome observed on both ranches. results from this study also indicate the existence of at least different gene segments among group a brv infecting cattle. keywords: rotavirus; cattle, rotavirus; diagnosis, rotavirus; diarrhoea; vaccination bovine rotavirus is an important etiological agent of neonatal calf diarrhea worldwide. economic losses due to brv infection in the cattle industry include losses associated with retarded growth, medication costs and animal death. rotavirus is a member of the reoviridae family and the viral genome is composed of segments of double-stranded rna which are surrounded by a double-shelled capsid (estes and cohen, ) . currently, rotaviruses are classified into groups (a through g) based on antigenic differences on the inner capsid protein (vp ). group a rotaviruses which are commonly associated with neonatal calf diarrhea (theil, ) , contain major outer capsid proteins, p or vp and g or vp encoded by gene segments and or , respectively (estes and cohen, ) . the p and g proteins contain antigenic epitopes which are involved in neutralization of viral infectivity (hoshino et al., ; offit et al., ) . based on antigenic differences in the p and g proteins, group a rotaviruses have been classified into different p-types and different g-types (estes and cohen ; isegawa et al., ; hussein et al. ; parwani et al. ) . at least different brv g-types have been associated with neonatal calf diarrhea (mebus et al. ; woode et al., ; matsuda et al., , snodgrass et al., , snodgrass et al., hussein et al. ; parwani et al., ) . in the united states, type g appears to be most widespread, with type g accounting for a lower percentage of infections ~ . limited information is available on the prevalence of brv p types in cattle. however, one study indicates different brv p-types in the united states, with p being the most prevalent . reference strains for each p-and gtypes known to infect cattle include strains ncdv-lincoln (pi:g ), united kingdom (uk) and b (p :g ), and b (p :g ) (woode et al., ; zheng et al., ; matsuda et al., ; hardy et al., , hardy et al., . a vaccine for prevention of rotavirus-associated neonatal calf diarrhea has been commercially-available in the united states for over years (sharpee et al., ; saif and jackwood, ) . however, brv continues to be routinely identified in feces of calves with diarrhea, often from herds where vaccination of the dams with brv vaccines is current (g. duhamel, personal observation). there has been no detailed studies on the genetic background of brv causing calf diarrhea on cattle ranches where breeding stock are vaccinated with the commercial vaccine. in this paper, we present serological and genotypic characterizations of brv strains isolated from newborn calves with diarrhea that were born to dams vaccinated with commercially-available brv vaccines in beef cattle ranches in nebraska, united states. case history. outbreaks of neonatal calf diarrhea occurred in the spring of on separate but adjacent beef cow-calf ranches in western nebraska. clinical aspects of these outbreaks including data on the vaccination schedule with commercially-available vaccines containing brv strain ncdv-lincoln have been described elsewhere (grotelueschen et al., ) . the age of the calves at the onset of diarrhea ranged from days to days on ranch , and morbidity associated with diarrhea was approximately % in calves from heifers and % in calves from cows. mortality records were not available from ranch . on ranch , . % of the calf crop developed diarrhea at approximately days of age and % of the affected calves died as a result. fecal samples obtained from calves with diarrhea on each ranch were submitted to the nebraska veterinary diagnostic laboratory system for routine laboratory examinations. rotavirus was the only significant pathogen identified. reference virus strains. strain ncdv-lincoln was obtained from american type tissue . after adsorption for h at °c, the inoculum was removed and the cell monolayers were washed twice with mem before adding serum free mem containing /xg/ml of trypsin, u/ml of penicillin, and /xg/ml of streptomycin. the inoculated cultures were maintained at °c and examined daily for cytopathic effect. after days, the cells were frozen and thawed times, and the lysate was passaged onto fresh monolayers more times. the presence of rotavirus was confirmed by staining infected monolayers with fluorescein-conjugated calf anti-brv antibodies (fitc-calf anti brv; national veterinary service laboratory, ames, ia). cytopathic brv isolates, designated nebraska scottsbluff (ns- ) and ns- from ranch and ranch respectively, were plaque purified after the third passage, as previously described (hoshino et al., ) . virus stock was kept at - °c until needed. the rna from each field brv strain was extracted with phenol-chloroform and their rna electrophoretic patterns were compared with reference brv strains after separation on a discontinuous, vertical sodium dodecyl sulfate-polyacrylamide gel (sds-page) ( % resolving, % stacking) run in tris buffer ( . m tris, . m glycine, . % sds [ph . ] ) at °c using a constant current of ma (biorad protean ii electrophoresis unit, biorad, richmond, ca). the electropherotype of each virus individually and after co-electrophoresis with reference brv strains b and b was visualized after staining with ethidium bromide. monoclonal and polyclonal antibodies. eight-to -week old balb/c ann mice were immunized intraperitoneally with fluorescent focus units of brv strain ncdv-lincoln in equal volumes of freund's complete adjuvant. identical booster injections were given weeks later. three weeks after the last booster injection and days prior to harvest of splenocytes, the mice were immunized intravenously with ncdv-lincoln diluted in pbs. mouse splenocytes were fused with ns- myeloma cells, and the culture supernatants from hybridomas were screened for the presence of neutralizing antibody against the homologous brv (coulson et al., ) . after subcloning twice by limiting dilution, ascites tumors were produced as previously described (galfre et al., ) . the vp specificity of mab / was confirmed by radioimmunoprecipitation (harlow and lane, ) . mab b -n , specific for vp of brv strain b was provided by dr. gerald n. woode (zheng et al., ) . guinea pig hyperimmune sera (gphs) were produced against brv strains b and b by repeated immunization of rotavirus-seronegative guinea pigs with gradient purified virus mixed in equal volumes of freund's complete (first immunization) or incomplete (subsequent boosters) adjuvant. serological characterization of field brv. the serological relationship between the brv field strain ns- , and reference strains b , ncdv-lincoln and b was determined using fluorescent focus neutralization assays (ffn; knowlton et al. ) with modifications. briefly, /xl of -fold dilutions of mab or heat-inactivated ( min at °c) gphs was mixed with /zl of virus ( tcidso ) in -well culture plates ( wells per dilution) and incubated for h at °c. the virus-antibody mixture then was transferred to fresh ma- cell monolayers grown in -well culture plates. the plates were centrifuged at g for h at room temperature. after h of incubation at °c in a co atmosphere, the cell monolayers were fixed with % acetone in water and stained with fitc-calf anti brv (nvsl). the neutralization titer was expressed as the reciprocal of the highest dilution of antibody which completely inhibited fluorescence. northern blot with whole genomic rna probes. whole genomic rna probes of field and reference brv strains were synthesized and labelled with a-~ p-gtp ( ci/mmol, amersham co., arlington heights, il) by in vitro transcription from purified single-shelled viral cores (flores et al., ) . rna from each virus was separated by sds-page, blotted onto nylon membranes (amersham), and fixed by irradiation with ultraviolet light ( nm) for rain. northern blot hybridizations were performed as previously described (hesse et al., ) , except that the membranes were prehybridized for h and hybridized for h. g-type classification offield brv. two primers, complementary to nucleotides to of the minus strand and nucleotides to of the plus strand of the vp gene of group a rotavirus (flores et al., ) were synthesized (national sciences, plymouth, mn). after denaturation at °c for min, purified viral rna from strains ns- , b , ncdv-lincoln and b was used as rna template and reverse transcribed (rt) into single stranded cdna by polymerase chain reaction (pcr) as described by the manufacturer (geneamp rna pcr kit, perkin elmer cetus, norwalk, ct) using a thermal cycler (geneamp tm , perkin elmer cetus). the final volume of the rt mixture was /xl [ . u//xl reverse transcriptase, mm mgc , × pcr buffer, mm dntps, u//zl rnase inhibitor and pmol of each primer]. incubation time and temperature for rt were min at °c, min at °c and min at °c. the resulting cdna was amplified by adding taq polymerase and the volume of the pcr reaction was adjusted to /zl (final concentration: mm mgc , × pcr buffer and . u/ /zl tag polymerase). amplification consisted of cycles with sec at °c for denaturing; min at °c for annealing and min at °c for extension in each cycle. the pcr products were separated by electrophoresis in % agarose gels (seaplaque gtg, fmc rockland, me) and transferred to nylon membranes by southern blotting. the pcr amplified cdna fragments from strains ns- and b were purified from agarose gel (bio , la jolla, ca), and labelled with a- p-dctp ( ci/mmol, amersham) using an oligo-labelling kit (pharmacia lkb biotechnology, piscataway, nj). each radioisotope labelled cdna probe was hybridized with a southem blot membrane using the same conditions as for northem blot hybridizations described above. virus isolation and identification. cytopathic brvs were isolated from fecal samples obtained from calves with diarrhea on both ranches. presence of brv was confirmed based on the ultrastructural appearance of the virus particles and positive fluorescence by direct fluorescent antibody staining. sds-page indicated that strains ns- and ns- had identical rna electropherotypes consisting of a - - - rna migration pattern characteristic of group a rotaviruses (fig. ) . comparison of electropherotypes with reference brv strains revealed that gene segment of strain ncdv-lincoln had a lower molecular weight than other brvs and gene segment of strain b moved slower than other brvs (fig. ) . by co-electrophoresis gene segments , , , , and of strain ns- migrated similarly to strain b , whereas gene segments , , and had migration patterns identical to strain b (fig. ) . in contrast, gene segments and of strain ns- migrated differently from either reference strain b and b , and gene segment could not be resolved. serological characterization offieldbrv. results of ffn assays of the field and reference brv strains are summarized in table . strain ns- was neutralized by the mab / and the gphs raised against brv strain b , but not by the mab b -n or the gphs raised against brv strain b . northern blot with whole genomic rna probes. northern blot studies with whole genomic rna probes indicated that each virus probe hybridized to all gene segments of its homologous virus (fig. ) . additionally, strain ns- hybridized at all gene segments with the ns- whole genomic rna probe. although, the field and reference strains of brv had a high degree of homology at gene segments , , , , and , gene segment of strain ns- hybridized only with gene segment of strain b , but not with strains ncdv-lincoln and b . additionally, gene segment of strain ns- hybridized with strain b , but not with strains ncdv-lincoln and b . gene segments , and migrated in close proximity, making it impossible to determine their origin. g-type classification offield brv. the pcr products obtained from brv strains ns- , b , ncdv-lincoln, and b consisted of a cdna fragment of approximately nucleotides (fig. ) . cross-hybridization experiments using ot-a p-dctp labelled g-spe- a b fig. . southern blot hybridization between reference and field brv strains using g-type specific cdna probes from strains ns- (a) and b (b). lanes: , ns-i; , b ; , ncdv-lincoln; , b . cific cdna probes showed that the g-specific cdna probe made from strain ns- hybridized with g strains b and ncdv-lincoln, but not with g strain b . conversely, the g -specific probe made from strain b hybridized only to itself, and not to strains ns- , b and ncdv-lincoln (fig. ) . two strains of brv, designated ns- and ns- , obtained from diarrheic calves from cow-calf beef cattle ranches where severe brv-associated calf diarrhea occurred despite vaccination of the dams with brv strain ncdv-lincoln (pi:g ) were characterized. page and northern blot analyses revealed that the field strains ns- and ns- had identical group a rotavirus electropherotypes and cross-hybridized completely at all gene segments, suggesting that a single strain of brv was responsible for the diarrheal syndrome observed on both ranches. group a rotavirnses have been assigned to different g-types on the basis of serological methods including neutralization, plaque reduction, and enzyme-linked immunosorbent assays using serotype-specific mabs and monospecific polyclonal antisera (estes and cohen, ) . more recently, nucleotide sequence analyses has shown that rotaviruses from the same g-type have a high degree of sequence homology in their variable regions, and this is the basis for production of g-type specific differential probes (flores et al., ) . the oligonucleotide primer pair used in this study generated a nucleotide cdna fragment representative of the variable region of each of the field and reference brv strains examined. specific hybridization of the cdna probe obtained from strain ns- with g brv strains ncdv-lincoln and b , but not with g brv strain b indicated that strain ns- belonged to the same g-type as the vaccine strain used on these ranches. serological studies confirmed this result. northern blot hybridization studies with whole genomic rna probes revealed that gene segment of strains ns- hybridized with strain b , but not with the vaccine strain ncdv-lincoln, suggesting that strains ns- and b belonged to the same p-type which is different from strain ncdv-lincoln. this result is in agreement with the sequence analysis data of hardy and co-workers ( ) . the results from our study also support recent reports indicating the existence of at least distinct gene segment among brv in the united states (hardy et al., ; parwani et al., ) . since gene segment of strain b has been shown to have a high degree of sequence homology with that of strain uk (hardy et al., ) , brv strains uk, b and ns- most likely belong to the same ptype. although strains b and ns- had the same p-and g-genes, co-electrophoresis and whole genomic rna hybridization studies revealed that they had different rna electropherotypes and gene segment , respectively. gene segment of strain ncdv-lincoln cross-hybridized with that of strain b , but not with strains b and ns- . conversely, gene segment of strain b hybridized with that of strain ns- . our results thus indicate the existence of at least different gene segment among group a brv. gene segment encodes the nonstructural protein ns (estes and cohen, ) , but the exact function of this protein in the biology of rotavirus infection is unknown. however, it has been noted that gene segment displays non-random segregation during rotavirus mixed infections in vivo and in vitro (gombold and ramig, ) . it is possible that strain ns- gained a selective advantage over other strains of brv carrying ncdv-lincoln gene segment . the segmented nature of the rotavirus genome allows reassortment of individual viral gene segment between or more different viruses during mixed infection (estes and cohen ) . rotavirus reassortants have been made experimentally using in vitro and in vivo methods (gombold and ramig, ; urasawa et al., ; hesse et al., ) . based on this evidence, a rotavirus strain potentially can contain any combinations of genes in nature. naturally-occurring rotavirus reassortants with gene segments derived from different human rotaviruses have been reported (matsuno et al. ; nakagomi and nakagomi, ) . results from our study suggest that strain ns- is a naturally-occurring brv reassortant with gene segments derived from brv strains b and b . currently, prevention of rotavirus-associated neonatal calf diarrhea is based on passive protection by maternal antibodies following immunization of the dam (sharpee et al., ; saif and jackwood, ) . calves are protected from rotavirus infection when receiving colostrum containing high titer of rotavirus-specific neutralizing antibodies. it is well established that the p and g proteins have antigenic epitopes involved in neutralization of viral infectivity in vitro and protection in vivo and can segregate and elicit neutralization antibodies independently (hoshino et al. ; offit et al., ) . although the information on homotypic and heterotypic immunity and protection is incomplete, it is likely that passive protection with monovalent vaccine is effective only against challenge with brv homologous to the vaccine strain at the p and g genes. in the united states, commerciallyavailable rotavirus vaccines for cattle contain only strain ncdv-lincoln. however, it is now becoming clear that in contrast to natural recovery from infection which results in high titers of p-specific neutralizing antibodies, parenteral immunization primarily elicits gspecific neutralizing antibodies (shaw et al., ; richardson et al., ; ward et al., ) . differences in p proteins has been proposed as a possible mechanism responsible for a lack of cross-protection in vivo between g brv strains ncdv-lincoln and b (zheng et al., ) . we have shown that strains ns- and ncdv-lincoln belong to the same g , but differ at gene segment encoding the p protein, and gene segment encoding protein ns . our results are in agreement with the assumption made by zheng and coworkers ( ) , and further suggest that failure of passive protection provided by monovalent vaccine could result from infection of susceptible calves with a brv containing a p gene different from the vaccine strain. the benefits from vaccination with monovalent vaccine for prevention of brv-associated diarrhea in neonatal calves may be less than optimal due to diversity of p-and g-types occurring in nature. neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates rotavirus gene structure and function use of transcription probes for genotyping rotavirus reassortants identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein vp antibodies to major histocompatibility antigens produced by hybrid cells lines analysis of reassortment of genomic segments in mice mixedly infected with rotaviruses sa and prv possible vaccination failure in beef cow herds caused by infection with rotavirus distinct from the vaccine virus: clinical observations comparative amino acid sequence analysis of vp for vp serotype bovine rotavirus strains ncdv, b and uk amino acid sequence analysis of bovine rotavirus b reveals a unique outer capsid protein vp and confirms a third bovine vp type in: antibodies -a laboratory manual production and characterization of vp /vp reassortant swine rotaviruses derived from gottfried and osu parental strains serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization independent segregation of two antigen specificities (vp and vp ) involved in neutralization of rotavirus infectivity detection of rotavirus g , g , g , and g in feces of diarrheic calves by using polymerase chain reaction-derived cdna probes a vp sequence highly conserved in human strain au- and feline rotavirus strain frv- development of an improved method for measuring neutralizing antibody to rotavirus presence of three types (vp serotypes) and two g (vp serotypes) among bovine rotavirus strains characterization of a human rotavirus strain which is possibly a naturally-occuring reassortant virus cell culture propagation of neonatal calf diarrhea (scours) virus molecular evidence for naturally occurring single vp gene substitution reassortment between human rotaviruses belonging to two different genogroups role of gene segments and in determining rotavirus virulence and protection against rotavirus challenge characterization of field strains of group a bovine rotaviruses by using polymerase chain reaction-generated g and p type-specific cdna probes analysis of homotypic and heterotypic serum immune responses to rotavirus proteins following primary rotavirus infection by using the radioimmunoprecipitation technique enteric virus vaccines: theoretical considerations, current status, and future approaches immunogenicity of a vaccine containing inactivated bovine rotavirus and coronavirus combined with an escherichia coli bacterin serotypic analysis of vp and vp neutralization escape mutants of rhesus rotavirus bovine rotavirus serotypes and their significance for immunization rotavirus serotype and predominate in cattle nongroup a rotaviruses genetic reassortment between two human rotavirnses having different serotype and subgroup specificities immunodominance of the vp neutralization protein of rotavirus in protective natural infections of young children antigenic relationships among rotavirns: serum neutralization and cross-protection in gnotobiotic calves comparative studies of the antigenic polypeptide species vp , vp , and vp of three strains of bovine rotavirus the authors thank dr. prem paul, veterinary medical research institute, iowa state university, ames, iowa, and mr. eric nelson, south dakota state university for their technical assistance. the research was supported in part by funds provided by the united states department of agriculture/csrs, animal health project no. neb. - , immunobiology of enteric diseases of swine and cattle. this paper is published as paper university of nebraska-lincoln, lincoln, ne. key: cord- -grndfx b authors: koopmans, m.; van den boom, u.; woode, g.; horzinek, m.c. title: seroepidemiology of breda virus in cattle using elisa date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: grndfx b two direct blocking enzyme linked immunosorbent assays (elisa) for the detection of antibodies to breda virus in sera of cattle were compared. an elisa with consecutive addition of antigen and test serum to an antibody-coated plate gave higher positive: negative absorbence ratios than an elisa in which antigen and test serum were added simultaneously. sera collected from breeding and fattening herds in the netherlands (n = ) and the f.r.g. (n = ) were tested, and antibodies to breda virus were demonstrated in % of adult cattle. ninety percent of newborn calves had high levels of maternal antibodies, which waned until the age of months. active seroconversion occurred between and months in most animals. during an outbreak in of neonatal calf diarrhea in a beef herd in breda, ia, u.s.a., a new virus was detected. this "breda virus" (brv) is morphologically and antigenically different from known bovine viruses and causes diarrhea in gnotobiotic and colostrum-deprived calves (woode et al., ) . an antigenic relationship of brv with berne virus (bev) was discovered (weiss et al., ) ; bev has been extensively characterized (weiss and horzinek, ; horzinek et al., a,b) and proposed as prototype of the new family toroviridae horzinek et al., a,b) . other breda viruses were found in the feces from a colostrum-deprived calf in ames, ia (woode et al., ) and in - -month-old diarrheic calves in ohio (saif et al., ) . all viruses shared antigens as determined by enzyme-linked im-munosorbent assay (elisa), with - -fold differences between the homologous and heterologous reactions. for this reason and on the basis of hemagglutination inhibition tests and immune electron microscopy the three isolates were assigned to two serotypes: breda virus serotype i (brv ) is represented by the original breda, ia isolate, and serotype (brv ) comprises the ohio isolate and the second iowa isolate (woode et al., ) . so far, all attempts to grow brv in vitro have failed (woode et al., ) ; recent observations by lamouliatte et al. ( ) need to be confirmed. for this reason, only limited studies on the characterization of the virus (koopmans et al., ) , its diagnosis, pathology and epidemiology (woode, ) were done. in neonatal calves, brv infections spread rapidly with diarrhea commencing as early as - days after birth (woode, ) . large amounts of virus ( viral hemagglutinating units ml- ) were detectable in feces - days after infection. in experimentally infected calves incubation periods ranged from to h (woode et al., ) . the role of older cattle and of other animal species in brv epidemiology is not known. in the present communication we describe the distribution of brv antibodies in the netherlands and the f.r.g. and some epidemiological aspects of the infection; the data were obtained using two modifications of the elisa method. we chose to use brv in our studies because it is relatively stable as compared with brv (koopmans et al., ) . a gnotobiotic calf (gc ) was fed a suspension of brv (iowa isolate at passage in gc) at day of age; diarrheic fecal samples were collected at and h. no other virus was observed to be present, by electron microscopy, hemagglutination and tissue-culture methods, including bovine coronavirus, rotavirus, astrovirus, parvovirus and bovine pestivirus (bvdv). a % suspension in phosphate buffered saline (pbs) was clarified by low speed sedimentation ( ×g for min) and virus from the supernatant was spun through a % (w/w) sucrose solution onto a cushion of % (w/w) sucrose. the interphase was collected, diluted fold in pbs, layered on top of a linear - % sucrose gradient and centrifuged to equilibrium at xg for h. the peak fractions ( . g m - ) were used as antigen in elisa. a gnotobiotic calf (gc ) was fed brv (iowa isolate at passage in gc) at day of age. breda virus was extracted from the diarrheic feces of this calf and semipurified by ultracentrifugation through % sucrose. at and days post-infection, the calf was vaccinated intramuscularly with brv with freund's incomplete adjuvant and at days post-infection with brv without adjuvant. the calf was bled days later. the hyperimmune serum was shown to be free of antibodies to rotavirus, bovine coronavirus, astrovirus and bovine pestivirus (bvdv) and by electron microscopy and tissue-culture isolation methods, no other virus was observed to be present in the feces. immunoglobulins were precipitated from the serum by salt fractionation (hudson and hay, ) and quantified using the lowry method. a fraction of the purified igg was conjugated with horseradish peroxidase as described by nakane and pierce ( ) . for the serosurvey, bovine sera were obtained from veterinary diagnostic institutes in arnsberg (n-- ), niirnberg (n-- ) and neumiinster (n-- ) and from the virology department of the veterinary school in hannover, f.r.g. (n-- ); additional samples were collected at farms in hessen and baden-wiirtemberg, f.r.g. (n= ). the sera representing the dutch cattle population were obtained from animal health services in overijssel (n= ), gelderland (n= ), friesland (n-- ) and west/midden nederland (n-- ), the central veterinary institute (cvi), lelystad (n-- ), and the department of large animal medicine of the veterinary faculty in utrecht (n-- ). the sera obtained from the dutch animal health services and the german veterinary diagnostic institutes were collected for regular screening purposes. the cvi sera included sera from specified pathogenfree (spf) calves and paired samples from milking cows from farms where a tentative diagnosis of winter dysentery was made, based on the following signs: outbreak of rapidly spreading scours in milking herds with a morbidity rate approaching %, lasting - days and resulting in a sharp drop in milk production (hoyer et al., ) . the sera from the veterinary faculty in utrecht included paired samples from an outbreak of acute respiratory disease in month-old calves and six sera from newborn calves with blood protein levels below g l- as a result of insufficient colostrum intake. the sera were coded and stored at - ° c until testing was done. in addition, colostrum samples from healthy dairy cows were tested. a direct blocking elisa was used for mass serology. advantages of this test are that antibodies of different ig classes and from different animal species can be detected, and that antibodies are detected also at low concentrations (ellens, ) . a modification of the method described by brown et al. ( ) was em-ployed. in short, the wells in a polystyrene plate were coated with purified bovine anti-brv immunoglobulin (capture antibody, babrv ig) diluted : in . m na cojnahco ph . by overnight incubation at ° c; all subsequent incubation steps were done in -zl volumes at ° c for i h unless indicated otherwise. to remove excess immunoglobulin, the plates were washed with a solution of . m nac containing . % tween . from this point onward, two different techniques were followed. in the first approach (consecutive method) a : dilution of the brv antigen preparation, purified from calf feces was added to the coated wells in elisa buffer (pbs supplemented with . m nac , mm edta ph . and . % tween ). the plates were washed and the field serum to be tested was added at a : dilution in elisa buffer (pbs supplemented with . m nac , i mm edta ph . and . % tween ). after another rinse, purified bovine anti-brv immunoglobulin conjugated to horseradish peroxidase (babrv ig-po) diluted : in buffer was added, and bound conjugate was visualized after adding the substrate ( . m citric acid, ph , containing . % h and . mm , '-azinodi-( -ethylbenzthiazoline sulfonic acid). high absorbence is seen in the absence of anti-brv antibodies whereas the reaction is blocked when the ~serum contains antibodies. hyperimmune sera against brv and cryptosporidia or rotavirus, prepared similarly to ba-brv ig, served as positive ( % blocking) and negative ( % blocking) reference preparations. all the sera from the netherlands were tested in this system. in the other type of test (simultaneous method), antigen and test serum were mixed in a separate microtiter plate. after i h of incubation, the mixture was pipetted into the babrv ig-coated plates. the remaining steps were similar to the consecutive test method. all sera from germany were tested in this way. a comparison was made between both methods by testing serum samples in parallel. the blocking results were grouped into one of the following clusters: - %, - %, >~ % inhibition of binding of babrv ig-po. positive to negative (p/n) ratios were determined for both elisa modifications by testing a known positive (gc ) and negative (gc ) serum times in parallel. the optimal combination of babrv ig concentration and brv dilution was determined by checkerboard titration: a microtiter plate was coated with serial -fold dilutions of babrv ig in coating buffer and serial -fold dilutions of the antigen in elisa buffer were added. we used a combination of dilutions giving p/n ratios of >/ in our routine testing. the optimal conjugate concentration was determined by testing serial -fold dilutions in elisa buffer at constant antigen and ig-coating concentrations. a dilution with a p/n ratio of >i was used in all subsequent experiments. the results are expressed as blocking percentages; they were calculated using the formula: (ane~ -at) percentage blocking-(aneg --apos) x at = mean absorbence at nm of the test serum; aneg----mean absorbence of the negative reference serum; apos = mean absorbence of the positive reference serum. to detect a possible correlation between percentage of blocking and antibody titer the sera were assigned to groups according to the percentage of blocking ( - %, - %, etc. ) at a dilution of : . from each group, sera were selected and titrated in serial -fold dilutions in the consecutive elisa. titers were calculated from the highest serum dilution resulting in >/ % blocking when compared with the positive and negative reference sera. the correlation coefficient was calculated using pearson's test (dixon and massey, ) . when using the consecutive method more animals were found negative for antibodies to brv or had low blocking percentages (table ) . a higher p/n ratio was also noted between positive and negative reference sera, when tested in the consecutive elisa method ( . + . ) as compared with the simultaneous method ( . + . ); this difference is significant (p< . , n-- , student's t-test). the correlation coefficient between the blocking percentages and the serum titers in the consecutive elisa was . (p< . , pearson's test; fig. ). as can be seen from fig. , the sera with blocking percentages of < and > do not follow the normal distribution pattern. expressed in serum titers, distributions of blocking percentages in sera from the f.r.g. and the netherlands are shown in fig. a and b ; since most sera came from the dutch provinces gelderland and overijssel we show the results obtained with these samples separately (fig. c and d ) . when disregarding the - % columns, a normal distribution can be recognized; median values are situated at - % blocking in fig. b and c (the netherlands and gelderland), and at - % in fig. a and d (germany and overijssel) . in fig. , the percentages of animals with antibodies to brv ( >~ % blocking, corresponding to a serum titer of/> ) are shown for different age groups (n-- ). ninety percent of calves < month old had antibodies, % at months of age; then a gradual decline was noted until at approximately - weeks of age most calves were seronegative. when the animals had reached the age of about months, seroconversion took place. from this age onward an increasing fraction of animals had antibodies to brv, with . % of adult animals detected in our populations; . % of adult animals had blocking percentages of >~ %, corresponding to titers of >/ . six sera from adult bulls of a closed artificial insemination center as well as sera from spf calves and six sera of hypoproteinemic newborn calves had blocking percentages of ~< %. all colostrum samples had high levels of brv antibodies ( - % blocking). in view of the regular occurrence of winter dysentery in the netherlands, we were interested to know whether seroconversion to breda virus antigens would occur. paired serum samples from adult milking cows were, therefore, collected during outbreaks and were tested. seroconversion was defined as > % rise in percentage blocking between sera collected as soon as possible after the onset of disease, as compared with sera collected - weeks later. antibody rises occurred in out of farms, with % (n= ), % (n= ), % (n = ) and % (n = ) seroconversion (average % ). in another group of paired sera, from -month-old calves with a history of acute respiratory disease, seroconversion was seen in four out of animals. three of these animals had low starting titers ( ~< % blocking). in our hands, the simultaneous elisa resulted in comparatively more animals being grouped into the high blocking percentage columns. partially antibody-covered brv antigen (as should be found after preincubation in the simultaneous elisa) will probably not bind as efficiently to the antibody-coated polystyrene plate as brv alone does. during the subsequent washing cycles these complexes may be rinsed away and no binding sites for the conjugate will be left, resulting in a false-positive blocking reaction. in combination with the higher p/n ratios determined for the consecutive elisa we decided to use this method in all further experiments. at this point all german sera had already been assayed in the simultaneous test. when the antibody concentration in a test serum is at equilibrium with the antigen, % blocking will occur. at higher antibody concentrations, however, the elisa results will be the same, leading to an accumulation of animals in the - % blocking group. this is likely to occur when tests are performed at a constant serum dilution ( : in our case ). when disregarding these groups in the frequency distribution of blocking percentages, a normal distribution can be recognized. the graph for the sera from germany has a maximum at lower values as compared with the graph from the netherlands. the different test system used for the german sera does not explain this lower median value: a higher percentage of seropositive animals should be expected, as discussed above, leading to a right shift of the distribution of blocking percentages. a similar difference was observed within the dutch sera between the provinces gelderland and overijssel. one reason for this finding may be a lower incidence of brv infections in farms in overijssel and germany, resulting in less frequent booster infections. a second possibility is that different sampling strategies between the animal health services would give similar results: if a relatively high percentage of animals between and months of age is tested, when most calves are seronegative, lower overall values may be calculated; the age was unknown for many animals. third, a torovirus serotype, different from that in our test, may exist in cattle in europe. when studying our elisa results per animal, lower average blocking percentages are found in the overijssel area; therefore, the presence of a different serotype in this province is a possibility. ninety percent of newborn calves had antibodies to brv. antibodies were absent in specified pathogen-free calves from the central veterinary institute, lelystad, and in -day-old diarrheic calves from the department of large animal medicine with low blood protein levels as a result of insufficient colostrum intake. this indicates that the brv antibodies in young calves are maternally derived as has been found for antibodies to other pathogens (snodgrass and wells, ) ; indeed all colostrum samples tested had high levels of antibodies to brv. the rise in percentage of seropositive animals at months of age indicates an infection in the -month age group. for rotaviruses, enteric infection in the presence of circulating antibodies has been proven (moerman et al., ; snodgrass and wells, ) . we have preliminary data indicating that a similar situation exists for brv. the sudden seroconversion of most calves at the age of - months coincides with the time they are stabled after the grazing period; this leads to closer contact with older cattle and exposure to infection. crouch and acres ( ) have shown that up to % of adult cows excrete rotavirus and % coronavirus with their feces. the role of fecal spread from seropositive cows in brv epidemiology remains to be studied. our findings of . % positive serum samples are in agreement with those of other authors reporting . % by elisa in the u.s.a. (woode et al., ) and % by neutralization test in switzerland ; in gt. britain, % of cattle was found seropositive (brown et al., ) . in that study, however, a serum was considered to contain brv-specific antibodies at > % blocking. when using the same threshold in the interpretation of our results a comparable % of dutch cattle (n-- ) would be found positive. the higher percentages, however, are more likely to reflect the true distribution, since they have also been found in neutralization tests with their intrinsically higher sensitivity. both brv serotypes have been isolated from outbreaks of diarrhea and are pathogenic under experimental conditions (woode, ) . the pathogenicity under farm conditions, however, remains to be proven. with this intention we have tested paired serum samples from field outbreaks of winter dysentery. in four out of the five tested farms, a varying percentage of animals ( - %) showed a distinct antibody rise at the time of the second testing. the seroconversion seen in cattle aged - months (fig. ) does not account for this high number of seroconversions, since only milking cows were tested. further studies are needed to examine the role of brv in this disease. serological evidence for infection with bovine coronavirus in outbreaks of winter dysentery in japan has been presented (takahashi et al., ) ; in the netherlands no such evidence was found (p.w. de leeuw, personal communication, ) . the seroconversions seen in sera from four out of calves, aged months and with a history of acute respiratory disease, may be coincidental although in experimental infections with brv , ocular discharge and hyperpnea were seen (woode et al., ) . detection of breda virus antigen and antibody in humans and animals by enzyme immunoassay prevalence of rotavirus and coronavirus antigens in the feces of normal cows introduction to statistical analysis. mcgraw-hill kogakusha laboratory diagnosis in neonatal calf diarrhoea toroviridae: a taxonomic proposal a new family of vertebrate viruses: toroviridae. intervirology toroviridae, a proposed new family of enveloped rna viruses practical immunology surface proteins of breda virus further studies on breda virus prevalence and significance of viral enteritis in dutch dairy calves enzyme labelled antibodies: preparation and application for localisation of antigens studies on an enteric "breda" virus in calves. nd passive immunity in rotaviral infections bovine epizootic diarrhea resembling winter dysentery caused by bovine coronavirus the proposed family toroviridae: agents of enteric infections purification and partial characterization of a new enveloped rna virus (berne virus) antibodies to berne virus in horses and other animals breda and breda-like viruses: diagnosis, pathology and epidemiology studies with an unclassified virus isolated from diarrheic calves diagnostic methods for the newly discovered "breda" group of calf enteritis inducing viruses comparative studies on three isolates of breda virus of calves we would like to thank the animal health centers of friesland, overijssel, gelderland, west/midden nederland (the netherlands), arnsberg, niirnberg, neumiinster (f.r.g.), the virology department of the veterinary school in hannover and the dutch central veterinary institute, lelystad, for sending us sera. key: cord- - mj isyl authors: chanter, n.; hall, g.a.; bland, a.p.; hayle, a.j.; parsons, k.r. title: dysentery in calves caused by an atypical strain of escherichia coli (s - ) date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: mj isyl dysentery lasting – days was produced in five -day-old colostrum-fed calves, after inoculation with an atypical strain of escherichia coli s - ; peak excretion of s - occurred during the period of dysentery. two calves were killed when clinical signs were most severe and bacteria were seen attached to the surfaces of enterocytes in the large intestine; microscopic lesions were seen in these areas. the lesions were identical to those previously reported in a natural outbreak of dysentery in calves, from which e. coli s - was isolated, and to those seen in gnotobiotic calves experimentally infected with s - . reinfection of the three surviving calves – days later with s - and primary infection of two calves aged and days did not cause dysentery. four of coliforms isolated from field outbreaks of calf diarrhoea resembled the atypical strain s - . these four isolates and s - did not produce heat-stable enterotoxin, but all produced a toxin cytopathic for vero and hela cells. two of the four isolates were inoculated alone into -day-old gnotobiotic calves deprived of colostrum; neither calf developed dysentery but microscopic lesions identical to those produced by s - were detected in the large intestines of both animals. an atypical strain of escherichia coli (designated $ - ) was isolated from the faeces and intestinal contents of farm calves aged -- days old during an outbreak of dysentery (hall et al., ) and reproduced the disease after experimental infection of gnotobiotic calves . the distinctive characteristics of $ - included an atypical colony morphology on macconkey agar, production of urease and anaerogenicity, but it was identified as e. coli in particular by its abilities to produce acid in macconkey broth at °c and indole at °c. $ - was shown by an immunoperoxidase method to adhere to the mucosae of the large bowel in gnotobiotic and farm calves with dysentery, and microscopic lesions were seen in the colonic and rectal mucosae (hall et al., ) . these were identical to those seen in piglets (moon et al., ) , man (rothbaum et al., ) and rabbits (takeuchi et al., ) infected with e. coli which do not produce the classical enterotoxins and which are not invasive. the objectives of the experiments reported here were firstly to establish the pathogenicity of e. coli s - for conventional calves, secondly, to determine whether e. coli with atypical characteristics were an important cause of calf diarrhoea by examining a culture collection of isolates from field outbreaks of diarrhoea, and thirdly, to investigate by experimental infection of gnotobiotic calves, the association between the atypical characteristics of these isolates and pathogenicity. electron microscopy was used to examine more precisely lesions produced by atypical e. coli and to compare them with those described previously. experimental design five friesian cross calves were infected with e. coli $ - and compared with two uninfected calves for signs of dysentery. two of the five infected calves were killed when clinical signs were maximum to investigate the presence of lesions. immunity and age resistance to infection were investigated by allowing the other three calves to recover and then reinfecting them and comparing the results with two primarily infected calves, one age matched and one days older. the nine calves were left with their dams for h, then moved to a loose box, separate from other calves, and fed on a milk replacer diet. on the fourth day, the calves were moved to rooms in an isolation unit; they were fed sterilised canned milk. infected and control animals were housed in separate rooms. calves were inoculated orally after the morning feed; ml of sterilised canned milk (carnation ltd.) containing . x -- . x ° colony forming units (cfu) of e. coli grown on bovine blood agar at °c for h, was given by syringe. calves -- were infected when days old; calves and were killed on days when clinical signs were judged to be most severe. calves -- were allowed to recover and then reinfected when , and days old, respectively. calves and were infected for the first time at and days old respectively and were controls for the reinfection of calves -- . calves and were housed together until and days of age, respectively. faeces were sampled daily. blood samples were taken at days, at reinfection and daily from calves with dysentery. rectal temperatures were measured twice daily. the strain of atypical e. coli s - ( :k-:h-) was used . rotavirus and coronavirus were detected by enzyme linked immunosorbent assays of faecal samples taken daily and calicivirus by electron microscopic examinations of faeces taken on the first day of illness (bridger and hall, ) . smears of faeces stained with giemsa were examined for cryptosporidial oocysts on the first and last day of illness. e. coli were enumerated in scrapings of mucosa from the ileum, caecum, colon and rectum, and in faeces and intestinal contents as described previously . the mucosae were washed, scraped off with a glass slide and ground to make - suspensions in saline. one gram of the mucosal suspensions, intestinal contents or faeces were serially diluted fold in saline and . ml of appropriate dilutions spread on to macconkey agar plates (mackie and maccartney, } in triplicate before incubation at °c for h. e. coli g- were enumerated according to the dilution of sample and the number of lactose fermenting colonies per dilution. atypical e. coli were initially identified by their characteristic colonies on mac-conkey agar; these had a red centre, a clear outer zone and the surrounding medium was clear. isolates from experimental infections were occasionally tested for anaerogenicity and production of urease. faeces taken from calves aged and days and at onset of diarrhoea or dysentery were examined for salmonellae (jones et al., ) . tissues were fixed, processed and examined for the presence of lesions as described previously (hall et al., ) . paraffin sections of mercuric formol fixed tissue were stained by an indirect immunoperoxidase method with primary antisera to bovine rotavirus prepared in calves (parsons et al., ) or by a peroxidase-antiperoxidase method (sternberger et al., ) with primary rabbit antisera to live e. coli s - produced by the method of sojka ( ) . immunoglobulins were detected in calf serum taken at days of age by the zinc sulphate turbidity test (mcewan et al., ) . serum samples were serially diluted in . -ml volumes of isotonic saline in a u-bottomed microtitre tray. a suspension of e. coli s ° ( . ml equivalent to browns tube no. ) was added to each well and to wells con-taining saline alone. tests were incubated at °c for h followed by h at °c. rabbit antisera to live e. cpli s - was used as a positive control. during a survey of field cases of calf diarrhoea in southern england during the winters of / and / isolates that grew on macconkey agar (mackie and maccartney, ) at °c and fermented lactose, were subinoculated into vials of ferrous metabisulphate pyruvate (fbp) semi-solid medium (oxoid) and were frozen at - °c. stored cultures (total ) were revived by scraping frozen medium from vials onto trypticase glucose extract (tgx) agar (orskov et al., ) , subsequently incubated at °c for h. e. coli strains k and b ( :k ) were used as controls in characterisation tests. tests described by cowan and steel { ) were used. e. coli b was used as a typical control. serotyping of e. coli rabbit antisera to live e. coli s - , produced by the method of sojka ( ) , was used in a slide agglutination test. further typing of somatic antigens was kindly undertaken by dr. b. rowe at the central public health laboratory, colindale, london. the microtitre method of burrows et al. ( ) was used. tests were incubated at °c or °c in the presence or absence of % d-mannose. cytotoxin test e. coli isolates which were to be tested for production of cytotoxin were inoculated into ml of evans medium (evans et al., ) which was incubated at °c for h and shaken at r.p.m. a . -ml aliquot of this culture was subinoculated into a second evans medium and treated in the same way as the first. cultures were then centrifuged at x g for min and the supernatant fractions filtered through an . -pm millipore membrane and the filtrate tested for the presence of toxin. aliquots of pl of l s chinese hamster ovary (cho) or hela cells m - or × l s vero cells my were placed into all wells of a flat-bottomed microtitre tray (intermed, nunc); pl of filtrate was placed in a duplicate set of wells and serially diluted. microtitre trays were sealed and incubated in a co cabinet at °c for days. cells were stained with a mixture of equal volumes of . % (w/v) crystal violet and % (v/v) neutral buffered formalin for h, washed, dried at °c and examined. toxin was prepared in evans medium and tested in the suckling mouse test (guinee et al., ) . bacteria were washed from an overnight culture on either % sheep blood agar or tgx medium with phosphate buffered saline (ph . ) and dropped onto formvar/carbon-coated grids. after s, excess fluid was removed with filter paper. the grids were rinsed in distilled water and then stained with % (w/v) phosphotungstate (ph . ), excess stain removed, air-dried and examined in a philips electron microscope. experimental infection of gnotobiotic calves with atypical e. coli isolates / or / bacteria two out of four atypical isolates from the culture collection were selected for experimental infections. e. coli / had been isolated from a calf with diarrhoea from which no other enteropathogen was detected and e. coli / had been isolated from a calf which did not have diarrhoea. gnotobiotic calves were derived by the methods of dennis et al. ( ) . one was infected with e. coli / and another with e. coli / . infection of calves, pathology and immunoperoxidase staining methods these methods were as described for the experimental infection of conventional calves with e. coli s - . the occurrence of dysentery in calves and the excretion of enteropathogens are summarised in table i . all five calves ( -- ) infected with e. coli s - , at days of age, developed dysentery, whereas older calves ( -- ) infected for the first time or reinfected ( -- ), and control calves ( -- ) did not. the dysentery was characterised by the presence in faeces of bright, fresh blood (sometimes copious) and large amounts of clear or faeces-stained mucus. the faecal consistency was soft or semi-liquid. peak excretion of s - (> cfu g-i of faeces) occurred during signs of dysentery in all calves ( -- ) infected at days of age. in contrast only ( and ) of the older calves { -- , and ) excreted these numbers; this occurred on the first day after reinfection. rotavirus or coronavirus were excreted by calves -- , but not at times infection occurrence of excretion excretion excretion of dysentery of s - when dysentery was seen. calves and , infected at days of age and necropsied at the peak of dysentery ( days), were excreting rotavirus whereas control calves ( -- ) excreted rotavirus at times when mild diarrhoea was seen. salmonellae, cryptosporidia and calicivirus were not detected. pyrexia or bacteraemia did not occur consistently during the period of dysentery. prior to infection, the serum of calves contained widely different amounts of immunoglobulin ( -- zinc sulphate turbidity units). prior to infection the amount of serum immunoglobulins of calf was the lowest recorded. calf alone responded to infection by the production of agglutinins to e. coli s - (titre of ), and this calf had the longest period of dysentery and the longest period of excretion of $ - . in calves and , necropsied at the peak of dysentery, e. coli $ - colonised the colonic and rectal mucosae in approximately equal or greater numbers than in the contents. in contrast e. coli of other types colonised the mucosae in fewer numbers than in the contents (table ii) . the mean number of s - in these mucosae was log . g- whereas for other e. coli it was log~ . g- . the same distribution of e. coli was seen in the caecum of calf . e. coli of all types colonised the mucosae of the small intestine in lower numbers than in the contents. petechial haemorrhages were seen in the rectal mucosae of calves and ; all of the rectal mucosa of calf and the longitudinal folds of calf were markedly reddened. the same changes were seen in the colon of calf and were most noticeable over the gut-associated lymphoid tissue of the anterior colon and along the tops of longitudinal folds. in the caecal mucosa of calf there were areas of reddening -- cm in diameter. in the caecum, colon and rectum of calves and foci of bacteria were seen adherent to clumps of irregularly arranged and exfoliated enterocytes and there were neutrophils in the lamina propria. in the caecum, neutrophils were in foci in the lamina propria; in the colon and rectum, neutrophils were more numerous and were also present in foci in an exudate on the mucosal surface with mucus and exfoliated enterocytes. lesions were most severe in the colon. immunoperoxidase staining of sections of tissue with antiserum to e. coli s - revealed microcolonies of bacteria adherent to the colonic and rectal rugae of calves and . all adherent bacteria that were seen were immunostained. rotavirus was detected in the ileal enterocytes by immunoperoxidase staining and in the small intestine there were lesions of a viral enteropathy (stunted and fused villi covered by cuboidal enterocytes). of isolates, including from calves with diarrhoea, four had an atypical colony morphology on macconkey agar indistinguishable from that of e. coli s - , were anaerogenic and produced urease. on the basis of other tests these isolates were identified as e. coli (table iii) . the four isolates originated from different farms and three were isolated from calves with diarrhoea. coronavirus was isolated from one of these calves and sa/monella typhimurium from another; the third isolate from a normal calf was the results of further characterisation of the four atypical isolates and s - are in table iv . preliminary serotyping of e. coli / and / revealed that they produced the same somatic antigen ( ) as s - . in addition to the four atypical isolates, another isolate, which was aerogenic but otherwise indistinguishable from e. coli s - , produced a toxin active on hela and veto cells but not cho cells. fifty-five isolates were detected with either or of the atypical characteristics of e. coli s - ; of these were tested for the production of cytotoxins. they comprised four isolates which produced urease, five anaerogenic isolates, five which agglutinated in antiserum to e. coli s - , one with an atypical colony morphology and one with an atypical colony morphology which agglutinated in antiserum to e. coli $ - . one isolate produced a cytotoxin active on veto cells; it also produced urease. in a preliminary investigation, two gnotobiotic calves were infected with either e. coli / or / ; they did not develop dysentery, although the calf infected with e. coli / produced mucoid liquid faeces from to days after infection, when it was killed. e. coli / was isolated from a normal calf and the faeces of the gnotobiotic calf infected with / were unchanged until days after infection, when the animal was killed. this absorption of antibody to s - a fimbriae (f) or haemagglutinins (h) produced on suggested that isolate / was avirulent. calves excreted between . x and . x l° e. coli g- of faeces until they were killed. three days after infection, faeces of the gnotobiotic calf infected with e. coli / inoculated onto macconkey agar revealed e. coli with two different colony morphologies in approximately equal numbers. one was atypical and of the kind produced by e. coli $ - , and the other was typical of most isolates of e. coli. e. coli of both types, however, were anaerogenic, produced urease and agglutinated in antiserum to e. coli s - . at necropsy of the two gnotobiotic calves there were more e. coli in the caecal, colonic and rectal contents (~ . x cfu g-l) than in the respective mucosae ( . x -- . x cfu g-l) which indicated that colonisation of the large intestinal mucosae was less than that seen with e. coli s - infections of gnotobiotic calves . in the mucosae of the colon and rectum of both calves there was marked congestion and occasional small haemorrhages. neutrophils were numerous in the lamina and they were present as foci on the mucosal surface forming an exudate, together with mucous and exfoliated enterocytes. these lesions were apparently as severe as those seen with e. coli s - infections of gnotobiotic calves {hall et al., ) . scanning electron microscopy revealed many short, rod-shaped bacteria attached to the mucosal surface of the large intestines of both calves. enterocytes to which bacteria were attached, and adjacent enterocytes, lacked microvilli or microvilli were abnormally orientated or were shortened or lengthened. changes to microvilli were confirmed by transmission electron microscopy which also showed that the bacteria were closely associated with the enterocyte cell membrane. at the point of attachment, enterocyte cytoplasm was often cup-shaped or arranged as a pedestal. immunoperoxidase staining revealed microcolonies of e. coli adherent to the colonic and rectal rugae of both calves. e. coli s - was pathogenic for conventional calves; all animals infected at days of age developed dysentery similar to that previously seen in gnotobiotic and farm calves hall et al., ) . the calves excreted other enteropathogens, but in two these were not associated with dysentery, and in a third they were not excreted during the major episode of dysentery. in two calves, slaughtered at the estimated peak of clinical signs, rotavirus was detected in the ileum and was not associated with lesions that could have been a source of fresh blood in faeces. lesions in the large bowel, which appeared to be the source of blood in faeces, were consistently associated with adherent e. coli and were identical in nature to those produced in gnotobiotic calves and those seen previously in naturallyaffected farm calves (hall et al., ) . primary infection of two older calves did not reproduce dysentery, which suggested that the calves had acquired resistance to the effects of infection. in this respect, resistance might be similar to that acquired with age by calves to infection with k ÷ e. coli (smith et al., ) . detection of four atypical e. coli out of isolates collected from natural outbreaks of calf diarrhoea suggested that they were not an important cause of enteric disease in calves in that survey. one isolate with these properties has previously been associated with diarrhoea in a calf in france (de ryke et al., ) . two isolates of atypical e. coli ( / and / ) caused microscopic lesions indistinguishable from those produced by e. coli s - (hall et al., ) . however, one ( / ) did not cause disease and the other ( / ) caused mucoid diarrhoea. failure of these strains to cause dysentery may have been related to the extent of bacterial colonisation and the lesions caused. there were approximately -fold fewer e. coli / or / in the mucosae of the large intestine than were previously reported for e. coli $ - in gnotobiotic calves . the extent of microscopic lesions in the gnotobiotic calves was difficult to assess, particularly because of their focal nature, which was apparently similar to that seen in calves infected with e. coli s - . consequently, the virulence of these e. coli may vary not only with the ability to colonise but also the extent of the lesions they cause or, alternatively, it may differ with the production of an unknown pathogenic determinant. in man infected with different strains of verotoxic e. coli, which cause the same type of lesion, signs of disease vary from mild diarrhoea to dysentery (anon, ). an alternative explanation for the failure of e. coli / and / to cause dysentery may have been a variation of animals in their susceptibility. moon et al. ( ) reported considerable animal to animal variation in the response of pigs to infection with e. coli enteropathogenic for humans; identical lesions were seen in the colons of pigs with or without diarrhoea. historically, the term enteropathogenic e. coli or epec was first applied to strains of e. coli implicated in epidemic infantile diarrhoea of humans. some of these strains and others virulent for a number of species have been shown to cause highly characteristic microscopic lesions of the gut mucosa and to produce a cytotoxin detectable in vitro and have been called epec (anon, ) . the microscopic lesions and production of cytotoxin may be unifying features of an emerging sub-group of enteropathogenic e. coli for which the term epec is not sufficiently descriptive. takeuchi et al. ( ) suggested that these lesions in rabbits were induced by a cytotoxin which showed similarities to the shiga toxin produced by shigella dysenteriae (o'brien et al., ) . e. coli enteropathogenic for man have been shown to produce a toxin (konowulchuk et al., ) which is biologically and serologically related to the shiga toxin (o'brien et al., ) . the e. coli described here may form a distinctive sub-group in view of their isolation from cattle, their atypical characteristics and their distinctive properties in tests for fimbriae and haemagglutinins. these e. coli produced a mannose-resis-tant haemagglutinin in anaerobic but not aerobic conditions which was of particular interest; apparently the haemagglutinin was not associated with fimbriae. in aerobic conditions these bacteria produced fimbriae without detectable haemagglutinating activity which, although previously described (isaacson, ) , are nonetheless unusual. the unusual colony morphology produced on macconkey agar might usefully serve as a primary selective characteristic for potentially pathogenic e. coli of the calf. however, strain / produced in vivo a variant with a colony morphology indistinguishable from that produced by most e. coli. furthermore, if in vitro the common characteristic of the enteropathogenic e. coli is cytotoxigenicity then it is probable that e. coli which can cause enteric disease in the calf may be found with otherwise typical properties. mechanisms in enteropathogenic escherichia coli diarrhoea effects of a calicivirus-like agent (the newbury agent) on gnotobiotic calves haemagglutinating and adhesive properties associated with k antigen of bovine strains of escherichia coli dysentery in gnotobiotic calves caused by atypical escherichia coli identification of medical bacteria evidence ofescherichia coli with bacteraemic properties in mucoid enteritis of newborn calves a simplified apparatus for the microbiological isolation of calves identification of enterotoxigenic escherichia coli and serum antitoxic activity by the vascular permeability factor assay escherichia coli associated with neonatal diarrhoea in piglets and calves dysentery caused by escherichia coli (s - ) in calves: natural and experimental disease pili of enterotoxigenic escherichia coli salmonella saint-paul infection in two dairy herds properties of an escherichia coli cytotoxin handbook of practical bacteriology, edinburgh a turbidity test for the estimation of immune globulin levels in neonatal calf serum attaching and effacing activities of rabbit and human enteropathogenic escherichia coli in pig and rabbit intestines production of shigella dysenteriae type l-like cytotoxin by escherichia coli the establishment of k , a thermolabile, transmissible escherichia coli k antigen, previously called 'kco', possessed by calf and lamb enteropathogenic strains localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase evaluation of elisa and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces a clinico-pathologic study of enterocyte-adherent escherichia coli: a cause of protracted diarrhoea in infants observations by the ligated intestinal segment and oral inoculation methods on escherichia coli infections in pigs, calves, lambs and rabbits the unlabelled antibody enzyme method of immunohistochemistry : preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in the identification of spirochaetes scanning and transmission electron microscopic study of escherichia coli ois (rdec- ) enteric infections in rabbits key: cord- -nvhu blo authors: richard dorn, c.; francis, david h.; angrick, elisabeth j.; willgohs, joann a.; wilson, richard a.; collins, james e.; jenke, bruce h.; shawd, stacey j. title: characteristics of vero cytotoxin producing escherichia coli associated with intestinal colonization and diarrhea in calves date: - - journal: vet microbiol doi: . / - ( ) -u sha: doc_id: cord_uid: nvhu blo isolates of escherichia coli which produce vero cytotoxin (vtec) were obtained during – from calves raised in north-central states of the usa. all of the calves experienced intestinal epithelial colonization by vtec, diarrhea or both; twelve of the calves had bloody diarrhea. twenty one isolates were serogroup o and the others were o , o , o , o , o , or non-typable ( isolates). all but one of the isolates hybridized with the cvd probe which identifies most vtec strains. thirty two isolates hybridized with the vt probe, with both the vt and vt probes, and one with neither probe. the culture filtrate of the vt probe negative isolate was partially neutralized by slt i monoclonal antibody. for the other isolates, the results of toxin neutralization by anti-slt i and anti-slt ii monoclonal antibodies corresponded exactly with the vt and vt probe hybridization results. three of the strains adhered in a localized manner to hep- cells and intestine cells. first reported that certain e. coli isolated from human diarrhea produced cytotoxins which acted in vitro on vero cells. two serologically distinct vero cytotoxins have been described: vt and vt , also called slt i and slt ii because of their similarity to the toxin produced by shigella dysenteriae (o'brien and holmes, ). e. coli producing vero cytotoxin (vtec) have been isolated from cattle, beef, other meats, milk and milk products (karmali, ; giffin and tauxe, ) . many of the bovine vtec have been isolated from cattle with diarrhea. pathological examination of the intestinal tracts of some of these animals revealed characteristic epithelial lesions described as attaching and effacing with loss of microvilli and pedestal and cup formations of the cell membrane (hall et al., ; moxley and francis, ) . vtec of animal and human origin did not hybridize with the enteropathogenic e. coli (epec) adherence factor (eaf) dna probe (dorn et al., ) . many of the vtec of bovine origin have characteristics in common with those proposed for the enterohemorrhagic e. coli (ehec) (levine, ) . these e. coli of human origin produce vero cytotoxin; they are associated with hemorrhagic colitis; they lack production of enterotoxins lt and st characteristic of enterotoxigenic e. coli (etec); and they are not invasive. a dna probe, designated cvd , has been developed which hybridizes with most e. coli o : h and other serotypes that fit the above criteria . the source of the cvd probe was serotype o :h which is associated with human outbreaks and sporadic illnesses. this serotype is a serious human pathogen capable of producing hemorrhagic colitis (hc), hemolytic uremic syndrome (hus) and thrombotic thrombocytopenic purpura (karmali, ) . the sources of infection and associated human illnesses have been traced to undercooked beef and unpasteurized milk; serotype o : h e. coli have been isolated from cattle (karmali, ; giffin and tauxe, ) . the isolates have been from healthy cattle, except for four reports of serotype o : h from diarrheic calves (kashiwazaki et al., ; orskov et al., ; blanco et al., ; yano et al., ) . none of these calves was reported to have blood present in the feces. the purpose of this study was to characterize the vtec isolates from cattle obtained by two veterinary diagnostic laboratories. this information may be used in the diagnosis and epidemiologic investigation of vtec infections of cattle, and in understanding their possible role in human infections. bacterial strains and tissue specimens vtec isolates from calves with diarrhea were collected by the south dakota animal disease research and diagnostic laboratory and the department of diagnostic investigation at the university of minnesota. entire calves and tissues collected at field necropsies were routinely submitted to the laboratories. historical and clinical information about each animal was obtained from submission forms and interviews with producers and veterinarians. intestines were processed for histopathological examination using routine methods. colonization was based upon observations under light microscopy of short bacilli which were intimately adherent to intestinal epithelial cells exhibiting irregular erosive lesions typical of attaching and effacing infections (moxley and francis, ; janke et al., ) . specimens of intestines were cultured aerobically on blood agar, tergitol- agar (difco laboratories, detroit, mi ) and e agar (francis et al., ) and on brilliant green agar plates (flow laboratories, mclean, va). the following biotyping tests were performed: indole, methyl red, voges-proskauer, citrate, h s, urease, phenylalanine deaminase, lysine decarboxylase, arginine decarboxylase, ornithine decarboxylase, motility, gelatin hydrolysis, salicin, malonate, esculin hydrolysis, nitrate, oxidase, and carbohydrate and sugar alcohol fermentations. isolates from the intestines identified as e. coli were examined for production of enterotoxin by methods previously described (francis, ) and serotyped by the e. coli reference center, pennsylvania state university, university park, penn. the vt probe was a hincli fragment of . kb specific for vt sequences cloned from a vt encoding phage carried by h , e. coli : h (willshaw et al., ) . the vt probe was a . kb avai-psti fragment specific for vt sequences cloned from a vt -encoding phage carried by e. coli strain e of serotype o :nm . the cvd probe was a . kb hindlii fragment cloned from strain , e. coli o : h . the probe fragments were cut from low melting point agarose gels and labelled with deoxyadenosine '-a [ s ] thiotriphosphate (amersham, arlington heights, il) using random primers (feinberg and vogelstein, ) . hybridization with the s-labelled probes was performed according to maniatis et al. ( ) . in all hybridizations, the following three controls were used: e , e. coli :h , vt positive, vt negative and cvd positive; e , e. coli o :nm, vt negative, vt positive and cvd positive; and r , e. coli k , negative with all of these probes. the vero cytotoxin assay used was a modification of the method of scotland et al. ( ) . five-fold dilutions of filtrates was tested on vero cell monolayers. the plates were resealed and incubated at °c, % co for days. a filtrate was considered positive if a : or greater dilution produced a cytotoxic effect on the monolayer and the corresponding heat-inactivated preparation was negative. neutralization tests were performed using two mouse ascitic fluid reagents containing monoclonal antibody (mab) c and mab bc -bb , respec-tively, obtained from dr. n. a. strockbine, centers for disease control, atlanta, georgia. mab c has approximately a : neutralizing titer against cdso of slt i. mab bc -bb has approximately a : neutralizing titer against cds of slt . the stock ascitic fluid preparations were diluted : and : for use in the neutralization tests. the respective ascitic fluid dilutions ( . ml) were mixed with an equal amount of filtrate in . ml supplemented mem and incubated in a well plate at °c for hrs. the neutralization mixtures were then transferred to well plates containing vero cell monolayers and incubated at °c, % c for days. neutralization of the original filtrate was recorded when the addition of anti-slt specific ascitic fluid resulted in a decrease in the cytotoxic titer by one or more dilutions from the cytotoxic titer of the original filtrate. the method of scotland et al. ( ) was used to test each strain for ability to adhere to hep- cells and intestine cells, in the presence of d-mannose, during a h incubation period. only localized adherence as described by nataro et al. ( ) was observed and it was scored as the percentage (mean of at least tests) of hep- or intestine cells with or more adherent bacteria. strains which adhered to less than % of the hep- or intestine cells were considered non-adhesive for that cell line. a total of vtec isolates, each from a different calf between and , were identified for study (table ) . they were isolated predominantly from calves designated as dairy or dairy-crossbred; only of the isolates were from beef or beef-crossbred calves. one calf was months old and all the other calves of known age were less than weeks of age. sixteen of the calves were from minnesota, from south dakota, from iowa, one from north dakota and one from nebraska. thirty two of the calves had bacteria colonizing the epithelium of their intestinal tract and diarrhea, had colonization with no observed diarrhea, and one had diarrhea but colonization was obscured by post-mortem autolysis. thirteen of these were observed by histopathology to have lesions of the colon. twelve of the calves were observed to have bloody diarrhea. all isolates fermented sorbitol within hours. the four :nm and two other isolates did not ferment raffinose. none of the isolates were positive for the k antigen. twenty one of the isolates were serogroup o , isolates were non-typable, isolates were and the other isolates were distributed among other except for isolate - , all of the isolates hybridized with the cvd probe (table ) . thirty two of the isolates hybridized with only the vt probe; two of the serotype o :nm isolates and one of the o :nm isolates hybridized with both vt and vt probes. none of the isolates hybridized with only the vt probe. the highest titer obtained for these vtec isolates was . filtrates of the other isolates had lower titers: were and the other were less than (table ) . for the vt and vt positive isolates, the results of neutralization of their filtrates by anti-slt i and anti-slt ii ascites fluid matched exactly the vt probe hybridization results. the filtrate of isolate - , which did not hybridize with either vt or vt , was partially neutralized by anti-slt i monoclonal antibody. the anti-slt i ascites fluid completely neutralized the filtrates of of the slt i positive isolates. incomplete neutralization of slt i was observed with filtrates from the remaining slt i positive isolates. anti-slt ii ascites fluid completely neutralized one of the filtrates of slt ii positive isolates. three isolates possessed the ability to adhere in a localized manner to greater than % of both hep- and intestine cells; two were serotype o :nm and one was serotype o : h , . the adherence test results for a particular isolate were similar for both hep- and intestine cells. other pathogenic agents were isolated from of the calves; were infected with two other pathogens besides vtec. fifteen co-infections involved cryptosporidia, coronavirus, rotavirus, one enterotoxigenic e. coli (etec) and one bredavirus. the absence of serotype o : h in this series of vtec isolates from calves is consistent with previous reports that o :h is not a frequent cause of illness in cattle. there are only reports of this serotype being isolated from ill cattle (kashiwazaki et al., ; orskov et al., ; blanco et al., ; yano et al., ) and in none of these was o :h confirmed as the cause of illness. the most common serogroup in this series, o , has been recorded and also identified as verotoxigenic in other studies of bovine isolates (sherwood et al., ; marques et al., ; mohammad et al., ; mainil et al., ; schoonderwoerd et al., ; smith et al., ; dorn et al., ) . other serogroups represented in this series have also been previously isolated from cattle and shown to be verotoxigenic: o (mohammad et al., ; d orn et al., ; gonzalez et al., ) : (kashiwazaki et al., ; sherwood et al., ; marques et al., ; mainil et al., ; smith et al., ; rietra et al., ; scotland et al., ; woodward et al., ) : and (chanter et al., ; hall et al., ; dorn et al., ) . no previous published reports of verotoxigenic isolates belonging to serogroups and were found. none of the vtec isolates showed delayed sorbitol fermentation, a unique characteristic of serotype o : h (farmer and davis, ) . twelve of the calves had bloody diarrhea. seven of these calves yielded cultures of serotype o i:nm, were non-typeable, one was o :h , and one was : nm. serotypes which were isolated from calves in this study and which have been reported from hc in humans include o i:nm, o :h , o :h and o :nm (bopp et al., ) other : nm strains of bovine origin have been associated with hemorrhagic enteritis in cattle. a serotype : nm e. coli was isolated from an outbreak of dysentery among to day old calves in england (chanter et al., ) . in both natural and experimentally reproduced disease with this strain, the calves passed copious bright red blood in the feces and developed diarrhea (hall et al., ) . this o :nm strain produced a toxin active on vero cells which was neutralized by anti vt and hybridized with the vt and cvd probes (dorn et al., ) . one of the serotype o :nm e. coli in the series ( - in table ) was previously reported by moxley and francis ( ) to produce bloody diarrhea in calves and lambs. in the present study, isolate - produced a toxin active on vero cells which was incompletely neutralized by anti slt i ascites fluid. however, it failed to hybridize with the vt , vt and cvd probes. it apparently produces another toxin active on vero cells, neutralizable by anti-slt i ascites fluid, but not mediated by the phage genes encoding slt i and slt ii. a similar observation has been made with porcine edema disease strains which produce a variant of slt ii that is chromosomally mediated and active on vero cells, but not on hela cells weinstein et al., ) . our observation that of vtec isolates from calves in north-central states hybridized with only the vt probe is consistent with reports that most of the non-o :h bovine vtec isolated by laboratories located in england were positive for only vt (smith et al., ; dorn et al., ; scotland et al., ) . however, few of the non-o :h vtec isolated from healthy cattle (usually mature) hybridized with only the vt probe in germany and in thailand, % and %, respectively (montenegro et al., ; suthienkul et al., ) . serotype o :h and other serotypes isolated from human hc and hus cases usually produce slt ii alone or both slt i and ii, but much less frequently slt i alone dickie et al., ; ostroff et al., ) . the slt production pattern of vtec isolates from human cases appears to be similar to that of vtec isolates from healthy cattle, but dissimilar to that of calves with diarrhea. it is possible that slt ii strains are more pathogenic for humans while slt i strains are more pathogenic for calves resulting in higher frequencies of these respective toxins among isolates from humans and from calves. it is also possible that slt i producing e. coli are more common in calves and slt ii e. coli infections are more common among mature cattle. the higher prevalence of slt iie. coli than slt i e. coli infections among mature cattle (montenegro et al., ; suthienkul et al., ) might result in more passive transfer of slt ii antibody than slt i antibody, resulting in lower incidence of clinical illness due to slt ii producing vtec strains in calves. this hypotheses should be explored in future studies. the vtec isolates of this study were from midwestern states with a substantial beef production industry, yet of vtec isolates came from dairy or dairy cross calves. the predominance of dairy calves as the source of vtec strains suggests that these animals may be more susceptible to life threatening illness by vtec than are beef calves. dairy calves are usually fed a milk replacement starter and many may either not receive or poorly absorb colostrum: they are thus more at risk than beef calves which are usually nursed. this is consistent with the observation of higher incidence of gastroenteritis in breast-fed children infected with e. coli serogroup o compared to that in formula-fed infants, similarly infected (belnap and o'donnell, ) . all but one of the vtec isolates in this study hybridized with the cvd probe (table ). in another study of vtec isolated from healthy cattle, only % hybridized with this probe. it is possible that the ehec probe is more specific for pathogenic vtec than for vtec strains associated with subclinical infections; however, additional studies of cattle with different ages and from different geographic areas are needed. epidemic gastroenteritis due to escherichia coli production of toxins by escherichia coli strains isolated from calves with diarrhea in galicia unusual verotoxin-producing escherichia coli associated with hemorrhagic colitis dysentery in calves caused by an atypical strain of escherichia coli purification of an escherichia coli serogroup o : h verotoxin and its detection in north american hemorrhagic colitis isolates properties of veto cytotoxin producing escherichia coli of human and animal origin belonging to serotypes other than o :h h antiserum sorbitol fermentation medium: a single tube screening medium for detecting escherichia coli o :h associated with hemorrhagic colitis a technique for radiolabeling dna restriction endonuclease fragments to high specific activity production of k , k and p antigens of escherichia coli cultured on synthetic and complex media use of immunofluorescence, gram's staining, histologic examination, and seroagglutination in the diagnosis of porcine colibacillosis the epidemiology of infections caused by escherichia coli o :h , other enterohemorrhagic e. coli, and the associated hemolytic uremic syndrome serotypes and antibiotic resistance of verotoxigenic (vtec) and necrotizing (ntec) escherichia coli strains isolated from calves with diarrhea dysentery caused by escherichia coli (s - ) in calves: natural and experimental disease attaching and effacing escherichia coli infection as a cause of diarrhea in young calves infection by vero cytotoxin-producing escherichia coli veto cytotoxin produced by escherichia coli strains of animal origin vero response to a cytotoxin of escherichia coli escherichia coli that cause diarrhea: enterotoxigenic enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent a dna probe to identify enterohemorrhagic escherichia coli of o :h and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome shiga-like toxin production and attaching effacing activity of escherichia coli associated with calf diarrhea molecular cloning: a laboratory manual production of shiga-like toxin by escherichia coli escherichia coli strains isolated from pigs with edema disease produce a variant of shiga-like toxin ii serotypes of verocytotoxic escherichia coli isolated from cattle and buffalo calf diarrhoea detection and characterization of fecal verotoxin-producing escherichia coli from healthy cattle natural and experimental infection with an attaching and effacing strain ofescherichia coli in calves patterns of adherence of diarrheagenic escherichia coli to hep- cells shiga and shiga-like toxins cattle as reservoir of verotoxin-producing escherichia coli o : h . lancet, ii toxin genotypes and plasmid profiles as determinants of systemic sequelae in escherichia colio : h infections comparison of vero-cytotoxin-encoding phages from escherichia coli of human and bovine origin colitis in calves: natural and experimental infection with a verotoxin-producing strain of escherichia coli o :nm. canad laboratory tests for enterotoxin production, enteroinvasion and adhesion in diarrhoeagenic escherichia coli properties of strains of escherichia coli belonging to serogroup o with special reference to production of vcro cytotoxins vti and vt properties of strains of escherichia coli : h in relation to their enteropathogenic or enterohemorrhagic classification shiga-like toxin production from escherichia coli associated with calf diarrhea vero cytotoxin production and presence of vt genes in escherichia coli strains of animal origin shiga-like toxin-producing escherichia coli in retail meats and cattle in thailand cloning and sequencing of a shiga-like toxin type ii variant from an escherichia coli strain responsible for edema disease of swine cloning of genes determining the production of vero cytotoxin by escherichia coli heterogeneity of escherichia coli phages encoding vero cytotoxins: comparison of cloned sequences determining vti and vt and development of specific gene probes dna probes for the detection of toxin genes in escherichia coli isolated from diarrhoeal disease in cattle and pigs fimbria-like adhesive factor (eaf ) from verocytotoxigenic escherichia coli the authors wish to thank g.a. willshaw of the division of enteric pathogens, central public health laboratory, colindale, london, uk for the cloned vt and vt probes. the cloned cvd probe was kindly provided by m.m. levine, center for vaccine development, university of maryland, school of medicine, baltimore; and the slt-specific monoclonal antibodies were kindly provided by n.a. strockbine, centers for disease control, atlanta, georgia. key: cord- -yxz nil authors: weingartl, h.m.; derbyshire, j.b. title: binding of porcine transmissible gastroenteritis virus by enterocytes from newborn and weaned piglets date: - - journal: vet microbiol doi: . / - ( ) -l sha: doc_id: cord_uid: yxz nil enterocytes were harvested by chelation in a series of seven fractions from the tips of the villi to the crypts of the jejunum of newborn or weaned piglets. binding of the low cell culture passaged miller- strain of transmissible gastroenteritis virus (tgev) to villous enterocytes from newborn piglets was at a high level, similar to that observed to culturedswine test is (st) cells. binding of the virus to cryptal enterocytes from newborn piglets or to villous or cryptal enterocytes from weaned piglets was significantly lower. in a competitive virus binding assay with radiolabelled virus, the binding of tgev to st cells was found to be saturable, while binding to mdbk cells, in which the virus fails to replicate, was at a lower level and was non-saturable. in the same assay, virus binding to the villous enterocytes from the jejunum of a newborn piglet was saturable, while binding to cryptal enterocytes from a newborn piglet, and to villous and cryptal enterocytes from a weaned piglet, was non-saturable. it was concluded that the high susceptibility of newborn piglets to tgev infection, and the tropism of the virus for villous enterocytes, may relate to the presence of specific, saturable binding sites on the plasma membrane of villous enterocytes. ) which infects the mature enterocytes of the small intestine, leading to villous atrophy, diarrhoea, malabsorption and death in newborn piglets (saif and heckert, ) . the virus does not infect the cryptal enterocytes, and there is also evidence that the enterocytes which replace those lost from the villi during the infection are also resistant to the virus (pensaert et al., ) . the tropism of the virus for mature villous enterocytes has not been explained, and a major objective of the present study was to determine whether this might relate to the ability of the virus to bind to these cells. a further feature of the pathogenesis of tge is that the severity of the disease is inversely related to the age of the piglet (moon et al., ) , and it has been postulated that the high sensitivity of the neonate may relate to the slow replacement rate of enterocytes in newborn piglets (moon, ) , to the facilitation of viral replication by deep cytoplasmic tubular invaginations in neonatal enterocytes (wagner et al., ) and by the lack of natural killer cell activity in newborn piglets (cepica and derbyshire, ) . the possibility that there might be a decline in enterocyte virus-binding activity with age has not been investigated. specific attachment sites for tgev have been demonstrated in porcine cell culture (nguyen et al., ) , and a to kda protein was subsequently identified as a receptor molecule on swine testis (st) cells (flakus et al., ) . recently, delmas et al. ( ) identified aminopeptidase n as a major receptor for tgev on both st cells and porcine enterocytes. aminopeptidase n is also a receptor for the human coronavirus e (yeager et al., ) while mouse hepatitis virus attaches to a glycoprotein of the carcinoembryonic antigen family on intestinal brush border and hepatocyte membranes (williams et al., ) . the s glycoprotein of the bovine coronavirus was shown recently to attach to a sialic acid receptor on the surface of red blood cells (schultze et al., ) , although this virus may also attach to the same receptor by means of the haemagglutinin-esterase glycoprotein (vlasak et al., ) , which is lacking in tgev. in this paper we describe the binding of tgev to susceptible porcine and resistant bovine cell cultures, and to enterocytes contained in a series of fractions collected from the tips of the villi to the crypts of the jejunum of neonatal and weaned piglets. as evidence of specific binding to these cells, the saturability of virus binding was determined in a competitive assay with radiolabelled virus. stable swine testis (st) cells (mcclurkin and norman, ) and madin-darby bovine kidney (mdbk) cells (american type culture collection) were cultivated by standard methods. enterocytes were harvested as described by weiser ( ) from the jejunum of eight newborn piglets at or days of age and from four weeks old weaned piglets which were killed with an intravenous overdose of sodium pentobarbitone. an approximately m length of jejunum was rinsed five times with . m nac , mm dithiothreitrol (dtt) and then closed at one end with surgical thread. the intestine was filled with citrate buffer ( . mm dtt, . mm kc , mm nac , mm sodium citrate, mm kh po , . mm na hpo , ph . ), closed with a clip and incubated in hanks' balanced salt solution (hbss) containing . mm dtt for min at °c. the contents of the intestine were then discarded and replaced with magnesium and calcium-free phosphate-buffered saline (pbs) containing . mm ethylenediamine tetraacetic acid (edta) and . mm dtt. the intestine was incubated for min at °c, when the released cells were collected by centrifugation at g for min and washed three times with pbs. the min cycles of chelation with edta were repeated six times to obtain seven fractions of enterocytes. samples of intestine were fixed and sections were stained with haematoxylin and eosin after each chelation cycle to monitor the progressive detachment of enterocytes. alkaline phosphatase activity was determined in each enterocyte fraction as described by weiser ( ) . a volume of . ml of packed cells was incubated at °c for min in . m tris-hc , ph . , . mm znc , mm hgc and . mm p-nitrophenyl phosphate. after incubation, . ml of . n naoh was added to the assay system, and the released p-nitrophenol was determined spectrophotometrically at nm and related to the protein content of the sample which was determined by the bradford ( ) method with the bio-rad protein assay kit ii (bio-rad, richmond, california). periodic acid-schiff staining of the cells was performed as described by gratecos etal. ( ) . the low cell culture passaged miller- strain of tgev (welch and saif, ) , kindly supplied by dr. l.j. saif, ohio agricultural research development center, wooster, ohio) was cultivated in st cells maintained in serum-free medium. plaque assays were also performed on st cells. for the preparation of radiolabelled virus, tgev was cultivated in st cells in the presence of /~ci ml- of s-methionine (amersham, oakville, canada ). the cell culture supernatant was clarified by centrifugation at × g for min at °c, and the labelled virus was pelleted through a % sucrose cushion in tes buffer ( . m tris-hc , mm edta, . m nac , ph . ) supplemented with . % tween , by centrifugation at × g for h at °c. enterocyte fractions i to vii, st cells or mdbk ceils were incubated at a concentration of l cells m - for min at °c with , or pfu ml- tgev. the cells were removed by centrifugation at × g for min, and the supernatants tested for residual activity by plaque assay in st cells. the % virus binding was calculated as pfu virus added -pfu residual virus~ ~ ~-~dadd~ ) × . each sample of ceils was tested in triplicate against each concentration of virus, and the mean % binding was calculated for each sample. the procedure was modified from that described by schlegel et al. ( ) . enterocytes harvested from the tips of the villi (fractions i and ii ) or from the crypts (fraction vii) of the jejunum of one newborn and one weaned piglet, st cells or mdbk cells, at a concentration of cells ml -~ were incubated with pfu m - of tgev, or with eagle's minimum essential medium (emem), for h at °c. to each sample, s-methionine labelled tgev was added, in concentrations ranging from - to pfu/cei , and incubation was continued for min at ° c. the cells in each sample were then pelleted by centrifugation at ×g for min, and the pellets resuspended in /tl of % triton x- (sigma chemical co., st. louis, missouri) in pbs. a portion ( /a) of the suspension was transferred into a scintillation vial with ml of universal scintillation cocktail (icn biomedicals, irvine, california) and the radioactivity measured as counts min-(cpm) in a liquid scintillatio~ counter. the assay was run in triplicate on each sample, and mean values obtained. the cpm obtained from the cells preincubated with emem represented total virus binding, while the counts obtained after preincubation with pfu ml-~ of unlabelled tgev represented non-saturable virus binding. saturable binding was obtained by subtracting the non-saturable binding cpm from the total binding cpm. virus binding to mdbk cells was less than % for each of the three concentrations of tgev, while for st cells the mean ___ sd % virus binding was + , + and +_ for concentrations of tgev of , and pfu ml- respectively. in the competitive virus binding assays, there was no evidence that the binding of tgev to mdbk cells was saturable, while for st cells a typical hyperbolic saturable binding curve was obtained (fig. ) , indicating the presence of saturable binding sites for tgev on these cells. histological examination of sections of intestine after each fraction was harvested revealed that the enterocytes were released progressively from the tips of the villi (fraction i) to the crypts (fraction vii) as chelation with edta proceeded. representative sections are shown in fig. remained in the jejunum of the newborn piglets after the harvesting of fraction vii, while in the weaned piglets, small numbers of cells remained deep in the crypts after the removal of this fraction. these could be released by chelation for a further min. the highest levels of alkaline phosphatase activity (mean ___ sd mu mg-l protein) were found in the enterocytes contained in fraction i from four piglets killed at days of age. the activity in the same fraction from the weaned piglets was + mu mg-~ protein. the remaining fractions, from both newborn and weaned piglets had lower levels of activity than fraction i, ranging from + mu to + mu in the newborn piglets and from ___ mu to _+ mu mg- protein in the weaned piglets, and there were no consistent differences in activity among fractions ii to vii. the periodic acid-schiff stained preparations of enterocytes showed a progressive reduction in staining intensity from fractions i to vii in both the newborn and weaned piglets. in the virus binding assays, similar results were obtained for each of the three concentrations of virus which were used, and the findings when pfu ml- of tgev were added to the enterocyte fractions are shown in fig. . high levels of virus binding were obtained with the enterocytes in fractions i and ii, derived from the tips of the villi of newborn piglets, with relatively low binding to enterocytes in the remaining fractions from the newborn piglets, and in all the enterocyte fractions from the weaned piglets. when analysed by the student's t-test, binding was significantly higher (p < . ) to enterocytes in fractions i and ii from the newborn piglets than in all other enterocyte fractions, among which significant differences in virus binding did not occur. in the competitive virus binding assays, st and mdbk cells were included ¢ . saturable binding of radiolabelled tgev to enterocytes harvested from the tips of the villi (•) or the crypts (•) of the jejunum from a newborn piglet. increasing amounts of labelled tgev were incubated with enterocytes and virus binding was determined as cpm for cells preincubated in emem (total binding) or in unlabelled virus (non-saturable binding). saturable binding was calculated by subtraction of non-saturable binding cpm from total binding cpm. in each assay as positive and negative controls for saturable virus binding, and the results obtained confirmed the findings reported above for these cells. of the enterocyte fractions harvested from the tips of the villi or the crypts of a newborn or a weaned piglet, and tested in the competitive virus binding assay, only the enterocytes harvested from the villi of the newborn piglet showed saturable binding of tgev (fig. ) , similar to that demonstrated for st cells. binding of tgev to the other three fractions, like the binding of the virus to mdbk cells, was non-saturable. binding of tgev to enterocytes from the crypts of the newborn piglet is shown in fig. , and similar results were obtained with enterocytes from the villi and crypts of the weaned piglet. studies on the attachment of tgev to st cells were first described by nguyen et al. ( ) , who concluded that the attachment was to specific sites on the cell surface since incubation of the cells with one strain of tgev inhibited the attachment of another strain of the virus. the present findings on the saturability of virus binding to st cells support this conclusion. binding of tgev to mdbk cells, in which the virus fails to replicate, was at a much lower level than to st cells, and since the binding to mdbk cells was nonsaturable, it is concluded that these cells lack specific receptors for tgev. the technique of release of enterocytes, by chelation, from the tips of the villi to the jejunal crypts, developed initially for use in rats (weiser, ) was successfully applied to piglets. histological examination of the intestine after each cycle of chelation confirmed the progressive release of enterocytes in each fraction. a high level of alkaline phosphatase activity was found only in the enterocytes harvested from the tips of the villi. this contrasted with findings in the rat (weiser, ) in which there was a progressive decline in enzyme activity from the villi to the crypts. however, in the present study there was a progressive decline in the intensity of periodic acid-schiff staining, from the villi to the crypts, as described in the rat (gratecos et al., ) , corresponding to the glycoprotein content of the plasma membrane, which increases with progressive differentiation of the enterocytes. binding of tgev to the villous enterocytes of newborn piglets occurred at a level comparable to that demonstrated for st cells, and significantly higher than virus binding to all other enterocyte fractions from both newborn and weaned piglets. the competitive binding assays indicated that saturable binding occurred only to the villous enterocytes of newborn piglets. since saturable binding is accepted as evidence of binding to specific receptors on the cell surface (tardieu et al., ) this finding would suggest that specific tgev receptors on enterocytes are restricted to neonatal villous enterocytes. this could be an important factor contributing to the susceptibility of villous, rather than cryptal enterocytes, to infection with tgev. the apparent lack of specific virus binding to villous enterocytes from weaned piglets was a somewhat puzzling finding since the virus is capable of infecting these cells in older piglets, although the latter are much less susceptible to infection than newborn piglets. our use of a cell culture adapted strain of tgev which is somewhat less virulent than some field strains of the virus may have contributed to this result, but it may be that specific receptors are far fewer in older piglets, and below the level of detection by the methods used in the present study. a low level of specific binding may have been masked by non-specific binding of the virus to the cell surface. it is also possible that the non-specific binding of tgev to villous enterocytes may lead to productive infection, but much less efficiently than binding to specific receptors. it has been shown that the dose of virus needed to infect an adult pig is times greater than that required to infect a neonate (witte and walther, ) and this may reflect the sparseness of receptors on the villous enterocytes of older pigs. it is concluded that the high susceptibility of newborn piglets to tgev, and the tropism of the virus, may be related to the presence of specific, saturable binding sites on the plasma membrane of the villous enterocytes in the neonate. shepherd et al. ( ) observed tge viral particles traversing the intact microvilli of mature enterocytes in the small intestine of an experimentally infected piglet, and postulated the synthesis of brush border receptors as the enterocytes differentiated during their migration from the crypts to the tips of the villi. the present findings provide the first evidence in support of such a hypothesis. the relationship of the aminopeptidase n receptor for tgev, demonstrated recently by delmas et al. ( ) , to our findings, remains to be explored by determining the distribution of this enzyme in different enterocyte fractions from newborn and weaned piglets. transmissible gastroenteritis virus (classical enteric variant) and transmissible gastroenteritis virus (respiratory variant) a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding antibody-dependent and spontaneous cell-mediated cytotoxicity against transmissible gastroenteritis virus infected ceils by lymphocytes from sows, fetuses and neonatal pigs aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev studies of transmissible gastroenteritis virus receptors on swine testicle cells plasma membranes from rat intestinal epithelial cells at different stages of maturation i. preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells studies on transmissible gastroenteritis of swine ii. selected characteristics of a cytopathogenic virus common to five isolates of transmissible gastroenteritis epithelial cell migration in the alimentary mucosa of the suckling pig age dependent resistance to tge of swine. . clinical signs and some mucosal dimensions in the small intestine transmissible gastroenteritis (tge) of swine: in vitro virus attachment and effects of polyanions and polycations transmissible gastroenteritis of swine: virus-intestinal cell interactions i. immunofluorescence, histopathology and virus production in the small intestine through the course of infection enteropathogenic coronaviruses saturable binding sites for vesicular stomatitis virus on the suface of vero cells the s protein of bovine coronavirus is a hemagglutinin recognising - -acetylated sialic acid as a receptor determinant the mucosal lesion in viral enteritis. extent and dynamics of the epithelial response to virus invasion in transmissible gastroenteritis of piglets interaction of viruses with cell surface receptors human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses electron microscopy of intestinal epithelial cells of piglets infected with a transmissible gastroenteritis virus intestinal epithelial cell surface membrane glycoprotein synthesis i. an indicator of cell differentiation monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated strains receptor for mouse hepatitis virus is a member of/he carcinoembryonic antigen family ofglycoproteins age-dependent susceptibility of pigs to infection with the virus of transmissible gastroenteritis human aminopeptidase n is a receptor for human coronavirus e this research was supported by the natural sciences and engineering research council of canada, and by the ontario ministry of agriculture and food. technical assistance was provided by marta dubisz, tricia horner and l~va varady. key: cord- -nn l rai authors: garcia-sanchez, j.; corral, c.; halaihel, n.g.; simon, m.c.; alonso, j.l.; muzquiz, j.l.; ortega, c.; girones, o. title: survey of rotavirus infection in a dairy herd: comparison between polycrylamide gel electrophoresis and two commercial tests date: - - journal: vet microbiol doi: . / - ( ) -e sha: doc_id: cord_uid: nn l rai a survey of rotavirus infection in a dairy herd with a history of neonatal diarrhoea was carried out. faecal samples taken from cows before and after calving as well as faeces taken from their calves daily from birth to two weeks of life were tested for rotavirus by polyacrylamide gel electrophoresis (page) and compared with an elisa and a latex agglutination commercial test. rotavirus excretion was not detected in faeces from cows around parturition by any of the three tests. however, all of their calves shed rotaviruses during the observation period. the onset of rotavirus excretion determined by page ranged from day to day of life (day . ± . on average) and lasted for to days ( . ± . days on average). chi-square test showed a significant association (p= . ) between the presence of rotavirus and the altered consistency of calves faeces. all the three tests showed similar results (overall agreement . %) but discrepancies were detected mainly at the beginning or at the end of the rotavirus excretion period. results obtained with both commercial kits closely paralleled each other and parameters other than sensitivity, specificity, diagnostic accuracy or predictive values have to be considered as selection criteria. neonatal calf diarrhoea is a major threat to the cattle industry due to mortality, medical costs and poor growth of calves in both dairy and beef herds. the syndrome has a great aetiological complexity and numerous pathogens have been reported in association with the disease (woode and bridger, ; woode et at., ; moon et at., ; tzipori, accepted to be an important cause of neonatal diarrhoea in calves throughout the world with its greatest frequency and severity within the first two weeks of life (acres et al., ; de leeuw et al., ) . large numbers of rotavirus particles are excreted in faeces of diarrheic calves and are highly resistant to inactivation. thus, the main source of rotavirus in an outbreak results from environmental contamination and the spread of infection can temporarilly be broken by thorough cleaning and disinfection of the premises (mcnulty and logan, ) . however, subclinically infected older calves and adult cows may play a role in the spread of infection to young calves as well as in the perpetuation of infection within a herd (goto et al., ) . various methods have been developed for rapid detection of rotaviruses in faecal samples; these include electron microscopy (considered for a long time as the reference method), immune electron microscopy, counter immuno-electrophoresis, complement fixation, fluorescent antibody tests, different immunoassays using antibodies labelled with radioisotopes or enzymes (elisa), latex agglutination and polyacrylamide gel electrophoresis (page). some of these tests are commercially available. this paper presents the results of a longitudinal survey of rotavirus infection from birth to two weeks of life in conventionally reared calves using the page technique and analyzes the relationship between rotavirus shedding and the consistency of faeces. faecal shedding of rotavirus in their dams around calving is also studied. the page results are compared with those obtained by two commercial kits, one using elisa and the other latex agglutination. the survey was carried out on a friesian dairy farm located in northeastern spain containing approximately heifers and cows. the herd had a history of recurrent neonatal diarrhoea for previous years. pregnant cows were vaccinated with a bacterin against e. coli k + (imocolibov, rhone-merieux laboratories) approximately three weeks before the expected calving date and were usually moved to a communal house one week before calving. the calves were separated from their dam a few hours after birth and moved to a communal pen next to the cows where they remained for at least days and then they were transferred to fattening units with - calves per unit. each calf was bucket-fed its mother's colostrum during the first day of life ( feedings, litres per feeding), the first feeding being usually within hours after birth, and pooled cows colostrum during the second day of life in the same conditions. thereafter, they were fed milk replacers from an automatic feeder. fifteen cows, chosen on the basis of their predicted calving dates, were included in the survey. calvings took place over the period january- february . faeces samples were collected from cows before calving (days , , and between days - pre-partum; more samples were taken but only those cited were tested), the day of calving and after calving (days , and post-parturn). faeces samples were also obtained from the rectum of the calves daily from birth (considered as day ) to days of life. at each sampling the faeces were recorded as being normal, abnormal (if not obviously diarrheic but changed with respect to consistency and/or presence of blood or mucus ) or diarrheic (if fluid or profuse ). faecal score was determined by the same person for reasons of uniformity of interpretation. none of the calves with diarrhoea was treated at all or isolated from other calves. all the faeces samples ( from cows and from calves) were tested for the presence of rotaviruses by polyacrylamide gel electrophoresis (page), a commercial elisa kit and a commercial latex agglutination kit. page. the extraction of viral rna, its resolution and staining was carried out as described by herring et al. ( ) with minor modifications. briefly, faecal specimens were diluted : by weight with extraction buffer containing % sodium dodecyl sulfate, an equal volume of : phenol-chloroform was added and the mixture was vortexed and centrifuged for min at g. the aqueous phase was removed. for electrophoresis, pl of the clear supernatant were mixed with # of blue marker ( % sucrose containing . % bromophenol blue) and loaded onto a discontinuous polyacrylamide gel ( % concentration for the stacking gel and . % for the running gel). the gels were assembled using a mini-protean ii cell (biorad laboratories, richmond, california, usa) and ran with a ma running current and constant voltage for . h using a / . model power supply (biorad). after that, gels were fixed, developed and silver-stained. a positive control sample was always included in each gel for comparison of the segmented viral rna migration pattern. commercial elisa. the enzygnost rotavirus (ag) monoclonal test (behring institute, behringwerke ag, marburg, germany) was performed according to the manufacturer's instructions. briefly, faeces taken using the dispenser provided were transferred to . ml of the dilution buffer and throughly mixed. then, . ml of the supernatant was transferred to the test plate wells coated with rabbit sa- rotavirus antiserum. following incubation for min at °c the plates were washed times and . ml of antirotavirus conjugate consisting of a mouse monoclonal antibody conjugated with peroxidase were added to each well. the plates were incubated and washed again in the same conditions, . ml of the substrate-chromogen were added per well and were incubated for min at room temperature protected from light. finally, the reaction was stopped and the plates were read at nm wavelength using a multiskan spectrophotometer (flow laboratories). the cut-off value was calculated by adding the mean value of the negative control samples to a threshold value of . .d. units. the retest limit was calculated by adding . o.d. units to the cut-off value. in this way results could be considered as negative (if the absorbance of the sample was less than the cut-off value ), positive (if it was greater than the retest limit) and gray zone (if it was between the cut-off and retest limits ). commercial latex agglutination. the slidex rota-kit monoclonal test (biomrrieux, marcy-l'etoile, france) was carried out according to the instructions enclosed with the kit. briefly, . gr of the faecal specimen were diluted in ml of the buffer supplied and mixed. the emulsions were centrifuged for min at g and then a drop of supematant from each specimen was placed in each of two circles on an agglutination card. a drop of reagent (latex coated with monoclonal antirotavirus antibody) was added to one and a drop of reagent (latex coated with rotavirus negative serum) to the other. following mixing, the cards were rotated gently for min. a positive reaction was considered when clear agglutination of the test latex with no agglutination of the control was seen. agglutination of both control and test latex indicated a non-specific reaction. all samples were read by the same person. in order to study the association between the variable "presence of rotavirus" and the variable "consistency of faeces", a × contingency table was done and the chi-square test was performed. diagnostic test results were analyzed in two ways: first, with page as a gold standard and secondly, on a comparative basis with each other. in the first case, results of the commercial tests were compared with those of page by using sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy. in the abscence of a standard, the kappa statistic was used for comparison. the comparative measures are defined as follows: sensitivity (true-positive rate ): is the proportion of samples in which the test is positive when page is positive. specificity (true-negative rate ): is the proportion of samples in which the test is negative when page is negative. positive predictive value: is the proportion of samples in which page is positive when the test is positive. negative predictive value: is the proportion of samples in which page is negative when the test is negative. diagnostic accuracy: was determined by dividing the number of positive and negative specimens in both the commercial kit and page by the total number of specimens tested. kappa statistic is an overall measure of agreement between two tests and is useful for measuring agreement in the absence of a standard. it compares the observed proportion of samples in which the tests agree with the proportion that would be expected to agree by chance. kappa calculation was carried out as described by goodman and kruskal ( ) . for interpreting kappa values the following benchmarks can be considered: a result between . and . indicates a slight agreement, between . and . is a fair agreement, between . and . is a moderate agreement, between . and . is a substantial agreement and between . and . is an almost perfect agreement. rotavirus shedding was not detected in any sample taken from cows before or after parturition. all samples were also negative for rotavirus when cold-ethanol precipitation of rna was performed. the results of rotavirus shedding in faecal samples taken from the calves are shown in table . during the first two weeks of life rotavirus was detected in the faeces of all calves. the presence of rotavirus was determined in out of samples ( %). thirteen out of calves showed a continuous shedding pattern and only two calves (calves no. and ) showed a discontinuous one. the onset ofrotavirus excretion in faeces ranged from day to day of life (day . +_ . as a mean) and the duration of that excretion ranged from to days (mean . _+ . days). only one electropherotype was found in the survey and it was characteristic of group a rotaviruses. all calves passed abnormal and diarrheic faeces along the two week observation period but no fatal cases were observed during that time (table ) . the onset of rotavirus excretion in faeces coincided with the onset of diarrhoea or of excretion of abnormal faeces in some of the animals (clearly in calves no. , , , and ) but that association was quite variable. however, when every sample was considered independently of the animal and of the chronological sequence, the chi-square test showed a high association be- tween the two variables in such a way that, when a sample was positive for rotavirus, the faeces tended to be diarrheic and when a sample was negative for rotavirus it tended to be normal (p= . ) ( table ) . as observed with page, rotavirus shedding was not detected in cows faeces taken around calving by any of the commercial tests. when calves faeces were tested, results obtained by elisa and latex agglutination commercial kits closely coincided with those by page: the agreement between page and latex agglutination was . %, % between page and elisa, . % between latex agglutination and elisa and the overall agreement between the three tests was . % (table ). the discrepant resuits fall into four categories and are shown in table . most of the discrepancies ( samples) were observed in the first or in the last sample positive for rotavirus by page in the calves rotavirus excretion period, that's to say, when the shedding pattern changed from negative to positive or vice versa and corresponded to samples negative by latex agglutination while negative (or gray zone) by elisa ( samples) or positive by elisa ( samples). another three discrepancies were detected in which the samples were positive by page and negative by both elisa and latex agglutination and all of them corresponded to two calves with a discontinuous shedding pattern (calf no. , days and ; calf no. , day ). the other two discrepant results corresponded to samples positive by elisa and negative by both page and latex agglutination; one of them was detected in a calf with a discontinuous shedding pattern (calf no. , day ) and the other one corresponded to the first positive sample in the rotavirus shedding pattern determined by elisa (calf no. , day ). in table sensitivity, specificity, positive and negative predictive values and diagnostic accuracy of both latex agglutination and elisa kits are shown considering page as a gold standard. latex agglutination showed a lower sensitivity ( %) and a higher specificity ( %) than elisa ( . % and . %, respectively) but the predictive values and the diagnostic accuracy were quite similar for both tests. the kappa statistic for each pair of tests (when no gold standard was considered) is given in table . it shows an almost perfect agreement between the three tests (kappa values between . and . ). all calves monitored in our survey were shown to be infected and shed rotaviruses in faeces within the first two weeks of life. this result seems to indicate that bovine rotavirus is enzootic at this farm and it could explain its history of recurrent outbreaks of neonatal diarrhoea. in the present study, rotavirus was detected in the faeces of some animals as early as - days of age with an increasing number of excretors thereafter. experimental infection of calves has shown that the period between infection and detection of excretion of the virus in faeces is about two days (de leeuw et al., ; logan et al., ) . that indicates that most calves were exposed to infection very soon after birth. excretion of rotavirus in calves faeces has been reported before hours of age but such an early detection is infrequent (acres and b abiuk, ; de leeuw et al., ) . the delay in excretion of the virus under field conditions is probably because of the presence of specific colostral antibody within the gut lumen and the elimination of this antibody renders the calf susceptible to overt infection as suggested by de leeuw et al. ( ) . so, the early detection of rotavirus in faeces detected in the present study could be due to an inadequate feeding of colostrum. in our study, rotavirus shedding in faeces started at . days of life on average and showed a mean duration of . days. in a similar survey, mcnulty et al. ( ) reported an onset of rotavirus excretion at . days old on average in an endemically infected dairy herd and similar excretion periods have been described in both beef and dairy herds by others (acres and babiuk, ; de leeuw et al., ; goto et al., ) . nevertheless, the exact onset and duration of rotavirus shedding is difficult to establish in all these studies because they lack a daily sampling design. rotavirus excretion coincided with abnormal or diarrheic faeces in some animals and chi-square test showed a statistically significant association (p= . ) between the presence of rotavirus and the altered consistency of faeces. this suggests that rotaviruses are involved in the aetiology of this outbreak of neonatal diarrhoea although no other agents were studied. de leeuw et al. ( ) described that rotavirus infection was associated with cases of "late diarrhoea" (diarrhoea from the th to the th day of life) whereas other agents appeared to be of minor importance, and schusser et al. ( ) reported that, although the correlation between rotavirus excretion and clin-ical diarrhoea in calves was poor during the first weeks of life, the severest form of watery diarrhoea was limited to the first three weeks of life, where rotavirus excretion peaked. the association between rotavirus infection and abnormal or diarrheic faeces has also been reported by several authors (mcnulty and logan, ; goto et al., ; bellinzoni et al., ; murakami et al., ) . the role that adult animals may play as a source of infection for calves has been a matter of controversy. in the present study, we did not detect the presence of rotavirus in any sample taken from cows around parturition by any of the three tests. our results agree with those of several authors that have also failed to detect rotaviruses in faeces of adult animals (dagenais et al., ; scherrer et al., ; sihvonen and miettinen, ) . on the contrary, there have been several reports describing the excretion of rotavirus in faeces of a small number of animals with or without diarrhoea and usually over a short period around parturition (mcnulty and logan, ; bellinzoni et al., ; murakami et al., ) and it has been suggested that adult carriers might be important in the epizootiology of the infection (woode, ; crouch and acres, ) . recently, kodituwakku and harbour ( ) reported an intermitent excretion of rotavirus in asymptomatic cows throughout pregnancy although rotavirus excretion and diarrhoea were detected in only one of their calves and goto et al. ( ) have indicated that the perpetuation of rotavirus infection within a herd may result from persistent or chronic infections in older calves and adult animals. however, it is generally accepted that once an outbreak is underway, young calves act as amplifiers of the infection and that environmental contamination is the major source of infection for newborn calves, whereas maternal infection is of minor significance (dagenais et al., ; mcnulty and logan, ; goto et al., ; murakami et al., ) . the rotavirus shedding pattern obtained with the three tests was quite similar, the overall agreement being . % among the three tests. most discrepant results were positive by page while negative by latex agglutination ( samples ) and negative (or gray zone ) by elisa ( samples ) and all of them except three corresponded to the first or last positive sample in the page shedding pattern, when the viral content in faeces was probably low. these results seem to indicate a higher sensitivity of the page test compared with that of latex agglutination and elisa. the same explanation may be given for the discrepant results observed in calf no. (days and ) and calf no. (day ) in which a discontinuous shedding pattern was detected by page but we have no certainty. the results observed in two samples (calf no. , day and calf no. , day ), in which rotaviruses were detected only by elisa, are more difficult to explain. they could be false-positive results but if the shedding patterns of that calves are considered, the true-positive result cannot be ruled out. latex agglutination for detection of rotavirus in faecal specimens quantitative observations on experimental reo-like virus (rotavirus) infection in colostrum-deprived calves an assessment of the sensitivity of three methods for the detection of rotavirus longitudinal survey of rotavirus infection in calves comparison of six commercial kits for the diagnosis of rotavirus infection in man and calves pathogenic relationships of rotavirus, escherichia coli and other agents in mixed infections in calves comparison of six methods for detecting human rotavirus in stools a survey on bovine rotavirus type l-associated neonatal calf diarrhea in a beef herd direct appraisal of latex agglutination testing, a convenient alternative to enzyme immunoassay for the detection of rotavirus in childhood gastroenteritis, by comparison of two enzyme immunoassays and two latex tests elisa for the detection of rotavirus and rotavirus/antibody complexes in faeces a follow-up study on bovine rotavirus dissemination among calves of a large dairy herd rotavirus and enterotoxigenic escherichia coli infections of calves on a closed finnish dairy farm evaluation of seven immunoassays for detection of rotavirus in pediatric stool samples the aetiology and diagnosis of calf diarrhoea epizootology of bovine rotavirus infection viral enteritis of calves morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals key: cord- -swx kqob authors: paul, prem s. title: applications of nucleic acid probes in veterinary infectious diseases date: - - journal: vet microbiol doi: . / - ( ) -z sha: doc_id: cord_uid: swx kqob nucleic acid probe technology is increasingly being used in basic research in veterinary microbiology and in diagnosis of infectious diseases of veterinary importance. this review presents an overview of nucleic acid probe methodology and its applications in veterinary infectious diseases. the major applications of nucleic acid probes include detection of pathogens in clinical samples, especially those organisms which are fastidious and difficult to cultivate, differentiation of virulent from a virulent organisms and vaccine strains from wild type isolates, typing of microorganisms mapping genes, screening libraries of cloned dna for specific genes, detection of latently infected or carrier animals, study of mechanisms of pathogenesis, epidemiological studies and food safety. nucleic acid probe technology is being increasingly used by veterinary medical researchers and diagnosticians. this has resulted from the increased awareness, commercial availability of reagents for the preparation of probes, the need for more sensitive methods for the detection of fastidious organisms, the need for more specific methods for the detection of nucleic acids, and recent advances in nucleic acid probe technology. the key to the development of nucleic acid probes is to identify nucleotide sequences which are unique to the particular organism of interest. these sequences may be genomic dna, plasmid dna or ribosomal rna. the nucleic acid which contains such sequences is isolated and amplified by recombinant dna technology. the unique nucleotide sequence is then cleaved and is labelled with a reporter molecule such as a radioactive ( p, s) or nonradioactive (biotin, digoxigenin ) molecule. alternatively, complementary dna, rna or synthetic oligonucleotides may be labelled and used as probes. the labelled dna or rna in single-stranded form is then hybridized to singlestranded nucleic acid (dna or rna) in tissues (in situ hybridization), on paper, or in solution. if the nucleotide sequences in the nucleic acid probe are complementary to those in the sample, hybridization occurs and results in double-stranded nucleic acid being formed. nonhybridized single strand probe is removed. hybridization is monitored by autoradiography (exposure to xray film) in the case of probes labelled with radioactive molecules, or colorimetrically or visually for probes labelled with nonradioactive molecules. the basic principles of nucleic acid hybridization and the effect of different hybridization conditions such as probe size, salt concentration, and temperature have been reviewed by gillespie ( ) . gillespie also presents an overview of different hybridization formats which have either been developed or are being developed. readers are also referred to reviews on nucleic acid probes by minson and darby ( ) , edberg ( ) , kulski ( ) , pereira ( ), norval and bingham ( ) , paul ( ) , and tenover ( and tenover ( , . major drawbacks of utilizing nucleic acid probes in the past have included sensitivity limits of hybridization technology, safety and short half-life of radioactive probes. the introduction of new technologies for nucleic acid amplification such as the polymerase chain reaction (oste, ) have revolutionized probe technology. sensitivity is no longer a major problem as target sequences can by amplified prior to hybridization. amplification of target sequences by the polymerase chain reaction and transcription amplification system (tas) is reviewed by gingeras et al. ( ) . alternative methods for amplification are covered by gillespie ( ) . a number of nonradioactive detection systems are currently available and some of them are reviewed by pereira ( ) ; however, improved technologies need to be developed for nonradioactive detection of nucleic acids in clinical samples, especially feces. some of the potential applications of nucleic acid probes in veterinary infectious diseases are presented in this review. conventional approaches of detecting etiologic agents of infectious disease include the isolation of organisms, direct detection of organisms in clinical samples by electron microscopy, and direct detection of antigens in tissue sections and clinical samples by immunofluorescence, immunocytochemistry or elisa. the availability of monoclonal antibodies has provided specific detection of many infectious agents in clinical samples. monoclonal antibodies will continue to play a significant role in diagnostics, however, in some instances monoclonal antibody based tests are unsuitable and alternative methods are needed. monoclonal antibody assays may be ineffective on samples contaminated with other organisms either due to the condition of the samples or the sensitivity of the assays. nucleic acid probes provide an alternative diagnostic tool for the detection of infectious agents. nucleic acid probes have been used to detect viruses, bacteria, and parasites in clinical samples. among viruses of veterinary importance detected by nucleic acid probes are adenovirus (jouvenne et al., ) , african swine fever virus (tabares, ) , blue tongue virus (dangler et al., ) , bovine viral diarrhea virus (brock and potgieter, ) , enterovirus (bruce et al., ) , infectious bursal disease virus (jackwood, ) , foot-and-mouth disease virus (rossi et al., ) , porcine parvovirus (ridpath et al., ; harding and molitor, ; oraveerakul et al., ) , pseudorabies virus (gutekunst, ; maes et al., ) , rhinovirus (gama et al., ) , rotavirus (dimitroy et al., ; johnson et al., ; rosen et al., ) , and transmissible gastroenteritis virus (shockley et al., ) . bacteria for which probes have been developed include campylobacter spp. (gebhart et al., ) , escherichia coli (moseley et al., ; maddox and wilson, ) , leptospira sp. (zuerner and bolin, ) , listeria sp. (wesley et al., ) , mycobacterium paratuberculosis (mcfadden et al., (mcfadden et al., , , mycobacterium tuberculosis (eisenach et al., ; mcfadden et al., ) , mycoplasma gallisepticum (geary et al., ) and other mycoplasma spp. (razin et al., ) . nucleic acid probes have also been developed to detect anaplasma marginale (eriks et al., ; aboytes-torres and buening, ; golf et al., ) and histoplasma capsulatum (keath et al., ) . the most important application of nucleic acid probes is the detection of fastidious organisms. many microorganisms, such as mycoplasma spp. and mycobacterium spp., cannot be cultivated easily or require weeks to months of culture. nucleic acid probes have been developed and are being successfully used for their detection either directly in clinical specimens or following a short culture period. probes have also been used to detect specific microorganisms in tissues by in situ hybridization (brigati et al., ; dunn et al., ; brown et al., ; collisson et al., ) . mycoplasmas, parvovirus and bovine viral diarrhea virus are frequently present as contaminants in cell cultures. their detection is sometimes difficult by inexperienced workers. nucleic acid probes offer standardized reagents for their detection and such probes have been developed for these microorganisms (mcgarrity and kotani, ; oraveerakaul et al., ) . the differentiation of virulent from avirulent organisms and vaccine strains from wild type isolates is extremely important in diagnostic medicine and epidemiological studies. this can be accomplished immunologically by monoclonal antibodies, and genetically by restriction endonuclease analysis, se-quence analysis and nucleic acid probes. treponerna hyodysenteriae, causative agent of swine dysentery, can not be easily distinguished from a nonpathogenic treponeme, treponema innocens, which is commonly present in normal swine. a nucleic acid probe prepared from a plasmid associated with treponema hyodysenteriae was successfully used to differentiate pathogenic and nonpathogenic treponemes (joens, ) . similarly, probes have been developed to detect virulence genes. moseley et al. ( ) used probes for the first time to identify organisms carrying genes for enterotoxins. such probes are now routinely being used to determine potential pathogenicity of escherichia coli by screening for genes of enterotoxins (moseley et al., ; maddox and wilson, ; karch and meyer, ) . typing of microorganisms has usually been based on biologic and antigenic characteristics. these classical procedures have been found to be unsatisfactory in many cases. monoclonal antibodies have provided useful methods for typing many organisms where conventional reagents have not worked. production of type specific monoclonal antibodies is often difficult, time consuming and sometimes an impossible task, especially when bacterial and parasitic organisms are used due to the large number of proteins. genetic tools have been useful in typing organisms where serologic tests were unsuccessful, tedius or gave ambiguous results. nucleic acid probes have been used to type pillus and enterotoxin gene types to differentiate campylobactor spp. (chevrier et al., ; gebhart et al., ) , escherichia coli (maddox and wilson, ) , leptospira spp. (zuerner and bolin, ) , mycobacterium spp. (mcfadden et al., ) , and rotavirus groups, subgroups and serotypes (dimitrov et al., ; johnson et al., ; rosen et al., ) . nucleic acid probes provide a powerful tool for evaluating homology between related dnas (howley et al., ) . this can be accomplished by dot blot, southern blot or northern blot hybridization. for example, homology between polyoma and papovavirus was analyzed by southern blot hybridization (howley et al., ) . similarly, homology between porcine and canine parvovirus was evaluated using southern blot hybridization (ridpath et al., ) . location of homologous sequences on the physical map of these viruses was also revealed in these studies by using probes from subgenomic fragments. hybridization has also been successfully used to identify genetic reassortants (midhun et al., ) and gene rearrangement (paul et al., ) among rotaviruses. these strategies can also be employed to map specific genes of interest using well studied genes from related organisms. a complete map of bovine adenovirus was generated by southern blot hybridization at low stringency with probes prepared from human adenovirus dna (hu et al., ) . transcriptional maps for specific genes and identification of microbespecific rnas can be identified by northern blot hybridization. additionally, coding assignments for genes can be established by a combination of northern blot hybridization and in vitro translation (collins et al., ) . probes have also been useful for screening libraries for specific genes (rigas et al., ) and cloning genes by oligonucleotide hybridization (mayaux et al., ) . the latter method is being extensively used for cloning specific genes or subcloning regions of interest. nucleic acid probes are an excellent tool for the determination of molecular mechanisms of pathogenesis. mechanisms of latency induction by herpesviruses have been studied by in situ hybridization. rock et al. ( ) found that with bovine herpesvirus- (bhv- ), causative agent of infectious bovine rhinotracheitis, the nucleic acid is present in ganglionic neurons of rabbits latently infected with bhv- . furthermore, only selected regions of the viral genome were transcribed in neurons of latently infected rabbits, whereas all regions of the genome were transcribed in lytically infected cells. these studies indicated that transcription of bhv- is restricted during the latent phase of infection and suggested that transcription of specific genes may be important in the establishment and maintenance of latency. the mechanism of persistent infection by equine infectious anemia virus (eiav) was determined using southern blot hybridization by rasty et al. ( ) . they found that the degree of provirus integration may be important in the establishment of persistent infection since % of provirus was randomly integrated in persistently infected cells, whereas - % ofprovirus was integrated in lytically infected cells. hybridization technology was also used to better understand the mechanisms of leukemogenesis by bovine leukemia virus (blv) (gregoire et al., ) . these studies revealed that blv-induced tumors in sheep were monoclonal in the same sheep, integration was observed at different sites in the animals and there was nonintegrated blv in tumors. hybridization procedures can provide information on organs and cell types harboring organisms, the number of genome copies per cell, whether genes are integrated, and the transcription status and regulation of the genes. these studies, combined with immunocytochemistry, can provide valuable information on the presence of viral nucleic acid and antigens in cells and can provide information on the status of virus expression. p.s, paul the development of specific probes specific for organisms, groups of organisms, or specific serotypes provides a valuable tool for epidemiological studies. such tests can be developed in a few reference laboratories and distributed to diagnostic laboratories to determine prevalence and incidence of a specific organism. probes will be especially useful for determining the epidemiology of microorganisms which are difficult to cultivate or type. there is increasingly more emphasis on the rapid detection and identification of microorganisms in foods. in the past, this has generally been accomplished by the isolation and identification of bacteria using conventional methods that are time consuming and require the presence of viable microorganisms. nucleic acid probes provide a more rapid method for the identification of microorganisms in food. nucleic acid probes have been developed and are commercially available for the detection of several microorganisms of public health importance e.g., toxigenic escherichia coli, listeria sp., salmonella spp., and yersinia enterocolitica (hill, ) . probes for additional microorganisms and improved methodology for their detection in foods will enhance their use in food safety. in this review, some of the applications of nucleic acid probes are highlighted. additional potential applications will become evident as newer technologies are developed. automation in nucleic acid probe technology will significantly enhance its acceptance in diagnostic medicine. in the meantime, nucleic acid probes will continue to serve as a powerful tool to researchers and to diagnosticians in selected cases. development of a recombinant anaplasma marginale dna probe detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes detection of bovine viral diarrhea virus in serum from cattle by dot blot hybridization assay detection of latent pseudorabies virus in swine using in situ hybridization detection ofenteroviruses using edna and synthetic oligonucleotide probes a new method for identifying ('aotpylobacter spp identification of a tenth mrna of respiratory syncytial virus and assignment of polypeptides to the viral genes detection of avian infectious bronchitis viral infection using in situ hybridization and recombinant dna rapid identification of bluetongue virus by nucleic acid hybridization in solution detection of rotaviruses by nucleic acid hybridization with cloned dna of simian rotavirus sa genes detection of bovine herpes virusspecific nucleic acids by in situ hybridization with biotinylated dna probes principles of nucleic acid hybridization and comparison with monoclonal antibody technology for the diagnosis of infectious diseases repetitive dna sequences as probes for mycobacterium tuberculosis detection and quantitation ofanaplasma marginale in carrier cattle by using a nucleic acid probe polymerase chain reaction amplification ofrhinovirus nucleic acids from clinical materials species-specific biotinylated dna probe for the detection ofmycoplasma gallisepticum species-specific dna probes for campylobacter species isolated from pigs with proliferative enteritis the magic and challenge of dna probes as diagnostic reagents methodologies for in vitro nucleic acid amplification and their applications comparison of a dna probe, complement-fixation and indirect immunofluorescence tests for diagnosing anaplasma marginale in suspected carrier cattle different bovine leukemia virus-induced tumors harbor the provirus in different chromosomes latent pseudorabies virus infection in swine detected by rna-dna hybridization porcine parvovirus: replication in and inhibition of selected cellular functions of swine alveolar macrophages and peripheral blood lymphocytes detection of bacteria in foods using dna hybridization a rapid method for detecting and mapping homology between heterologous dnas restriction analysis and homology studies of the bovine adenovirus genome development and characterization of nucleic acid probes to infectious bursal disease viruses the diagnosis of swine dysentery using a labeled nucleic acid probe development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes cloning, physical mapping and cross hybridization of the canine adenovirus types and genomes evaluation ofoligonucleotide probes for identification ofshigalike-toxin-producing escherichia coli nucleic acid probes in diagnosis of viral diseases of man dna probe for the identification ofhistoplasma capsulatum high technology diagnostics: detection of enterotoxigenic escherchia coli, using dna probes polymerase chain reaction amplification ofpseudorabies virus dna from acutely and latently infected cells cloning e. coli genes by oligonucleotide hybridization analysis by rna-rna hybridization assay of intertypic rotaviruses suggests that gene reassortment occurs in vivo hybridization techniques crohn's diseaseisolated mycobacteria are identical to mycobacterium paratuberculosis, as determined by dna probes that distinguish between mycobacterial species dna probes to identify and detect mycobacterium paratuberculosis in clinical and veterinary samples detection of cell culture mycoplasmas by a genetic probe identification of enterotoxigenic escherichia coli by colony hybridization using three enterotoxin gene probes advances in the use of nucleic acid probes in diagnosis of viral diseases of man detection of porcine parvovirus using non-radioactive nucleic acid hybridization polymerase chain reaction applications of recombinant dna technology in the development of vaccines, reagents and dna probes isolation of a bovine rotavirus with a "super-short" rna electrophoretic pattern from a calf with diarrhea non-radioactive nucleic acid probes for the diagnosis of virus infections proviral integration and transcriptional patterns of equine infectious anemia virus during persistent and cytopathic infections dna probes for detection and identification of mycoplasmas (mollicutes) comparison of porcine parvovirus to other parvoviruses by restriction site mapping and hybridization analysis of southern blots rapid plasmid library screening using reca-coated biotinylated probes mapping bovine herpesvirus type latencyrelated rna in trigeminal ganglia of latently infected rabbits hybridization probes for the detection and differentiation of two serotypes of porcine rotavirus detection of foot-and-mouth disease virus with dna probes in bovine esophageal-pharyngeal fluids diagnosis of porcine and bovine enteric coronavirus infections using cloned cdna probes detection of dna viruses by radioactive and non-radioactive dna probes: application to african swine fever virus diagnostic deoxyribonucleic acid probes for infectious diseases dna probes for infectious diseases characterization of listeria monocytogenes isolates by restriction enzyme analysis and southern blot hybridization nucleic acid probe characterizes leptospira interrogans serovars by restriction fragment length polymorphisms key: cord- - w crrf authors: fey, h. title: proceedings of minisymposium on neonatal diarrhea in calves and pigs: saskatoon, sask., – may . veterinary infectious diseae organization (vido), university of saskatchewan, saskatoon, university publications office, , pp., $ . , isbn - - - date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: w crrf nan economic importance but also of great relevance for the understanding of the pathogenesis of intestinal disorders in man. this symposium proved to be very fruitful and the study of its proceedings offers a great deal of information. it was pointed out (s.d. acres, c.a. mebus and m. morin) that neonatal diarrhea is a multifactorial disease with the preponderance of some bacterial, viral and parasitic agents (enteropathogenic e. coil, coronavirus, reoviruslike organisms) and even the -- % of cases with unidentified etiology are considered to be infectious processes. the influence of the environment, i.e., climate, housing, nutrition, is enormous. c.l. gyles gave a very competent and impressive outline on the genetics of e. coll. this is no longer a science to be anxiously avoided by the practising clinician because of the hygienic and epidemiologic significance of the plasmid-born multiple resistance. the problem of the control of neonatal diarrhea by immunization procedures has been amply discussed by l.l. myers, m.r. wilson, i. mccallum and g. khachatourians and the clinical treatment by o.r. radostits as well. it seems that considerable progress has been made, for instance by the use of the k antigen as an immunogen. the antibody thus produced should be protective by prevention of the adhesion of enteropathogenic coli strains to the epithelial cell. also bacterins and e. coil vaccines of minicell cultures and immunoglobulin preparations have been successfully used. other authors presented their recent experiments on e. coil enterotoxins and offered pathogenetic considerations. a _fine _and very clear summation aiming at recommendations for future research and for measurements to be taken by veterinarians and the authorities, is given by h.w. moon, who is himself one of the most competent investigators in this field. this kind of symposium is very useful. it brings people working on different aspects of a given entity together ~.~d reflects the present standard of knowledge. it is a pleasure to be informed in such a concise way by this booklet. 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the latter preferably as sentences at the end of existent paragraphs or as mw paragraphs. . fifty reprints will be supplied free of charge. . additional reprints can be ordered on a reprint order form, which is included with the proofs. . unesco coupons are acceptable in payment of extra reprints. papers for consideration should be submitted to: the editorial secretariat, veterinary microbiology, p.o. box , ah amsterdam, the netherlands. key: cord- -gpz gkv authors: weiss, m.; steck, f.; kaderli, r.; horzinek, m.c. title: antibodies to berne virus in horses and other animals date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: gpz gkv after inoculation into foals, berne virus induced neutralizing antibody, but did not cause clinical symptoms. in a horizontal study of seropositive mares and their offspring, a decline of maternal antibodies and a sudden synchronous seroconversion in all foals were observed, again without clinical symptoms. the virus is widespread in the swiss horse population and has been so during the last decade; rises in antibody titers were noted in % of paired sera sampled at random. positive reactions were also obtained in serum neutralization tests and elisa using small numbers of horse sera from germany, france and the u.s.a. the results of neutralization tests and elisa were correlated in % of random samples tested; % were neutralization-positive and elisa-negative and in % the inverse was observed. neutralizing activity was found in the sera of other ungulates (cattle, goat, sheep and pig), laboratory rabbits and species of wild mice (clethrionomys glareolus and apodemus sylvaticus). inconclusive results were obtained with feline and human sera; those from dogs and foxes (vulpes vulpes) were consistently negative. the probable occurrence of antigenic variants in berne-type viruses is discussed. the purification and partial characterization of a new enveloped rna virus isolated during routine diagnostic work from a horse in berne, switzerland has been reported (weiss et al., ) . 'berne virus' measures -- nm in its largest diameter and consists of a peplomer-bearing envelope and an elongated core which is usually bent into an open torus within the membrane. the core is tubular in appearance and has a morphology indicative of helical nucleocapsid symmetry. the virus possesses an rna genome since its growth is not affected by dna nucleotide analogues; actinomycin d and alpha-amanitin on the other hand do inhibit replication, as does uv irradiation of the cells before infection. a unique pattern of viral polypeptides was reported (horzinek et al., ) . berne virus was shown to be serologically unrelated to known equine viruses as well as to representatives of the main antigenic clusters of the coronaviridae family (infectious bronchitis, mouse hepatitis and transmissible gastroenteritis viruses), which it resembles superficially in negatively stained preparations. however, an antigenic relationship was detected with the breda group of viruses recently discovered by workers in ames, ia (woode et al., ) . in this paper, results of seroepidemiological studies in horses and other animal species are presented, with the aim of showing the distribution and possible significance of this virus in the domestic and wild animal population. berne virus (strain p / ) was originally isolated in secondary horse kidney cells and our neutralization tests early in this study (before ) were also performed in these cells. later on, virus propagation and assay was done in embryonic mule skin cells (ems line) grown in eagle's minimum essential medium supplemented with non-essential amino acids ( %), lglutamine ( mm), sodium bicarbonate, antibiotics and -- % foetal calf serum; the serum had been pre-screened for the absence of antibody against berne virus by neutralization tests. a total of randomly selected adult horses from various regions of switzerland were sampled two times, in order to show seroconversion. the period between the bleedings ranged between and days, with about % of the second samples taken -- days after the first ones. additional ungrouped random serum samples were obtained from berlin (germany), the lyon area (france) and from various regions of the usa. in the swiss "haras federal avenches" mares and their foals were followed serologically over a period of months (table i) . a limited serosurvey was attempted using random samples from cattle, goats, sheep, pigs, dogs, cats and laboratory rabbits; most sera were from switzerland, supplemented with some cat and rabbit specimens from the netherlands. in addition, red fox (vulpes vulpes)sera were tested. from the same source, mouse sera from wild-caught specimens of the species clethrionomys glareolus, apodemus sylvaticus and a. flavicollis were obtained. infectious doses (ids ) were routinely determined in flat bottom microtiter trays (greiner and soehne, nuertingen, germany) by adding /a volumes of serial -fold virus dilutions to wells containing a monolayer of × ems cells. the spearman-kaerber formula was applied for the calculation of infectivity titers after reading the plates days after infection. neutralization titers were determined after reacting serial -fold dilutions of the inactivated serum with ids units of native berne virus for min at °c. subsequently, ems cell monolayers (in microtiter wells) per dilution were inoculated and the plates were examined for cpe days after infection. an indirect elisa procedure (engvall and perlmann, ) was used for monitoring antibody in sera from horses, cattle and goats. antigen semipurified by ammonium sulphate precipitation and subsequent interphase centrifugation ( / % sucrose; weiss et al., ) was adsorbed to polystyrene dynatech immulon flat bottom micro-elisa plates in the presence of a . m carbonate/bicarbonate buffer, ph . . prediluted horse sera ( / or / ) and an anti-horse total igg preparation conjugated to horse radish peroxidase (sigma, st. louis, mo, u.s.a.)by a -step glutaraldehyde coupling procedure (avrameas et al., ) were employed. in the substrate mixture . '-azinodi-( -ethyl-benzthiazolin-sulphonate( )) (abts, boehringer, mannheim west germany) was used at a concentration of mm in . m acetate buffer, ph . , containing mm nah po and mm h as described previously (horzinek et al., ) . absorption measurements were made at nm using a titertek multiscan recording photometer (flow labs., glasgow, u.k.). since berne virus had been isolated from a horse with pseudomembraneous enteritis and miliary granulomas with necrosis in the liver (weiss et al., ) , the pathogenic properties of the agent were studied in foals. the animals, which were about months old, received approximately ~ ids units of p / material by the intravenous route. they were examined daily for disease symptoms. serum samples taken from the foals before inoculation of berne virus had neutralization titers of ~< (fig. ) . a distinct rise in titer could be detected on days and post-infection (p.i.) and maximum values were reached at days and p.i., respectively; afterwards the titers tended to decline. neither clinical signs nor abnormal laboratory data were observed in the animals during the whole experiment. it can be seen in table i that the antibody titers in foals initially reflected those of their mothers with whom they shared separate boxes; when they were to weeks old the titers had dropped below detection level. in december--january, when the animals were between and months of age a sudden seroconversion was noted in the whole herd which had been housed together in one stable months previously (beginning of october). endemic infection had obviously taken place; however, enteric disease symptoms were not reported for this period. the animals remained seropositive at subsequent tests. the activity of berne virus in the swiss adult horse population was eval- positive reactions in serum neutralization of small numbers of randomlycollected equine sera from germany ( / ) and the usa ( / ) and in elisa of samples from southern france ( / ) and the usa ( / ) were recorded. the correspondence between the results of the serum neutralization test and elisa was studied. one hundred randomly-selected horse sera were assayed in both tests and correlation of the results was obtained in samples. in cases, the neutralization test was positive (titers approaching ) whereas elisa was negative. the inverse was observed in instances where neutralization-negative sera were weakly ( cases) or distinctly positive ( case) by elisa (table iii) . as shown in table iv , high percentages of serum samples with elevated neutralization titers were encountered in all ungulates studied (horse, cattle, goat, sheep and pig). no antibodies were detected in dogs and foxes; in cats, one sample from the netherlands and one from switzerland reproducibly showed titers of and , respectively. low, but reproducible titers, were also obtained with sera from laboratory rabbits. all sera of the wild mouse species apodemus flavicollis had titers < whereas the ( ) clethrionomys samples had tlters of and , respectively. of the samples from a. sylvaticus, only one had a titer value below . testing of the human sera at low dilutions resulted in neutralization values of <= in all samples; of these samples showed values of < upon re-examination. erratic inhibition of virus growth was observed with sera (calculated "titers" between and ). our study shows that: a high percentage of the adult horses in switzerland has experienced an infection with berne virus; in the very few experimental and natural cases observed by us, the infection was not accompanied by overt clinical symptoms; berne virus is not "new" since antibody incidence has not changed significantly during the last years; berne virus is present in other european countries and in north america. however, it is obvious that the horse, from which the virus was originally isolated, is not the only host species of berne virus or antigenically related viruses. the high prevalence of antibody in other ungulates and the previously demonstrated serological relationship of berne virus with the breda viruses and lyon- virus (weiss et al., ) demonstrated in cattle with diarrhea (woode et al., ) indicate that agents of this new taxonomic cluster may be of significance as pathogens. we have preliminary evidence (from neutralization tests using randomly collected cattle sera) that a berne-related virus is active also in the dutch cattle population; in only samples titers were < and in sera values of > were recorded. also, cattle sera from austria ( / ) and the u.s.a. ( / ) showed neutralization in a titer range of to > (m. weiss and m.c. horzinek, ) . earlier, steck et al. ( ) have reported similar results from switzerland. virus neutralization is characterized by its high sensitivity and specificity. these properties should be kept in mind when interpreting findings of antibody in different species of animals. in ruminants and the pig where high neutralization titers occur, the causative viruses are expected to be closely related to our equine isolate. the marginal values and erratic inhibitions obtained with feline, rabbit and human sera may reflect low affinity antibody induced by a more distant serotype; murine sera would assume an intermediate position. using hemagglutination-inhibition tests, woode et al. ( ) compared isolates (iowa and ) with another strain from ohio (saif et al., ) and arrived at the conclusion that serotypes exist in breda virus. in its exclusivity (recognition of determinants on the virion membrane surface), hemagglutination-inhibition is similar to neutralization; elisa, however, would also detect internal (nucleocapsid) antigens which tend to be evolutionary more conserved and broadly cross-reactive. the discrepancies observed (table iii) in the sense that elisa-negative horse sera gave positive results in neutralization are explained by the superior sensitivity of the latter test. it can be assumed that in those cases where neutralization-negative sera have been found elisa-positive, internal virion antigens are detected. experiments are under way to examine sera from different animal species using elisa and radioimmune precipitation. berne virus did not induce overt disease symptoms in the horse in the few experimental and natural infections observed. it cannot be excluded, however, that the virus may cause inconspicuous or chronic degenerative changes after an apparent primary infection. the pathogenic significance of related viruses has been established in cattle (saif et al., ; woode et al., ) and electron microscopic evidence of coronavirus-like particles in association with enteric disease (see e.g., pass et al., ) should alert the investigator to this new family of viruses (weiss et al., ; horzinek et al., ) . for the time being, however, berne virus in horses must be considered as a "virus in search of disease". coupling of enzymes to antibodies and antigens enzyme-linked immunosorbent assay, elisa. ill. quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigencoated tubes antigenic relationships among homologous structural polypeptides o~ porcine, feline, and canine coronaviruses berne virus is not 'coronavirus-like intestinal coronavirus-like particles in sheep with diarrhoea studies of an enteric "breda" virus in calves. nd immune responsiveness in cattle fatally affected by bovine virus diarrhea-mucosal disease purification and partial characterization of a new enveloped rna virus (berne virus) studies with an unclassified virus isolated from diarrheic calves key: cord- -d h k l authors: nan title: author index, volumes - date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: d h k l nan diaz, a., see alfonso, r., ( ) ( ) dienglewicz, r.l., see shamblin, c.e., ( ) ( ) dizier, i., see thomas, a., ( ) ( ) revised definition of actinobacillus sensu stricto isolated from animals. a review with special emphasis on diagnosis characterisation of bovine strains of pasteurella multocida and comparison with isolates of avian, ovine and porcine origin adhesion of outer membrane proteins containing tandem repeats of anaplasma and ehrlichia species (rickettsiales: anaplasmataceae) to tick cells anaplasma infection in free-ranging iberian red deer in the region of castilla influenza surveillance in birds in italian wetlands ( - ): is there a host restricted circulation of influenza viruses in sympatric ducks and coots? in vitro growth inhibition of major mastitis pathogens by staphylococcus chromogenes originating from teat apices of dairy heifers piscine mycobacteriosis: a literature review covering the agent and the disease it causes in fish and humans first isolation of mycobacterium microti (llama-type) from a dog antimicrobial resistance of commensal escherichia coli from dairy cattle associated with recent multiresistant salmonellosis outbreaks attenuation of a virulent type bovine viral diarrhea virus recombinant lipl antigen-based single serum dilution elisa for detection of canine leptospirosis oxytetracycline as a predisposing condition for chalkbrood in honeybee argentine strain of equine herpesvirus isolated from an aborted foetus shows low virulence in mouse respiratory and abortion models identification of an alkaline ceramidase gene from dermatophilus congolensis evaluation of molecular and immunological techniques for the diagnosis of mammary aspergillosis in ewes differential expression of the msp a gene of anaplasma marginale occurs in bovine erythrocytes and tick cells role of catalase in the virulence of brucella melitensis in pregnant goats characterization of two proteins of staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde- -phosphate dehydrogenase the transmission of phocine herpesvirus- in rehabilitating and free-ranging pacific harbor seals (phoca vitulina nanoviruses: genome organisation and protein function transmission of multiple antimicrobial-resistant staphylococcus intermedius between dogs affected by deep pyoderma and their owners genetic characterization of orf viruses isolated from various ruminant species of a zoo efficacy of vaccines against bacterial diseases in swine: what can we expect? correlation between invasion of caco- eukaryotic cells and colonization ability in the chick gut in campylobacter jejuni the clinical expression and emergence of porcine circovirus effect of acidified feed on susceptibility of broiler chickens to intestinal infection by campylobacter and salmonella analysis of differential protein expression in actinobacillus pleuropneumoniae by surface enhanced laser desorption ionisation-proteinchip tm (seldi) technology recombinant major outer membrane protein (momp) of chlamydophila abortus, chlamydophila pecorum, and chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera occurrence of chlamydiaceae spp. in a wild boar (sus scrofa l.) population in thuringia (germany) multiplex pcr for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses detection of mutations in the gyra gene and class i integron from quinolone-resistant salmonella enterica serovar longitudinal study of interferon-gamma, serum antibody and milk antibody responses in cattle infected with mycobacterium avium subsp pcrbased identification of serotype isolates of actinobacillus pleuropneumoniae biovars i and ii risk assessment of transmission of capsule-deficient routine diagnostics of lawsonia intracellularis performed by pcr, serological and post mortem examination, with special emphasis on sample preparation methods for pcr differentiation of actinobacillus pleuropneumoniae by pcr-rea based on sequence variability of the apxiva gene and by ribotyping enterotoxigenic k þ escherichia coli attachment to host cell receptors inhibited by recombinant pili protein antimicrobial susceptibility of swedish, norwegian and danish isolates of clostridium perfringens from poultry, and distribution of tetracycline resistance genes molecular epidemiology of rabies in botswana: a comparison between antibody typing and nucleotide sequence phylogeny a lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model an experimental mouse model of progressive atrophic rhinitis of swine development of maternal antibodies after oral vaccination of young female wild boar against classical swine fever occurrence and characteristics of enterohemorrhagic escherichia coli o in calves associated with diarrhoea resistance of broiler chickens to escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies identification of a novel collagen-like protein, sclc, in streptococcus equi using signal sequence phage display protection of pigs from swine dysentery by vaccination with recombinant bmpb, a . kda outer-membrane lipoprotein of brachyspira hyodysenteriae characterization of bohv- ge envelope glycoprotein mimotopes obtained by phage display multiple genetic typing of salmonella enteritidis phage-types , , , and a isolates from animals and humans in the uk pcr detection of a putative n-acetylmuramidase gene from listeria ivanovii facilitates its rapid identification paratuberculosis in farmed and free-living wild ruminants in the czech republic identification of bartonella strains isolated from wild and domestic ruminants by a single-step pcr analysis of the s- s intergenic spacer region erratum to ''identification of bartonella strains isolated from wild and domestic ruminants by a single-step pcr analysis of the s- s intergenic spacer region molecular biology of porcine circovirus: analyses of gene expression and viral replication occurrence, distribution, and role in abortion of coxiella burnetii in sheep and goats in development of a pcr assay for streptococcus iniae based on the lactate oxidase (lcto) gene with potential diagnostic value impact of sawdust and wood shavings in bedding on pig tuberculous lesions in lymph nodes, and is rflp analysis of mycobacterium avium subsp. hominissuis of serotypes and isolated from pigs and environment erratum to ''recent advances in molecular epidemiology and detection of taylorella equigenitalis associated with contagious equine metritis (cem) contagious bovine pleuropneumonia (cbpp) caused by vaccine strain t / of mycoplasma mycoides subsp. mycoides sc mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis molecular characterization of porcine tt virus, an orphan virus, in pigs from six different countries comparison of an interferon-g to a phospholipase d enzyme-linked immunosorbent assay for diagnosis of corynebacterium pseudotuberculosis infection in experimentally infected goats intestinal colonisation-inhibition and virulence of salmonella phop, rpos and ompc deletion mutants in chickens genetic typing of bovine viral diarrhoea virus isolates from india detection of specific antibodies against deflagellated salmonella enteritidis and s. enteritidis flicspecific kda polypeptide prevalence and epidemiological features of bovine viral diarrhoea virus infection in lithuania molecular conservation of msp and msp in anaplasma marginale and a. centrale vaccine strain prevalence of bartonella infection in wild african lions (panthera leo) and cheetahs (acinonyx jubatus) detection of carriers of foot-and-mouth disease virus among vaccinated cattle comparable sensitivity and specificity in three commercially available elisas to differentiate between cattle infected with or vaccinated against foot-and-mouth disease virus infection of endothelial cells with anaplasma marginale and a. phagocytophilum evaluation of a lam elisa for diagnosis of paratuberculosis in sheep and goats pulsed-field gel electrophoresis-based subtyping of dna degradation-sensitive salmonella enterica subsp. enterica serovar livingstone and serovar cerro isolates obtained from a chicken layer farm identification and differentiation of avirulent and virulent rhodococcus equi using selective media and colony blotting dna hybridization to determine their concentrations in the environment attaching and effacing escherichia coli isolated from dogs in brazil: characteristics and serotypic relationship to human enteropathogenic serological relationship between cattle exposed to brucella abortus enzyme immunoassay for the diagnosis of brucellosis: chimeric protein a-protein g as a common enzyme labeled detection reagent for sera for different animal species the effect of a killed porcine reproductive and respiratory syndrome virus (prrsv) vaccine treatment on virus shedding in previously prrsv infected pigs chicken anemia virus induced apoptosis: underlying molecular mechanisms phenotypic characterization of brucella strains isolated from livestock in nigeria erratum to ''phenotypic characterization of brucella strains isolated from livestock in nigeria detection of genomic dna of the crayfish plague fungus aphanomyces astaci (oomycete) in clinical samples by pcr haemophilus parasuis: new trends on diagnosis a novel is element, ismpa effect of porcine parvovirus vaccination on the development of pmws in segregated early weaned pigs coinfected with type porcine circovirus and porcine parvovirus fecal shedding of helicobacter spp. by co-housed australian sea lions (neophoca cinerea) and australian fur seals (arctocephalus pusillus doriferus) serotypes and virulence genes of bovine shigatoxigenic escherichia coli (stec) isolated from a feedlot in argentina the high prevalence of helicobacter sp. in porcine pyloric mucosa and its histopathological and molecular characteristics phage types, ribotypes and tetracycline resistance genes of salmonella enterica subsp. enterica serovar typhimurium strains isolated from different origins in italy prevalence and antimicrobial resistance of campylobacter coli isolated from fattening pigs in france viremia and effect of fetal infection with porcine viruses with special reference to porcine circovirus infection higher incidence of malassezia pachydermatis in the eyes of dogs with corneal ulcer than in healthy dogs prevalence and deletion types of the pathogenicity island ett among escherichia coli strains from oedema disease and colibacillosis in pigs safety and efficacy of a modified-live canine coronavirus vaccine in dogs been to the library lately? veterinary microbiology hits a century colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes interaction between attaching and effacing escherichia coli serotypes o :h and o :k in cell culture antimicrobial susceptibility of mastitis pathogens from first lactation and older cows antimicrobial resistance of salmonella isolated from finishing swine and the environment of alberta swine farms efficacy of live chlamydophila abortus vaccine b in protecting mice placentas and foetuses against strains of chlamydophila pecorum isolated from cases of abortion the effect of mutation on rhodococcus equi virulence plasmid gene expression and mouse virulence comparison of methods for antimicrobial susceptibility testing and mic values for pleuromutilin drugs for brachyspira hyodysenteriae isolated in germany evaluation of variable number tandem repeat (vntr) loci in molecular typing of mycobacterium bovis isolates from ireland physiological and genetic characterisation of some new aphanomyces strains isolated from freshwater crayfish evaluation of three serum i-elisas using monoclonal antibodies and protein g as peroxidase conjugate for the diagnosis of bovine brucellosis influence of sampling time on bacteriological diagnosis of goat intramammary infection phenotypic and genotypic characteristics of staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep immunosuppression in postweaning multisystemic wasting syndrome affected pigs pathological findings associated with naturally acquired porcine circovirus type associated disease comparative analysis of marek's disease virus (mdv) glycoprotein-, lytic antigen pp -and transformation antigen meq-encoding genes: association of meq mutations with mdvs of high virulence pathogen carriage by the cat flea ctenocephalides felis (bouche´) in the united kingdom development of a monoclonal antibody based competitive-elisa for detection and titration of antibodies to peste des petits ruminants (ppr) virus influence of porcine intestinal ph and gastric digestion on antigenicity of f fimbriae for oral immunisation inhibition of adhesion of f þ escherichia coli to piglet intestinal villous enterocytes by monoclonal antibody against blood group h- antigen the correlation between salmonella serology and isolation of salmonella in danish pigs at slaughter subviral dnas associated with geminivirus disease complexes infection dynamics of lawsonia intracellularis in pig herds a long-term study in merino sheep experimentally infected with mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies analysis of variation of bovine viral diarrhoea virus e sequence following transplacental infection of cattle selection of enterococci for potential canine probiotic additives intestinal translocation of streptococcus suis type ef þ in pigs bovine viral diarrhea virus is classified into different subgenotypes depending on the analyzed region within the viral genome genetics and geographical variation of porcine reproductive and respiratory syndrome virus (prrsv) in thailand different approaches to the vaccination of free ranging village chickens against newcastle disease in qwa-qwa the p * adhesin pseudogene of mycoplasma bovis characterization of streptococcus suis serotype isolates from diseased pigs in denmark avian circovirus diseases: lessons for the study of pmws genetic typing of bovine viral diarrhoea virus: most slovenian isolates are of genotypes d and f pseudomonas aeruginosa from canine otitis externa exhibit a quorum sensing deficiency erratum to ''pseudomonas aeruginosa from canine otitis externa exhibit a quorum sensing deficiency antigenic and genotypical characterization of newcastle disease viruses isolated in taiwan between van der meulen characterization of the pcs region of different ehrlichia ruminantium isolates genotypic and phenotypic screening of high and low virulence staphylococcus aureus isolates from rabbits for biofilm formation and mscramms antimicrobial resistance and resistance genes in staphylococcus aureus strains from rabbits conservation of deduced amino acid sequence of fimh among escherichia coli of bovine, porcine and avian disease origin immunoblotting, elisa and culture evidence for chlamydiaceae in sows on belgian farms newly developed primers for the detection of mycobacterium avium subspecies paratuberculosis enhanced efficacy of recombinant brucella abortus rb vaccines against b. melitensis infection in mice conserved regions in the sequence of the f (k ) fimbrial adhesin faeg suggest a donor strand mechanism in f assembly virulenceassociated genes in escherichia coli isolates from poultry with colibacillosis: correction differential clustering of mycoplasma mycoides subsp. mycoides sc strains by pcr-rea of the bgl locus correlation between production of acyl homoserine lactones and proteases in an aeromonas hydrophila aroa live vaccine intrapreputial infection of young bulls with bovine herpesvirus type . (bhv- . ): acute balanoposthitis, latent infection and detection of viral dna in regional neural and non-neural tissues days after experimental reactivation characterisation of a type bovine viral diarrhoea virus isolated from cattle in the uk oxalate degradation by intestinal lactic acid bacteria in dogs and cats comparison of pulsed field gel electrophoresis and repetitive sequence polymerase chain reaction as genotyping methods for detection of genetic diversity and inferring transmission of salmonella excessive porcine circovirus type antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study a specific pcr for the identification of mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (ccpp) expression of apoptosis-related gene mrnas in feline t-cells infected with feline immunodeficiency virus (fiv) experimental dual infection of pigs with an h n swine influenza virus (a/sw/hok/ / ) and mycoplasma hyopneumoniae the effect of maternal antibodies on the detection of bovine virus diarrhoea virus in peripheral blood samples evaluation of the german cockroach (blattella germanica) as a vector for verotoxigenic escherichia coli f in confined swine production key: cord- - i hwn authors: nan title: contents vol. , date: - - journal: vet microbiol doi: . / - ( ) -x sha: doc_id: cord_uid: i hwn nan application of the polymerase chain reaction for the detection of ehrlichia canis in tissues of dogs tinidazole treatment of experimentally induced summer mastiffs -effect on elimination rates of bacteria and outcome of the disease j. hirvonen, s. pyt~r~l~, a. hein~uo (hautj~irvi, finland) and h. jousimies-somer (helsinki. finland) use of bovine myeloperoxidase as an indicator of ma,stitis in dairy cattle r. cooray (uppsala, sweden) development of a selective polymerase chain reaction assay for the detection of rnycoplasma rnycoides subsp. mycoides s.c. (contagious bovine pleuropneumonia agent) antibody titers to pseudorabies virus in piglets immunized with glii deleted pseudorabies vaccine in a pseudorabies infected herd f. elvinger a retrospective study of clinical and laboratory characteristics of ovine footrot experimental infections of pregnant sows with ovine ctdaraydia psittaci strains c. vazquez-cisneros, a.j. wilsmore (potters bar, uk) and e. bollo (torino. italy) key: cord- -n knqj z authors: su, yunfang; hou, yixuan; wang, qiuhong title: the enhanced replication of an s-intact pedv during coinfection with an s ntd-del pedv in piglets date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: n knqj z porcine epidemic diarrhea virus (pedv) variants having a large deletion in the n-terminal domain of the s subunit of spike (s) protein were designated as s ntd-del pedvs. they replicate well in experimentally infected pigs. however, on farms they often co-infect pigs with the pedv containing an intact s protein (s-intact pedv). we aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of pedv using two recombinant pedvs: icpc a and its s ntd-del form icpc a-s Δ . additionally, viral replication was compared in vero and ipec-dq cells at the presence of bovine mucin (bm), porcine gastric mucin (pgm), swine bile and bile acids during inoculation. in the pigs coinfected with icpc a and icpc a-s Δ , icpc a replicated to a higher peak titer than its infection of pigs without the presence of icpc a-s Δ . the severity of diarrhea and intestinal atrophy were similar between icpc a and the coinfection groups, but were significantly higher than icpc a-s Δ group. in vero and ipec-dq cells, certain concentrations of bm, pgm, bile and bile acids increased significantly the infectivity of icpc a but had no or negative effects on icpc a-s Δ . these results indicated that the replication of the s-intact pedv was enhanced during coinfection in piglets. this observation may be explained partially by the fact that mucin, bile and bile acids in gastrointestinal tract had facilitating effects on the infection of s-intact pedv, but no/inhibition effects on s ntd-del pedv. porcine epidemic diarrhea virus (pedv) belongs to the genus alphacoronavirus in the family coronaviridae. it causes severe gastroenteritis in neonatal pigs worldwide. the genome size of pedv is kb. it contains orf a and orf b encoding - non-structural proteins, an orf encoding an accessory protein, and four orfs encoding structural proteins: spike (s), membrane (m), envelop (e), and nucleocapsid (n) proteins. pedv s protein mediates the essential functions of receptor binding (via s subunit) and subsequent fusion of the viral and cellular membranes (via s subunit) (li, ) . s subunit contains two domains, the n-terminal domain (s ntd, residues - based on pedv cv ) and the c-terminal domain (s ctd, residues - ). however, the cellular receptor of pedv is still unknown (li et al., b; shirato et al., ) . although the sialic acid binding activity of s -ntd was observed in many coronaviuses and facilitated virus replication (schwegmann-wessels et al., ; li et al., ; desmarets et al., ) , s -ntd is dispensable for some mutants of transmissible gastroenteritis coronavirus (tgev) and pedv (schwegmann-wessels et al., ; oka et al., ; suzuki et al., ; hou et al., ) . pedv variants with a big deletion in the s -ntd have been designated as s ntd-del pedvs (hou et al., ) . they have been detected in field pig samples, indicating sufficient replication of these s ntd-del pedvs in vivo (suzuki et al., ; diep et al., ; zhang et al., a; su et al., ) . interestingly, most field s ntd-del pedvs exist as a member of coinfection of pigs with the s-intact pedv (diep et al., ; su et al., ) . however, the mechanisms and consequences of the coinfection have not been investigated. studies showed that under unfavorable conditions as encountered in the intestinal tract, sialic acid-binding of tgev played an important role in viral replication. when absorption time was reduced, tgev infectivity was also reduced in the cells whose sialic acids were removed (schwegmann-wessels et al., ) . consequently, sialic acid binding activity may facilitate the infection of tgev by helping the virus resist detergent-like substances encountered during passing through the gastrointestinal tract (krempl et al., ) . therefore, we hypothesized that mucin, rich in sialic acid, may enhance the infection of pedv. interestingly, bile acids can induce or promote the replication of some enteric viruses like a porcine sapovirus (chang et al., ) and human noroviruses (ettayebi et al., ) . since intestinal epithelial cells are exposed to bile acids, we hypothesized that bile acids promote pedv infection. previously, we generated two recombinant pedvs, pedv icpc a and icpc a-s Δ (hou et al., ) . pedv icpc a is an infectious cdna clone-derived virus of the emerging highly virulent pedv pc a strain. on the other hand, pedv icpc a-s Δ is a mutant version of icpc a bearing a -aa deletion (residues - ) in the s ntd region, corresponding to the s domain (s °) of the spike protein (li et al., a; hou et al., ) . in this study, we investigated the replication and pathogenesis of the two viruses during coinfection in neonatal gnotobiotic (gn) piglets. some intestinal substances (mucin, bile and bile acids) were tested for their effects on the infection of the individual viruses in vitro. we selected two cell lines, vero and ipec-dq cells. vero cells are the most commonly used cell line for pedv isolation and propagation (teeravechyan et al., ) . ipec-dq cell is a subclone of porcine small intestinal cell line ipec-j (zhang et al., b) . although pedv grows less efficiently in ipec-dq cells than in vero cells, pedv infection of ipec-dq cells is more relevant physiologically to pedv natural infection in the intestines of pigs. vero cells (atcc number ccl ) were cultured in dulbecco's modified eagle's medium (dmem, gibco, carlsbad, ca, usa) supplemented with % fetal bovine serum (fbs, hyclone, logan, ut, usa) and % penicillin/streptomycin (gibco). for virus propagation in vero cells, the maintenance medium was dmem supplemented with . % tryptose phosphate broth solution (tpb, sigma, st. louis, mo, usa), % penicillin/streptomycin (gibco) and μg/ml trypsin ( . % trypsin, gibco). ipec-dq cell line was a gift from dr. dongwan yoo, university of illinois at urbana-champaign, urbana il, usa. it was a subclone of ipec-j cells, a porcine small intestinal cell line, and maintained in roswell park memorial institute medium (rpmi- , gibco) supplemented with % fbs and % penicillin/streptomycin (zhang et al., b) . for virus propagation in ipec-dq cells, maintenance medium was rpmi- containing . % tpb, % penicillin/streptomycin and μg/ml trypsin. two recombinant pedv, icpc a and icpc a-s Δ , were generated previously by our lab using the infectious clone of pedv pc a strain (hou et al., ) . neuraminidase (na, from clostridium welchii), porcine gastric mucin (pgm) and several bile acids [(glycochenodeoxycholic acid (gcdca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), and ursodeoxycholic acid (udca)] were all purchased from sigma. bovine mucin (bm) was purchased from worthington (worthington biochemical corp., lakewood, nj, usa). swine bile was collected from a pregnant sow after c-section surgery. the mouse monoclonal antibodies (mab) h , f and a targeting the s °domain of pedv s protein (fig. s a ) was provided by dr. berend jan bosch, utrecht university, utrecht, netherlands (li et al., a) . the mab sd - targeting pedv nucleocapsid (n) protein was provided by drs. eric nelson and steven lawson, south dakota state university. the guinea pig polyclonal antibody (pab) gp targeting the s subunit of the s protein of pedv pc strain (s Δ ) (fig. s a) , rabbit pab rb targeting pedv n protein and swine pedv-positive serum (virus neutralization titer : ) were generated by our laboratory previously (hou et al., ) . . . duplex rt-qpcr method for the detection and differentiation of pedv icpc a and icpc a-s Δ based on the nucleotide sequence differences in the s ntd coding region between the icpc a and icpc a-s Δ , a duplex taqman real-time reverse-transcription quantitative pcr (rt-qpcr) was developed. we designed the primers and probes (table , fig. s b ) and had them synthesized by integrated dna technologies (skokie, il, usa). the rna of the two viruses was extracted using rneasy mini kit (qiagen, valencia, ca, usa). to generate a standard dna, a kit of superscript™ iii first-strand synthesis supermix (thermo fisher scientific, carlsbad, ca, usa) was used for cdna synthesis following manufacturer's instructions. primer set pedv- f and pedv- r (oka et al., ) was used to amplify a . kb or a . kb fragment covering pedv partial nsp gene and s gene of icpc a and icpc a-s Δ , respectively. the pcr products were purified using gel extraction kit (qiagen) and used as standard dna. the reaction consisted with μl × pcr buffer, . μl mm dntps (qiagen), . μl each primer ( μm), . μl each probe ( μm), . μl rnasin (promega, wi, usa), . μl enzyme mix (qiagen), . μl each dna/rna and . μl sterilized water. the rt-qpcr procedure was: °c for min, °c for min and cycles of °c for s and °c for s. we generated two standard curves using light-cycler® system (roche life science, indianapolis, in, usa). the two standard curves for the serially diluted dna fragments of icpc a and icpc a-s Δ showed regressions with coefficients of - . and - . , respectively, and the corresponding correlation coefficient (r ) of . and . , respectively. the primers and probes were further tested using individual and the mixture of two viruses. we tested the correlation between s gene titers and infectious titers of pedv icpc a and icpc a-s Δ using serially diluted, cell cultured viruses (table s and s ). for the samples of mixed viruses, the duplex rt-qpcr was performed to differentiate the s gene of each virus. the detection limit was log s gene copies/ml for both icpc a and icpc a-s Δ . the gn piglets were derived and raised as described previously (saif et al., ) . thirty-one gn piglets were assigned to five experimental groups: ( ) icpc a (n = ; pfu/pig); ( ) icpc a-s Δ (n = ; pfu/pig); ( ) coinfection of icpc a and icpc a-s Δ (n = ; pfu/pig of each virus); ( ) mock (n = ; pbs); ( ) second passage of coinfection [n = ; small intestinal contents (sic) (sample #pe ) of one piglet in group collected at day post inoculation (dpi), at a dose of log copies/ml based on the n gene, log copies/ml and log copies/ml based on the icpc a-specific s gene and icpc a-s Δ -specific s gene, respectively, corresponding to table primers and probes of the duplex rt-qpcr. log pfu/ml of infectious pedv] . at days of age, piglets in groups - were orally inoculated with individual or the mixture of the viruses. the group pig was inoculated at days of age and was euthanized at dpi. to examine histopathological changes, one or two piglets of groups - were euthanized at . dpi and - dpi, respectively. after inoculation, all piglets were evaluated daily for clinical signs, such as diarrhea, vomiting, anorexia, and depression. fecal consistency was examined by collecting rectal swabs and scored as follows: , solid; , pasty; , semiliquid (mild diarrhea); and , liquid (severe diarrhea). total fecal rna was extracted using magmax- rna isolation kit (thermo fisher scientific), and the viral rna was titrated by the rt-qpcr targeting the n gene and the duplex rt-qpcr targeting the s gene of each virus. fecal infectious pedv shedding titers were tested using vero cells in -well plates and determined as % tissue culture infective doses (tcid ) (reed and muench, ) . at necropsy, three sections of the jejunum (proximal, middle, and distal) and one section of the ileum were collected and fixed in . % formalin (fisher scientific co llc, florence, ky, usa). all tissues were trimmed, processed, embedded in paraffin, and sectioned using routine procedures . to compare the pedv-specific histopathological changes among different groups of pigs, ihc staining was performed as described previously for the detection of pedv n proteins using mab sd - as the primary antibody (hou et al., ) . the ihc staining was carried out using a nonbiotin polymerized horseradish peroxidase system (biogenex laboratories, san ramon, ca, usa). finally, tissues were counterstained with hematoxylin. images of tissues were observed and captured by olympus ix- fluorescent microscope (center valley, pa, usa). ratios of villous height to crypt depth (vh/ cd) of the jejunum and ileum of individual piglets were measured using a computerized imaging system (metamorph, olympus, japan) as described previously . for each intestinal section, villi and crypts were measured. to detect and differentiate icpc a-and icpc a-s Δ -infected cells in the coinfected pigs, if staining was performed. the slide preparation steps were as same as those of the above ihc procedure. the mixture of mouse mabs h , f and a ( : in pbs) (li et al., a) targeting the s °domain of pedv s protein was used as the primary antibody for the staining of icpc a-infected cells only. tissues were incubated at ℃ overnight, after three washings with pbst (pbs with . % tween ), alexa fluor -conjugated goat antimouse igg (h + l) (thermo fisher scientific) ( : diluted in pbs) was used as the second antibody and tissues were incubated at room temperature for h. after washing with pbst, guinea pig pab gp ( : diluted in pbs), targeting the s Δ and was for the staining of both viruses, was added. then, the slides were incubated at room temperature for h. after washing three times with pbst, alexa fluor -conjugated goat anti-guinea pig igg (h + l) (thermo fisher scientific) ( : diluted in pbs) was used as the nd antibody and the slides were incubated at room temperature for h. after washing with pbst, mg/ml dapi ( ′, -diamidino- -phenylindole, dihydrochloride, roche life science, indianapolis, in, usa) was added for nucleic acid staining. finally, an autofluorescence quenching kit (vector® trueview™, burlingame, ca, usa) was used to quench the autofluorescence of the tissues. images of stained tissues were captured by olympus ix- fluorescent microscope. red, green and blue fluorescence staining was merged by imagej software (https://imagej.nih. gov). . . isolation of the pig-passaged icpc a and icpc a-s Δ from the sic of a pig coinfected with the viruses the sic (sample #pe ) of a coinfected piglet in group was positive for both viruses by the duplex rt-qpcr. virus isolation was performed using plague assays (oka et al., ) . the sic was diluted in dmem and the % suspension was vortexed briefly followed by centrifugation at , ×g for min at ℃. a series of -fold dilutions ( − - − ) of the supernatants were prepared in dmem (containing . μg/ml trypsin) and were used immediately for inoculation of vero cell monolayers in -well plates ( μl per well). after incubating at ℃ for h, the inoculum was removed and the monolayers were washed once with pbs. then ml/well of agarose overlay was added. the plate was kept for days before picking up clones. for propagating each clone, vero cell monolayers in -well plates were prepared and individual plagues were picked and added into individual wells. at dpi, rna was extracted and the duplex rt-qpcr method specific for both viruses was performed. . . multi-step growth kinetics and if staining of coinfection of pedv icpc a and icpc a-s Δ in vero and ipec-dq cells the growth kinetics of icpc a-s Δ and icpc a during the coinfection of the two viruses were investigated in vero and ipec-dq cells. cell monolayers on -well plates were inoculated with one of the three inocula: ( ) . multiplicity of infection (moi) of icpc a; ( ) . moi of icpc a-s Δ ; and ( ) . moi of icpc a and . moi of icpc a-s Δ . viruses were diluted in dmem containing μg/ml trypsin and were added to cell monolayers. after incubating at ℃ for h, the inoculum was removed and the cell monolayers were washed for three times. then the plates were added with the maintenance medium and incubated at ℃. the samples were collected at different time points ( h, h, h, h, h) and frozen (- ℃) and thawed once before testing. rna was extracted using the magmax- rna isolation kit (thermo fisher scientific) and virus titers were tested by the duplex rt-qpcr specific for both viruses. for if staining of s proteins at h post infection (hpi), the procedure in . section was performed. . . infection of pedv icpc a and icpc a-s Δ in na-treated vero and ipec-dq cells vero/ipec-dq cell monolayers in -well plates were treated with mu na in dmem/rpmi- or mock dmem/rpmi- at ℃ for h. after three washings with dmem/rpmi- , cells were inoculated with icpc a or icpc a-s Δ at a moi of . with μg/ml trypsin for h. next, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added to prevent virus spreading. cells were fixed with acetone-methanol ( : ) at hpi. the numbers of pedv-infected cell foci were determined by if assays as described for the staining of pedv n proteins (hou et al., ) . briefly, rabbit pab rb ( : ) was used as the primary antibody. after incubating at ℃ overnight, cells were washed three times. then alexa fluor -conjugated goat anti-rabbit igg (h + l) (thermo fisher scientific) ( : ) was used as the nd antibody and the cells were incubated at room temperature for h. after washing and adding mounting medium, cells were observed and picture were captured by using olympus ix- fluorescent microscope. the numbers of fluorescent focus units (ffu) were counted by imagej software. the percentages of ffu were calculated by the value of mock group as %. . . effect of mucin, bile and bile acids on the infection of pedv icpc a and icpc a-s Δ in vero and ipec-dq cells viruses (icpc a or icpc a-s Δ ) were mixed with different concentrations of bm ( , . , . , . mg/ml) or pgm ( , . , . , . , . mg/ml). then μg/ml trypsin were added to the mixture. monolayer of vero/ipec-dq cells in -well plates were inoculated with each virus mixture at a moi of . . the plates were incubated at ℃ for h, then the inoculum was removed. after three washings, dmem/rpmi- containing μg/ml sbti and swine pedv positive serum ( : dilution) was added. cells were fixed at hpi. the numbers of ffu were determined by if assay for the staining of pedv n proteins (see section . ). the percentages of ffu were calculated by the value of mock group as %. similarly, different concentration of swine bile ( , . , . , . , . %) and bile acids (gcdca, cdca, dca, and udca) at , , , , μm were tested. statistical analysis was performed using graphpad prism software (graphpad software, la jolla, ca, usa). the experimental data were analyzed by one-way anova and student`s t-test. data are shown as m ± sd, and a p < . was considered as significant. . . the replication of icpc a was eventually enhanced during coinfection with icpc a-s Δ in neonatal gn piglets as shown in fig. , all pedv-inoculated pigs but no pigs in the mock group had diarrhea and shed pedv. the fecal consistency scores and peak fecal pedv n and s rna shedding titers of piglets in the coinfection group reached the peak at . dpi, which was delayed half a day compared with the icpc a group ( dpi) but earlier than the icpc a-s Δ group (> dpi). by dpi, all pigs in the icpc a and coinfection groups but no pigs in the icpc a-s Δ group died or showed moribund. compared with the peak fecal pedv n gene shedding titer ( . ± . log copies/ml) of piglets in the icpc a group ( dpi), pigs in the coinfection group had a significantly higher peak titer ( . ± . log copies/ml) ( fig. b and table ) at a delayed time point ( . dpi). in addition, the peak fecal infectious pedv shedding titer of the coinfection group ( . ± . log tcid /ml) were significantly higher than that of the single infection groups ( . ± . and . ± . log tcid /ml of icpc a and icpc a-s Δ groups, respectively) ( table ) . interestingly, the peak s gene shedding titers of icpc a ( . ± . log copies/ml) in the coinfection group at . dpi was significantly higher than that of icpc a group ( . ± . log copies/ml) at dpi (fig. c, table ). however, the peak s gene shedding titers ( . ± . log copies/ml) of icpc a-s Δ in the coinfection group was significantly lower than that of the icpc a-s Δ group ( . ± . log copies/ml) (fig. c , table ). in the second passage of coinfection, only icpc a but not icpc a-s Δ s gene was detected in the large intestinal contents (lic) of the piglet, which was orally inoculated with the sic (sample #pe ) of one pig in the coinfection group and was positive for both viruses. the lic had a virus titer of . log tcid /ml for infectious pedv, . log s gene copies/ml and . log n gene copies/ml for icpc a. histopathological examination was performed and villous atrophy was observed in the jejunum and ileum of piglets in all pedv-inoculated groups ( fig. a and b ). the small intestinal villi in the icpc a-s Δ group had a milder villous atrophy than those in the icpc a and coinfection groups. the vh/cd ratios of jejunum and ileum of all the pedv-inoculated groups were significantly lower than those of the mock group (fig. b) . the ratios of the icpc a and coinfection groups were similar and were significantly lower than that of the icpc a-s Δ group. ihc staining showed that no pedv-positive intestinal epithelial cells were observed in all pedv-inoculated pigs fig. . evaluation of the replication of icpc a and icpc a-s Δ in gn pigs infected with individual or both viruses. (a) fecal consistency scores of individual gn piglets and the mean of each group were shown. the scores were named as follows: , solid (normal); , pasty (normal); , semiliquid (mild diarrhea); and , liquid (severe diarrhea). two piglets from icpc a, icpc a-s Δ and coinfection groups, and one pig from mock group were euthanized at hpi and - dpi, respectively, for histopathological examination. (b) fecal pedv n gene rna shedding titers (for both icpc a and icpc a-s Δ ) of each group. data are shown as mean (m) ± standard deviations (sd) of pigs in each group. (c) fecal pedv s gene rna shedding titles of each virus. data are shown as m ± sd. at . dpi (data not shown). at - dpi, a few pedv n antigens were observed in the small intestine of the icpc a-s Δ -infected piglets, while extensive antigens were stained in those of the icpc a and coinfection piglets ( fig. a) . by if staining for the pedv s proteins, a few pedv antigens were observed in the jejunum of the icpc a-s Δ -inoculated piglets (green alone, positive for s Δ only), while many antigens were observed in those of the icpc a-inoculated piglets [yellow, representing positive for both s Δ (green) and s °d omain (red)] and the coinfection piglets (yellow) (fig. c) . . . icpc a alone, but not icpc a-s Δ alone was isolated from the sic of one of the pigs infected with the two viruses simultaneously the sic of the one piglet in the coinfection group, which was positive for both viruses by the duplex rt-qpcr were used for virus isolation in vero cells using plague assays. we picked up plagues and found that plagues were positive for icpc a and plagues were positive for both viruses by the duplex rt-qpcr. no plagues were positive for s ntd-del pedv alone. during coinfection in vero cells, icpc a replicated to a titer of . ± . log copies/ml at hpi, which was similar to that of the single infection ( . ± . log copies/ml) (fig. a) . however, icpc a-s Δ s gene peak titer was . ± . log copies/ ml, which was significantly lower than that of the single infection ( . ± . log copies/ml). in vero cells, icpc a alone and icpc a-s Δ alone replicated to similar titers. similarly, during coinfection in ipec-dq cells, icpc a replicated to a similar titer to its single infection ( . ± . vs. . ± . log copies/ml at hpi (fig. c ). icpc a-s Δ s gene peak titer was . ± . log copies/ml, which was significantly lower than that of the single infection ( . ± . log copies/ml). in contrast to those results in vero cells, icpc a alone replicated to significantly higher titers than icpc a-s Δ alone in ipec-dq cells. by if staining, more icpc a-s Δ -infected cells (green) were observed in vero cells than in iepc-dq cells in both icpc a-s Δ alone and co-infection conditions ( fig. b and d) . at the second passage of the coinfection, icpc a rna and antigens were detected in both cells; however, icpc a-s Δ rna and antigens were detected exclusively in vero cells but not in ipec-dq cells (data not shown). . . the percentage of infectivity of icpc a was significantly lower than that of icpc a-s Δ in na-treated vero and ipec-dq cells after removing sialic acids from the cell surface using mu na in vero and ipec-dq cells, the percentages of infectivity of icpc a and icpc a-s Δ were reduced significantly (fig. ) . however, in both cells treated with na, the percentages of infectivity of icpc a were significantly lower than those of icpc a-s Δ . . . mucin enhanced the infectivity of icpc a but had no or inhibition effects on icpc a-s Δ in both vero and ipec-dq cells in vero cells, . - . , . and . mg/ml bm increased, had no effects and decreased the infectivity of icpc a, respectively (fig. a ). . - . mg/ml pgm increased the infectivity of icpc a . - . fold (fig. b) . however, the infectivity of icpc a-s Δ was unaffected or inhibited significantly by bm and pgm at the tested concentration ( fig. a and b) . in ipec-dq cells, the infectivity of icpc a was increased . - . fold and . - . fold by bm ( . - . mg/ml) and pgm ( . - . mg/ ml), respectively. however, there was no effects at the highest pgm concentration tested ( . mg/ml). on the other hand, low to medium concentrations of bm ( . - . mg/ml) and pgm ( . - . mg/ml) had no effects on the infectivity of icpc a-s Δ . only high concentrations of bm ( . mg/ml) and pgm ( . - . mg/ml) reduced the infectivity of icpc a-s Δ significantly ( fig. c and d ). . . bile and bile acids enhanced the infectivity of icpc a but had no or inhibition effects on icpc a-s Δ in vero cells, low to medium concentrations of swine bile ( . - . %) increased . - . fold of the infectivity of icpc a (fig. a) . (fig. b-e) . however, the infectivity of icpc a-s Δ was unaffected ( . - . %) or inhibited significantly by bile ( . - . %) by bile (fig. a) . bile acids a piglets in group were inoculated with pfu/pig of icpc a; piglets in group were inoculated with pfu/pig of icpc a-s Δ ; piglets in group were inoculated with pfu/pig of each virus; piglets in group were inoculated with pbs; piglets in group were inoculated with the sic of piglet in group with a dose of log pfu/ml targeting n gene of pedv. b fecal consistency (fc) was scored as follows: , solid, , pasty, , semiliquid; and , liquid. an fc score of and were considered diarrhea and severe diarrhea, respectively. c values in parentheses are the number of positive results/number of animals tested. d the detection limit of pedv rt-qpcr targeting the s gene of icpc a or icpc a-s Δ is log copies/ml. e the detection limit of pedv rt-qpcr targeting the n gene of both icpc a and icpc a-s Δ is . log copies/ml . f the pig number for diarrhea observation is or less than the total pig number because or pigs of each group were euthanized at hpi. g the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < . ). '-', not detected. y. su et al. veterinary microbiology ( ) - at the tested concentrations had no effects on the infection of icpc a-s Δ (fig. b-e) . similarly, in ipec-dq cells, the infectivity of icpc a was increased significantly by . - . % bile but was decreased significantly by the concentration of . % (fig. f) . however, . - . % bile decreased the infectivity of icpc a-s Δ significantly. bile acids gcdca (fig. g-j) . no significant effects of bile acids on icpc a-s Δ were observed except for that the highest concentration ( μm) of gcdca and dca decreased its infectivity significantly (fig. g-j) . fig. . histopathological examination of the gn piglets infected with individual or both icpc a and icpc a-s Δ . (a) ihc staining of pedv n proteins in the jejunal and ileal sections of piglets that died or were euthanized at - dpi (magnification, ×) . the brown signals represented the pedv n antigens in enterocytes. (b) villous height to crypt depth (vh/cd) ratios of jejunum and ileum of the gn piglets euthanized at - dpi. for each intestinal section, villi and crypts were measured. data are shown as the m ± sd. number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). 'ns', not significant. (c) if staining of pedv s proteins in the jejunal sections of piglets that died or were euthanized at - dpi (magnification, x). tissue sections were stained for the detection of icpc a and icpc a-s Δ antigens targeting the s Δ protein (green), and for icpc a antigens targeting the s °domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc a-infected (or co-infected in coinfection group) cells (merged from red and green) and the green dots represented icpc a-s Δ infection alone. (magnification, x) . cells were stained for the detection of icpc a and icpc a-s Δ antigens targeting the s Δ protein (green), and for icpc a antigens targeting the s °domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc a-infected (or co-infected in coinfection condition) cells and the green dots represented icpc a-s Δ infection alone. the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < . ). previously, our laboratory isolated the first s ntd-del pedv strain, pc , from vero cell culture (oka et al., ) . recent studies revealed that s ntd-del pedvs naturally evolved in the field and often coinfected pigs with the s-intact pedv (suzuki et al., ; diep et al., ; zhang et al., a; su et al., ) . the s ntd-del pedv had no tissue tropism change compared with the s-intact pedv (suzuki et al., ; hou et al., ) . this differs from porcine respiratory coronavirus (prcv) that is a s ntd-del version of tgev and changes the major tissue tropism from intestines to respiratory tract (zhang et al., ) . in this study, we investigated the replication of s ntd- fig. . infection of pedv icpc a and icpc a-s Δ in neuraminidase (na)-treated vero and ipec-dq cells. vero or ipec-dq cell monolayers in -well plates were treated with mu na or mock at ℃ for h. after three washings with dmem/rpmi- , cells were inoculated with equal amounts of virus at a moi of . diluted in the medium containing μg/ml trypsin. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as the m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). fig. . effect of bovine mucin (bm) and porcine gastric mucin (pgm) on the infection of pedv icpc a and icpc a-s Δ in vero (a and b) and ipec-dq cells (c and d). monolayers of vero or ipec-dq cells in -well plates were inoculated with a moi of . icpc a or icpc a-s Δ in medium containing μg/ml trypsin and different concentrations of bm or pgm. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). del and s-intact pedvs during coinfection in pigs. we developed a duplex rt-qpcr targeting the s gene of pedv to differentiate the two viruses. we found that the duplex rt-qpcr assay had different sensitivities for the detection of infectious icpc a and icpc a-s Δ (table s ) although it had a similar detection limit ( log copies/ml) for both viruses. for example, log s gene copies/ ml corresponded to . and . log ffu/ml for icpc a and icpc a-s Δ , respectively. it may reflect the fact that icpc a-s Δ replicates to lower peak infectious titers (∼ . log ffu/ml) than that (∼ . log ffu/ml) of icpc a in vero cells although both viruses replicated to similar peak titers based on the s gene (∼ . log copies/ml) (fig. a) . in the mixture of two viruses, the detection fig. . effect of bile and bile acids on the infection of pedv icpc a and icpc a-s Δ in vero (a-e) and ipec-dq cells (f-j). monolayers of vero or ipec-dq cells in -well plates were inoculated with a moi of . icpc a or icpc a-s Δ in medium containing μg/ml trypsin and different concentrations of bile or bile acids. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml sbti and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). sensitivity of each virus differed significantly (tables s ). for the detection of icpc a at high titers ( . log ffu/ml) and as the predominant virus in the mixture of two viruses (icpc a: icpc a-s Δ = - : ), the s gene titers (∼ . - . log s gene copies/ ml) (table s ) were similar to that (∼ . log s gene copies/ml) in the single virus alone (table s ). because icpc a replicated to high titers and was the predominant virus during the coinfection in pigs, we can predict that the peak infectious titer of icpc a in coinfection was ∼ . log -higher than that in its single infection based on the duplex rt-qpcr results (∼ . vs ∼ . log s gene copies/ml) (table ) . so, we conclude that the replication of icpc a was interfered at the beginning based on delayed timing ( . vs . dpi) to reach peak titers but enhanced eventually in coinfection compared with its single infection in pigs. on the other hand, the sensitivity for the detection of icpc a-s Δ decreased significantly and icpc a-s Δ became undetectable when icpc a was high ( . log ffu/ml) and icpc a-s Δ infectious titers were lower than . log ffu/ml in the mixture of two viruses (table s ). therefore, the decreased peak s gene titer (∼ . log copies/ml) of icpc a-s Δ in coinfection of pigs compared with that (∼ . log copies/ml) in its single infection may be due to inhibition of replication or the decreased detection sensitivity of the duplex rt-qpcr for the detection of icpc a-s Δ during coinfection (table ). in the first passage of coinfection, we observed much lower numbers of s ntd-del pedv-infected cells than the s-intact pedv-infected cells in the small intestines of pigs infected with the same dose of both viruses simultaneously. in addition, plaque assays were performed to isolate and purify individual viruses from the sic of one of the coinfection pigs. however, only s-intact pedv was isolated alone. in contrast, s ntd-del pedv was exclusively detected together with the sintact pedv from the plagues. in the second passage of the coinfection in pigs, s-intact pedv but not s ntd-del pedv rna was detected in the lic of the pig inoculated with the sic of the pig inoculated with both viruses. this finding suggests that s ntd-del pedv had no replication advantage and was either outcompeted or coexisted with sintact pedv in pigs. this conclusion is in agreement with what were observed in the field (diep et al., ; su et al., ) . s ntd-del pedv replicated to a lower peak titer in coinfection than that in single virus infection in both vero cells and ipec-dq cells. these in vitro results were similar to those in the pig studies. in the second passage of coinfection, s ntd-del pedv rna was detected in vero cells but not in ipec-dq cells, probably due to the lower replication efficiency of the s ntd-del pedv in ipec-dq cells than in vero cells ( fig. a and c) . similar to other sialic acid-binding coronaviruses, the s ntd of pedv has a sialic acid binding activity, and the sugar-binding activities of a field isolate pedv variant chgd- was stronger than that of the prototype strain pedv cv using bm (deng et al., ) . the sialic acid binding activity occurred within the n-terminal residues and the capacity of sialic acid binding differs among pedv strains using an hemagglutination assay . the recombinant virus used in this study, pedv icpc a-s Δ , is a s ntd-del version of icpc a. it lost most sialic acid binding activity tested in vero cells (hou et al., ) . in this study, we comparatively tested icpc a-s Δ and icpc a in vero and ipec-dq cells treated with na. similar to the previous reports, our results confirmed that the binding of s ntd of pedv to sialic acids on cell surface enhanced virus infectivity. bm is a mixture of highly glycosylated proteins containing sugar moieties, such as -n-acetyl- -o-acetylneuraminic acid (neu , ac ), -n-glycolylneuraminic acid (neu gc), and -n-acetylneuraminic acid (neu ac). among them, neu gc and neu ac can serve as receptors or co-receptors for some alphacoronaviruses (e.g., tgev) and gammacoronaviruses [e.g., infectious bronchitis virus (ibv)] (cavanagh and davis, ; krempl et al., ) . similar to tgev, which uses neu gc and neu ac as co-receptors (krempl et al., ; schultze et al., ; schwegmann-wessels and herrler, ) , the s ntd of pedv interacted with sugar (deng et al., ) . pgm contains . - . % n-acetylneuraminic acid (neuac). our data indicated that low-medium concentrations of bm or pgm enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. in contrast, the s ntd-del pedv was not affected or inhibited, probably due to the covered mucin that blocked virus binding to the receptors. the intestinal epithelial cells are covered by a layer of mucus that is rich in sialic acids. these results suggest that low-medium amount of mucin binding to s-intact pedv may help the virus particles approach the receptors via sticking to the sialic acids on the cell surface. however, the receptor binding domain (rbd) on the s protein of s ntd-del pedv, which lacks most sialic acid binding activity, is probably covered with mucin and blocked its binding to the receptors. when high concentration of mucin saturates the s ntd of s-intact pedv, it can also cover viral rbd and block its binding to the receptors, resulting in decreased infectivity as observed in . mg/ml bm effects on s-intact pedv in vero cells (fig. a) . the major components of bile include bile acids, cholesterol, lipids, bilirubin, proteins and carbohydrates. the bile acids are stored in the gall bladder (at ∼ mm) and released into the small intestine (mcleod and wiggins, ) . in the small intestine, the total bile acids have a concentration of - mm (dowling, ) . we selected two primary bile acids (gcdca and cdca) and two secondary bile acids (dca and udca) to test the effects of bile acids on pedv infection. the secondary bile acids are dehydroxylated ones from the primary bile acids by intestinal bacteria (björkhem, ) . our data indicated that a broad range of concentrations of bile and bile acids enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. however, bile and bile acids cannot increase the infectivity of s ntd-del pedv. these results suggest that the bile-and bile acid-mediated promotion of pedv infection is related to the s ntd. it was reported that gcdca may facilitate the adaptation of a s-intact pedv to trypsin-free growth in vero cells at the early passages (< p ) (kim et al., ) . however, the mechanisms of the bile acid function on the pedv infection is unknown and consequently need to be further investigated. the inability of the s ntd-del pedv to outcompete the s-intact pedv may account for the fact that s ntd-del pedvs were exclusively detected from coinfection with the s-intact pedv (diep et al., ; su et al., ) . consequently, the disease caused by the coinfection was as severe as that by the highly virulent s-intact pedv alone. this situation is quite different from what occurred for prcv and tgev: the wild spread of clinically mild prcv, which often infects pigs asymptomatically and repeatedly, inducing protective herd immunity against tgev outbreaks (vancott et al., ; brim et al., ) . prcv functions as a natural effective vaccine against tgev. however, for pedv, the clinically mild s ntd-del pedv variants do not infect pigs alone. therefore, safe and effective vaccines are still desired to control the deadly pedv infection in neonatal piglets. due to the % mortality rates of piglets in the icpc a-infected pigs (hou et al., ) and coinfection groups, we currently cannot determine whether the pathogenicity of pedv icpc a is enhanced during the coinfection. that should be investigated in older pigs (weaned pigs, sows and boars) which are more resistant to pedv infection and diseases (niederwerder and hesse, ) . in addition, the emergence of s ntd-del pedv variants may complicate pedv disease pattern on farms, which needs to be further investigated. in this study, we showed that the replication of the s-intact pedv was enhanced during coinfection with an s ntd-del pedv in pigs. we found that mucin, bile and bile acids can all increase the infection of sintact pedv but not the s ntd-del pedv. this feature may help explain why s-intact pedv outcompetes s ntd-del pedv in vivo. further studies are needed to understand the mechanisms of mucin-, bile-and bile acid-mediated enhancement or inhibition effects on the infection of different pedv variants. all animal experiments were performed according to the protocols approved by institutional animal care and use committee (iacuc) of the ohio state university (osu). mechanism of bile acid biosynthesis in mammalian liver cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus coronavirus ibv: removal of spike glycopolypeptide s by urea abolishes infectivity and haemagglutination but not attachment to cells bile acids are essential for porcine enteric calicivirus replication in association with downregulation of signal transducer and activator of transcription identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains role of sialic acids in feline enteric coronavirus infections novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation the enterohepatic circulation of bile acids as they relate to lipid disorders replication of human noroviruses in stem cell-derived human enteroids deletion of a -amino-acid region in the n-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets the deletion of both tyrosinebased endocytosis signal and endoplasmic reticulum-retrieval signal in the cytoplasmic tail of spike protein attenuates pedv in pigs pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs trypsin-independent porcine epidemic diarrhea virus us strain with altered virus entry mechanism point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants structure, function, and evolution of coronavirus spike proteins cellular entry of the porcine epidemic diarrhea virus cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry bile-salts in small intestinal contents after ileal resection and in other malabsorption syndromes swine enteric coronavirus disease: a review of years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the united states and canada cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene a simple method of estimating fifty percent endpoints the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity sialic acids as receptor determinants for coronaviruses the sialic acid binding activity of the s protein facilitates infection by porcine transmissible gastroenteritis coronavirus porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity new variants of porcine epidemic diarrhea virus with large deletions in the spike protein, identified in the united states molecular characterization of pig epidemic diarrhoea viruses isolated in japan from pig epidemic diarrhoea virus s gene variant with a large deletion non-lethal to colostrum-deprived newborn piglets deciphering the biology of porcine epidemic diarrhea virus in the era of reverse genetics contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the united states type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling the authors declare no conflict of interest. none. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -adpn fb authors: pan, yongfei; tian, xiaoyan; qin, pan; wang, bin; zhao, pengwei; yang, yong-le; wang, lianxiang; wang, dongdong; song, yanhua; zhang, xiangbin; huang, yao-wei title: discovery of a novel swine enteric alphacoronavirus (seacov) in southern china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: adpn fb outbreaks of diarrhea in newborn piglets without detection of transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov), have been recorded in a pig farm in southern china since february . isolation and propagation of the pathogen in cell culture resulted in discovery of a novel swine enteric alphacoronavirus (tentatively named seacov) related to the bat coronavirus hku identified in the same region a decade ago. specific fluorescence signal was detected in vero cells infected with seacov by using a positive sow serum collected in the same farm, but not by using tgev-, pedv- or pdcov-specific antibody. electron microscopy observation demonstrated that the virus particle with surface projections was – nm in diameter. complete genomic sequencing and analyses of seacov indicated that the extreme amino-terminal domain of the seacov spike (s) glycoprotein structurally similar to the domain of the alphacoronavirus nl , whereas the rest part of s structurally resembles domains b to d of the betacoronavirus. the seacov-s domain associated with enteric tropism had an extremely high variability, harboring -amino-acid (aa) substitutions and a -aa insertion, compared to that of hku , which is likely responsible for the extended host range or cross-species transmission. the isolated virus was infectious in pigs when inoculated orally into -day-old newborn piglets, leading to clinical signs of diarrhea and fecal virus shedding. these results confirmed that it is a novel swine enteric coronavirus representing the fifth porcine coronavirus. coronavirus (cov) is an enveloped, single-stranded, positive-sense rna virus of the order nidovirales, family coronaviridae, subfamily coronavirinae, which comprises four genera, alpha-, beta-, gamma-, and delta-cov. covs infect humans, other mammals, and birds, causing subclinical or respiratory and gastrointestinal diseases (de groot et al., ; woo et al., ) . as of date, three types of swine enteric covs (secovs): transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov), have been identified to induce clinical diarrhea in young pigs (jung et al., ; pensaert and de bouck, ) . in particular, emergences of variant pedv fatal to newborn piglets in china in late (pan et al., ) , and later in the united states in tian et al., ) , have posed a serious threat to the pork industry. most recently, several chimeric secov strains with a tgev genomic backbone replaced by a pedv spike (s) gene were identified from swine fecal samples in europe (akimkin et al., ; belsham et al., ; boniotti et al., ) , implying that novel secov pathogens could emerge by inter-cov recombination under co-infection. the s gene encodes a glycoprotein, forming trimer projections on the viral surface, which is a major structural protein critical for cov attachment and entry into the host cell (hulswit et al., ) . in addition to recombination events between two distinct covs, amino acid (aa) mutations in the s protein may alter the tropism of the virus. for example, -aa substitutions and a -aa insertion in the amino-terminal domain (ntd) of the s glycoprotein of a murine hepatitis cov (mhv) variant confer the ability to bind and in some cases infect cells of nonmurine species including swine cells (schickli et al., ) . in this study, we report the isolation and genetic characterization of a novel swine enteric alphacoronavirus (tentatively named seacov), related to a bat enteric coronavirus, from a pig farm that reported newborn-piglet diarrhea in southern china in . this is yet another example to corroborate that the extended host range of cov, here from bat to pig, is likely associated with aa substitutions at the ntd of the s glycoprotein. furthermore, we conducted a pilot experimental infection study with this novel seacov, confirming its infectivity and ability to induced clinical signs of diarrhea in piglets. baby hamster kidney fibroblast cell line bhk- (atcc ccl- ), swine testis cell line st (atcc crl- ), porcine kidney epithelial cell line llc-pk (atcc cl- ), and african green monkey kidney epithelial vero cell (atcc ccl- ) were individually grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and % antibiotics (penicillin, streptomycin, w/v). a vero cell line stably expressing the tgev receptor porcine aminopeptidase n (vero-papn) was cultured in dmem supplemented with μg/ml puromycin and antibiotics (unpublished data). all cells were grown at °c with % co . a pan-cov rt-pcr assay was used to detect the unknown pathogen with a pair of primers: cor-fw ( ′-acwcarhtvaayytnaartaygc- ′) and cor-rv ( ′-tcrcayttdggrtartccca- ′) as described (moes et al., ) . after the pathogen (seacov) was identified, specific primers targeting the seacov-nucleocapsid (n) gene (the forward primer seaf: ′-atggataaacctgaatggaagcg- ′, and the reverse primer sear: ′-caccatctcaacctcttcctcag- ′) were used for virus detection during isolation and subsequent passages. fecal specimens collected from diarrheic piglets and positive for seacov rna were homogenized in dmem containing antibiotics followed by centrifugation at × g for min. the supernatants was inoculated onto confluent monolayers of bhk- , st, llc-pk or vero cells cultured with the maintenance medium plus trypsin (mmt) at °c and % co . the mmt consisted of dmem supplemented with % fbs, % antibiotics and μg/ml trypsin (sigma). cells were observed daily to record the development of cytopathic effect (cpe) as described previously (pan et al., ) . the virus strain isolated in vero cells with mmt, designated as ch/ gd- / , was plaque-purified in the presence of trypsin using neutral red staining as described (qin et al., ) . it was passaged serially using the culture supernatant and the viral titer was determined by plaque assay. supernatant from purified seacov-infected cell cultures showing cpes was negatively-stained. grids were stained with % sodium phosphotungstic acid (ph . ) for . min and examined using a hitachi model h- tem. vero cells infected with seacov on -well plates were washed twice with phosphate-buffered saline (pbs) and fixed with acetone. one hundred and fifty microliters of the collected sow serum samples at a : dilution in pbs was added to the cells in each well and incubated for h at room temperature. cells were washed thrice with pbs followed by addition of μl fitc-labeled rabbit anti-pig igg (thermo fisher scientific, usa) at : dilution. after incubation for h at room temperature, the cells were washed with pbs, stained with μl ′, -diamidino- -phenylindole (dapi) at : dilution and visualized under a fluorescence microscope. for antibody cross-reactivity test, vero cells infected with seacov or pedv (zju/g / strain; genbank accession no. ku ), vero-papn cells infected with tgev (purdue strain; a gift from dr. rong ye at shanghai medical college of fudan university), and llc-pk cells infected with pdcov (hunan strain; genbank accession no. ky ) were stained with the anti-pedv-n, anti-tgev-n and anti-pdcov-n monoclonal antibody (purchased from medgene labs, brookings, sd, usa), respectively. the fitc-conjugated goat anti-mice igg (thermo fisher scientific, usa) was used as the secondary antibody followed by dapi staining. total rna was extracted from the isolated virus with trizol reagent, and cdnas were subsequently amplified by superscript ii with specific primers according to the manufacturer's instructions (thermo fisher scientific). a total of primer pairs based upon the bat cov hku strain gd - (genbank accession no. ef ; supplemental table s ) were designed to amplify the complete genome of seacov. pcr products were purified and cloned into a pcr-blunt vector (thermo fisher scientific). for each amplicon, three to five individual clones were sequenced to determine the consensus sequence. the sequences were assembled and analyzed using the dnastar program. multiple alignments of the full-length genomes, non-structural protein genes and s genes with representative cov sequences and phylogenetic analyses were performed using the neighbor-joining method in mega . , respectively. structure homology-modeling of seacov s glycoprotein was performed by the swiss-model server (https://www.swissmodel.expasy.org/). a pilot animal experiment was approved by the experimental animal ethics committee of zhejiang university (approval no. zju ). briefly, ten -day-old conventional piglets, free of seacov, pedv, tgev, and pdcov rna in the feces, were assigned into two groups with in each. piglets in each group were housed with their mothers (seacov rna and serum antibody negative as determined by ifa) without any artificially supplemental colostrum or milk. piglets in group one were each challenged orally with a seacov/ch/gd- / /p isolate at a dose of × plaque-forming units (pfu)/ml ( ml per pig), whereas piglets in group two each received ml of dmem orally as negative controls. all the piglets were monitored daily for any signs of illness. two piglets in each group were euthanized at days post-infection (dpi) while the remaining three in each group were necropsied at dpi. the duodenum, jejunum and ileum samples were subjected to histological examinations by hematoxylin and eosin (he) staining, respectively. the villous height (vh) and the crypt depth (cd) were measured on a minimum of eight different sites per small intestinal segment, and the ratios of vh to cd were then calculated to quantify the villous atrophy according to previously described (jung et al., ) . fecal swabs for viral rna detection were collected at , , , , and dpi from all five pigs until they were alive. beginning from february , a remarkable increase in outbreaks of newborn-piglet diarrhea occurred in a commercial pig farm located in guangdong province of southern china. clinical signs of affected pigs were characterized by acute vomiting and watery diarrhea (fig. a) . the mortality rate was over % in piglets less than days old during february-may . in addition, the small intestine of the diseased pigs y. pan et al. veterinary microbiology ( ) - displayed thin walls and contained yellow watery feces (fig. b) , which was indistinguishable from that of pedv infection described previously pan et al., ) . fecal and small intestinal samples collected from affected piglets in this farm were submitted to our labs at zhejiang university and hog production division of wen's foodstuffs group, respectively, for routine laboratory diagnostics. upon laboratory analysis by rt-pcr, rna of pedv, tgev, pdcov or porcine hemagglutinating encephalomyelitis virus (phev), was not detected in these samples (data not shown). other possibly known viral pathogens associated with piglet diarrhea such as porcine enterovirus, rotavirus or mammalian orthoreovirus (qin et al., ) also could not be detected. subsequently, samples were tested by a pan-cov rt-pcr assay designed to amplify a conserved region of -bp in the orf b gene for all cov members (moes et al., ) . this test was positive for all the selected samples collected during february to may (data not shown). sequencing of the pcr products revealed that they were % identical to the corresponding region (nucleotide [nt] positions - ) of four known bat enteric alphacoronavirus hku strains (genbank accession nos. ef to ef ) identified from guangdong province and hong kong in and (lau et al., ) . the prevalence rate of bat cov hku from these two regions was reported to be . % ( / ) and . % ( / ) in chinese horseshoe bats (rhinolophus sinicus), respectively (lau et al., ) . hku infection associated with the other animal species has never been investigated. the results from pan-cov rt-pcr detection indicated that an hku like viral pathogen might be responsible for outbreaks of diarrhea in the pig farm. in an effort to isolate the novel swine enteric hku -related cov (seacov), suspension supernatants of selected hku -positive samples were prepared and inoculated in a panel of bhk- , st, llc-pk and vero cell lines routinely used to isolate porcine covs. cultured supernatants from each inoculated cell line were blind-passaged serially. from vero cell culture, we successfully isolated one seacov strain with cpe characterized by syncytia formation at h post-infection, beginning from passage two (p ) and in the following passages after plaque purification (fig. c) . furthermore, viral antigens were demonstrated in seacov-infected vero cells by ifa, with a serum sample collected from a sow mothering the diseased piglets (fig. d ), but not with the specific monoclonal antibodies against the n protein of pedv, tgev or pdcov (fig. ) , suggesting that seacov are probably antigenetically distinct from the three known porcine covs. seacov antibody-negative sera from the same farm were also found, as staining with these sera in seacov-infected cells displayed no fluorescent signal (fig. e) . electron microscopy of a negatively stained sample from the supernatant of virus-infected vero cells demonstrated that the virus particle was to nm in diameter, and had surface projections typical of cov (fig. f) . seacov rna was detected in supernatants from all virus passages to date (p to p ) by rt-pcr with primers seaf and sear. the virus titer reached up to × pfu/ml at p . this isolated cov strain was designated as seacov/ch/gd- / . we next determined the complete genome of p of ch/gd- strain by rt-pcr amplification of regions covering the entire seacov, as described previously for pedv or pdcov genomic cloning wang et al., b) . the complete genome sequence of the ch/ gd- / /p strain has been deposited in genbank under accession no. mf . the genomic sequence of ch/gd- / /p is , nt in length, excluding the poly(a) tail. the genome organization is similar to those of the four hku strains and a bat cov identified in yunnan province in southwestern china (btrf-alphacov/yn , genbank no. kj ), with the typical gene order ′-orf a/ b (orf ab)-s-orf -e-m-n-ns a- ′ (fig. ) . the ch/gd- / /p strain is -nt longer than hku ( , nt), including a -nt (ttg) insertion at nt , - , (corresponding to the hku /gd sequence) in the nonstructural protein (nsp) region, a -nt (ggcctc) insertion at nt , - , in the s gene, and a -nt (gta) deletion at nt , - , in the m gene (fig. ) . however, these insertions/deletion are not unique for seacov since they are also present in the btrf-al-phacov/yn genome in comparison with hku . seacov shared . % nt sequence identity with the four hku strains, and exhibited . % nt identity with btrf-alphacov/yn . accordingly, seacov is phylogenetically located between hku and btrf-alphacov/yn , together forming a sublineage closely related to the proposed alphacov group- b lineage, including pedv and human covs nl and e, at the complete genome level (fig. a) . however, analysis of the phylogenetic tree constructed based on the s genes (fig. b ) indicated that these six hku -related cov strains along with a newly identified rat alphacov, lrnv (wang et al., a) , formed a separate lineage clustered within the betacovs. the previous studies have suggested that hku and the related lrnv probably resulted from an ancient recombination event with an alphacov genomic fig. . ifa results of vero cells infected with seacov or pedv, vero cells stably expressing porcine aminopeptidase n (vero-papn) infected with tgev, and llc-pk cells infected with pdcov at h post-infection. seacov-infected vero cells were stained with the anti-pedv-n, anti-tgev-n or anti-pdcov-n monoclonal antibody, respectively (left panels). cells infected with pedv, tgev or pdcov were stained with the respective virus-specific antibody as the controls (right panels). the fitc-conjugated goat anti-mice igg was used as the secondary ab in ifa. magnification = ×. fig. . schematic diagram of the genomic structure of seacov and the proposed domain organization of the seacov spike protein s subunits according to the structure similarity analysis with nl and mhv that are both structure available. numbers indicate amino acid positions in s glycoprotein of seacov, nl or mhv, respectively. see supplemental fig. s for the detailed sequence alignment. nucleotide insertion/deletion at three locations (nsp , s and m genes) in seacov compared to the consensus sequences of four bat-cov hku strains (genbank accession nos. ef to ef ) are marked by "*". y. pan et al. veterinary microbiology ( ) - backbone replaced by a betacov s gene (lau et al., ; wang et al., a) . furthermore, pairwise comparison of seacov genomic sequence with hku indicated that the most dissimilar region was in the s gene, particularly, in the extreme ntd (aa - ). the entire seacov s protein had . % aa identical with s of the hku /gd strain, but there was only a . % identity in the extreme ntd of the s protein (s-ntd) between seacov and hku . we identified a total of -aa substitutions plus a -aa insertion (resulting from a -nt insertion as mentioned above) within the seacov s-ntd compared to hku . in contrast, only aa substitutions were found in the remaining part of the s protein. the extreme ntd changes in seacov are likely to be associated with the extended host range, similar to a previously reported mhv variant that was able to expand nonmurine-species tropism, with the phenotype mapped to substitutions and a -aa insert in ntd of s subunit (schickli et al., ) . during the time of this manuscript preparation, a sequence of another hku -related seacov strain gds , identified in the same region, was reported online but it did not give in-depth analyses (gong et al., ) . it remains unknown if gds can be isolated in cell culture. moreover, neither detection of serum anti-seacov antibodies nor observation of virus morphology was demonstrated. nevertheless, comparative sequence analysis showed that the gds strain, having the same genomic size ( , nt), shared . % nt homology with gd- / /p at the complete genome level. however, the gds sequence was determined by the next generation sequencing, which should theoretically be less accurate than the gd- / /p sequence determined based upon the consensus sequences from different short rt-pcr fragments covering the full-length genome. the s-ntd of gds also contains -aa substitutions and a -aa insertion compared to that of hku . there are only three aa differences at the positions (d/g), (m/r) and (a/v) in the s-ntd between gds and gd- / /p . the corresponding aa in bat cov hku at these positions are g, m and m. for nonstructural protein genes analysis, the seacov gd- / /p exhibited . % and . % nt identities, . % and . % nt identities, or . % and . % nt identities with gds and hku based on the orf ab, the orf a, or the orf b genes, respectively. these sequence analyses suggested that the seacov strains gd- / and gds could have the same origin. the s glycoprotein of seacov or hku is unique and not related to any currently known betacovs at the aa sequence level. most recently, the structures of several cov s glycoprotein trimmers have been resolved (walls et al., a (walls et al., , b . the betacovs comprise of four domains (domains a-d) in the s subunit whereas human alphacov nl shows an additional domain, named domain (equivalent to the extreme ntd) compared to betacovs (fig. ) . therefore, a structure homology-modeling was performed in order to better understand the evolutionary origin of the s glycoprotein of seacov/hku in the protein structure level. surprisingly, the result suggested a hybrid structure of seacov-s: the extreme ntd (domain ) of seacov-s is structurally similar to that of nl , whereas the rest part in s structurally resembles domains b to d of the betacov mhv ( fig. and supplemental fig. s ). the s subunits of seacov and mhv also have a similar structure (supplemental fig. s ). the deduced structure of the linking region between domain and domain b of seacov is uncertain. we hypothesize that the domain a is likely not present, which may be a unique feature of seacov/hku s subunit. since domain and domain a are structurally similar and might come from a gene-duplication event (walls et al., b) , we also hypothesize that either of them is likely dispensable in the s of covs. in addition, the presence of domain in seacov/hku is in line with the enteric tropism of these viruses since pedv and tgev also possess this domain (hulswit et al., ) . future study on developing the seacov infectious clone and resolving the alphacov/betacov-hybrid seacov-s glycoprotein structure are warranted to confirm these findings. in order to test whether or not, seacov is able to infect pigs, we performed a pilot challenge experiment using the cell-cultured seacov/ch/gd- / isolate. as expected, the five piglets in dmem-inoculated group neither showed clinical sign nor shedding y. pan et al. veterinary microbiology ( ) - virus in the feces throughout the experimental period (data not shown). in contrast, clinical signs characterized by acute vomiting and watery diarrhea (similar to fig. a) were observed in the five seacov-infected piglets at to h post-infection, and thereafter lasted until necropsy. fecal virus shedding was detected in five seacov-infected pigs at , and dpi, and in three remaining pigs at and dpi by rt-pcr with the primers seaf and sear (data not shown). sequencing of the pcr products indicated that they were identical with the seacov n gene sequence, confirming that the infectious virus was originated from the seacov isolate. upon histopathological analysis, no intestinal lesions were observed in control pigs (fig. ) ; the mean duodenal, jejunal and ileal vh/cd were . ( ± . ), . ( ± . ) and . ( ± . ), respectively. typical microscopic lesions, showing gradual atrophy with significantly reduced vh/cd (the mean jejunal or ileal value was . [ ± . ] or . [ ± . ]), diminishing capillaries and central lacteals of the intestinal villous (fig. ) , were detected in the jejunum and ileum of seacov-infected piglets. the duodenal sections displayed only mild microscopic lesions (the mean duodenal vh/cd = . [ ± . ]) in all seacov-infected pigs (fig. ) . it was different from the result observed for the experimental infection using the virulent chinese pedv strain, in which marked microscopic lesions in all the three parts of the small intestine were found (zhang et al., ) . the results indicated that the seacov isolate is actually infectious and causes diarrhea in pigs. since the specific non-swine antibodies against the structural proteins of seacov are not available currently, further comprehensively pathological studies by immunohistochemistry and serological assays, which is not the scope of this study, are warranted to provide more information on seacov infection. in summary, we have isolated, sequenced and genetically characterized a novel swine enteric alphacoronavirus, which is probably distinct from pedv, tgev and pdcov antigenetically, from diarrheal samples in a pig farm of southern china in . the isolated seacov can actually infect and cause diarrhea in pigs, and should represent the fifth porcine coronavirus in addition to pedv, tgev (considering that porcine respiratory virus, prcv, is a variant of tgev), pdcov and phev. to our knowledge, this is also the first study describing seacov related to the bat coronavirus hku that could be isolated and propagated in cell culture. however, infection of vero cells (a monkey cell line) with seacov also raises concerns about its potential host range other than swine. we also identified that the extreme ntd (aa - ) of seacov spike protein consists of -aa substitutions and a -aa insertion compared to that of hku , which is likely to be responsible for the cross-species transmission. moreover, this region but not the other betacov-related domains of seacov s subunit is structurally similar to the alphacov domain , implying that these viruses gained enteric tropism through this domain. the results provide much needed information on seacov and hku evolution, and the availability of seacov in cell culture will guide future efforts to develop effective vaccines against seacov. . representative histological examinations of the duodenum, jejunum and ileum samples collected at days post-infection from piglets inoculated with seacov or dmem in the animal challenge experiment. sections of jejunum and ileum in the seacovinfected group showed scattered areas of villi atrophy, whereas the section of duodenum showed mild microscopic lesions as compared to the dmem control group. y. pan et al. veterinary microbiology ( ) - new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus virus taxonomy: ninth report of the international committee on taxonomy of viruses a new bat-hku -like coronavirus in swine origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states coronavirus spike protein and tropism changes pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis complete genome sequence of bat coronavirus hku from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium isolation and characterization of a variant porcine epidemic diarrhea virus in china a new coronavirus-like particle associated with diarrhea in swine genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus (mrv ) from a diarrheic piglet and seroepidemiological survey of mrv in diarrheic pigs from east china the n-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells evidence of recombinant strains of porcine epidemic diarrhea virus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy discovery, diversity and evolution of novel coronaviruses sampled from rodents in china complete genome sequence of porcine deltacoronavirus strain ch/sichuan/s / from mainland china discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence this work was supported by the national key research and developmentprogram of china ( yfd and yfd ), and the national natural science foundation of china ( ). we thank dr. narayan paudyal for conducting english language review. supplementary data associated with this article can be found, in the online version, at https://doi.org/http://dx.doi.org/ . /j.vetmic. . . . key: cord- -dh jvbmi authors: van kasteren, puck b.; knaap, robert c.m.; van den elzen, paul; snijder, eric j.; balasuriya, udeni b.r.; van den born, erwin; kikkert, marjolein title: in vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: dh jvbmi arteriviruses are a family of positive-stranded rna viruses that includes the prototypic equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv). although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of prrsv. the ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. we have recently shown that the deubiquitinase (dub) activity of eav papain-like protease (plp ) is important for the inhibition of innate immune activation during infection. a vaccine virus lacking plp dub activity may therefore be more immunogenic and provide improved protection against subsequent challenge than its dub-competent counterpart. to test this hypothesis, twenty shetland mares were randomly assigned to one of three groups. two groups were vaccinated, either with dub-positive (n = ) or dub-negative (n = ) recombinant eav. the third group (n = ) was not vaccinated. all horses were subsequently challenged with the virulent ky strain of eav. both vaccine viruses proved to be replication competent in vivo. in addition, the dub-negative virus provided a similar degree of protection against clinical disease as its dub-positive parental counterpart. owing to the already high level of protection provided by the parental virus, a possible improvement due to inactivation of plp dub activity could not be detected under these experimental conditions. taken together, the data obtained in this study warrant further in vivo investigations into the potential of using dub-mutant viruses for the improvement of arterivirus vaccines. arteriviruses are a family of animal viruses that includes the prototypic equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv). whereas outbreaks of equine viral arteritis (eva) are only occasionally reported, infections with prrsv pose a major threat to swine-farming industries worldwide. especially the emergence of highly virulent strains of prrsv in china since has been a major concern (zhou and yang, ) . although current vaccine-based control strategies for eav are considered adequate, some concerns regarding efficacy (i.e. limited cross protection) and safety (i.e. shedding of vaccine virus) provide room for improvement (balasuriya et al., ; mccollum et al., ) . in contrast, whereas currently available vaccines against prrsv generally provide protection against clinical disease, they do not consistently prevent replication of field strains and shedding of infectious virus, and especially the limited protection against heterologous field strains is a major issue (kimman et al., ) . the design of (novel) prrsv vaccines that provide improved protection against both homologous and heterologous field strains is therefore of significant importance. it has been suggested that the immune-evasive capabilities of prrsv play an important role in reducing vaccine efficacy (kimman et al., ; wang et al., b) . one of the arterivirus protein domains that have been suggested to be involved in downregulating the innate immune response is papain-like protease (plp ) (frias-staheli et al., ; sun et al., ; van kasteren et al., ) , which functions both as a deubiquitinase deubiquitinase interferon vaccine eav prrsv arterivirus arteriviruses are a family of positive-stranded rna viruses that includes the prototypic equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv). although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of prrsv. the ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. we have recently shown that the deubiquitinase (dub) activity of eav papain-like protease (plp ) is important for the inhibition of innate immune activation during infection. a vaccine virus lacking plp dub activity may therefore be more immunogenic and provide improved protection against subsequent challenge than its dub-competent counterpart. to test this hypothesis, twenty shetland mares were randomly assigned to one of three groups. two groups were vaccinated, either with dub-positive (n = ) or dub-negative (n = ) recombinant eav. the third group (n = ) was not vaccinated. all horses were subsequently challenged with the virulent ky strain of eav. both vaccine viruses proved to be replication competent in vivo. in addition, the dub-negative virus provided a similar degree of protection against clinical disease as its dub-positive parental counterpart. owing to the already high level of protection provided by the parental virus, a possible improvement due to inactivation of plp dub activity could not be detected under these experimental conditions. taken together, the data obtained in this study warrant further in vivo investigations into the potential of using dub-mutant viruses for the improvement of arterivirus vaccines. ß elsevier b.v. all rights reserved. (dub) and plays an essential role in the [ _ t d $ d i f f ] autoproteolytic maturation of the viral replicase polyproteins. importantly, we have recently been able to show that the dub activity of eav plp inhibits the innate immune response in infected cells, via the structure-based design of a viable mutant virus lacking this activity (van kasteren et al., ) . the aim of the current study was to determine in vivo whether this dub-negative virus provides better protection against subsequent challenge infection than its parental dubcompetent counterpart. since to date the separation of dub and polyprotein processing functions of arterivirus plp has only been successful for eav, we have used this virus for the present animal study. bhk- cells were cultured in glasgow minimum essential medium (lonza) supplemented with % foetal bovine serum (fbs), % tryptose phosphate broth, and mm hepes (ph . ). primary equine lung fibroblasts (elfs) were cultured in minimum essential medium (lonza) supplemented with % fbs and grown on collagen-coated plastics for a maximum of passages. vero cells were cultured in proprietary cell culture medium (msd animal health) supplemented with % fbs. all culture media contained u/ml of penicillin and mg/ml of streptomycin or neomycin. the cloning and production of the dub-competent (in our previous publication referred to as ''wild-type'') and dub-negative (t a/i v/i r, amino acid numbering based on polyprotein) viruses (strain bucyrus; ean ) that were used for vaccination were described previously (van kasteren et al., ) . both viruses have been thoroughly characterized in cell[ _ t d $ d i f f ] culture experiments, revealing no differences in replication kinetics, yet a strongly decreased dub activity and an enhanced induction of interferon beta mrna expression (a hallmark of innate immune activation) of the mutant virus compared to its parental counterpart (van kasteren et al., ) . viral titres were determined by standard plaque assay on elfs. for experimental challenge, we used the virulent kentucky (ky ) strain of eav, which has been previously described (zhang et al., ) . to confirm the use of the correct virus for vaccination, the presence or absence of plp mutations was established, both before and after vaccination. before vaccination, viral rna was isolated from the produced virus stocks using the qiaamp viral rna mini kit (qiagen) and converted to cdna using revertaid h minus reverse transcriptase (rt) (fermentas) and random hexamer primers. the plp -encoding region was subsequently pcr amplified using pfu dna polymerase (fermentas) and sequenced. after vaccination, viral rna present in the blood of four horses from each of the vaccinated groups at days post vaccination was subjected to sequencing. this was done essentially as described above with the exception that the rt reaction on whole blood total rna (see below) was performed using a primer that recognizes the eav genome and thus specifically converts the limited amount of viral rna present in the samples to cdna. primer sequences are available upon request. the experiment was performed in accordance with european community guidelines and national laws on animal experiments. the design of the experiment was approved by the msd animal health's committee on the ethics of animal experiments (dierexperimentencommissie), which is required by national legislation to include both msd animal health employees and independent members, prior to the start of the trial (permit number: exp . ). all efforts were made to minimize animal discomfort. twenty female shetland horses (equus ferus caballus; average age . ae . years) that tested negative for eav-neutralizing antibodies (antibody titres were determined as described previously, zhang et al., ) before the start of the experiment were randomly assigned to one of three treatment groups. after a one-week acclimatization period, horses in group (n = ) and group (n = ) received an intramuscular (cervical muscle) vaccination of ml phosphate-buffered saline containing  plaque-forming units (pfu) of parental or plp dub-negative eav[ _ t d $ d i f f ] , respectively. horses in group (n = ) were not vaccinated and were included in the study one week before challenge. at days post vaccination (dpv), all horses were challenged by intranasal inoculation with  pfu of eav ky in a total volume of ml phosphate-buffered saline. given the fact that the viruses used for vaccination qualify as genetically modified organisms (gmo), vaccinated horses were kept in vbsl containment during the entire experiment. all horses were housed in groups, but group horses were kept separate from group horses to prevent any cross-contamination. horses from group were divided among the two stables upon inclusion, without having direct contact with group or horses. water was provided ad libitum and standard feeding procedures were applied. the general health status of the animals was checked by a veterinarian before vaccination as well as before challenge, and daily by animal care-takers during the entire course of the experiment. in addition, clinical signs were recorded daily from to days post challenge (dpc) and scored according to table . rectal temperatures were taken daily from to , and at and dpv, and daily from to , and at and dpc. blood samples for serum and total rna isolation were taken every other day between and , and at and dpv, and every other day between and , and at and dpc. animals were euthanized according to standard procedures at dpv ( dpc). for a schematic overview of the experimental set-up see fig. . blood for serum neutralizing antibody analysis was collected in ml vacuette serum clot activator tubes (greiner bio-one) and incubated for at least h at room temperature to allow for clotting. serum samples were subsequently collected by centrifugation at plates were subsequently incubated for days at c after which each well was scored (positive or negative) for cytopathic effect (cpe) by visual inspection. the eav neutralizing antibody titre was finally determined as the reciprocal value of the highest serum dilution at which no cpe was observed. total rna was isolated from whole blood collected in tempus blood rna tubes (greiner bio-one) using the magmax for stabilized blood tubes rna isolation kit (life technologies) according to the manufacturer's instructions. isolated rna was converted to cdna using revertaid h minus reverse transcriptase (fermentas) and oligo(dt) primer. samples were subsequently analyzed by quantitative reverse-transcriptase (qrt) pcr on a cfx touch real-time pcr detection system (biorad) using itaq sybr green supermix (biorad). primers targeting mrnas encoding equine b-actin (nm_ , ccacgccatcctgcgtctgg- , accgctcgttgccgatggtg- ), isg (xm_ , g-gaattcctggtgcccctgaa- , cagttctgcacgacaagcac- ), and mx (nm_ , cggccagcagctgcagaagt- , g-ggcctccgctccctggagat- ), or eav rna (nc_ and af , ggttcgcggcaacgggtaca- , ggtggcgcgctcc-ggtggcgcgctcctgttgat- ) were designed using primer (rozen and skaletsky, ) . the eav-specific primer set amplifies cdna derived from both genomic and subgenomic viral mrnas and the forward primer includes one mismatch with ky . the real-time pcr program was performed in triplicate, starting with min at c, s at c, followed by cycles at c for s, c for s, and c for s. all runs included a standard dilution series and were followed by a melting-curve analysis to confirm the identity of the reaction products. to assess whether a vaccine virus lacking plp dub activity would provide better protection against heterologous challenge than its dub-positive parental counterpart, we performed a vaccination-challenge trial in horses. twenty female shetland horses were randomly assigned to one of three groups. animals from two groups were subsequently vaccinated intramuscularly with cell culture-adapted dub-competent (group , n = ) or dubnegative (group , n = ) eav. horses from group (n = ) were not vaccinated and served as challenge controls. all horses were intranasally challenged with[ _ t d $ d i f f ] virulent eav ky at dpv. a schematic representation of the experimental set-up including timing of vaccination, challenge, and sampling is depicted in fig. . the identity of the virus used for vaccination was confirmed by sequencing of viral rna isolated from whole blood at dpv for a subset of four horses per group from groups and (data not shown). upon vaccination, animals from groups and (dubcompetent and dub-negative virus, respectively) developed comparable levels of mild fever which reached an average maximum of . c at dpv and subsided approximately dpv in both groups ( fig. a) . upon challenge, only the non-vaccinated animals in group developed a fever, which reached higher levels (average maximum . c) and lasted approximately two days longer than the mild temperature increase observed after vaccination. neutralizing antibodies could be detected in both groups and from dpv onwards and no difference in titres was observed between the two groups (fig. b) . titres remained stable during the course of the experiment and did not show an increase upon challenge. in the unvaccinated controls, neutralizing antibody titres could be detected at days post challenge (dpc) and reached similar titres as observed after vaccination. from to dpc, clinical signs (including for example fever, nasal secretions, and loss of appetite) were recorded daily for each animal. an overall clinical signs score was subsequently determined for each animal by scoring these clinical signs according to table . for example, a horse that has reduced appetite on day , mucopurulent eye discharge and a temperature of c on day , and no abnormalities on any of the other days has an overall clinical signs score of ( + + ). this resulted in an average score per animal per day of . (ae . , group ) and . (ae . , group ), which did not differ significantly between the two groups (student's t-test; p > . ; table ). the horses in group reached an average score of . (ae . ) per animal per day, which is consistent with the fact that these animals had not been vaccinated. thus, vaccination with either virus provided a similar high degree of protection against clinical disease. we then assessed viral replication from to dpv by performing real-time qrt-pcr analysis on total rna isolated from whole blood. as can be seen in fig. a , all vaccinated horses showed viral replication which, on average, peaked at dpv in both [ ( f i g . _ ) t d $ f i g ] fig. . schematic representation of the animal trial. twenty female shetland horses were randomly assigned to one of three groups. at the start of the experiment, horses from groups and (n = each) were vaccinated with plp dub-competent or dub-negative eav[ _ t d $ d i f f ] , respectively. horses in group (n = ) were not vaccinated. at dpv, all horses were challenged with moderately virulent eav ky . the experiment ended at dpv. blood samples for serum and total rna isolation were taken at the indicated days (lower arrows). groups. whereas viral rna could be detected on multiple days in all group horses (dub-competent vaccine), in four group horses (dub-negative vaccine), viral rna could only be detected on a single day, i.e. dpv. in addition, when comparing the average amounts of viral rna between groups at each day post vaccination, these were consistently lower in group compared to group . however, this difference was only statistically significant at dpv (student's t-test; p < . ). upon challenge with the[ _ t d $ d i f f ] virulent eav ky strain, both nonvaccinated control horses (group ) showed virus replication, with amounts of viral rna reaching levels comparable to those produced by the vaccine viruses (fig. a) . in contrast, amounts of challenge virus rna remained below the detection limit of the assay in all vaccinated horses from groups and . this result was consistent with the observed appearance of neutralizing antibodies after vaccination in all horses and the low clinical sign scores in both vaccinated groups after challenge. finally, we assessed the induction of an innate immune response by parental and plp dub-negative virus from to dpv by means of real-time qrt-pcr on rna extracted from whole blood. since we were unable to detect mrna encoding type i interferons (a hallmark of innate immune activation), we instead focussed on two highly expressed interferon-stimulated genes encoding isg and mx , respectively. both of these genes showed an approximately -log increase of expression at dpv, which did not significantly differ between animals from groups and ( fig. b and c) . at and dpv, the expression of isg and mx mrna was significantly lower in the animals vaccinated with the plp dub-negative virus (group ) than in those vaccinated with the parental virus (group ) (student's t-test; p < . ), which [ ( f i g . _ ) t d $ f i g ] might be explained by the slight decrease in replication efficiency of the former virus. we have previously shown that mutant eav lacking plp dub activity induces a more potent innate immune response than its dub-competent parental virus upon infection in cell culture (van kasteren et al., ) , and hypothesized that this feature might be included in an improved arterivirus vaccine. in order to assess whether a plp dub-negative vaccine virus provides better protection than its dub-competent counterpart against challenge with a virulent eav strain, we performed a vaccination-challenge trial in horses. the data obtained in this study showed that in contrast to what was previously seen in cell[ _ t d $ d i f f ] culture experiments (van kasteren et al., ) , the plp mutant virus appears to replicate slightly less efficiently than its parental virus in vivo (fig. a) . even though the dub-negative virus is clearly replication competent, this finding does suggest a role for plp dub activity in supporting replication in vivo, e.g. by inhibiting the innate immune response. in addition, despite this slight decrease in replication efficiency, the levels of protection against clinical disease (table ) , the antibody response (fig. b) , and the innate immune response (assessed based on the expression of two interferon-stimulated genes; fig. b and c) induced by the plp dub-negative virus was comparable to that induced by the parental virus. surprisingly, we did not observe an increase in eav-neutralizing antibody titres upon challenge infection of vaccinated animals (fig. b) . a possible explanation for this finding is that the virus dose used for challenge was too low, resulting in its efficient clearance before (re-)activation of the antibody response. taken together, the present study could not be used to substantiate (or refute) our hypothesis that a dub-negative virus provides better protection against challenge than its dub-positive counterpart. the main reason for this seems to be the fact that the parental virus already provides a very high degree of protection, which as it turned out could not be detectably improved using this experimental set-up. in hindsight, one solution might have been to perform a more severe challenge, for example by using a higher virus dose for inoculation or a more heterologous and/or virulent strain than ky . as prrsv vaccines are in general less efficacious than eav vaccines, another option would be to perform a similar trial with a prrsv vaccine in pigs, but this will first require the design of viable prrsv plp dubnegative mutants. in contrast to what was previously found in cell culture-based assays (van kasteren et al., ) , we did not detect an increased activation of innate immunity by the plp dub-negative virus compared to its parental virus in vivo. however, it needs to be noted that we have thus far assessed the expression of only two interferon-stimulated genes, whereas many more exist. it therefore remains a possibility that differences do exist in the expression of other interferon-stimulated genes. similarly, the induction of interferon-stimulated genes is only one of several consequences of the activation of innate immune signalling. it therefore remains possible that the dub-negative vaccine virus differs from the parental virus in some other respect pertaining to immunity, for example the activation of cell-mediated adaptive immunity. also, plp dub activity likely constitutes only one of several innate immune evasion strategies employed by arteriviruses. for example, nonstructural protein (nsp ) of eav was recently shown to inhibit the induction of interferon beta in a luciferase reporter assay (go et al., ) and the prrsv nsp main protease was recently shown to cleave the innate immune signalling factor nemo (huang et al., ) . also [ ( f i g . _ ) t d $ f i g ] fig. . quantitative rt-pcr analysis of viral rna and cellular mrna encoding isg and mx . (a) viral replication from to dpv (groups and ) or to dpc (group ) was assessed by real-time qrt-pcr analysis on total rna isolated from whole blood using eav-specific primers. white, black, and grey circles represent animals from group , , and , respectively. horizontal bars represent the mean for all animals on that day and the dotted line represents the limit of detection. activation of the innate immune response was assessed by real-time qrt-pcr on total rna isolated from whole blood using primers specific for b) isg -or (c) mx -encoding mrna. values obtained for isg and mx mrna levels were normalized to the amount of b-actin mrna. the dotted lines represent average baseline levels, error bars represent standard deviations, and asterisks indicate statistical significance (student's t-test; p < . ). abbreviations: au, arbitrary units; dub+, deubiquitinase-competent vaccine virus; dubÀ, deubiquitinase-negative vaccine virus. nsp a han et al., ; kim et al., ; song et al., ) , nsp b (beura et al., ; chen et al., ; patel et al., ; wang et al., a) , and the nucleocapsid protein (sagong and lee, ) have been proposed to counteract innate immune activation. taken together, the plp dub activity appears to constitute only a part of the total repertoire of arterivirus innate immune evasion strategies. for this reason, its contribution to the inhibition of innate immunity perhaps cannot be readily detected in the in vivo experimental set-up used here. for example, detecting more prominent consequences of its inactivation may depend on the details of the animal study, including specific properties of viruses and horses used. nevertheless, the apparently limited negative effect on viral replication of plp dub-mutations does open up possibilities for combining mutations in multiple domains involved in immune evasion, thereby potentially synergistically increasing the overall immunogenicity of the virus. notably, arteriviruses are not the only group of viruses that were shown to harbour dub activity. for example, coronaviruses (including sars-and mers-cov) encode papain-like proteases that display dub activity (for a review, see mielech et al., ) . the otu proteases of nairoviruses have also been found to possess dub activity (frias-staheli et al., ) . like arterivirus plp , these viral dubs have been implicated in innate immune evasion (frias-staheli et al., ; mielech et al., ) and are therefore potential targets for the design of novel vaccines. interestingly, we have recently been able to show that the dub and polyprotein processing functions of the papain-like protease encoded by mers-cov can also be separated by targeted mutagenesis (bailey-elkin et al., ) . taken together, the data obtained in this study definitely warrant further in vivo examination of the consequences of knocking out arterivirus plp dub activity as well as other immune-evasive activities to improve vaccine efficacy. the data obtained in such studies could subsequently also be used as preliminary proof of principle for the design of novel vaccines for other viruses, including corona-and nairoviruses. mk, ejs, and pbk have filed a provisional patent application that relates to some aspects of this work. crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression equine arteritis virus porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation identification of two auto-cleavage products of nonstructural protein (nsp ) in porcine reproductive and respiratory syndrome virus infected cells: nsp function as interferon antagonist ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein as a main interferon antagonist degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp alpha subunit porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes ifnbeta expression by targeting nemo modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology response of vaccinated and non-vaccinated mares to artificial insemination with semen from stallions persistently infected with equine arteritis virus nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deis-gylating activities porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation primer on the www for general users and for biologist programmers porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf activation in immortalized porcine alveolar macrophages nonstructural protein alpha subunit-based inhibition of nf-kappab activation and suppression of interferon-beta production by porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling porcine reproductive and respiratory syndrome virus nsp beta inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-alpha degradation enhancing neutralizing antibody production by an interferoninducing porcine reproductive and respiratory syndrome virus strain development and characterization of an infectious cdna clone of the modified live virus vaccine strain of equine arteritis virus porcine reproductive and respiratory syndrome in china we would like to thank the animal caretakers at the msd-ah animal facility for their efforts. this research was supported in part by the division of chemical sciences of the netherlands organization for scientific research (nwo-cw) through echo grant . . to mk and ejs. key: cord- -s f sb authors: wattrang, eva; mcneilly, francis; allan, gordon m.; greko, christina; fossum, caroline; wallgren, per title: exudative epidermitis and porcine circovirus- infection in a swedish spf-herd date: - - journal: vet microbiol doi: . /s - ( ) -x sha: doc_id: cord_uid: s f sb an outbreak of exudative epidermitis (ee) among piglets in a swedish spf-herd initiated a survey for indications as to the cause of disease. the herd was established by caesarean section and has been closed to all new animal material, with the exception of semen for artificial insemination (ai). the study comprised serum samples from the spf-herd over a -year period (n= ) and a close monitoring of animals in the herd during the period after the ee outbreak. serum samples from conventional boars at the ai-station servicing the herd were also included (n= ). all serum samples were tested for antibodies to porcine circovirus- (pcv- ). in addition, -week-old piglets from three litters (n= ) farrowed close after the initial ee outbreak were closely monitored for clinical signs of skin disease, sampled for staphylococcus hyicus, tested for antibodies to porcine parvovirus and in sequentially collected serum samples tested for interferon-α (ifn-α) and interleukin- . the pvc- serology showed that animals in the herd were sero-negative at least until months prior to the ee outbreak. during the period close after the ee outbreak the animals showed varying levels of antibodies to pcv- but all the tested animals had sero-converted months later. the ai boars were also sero-positive to pcv- at the time of the ee outbreak. animals in the spf-herd remained sero-positive to pcv- during the following years. in the monitored litters, one piglet had clinical ee and piglets displayed defined erythemas on the abdomen. fourteen of the piglets also had ifn-α in serum on one or more occasions during the study, indicating viral activity among the animals. s. hyicus was isolated from all of the piglets from the earliest sampling point ( days of age) and onwards, irrespective of clinical signs. pcv- was isolated from lymphnode tissue collected from one of the ee affected pigs. further, increases in the number of stillborn piglets, small litters (< piglets) and repeat breeders could be correlated to the time of pcv- sero-conversion. coincidence of active viral infection and sero-conversion to pcv- points to the virus as the cause of the ee outbreak and reproductive disturbances. porcine circoviruses (pcvs; family circoviridae) are small, non-enveloped, dna viruses containing an unique single-stranded circular genome (for reviews, see lukert, ; allan and ellis, ) . pcv- is considered apathogenic and ubiquitous throughout the world. pcv- on the other hand, has been associated with various disease syndromes in pigs, including congenital tremors, post-weaning multisystemic wasting syndrome (pmws) and recently also dermatitis/nephropathy syndrome (allan et al., b; rosell et al., ; thomson et al., ) . the syndrome pmws was ®rst identi®ed in in a canadian spf-herd and pcv- was subsequently associated with the disease . pcv- has since been identi®ed in association with cases of pmws from other countries in northern america, europe and asia choi et al., ; fenaux et al., ; mankertz et al., ; mori et al., ; wellenberg et al., ) but to date information on sero-prevalence and modes of virus transmission is limited. however, serum antibodies to pcv- seem to be widespread in the swine populations of those countries where testing has been carried out . in the case of pmws, other viruses such as porcine parvovirus (ppv) or porcine reproductive and respiratory syndrome virus (prrsv; allan and ellis, ) as well as non-speci®c activation of the immune system (allan et al., a; krakowka et al., ) have been demonstrated as cofactors in the full expression of disease syndromes. exudative epidermitis (ee) is a generalised skin infection with greasy exudation and exfoliation, affecting mainly suckling piglets (reviewed by wegener and skov-jensen, ) . exfoliative-toxin producing strains of staphylococcus hyicus are considered the causative agents of ee, but predisposing factors are probably necessary for the disease (andersen et al., ; tanabe et al., ; aarestrup and wegener, ) . for instance ppv has been suggested to be involved in the pathogenesis of ee (whitaker et al., ) . in april , a swedish spf-herd was inexplicably struck by an outbreak of ee. an extensive survey was initiated to determine the cause of the disease and possible mode of entry into the herd, but at the time no plausible explanation was found. in , a retrospective sero-survey for antibodies to pcv- among swedish pigs, revealed that the spf-herd in question was sero-negative to the virus early in but later this year animals in the herd had sero-converted to pcv- . therefore, samples from the outbreak of ee were re-analysed with respect to pcv- and the results are presented herein. the spf-herd was established in june by hand-rearing of colostrum deprived swedish yorkshire piglets delivered by caesarean section. once established, the herd has been completely closed to new animal material, but semen collected from conventional boars free from aujeszky's disease virus (quality genetics, ka Èvlinge, sweden) has continuously been used for arti®cial insemination (ai). a teaser boar was kept and this boar was occasionally also used for coverings. due to the risk of ppv transmission via ai semen, sows were vaccinated against this virus and due to conventional handling of feed and straw sows were also vaccinated against the bacteria erysipelothrix rhusiopatie. the herd is tested free from a number of pathogens (wallgren and vallga Êrda, ; wallgren et al., ) and the overall pathogen load is considered to be low indicated by a high weight gain, a low incidence of medical treatments and a low mortality (wallgren, (wallgren, , . in this farrow to ®nish herd comprised sows and the production system was continuous, but with batchwise farrowings. during the herd increased to sows and an age segregating system was introduced. the herd subsequently increased further and since it has comprised around sows. reproductive parameters in the spf-herd were documented since its establishment. repeat breeders within an interval of ± days were de®ned as having a normal oestrous cycle, whereas returns at other intervals were regarded as abnormal. due to the housing of dry sows, untied in deep litter, aborted foetuses are seldom discovered and therefore no distinction between abortion and non-pregnant in late gestation was made. piglet mortality was expressed as deaths pre-weaning (days ± ) and post-weaning (days ± , corresponding to kg body weight). the present study comprised a retrospective survey of serum samples from pigs in the spf-herd during the period ± and from ai boars servicing the herd in (table ). in addition, a closer survey was performed on the ®rst sows to farrow after the initial observation of ee among piglets, i.e., weeks after the outbreak. three sows (nos. , aged year; , aged years and , aged years) and their offspring were monitored clinically and with serum samples. piglets in litter were numbered ± ; litter , ± and litter , ± , however, piglets nos. and were the offspring of sow no. and received the spf-herd is free from the following exotic micro-organisms: african swine fever virus, aujeszky's disease virus, foot and mouth disease virus, hog cholera virus, japanese b encephalitis virus, porcine epidemic diarrhoea virus, prrsv, rabies virus, swine vesicular disease virus, transmissible gastro-enteritis virus and brucella abortus, and the indigenous: mycoplasma hyopneumoniae, actionobacillus pleuropneumoniae, toxin producing strains of pasteurella multocida, brachyspira spp., leptospira spp., haematopinus suis, salmonella spp., mycobacterium spp., porcine respiratory coronavirus, swine influenza virus and sarcoptes scabiei. colostrum from this sow. sows and piglets were sampled days after farrowing (day ) and piglets were sampled daily from the day of the ®rst clinical signs of ee (day ) until day . seven of these piglets remained in herd at the age of weeks and were sampled at that age. skin swabs for isolation of s. hyicus were collected from sows ± and their offspring on day . in addition, samples were collected from piglets with and without clinical signs between days and . all sampled pigs, irrespective of symptoms, were swabbed on an area of  cm on at least three different sites. the ®bre swabs were placed in modi®ed stuart medium for transport (culturette ). samples were cultured within hours of sampling on blood-agar ( % horse blood, v/v) and on a selective and elective medium modi®ed after devriese ( ) . brie¯y, the latter medium consisted of a blood-agar base (oxoid no. ) supplemented with polymyxin b (sigma chemicals), mg/l, polysorbate , ml/l, and cacl Á h o, mg/l. both agar plates were incubated for h at c. colonies with morphology consistent with s. hyicus were subcultured and tentatively identi®ed according to barrow and feltham ( ) . all serum samples were analysed for presence of antibodies to pcv- by elisa (walker et al., ) and indirect immuno-¯uorescence (iif) technique (allan et al., ) was also applied to some samples. a sonicated preparation of pcv- infected cells was used as antigen in the elisa and results were expressed as percent inhibition (pi), where < % was considered negative, > % considered positive and ± % considered table description of animals included in the retrospective survey a for antibodies to pvc- in the spf-herd intermediate. the iif technique detects antibodies binding to a pcv- infected cell-line and the results were graded as: () strong positive to () weak positive and (À) indicating a negative result. serum samples collected from animal nos. ± were also analysed for presence of antibodies to ppv, using a competitive elisa technique (svanovir ppv-ab, svanova biotech, uppsala, sweden). ifn-a was detected with a delfia technique described by artursson et al. ( ) . in this assay the detection of ! . units ifn-a/ml serum was considered a positive result. il- activity was measured in a bioassay using the murine b cell-line as described by fossum et al. ( ) . serial dilutions of a porcine recombinant il- preparation (endogen, woburn, ma, usa) was included on every test plate and used as positive control. some surviving pigs from the litters affected by ee were kept under spf conditions and included in another study which also comprised pigs born in the spf-herd the following year, i.e., (wattrang et al., ) . cryopreserved (liquid n ) samples of lymphnodes collected at euthanasia, from one of the ee pigs at months of age and from one pig born in aged months, were used for detection of pcv- dna and virus isolation according to (allan et al. ( c) . in april , piglets in two litters in the same farrowing batch, unexpectedly displayed exudative skin disease with blackening crusts, i.e., signs of ee. both dams of the affected litters were years of age and the litters were fairly big, and piglets, respectively. most piglets in the litters were affected by clinical ee and were treated with antibiotics (fucidic acid locally; fucidin, leo, denmark, and/or penicillin parentally, penovet, boehringer ingelheim, germany). however, due to the ee and four piglets, respectively, died before weaning in these litters. the ®rst batch of sows to farrow after this outbreak was therefore monitored closely for clinical signs of ee, and samples collected, as described below. four sows were due to farrow in the monitored batch (see above), one gave birth to three stillborn piglets while the other sows gave birth to four (sow ) and (sows and ) live piglets, respectively. when the piglets in the three remaining litters were days of age, blackening crusts where noticed on the abdomen of piglet no. in litter . this initiated the close survey of the piglets regarding general health and monitoring of clinical signs of ee (table ). piglet no. had clinical ee and was treated with penicillin from the age of days (after sample collection) until remission days later. none of the other piglets monitored showed clinical ee, but on close examination some piglets showed de®ned red erythemas on the abdomen. when piglets showed very small or unde®ned reddish skin lesions, these were recorded as tendencies to/suspected erythemas. in total, piglets displayed de®ned erythemas during the study, two in litter , ®ve in litter and eight in litter . apart from the skin symptoms all piglets were generally bright and healthy apart from piglet no. which showed lethargy and pyrexia on day and piglet no. which was treated with penicillin for suspected septic arthritis on day . ± ± * ± ± ? ? ± ± ± ± ± ± ± a ee: exudative epidermitis (clinical); : defined erythemas on abdomen; (): remission of erythemas; ?: tendencies to/suspected erythemas and ±: no clinical sings. b piglet nos. and were the offspring of sow no. . * indicates samples collected for isolation of s. hyicus. the bacteria were demonstrated in all of these animals. among the swab samples collected when the piglets were days old, s. hyicus was isolated from at least one of the sites sampled on each of the sows as well as on each of their offspring. over the -week period when the piglets were closely monitored for signs of ee, piglets displaying varying degrees of clinical signs and four piglets with no clinical signs, were swabbed for bacteriology on one or more occasions (in total sets of swabs, table ). s. hyicus was isolated from all of these individuals regardless of time point or clinical signs. thus, the bacteria were present on the skin of all pigs prior to the onset of ee and no clear correlation to clinical signs could be made. all tested serum samples from the spf-herd collected in , and february , i.e., up to months prior to the ee outbreak, were negative for antibodies to pcv- both in elisa n and iif (n , february samples). at weeks of age, piglets in one of the litters initially affected by ee were either clearly sero-negative or intermediate for antibodies to pcv- (pi ranging ± %, n out of survivors). pigs in the second of these litters were all sero-positive to pcv- (pi ranging ± %, n out of survivors). at this time point, other young pigs in the herd ranged from intermediate to sero-positive (pi ± %, n ). thus, after being sero-negative to pcv- earlier in , pigs in the spf-herd had varying levels of antibodies to this virus weeks after the ®rst outbreak of ee. further, all other serum samples collected from pigs in the spf-herd after april , i.e., august ± n were positive for antibodies to pcv- with titers ranging from : to : , (median : ). among the adult animals, the three sows farrowing weeks after the ®rst outbreak of ee showed different levels of antibodies to pcv- at farrowing. sow no. was negative both in elisa and in iif (pi %, iif À), sow no. showed a strong positive reaction (pi %, iif ), and sow no. a weak positive reaction (pi %, iif ). the boars at the ai-station servicing the spf-herd were all clearly sero-positive to pcv- (pi ± %) at this time point. the serological status to pcv- among the dams was also re¯ected by the transfer of maternal antibodies to their offspring (table ). in litter , the offspring of sow no. were all sero-negative to pcv- at days of age, while the offspring of sow no. raised in this litter (piglet nos. and ) showed the same high antibody levels as their siblings in litter . the offspring of sow no. remained sero-negative at least until the age of days but were strongly sero-positive to pcv- at the age of weeks. the offspring of sow no. were all strongly sero-positive to pcv- both during suckling and at the age of weeks. piglets in litter were also sero-positive to pcv- at days of age but had lower levels of antibodies compared to the offspring of sow no. . the maternal antibodies to pcv- in litter quickly declined and by the age of days all but one of the piglets were sero-negative. however, at the age of weeks pigs from litter were again strongly sero-positive to pcv- . thus, these observations show that maternal antibodies to pcv- were transferred to the offspring. the seven piglets remaining in the herd until weeks of age were all strongly sero-positive to pcv- at that time, regardless of their antibody status during their new-born period. taken together, these results point to an introduction of infectious pcv- into the herd in and a subsequent persistence of the virus in the herd. among the piglets tested for antibodies to ppv, all were sero-positive to this virus at three days of age. the offspring of sows nos. and all had similar levels of antibodies to ppv at this time point (a : : ae : , n ), while the offspring of sow no. showed lower antibody levels (a : : ae : , n ). the ppv antibodies then constantly declined at days and for all of the animals. at weeks of age, the seven pigs remaining in the herd were all sero-negative to ppv. thus, transfer of ppv antibodies induced by vaccination of dams to the piglets occurred but there were no indications of an active infection with ppv in the spf-herd. na na na na na na na a antibodies to pcv- were detected with elisa (expressed as pi where < % is negative, > % is positive) and indirect immuno-fluorescence (iif; () strong positive to () weak positive and (À) negative), for details, see section . na: no sample available. b piglet nos. and were the offspring of, and received colostrum from, sow no. . a for details on the definition of production and fertility parameters, see section . due to the sale of pregnant animals the number of matings may occasionally exceed the number of farrowings. b the results for are given quarterly. ifn-a in serum was used as an indicator of ongoing viral infections and serum il- as an indicator of acute bacterial infections. the piglets were tested at days of age and daily during the period of clinical signs of ee (days ± ). regarding serum ifn-a, none of the piglets in litter displayed this cytokine at any of the time points tested. in litter , four out of the eight piglets had ifn-a in serum at days of age, and three piglets had ifn-a in serum on one or more occasions during the rest of the study. all of the piglets in litter were positive for ifn-a in serum at days of age and ®ve of them also had ifn-a in serum on one or more occasions during the rest of the study. in total, out of the piglets in litters and were positive for ifn-a at days of age, one on day , one on day , ®ve on day , ®ve on day and three on day . the sows were tested for ifn-a in serum when the piglets were days old and sows nos. and were negative while sow no. showed . units ifn-a/ml serum. none of the piglets displayed il- in serum at any of the time points tested. pcv- immuno-histochemistry was negative for the virus when applied on the cryopreserved lymphnodes. however, using pcr technique, the sample from the ee affected pig was positive for pcv- dna. pcv- was also isolated at the third passage of tissue homogenates from the ee pig through cell cultures. the sample from the pig born in was negative for pcv- in all tests. reproduction parameters and pre-weaning piglet mortality for ± are summarised in table . during this time period no piglet mortality was observed in postweaning. with some exceptions during the years immediately after the establishment of the herd, piglet production remained at an even and high level with approximately . weaned piglets per litter. the percentage of small litters (< piglets) and repeat breeders were also on a fairly low and uniform level. during the second quarter of pre-weaning piglet mortality increased due to the ee outbreak. the number of stillborn piglets and the percentage of small litters were also unusually high during this quarter. further, aborting sows and sows found non-pregnant during late stage gestation was increased for animals covered during the ®rst three quarters of . despite a previous normal fertility of the heard teaser boar, none of the sows covered by him during the ®rst two quarters of n farrowed. taken together, these results are indicative of a fertility disturbance occurring around the second quarter of . an unexpected outbreak of ee in a spf-herd initiated an extensive survey into possible causes of the disease. the causative agent of ee, i.e., s. hyicus, is a natural part of the porcine skin¯ora (aarestrup and wegener, ) and was indeed also present in this spf-herd. it is well known that even virulent strains of s. hyicus may be present on healthy as well as diseased pigs (devriese, ; tanabe et al., ; wegener and skov-jensen, ) . it is, however, also believed that predisposing factors causing skin trauma, disrupting the epidermis and exposing the dermis to s. hyicus, are needed for the development of ee (aarestrup and wegener, ; wegener and skov-jensen, ) . among viruses, ppv has been put forward as causing skin lesions (kresse et al., ; choi et al., ; whitaker et al., ) , but this virus was not present in the herd in question. interestingly, pcv- has also been associated with skin lesions in the porcine dermatitis and nephropathy syndrome (allan et al., b; rosell et al., ; thomson et al., ) . the pcv- serology revealed that the spf-herd was sero-negative at least until february . two months lather, around the ee outbreak, the pigs showed varying levels of antibodies to pcv- . all the monitored piglets sero-converted later that year and the herd has remained sero-positive since. the serological evidence thus reveal an active infection with pcv- . further, the presence of serum ifn-a in the young pigs also indicates viral activity amid the piglets. we therefore hypothesise that pcv- replicating in the naõ Ève piglets caused the skin lesions necessary for s. hyicus to gain access to the dermis, and thereby predisposed for the ee outbreak. indeed, pcv- was also isolated from one of the ee affected piglets which is the ®rst time this virus has been isolated in sweden. moreover, the introduction of pcv- in the herd coincided with the most pronounced reproductive disturbance ever recorded in the herd. indeed, pcv- has been associated with reproductive problems (west et al., ) , and the observed increases in number of stillborn piglets, percentage of small litters and repeat breeders may therefore have been caused by the introduction of the virus into the herd. further, it is likely that the fertility problems were due to pcv- infection of naõ Ève or insuf®ciently immune pregnant sows and gilts as this type of reproductive signs at least became less evident when pcv- became established in the herd. the spf-herd is closed to live animals, geographically isolated and practices strict management routines. therefore, the sero-positive boars at the ai-station point out a plausible route of how pcv- was introduced into the herd. indeed, pcv- dna has been detected in semen of experimentally infected boars (larochelle et al., ) . further, transmission of virus via semen has been shown for chicken anaemia virus (hoop, ) which is another circoviridae family member. no cases of pmws have to date been reported in sweden. this disease has been closely linked to pcv- , but it has also been emphasised that other pig husbandry practices and/or infectious agents such as ppv or pprs may be required for a full expression of the syndrome . the present spf-herd is free from both ppv and prrs, and the overall pathogen load is low. this may well explain why the presence of pcv- in this spf-herd has passed relatively unnoticed. taken together, these data indicate that the introduction of pcv- into the spf-herd coincided in time with the ee outbreak. hence, this newly introduced virus may well have been the predisposing factor initiating the s. hyicus skin lesions. j. vallga Êrda for their kind co-operation as well as i.-l. wile Ân and l. fuxler for their skilled technical assistance. association between production of fibrinolysin and virulence of staphylococcus hyicus in relation to exudative epidermitis in pigs production, preliminary characterisation and applications of monoclonal antibodies to porcine circovirus isolation and characterisation of circoviruses from pigs with vasting syndromes in spain, denmark and northern ireland immunostimulation, pcv- and pmws pcv- -associated pdns in northern ireland in a sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation staphylococcus hyicus-skin reactions in piglets caused by crude extracellular products and by partially purified exfoliative toxin interferon-a production and tissue localisation of interferon-a/b producing cells after intradermal administration of aujeszky's disease virusinfected cells in pigs cowan and steele's manual for the identification of medical bacteria pathogenicity of a skin isolate of porcine parvovirus in swine fetuses porcine postweaning multisystemic wasting syndrome in korean pig: detection of porcine circovirus- infection by immunohistochemistry and polymerase chain reaction genetic characterization of type porcine circovirus (pcv- ) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of north america and development of a differential pcr-restriction fragment length polymorphism assay to detect and differentiate between infections with pcv- and pcv- evaluation of various cytokines (il- , ifn-a, ifn-g, tnf-a) as markers for acute bacterial infection in swineÐa possible role for serum interleukin- transmission of chicken anaemia virus with semen activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus (pcv- ) parvovirus infection in pigs with necrotic and vesicle-like lesions pcr detection and evidence of shedding of porcine circovirus type in boar semen porcine circovirus characterisation of pcv- isolates from spain retrospective study of porcine circovirus- infection in japan: seven cases in identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of staphylococcus hyicus pdns, pmws and porcine circovirus type in scotland development and application of a competitive elisa for the detection of serum antibodies to porcine circovirus type the importance of diseases for daily growth of pigs ethical, ecological and economical aspects on diseases among pigs in sweden (etiska, ekologiska och ekonomiska synpunkter pa Ê sjuklighet bland grisar i sverige) spf-pigsÐpresentation, definition and specification of regulations (serogrisenÐpresentation, definition och kravlista) experimental infections with actinobacillus pleuropneumoniae in pigs. i. comparison of five different parenteral antibiotic treatments tissue chambersÐa useful model for in vivo studies of cytokine production in the pig exudative epidermitis isolation and characterization of porcine circovirus type from pigs showing signs of postweaning multisystemic wasting syndrome in the netherlands myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus parvovirus infection in pigs with exudative skin disease this study was supported by grants from the swedish council for forestry and agricultural research. the authors wish to thank p. so Èderstro Èm, s. johansson and key: cord- -n joeda authors: chang, long; yuan, weifeng; zhu, liqian title: β-cantenin is potentially involved in the regulation of c-jun signaling following bovine herpesvirus infection date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: n joeda c-jun, activated by various extracellular signals, is important for cell differentiation, proliferation, apoptosis, and inflammatory responses. we have previously reported that bovine herpesvirus (bohv- ) infection in mdbk cells stimulates the c-jun nh -terminal kinase (jnk)/c-jun cascade for efficient replication. however, the mechanisms regarding the regulation of c-jun following bohv- infection remain unknown. in this study, we show that virus infection increases accumulation of p-c-jun(s ) (phosphorylated c-jun at ser ) and p-β-catenin(s ) in the nucleus, resulting in relocalized nuclear p-c-jun(s ) to assemble in highlighted punctum via a confocal microscope assay. an association between β-catenin and c-jun in the nucleus was readily detected in virus-infected, but not mock-infected cells. interestingly, β-catenin was found to be involved in the regulation of c-jun signaling in virus-infected cells as icrt , a β-catenin-specific inhibitor that can inhibit β-catenin-dependent transcriptional activity, was able to decrease protein expression and phosphorylation of c-jun. furthermore, we suggest that bohv- infection stimulates c-jun phosphorylation regulated by β-catenin via both c-jun nh -terminal kinase (jnk)-dependent and jnk-independent mechanisms. these data add to our knowledge regarding the regulation of c-jun following virus infection and further support the important roles of β-catenin signaling playing in bohv- infection. similar to herpes simplex virus (hsv- ), bovine herpesvirus (bohv- ) is an enveloped dna virus which belongs to the family herpesviridae and the subfamily alphaherpesvirinae (muylkens et al., ; tikoo et al., ) . bohv- can infect cattle of all ages and breeds. acute infection of cattle with bohv- generally results in inflammatory responses in distinct tissues, including the upper respiratory tract, nasal cavity, and ocular cavity, and leads to erosions in the mucosal surface (hodgson et al., ) . bohv-s infection suppresses the immune response, which may result in secondary infection by other pathogens, such as bovine viral diarrhea viruses (bvdv), bovine respiratory syncytial virus (brsv), parainfluenza- virus (pi v) and bovine coronaviruses, and bacteria including mannheimia haemolytica and pasteurella multocida, ultimately leading to life-threatening pneumonia known as bovine respiratory disease complex (brdc) (fulton et al., ; muylkens et al., ; tikoo et al., ) . bohv- is regarded as a critical co-factor for brdc development (neibergs et al., ) . it has been suggested that bohv- infection costs the us cattle industry approximately billion dollars in losses annually (jones and chowdhury, ) . j o u r n a l p r e -p r o o f as an activator protein (ap- ) family member, c-jun plays essential roles in multiple cellular processes, including cell proliferation, survival, and death (angel and karin, ; jochum et al., ; kovary and bravo, ) . c-jun can be activated by various extracellular signals such as growth factors, cytokines, and extracellular stresses, typically through a serine/threonine kinase c-jun nh -terminal kinase (jnk) (davis, ; liu et al., ; yung and giacca, ) . studies have indicated that c-jun facilitates the production of numerous inflammatory cytokines and chemokines. for example, c-jun facilitates h n influenza virus replication in human lung epithelial cells and expression of cytokines including tumor necrosis factor (tnf)-α, interferon (ifn)-β, interleukin (il)- , and il- (xie et al., ) . additionally, c-jun is able to mediate inflammatory responses triggered by these cytokines, resulting in establishment of a potential feedback loop to amplify inflammatory effects following virus infection (riesenberg et al., ; schonthaler et al., ) . targeting c-jun is regarded as an effective avenue for therapeutic interventions of inflammatory diseases. as such, studying the interplay between c-jun and bohv- infection is important to understand mechanisms of virus pathology. it has been reported that c-jun-dependent trans-activation supports hsv- replication in cell cultures (mclean and bachenheimer, ) . similarly, bohv- infection activates the jnk/c-jun cascade, with inhibition of this pathway via the chemical inhibitor sp significantly blocking productive infection in cell cultures (zhu et al., ) . however, how c-jun signaling is affected by virus infection remains poorly understood. j o u r n a l p r e -p r o o f the wnt/β-catenin signaling pathway is required for the regulation of cell proliferation, cell survival, development and tissue regeneration (clevers and nusse, ; gough, ) . furthermore, it can both regulate the production of multiple inflammatory cytokines, and be activated by inflammatory cytokines to form a feedback loop (silva-garcia et al., ; yang et al., ) . the β-catenin signaling pathway is additionally activated to support bohv- latent infection in sensory neurons and productive infection in cell culture (liu et al., ; workman et al., ; zhu and jones, ; zhu et al., a; zhu et al., b) . in quiescent cells, β-catenin in the cytoplasm is constitutively associated with adenoma polyposis coli (apc), axin, glycogen synthase kinase β (gsk- β), and casein kinase i, resulting in the assembly of a β-catenin destruction complex, which leads to polyubiquitination of β-catenin and degradation by the proteasome (clevers and nusse, ) . upon activation by wnt, the intracellular phosphoprotein disheveled (dvl) is activated, causing the disassociation of the β-catenin destruction complex. the activated β-catenin is stabilized and accumulates in nucleus, where it interacts with members of the t-cell factor (tcf) family of dna-binding proteins specifically bound to the consensus site agatcaagg (behrens et al., ; van de wetering et al., ) . the binding of β-catenin to tcf family members displaces the bound corepressors and recruits coactivators such as cyclic amp-response element-binding protein (creb)-binding protein (cbp) and its close relative p to the carboxy-terminal transactivation domain of β-catenin to activate wnt target genes (clevers and nusse, ; mosimann et al., ). c-jun is able to heterodimerize j o u r n a l p r e -p r o o f with creb family members such as activating transcription factor- (atf ) and forms a complex which subsequently binds to cyclic amp-responsive element (cre), to drive cre-dependent transcription (angel and karin, ; mclean and bachenheimer, ) . it is likely that creb/cre connects the interplay between c-jun and β-catenin. c-jun promoter can be activated by c-jun itself because this promoter also contains cre sites (gupta et al., ; kappelmann et al., ; karin et al., ; mclean and bachenheimer, ) . importantly, it has been reported that nuclear dvl, c-jun, β-catenin, and tcf form a complex on the promoters of wnt target genes and regulate gene transcription by stabilization of β-catenin-tcf interactions (gan et al., ) , providing evidence that c-jun physically associates with β-catenin and regulates β-catenin-dependent transcription. however, whether β-catenin has an effect on c-jun expression remains unknown. here, we hypothesized that β-catenin is involved in the regulation of c-jun expression during bohv- infection in vitro. in this study, we report that bohv- infection stabilized the association between β-catenin and c-jun in mdbk cells, and that association was readily detected in the j o u r n a l p r e -p r o o f madin-darby bovine kidney (mdbk) cells (purchased from chinese model culture preservation center, shanghai, china) were cultured in dmem containing % fetal bovine serum (fbs). bohv- (nj- - isolated from bovine semen samples (zhu et al., c) was propagated in mdbk cells. aliquots of virus stocks were titered in mdbk cells and stored at - °c. the following chemical reagents were used in this study: icrt (medchemexpress, cat# hy ), sp (cell signaling technology, cat# ). the following antibodies were used in this study: phospho(p)-c-jun (ser ) rabbit . goat anti-bohv- serum was purchased from vmdr inc (cat# pab-ibr). mdbk cells were seeded into mm dishes and cultured overnight. cell cultures were treated with either dmso vehicle or icrt at a concentration of μm for hour at o c in a humidified incubator with % co . cells were infected with bohv- (moi = . ) for hour in the presence of the chemicals indicated. after washing three times with pbs, fresh medium containing either dmso or icrt was replaced. at hours post infection (hpi), cell lysates were prepared using lysis buffer ( % triton x- , mm sodium chloride, mm edta, mm egta, mm sodium fluoride, mm sodium pyrophosphate, mm phenylmethylsulfonyl fluoride, . g/ml leupeptin, mm benzamidine, and mm sodium orthovanadate in mm tris-hcl, ph . ). after centrifugation at , rpm for min at °c, clarified supernatants were collected and boiled together with laemmli sample buffer for min; samples were subsequently separated by % or % sds-page and proteins were transferred onto pvdf membranes (bio-rad, cat# ). after blocking with % nonfat milk in pbs for hour (h) at room temperature, the membranes were incubated with primary antibodies diluted in % bovine serum albumin in pbs, overnight at °c. after extensive washing with pbst ( . % tween- in pbs), membranes were incubated with either anti-rabbit or anti-mouse secondary antibodies for hour at room temperature. after extensive washing with pbst, protein bands were developed onto film by using clarity western ecl substrate (bio-rad, cat# ). bohv- (moi = . ) for hour. after washing three times with pbs, fresh medium was replaced. at hpi, cells were lysed with μl of ripa buffer ( x pbs, % np- , . % sodium deoxycholate, . % sds) supplemented with the aforementioned protease inhibitors. cell lysates were then clarified by centrifugation at , rpm for minutes and incubated with dynabeads protein a beads (life technologies, cat# d), which were precoated with primary antibodies or isotype igg by incubation for h at room temperature with rotation. after overnight incubation at °c with rotation, beads were collected using a magnet (dynamag™) (life technologies, cat# d). after three washings with pbs, beads were boiled in sds-loading buffer and western blots were performed using the indicated antibodies. mdbk cells seeded into -well chamber slides (nunc inc., il, usa) were mock infected or infected with bohv- (moi = . ) for h. cells were fixed with % paraformaldehyde in pbs for min at room temperature, permeabilized with . % triton x- in pbs for min at room temperature, and blocked with % bsa in pbst for h followed by incubation with the antibodies indicated in % bsa in pbst overnight at °c. after three washings, cells were incubated with alexa fluor ®-conjugated goat anti-rabbit igg (h+l) (invitrogen, cat# a- , : dilution) for h in the dark at room temperature. after three washings, dapi j o u r n a l p r e -p r o o f ( ′, -diamidino- -phenylindole) staining was performed to visualize nuclei. slides were covered with coverslips by using antifade mounting medium (electron microscopy sciences, cat# - - ). images were captured using a confocal microscope (leica). phosphorylation of β-catenin at ser (s ) increases transcriptional activity (fang et al., ; hino et al., ; taurin et al., ) . c-jun protein has a c-terminal dna-binding domain and an n-terminal transactivation domain. the phosphorylation of serine (s ) in the transactivation domain is closely associated with its transcriptional activity (pulverer et al., ; zhu et al., ) . to address our hypothesis, we initially investigated the effects of bohv- infection on the accumulation of β-catenin, p-β-catenin(s ), c-jun, and p-c-jun(s ) in the cytosol and nucleus. mdbk cells were mock infected or virus-infected for h, after which fractions of both cytosol and nucleus were isolated by using a commercial kit, and subjected to western blotting. we found that relative to the mock infected control, higher levels of p-β-catenin(s ) were readily detected in virus infected cells (whole cell extracts) ( figure a ), as well as in fractions of both the cytosol ( figure a ) and nucleus ( figure c ). steady state β-catenin protein expression was decreased in whole cell extracts ( figure b ) and cell fractions of both cytosol ( figure b ) and nucleus ( figure d ) following virus infection. in addition, lamina/c in the cytosol j o u r n a l p r e -p r o o f fraction and tubulin in the nucleus fraction were rarely detected, indicating that these cellular fractions were not contaminated ( figure e) . the host-shutoff effects of bohv- at late stages of virus infection may account for the decreased protein levels of β-catenin. increased levels of p-β-catenin(s ) were not due to the increased protein expression of total β-catenin. p-c-jun(s ) was readily detected at higher levels in whole cell extracts ( figure a ) and the nucleus fractions following virus infection ( figure c ), but not in the cytosol fractions of either mock-infected or virus-infected cells ( figure a ). c-jun was detected at higher levels in whole cell extracts ( figure b ) and cell fractions of both cytosol ( figure b ) and nucleus ( figure d ) following virus infection. increased c-jun steady state protein expression may partially account for the increased phosphorylation levels of c-jun. to validate the association between virus infection and these identified modulations in protein expression and cellular relocalization, the protein expression of virion-associated proteins was detected by western blot. as can be seen in figure f , three bands indicative of virion-associated proteins were readily detected after infection for hours. collectively, these data suggest that bohv- infection activates β-catenin and c-jun, and promotes nuclear accumulation of these phosphorylated proteins. since virus infection promoted nucleus accumulation of both p-β-catenin(s ) and p-c-jun(s ), we next investigated if they were relocated. ifa assay indicated that following virus infection, β-catenin(s ) partly translocated into the nucleus, in j o u r n a l p r e -p r o o f agreement with results obtained via western blot ( figure c ). in the mock infected cells, pronounced staining of p-β-catenin(s ) was mainly located at sites of cell-cell contacts, with faint staining observed in the nucleus ( figure a ). p-β-catenin(s ) was readily detected in virus infected cells, and highlighted staining was located at sites of cell-cell contacts ( figure b ). interestingly, following virus infection p-c-jun(s ) predominantly located in the nucleus, and clearly formed foci which was not readily observed in uninfected cells ( figure c it has been reported that nuclear c-jun and β-catenin form a complex in hek t cells (gan et al., ) . to detect whether this association was affected by bohv- infection, immunoperecipitation (ip) was performed by using antibody against c-jun and β-catenin, respectively. when c-jun was immunoprecipitated from whole cell lysates, β-catenin was consistently detected, but levels of β-catenin were reduced in precipitates from bohv- -infected cells in comparison to that of uninfected control ( figure a, left panel) . vice versa, c-jun could be detected in immunoprecipitates when ip was performed by using β-catenin antibody ( figure a after infection for hours, the activated molecules of both p-c-jun(s ) and p-β-catenin(s ) were promoted to translocate into the nucleus (figure ) . we further investigated whether the association between jun and β-catenin could be detected in the nucleus, by performing ip assays using nucleus fractions. these j o u r n a l p r e -p r o o f experiments revealed that β-catenin could be clearly detected in the c-jun immune precipitates from virus infected nucleus fractions but not in those of the uninfected nucleus (figure ) , suggesting that the activated β-catenin was also associated with the activated c-jun in the nucleus. it is likely that undetectable associations between β-catenin and c-jun in the mock-infected nucleus are attributed to the relatively lower levels of c-jun that are nearly undetectable in the uninfected nucleus fractions via western blotting (figure ). taken together, the association between β-catenin and c-jun was maintained in the bohv- -infected nucleus, which further confirms the physical interaction between β-catenin and c-jun, providing supporting evidence that β-catenin may affect c-jun signaling. in order to understand the effects that β-catenin had on the activation of c-jun signaling stimulated by bohv- infection, we detected the protein levels of both c-jun and p-c-jun(s ) in the presence of β-catenin-specific inhibitor icrt via western blot. using this approach, we found that c-jun protein expression levels were reduced by icrt either in the presence or absence of virus infection in comparison to the individual control ( figure a ). it is known that icrt inhibits β-catenin-dependent transcription by interfering with the interaction between β-catenin and tcf family members (gonsalves et al., ; zhu et al., a) . it has been reported that icrt blocks β-catenin-dependent transcription stimulated by bohv- infection (zhu et al., a) . as such, it is reasonable that icrt reduces j o u r n a l p r e -p r o o f c-jun protein expression by disruption of β-catenin-dependent transcriptional activity essential for c-jun transcription. however, we found that the phosphorylation of c-jun at ser was significantly inhibited by the treatment of icrt ( μm) in the absence or presence of virus infection ( figure b ). this was unexpected because broad spectrum anti-kinase activity of icrt has not been documented, and was not associated with β-catenin phosphorylation ( figure c ). icrt at a concentration of μm was not cytotoxic in uninfected cell cultures analyzed by trypan-blue exclusion test as described elsewhere (fiorito et al., ) ( figure d ). when cell morphology was examined, µm icrt reduced the effects of virus infection-induced cytopathology ( figure e ), suggesting that treatment with µm icrt did not exert cytotoxicity in virus-infected cells. collectively, these data indicate that the observed inhibitory effects of icrt on either c-jun expression or phosphorylation in the virus infected cells was not due to cytotoxicity effects. taken together, we suggest a role for β-catenin in the activation of c-jun signaling pathway stimulated during late-stage bohv- infection. jnk is a canonical upstream activator of c-jun phosphorylation. to understand how c-jun signaling was inhibited by icrt , the effects of icrt on jnk signaling activity were investigated. there are three jnk genes (jnk , jnk and jnk ), each of which undergoes alternative splicing, resulting in numerous isoforms, which allows for different jnk activities in specific tissue types (gupta et al., ; j o u r n a l p r e -p r o o f kyriakis and avruch, ) . two isoforms of jnk, jnk-p and jnk-p , could be detected with the antibody used in this study. treatment with icrt led to the depletion of jnk-p expression in uninfected cells but not in virus-infected cells ( figure a ). phosphorylation of both jnk-p and jnk-p at thr /tyr stimulated by virus infection was blocked by icrt in cell cultures ( figure b ), corroborating our results that icrt inhibited the phosphorylation of c-jun stimulated by virus infection. jnk specific inhibitor sp could efficiently block jnk phosphorylation. to validate the inhibitory effects of icrt on jnk phosphorylation, virus-infected cells were treated with either icrt or sp , and the phosphorylation of c-jun was detected by western blotting. we found that the phosphorylation of both jnk-p and jnk-p stimulated by bohv- infection could be blocked by both icrt ( μm) and sp ( μm) ( figure c ). in addition, the phosphorylation of c-jun at ser was also inhibited by both icrt and sp with distinct intensity ( figure d ). as determined by a trypan-blue exclusion text, sp at a concentration of μm was not cytotoxic to uninfected cell cultures ( figure e ). when cell morphology was examined, treatment with μm sp reduced the effects of virus infection-induced cytopathology ( figure f ). as such, it was unlikely that the observed inhibitory effects of sp on the phosphorylation of jnk and c-jun in the virus-infected cells was due to cytotoxicity effects. of note, even though both sp and icrt possessed a similar capacity to depress jnk phosphorylation, higher levels of p-c-jun(s ) were still observed following sp treatment, but not following icrt treatment ( figure d ). therefore, icrt may inhibit c-jun phosphorylation with either jnk-dependent or jnk-independent mechanisms. taken together, these data suggest that in bohv- -infected mdbk cells, β-catenin is potentially involved in the phosphorylation of c-jun signaling partially through the activation of jnk. c-jun, a known transcriptional activator, regulates gene expression via binding to a cre motif in the promoter region of targeted genes, which is implicated in a diversity of virus infections. for instance, the increased phosphorylation of c-jun in promoter via a cre motif, which is important for ebv lytic infection (feng et al., ) . the binding of c-jun to the u region of long terminal repeats (ltr) in human foamy virus (hfv) maintains the optimal activity of the hfv promoter (maurer et al., ) . c-jun together with another ap- family member, c-fos, binds to early and late promoters of human polyomavirus jc (jcv), resulting in the activation of these promoters. consequently, the phosphorylation and protein expression of c-jun are substantially increased at the late phases of jcv infection cycles (sadowska et al., ) . these reports suggest a possible versatile mechanism whereby c-jun directly associates with the viral genome to regulate viral gene expression. c-jun has been shown previously to physically bind to the consensus sequence ( '-tgac/gtca- ' and '-tgacgtca- ') in k cells (li et al., ). an analysis of the bohv- j o u r n a l p r e -p r o o f genome sequence (acc. no. aj . ) indicated that it contains three potential c-jun binding sites, located at - nt, - nt and - nt, respectively (data not shown). here we found that nuclear p-c-jun(s ) was relocalized following bohv- infection, and formed numerous c-jun foci ( figure b) , reminiscent of the virus replication compartment as characterized in hsv- by either bromodeoxyuridine (brdu)-pulse-labeled dna synthesis initiation sites or icp staining (xu and roizman, ; zhong and hayward, ) . it is possible that c-jun can bind to the bohv- genome in the virus replication compartment to regulate virus replication and gene transcription, which is an interesting question that warrants further study. apart from the canonical stimulator jnk, c-jun can be activated by various extracellular signals such as growth factors, cytokines and extracellular stresses, employing diverse mechanisms (davis, ; liu et al., ; yung and giacca, ) . for example, c-jun can be activated by the protein kinase c (pkc)/p mapk cascade in a jnk-independent manner, in human primary t lymphocytes (humar et al., ) . epidermal growth factor (egf) induces c-jun expression via both extracellular signal-regulated kinases / (erk / ) and erk in mouse embryonic fibroblasts (mefs) (kayahara et al., ) . radiation-induced c-jun activation depends on the mitogen-activated protein kinase kinase (mek )-erk / signaling pathway in microglial cells (deng et al., ) . hepatitis c virus infection stimulates c-jun signaling via protein kinase r to promote proliferation of hepatocellular carcinomas (watanabe et al., ) . we have previously shown that bohv- infection stimulates the jnk/c-jun cascade in growth arrested mdbk cells for efficient replication (zhu et al., ) . in this study, we showed that β-catenin was involved in the regulation of c-jun expression following bohv- infection in mdbk cells ( figure a ), which provides a novel mechanism underlying the regulation of the c-jun pathway. moreover, our data show that β-catenin is potentially involved in the phosphorylation of both jnk and c-jun stimulated by virus infection (figure b and b) . broad spectrum anti-kinase activity of icrt has not been documented, and in this study it did not appear to modulate the phosphorylation of either β-catenin itself ( figure c ) or plc-γ (data not shown). to our knowledge, similar anti-phosphorylation activity of icrt has not be documented previously. while the precise mechanism of how the β-catenin inhibitor icrt depresses phosphorylation of jnk/c-jun stimulated by virus infection remains unclear which need further study in the future, these data nonetheless extend our understanding on the biological functions of β-catenin signaling pathway. it is well known that activated β-catenin is transported into the nucleus to activate β-catenin-dependent transcription (herbst et al., ) . here, we found that accumulation of nuclear p-β-catenin(s ) was increased following bohv- infection ( figure c ), which corroborates a previous report that bohv- infection enhanced β-catenin-dependent transcriptional activity (zhu et al., a) . since c-jun transcription is autoregulated by its own product (angel et al., ) , the physical association between β-catenin and c-jun in the bohv- -infected nucleus (figure ) provides a possibility for increased c-jun transcriptional activity by β-catenin, as (figure , b and d) . we noticed that in our previous report that bohv- infection lead to an increased phosphorylation of c-jun, consistent with our data demonstrated here ( figure a) , while increased expression of c-jun was not observed (zhu et al., ) . in that study, mdbk cells were of different origination, cultured in horse serum, and subjected to serum starvation overnight before infection with a high moi of . in this study, mdbk cells were cultured in fetal bovine serum without serum starvation and infected at a moi of . . it is highly possible that the distinct cell origins, different serum used for cell culture, and with or without serum starvation contributed to this discrepancy in results. taken together, in this study for the first time we show that in bohv- -infected mdbk cells, β-catenin was associated with c-jun, β-catenin was involved in regulation of c-jun expression and phosphorylation, and nucleus c-jun was relocalized to form highlighted foci in an ifa assay. this study expands our understanding of how c-jun signaling is regulated in response to bohv- infection, and provides a novel cross talk between β-catenin and c-jun, which further addresses the implications of β-catenin in bohv- infection and pathogenesis. the jun proto-oncogene is positively autoregulated by its product the role of jun, fos and the ap- complex in cell-proliferation and transformation functional interaction of beta-catenin with the transcription factor lef- beta catenin-independent activation of myod in presomitic mesoderm requires pkc and depends on pax transcriptional activity wnt/beta-catenin signaling and disease signal transduction by the jnk group of map kinases an rnai-based chemical genetic screen identifies three small-molecule inhibitors of the wnt/wingless signaling pathway focus issue: wnt and beta-catenin signaling in development and disease selective interaction of jnk protein kinase isoforms with transcription factors transcription factor atf regulation by the jnk signal transduction pathway comprehensive analysis of beta-catenin target genes in colorectal carcinoma cell lines with deregulated wnt/beta-catenin signaling phosphorylation of beta-catenin by cyclic amp-dependent protein kinase stabilizes beta-catenin regulation of wnt target gene activation bovine herpesvirus infection and infectious bovine rhinotracheitis susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves phosphorylation of c-jun mediated by map kinases mitf and c-jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment regulation of human polyomavirus jc virus gene transcription by ap- in glial cells akt and bmpr , are expressed at higher levels during latency regulatory roles of c-jun in h n influenza virus replication and host inflammation the sp component of nd enhances accumulation of pml and suppresses replication and the assembly of hsv replication compartments activation of the wnt/beta-catenin pathway by an inflammatory microenvironment affects the myogenic differentiation capacity of human laryngeal mucosa mesenchymal stromal cells role of c-jun n-terminal kinase (jnk) in obesity and type diabetes assembly of complete, functionally active herpes simplex virus dna replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays the canonical wnt/beta-catenin signaling pathway stimulates herpes simplex virus productive infection the beta-catenin signaling pathway stimulates bovine herpesvirus productive infection potential role for a beta-catenin coactivator (high-mobility group at-hook protein) during the latency-reactivation cycle of bovine herpesvirus first report of bovine herpesvirus isolation from bull semen samples in china the activation of p mapk and jnk pathways in bovine herpesvirus infected mdbk cells. veterinary research , . j o u r n a l p r e -p r o o f icrt ( um) or sp ( um) for hours, after which they were mock infected or infected with bohv- at an moi of . in the presence of indicated inhibitors or dmso control. after infection for hours the cell lysates were prepared to detect both after a pretreatment for hours with sp ( um), mdbk cells were mock infected or infected with bohv- (moi = . ) in the presence of either sp or dmso control for hours, and cell morphology was observed under a light microscope lc generated the figures, wfy performed data analysis. lqz designed and supported this study and prepared the manuscript. the authors declare no potential conflicts of interest. key: cord- - fxfmn i authors: chang, ji-tao; li, xin; liu, hai-jun; yu, li title: ovine rotavirus strain llr- -based bovine rotavirus candidate vaccines: construction, characterization and immunogenicity evaluation date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: fxfmn i group a bovine rotaviruses (brvs) are the most important cause of diarrheal diseases in neonatal calves and cause significant morbidity and mortality in the young animals, and epidemiologic surveillance of bovine rotavirus g genotypes conducted in various cattle populations throughout the world has shown that approximately % of the bovine rotavirus isolates belong to g and g . based on the modified jennerian approach to immunization, we constructed and characterized a reassortant rotavirus stain, which bears a single bovine rotavirus vp gene encoding g genotype specificity while the remaining genes are derived from the ovine attenuated rotavirus llr- . the reassortant rotavirus strain, named as r , and its parental virus strain llr- were combined as bivalent vaccine candidates to inoculate the colostrums-deprived neonatal calves for evaluation of the immunogenicity. the calves were orally inoculated with the reassortant r (group ), the parental rotavirus llr- (group ), or combined the r and llr- (group ), and serum specimens were detected to determine the immune response of igg and iga antibodies. results showed that seroconversion to positivity for igg and iga antibodies occurred at postinoculation day (pid) in all of the inoculated calves, and the highest titers of the serum igg (range : to : ) and iga (range : to : ) antibodies were obtained at pid for all calves. meanwhile, virus shedding was detected after inoculation, showing that the inoculated virus was positive in of fecal specimens ( . %) collected from the inoculated calves during the first days of oral inoculation with the rotavirus vaccine candidates. the results suggested that the rotavirus strains r and llr- are promising bivalent vaccine candidates for the prevention of bovine g and g rotavirus infection. the rotaviruses (rvs) compose a genus within the family reoviridae, and comprise seven distinct groups (a-g), of which groups a-c rotaviruses are those currently found in both humans and animals, whereas viruses in groups d-g have been found only in animals to date (knipe and peter, ) . the rotavirus genome is composed of segments of double-stranded rna and can undergo genetic reassortment during mixed infections, leading to progeny viruses with novel or atypical phenotypes. there were numerous descriptions of rotavirus strains isolated from human and animals that share genetic and antigenic features of viruses from heterologous species (matthijnssens et al., ; rahman et al., ; santos and hoshino, ) . recently, there were reports of novel rotaviruses that are apparently derived from transmission between human and sheep (ciarlet et al., ; matthijnssens et al., ) . the emergence of novel strains derived from interspecies transmission has implications for design and implementation of successful reassortant rotavirus vaccine strategies. group a bovine rotaviruses (brvs) are the most important cause of diarrheal diseases in neonatal calves and cause significant morbidity and mortality in the young animals, and epidemiologic surveillance of bovine rotavirus g genotypes conducted in various cattle populations throughout the world has shown that approximately % of the bovine rotavirus isolates belong to g and g . based on the modified jennerian approach to immunization, we constructed and characterized a reassortant rotavirus stain, which bears a single bovine rotavirus vp gene encoding g genotype specificity while the remaining genes are derived from the ovine attenuated rotavirus llr- . the reassortant rotavirus strain, named as r , and its parental virus strain llr- were combined as bivalent vaccine candidates to inoculate the colostrums-deprived neonatal calves for evaluation of the immunogenicity. the calves were orally inoculated with the reassortant r (group ), the parental rotavirus llr- (group ), or combined the r and llr- (group ), and serum specimens were detected to determine the immune response of igg and iga antibodies. results showed that seroconversion to positivity for igg and iga antibodies occurred at postinoculation day (pid) in all of the inoculated calves, and the highest titers of the serum igg (range : to : ) and iga (range : to : ) antibodies were obtained at pid for all calves. meanwhile, virus shedding was detected after inoculation, showing that the inoculated virus was positive in of fecal specimens ( . %) collected from the inoculated calves during the first days of oral inoculation with the rotavirus vaccine candidates. the results suggested that the rotavirus strains r and llr- are promising bivalent vaccine candidates for the prevention of bovine g and g rotavirus infection. ß elsevier b.v. all rights reserved. in prior studies, a dual classification system based on the two major outer capsid proteins defines several g and p genotypes of group a rotaviruses. to date, g and p genotypes have been identified based on the glycosylated vp and the protease-sensitive vp (schumann et al., ; ursu et al., ) . recently, a novel classification system based on the nucleotide sequences of all rotavirus genome segments has been suggested (matthijnssens et al., a,b) enabling a comprehensive characterization of rotavirus strains. based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. the calculated cut-off values for the rotavirus rna segments vp , vp , vp , vp , vp , vp , nsp , nsp , nsp , nsp and nsp / are %, %, %, %, %, %, %, %, %, % and %, respectively. to designate the complete genetic makeup of a virus, the schematic nomenclature was proposed: gx-p[x]-ix-rx-cx-mx-ax-nx-tx-ex-hx, representing the genotypes of respectively the vp -vp -vp -vp -vp -vp -nsp -nsp -nsp -nsp -nsp / encoding gene segments, with x indicating the number of the corresponding genotypes. group a rotaviruses, which are transmitted by the fecal-oral route and selectively infect the mature villous absorptive epithelial cells (holland, ) , cause disease that varies in severity, including asymptomatic infection, mild and self-limiting diarrhea, or severe diarrhea with excessive fluid loss and severe electrolyte imbalance. diarrhea is one of the most important diseases of neonatal dairy and beef calves, and substantial economic loss occurs due to increased morbidity and mortality, treatment costs, and reduced growth rates (house, ) . for example, livestock and poultry production with $ billion per year industry in the united states is estimated to suffer a - % annual loss in potential productivity from disease and environmental problems (united states department of agriculture, ) , in which, calf loss from diarrhea is an important segment of total loss in cattle industry. it was reported in the late s that enteric pathogens killed up to % of calves per year, resulting in more than $ million in losses (hunt, ) . rotavirus, coronavirus, escherichia coli strain k , coccidia and cryptosporidium parvum are the main infectious agents inducing enteric infections in neonatal calves less than months of age. among the pathogens described above, rotavirus as a major cause of neonatal bovine diarrhea (bellinzoni et al., ; bendali et al., ; de la fuente et al., ; maes et al., ; reynolds et al., ) was found to be responsible for approximately % of the scours cases in dairy calves (snodgrass et al., ) . in addition to causing economic losses, diarrhea in livestock is important because of the public health implications. thus, the availability of a safe and effective bovine rotavirus vaccine capable of preventing this enormous economic and public health burden would represent a global goal. the advent in of the first licensed human rotavirus vaccine, a rrv (rhesus rotavirus) -based quadrivalent vaccine (rotashield tm , wyeth-lederle vaccines and pediatrics, philadelphia, pa) (kapikian, ) , provided an impetus for the expansion of programs of global human rotavirus strain surveillance and assessment of the rotavirus disease burden. in addition, availability in the early s of a reliable and relatively easy methodology for rotavirus g and p genotyping accelerated this trend (das et al., ; fischer and gentsch, ; gentsch et al., ; gouvea et al., ) . such information has indeed influenced the approaches to the development of an effective rotavirus vaccine. for example, additional candidate human rotavirus vaccines have been constructed that would give antigenic coverage not only for g -g but also g , g and g - as well as p a[ ], p b[ ] and p a[ ] . however, there was limited research in the field of animal rotavirus vaccine. the bovine rotavirus isolate (the lincoln strain, g genotype) was adapted to serial propagation in cell culture (fernelius et al., ; mebus et al., ) , which resulted in an attenuated virus for calves (mebus et al., ) . this attenuated strain was incorporated into a vaccine licensed by the us department of agriculture (usda) in for oral inoculation of newborn calves or intramuscular inoculation of pregnant cows to provide passive protection to their calves. apart from this, there was no advent of new bovine rotavirus vaccine in the world. field surveys have demonstrated that, so far, g genotype g and g are the epidemiologically most important bovine rotavirus genotypes worldwide, accounting for % of the rotavirus-caused diarrhea approximately. next to the g and g genotypes, g is the third typical bovine rotavirus genotype (alfieri et al., ; chang et al., ; falcone et al., ; garaicoechea et al., ; monini et al., ; parwani et al., ; reidy et al., ; snodgrass et al., ) . in china, our epidemiologic surveillance has shown that rotavirus diarrhea is one of the most important diseases of neonatal calves, and that g and g were also determined to be the most prevalent genotype of the bovine rotaviruses using nested rt-pcr and sequencing (chang et al., ) . since monovalent rotavirus vaccine cannot completely protect against heterologous rotavirus infection (feng et al., ) , the multivalent rotavirus vaccine candidates are the most important research goal for the protective immunity of rotaviruses. in addition, the reassortant rotavirus vaccine approaches have been successfully employed to develop various human rotavirus candidate vaccines that include rhesus rotavirus based and bovine rotavirus based multivalent vaccines listed earlier (hoshino et al., (hoshino et al., , midthun et al., midthun et al., , . in this study, an ovine rotavirus strain llr- -based bovine rotavirus reassortant was constructed and combined with its parental virus llr- as bivalent vaccine candidates for providing an attenuation phenotype of an ovine rotavirus in bovines and antigenic coverage of the main bovine rotavirus genotypes for g and g . our study showed that the bivalent vaccine candidates can induce immune response of igg and iga antibodies in the sera of the inoculated colostrum-deprived neonatal calves, and fecal shedding of rotavirus is uncommon following oral inoculation of calves with the rotavirus vaccine candidates. the bovine rotavirus strain ncdv (g p[ ]), which was originally isolated in the stool of a neonatal calf with diarrhea in nebraska, usa, in (fernelius et al., , was obtained commercially from china institute of veterinary drug control, beijing. the ovine rotavirus strain llr- (g p[ ]) (chen et al., ; knipe and peter, ) , which was initially derived from a lamb with diarrhea in qinghai province, china, in (zhou et al., , was propagated from monovalent human rotavirus vaccine llr (lanzhou institute of biological products, china) (bai et al., ) . these virus strains were adapted to growth in ma cell line, and passaged times on dmem medium (gibco) before a triple plaque purification. colostrum-deprived newborn calves without antibodies to rotavirus were divided into three groups to orally inoculate candidate vaccines within h after their birth. the calves were kept separate from each group and fed the substituted milk. primers for the amplification of gene segments vp , vp , nsp , nsp , nsp and nsp were synthesized (table ) based on the previous study (park et al., ) . rt-pcr was carried out with an initial reverse transcription step of min at c, followed by pcr for activation at c for min, cycles of amplification ( s at c, s at c, and s at c for vp , vp , nsp , and nsp and s at c, s at c, and s at c for nsp and nsp ), with a final extension of min at c. the pcr amplicons were purified with the gel extraction kit (tiangen biotech co., ltd., china) and sequenced by shanghai sangon biological engineering technology & services co., ltd., china. the sequences were analyzed with lasergene software, version . . rna polyacrylamide gel electrophoresis (page) was done as previously described (chudzio et al., ) . briefly, the supernatant of the culture lysates were mixed with an equal volume of rna extraction buffer ( . m trishydrochloride [ph . ], . m nacl, . m mgcl , . % sodium dodecyl sulfate, . m edta, % sucrose and . % bromophenol blue), and each preparation was mixed with an one-tenth volume of phenol-chloroform ( : ) and vortexed for s to yield a homogeneous suspension followed by spinning in a microcentrifuge at ,  g for min. the upper layer (dark blue solution) containing the double-stranded segments of rna was collected and stored at c or À c for electrophoretic analysis by the standard % polyacrylamide gel electrophoresis. genomic rnas were electrophoresed at ma for h and the resulting migration patterns were visualized by staining of gel with silver nitrate. confluent cell line of fetal rhesus monkey kidney, ma , was co-infected at a multiplicity of infection (m.o.i.) of one with bovine rotavirus strain ncdv and ovine rotavirus strain llr- . when approximately % of the infected cells exhibited cytopathic effects, the cultures were frozen and thawed three times and the lysate was plated on ma cells in a six-well plate for selection of the desired reassortant. each desired reassortant was plaque purified three times in ma cells and then its genomic rnas were analyzed by page to confirm the origin of genes of each reassortant. when the parental origin of a gene was questionable by page, the indistinguishable rna segments were analyzed by sequence analysis for confirmation. the plaque assay was performed with techniques similar to those of matsuno et al. ( ) . briefly, ma- cell monolayers were prepared in six-well tissue culture dishes and washed three times with pbs buffer. serial tenfold dilutions of virus prepared in dmem (without fbs) were inoculated in . -ml amounts. after h of adsorption at c, each culture received ml of agar overlay medium, which consisted of % purified agar in dmem containing mg of trypsin per ml. after the agar overlay solidified, the dishes were placed in an inverted position in a co incubator at c for - days. then an equal amount of a second agar overlay medium, containing % purified agar in dmem and . % neutral red was added, and plaques were counted the next day. two calves were orally inoculated with .  tcid of the attenuated llr- , two calves were orally inoculated with .  . tcid of the reassortant r , and seven calves were orally inoculated simultaneously with .  tcid of the attenuated llr- and .  . tcid of the reassortant r (table ) . rectal swab specimens were collected each day over the -day experimental period postinoculation to detect rotavirus. blood samples were collected at postinoculation days (pids) , , and , processed for serum, and kept at À c until they were tested. plates were coated overnight at c with semipurified brv or mock-infected cell culture supernatant in . m carbonate-bicarbonate buffer (ph . ), followed by a blocking step of h at c with % nonfat dry milk. the plates were washed three times with . % tween in pbs, ph . . serial twofold dilutions, starting at : , of each test sample were added to duplicate wells and incubated for h at c. after washing, the plates were incubated with horseradish peroxidase-conjugated monoclonal antibodies against bovine igg or iga (with dilution of : and : , respectively) for h at c. after washing, the plates were incubated with . % tmb ( , , , -tetramethylbenzidine) substrate for min at room temperature. the reaction was stopped with m h so . the a was read by using an elisa plate-reader (zenyth , hvd anthos). the elisa antibody titer of each sample was expressed as the reciprocal of the highest dilution that had a corrected a value (sample absorbance in the virus-coated well minus sample absorbance in the mock antigen-coated well) greater than the cut-off value (mean corrected a of negative controls + sd) (parreno et al., ; to et al., ) . in view of rotavirus genotypes g and g predominating in cattle worldwide, and the jennerian and modified jennerian approaches being successfully employed to develop various rotavirus candidate vaccines, we constructed g -based bovine rotavirus reassortant followed by molecular and biological characterization. ma cells were co-infected with bovine rotavirus strain ncdv and ovine rotavirus strain llr- at an m.o.i. of one, and the progeny viruses grew without selective pressure for generating resssortant. plaques were picked to screen for llr- -based reassortant with single ncdv-vp gene substitution. all plaques were routinely picked for initial screening by rna page analysis, and the indistinguishable rna segments were further analyzed by sequence analysis for confirmation. as indicated in fig. , electrophoretic migration patterns of genomic rnas of the parental viruses table inoculation of colostrums-deprived calves with rotavirus vaccine candidates and detection of rotavirus in the fecal specimens. calf no. inoculum postinoculation day on which rotavirus was detected r À À À À À À À À À À À À À À llr- À À À À À À À À À À À À À À llr- + r À À À À À À À À À À À À À À + À + À À À À À À À À À À À À À À À À À À À À À À À À À À À À À À À À ''À'' refers to being negative for rotavirus by rt-pcr. ''+'' refers to being positive for rotavirus by rt-pcr. . ) , indicating that the replication efficiency of the reassortant r in ma cells was not influenced by substitution of the vp gene. to evaluate the humoral immune responses induced by the oral administrations of the reassortant rotavirus r and one of its parental viruses llr- , the colostrumsdeprived newborn calves without antibodies to rotavirus were orally inoculated with the reassortant r and/or the parental virus llr- within h after their birth as described in table . two calves in the group were inoculated with the reassortant r , another two calves in the group were inoculated with the parental rotavirus llr- , and seven calves in the group were simultaneously inoculated with the reassortant r and the parental rotavirus llr- . blood samples were collected at pids , , and to prepare serum. all sera were diluted hundredfold to detect igg and iga antibodies to group a rotavirus by indirect elisa. the kinetics of rotavirus-specific igg and iga antibodies in sera of the calves inoculated with the rotavirus vaccine candidates were depicted in fig. . results showed that seroconversion of igg and iga antibodies to the rotavirus occurred at pid in all of the inoculated calves, and increased to the peak at pid . serum samples at pid in all of the calves were serially twofold diluted starting at a dilution of : followed by detection of the titers of rotavirus-specific igg and iga antibodies to group a rotavirus by indirect elisa. the results showed that the titers of serum igg antibodies were between : and : and the titers of serum iga antibodies were between : and : (table ) . in order to determine the profile of virus shedding after inoculation of the rotavirus vaccine candidates, fecal rotavirus shedding was investigated each day over the -day experimental period postinocuation. low-speed centrifugation supernatants of suspensions of rectal swab specimens were detected and typed by rt-pcr (gouvea et al., (gouvea et al., , , and results are described in table . results showed that in the three inoculated groups during the first days of oral inoculation with the rotavirus vaccine candidates, of fecal specimens ( . %) collected from the inoculated calves contained the inoculated rotavirus. the two rotavirus-positive specimens were collected from the same calf (no. ) in the group at the first and third day postinoculation. no inoculated rotavirus was detected in other fecal specimens collected from the inoculated calves between and days postinoculation. in prior study (theil and mccloskey, ) , rotavirus was detected in of daily fecal specimens collected from the calves following oral inoculation with a usda-licensed modified live bovine rotavirus-coronavirus vaccine. in our study, the low shedding rate ( / , . %) was similar to the result ( / , . %) of previous study, indicating that our vaccine candidates are safe and well tolerated in the colostrumsdeprived neonatal calves. in , a previously unrecognized virus recovered from diarrheic calves in nebraska, usa, was described and shown to induce diarrhea in experimentally inoculated, hysterectomy-derived, colostrum-deprived calves (mebus et al., ) . this virus was initially referred to as neonatal calf diarrhea virus, nebraska calf diarrhea virus, reo-like virus, or reovirus-like agent but is now known as bovine rotavirus; the name rotavirus was ultimately chosen because of the wheel-like appearance of the virus on transmission electron microscopy (tem). today rotaviral infections are proven to be a common, economically important cause of calf diarrhea throughout the world. genotypic analyses of a major neutralization protein vp of a bovine rotavirus in diarrheal stools collected in different cattle populations throughout the world have shown that the majority of typeable rotavirus isolates belong to g genotypes and (alfieri et al., ; chang et al., ; falcone et al., ; garaicoechea et al., ; monini et al., ; parwani et al., ; reidy et al., ; snodgrass et al., ) . since the first licensed bovine rotavirus vaccine was a monovalent one, monovalent rotavirus vaccine cannot completely protect against heterologous rotavirus infection (feng et al., ) . we constructed a reassortant rotavirus strain r that contains a single vp gene encoding genotype g specificity of bovine rotavirus ncdv strain and the remaining genes of ovine rotavirus strain llr- . the reassortant r and its parental rotavirus strain llr- were used as bivalent vaccine candidates to provide: (i) an attenuation phenotype of an ovine rotavirus in bovines; and (ii) antigenic coverage for g and g of the main bovine rotavirus genotypes. the attenuated virus strains r and llr- were shown experimentally in colostrum-deprived neonatal calves to be able to induce high levels of serum igg and iga antibodies to rotavirus, whereas fecal shedding of the rotaviruses was uncommon in the inoculated calves, and therefore are promising bivalent vaccine candidates for prevention of bovine g and g rotavirus infection. the benefits for using the llr- strain instead of an attenuated bovine rotavirus strain in production of the reassortant for vaccine purposes are that the former is attenuated in calves due to host range restriction, but the latter is often unstable and may develop back mutation, thus the llr- is more safe to calves; secondly, the passage history of the llr- is clear, it is an attenuated strain to lambs and children (bai et al., ) therefore should be safe to people and there is no public health problem; thirdly, the rna segments of the llr- are easily distinguished on the page electrophoretic migration pattern, which is useful to identify the reassortant; lastly and importantly, the llr- strain is highly immunogenic to neonatal calves. some nucleotide mutations of the vp , nsp , vp and nsp gene segments of the r occurred in the process of igg iga a the calves in the group were inoculated with the r , the calves in the group were inoculated with the llr- and the calves in the group were simultaneously inoculated with the r and llr- . generation and purification of the reassortant. there existed the insignificant differences of nucleotides (nt), nt, nt and nt between the r and the parental llr- for the vp , nsp , vp and nsp gene segments, respectively. obviously, the possible reasons to induce these nucleotide mutations were serial tissue culture passage for the six-time plaque purifications of the reassortant r on ma cells. the mechanisms responsible for immunity to rotavirus infections and illness are not completely understood in animals (knipe and peter, ) . animal models have been particularly instructive in elucidating the role of antibodies and in dissecting the relative importance of systemic and local immunity. initially, the observations of some related studies suggested that antibodies in the lumen of the small intestine were the primary determinant of resistance to rotavirus illness, whereas circulating serum antibodies failed to protect offit and clark, ; snodgrass and wells, ; woode et al., ) . but the subsequent study showed that systemic rotavirus antibodies are present in the lumen of the gastrointestinal tract of neonatal calves if the level of circulating antibodies is sufficiently high. such serum-derived mucosal antibodies can provide protection against experimentally induced infection and diarrhea (besser et al., ) . the association between the titers of serum anti-rotavirus igg and iga antibodies and protection against infection and illness was investigated in children under months of age attending day care centers in the united states for one or two rotavirus seasons (o'ryan et al., ) . these studies showed that preexisting serum anti-rotavirus iga titer of greater than : or igg titer of greater than : was associated with protection against infection. a similar study of mexican infants also demonstrated that both serum iga and igg were protective, but the titers needed to achieve significant protection were higher than those found in the us study (velazquez et al., ) . these previous studies about the association between serum anti-rotavirus antibodies and protection against infection and illness suggested that serum antibodies, if present at critical levels, are either protective themselves or are an important and powerful correlate of protection against rotavirus disease, even though other host effectors may play an important role as well. in this study, we want to evaluate the immunogenicity of the rotavirus vaccine candidates, so we used the major structural protein in virus particles of the semipurified brv as antigen to detect the igg and iga antibodies against the group a rotavirus in serum of the inoculated calves. and the results showed that the highest titers of serum igg and iga antibodies were obtained at pid , the calves in all inoculated groups developed high levels of serum anti-rotavirus igg antibodies (range : to : ) and serum anti-rotavirus iga antibodies (range : to : ). these results suggested that the animals inoculated with llr- -based vaccine candidates would develop antibodies to the inoculated viruses, and indicated that the attenuated virus strains llr- and r were well immunogenic to neonatal calves, and preexisting serum anti-rotavirus iga titers (range : to : ) reached to level of protective immunity. however, further evaluation is needed for a protective immune response of the llr- based candidate vaccines in future trials. to date, there is no criterion for fecal shedding rate of the rotavirus vaccine strains. in prior study, vaccine rotavirus was detected in only of daily fecal specimens collected from the calves following oral inoculation with a usda-licensed modified live bovine rotavirus-coronavirus vaccine by using negative stain electron microscopy. in contrast, rotavirus was demonstrable by the same negative stain electron microscopic examination procedure in of fecal specimens collected from the calves after inoculation with virulent bovine rotavirus field strains (theil and mccloskey, ) . in our study, vaccine rotavirus was detected in only of daily rectal swab specimens collected from neonatal calves following oral inoculation with the rotavirus vaccine candidates. this low shedding rate ( / , . %) was similar to the result of previous study ( / , . %), suggesting that the r and llr- are safe to calves based on the uncommon fecal shedding of the rotavirus. the procedure that we have used in the present study for the successful construction and selection of the single vp gene substitution reassortant rotavirus vaccine is relatively easy to perform. in recent years, rare genotypes of bovine rotavirus (such as g and g .) were also isolated in calves (ghosh et al., ; park et al., ) . so with this procedure, additional llr- -based reassortant rotavirus vaccine candidates that carry vp gene encoding a desired bovine rotavirus g genotype specificity can be generated readily in the future when the need for such g genotype(s) is warranted from epidemiologic studies. g and p genotypes of group a rotavirus strains circulating in calves in brazil selection and characterization of strain llr- for oral rotavirus live vaccine microbiology of diarrhoea in young beef and dairy calves in argentina pattern of diarrhoea in newborn beef calves in south-west france passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen neonatal calf diarrhoea: identification of a reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy etiologic investigation of bovine rotavirus in the some regions of china and isolation of a g p[ ] rotavirus the characterization of vp (g type) and vp (p type) genes of bovine group a rotaviruses from field samples using rt-pcr and rflp analysis whole genome sequencing of lamb rotavirus and comparative analysis with other mammalian rotaviruses rapid screening test for the diagnosis of rotavirus infection genomic characterization of a novel group a lamb rotavirus characterization of rotavirus strains from newborns in proportional morbidity rates of enteropathogens among diarrheic dairy calves in central spain determination of bovine rotavirus g and p serotypes in italy by pcr comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in nebraska. arch. gesamte virusforsch rotavirus typing methods and algorithms molecular characterization of bovine rotavirus circulating in beef and dairy herds in argentina during a -year period identification of group a rotavirus gene types by polymerase chain reaction molecular characterization of rare bovine group a rotavirus g p[ ] and g p[ ] strains from eastern india: identification of simian sa -like vp genes in g p[ ] strains polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens identification of bovine and porcine rotavirus g types by pcr some infectious causes of diarrhea in young farm animals generation and characterization of six single vp gene substitution reassortant rotavirus vaccine candidates: each bears a single human rotavirus vp gene encoding p serotype a[ ] or b[ ] and the remaining genes of rhesus monkey rotavirus mmu or bovine rotavirus uk construction and characterization of rhesus monkey rotavirus (mmu )-or bovine rotavirus (uk)-based serotype g , g , g or g single vp gene substitution reassortant candidate vaccines porcine rotavirus strain gottfried-based human rotavirus candidate vaccines: construction and characterization economic impact of rotavirus and other neonatal disease agents of animals symposium on calf diarrhea molecular characterization of vp genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation a rotavirus vaccine for prevention of severe diarrhoea of infants and young children: development, utilization and withdrawal fields virology. lippincott willians & wilkins nsp gene analysis of rotaviruses recovered from infected children with and without diarrhea evaluation of a human group a rotavirus assay for on-site detection of bovine rotavirus plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human sera 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 recommendations for the classification of group a rotaviruses using all genomic rna segments are human p[ ] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order artiodactyla? full genomic analysis of human rotavirus strain b and lapine rotavirus strain / provides evidence for interspecies transmission cell culture propagation of neonatal calf diarrhea (scours) virus calf diarrhea (scours): reproduced with a virus from a field outbreak immunity to neonatal calf diarrhea virus reassortant rotaviruses as potential live rotavirus vaccine candidates single gene substitution rotavirus reassortants containing the major neutralization protein (vp ) of human rotavirus serotype molecular characterization of bovine rotavirus strains circulating in northern italy anti-rotavirus g type-specific and isotype-specific antibodies in children with natural rotavirus infections protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies molecular characterization of novel g bovine rotavirus strains serum and intestinal isotype antibody responses to wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies characterization of field strains of group a bovine rotaviruses by using polymerase chain reaction-generated g and p type-specific cdna probes characterization of a novel p[ ],g human group a rotavirus molecular characterisation and analysis of bovine rotavirus strains circulating in ireland microbiology of calf diarrhoea in southern britain global distribution of rotavirus serotypes/ genotypes and its implication for the development and implementation of an effective rotavirus vaccine evidence of interspecies transmission and reassortment among avian group a rotaviruses rotavirus serotypes and predominate in cattle aetiology of diarrhoea in young calves rotavirus infection in lambs: studies on passive protection rotavirus shedding in feces of gnotobiotic calves orally inoculated with a commercial rotavirus-coronavirus vaccine serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease national animal health monitoring system. a concept for the future. usda, animal health information staff molecular analysis of the vp gene of pheasant rotaviruses identifies a new genotype, designated g molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including vp and nsp serum antibody as a marker of protection against natural rotavirus infection and disease levels of colostral antibodies against neonatal calf diaahoea virus diagnosis of diarrhoea in lamb caused by rotavirus infections this study was supported by the grant from national public welfare of china for agricultural special purpose (no. ). key: cord- - wj es authors: decaro, nicola; campolo, marco; lorusso, alessio; desario, costantina; mari, viviana; colaianni, maria loredana; elia, gabriella; martella, vito; buonavoglia, canio title: experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: wj es a pantropic canine coronavirus (ccov) strain (cb/ ) has been recently associated to a fatal outbreak of systemic disease in young dogs. we report the clinical, virological and serological findings in dogs experimentally infected with strain cb/ . the dogs, three . -month-old and two -month-old pups, were successfully infected, shedding viral rna with their faeces for the entire observation period ( days) and displaying systemic clinical signs resembling those observed during the course of natural infection. leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below % of the initial counts. considering the severity of the cb/ -induced disease, two of the youngest pups were euthanized for ethical reasons at days – postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. at postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for ccov by real-time rt-pcr and virus isolation on cell cultures. all pups seroconverted for ccov, as shown by the high optical density values and antibody titres detected by elisa and virusneutralisation tests, respectively. the present study confirms that strain cb/ is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions. coronaviruses (covs) are enveloped, singlestranded rna viruses which included in three different antigenic groups. covs infecting dogs comprise canine enteric coronavirus (ccov) (enjuanes et al., ) and www.elsevier.com/locate/vetmic available online at www.sciencedirect.com veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the newly recognised canine respiratory coronavirus (crcov) (erles et al., ; decaro et al., a) , belonging to group and group covs, respectively. two ccov genotypes have been identified so far, namely ccov type i and ccov type ii, which are responsible for the occurrence enteritis in dogs and are frequently associated in mixed infections decaro et al., c) . although its tropism is restricted to the gastroenteric tract, ccov has been recently associated to systemic disease followed by fatal outcome in pups . severe clinical signs were observed in the affected pups, whereas necropsy examination revealed remarkable gross lesions in lungs, liver, spleen and kidneys. virological and bacteriological investigations failed to detect common canine pathogens. unexpectedly, ccov type ii rna was detected at very high titres in the internal organs of the dead pups and the virus (strain cb/ ) was isolated on canine cell cultures. the association of strain cb/ to a severe, sometimes fatal disease of dogs, together with the isolation of the virus from organs with severe lesions, strongly suggests that ccov has changed its tropism, acquiring the ability to spread from the enteric tract to the internal organs (decaro et al., b) . in this study, we report the results of the experimental infection with isolate cb/ in pups with different age, showing that in contrast with classical ccovs, this virus is able to cause systemic disease followed by fatal outcome in younger pups. canine fibroma a- cells were grown in dulbecco's minimum essential medium supplemented with % foetal calf serum. strain cb/ was isolated from the lungs of a dead pup ( / -c) and adapted to growth on a- cells. at the rd passage, the virus was titrated on cell cultures and inocula containing . tcid /ml of viral suspension were stored at À c. contaminations by other canine pathogens, such as canine parvovirus type (cpv- ), canine distemper virus (cdv) and canine adenoviruses (cadvs), were ruled out by specific molecular assays (hu et al., ; decaro et al., b; elia et al., ) . the experimental study was performed according to the animal health and well-being regulations and was authorised by the ministry of health of italy (authorization no. / -c) . six mixed-bred female dogs including four . -month-old (n = - ) and two -month-old (n = , ) pups were housed at the ''infectious disease unit'' of the animal hospital, faculty of veterinary medicine of bari. the dogs had tested negative for ccov rna by a real-time rt-pcr assay carried out on the faeces and for ccov antibodies by an elisa test (pratelli et al., ) carried out on serum samples. all dogs were housed individually in separate boxes, fed twice daily with a commercial dry dog food and provided water ad libitum. after an acclimatization period of days, animals (n = , , , , ) were administered oronasally ml of a viral suspension of strain cb/ , with a titre of . tcid and .  rna copies per ml, whereas one dog (n = ), . -monthold, was maintained uninfected by oronasal administration of ml of sterile saline solution. the clinical condition of each dog was monitored daily for days. a scoring system was devised taking into account rectal temperatures, total white blood cell (wbc) counts, appearance of clinical signs (vomiting, diarrhoea, depression, loss of appetite, dehydration), following the scheme adopted in a previous study (decaro et al., a) and derived by nakamura et al. ( ) , with some modifications (table ). due to ethical reasons, dogs whose total clinical score reached a value ! were euthanized by intravenous administration of mg/ kg of body weight of zoletil (virbac s.r.l., italy) followed by . ml/kg body weight of tanax (intervet italia, italy). edta-treated blood samples were collected daily for total and differential wbc counting and for testing for ccov rnaemia by real-time rt-pcr . the presence of the viral rna in the blood was also evaluated at hours , , , and after inoculation. plasma samples were prepared daily to evaluate free viral rnaemia and weekly to determine ccovantibody titres by virus neutralisation (vn) and elisa tests (pratelli et al., ) . to evaluate the viral shedding in the faeces, the rectal swabs collected daily from the control dog and from the dogs inoculated with strain cb/ were subjected to rna extraction using qiaamp viral rna mini kit (qiagen s.p.a.). in addition, tissue samples from parenchymatous organs were withdrawn from the two dead pups (table ) . rna was extracted from the wbc pellets using qiaamp rna blood mini kit (qiagen s.p.a.), from the plasma samples using qiaamp viral rna mini kit and from the tissue samples using qiaamp rneasy mini kit. attempts to isolate the virus in a- cells were carried out on rectal swabs of all infected pups and on organs of the sacrificed animals as described previously (decaro et al., b) . real-time rt-pcr targeting the m gene of ccov type ii (genbank accession number d ) was carried out on the rna extracts as described elsewhere (decaro et al., c) . the genotypespecific rt-pcr assays were undertaken in an i-cycler iq tm real-time detection system (bio-rad laboratories srl, milan, italy) and the data were analyzed with the appropriate sequence detector software (version . ). after reverse transcription, triplicates of the ccov type ii standard dilutions and rna templates were simultaneously subjected to realtime analysis. the ml reaction mixture contained ml of iq tm supermix (bio-rad laboratories srl), nm of each primers ccovii-f (tagtgcat-taggaagaagct) and ccovii-r (agcaatttt-gaacccttc), nm of probe ccovii-pb (fam-cctcttgaaggtgtgcc-tamra) and ml of c-dna. the thermal profile consisted of activation of itaq dna polymerase at c for min, followed by cycles of denaturation at c for s, annealing at c (type ii-specific assay) for s and extension at c for min. plasma samples from inoculated dogs were tested in parallel by virus neutralisation (vn) and elisa tests (pratelli et al., ) . for vn test, duplicates of serial twofold dilutions of heat-inactivated plasmas (starting from dilution : ) were mixed with tcid of the isolated strain cb/ in -well microtitre plates. after preincubation at room temperature for min, , a- cells were added to each well. the plates were read after days of incubation at c. vn titres were calculated with the karber method and expressed as the highest plasma dilution that was able to neutralise the virus. for elisa, microtitre plates were coated with ccov antigen (enteric strain s- ) and, after treatment with blocking solution ( . % gelatin in carbonate buffer [ mm na co , mm nahco , ph . ]) and repeated washing, the : dilutions of the plasma samples were added to each well. then the plates were incubated for min at c, washed positive and negative controls were used as described previously (pratelli et al., ) . neither clinical signs or viral shedding were observed in the control dog, whose leukocyte counts did not draw away from the baseline values. all the inoculated animals displayed severe clinical signs similar to those observed in dogs infected naturally, although the outcome of the disease was different on the basis of the age (fig. ) . in fact, two out of the three youngest pups (dogs and ) had to be euthanized for ethical reasons, at days and postinfection (p.i.), respectively, while both the -month-old pups recovered from the disease, albeit very slowly. irrespective of the final outcome, i.e, euthanasia or recovery, clinical signs were remarkably similar in all inoculated animals. the . -month-old dogs that underwent a fatal outcome (fig. a) showed fever at days - p.i., with a peak of . c at day p.i. (dog ), and from days to p.i., with a peak of . c at day p.i. (dog ). depression (days - p.i.), anorexia/dysorexia (days - p.i.), haemorrhagic diarrhoea (days - p.i.) and vomiting (days - p.i.) also occurred. leukopenia appeared at day (dog ) or p.i. (dog ), with total wbc counts remaining below % of the baseline values until euthanasia. acute lymphopenia was observed in both dogs, with lymphocyte numbers dropping below % of the initial counts from day p.i. until death (mean, . %; .  lymphocytes/ ml at day p.i.). postmortem examination revealed severe changes in the intestines and major organs, which were very similar to those observed in dogs infected naturally (data not shown). the . -month-old dog that survived (fig. b ) displayed less severe symptoms, consisting of fever (up to . c) from days to p.i. and at days - p.i., and mucoid diarrhoea (days - p.i.). total wbc counts dropped below the % of the initial counts from days to p.i., whereas severe lymphopenia (below % of baseline values) was registered from days to p.i., with a peak at day ( %; .  lymphocytes/ml). subsequently, the total number of peripheral blood lymphocytes started to rise again and the clinical signs subsided. a mild loss of appetite occurred from days to p.i. in the two -month-old pups (fig. c) , fever showed a biphasic course, with a first peak at day p.i. of . and . c in dogs and , respectively. a transient remission was observed from days to p.i. (dog ) and at day p.i. (dog ), and a second episode of pyrexia occurred from days to p.i. with a peak of . c at day p.i. (dog ), and from days to . dogs inoculated oronasally with cb- strain were monitored for up to days for total wbc, lymphocyte and polymorphocyte counts (top graphs). in addition, fever, viral rna shed in faeces and clinical score where determined (bottom graphs). total wbc, lymphocyte and polymorphocyte counts are presented as percentages of the cell counts determined at day . viral rna titres as determined by real-time rt-pcr are expressed as log copy numbers (log ) per ml of template. clinical scores were calculated as shown in table and are reported for each day in correspondence of the temperature curves. p.i. with a peak of . c at day p.i. (dog ). depression was observed between days and p.i., whereas vomiting appeared only sporadically in the same period (days , , p.i. in dog ; days , , p.i. in dog ), with - episodes per day. both dogs displayed anorexia (days - p.i.), mucoid or fluid diarrhoea (days - ), and leucopenia, with wbc values dropping below % of the baseline from days to (dog ) or to p.i. (dog ). lymphocytes dropped below % of the initial cell counts from days to p.i. in dog and from days to p.i. in dog (mean, . %; .  lympholymphocytes/ml at day p.i.). starting from day (dog ) or p.i. (dog ), the clinical conditions of the dogs improved progressively, with a complete recovery at days - p.i. the control (uninfected) pup (fig. d ) remained in a good clinical status during the entire observation period and no variations in total wbc and lymphocyte counts were observed. the uninfected dog tested constantly negative for ccov rna. all the ccov-infected dogs tested negative for other common pathogens of dogs, including ccov type i (decaro et al., c) , cdv , cadvs (hu et al., ) and cpv- (decaro et al., b) . the faecal shedding of the infected dogs followed the similar pattern, although higher viral rna titres were obtained from the two sacrificed animals in comparison to survivors (fig. ) . the pups that succumbed shed virus starting between days and p.i. and lasting until the day of death, with a peak at day (titre of .  rna copy numbers/ml of template) or (titre of .  rna copy numbers/ ml of template). after their death, ccov type ii rna was detected in the organs at titres slightly lower than those observed in the dogs of the natural outbreak ( table ). the . -month-old pup that recovered shed ccov rna starting from day p.i. (titre of .  rna copy numbers/ml of template) and lasting for the entire observation period ( days), with a peak at day p.i. ( .  rna copy numbers/ml of template). shedding of ccov in the faeces of the two -month-old dogs was observed from day p.i. (mean titre, .  rna copy numbers/ ml of template) to day p.i. (last day of observation) reaching the maximal mean value of .  rna copy numbers/ml of template at day p.i. surprisingly, ccov rna was never detected in the blood of the -month-old pups, as well as in the euthanized animals, in whose organs remarkable viral rna titres were found. traces of ccov rna were detected only in the blood of the survived . -monthold pup between days and p.i., with plasma viral titres ranging from .  to .  rna copies/ml of template. the virus was successfully isolated on cell cultures from the rectal swabs of all inoculated dogs (data not shown) and from some organs of the euthanized pups ( table ) . all the infected animals seroconverted for ccov, whereas antibodies were not detected in the control dog (fig. d ). in the dogs that were euthanized the antibody titres were determined only at day p.i., with vn titres of : and od values of . (geometric means, fig. a ). in survivors, the maximal antibody titres were reached at days and p.i. by vn and elisa test, respectively ( fig. b and c) . in a previous study, a pantropic variant of ccov was associated to a fatal disease of dogs, characterised by leucopenia, gastroenteritis and severe changes in the internal organs . the disease induced by strain cb/ in pups of the natural outbreak was reproduced in dogs infected experimentally. all inoculated dogs were successfully infected, as shown by the occurrence of faecal shedding, seroconversion and severe clinical signs. the viral excretion was similar to those observed during enteric ccov infection (decaro et al., , c , but the course of disease was more severe, as clinical signs characteristic of systemic infection were observed in the infected dogs. the pantropism of the virus was confirmed by the presence of gross lesions in the internal organs of the dead dogs, as well as by the detection of viral rna in those tissues. interestingly, ccov rna was detected also in the brain of the dead dogs. in contrast, enteric ccov has been never associated to systemic infection, although the virus has been isolated previously from some tissues (tonsils, lungs and liver) of experimentally infected pups (tennant et al., ) . two of the three inoculated . -month-old pups had to be euthanized after few days of postinfection, whereas the other dogs (the remaining dog of the same age and the two dogs -month of age) recovered after a severe disease. considering that dogs infected naturally were all between and days of age, it could be hypothesised that the age of the infected animals plays a role in determining the fate of cb/ infection, with a very severe clinical course in the youngest pups. moreover, in the experimental infection, the organs of the dead dogs contained lower ccov rna titres with respect to dogs infected naturally, so that virus isolation was not obtained from all pcr-positive tissues. despite the drop of the wbc counts registered in all infected dogs and the detection of the viral rna in the internal organs of the sacrificed dogs, free or cell-associated ccov rnaemia was not found at any time either in euthanized or survived dogs, with the exception of the recovered . -month-old pup which showed very low rna viral titres in the plasma between days and p.i. thus, at this moment, the mechanisms of lymphopenia and viral spread to internal organs remained unknown. albeit strange, our findings are compatible with those obtained from cats experimentally infected with feline infectious peritonitis virus (fipv). cats succumbed to fipv display very high-viral titres in the haemolymphatic tissues (kipar et al., ) , in contrast with the low loads detected in the blood (de groot-mijnes et al., ) . in our study, cb/ -infected dogs were found to contain lower viral loads in the lymphoid tissues in comparison to fipv-infected cats, thus likely accounting for the undetected viral rnaemia in euthanized dogs. in conclusion, we have confirmed the pantropism of strain cb/ , reproducing the natural disease even in experimental conditions. further studies will contribute to better understand the epidemiological distribution and the pathogenetic mechanisms of the virus, including the possible involvement of the different lymphocyte classes. canine coronavirus highly pathogenic for dogs natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr maternally-derived antibodies in pups and protection from canine parvovirus infection a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs serological and molecular evidence that canine respiratory coronavirus is circulating in italy molecular characterisation of the virulent canine coronavirus cb/ strain detection of canine distemper virus in dogs by real-time rt-pcr family coronaviridae detection of a group coronavirus in dogs with canine infectious respiratory disease detection and differentiation of cav- and cav- by polymerase chain reaction natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats pathogenic potential of canine parvovirus types a and c in domestic cats prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of italy two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea canine coronavirus infection in the dog following oronasal inoculation key: cord- - h l nh authors: kuo, shu-ming; kao, hsiao-wei; hou, ming-hon; wang, ching-ho; lin, siou-hong; su, hong-lin title: evolution of infectious bronchitis virus in taiwan: positively selected sites in the nucleocapsid protein and their effects on rna-binding activity date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: h l nh rna recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (ibv), a highly contagious avian pathogen. we have demonstrated that rna recombination can give rise to a new viral population, supported by the finding that most isolated taiwanese (tw) ibvs, similar to chinese (ch) ibvs, exhibit a genetic rearrangement with the american (us) ibv at the ’ end of the nucleocapsid (n) gene. here, we further show that positive selection has occurred at two sites within the putative crossover region of the n-terminal domain (ntd) of the tw ibv n protein. based on the crystal structure of the ntd, the stereographic positions of both predicted selected sites do not fall close to the rna-binding groove. surprisingly, converting either of the two residues to the amino acid present in most ch ibvs resulted in significantly reduced affinity of the n protein for the synthetic rna repeats of the viral transcriptional regulatory sequence. these results suggest that modulating the amino acid residue at either selected site may alter the conformation of the n protein and affect the viral rna–n interaction. this study illustrates that the n protein of the current tw ibv variant has been shaped by both rna recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the rna-binding capacity of the n protein. among rna viruses, the coronavirus has the largest genome, consisting of a . kb, single-stranded, positivesense rna. the structural genes of the coronavirus genome encode spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins. frequent point mutations in the hypervariable regions of the spike (s ) gene contribute to most of the antigenic determinants of ibv. the n protein participates in binding to the viral rna genome and forms a ribonucleoprotein (rnp) complex. the n-and c-terminal domains (ntd and ctd) of the n protein are mainly involved in rna binding and oligomerization, respectively (jayaram et al., ) . recent evidence indicates that the ntd shows strong affinity for the transcriptional regulatory sequence (trs), which lies within the viral leader sequence and also precedes each viral open reading frame (spencer and hiscox, ) . this ntd-trs binding is critical for the control of viral life cycle, including viral packaging, genomic replication and viral transcription (grossoehme et al., ; hurst et al., ) . rna recombination has been shown to underlie the sporadic emergence of new variants of coronavirus, including the infectious bronchitis virus (ibv), a highly contagious avian pathogen. we have demonstrated that rna recombination can give rise to a new viral population, supported by the finding that most isolated taiwanese (tw) ibvs, similar to chinese (ch) ibvs, exhibit a genetic rearrangement with the american (us) ibv at the end of the nucleocapsid (n) gene. here, we further show that positive selection has occurred at two sites within the putative crossover region of the n-terminal domain (ntd) of the tw ibv n protein. based on the crystal structure of the ntd, the stereographic positions of both predicted selected sites do not fall close to the rna-binding groove. surprisingly, converting either of the two residues to the amino acid present in most ch ibvs resulted in significantly reduced affinity of the n protein for the synthetic rna repeats of the viral transcriptional regulatory sequence. these results suggest that modulating the amino acid residue at either selected site may alter the conformation of the n protein and affect the viral rna-n interaction. this study illustrates that the n protein of the current tw ibv variant has been shaped by both rna recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the rnabinding capacity of the n protein. ß elsevier b.v. all rights reserved. in addition to maintaining the viral rna in an ordered conformation for replication and transcription, the n protein is also involved in the regulation of cellular transcription, actin reorganization and apoptosis (kopecky-bromberg et al., ; surjit et al., ) . recently, an antibody against the n protein was used as a diagnostic marker of coronaviral infection (mourez et al., ) . moreover, immunization of the n protein alone can elicit sufficiently protective cellular immunity against lethal ibv challenges, indicating that the n protein is an immune-dominant antigen (cowley et al., ) . both genetic mutation and recombination contribute to the natural evolution of rna viruses. genetic variants attributed to rna recombination are often found in coronaviruses such as mouse hepatitis virus (mhv) (keck et al., ) and infectious bronchitis virus (ibv) (cavanagh, ; dolz et al., ) . ibv is an important chicken pathogen that causes severe economic losses for global poultry industry (cavanagh, ) . genetic recombination in the ibv s and n genes has been documented in different field isolates, based on sequence comparisons and phylogenetic incongruence (cavanagh, ) . the high frequency of rna recombination in coronaviruses is likely caused by their unique mechanism of rna synthesis, which involves discontinuous transcription and polymerase jumping (makino et al., ) . in addition to the sporadic generation of new variants, rna recombination may also give rise to new viral populations, as suggested by our study of taiwanese (tw) ibvs (kuo et al., ) . in general, tw ibvs are genetically closer to chinese (ch) isolates than to american (us) strains. the only exception is the n gene of the tw ibv, which shows higher similarity to that of us strains. in particular, recombination in the ntd of the n gene has been detected in most analyzed tw ibvs. this phylogenetic incongruence suggests that rna recombination can drive viral evolution at a population level. however, tw ibvs that carry the recombinant ntd may present the same antigenic epitopes, at least in part, as the us massachusetts-or connecticut-serotype vaccine strains, which are routinely administrated in chicken to control ibv in taiwan. this sequence rearrangement may therefore pose a survival disadvantage for the tw recombinants by increasing their susceptibility to vaccine-induced immune surveillance (chua et al., ) . whether this recombination event in the tw ibv enables the offspring of the recombinant founder to become dominant and evolutionally preserved is rather unclear. in this study, we show that tw ibv recombinants have undergone positive selection at two amino acids within the putative crossover region of the ntd. replacing either of these two residues with the amino acid present in most ch ibvs significantly attenuated the binding capacity of the n protein to synthetic rna repeats of the viral trs. these findings suggest that the fitness of recombinant tw ibvs has been increased by positive selection conferring a replication/ transcription advantage, even though these variants have been exposed to immune pressure in fowl vaccinated against us-like strains. detail information of the recruited ibv strains is provided in supplementary table (table s ) , including the genbank accession number and the place where the strain was isolated. we analyzed ibv strains, including tw strains, us strains and ch strains. the criteria of recruiting the ibv strains based on the decoded both full-length s and n genes. china sd strain is the only exception, whose s gene is still not sequenced. among the ibv strains, ibvs in the same clade of me tree in fig. b were chosen from each tw, ch and us groups for the bi analysis and positive selection. tw / and / were excluded because of their phylogenetic discordance in s and / was excluded due to the discordance in n. the length of the tested genes is all the same. notably, tw / is completely sequenced and is the most studied strain in taiwan (kuo et al., ) . phylogenetic trees based on me algorithms on the fulllength s and full-length n genes were analysis by mega . . bootstrap values, estimated from replicates of the me analysis, are given. for bi analysis, we compiled the n genes of various ibv strains, including a taiwanese group (tw / , tw / , tw / , tw / , tw / , tw / , tw - and tp/ ), an american group (mass , h , cal , cu-t , beaudette, gray, connecticut and arkdpi) and a chinese group (ldt , s , bj, lx , ck/ch/ltj/ i, ck/ch/lhb/ i, sh and sd ). multiple sequence alignment was performed using the clustalw program. the dna sequences were translated into amino acid sequences using the software dambe . . . phylogenetic trees were constructed with the amino acid sequences of the n genes by bi analysis. the best-fit models and parameters for initial settings of the phylogenetic programs were selected by prottest . . for bi algorithms on the basis of the bayesian information criterion. for the ibv n - data set, the best-fit model of substitution was the jtt model with a gamma substitution parameter of . . for the ibv n - data set, the best-fit model was the jtt model with a gamma substitution parameter of . . mrbayes . . was used for bi analysis. random starting trees were used. a total of million generations of markov chains were run. trees were saved every generations, resulting in , trees in the initial samples. the burn-in (number of initial trees that were discarded) was set to , . a majority-rule consensus tree was generated from the remaining samples ( , trees), and the percentage of samples recovering any particular clade represented the clade's posterior probability. the best-fit model of gtr + i + g was selected for bayesian analysis by the program of modeltest . using akaike information criterion. in the analyses, , , generations of markov chains were run. trees were saved every generations, yielding initial samples with a total size of , . the stationary phase of log-likelihood was reached within , generations. thus, burin-in (the numbers of the initial trees were discarded) was set to . majority rule consensus trees, constructed from the , remaining trees, were used to determine the posterior probabilities of each node. genetic recombination was evaluated by the recombination detection program (rdp) . and the genetic algorithms for recombination detection (gard) program to predict the putative cross-over region and single breakpoint position. detailed information was provided in our previous study (kuo et al., ) . based on bi analysis of the ibvs, variable selective pressures were evaluated at individual codon positions of the tw n protein. we applied paired models of variable d n / d s distribution among amino acid sites, including m (discrete) versus m (one ratio), m a (positive selection) versus m a (nearly neutral) and m (beta and d n /d s ) versus m (beta), in the codon-based phylogenetic models (codeml) program within phylogenetic analysis by maximum likelihood (paml) . the likelihood ratio test statistic was calculated as twice the log likelihood (l) difference between the two models and labeled as (l À l ) in table . the value was compared with a chisquare test with one degree of freedom (d.f.), which is equal to the difference in the number of free parameters between the compared models. a homology model of the ntd of the ibv n protein (tp/ ) was built using the automated mode of the swiss-model program (arnold et al., ) . we used the crystal structure of the ntd of the ibv beaudette strain (pdb no. btl) as a template to construct a plausible conformation. the deduced models and template were superimposed, showing the overall structure with root mean square deviations of $ . Å . model fidelity was confirmed using the procheck and adaptive poisson-boltzmann solver modules to examine the main-chain torsion angles and electrostatic distribution, respectively (laskowski et al., ) . the ibv n gene was cloned into pet- a(+) (merck-novagen, usa) via bamh i and xho i sites to synthesize a recombinant ibv n protein with a histidine tag. mutagenesis of n gene was performed according to the instructions provided with the quikchange site-directed mutagenesis kit (stratagene, usa). the sense oligonucleotides used for mutagenesis are as follows: -gat aat gaa aat ctt aaa cca agc cag cag cat gg- , thr to pro at aa ; -cct gat aat gaa aat ctt aaa aat agc cag cag cat gga tac tgg- , thr to asn at aa ; -gct gca aag ggt gct gat gtt aaa tct aga tct aat c- , thr to val at aa . these mutants were confirmed by nucleotide sequencing. the pet- plasmid containing the full-length n protein was transferred into e. coli bl (de ) competent cells (merck-novagen), and the transformed bacteria were selected and cultured at c in lb broth containing mg/ml kanamycin. n protein expression was induced by adding mm iptg for h when the od value reached . - . . to prevent protein degradation, the induction condition was carried out at c and supplemented with cocktailed protease inhibitors (sigma-aldrich). the cells were collected, suspended in lysis buffer ( mm sodium monobasic phosphate, mm sodium chloride, mm imidazole, mg/ml lysozyme) with protease inhibitor and then sonicated. the crude cell lysates were applied to resin affinity columns through a fast protein liquid chromatography (fplc) system (akta prime plus, amersham pharmacia) with buffer containing m urea. resinprepacked columns (amersham pharmacia) were equilibrated with a buffer consisting of . m nah po , . m tris-hcl, m urea, and mm imidazole (ph . ) and then washed with a buffer consisting of . m nah po , . m tris-hcl, m urea, and mm imidazole (ph . ). n protein fractions were eluted with a buffer containing . m nah po , . m tris-hcl, m urea, and mm imidazole (ph . ) at a flow rate of . ml/ min. purified proteins were further dialyzed and refolded with refolding buffer ( mm tris-hcl, mm nacl, . m l-arginine, mm edta, . mm phenylmethanesulfonyl fluoride, . mm oxidized glutathione, mm reduced glutathione, ph . ). samples were slowly refolded at c for four days as the urea concentration was lowered from m to m using a dialysis membrane with a molecular cutoff - kda (millipore). concentrations of the purified n proteins were determined by the bradford assay (bio-rad, usa). in addition, the n proteins were examined by % sds-page and stained with coomassie brilliant blue r (usb-affymetrix). the binding capacity of n proteins for viral rna was performed on a biacore a spr instrument (amersham pharmacia) equipped with a research-grade sensorchip sa . this apparatus measures binding capacity by monitoring changes in the refractive index of the sensor chip surface. these changes, recorded in resonance unites (ru), are assumed to be proportional to the mass of the molecules bound to the chip. oligomers of the repeated trs sequence, -(cuuaacaa) - , were synthesized using an automated rna synthesizer, labeled with biotin and purified by gel electrophoresis. the oligomer probes were manually immobilized to the streptavidin-coated biosensor chip. the purified n proteins were dissolved in a solution consisting of mm tris-hcl, mm nacl, mm edta, . mm oxidized glutathione, and mm reduced glutathione at ph . . the protein was applied to the chip surface at a flow rate of ml/min for s to reach equilibrium. before fitting to the : langmuir model, binding data were corrected by subtracting the control to account for simple refractive index differences. sensorgrams depicting interactions between rna and n proteins were obtained using bia evaluation software (version ). one-way analysis of variance (anova) was performed to determine significant differences in n protein rnabinding capacity in three independent experiments. the tukey's post hoc test was further used for multiple comparisons among the data collected for the wild type and variant n proteins. statistical significance was set at p < . . we analyzed ibv strains from taiwan, china and us in this study. the available full-length s and n gene sequences were the criteria for recruiting these strains (genbank, accessed on april th, ). based on a minimum evolution (me) analysis using mega . , the phylogenetic topology of the full-length s gene revealed that the most tested tw ibvs are similar to the ch ibvs (fig. a) . the tw / and tw / strains were the only exceptions, showing greater similarity to the us group. in contrast, analysis of the full-length n genes of the ibv strains by me ( fig. a and b) and baysian analyses (supplementary fig. s ) demonstrated that the tw ibvs are phylogenetically closer to the us ibvs than to the ch ibvs (fig. b) , indicating phylogenetic incongruence between the results for the s and n genes. twenty-four strains with complete sequences for the n gene were randomly chosen from the three tw, us and ch viral pools and subjected to phylogenetic analysis on the n protein. putative sporadic recombinant strains, such as tw / and tw / , are excluded to avoid misinterpretation of further analytic results. one candidate crossover region between the tw and us ibv strains is located between amino acid (aa) and of the n protein (kuo et al., ) , as predicted by the recombination detection program (rdp) (martin and rybicki, ) used in our previous study (fig. c) (kuo et al., ) . the p values of the rdp and bootscan algorithms for this recombination were .  À and .  À , respectively (kuo et al., ) . because aligning the n genes of severe acute respiratory syndrome (sars) virus, human coronavirus-oc (hcov-oc ) and mhv with ibv n sequences gives many gaps across these sequences, an ancestor strain as an outlier sequence was absent in fig. a and b (data not shown) . to further confirm our hypothesis of rna recombination, unrooted bayesian inference (bi) algorithm was applied in this study (fig. d and e) . bi analysis revealed that the putative crossover region (fig. d) in the tw n protein, but not the non-recombinant region (fig. e) , belongs to the monophyletic group of us n proteins. moreover, to demonstrate that the trees of fig. d and e are representative, we included all the strains in fig. b and constructed their bayesian trees (fig. s ) according to the two segments of n-terminal and c-terminal amino acid sequences of ibv n protein (fig. c) . the results in fig. s illustrated similar topologies as those in fig. d and e, strongly supporting the presence of rna recombination between the tw and us ibvs in the n gene sequence (fig. d and e and fig. s ). to further clarify whether the phylogenetic incongruence is caused by different evolutionary rates in different parts of the n gene, a ml test that is not biased by evolutionary rate variation was applied to recheck the phylogenetic relationships (holmes and rambaut, ) . no significant difference was observed between the results from the bi and those from the ml test (supplementary fig. s ), confirming that rna recombination probably occurred between tw and us ibvs in the -terminal region of the n gene and that the phylogenetic incongruence was not caused by point mutation or variation in local evolutionary rates. although the n protein is evolutionally conserved among ibvs, positive selection may occur at individual amino acid (aa) residues. to investigate this possibility, full-length n genes of the ibvs (shown in fig. d and e) were subjected to codon-based phylogenetic models (codeml) within the phylogenetic analysis by maximum likelihood (paml) programs (yang, ) . based on bayesian analyses, neutral and positive selection models were compared using likelihood ratio tests. the neutral models (m , m a and m ), selection models with a proportion of selected codons (m a and m ) and a model for d n /d s heterogeneity among aa residues (m ) were applied for tests of selective pressure on n protein residues. the log likelihood values (l n in table ) indicated that positive selection models (m a, m , m ) fitted the tested region better than neutral models (m , m a, m ). the nested comparisons between neutral and positive models, including m versus m (m /m ), m a/ m a and m /m , confirm the better fitness of positive models, suggesting that positive selection occurs at certain sites during the evolution of the tw n protein (p < . in these three comparisons, chi-square test) (table ). both aa and aa of the tw n protein were consistently highlighted by positive selection models (m a, m , m ) as putative selection sites with high posterior probability values (p a and p b values of both sites > . in m and m models, respectively) ( table ) . the detail profile of these two positively selected sites of all tested ibvs in this study was summarized in table . using the available database of fully sequenced n proteins ( ibv strains), we determined that thr residues are present at the aa and positions in . % ( / ) and . % ( / ) of the tw ibv strains, respectively (table ) . however, thr residues are present at the aa and positions in only . % ( / ) and . % ( / ) of the us ibv strains, respectively. in the ch ibvs, the prevalence of thr residues at these two positions is further reduced to . % ( / ) and . % ( / ), respectively. this information supports the codeml prediction that both positions have undergone positive selection in tw ibvs. to further demonstrate the result of positively selected residues in table ( ibv strains), ibv strains in table were recruited and few strains with partial sequences were excluded for codeml analysis. as shown in table s , both aa sites at and are predicted to be positively selected using m model (probability > . ), strengthening the finding of the occurrence of positive selection at aa and aa of n protein among tw ibvs. notably, the selected sites are located within the putative crossover region of the n protein in the tw ibvs (fig. c) , linking the rna recombination with the positive selection events. this observation also reflects the notion that particular residues of tw ibv recombinants have evolved under positive selection pressure in vaccinated flocks. in addition, the putative positively selected residues are located in the ntd, suggesting that viral progeny with strong rna-binding affinities may have been selected during the adaptive evolution of ibv in taiwan. the ntd of the ibv n protein participates in the binding to viral rna and the formation of the rnp complex. the crystal structure of the ntd, based on the beaudette strain, shows a u-shaped conformation composed of a fivestranded antiparallel b sheet with positively charged amino acids clustered throughout the groove (fan et al., ) . flexible loops and turns are around the inner core of the b sheet of the ntd. the positively selected sites, aa and , are located in the external a turn and loop, respectively (arrow heads, fig. a) . notably, neither site is close to the rna-binding groove (arrow, fig. a ). the locations of these two residues indicate that they do not directly participate in viral rna binding. software predictions by rnabindr (terribilini et al., ) , including ensemble, pssmseq and pssmstruct algorithms, also support this observation (data not shown). regarding the aa position, pro is present in the beaudette model strain, and thr is present in the tw tp/ strain. interestingly, replacing pro with thr at residue transforms the a-turn conformation into a looped structure after protein modeling (fig. b) , indicating the structural flexibility of this ntd residue. this result also suggests that the epitope character of this region of the tw t t bj aap p v arkdpi aax t v tw / aat t t ck/ch/lhb/ abc s v beaudette aaa p t tw / abg strains in gray are recruited for bi and codeml analyses. ibv n protein may be vulnerable to alteration under immunological pressure. for the other selected site, aa , where a thr is present in the tw tp/ strain and a val is present in the beaudette strain, no obvious conformational change was noted after homology modeling. nevertheless, a single mutational change from a hydrophilic amino acid (thr) to a hydrophobic one (val) could modulate the surface charge of the protein and its efficiency at forming high-order oligomers (fan et al., ) . to investigate the importance of the selected amino acid residues, the tw tp/ strain, which was the first ibv strain to be identified in taiwan in , was chosen to represent wild type tw ibv and subjected to an analysis of rnabinding activity. the tp/ strain shares high genetic similarity to most tw ibv isolates (fig. a and b and table ). three n protein mutants, including t p (aca to cca, thr to pro), t n (aca to aat, thr to asp), and t v (act to gtt, thr to val), were generated by site-directed mutagenesis and confirmed by sequencing (fig. a) . the production of full-length n protein of tp/ (arrow head in fig. b ) was successfully induced by adding isopropyl b-d- -thiogalactopyranoside (iptg) in e. coli (fig. b) . the n protein of tp/ (wild type, wt) and the mutant n proteins (t p, t n, t v) were purified by ni-column and further examined by the staining with coomassie blue in sds-page (fig. c) . the detected size of full-length n protein was around kda ( fig. b and c) , higher than the theoretical prediction ( kda). the upper shift of band location of expressed n protein might be caused by the intrinsic charged aa components of the n protein but not post-translational modification in e. coli due to the same detected molecular weight (mw) of n protein produced by an in vitro transcription and translation system (fig. s ). in addition, this upper shift of the n protein in sds-page were reported in a previous ibv n protein study (yu et al., ) and other coronaviral n proteins (hurst et al., (hurst et al., , . the rna-binding capacity of the n protein was determined by surface plasmon resonance (spr) analysis. previous studies showed that the coronaviral n protein has a high affinity for the trs sequence (grossoehme et al., ) . a repeated trs sequence has been used as a probe in the spr experiments to measure the interaction of the n protein with viral rna (huang et al., ; nelson et al., ) . the applied viral rna probe consisted of repeated ibv trs sequences, -(cuuaa-caa) - and was biotin-labeled. one hundred resonance units (ru) were immobilized onto a streptavidin-coated biosensor chip for detecting the binding capacity of purified ibv n proteins. compared to the wild type n protein, all three mutants showed significantly reduced trs-binding capacity at . and . mm protein concentrations ( fig. a -c,) (p < . , one-way anova, tukey's post hoc analysis). when the protein concentrations were elevated from . mm to . mm, proportional increases in their rna-binding capacities were detected (fig. c) . the t p variant, which mimicked the aa position of us beaudette strain, showed about half the rna-binding capacity of the wild type tw n protein (fig. c ) (p < . , one-way anova, tukey's post hoc analysis). likewise, the t n and t v variants, which represented the most common configurations of aa and aa in the ch n protein ( . % and . %, respectively) ( table ) , showed only - % of the rna-binding capacity of the tw tp/ strain (fig. c ) (p < . , one-way anova, tukey's post hoc analysis). the anova analyses also revealed that the rna-binding activity between the pairs of mutants (t p, t n and t v) has no significant difference. taken together, these results indicate that both aa and are critical for -(cuuaacaa) - binding, and the modulation of these two residues may affect the binding affinity of the n protein for viral genomic or subgenomic rna. while the ibv n protein has generally been conserved and negatively selected during viral evolution, we have identified two positively selected sites at aa and in the n protein of ibvs. these two residues are located in the putative recombinant region of the ntd domain and are critical for binding to trs repeats. to our knowledge, this is the first report on viral evolution linking an rna recombination event with positive selection. this study also provides the first functional assay to illustrate the importance of positively selected sites in the n protein for coronaviral rna binding. sporadic genetic recombination in the sequences of interests will change branch lengths and the topology of phylogenetic tree (yang et al., ) . these two parameters are assumed to be constant across the tested sequences for the analyses of codeml, especially when the positive selection is evaluated by branch-site method based on a likelihood ratio test (scheffler et al., ) . to avoid false result of the positive selection in this study, the possible recombinant ibv strains, such as tw / , tw / , tw / and tw / , are excluded ( fig. a and b ) and the selected strains are chosen from the population in a same clade. in addition, the candidate of positive selected sites (table ) is evaluated by a codon-substitution model but not a branch-site likelihood model. finally, the fidelity of the mathematic prediction by codeml is further validated by the experimental function assay, demonstrating that the positively selected sites are critical for the viral rna-binding activity. in addition to forming part of the rnp, the coronaviral n protein participates in the formation of the replicationtranscriptional complex. specifically, the n protein's ntd binds to the trs of the leading sequence and regulates trs-ctrs (complementary trs) helical unwinding (grossoehme et al., ) , suggesting its critical role in genomic duplication and subgenomic expression. in this study, we showed that substituting either of the positively selected residues with the amino acid present in most ch ibvs dramatically reduced the binding capacity of the n protein for synthetic trs repeats (fig. ) . although neither selected site is located in the rna-binding groove of the ntd, the modified residues may alter the secondary structure or surface charge distribution of the n protein and consequently affect rna-ntd interactions. here, phylogenetic evaluation of viral proteins not only helps us to reconstruct the evolutionary paths of viral species but also provides new insights into functional residues in viral proteins. during viral propagation, variants with enhanced cellular tropism, viral transmission or replicative advantage show enhanced fitness in infected hosts (domingo and holland, ) . amino acid mutations in human immunodeficiency virus (hiv) gag (banke et al., ) and pol genes (huang et al., ) , which are mainly involved in genomic duplication, were positively selected in patients and conferred fitness under the pressure of anti-hiv drug treatment. in addition, positively selected residues in capsid proteins have been reported in rabbit hemorrhagic disease virus (esteves et al., ) , hepatitis c virus (kurbanov et al., ) and foot-and mouth disease virus (haydon et al., ) . however, the importance of these selected sites has not yet been functionally evaluated. in this study, spr binding assays of point-mutant ibv n proteins demonstrated that the positively selected residues in the tw ibv n protein may improve binding efficiency for viral trs repeats. sophisticated approaches using viral replicons or recombinant infectious clone should further illuminate the detailed roles of these selected sites in coronaviral propagation. given that vaccine-based immunization imposes strong selective pressure on viral evolution, we did not rule out the possibility that the avian immune system has reshaped the recombinant n protein of tw ibv through positive selection. both s and n proteins are known to be major antigenic determinants of ibv (cavanagh, ) . administration of n proteins via intraperitoneal injection can elicit protective adaptive immunity against an ibv challenge (cavanagh, ) . we speculate that the tw ibv, sharing epitopes in the ntd of the n protein with the us-serotype ibv because of an rna recombination event, may have become more vulnerable to attack by the adaptive immune system in fowl vaccinated against the connecticut or other us-like ibv strains. to counteract this adverse effect, it is possible that residues in the a-turn (aa - ) and surrounding peptides located in an externally exposed loop (or a turn) have been positively selected to attenuate antigenic recognition by host b lymphocytes. this speculation is supported by the fact that almost all the test tw ibvs were isolated from vaccinated flocks and were under immune selection pressure. in addition, a recent study on mhv infection, which emphasized that epitope-escape coronaviral strains can be quickly selected by genetic deletion or mutation under strong immunological pressure (chua et al., ) . in addition, it has been suggested that the a-turn region (aa - ) may form an antigenic epitope in the ibv n protein (ignjatovic and sapats, ) . future works will aim to determine whether the mutations at aa and of the n protein in adapted strains result in altered epitope determinants and consequently provide competitive benefits for quasispecies in hosts by allowing the selected tw strains to escape immune surveillance. taken together, our data support the conclusion that positive selection has occurred in specific residues of the recombinant ibv n protein. this selection may promote the viral fitness in infected hosts, at least in part, by modulating the rna-binding capacity of the n protein's ntd. diagram illustrating the proposed evolution of ibv in taiwan is provided in fig. . further investigation is required to illustrate the details that how the recombinant configuration of the n gene in the original founder was maintained and consequently fixed in the current tw ibv population. the swiss-model workspace: a web-based environment for protein structure homology modelling positive selection pressure introduces secondary mutations at gag cleavage sites in human immunodeficiency virus type harboring major protease resistance mutations severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus effects of an epitope-specific cd + t-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants the murine coronavirus nucleocapsid gene is a determinant of virulence molecular epidemiology and evolution of avian infectious bronchitis virus in spain over a fourteen-year period rna virus mutations and fitness for survival detection of positive selection in the major capsid protein vp of the rabbit haemorrhagic disease virus (rhdv) the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes evidence for positive selection in foot-and-mouth disease virus capsid genes from field isolates viral evolution and the emergence of sars coronavirus elucidation of the stability and functional regions of the human coronavirus oc nucleocapsid protein the reverse transcriptase sequence of human immunodeficiency virus type is under positive evolutionary selection within the central nervous system identification of in vivointeracting domains of the murine coronavirus nucleocapsid protein an interaction between the nucleocapsid protein and a component of the replicase-transcriptase complex is crucial for the infectivity of coronavirus genomic rna identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation multiple recombination sites at the -end of murine coronavirus rna severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists evolution of infectious bronchitis virus in taiwan: characterisation of rna recombination in the nucleocapsid gene positive selection of core q variant genotype b hepatitis c virus strains induced by pegylated interferon and ribavirin pro-check: a program to check the stereochemical quality of protein structures high-frequency rna recombination of murine coronaviruses rdp: detection of recombination amongst aligned sequences baculovirus expression of hcov-oc nucleocapsid protein and development of a western blot assay for detection of human antibodies against hcov-oc high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna robust inference of positive selection from recombining coding sequences characterisation of the rna binding properties of the coronavirus infectious bronchitis virus nucleocapsid protein amino-terminal region the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells rnabindr: a server for analyzing and predicting rna-binding sites in proteins paml : phylogenetic analysis by maximum likelihood codon-substitution models for heterogeneous selection pressure at amino acid sites a novel b-cell epitope of avian infectious bronchitis virus n protein we are grateful to dr. drena dobbs and michael terribilini at iowa state university for supplying rnabindr rna-binding predictions for the ntd of the tw ibv n protein. this work was supported by national science council in taiwan (nsc - -b- - -my ). this work was also supported in part by the ministry of education, taiwan, r.o.c. under the atu plan. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . / j.vetmic. . . . key: cord- -r j yzfc authors: mcdonagh, phillip; sheehy, paul a; norris, jacqueline m title: identification and characterisation of small molecule inhibitors of feline coronavirus replication date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: r j yzfc feline infectious peritonitis (fip), a feline coronavirus (fcov) induced disease, is almost invariably fatal with median life expectancy measured in days. current treatment options are, at best, palliative. the objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against fcov in vitro to determine viable candidates for therapy. a resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against fcov. plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and cpe inhibition and ifa-based time of addition assays. three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced cpe at low micromolar concentrations. orthogonal assays confirmed inhibition of cpe was associated with significant reductions in viral replication. selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were , , and for chloroquine, mefloquine, and hexamethylene amiloride respectively. preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. these results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with fip. feline infectious peritonitis (fip), a feline coronavirus (fcov) induced disease, is almost invariably fatal with median life expectancy measured in days. current treatment options are, at best, palliative. the objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against fcov in vitro to determine viable candidates for therapy. a resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against fcov. plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and cpe inhibition and ifa-based time of addition assays. three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced cpe at low micromolar concentrations. orthogonal assays confirmed inhibition of cpe was associated with significant reductions in viral replication. selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were , , and for chloroquine, mefloquine, and hexamethylene amiloride respectively. preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. these results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with fip. ß elsevier b.v. all rights reserved. a number of compounds have demonstrated an inhibitory effect on the virus in vitro (barlough and shacklett, ; hsieh et al., ; keyaerts et al., ) , but there is little or no published data regarding their use in treating fip. the broad spectrum antiviral ribavirin demonstrated in vitro efficacy but provided limited clinical benefit and produced toxicity in cats (weiss et al., ) . more recently in a small study involving experimentally infected cats treatment with chloroquine, a drug with demonstrated in vitro antiviral efficacy, was associated with mild improvements in clinical signs, however there was no statistically significant difference in survival time compared to untreated cats (takano et al., ) . efficacious and safe antiviral therapeutics are desperately needed for fip treatment. modern antiviral drug discovery often involves high throughput screening of vast chemical libraries. these large scale unfocused screens are expensive and beyond the reach of companion animal medicine. an alternative approach is to utilise a more focused screening strategy, enriching the screening library with compounds considered likely to have an antiviral effect based on a prior knowledge of their pharmacodynamics and the viral life cycle. focused screening panels may consist of compounds related to those demonstrated effective against the challenge virus or those demonstrated effective against closely related viruses. in the current study we screened compounds with previously demonstrated antiviral activity against coronaviruses or other rna viruses, for antiviral activity against fcov using an optimised resazurin-based cpe inhibition assay. cytotoxicity of compounds was determined prior to screening using sequential resazurin-and srb-based assays to determine the optimal minimally toxic test concentration and to enable calculation of selectivity indices. the antiviral effects of compounds identified during screening were confirmed with plaque reduction and virus yield reduction assays. virucidal suspension assays and time of addition assays provided initial information on the stage of viral replication targeted and the potential mechanism of action. crandell rees feline kidney (crfk) cell line was propagated in dulbecco's modified eagle's medium (dmem; sigma-aldrich, castle hill, nsw, australia) supplemented with % fbs (sigma-aldrich) (dmem- ) in a humidified incubator at c in % co in air. two strains of fcov, fipv wsu - (fipv ) and fecv wsu - (fecv ), acquired from the american type culture collection (virginia, usa), were used. fcov fecv was originally isolated from mesenteric lymph nodes and intestinal washes of a . year old female domestic shorthaired cat that died of acute haemorrhagic gastroenteritis while fcov fipv was originally isolated from the liver, spleen, and lungs from a case of neonatal death in a -day-old male persian kitten (mckeirnan et al., ) . pathogenicity studies of these two isolates have shown that fipv is highly virulent and reliably causes signs of classic fip following oronasal inoculation, while fecv causes a low grade fever and mild enteritis, but no signs of fip (pedersen, ) . despite the dissimilar in vivo biological properties of the two isolates, the two have similar in vitro properties in immortalised cell lines. compounds were selected for the test panel based on their reported in vitro antiviral properties against coronaviruses or other rna viruses (see supplementary material for details). the compounds tested and their screening concentrations are shown in table . stock solutions were prepared by dissolving compounds in ultrapure water or dmso (sigma-aldrich). compounds were sterile filtered with a . mm regenerated cellulose filter (corning inc., corning, ny, usa), aliquoted into sterile single use microtubes (sarstedt, numbrecht, germany), and stored for a maximum of months at À c until use. to determine an appropriate screening concentration, cytotoxicity of test compounds was determined using sequential resazurin and sulforhodamine b assays. the resazurin-based assay was performed as for the antiviral screening assay except compounds were added in ml volume and there was no infection step. to perform the srb assay, cells were immediately fixed post fluorescent data acquisition by decanting culture media by inverting plates and adding % trichloroacetic acid for h at c. srb staining was as previously described by (vichai and kirtikara, ) except that . % srb was used for staining. following solubilisation of bound dye, od was measured using the fluostar omega microplate reader (bmg labtech, mornington, australia). viability was compared to untreated controls. test compound concentrations selected for subsequent antiviral screening were those resulting in cell viability of % or greater. compounds showing marked, moderate, or mild antiviral effects were defined as those showing - %, - %, and - % inhibition of cpe respectively. compounds demonstrating marked cpe inhibition were classified as candidate compounds and were selected for further characterisation. using the resazurin-based cpe inhibition assay a concentration-response experiment was conducted with serial dilutions of identified candidate compounds (nine concentrations per compound). to enable calculation of the selectivity index, a repeat cytotoxicity screen was performed concurrently. each treatment was performed in triplicate and repeated in three independent experiments. data were exported to microsoft excel for calculation of cell viability and cpe inhibition according to the formulae described above. data analysis were conducted in graphpad prism, with the % inhibitory concentration (ic ) and % cytotoxic concentration (cc ) values calculated using the inbuilt non-linear curve fitting functions following log transformation of compound concentrations. the selectivity index (si) for each compound was calculated according to the following formula: . . confirmatory assays plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified using the cpe inhibition assay. virus yield reduction assays were performed in -well plates (sarstedt). wells were seeded with .  cells well À in ml dmem- . plates were kept at room temperature for min and then at c in % co in air for h prior to the addition of test compounds. compounds were diluted in dmem to the required concentrations with ml added to each well. cells were incubated at c in % co in air for an additional h prior to infection with fcov fipv at moi . in ml dmem. cells were incubated for a further h at c in % co . at and h post-infection (hpi) cell monolayers were visually assessed for cpe using an olympus ckx inverted phase-contrast microscope (olympus, melville, ny, usa) and culture media was collected and stored at À c for virus titration. untreated infected cells, untreated uninfected cells, and treated uninfected cells were included as controls. this latter control was included to allow assessment of morphological changes to cells due to compound treatment. titration of extracellular virus harvested at and hpi was performed using the tcid method as described by mcdonagh et al. ( ) . each treatment and time point was performed in triplicate and repeated in two independent experiments, with results representing mean ae se. plaque reduction assays were performed in -well plates (corning). cells seeded at a density of  cells well À in ml dmem- were held at room temperature for min prior to incubation at c in % co in air for h, by which time monolayers were approximately % confluent. culture media was discarded and replaced with ml dmem supplemented with % fbs plus ml of various concentrations of test compounds in dmem (or ml dmem only for control wells) using five or six concentrations per compound. after exposure to the compound for h, cells were infected with pfu well À fcov fipv in ml dmem. virus was allowed to adsorb for min with plates rocked every min to ensure an even distribution of inoculum. culture media was discarded after min and cells overlaid with ml . % carboxymethylcellulose, % fbs in dmem containing the same concentration of compound as present prior to and during infection. cells were fixed and stained with . % (w/v) crystal violet hpi prior to manual plaque counting. the relative plaque number was calculated for each treatment, with the value of untreated control defined as %. each treatment was performed in duplicate, and repeated in three independent experiments, with data representing mean ae se. a virucidal suspension assay was performed to assess virucidal effects of test compounds. the assay was performed as above with the exception that virus was mixed and incubated with test compounds prior to infection. stock fcov fipv , diluted in dmem to  pfu ml À , was mixed with an equal volume of test compound diluted in dmem to  the test concentration used during screening. the control virus suspension was mixed with dmem containing an equal concentration of dmso as the test samples. virus suspensions were incubated for h at room temperature before serial dilution in dmem to infect cells with pfu well À in ml. following serial dilution of the virus, cells were exposed to test compounds at concentrations greater than log lower than concentrations previously shown to have no antiviral effect. the experiment was performed in triplicate and repeated in two independent experiments. data represent mean ae se. a modification of the resazurin-based cpe inhibition assay was performed to assess the effect of time of compound addition on the antiviral efficacy of identified compounds. the cpe inhibition assay was performed as previously described with the exception that test compounds were added at various time points before and after infection. the selected time points were h prior to infection, concurrent with infection, and , , or h postinfection. treatments were performed in triplicate and repeated in three independent experiments. data represent mean ae se. to further elucidate the stage of viral replication affected by each compound the effect of time of addition on viral antigen expression was examined. cells were seeded at a density of .  cells well À in ml dmem- in -well plates (mclear , greiner bio-one). after seeding plates were kept at room temperature for min and then incubated at c in % co in air for h prior to the first time-point of compound addition. compounds were added in ml to duplicate wells at different time points prior to, concurrent with, or postinfection. cells were infected with fcov fipv at moi . in ml or mock infected with ml dmem for an infection period of h. an infection period of h was selected based on the reported one step growth curve of fcov (rottier et al., ) . at hpi (measured from the end of the infection period) cells were fixed in % formaldehyde in pbs and permeabilised in ice cold methanol. viral antigen was detected with a biotinylated anti-fcov antibody (ccv - ; custom monoclonals international, sacramento, ca, usa) and visualised with streptavidin-conjugated alexafluor (life technologies, mulgrave, vic, australia). to enable accurate segmentation, cells were stained with the whole cell stain hcs cell mask blue (life technologies) and dapi (life technologies) to enhance nuclear visualisation. fluorescent imaging was performed using the bd pathway bioimager (bd bioscience, franklin lakes, nj, usa). images of wells were acquired using a  objective (na . ) using a  montage with laser autofocus performed for each montage frame. hcs cell mask blue/dapi, images were acquired with ex / bp and em lp filters, and alexa fluor images acquired with ex / bp and em lp filters. image analysis was performed using the free opensource image analysis software cellprofiler (r , www.cellprofiler.org) with data exported to fcs express image cytometry (version . . , de novo software, los angeles, ca, usa) for analysis. each treatment was performed in duplicate, and data represents mean ae sd. to assess efficacy against different fcov strains, identified candidate compounds were tested against fcov fecv using the resazurin-based cpe inhibition assay. the assay was performed as described, except that cells were infected with either fcov fipv or fecv at moi . . each treatment was performed in triplicate and repeated in three independent experiments, with data representing mean ae se. three of nineteen tested compounds showed marked inhibition of virus induced cpe (fig. ) and were selected for further characterisation. pre-treatment with chloroquine at mm, mefloquine at mm, and hexamethylene amiloride at mm resulted in . %, . %, and . % inhibition of cpe respectively. a further two compounds, glycyrrhizic acid at mm and cinanserin at mm displayed a mild antiviral effect with a . % and . % reduction in cpe respectively. all other compounds demonstrated limited or no inhibitory effect on cpe. included among these ineffective compounds was ribavirin, a broad spectrum antiviral compound that had previously shown in vitro (barlough and scott, ; weiss and oostrom-ram, ) , and to a limited extent in vivo efficacy against fcov (weiss et al., ) , as well as rfeifn-v which had previously shown in vitro efficacy against fcov (mochizuki et al., ; truyen et al., ) . a concentration-response study was conducted with chloroquine, mefloquine, and hexamethylene amiloride. a repeat cytotoxicity screen was concurrently performed for these compounds to allow calculation of selectivity indices. all compounds demonstrated a clear concentration-response effect over the tested range (fig. ) . calculated ic , cc , and si values for the compounds are shown in table . virus yield reduction assays confirmed the cpe inhibition identified during screening was associated with a marked reduction in extracellular viral titre. determination of extracellular virus titre was performed at and hpi with results shown in fig. . for chloroquine and mefloquine there was a considerable difference in the resulting concentration-response curves at and hpi, while for hexamethylene amiloride the shape of the curve was similar at both time points. differences in concentration-response curves between the two time points is reflected in the ic values, with increased ic values for chloroquine and mefloquine at hpi compared to hpi, while for hexamethylene amiloride ic values were similar at both time points (table ) . plaque reduction assays confirmed the findings of the cpe inhibition and virus yield reduction assays. pre-treatment with chloroquine, mefloquine, or hexamethylene amiloride resulted in a concentration-dependent decrease in plaque number, with high concentrations completely inhibiting macroscopic plaque formation. for all compounds plaque morphology was similar between treated and untreated wells however plaque size was smaller in treated versus untreated wells. during the virus yield reduction assay cells were monitored for the development of cpe using phase contrast microscopy. it was noted that infected and uninfected cells treated with chloroquine, mefloquine, or hexamethylene amiloride displayed characteristic morphological changes. these changes consisted of a large number of variably sized cytoplasmic (predominantly perinuclear) inclusions in addition to the presence, in some cells, of an increased number of cytoplasmic vacuoles. to investigate the nature of these inclusions, separate wells were stained with mg ml À neutral red in dmem for h. these inclusions appeared to accumulate the vital dye neutral red following suggesting they were likely dilated endosomes/lysosomes (fig. ) . using a virucidal suspension assay no virucidal effects were seen for chloroquine, mefloquine, or hexamethylene amiloride, with the infectivity of virus suspensions exposed to the compounds not significantly different from virus incubated with media alone. the effect of time of addition on the antiviral activity of selected compounds was assessed using a modification of the resazurin-based cpe inhibition assay and through ifa of viral protein expression. based on the cpe inhibition assay maximum antiviral effect was seen when compounds were added prior to or concurrent with infection, following which there was a time-dependent reduction in cpe inhibition (fig. ) . for all tested compounds cpe inhibition remained greater than % when compounds were added at the latest tested time point of h postinfection. the cpe inhibition assay encompasses multiple rounds of viral replication. to further elucidate the stage of viral replication affected by test compounds a single replication cycle ifa-based assay was conducted which confirmed that, based on viral antigen expression, all three compounds possess antiviral properties when added prior to, or at the time of infection. furthermore all compounds displayed a time of addition dependent reduction in antiviral effect; however the extent and timing of this reduction varied. the inhibitory effect of chloroquine was reduced, based on an increase in the percentage of fcov antigen positive cells, when added at any time postinfection (fig. ) . a similar result was seen for hexamethylene amiloride, although in this case a significant increase in the number of infected cells was not seen until compound addition was delayed until hpi. in contrast, mefloquine remained effective when added up to hpi suggesting it may act at a later stage of viral replication than chloroquine and hexamethylene amiloride. the efficacy of the three identified candidate compounds was tested against fcov fecv , a serotype ii enteric biotype fcov. comparison of the virus control (no treatment) wells showed fcov fipv infection resulted in more pronounced cpe over the h infection period compared to fcov fecv . pre-treatment with chloroquine, mefloquine, or hexamethylene amiloride provided a degree of protection against strain fcov fecv . pretreatment with hexamethylene amiloride provided protection against virus induced cpe that was similar for the two strains, with a reduction in cpe of . % and . % for fcov fipv and fecv respectively. both chloroquine and mefloquine however were more effective against fcov fipv than fecv , with cpe inhibition for chloroquine of . % for versus . %, and for mefloquine . % versus . % for strains fipv and fecv respectively. this study identifies three compounds (chloroquine, mefloquine, and hexamethylene amiloride) demonstrating a marked inhibitory effect on fcov replication in vitro by significant reductions in virus induced cpe and viral titres at low micromolar concentrations when present during the early stages of viral replication. an antiviral effect of chloroquine had previously been demonstrated against fcov, and hexamethylene amiloride had previously demonstrated efficacy against other coronaviruses, however this is the first demonstration of antiviral efficacy of mefloquine against a coronavirus. initial compound screening was performed using a cpe inhibition assay, with subsequent virus yield reduction assays and plaque reduction assays used for confirmatory testing. for the effective compounds the ic values, and corresponding selectivity index, varied with the assay method utilised. this is not unexpected given the assays measure different endpoints, and has been reported for other antiviral drugs such as the retroviral protease inhibitor saquinavir where the reported ic calculated based on production of viral p antigen is approximately -fold lower than that based on production of mature virions (buss and cammack, ) . similarly variation in assay conditions may result in the calculation of significantly different ic values. the concentration-response curve of chloroquine against sars-cov determined using a pcr based virus yield reduction assay was shown to shift considerably to the right when viral genome copies were assayed days post-infection compared to day postinfection (keyaerts et al., ) . a similar finding was noted in the current study for both chloroquine and mefloquine, with differences in potency reported with the tcid based virus yield reduction assay performed at and hpi, however this was not seen for hexamethylene amiloride. two compounds, ribavirin and rfeifn-v, which had previously demonstrated in vitro efficacy against fcov, failed to demonstrate significant inhibition of cpe during screening. for both compounds these discordant results are likely attributable to testing at concentrations below their useful therapeutic range and variations in assay conditions and sensitivity compared with previous work. the screening concentration of compounds used in this study was determined based on cytotoxicity testing to achieve cell viability greater than %. previous studies with ribavirin demonstrated ic values of . mg ml À ( mm) (barlough and scott, ) based on a visual assessment of protection from cytopathic effect and . mg ml À ( . mm), based on the reduction of extracellular viral titre (weiss and oostrom-ram, ) . the concentration used for screening ( . mm) was therefore more than times lower than the ic previously calculated based on a similar assay endpoint. from the results of the current study, virus yield reduction assays appear to provide a more sensitive assessment of antiviral efficacy than cpe inhibition assays, with the ic values calculated based on viral titre reduction significantly lower than those calculated based on cpe inhibition for all compounds. a small antiviral effect of ribavirin cannot therefore be ruled out based on the current findings, as although the tested concentrations did not provide protection against virus induced cpe, it may have been associated with a reduction in extracellular viral titre. the practical relevance of such a small antiviral effect is questionable, particularly given the known toxicity profile of this compound in cats. for rfeifn-v reductions in viral titres of . - . logs have been reported when crfk cells were treated with , u ml À h post-infection (truyen et al., ) and . - . logs when fcwf cells were pre-treated with - , u rfeinf-v (mochizuki et al., ) . protection from cpe was not seen in the current study when cells were pre-treated with rfeinf-v at u ml À , a concentration significantly lower than that previously shown to be effective using the same virus strain and cell line (truyen et al., ) . the tested concentration was however similar to that used by mochizuki et al. ( ) . this apparent lack of efficacy in this case may reflect differences in the drug exposure and infection conditions, the viral isolate tested, or an intrinsic enhanced susceptibility to the antiviral effects of interferon in fcwf cells compared to crfk cells as used in this study (weiss and toivio-kinnucan, ) . alternatively it may be that, as suggested for ribavirin, virus yield reduction assays provide a more sensitive assessment of antiviral effects than cpe inhibition assays, and that the screening method utilised failed to identify mild antiviral effects. a number of different mechanisms of action have been suggested to account for the antiviral properties of the compounds identified in this study against other viruses. for chloroquine antiviral effects have been ascribed to inhibition of glycosylation of viral proteins (savarino et al., ) or cellular receptors for viral attachment (vincent et al., ) , inhibition of glycoprotein expression (dille and johnson, ) , or inhibition of endosome mediated viral entry (savarino et al., ) . the antiviral effect of mefloquine against jc virus has been postulated to be due to its action as an adenosine mimetic (brickelmaier et al., ) , while for hexamethylene amiloride it has been suggested antiviral properties against different viruses may arise through competitive inhibition of viral rna polymerase (gazina et al., ) , an indirect mutagenic effect (levi et al., ) , or inhibition of viroporins (wilson et al., ) . interestingly all three compounds showing marked antiviral efficacy against fcov in this study resulted in similar morphological changes in cells exposed to sub-toxic concentrations. increased numbers of variably size cytoplasmic inclusions that accumulate the viral dye neutral red suggests these compounds result in a perturbation of the normal endocytic pathway in crfk cells. alterations in the endocytic pathway have previously been reported for chloroquine (dean et al., ) , mefloquine (labro and babin-chevaye, ) , and for amiloride and some of its derivatives (dutta and donaldson, ). this suggests a common physiological effect on treated cells for all three candidate antivirals and possibly a shared mechanism of action. viruses are known to usurp a variety of host endocytic pathways for cell entry and intracellular movement and inhibition of these pathways may be a useful therapeutic approach. although targeting a cellular pathway may be associated with an increased risk of toxicity, if that pathway is critical for viral replication this approach may slow or limit the development of resistance. time of addition studies demonstrated all compounds were most effective when added prior to infection, suggesting a mechanism of action involving early stages of viral replication. the cpe inhibition based time of addition assay involved infection at low moi with a h infection period, allowing for multiple rounds of viral replication. as a result of this, even with the delayed addition of compounds, cells uninfected by the original inoculum are effectively pre-treated prior to challenge with progeny virions produced during the primary replication cycle. using an ifa-based time of addition study involving a single replication cycle we were able to further clarify of the effect of time of addition, and refine the possible stage of the viral life cycle targeted by each compound. based on the ifa results chloroquine was effective only if present at the time of infection, supporting the hypothesis that chloroquine acts during cell entry for fcov fipv , possibly through inhibition of endosomal ph (takano et al., ) . hexamethylene amiloride and mefloquine provided significant antiviral effects when compound addition was delayed for up to and hpi respectively, suggesting that if the antiviral effects of these compounds arise through perturbation of endosomal function, the effects occur at different stages of the viral life cycle. alternatively distinct mechanisms of action may account for the observed effects of these compounds, as suggested for other viruses. there is limited published pharmacokinetic or safety data to inform the potential therapeutic application of the identified compounds in cats and given the relatively low si of all three compounds consideration must be given to their in vivo safety in this species. the human approved pharmaceuticals chloroquine and mefloquine are generally considered well-tolerated drugs, albeit with a narrow therapeutic index, while the clinical use of hexamethylene amiloride has not been reported. pharmacokinetic data available for chloroquine and mefloquine in humans would suggest that effective plasma concentrations could be achieved at standard therapeutic doses (pussard and verdier, ; simpson et al., ) . chloroquine has been shown to accumulate in leukocytes, where the concentration may be two orders of magnitude greater than that of plasma (mackenzie, ) , with the highest concentration reported in monocytes (french et al., ) . thus therapeutic concentrations may be attained in the target cells of virulent biotype fcovs at relatively low plasma concentrations, minimising the risk of dose-dependent adverse effects. mefloquine is known to accumulate within brain parenchyma at concentrations approximately - times higher than found in serum, with tissue concentrations of up to mm reported (nevin, ; pham et al., ) . mefloquine may therefore be useful in the treatment of dry (non-effusive) fip, where cns lesions are common (pedersen, ) although the potential for neurotoxicity must be considered. although the concentration of mefloquine achieved in the cns is greater than the cc of this compound in immortalised feline kidney cells, in humans this tissue concentration is achievable at therapeutic doses, despite in vitro data in human cells showing a cc approximately equal to that determined in the current study (brickelmaier et al., ) . it may be therefore that the more static cell population of the cns is more refractory to the toxic effects of mefloquine than mitotically active immortalised cells. this study has identified three compounds demonstrating marked in vitro inhibition of fcov in an immortalised cell line at low micromolar concentrations, including the first demonstration of antiviral effects of mefloquine against a coronavirus. although the low si of the three compounds may limit their therapeutic utility, these preliminary studies open the way for further investigation and potential optimisation of these compounds as antiviral agents. effectiveness of three antiviral agents against fip virus in vitro antiviral studies of feline infectious peritonitis virus in vitro identification and characterization of mefloquine efficacy against jc virus in vitro. antimicrob measuring the effectiveness of antiretroviral agents effects of exogenous amines on mammalian cells, with particular reference to membrane flow inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine search for inhibitors of endocytosis: intended specificity and unintended consequences randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis uptake of chloroquine and hydroxychloroquine by human blood leucocytes in vitro: relation to cellular concentrations during antirheumatic therapy amiloride is a competitive inhibitor of coxsackievirus b rna polymerase treatment of cats with feline infectious peritonitis synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle effects of amodiaquine, chloroquine, and mefloquine on human polymorphonuclear neutrophil function in vitro fidelity variants of rna dependent rna polymerases uncover an indirect, mutagenic activity of amiloride compounds pharmacologic actions of -aminoquinoline compounds in vitro inhibition of feline coronavirus replication by small interfering rnas isolation of feline coronaviruses from two cats with diverse disease manifestations inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro epileptogenic potential of mefloquine chemoprophylaxis: a pathogenic hypothesis a review of feline infectious peritonitis virus infection: - cerebral uptake of mefloquine enantiomers in fatal cerebral malaria antimalarial -aminoquinolines: mode of action and pharmacokinetics effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein effects of chloroquine on viral infections: an old drug against today's diseases anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors population pharmacokinetics of mefloquine in patients with acute falciparum malaria&ast analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase n is not important and a process of acidification of the endosome is necessary effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo antiviral potency of interferon-omega (ifn-omega) against selected canine and feline viruses sulforhodamine b colorimetric assay for cytotoxicity screening chloroquine is a potent inhibitor of sars coronavirus infection and spread evaluation of free or liposomeencapsulated ribavirin for antiviral therapy of experimentally induced feline infectious peritonitis inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication this study was supported by donations from the rex cat club (especially sharon barton and tracey gleeson), participants of the national annual feline health seminars, christine atkins, ruth thurling, cat fanciers association of nsw, and the cat protection society of nsw. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . / j.vetmic. . . . key: cord- -ytlnhyxr authors: zhao, kuan; li, li-wei; jiang, yi-feng; gao, fei; zhang, yu-jiao; zhao, wen-ying; li, guo-xin; yu, ling-xue; zhou, yan-jun; tong, guang-zhi title: nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of trim by interfering with trim -mediated rig-i ubiquitination date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ytlnhyxr porcine reproductive and respiratory syndrome (prrs) is caused by prrs virus (prrsv), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. identification of sustainable and effective measures to mitigate prrsv transmission is a pressing problem. the nucleocapsid (n) protein of prrsv plays a crucial role in inhibiting host innate immunity during prrsv infection. in the current study, a new host-restricted factor, tripartite motif protein (trim ), was identified as an inhibitor of prrsv replication. co-immunoprecipitation assay indicated that the prrsv n protein interferes with trim –rig-i interactions by competitively interacting with trim . furthermore, n protein inhibits the expression of trim and trim -mediated rig-i ubiquitination to suppress interferon β production. furthermore, with increasing trim expression, the inhibitory effect of n protein on the ubiquitination of rig-i diminished. these results indicate for the first time that trim inhibits prrsv replication and that the n protein antagonizes the antiviral activity by interfering with trim -mediated rig-i ubiquitination. this not only provides a theoretical basis for the development of drugs to control prrsv replication, but also better explains the mechanism through which the prrsv n protein inhibits innate immune responses of the host. the tripartite motif protein (trim ) is an e ubiquitin ligase involved in various cellular processes, including regulating the innate immune response against viruses (heikel et al., ) . immediately after a viral infection, pathogen-associated molecular patterns are sensed by host pattern recognition receptors (prrs), thereby allowing cells to distinguish self from non-self and activate an immune response (sparrer and gack, ) . for rna viruses, the main cytoplasmic prr is retinoic acid-inducible gene i (rig-i), which can directly recognize and bind viral ′-ppp rna and short double-stranded rna (chan and gack, ) . after recognition, the n-terminal caspase recruitment domains (cards) of rig-i are modified by ubiquitin which is mediated by trim . such modification is essential for activating a signaling cascade, ultimately resulting in the transcriptional activation of type i and iii interferons (ifns), mediating viral clearance, and inhibiting viral replication and spread (gack et al., ; martin-vicente et al., ) . various viruses are inhibited by trim , e.g., coxsackie b virus and poliovirus (schoggins et al., ) . porcine reproductive and respiratory syndrome (prrs) is endemic in most pig-producing countries (han and yoo, ) , and is caused by prrs virus (prrsv). in , a highly pathogenic prrsv (hp-prrsv) emerged in china, causing high fever, high morbidity, and high mortality . prrsv is an enveloped, single-stranded positive-sense rna virus. its genome is approximately . kb and encodes at least open reading frames (orfs) (han and yoo, ) . orf a and orf b encode two large nonstructural polyproteins, ppla and pplb, which are processed into at least nonstructural proteins (snijder and meulenberg, ; ziebuhr et al., ) . orf a-orf encode structural proteins, including four membrane-associated glycoproteins (gp a, gp , gp , and gp ), three unglycosylated membrane proteins (e, orf a, and m), and a nucleocapsid protein (n) (dea et al., ; snijder and meulenberg, ) . among these, nsp , nsp , nsp , nsp , and been identified and characterized as ifn antagonists (lunney et al., ) . further, the n protein is the most abundant protein during infection, accounting for approximately % of virion proteins (snijder and meulenberg, ) . it plays essential roles in the virus life cycle, including encapsidation of viral rna (spilman et al., ) . currently, it is only known that n protein suppresses ifn-β induction by antagonizing irf activation (sagong and lee, ) . however, other mechanisms through which n protein inhibits ifn-β production are not clear. the nucleocapsid protein of severe acute respiratory syndrome (sars) inhibits type i ifn production by interfering with trim mediated rig-i ubiquitination (hu et al., ) . whereas prrsv and sars both belong to the nidovirales order, whether prrsv n protein inhibits the host innate immune response by interfering with trim mediated rig-i ubiquitination is not clear. herein, we show that trim plays an important role in inhibiting prrsv replication. the n protein interacts with trim and suppresses the ubiquitination of rig-i. further, trim expression is reduced upon co-transfection with the n protein or prrsv infection. together, these findings suggest a novel mechanism through which prrsv n protein inhibits host innate immunity, and provide improved understanding of the mutual regulatory mechanism between prrsv and the host. human embryonic kidney (hek t) cells and african green monkey kidney (marc- ) cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum (gibco, thermo fisher scientific, waltham, ma). cells were maintained at °c with % co . the highly pathogenic prrsv strain hun (genbank no. ef ) was used for all the experiments (tong et al., ) . total rna was extracted from porcine alveolar macrophages (pams) using rneasy mini kit (qiagen, hilden, germany), following the manufacturer's instructions. reverse transcription reactions were performed at °c for min and °c for h using the m-mlv reverse transcription polymerase system (takara, dalian, china). full-length trim -and rig-i-encoding sequences were amplified with specific indicated primers. the sequences were then cloned into pcaggs vector to generate pcaggs-trim -ha, pcaggs-trim -flag, pcaggs-trim -myc, pcaggs-rig-i-ha, pcaggs-rig-i-flag, or pcaggs - card-flag. orf of prrsv hun was cloned into pcaggs to generate pcaggs-n-ha, pcaggs-n-flag, or pcaggs-n-myc. all plasmids were constructed by homologous recombination using the nebuilder® hifi dna assembly master mix (new england biolabs; ipswich, ma) according to the manufacturer's instructions. sequences of all primers used for gene amplification will be made available upon request. to investigate the effect of trim on prrsv replication, marc- cells cultured in -well plates were transfected with μg of pcaggs-trim -flag using x-treme gene hp dna reagent (roche applied science, penzberg, germany). next, h post-transfection (hpt), the cells were infected with prrsv hun (multiplicity of infection, moi, of . ). after inoculation for h at °c, the supernatants were discarded, and cells were washed three times with phosphate-buffered saline (pbs). the supernatant was harvested , , , and h post-infection (hpi), and the cells were lysed using ripa lysis buffer (thermo fisher scientific). viral titers in the supernatants were determined using a microtitration assay, according to the method of reed and muench (reed and muench, ) . the amount of the n protein was then detected in cell lysates by western blotting (wb) using a mouse anti-n polyclonal antibody ( : ) produced by the authors. small interfering rnas (sirnas) against macaque trim (genbank no. xm_ . ) were synthesized by genepharma (shanghai, china). the sirna molecules used were ccu gga gua uua cgu uaa att (sirna-trim - ) and gca ucu acc aua gca ccu utt (sirna-trim - ). the control sirna (nc) sequence was uuc ucc gaa cgu guc acg utt. marc- cells were seeded in well plates and transfected with pm sitrim or nc using lipofectamine™ rnaimax transfection reagent (cat. no. , , ; thermo fisher scientific) . the cells were lysed in ripa lysis buffer after h of transfection and the effects of sirnas were analyzed by wb using an anti-trim monoclonal antibody (cat. no. , ; cell signaling technology; danvers, ma; : ). the knockdown efficiency of sirna was analyzed by grayscale scanning with image j software. next, efficient sirna and nc were selected for transfection; hpt, the cells were infected with prrsv hun at an moi of . . the supernatant and cells were harvested , , , and hpi, and analyzed based on virus titers and by wb. cell lysates were prepared by harvesting virus-infected or plasmidtransfected cells in ripa or ip-lysate buffer( mm tris•hcl ph . , mm nacl, % np- , mm edta, % glycerol) at °c for min containing mm phenylmethylsulfonyl fluoride and mg/ml of protease inhibitor cocktail (roche). after centrifuging at , × g for min, the supernatants of cell lysates were mixed with × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample loading buffer (beyotime), and placed in boiling water for min. the proteins were then separated by sds-page and transfected onto a nitrocellulose membrane. the membrane was blocked in % skim milk at room temperature for h and then incubated with the indicated primary antibody at °c overnight. after washing three times with trisbuffered saline with . % tween , the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse igg(h + l) and/or goat anti-rabbit igg(h + l) secondary antibody ( : ) for h at room temperature. the target proteins were visualized by treating the membrane with pierce ecl wb substrate (thermo fisher scientific). for the quantification of target proteins, their levels were normalized to the levels of β-actin. to detect the interaction between trim and prrsv n protein, hek t cells grown in -well plates were co-transfected with pcaggs-trim -flag and pcaggs-n-ha ( μg each) using x-tremegene dna transfection reagent. at hpt, the cells were lysed with ice-cold ip lysis buffer containing mm phenylmethylsulfonyl fluoride and mg/ml of protease inhibitor cocktail (roche) at °c for min. approximately % of the lysate supernatant was used as an input control and the remaining lysates were incubated with anti-ha agarose beads (cat. no. a ; sigma-aldrich; st. louis, mo) or ezview™ red anti-flag ® m affinity gel (cat.no. m ; sigma-aldrich) for h at °c. the beads were washed five times with the ip lysis buffer and then analyzed by wb using rabbit anti-flag monoclonal antibody (cat. no. f ; sigma-aldrich; : ) or mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ). the interaction between trim and rig-i was tested using the same methods with anti-ha agarose beads and wb using mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ) and mouse anti-myc monoclonal antibody (cat. no. ; cell signaling technology; : ). in order to investigate whether the n protein k. zhao, et al. veterinary microbiology ( ) - competitively affects the interaction between rig-i and trim , hek t cells grown in -well palate were co-transfected with pcaggs-rig-i-ha ( μg) and pcaggs-trim -myc ( μg) with or without pcaggs-n-flag ( μg) for immunoprecipitation. further, to detect the interaction between endogenous (host) trim and n protein in prrsv-infected cells, pam cells were grown in -cm-diameter dishes and infected with prrsv at an moi of . for h. the cells were lysed in μl of ip lysis buffer and the supernatants were precleared with protein g-agarose beads (cat. no. ; roche). the supernatant was incubated with μg of anti-n polyclonal antibody for h. next, μl of protein g-agarose beads was added, followed by an additional h incubation. ip pellets were washed five times with μl of ip lysis buffer, boiled in sds-page sample loading buffer, and analyzed by wb with rabbit anti-trim monoclonal antibody (cat. no. ab ; abcam; : ) and anti-n polyclonal antibody. hek t cells were co-transfected with pcaggs-n-ha and pcaggs-trim -flag, or pcaggs-rig-i-ha and pcaggs-trim -flag. at hpt, the cells were fixed in % paraformaldehyde for min, blocked with % bovine serum albumin for h, and permeabilized with . % triton x- for min. the transfected cells were incubated with mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ) and rabbit anti-flag monoclonal antibody (cat. no. f ; sigma-aldrich; : ) for h at °c, and then washed three times with pbs. the cells were then incubated at °c for h with donkey anti-mouse igg(h + l) antibody conjugated with alexa fluor (cat. no. ; life technologies; : ) and goat anti-rabbit igg(h + l) antibody labeled with alexa fluor (cat. no. ; life technologies; : ). samples were then stained with μg/ml of ′, ′-diamidino- -phenylindole (dapi) for min and examined using a zeiss confocal system. besides, in order to investigate the colocalization of endogenous (host) trim and n protein in prrsv-infected cells, the pam cells infected with prrsv were send to colocalization detection with anti-trim and anti-n protein antibody. hek t cells seeded in a -well plate were co-transfected with control plasmids or the indicated expression plasmids ( . μg per well) together with ng of a luciferase reporter plasmid; prl-tk plasmid ( ng) was used as a control of transfection efficiency (huang et al., ) . next, hpt, cells lysates were harvested and luciferase activity in the lysates was analyzed using a dual-luciferase reporter assay kit (promega, madison, wi), following the manufacturer's instructions. the results are expressed as the means ± standard deviation (sd) from three independent experiments. statistical analysis was performed using graphpad prism (graphpad, la jolla, ca). the measured values are expressed as the mean with sd. the statistical significance was assessed using student's t-test, with p < . considered statistically significant. to examine the effects of trim on prrsv replication, specific sirna molecules were synthesized to knockdown trim expression in marc- cells and efficiently reduce trim expression. using sirna- , the knockdown efficiency was approximately % (fig. a) . this sirna molecule was used in the subsequent interference experiments. as shown in fig. b , n protein levels increased upon transfection with sirna- , especially and hpi, compared with those in nctransfected cells. virus titers in the culture supernatants of cells transfected with sirna- were also increased, which was consistent with the expression levels of the n protein, with a significant difference hpi (p < . ; fig. c ). by contrast, when trim was overexpressed, the n protein levels of hun were lower than those in the control cells (fig. d) . furthermore, there was a significant difference in virus titers between cells transfected with pcaggs-trim -flag or pcaggs, with an approximate . log decrease in virus titers from to hpi (p < . ; fig. e ). these observations suggested that trim is a cellular antiviral factor that represses prrsv infection. as shown in fig. d , the expression of trim -flag decreased gradually upon prrsv infection. therefore, we speculated that the virus might inhibit the expression of trim . to verify this hypothesis, marc- cells infected with prrsv or uninfected cells were lysed , , , and hpi, and analyzed by wb. the analysis revealed that the expression of trim decreased in prrsv-infected cells at every time point, as compared with the uninfected cells ( fig. a) . furthermore, trim expression was significantly suppressed when pcaggs-trim -myc and pcaggs-n-ha were co-transfected into hek t cells. these findings indicated that the n protein inhibits the expression of trim . furthermore, the n protein inhibited trim expression in a dose-dependent manner (fig. b) . since we demonstrated that prrsv infection and n protein accumulation suppress the expression of trim , we next investigated whether the n protein of prrsv exerts an inhibitory effect on trim expression by interacting with it. to test this, the interaction between trim and n protein was investigated by co-ip. based on precipitation with anti-flag agarose, flag-tagged trim interacted with hatagged n protein (fig. a) . furthermore, n protein was efficiently coimmunoprecipitated with trim using anti-ha agarose (fig. b) . we also investigated whether trim co-localizes with the n protein in hek t cells co-transfected with pcaggs-trim -flag and pcaggs-n-ha. trim was mainly located in the cytoplasm ( fig. c-a) , whereas the n protein localized in the cytoplasm and nucleus ( fig. c-b) . as shown in fig. c -d, trim and n protein colocalized in the cytoplasm. to examine the interaction of n protein and endogenous trim in the context of prrsv infection, virus-infected pam cells were stained with anti-n polyclonal antibody and anti-trim monoclonal antibody or immunoprecipitated using anti-n polyclonal antibody and the pellets were detected with anti-trim and n antibody. as shown in fig. d and e, endogenous trim was co-localized with n protein and coimmunoprecipitated with the n protein. these observations confirmed that the endogenous trim indeed interacts with the n protein of prrsv in prrsv-infected cells. in the current study, we demonstrated that the n protein of prrsv could interact with trim . further, it has been reported that trim interacts with rig-i n-terminal cards and e ubiquitin-conjugating enzymes (gack et al., ) . whether the n protein can competitively regulate the interaction between trim and rig-i was unknown. we first detected the interaction between trim and rig-i by co-ip, confirming that trim could be immunoprecipitated with rig-i (fig. a ). in addition, co-localization analysis demonstrated that rig-i and trim co-localize in the cytoplasm (fig. b) . further, we k. zhao, et al. veterinary microbiology ( ) - validated the effect of n protein on the interaction between rig-i and trim . when rig-i-ha and trim -myc were co-transfected with n-flag, the interaction between trim and rig-i was markedly diminished. meanwhile, when trim -myc and rig-i-ha were co-transfected without n-flag, the interaction between trim and rig-i was not affected (fig. c) . these results suggested that the n protein inhibits the interaction between trim and rig-i via competitive binding to trim . to investigate whether trim -mediated rig-i ubiquitination is regulated by the prrsv n protein, hek t cells grown in -well plates were co-transfected with pcaggs-flag-rig-i ( . μg per well) and ha-ubiquitin ( . μg per well), and the indicated amounts of the myc-n expression plasmids. cells lysates were immunoprecipitated using mouse anti-ha monoclonal antibody or rabbit anti-flag monoclonal antibody. the experiment revealed that trim -mediated rig-i ubiquitination was potentiated by sendai virus (sev) infection but was substantially suppressed by increasing the prrsv n protein expression, in a dose-dependent manner (fig. ) . when viral rna is recognized by rig-i, this protein is modified by ubiquitin, which is mediated by the e ligase trim . hence, this enzyme is essential for activating a signaling cascade that ultimately results in the transcriptional activation of type i and iii ifns. to examine regulation of rig-i activity by the prrsv n protein, a luciferase reporter under the control of the ifn-β promoter (ifn-β-luc) was used to quantify promoter activation. consistent with the inhibition of rig-i ubiquitination by the n protein, ifn-β promoter activation induced by rig-i or rig-i card domain overexpression was significantly inhibited by prrsv n expression, in a dose-dependent manner (fig. a, b) . however, co-expression of trim with prrsv n significantly counteracted this inhibitory effect mediated by the n protein (p < . ) (fig. c ). in addition, suppression of rig-i ubiquitination by the prrsv n protein was partially rescued by trim overexpression (fig. d) . these observations indicated that the n protein inhibits rig-i ubiquitination and that ifn production can be restored by trim overexpression. prrsv has been a major threat to global industrial pig farming ever since its emergence in the late s (shi et al., ) , and especially or pcaggs ( μg) were inoculated with prrsv at an moi of . . the cells and supernatant were harvested at the indicated times, and the replication of prrsv was evaluated by wb with an anti-n polyclonal antibody (d) and tcid assay (e). the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using student's t-test (*p < . ). since the outbreak of hp-prrsv in . to date, although there is some understanding of the mechanisms through which prrsv escapes innate immunity, very little is known about how viral proteins of prrsv antagonize the host innate immune response. therefore, understanding the interactions between prrsv and host proteins will be beneficial for the development of effective therapies to control outbreaks of this disease in the future. in the current study, we demonstrated that trim functions by restricting prrsv replication, and that it could act as a host-derived inhibitor of the virus. previously, many host factors that result in cellular resistance to prrsv via different mechanisms were identified song et al., ; wang et al., ; zhao et al., zhao et al., , . for example, cholesterol -hydroxylase protects against prrsv infection by converting cholesterol to -hydroxycholesterol, which can be used as a natural antiviral agent to combat prrsv infection (song et al., ) . further, galectin- inhibits replication prrsv by interacting with viral nsp in vitro . in the current study, using marc- cells, we demonstrated that the overexpression of trim restricts the replication of prrsv, whereas knocking down this protein promotes the replication of prrsv (fig. ) . therefore, trim acts as a novel host factor that inhibits the replication of prrsv. trim is an e ubiquitin ligase enzyme that can regulate the innate immune response against viruses (martin-vicente et al., ) . trim -mediated ubiquitination of the cytosolic prr rig-i is an essential step for the initiation of an intracellular antiviral response (gack et al., ) . data presented herein revealed that upon trim overexpression, the n protein-mediated inhibition of rig-i ubiquitination and ifn-β promoter activity were diminished (fig. ) . the host innate immunity was hence activated, leading to a series of signaling cascades and thereby inhibiting prrsv replication. trim can activate the host innate immune system and simultaneously induce a series of antiviral responses by promoting the ubiquitination of rig-i and activation of ifn-β promoter activity. however, in the course of natural infection, prrsv can complete the replication cycle and efficiently spread. hence, prrsv has evolved several general strategies to evade the innate immune response. it has been reported that some viral proteins interact with trim and inhibit rig-i activation. for example, the non-structural protein (ns ) of influenza a virus interacts with the cc domain of trim preventing its dimerization and the k -linked ubiquitination of rig-i cards, thereby suppressing rig-i signal transduction (gack et al., ) . further, trim interacts with the n protein of sars-cov, thereby inhibiting the activation of rig-i (hu et al., ) . in the current study, we found that the n protein of prrsv inhibits the ubiquitination of rig-i by competitively interfering with the interaction between rig-i and trim . this might be the mechanism through which prrsv inhibits the antiviral effect of trim . furthermore, trim levels decreased when the cells were infected with prrsv. in addition, when plasmids expressing trim and the n protein of prrsv were cotransfected into cells, the expression of trim was significantly suppressed. based on this, it would be difficult for trim to exert an antiviral effect upon prrsv infection. this might represent another mechanism through which prrsv antagonizes the antiviral response of trim . besides, the n protein of pedv, another coronavirus, is also able to antagonize ifn-β production (ding et al., ) . since prrsv, sars, and pedv all belong to nidovirales, we speculate that the respective n proteins may exert a similar effect of inhibiting trim mediated ubiquitination of rig-i. however, the effect of pedv n protein on the inhibition of rig-i ubiquitination requires further research. in the present study, we confirmed that trim inhibits prrsv replication. further, prrsv can antagonize the antiviral activity of this protein by decreasing its expression and modulating the trim mediated ubiquitination of rig-i. in addition, the n protein of prrsv inhibits ifn-β production. all these mechanisms improve the understanding of the effect of trim on prrsv replication and will further co-ip experiment was performed using agarose beads conjugated with anti-flag monoclonal antibody or (b) agarose beads conjugated with anti-ha monoclonal antibody. the precipitated proteins were analyzed by wb using antibodies against the flag and ha tags. (c) co-localization of the n protein with trim . hek cells were transfected with plasmids expressing ha-n protein and flag-trim . after h, the cells were fixed and analyzed by ifa to detect tagged n protein and trim , using mouse anti-ha and rabbit anti-flag monoclonal antibodies, respectively. the nucleus is stained with dapi (blue) in the merged images. (d and e) colocalization and interaction of prrsv n and endogenous trim . pam cells infected with prrsv were fixed h after infection, and then subjected to indirect immunofluorescence to detect n protein and trim . in addition, cell lysates from prrsv-infected or mock-infected pam cells were co-ip with mouse anti-n polyclonal antibody, and the precipitates were immunoblotted with the indicated antibodies (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). k. zhao, et al. veterinary microbiology ( ) - help to understand how prrsv evades the trim -mediated innate immune response via the n protein. hence, the current study not only offers a new target for the development of drugs to control prrsv spread but also provides an explanation of the mechanism through which prrsv n protein modulates host innate immune responses. in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. . the prrsv n protein suppresses the trim -mediated rig-i ubiquitination. hek t cells were transfected with the indicated plasmids for h, and were infected (with or without sev) for h. anti-flag immunoprecipitates prepared from the cell extracts were analyzed by wb using the indicated antibodies. k. zhao, et al. veterinary microbiology ( ) - viral evasion of intracellular dna and rna sensing current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i engineering the prrs virus genome: updates and perspectives the role of trim in development, disease and rna metabolism the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-κb essential modulator galectin- inhibits replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp in vitro porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system trim in the regulation of the antiviral innate immunity. front. immunol. , . reed porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf activation in immortalized porcine alveolar macrophages pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity molecular epidemiology of prrsv: a phylogenetic perspective the molecular biology of arteriviruses cholesterol -hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection intracellular detection of viral nucleic acids cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome the interferon-induced mx inhibits porcine reproductive and respiratory syndrome virus replication porcine ', '-oligoadenylate synthetase inhibits porcine reproductive and respiratory syndrome virus replication in vitro mov inhibits replication of porcine reproductive and respiratory syndrome virus by retaining viral nucleocapsid protein in the cytoplasm of marc- cells virus-encoded proteinases and proteolytic processing in the nidovirales a and b) hek t cells seeded in -well plates were co-transfected using the firefly luciferase reporter plasmid ifn-β-luc and the renilla luciferase control reporter plasmid prl-tk. for the experiment, pcaggs-rig-i-flag ( . μg), or pcaggs - card ( . μg), pcaggs-n-ha were co-transfected. (c) pcaggs - card-flag ( . μg), pcaggs-n-falg ( . μg) and pcaggs-trim -myc ( . μg) plasmids were cotransfected for the experiment, hpt, the cells were infected with sev, and hpi, whole-cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of rig-i-card. the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using key: cord- -er orx s authors: ladekjær-mikkelsen, a.-s; nielsen, j; stadejek, t; storgaard, t; krakowka, s; ellis, j; mcneilly, f; allan, g; bøtner, a title: reproduction of postweaning multisystemic wasting syndrome (pmws) in immunostimulated and non-immunostimulated -week-old piglets experimentally infected with porcine circovirus type (pcv ) date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: er orx s postweaning multisystemic wasting syndrome (pmws) in swine is causally associated with the newly recognised pathogen, porcine circovirus type (pcv ). in this study, -week-old spf pcv -seronegative piglets were inoculated intranasally with pcv . the effect of immunostimulation on the induction of pmws was investigated by immunisation with keyhole limpet hemocyanin (klh) emulsified in incomplete freunds adjuvant. the study was terminated weeks after inoculation. while disease was not observed in the age-matched controls, two out of five non-immunised pcv -infected piglets died on postinoculation day (pid) , and one was euthanized on pid in moribund condition. these animals had appeared lethargic with persistent fever from pid onwards. the euthanized pig appeared smaller than littermates and suffered from jaundice. at postmortem examination, gastric ulceration, icterus, and liver and thymus atrophy were observed. furthermore, histological lesions of degenerating hepatocytes and hepatitis in combination with lymphoid depletion and syncytial cells in lymph nodes were consistent with the diagnosis of pmws. one out of five immunostimulated pcv -infected piglets was euthanized on pid with convulsions after a period with wasting. this pig was lethargic from pid onwards with persistent fever from pid and transient dyspnoea. no differences in clinical signs, gross pathologic or histological findings were observed for the remaining non-immunostimulated and immunostimulated pcv -infected piglets. all pcv -inoculated piglets seroconverted to pcv within days after inoculation. by virus isolation, quantitative polymerase chain reaction (q-pcr), and immunostaining of cryostat sections, it was demonstrated that lymphoid tissue contained abundant pcv antigen. viral dna load in serum samples was assessed by q-pcr. all four pmws-affected piglets had high levels of pcv dna in serum, suggesting that there was a correlation between high levels of viral dna in serum and the development of pmws. in conclusion, infection with pcv caused pmws in spf piglets, however, the immunostimulation did not seem to play a critical role. reproduction of postweaning multisystemic wasting syndrome (pmws) in immunostimulated and non-immunostimulated -week-old piglets experimentally infected with porcine circovirus type (pcv ) the small single-stranded dna virus, porcine circovirus (pcv), has recently been recognised as a pathogen of swine. two types of pcv have been characterised and designated pcv type (pcv ) and pcv type (pcv ), respectively (allan and ellis, and references therein) . pcv is considered to be a non-pathogenic virus (tischer et al., ; allan et al., ) , whereas infection of swine with pcv is causally associated with development of postweaning multisystemic wasting syndrome (pmws) in weaned -to -week-old piglets (allan et al., ; ellis et al., ) . pmws is characterised by weight loss, dyspnoea, and jaundice combined with the pathological ®ndings of interstitial pneumonia, generalised enlarged lymph nodes, hepatitis, and nephritis (allan et al., ; ellis et al., ) . typical histological lesions include degeneration and necrosis of hepatocytes, multifocal lymphohistiocytic pneumonia, and lymphocytic depletion and multinucleated giant cell formation in lymph nodes (clark, ; ellis et al., ) . pcv antigen is typically localised within the multisystemic lesions in affected piglets (clark, ) . pmws was ®rst identi®ed within high health herds in western canada in (clark, ) but has since been reported in swine throughout the world (allan et al., ; ellis et al., ; onuki et al., ; choi et al., ) . interestingly, serological surveys have shown that pcv was present in swine before pmws was ®rst reported (magar et al., b; walker et al., ) . furthermore, serologic studies have revealed that many herds are seropositive to pcv without clinical signs of pmws (labarque et al., ; rodriguez-arrioja et al., ) . a number of experimental studies have indicated that infection with pcv alone is not suf®cient to induce clinical pmws. as such, several inoculation studies using pcv alone have resulted in asymptomatic infection with mild to moderate histopathologic lesions magar et al., a; pogranichyy et al., ) . in contrast, dual infection with pcv and porcine parvovirus (ppv) or porcine reproductive and respiratory syndrome virus (prrsv) potentiates the replication and distribution of pcv and induces clinical disease in addition to more severe histopathological lesions kennedy et al., ; krakowka et al., ; harms et al., ) . these results are in concordance with the observation of pcv and ppv coinfection in ®eld cases of pmws and suggest that the presence of another pathogen may augment the pathogenic effects of pcv . similarly, gnotobiotic piglets inoculated with pcv alone remained clinically asymptomatic while gnotobiotic piglets inoculated with tissue homogenates from pmws-affected piglets containing both pcv and ppv developed lesions typical of pmws with severe clinical disease krakowka et al., ) . recently , it has been demonstrated by krakowka et al. ( ) that activation of the immune system is a key component of the pathogenesis of pcv -associated pmws in gnotobiotic pigs. in that study, immune activation was achieved by injection of keyhole limpet hemocyanin (klh) in incomplete freund's adjuvant (icfa) into gnotobiotic piglets infected with pcv at day of age. by incorporation of bromo-desoxiuridine (brdu), upregulation of virus production was roughly correlated to increased numbers of replicating cells. in concordance, increased amounts of infectious virus was recovered from draining lymph nodes by quantitative viral titrations. in that study, all immunised piglets developed pmws whereas none of the non-immunised piglets inoculated with pcv alone developed pmws and extensive viral replication of pcv was observed in pmws-affected piglets, only . these observations also suggest why all pigs infected with pcv do not develop pmws during an outbreak or an experimental infection since differences in immunological status of pigs are bound to exist both between and within herds or experimental groups. the gnotobiotic model for experimental infections cannot be directly compared to ®eld conditions because of the immature immunological status of gnotobiotes and the obvious differences in husbandry conditions. furthermore, under ®eld conditions, pmws is a problem in -to -week-old piglets indicating that the piglets are infected with pcv after weaning. therefore, the objective of the present study was to investigate the signi®cance of immunostimulation on the development of pmws in -week-old spf piglets infected with pcv immediately after weaning. the virus inoculum was prepared by three passages in gnotobiotic pigs of a th cell culture passage of a canadian ®eld isolate ( ) of pcv . the cell culture passages were performed in pcv-free porcine kidney (pk- ) cells and con®rmed to be pcv by nucleotide sequence analysis and reactivity with pcv -speci®c mab . the inoculum (osu ) was a % (w/v) lymphoid tissue homogenate of lymphoid tissue collected from the inoculated gnotobiotic pigs. the titer of the pcv inoculum was . tcid /ml as determined by titration on pcv-free pk- cells. the piglets used in this experiment originated from the spf herd at the danish veterinary institute for virus research (dvivr). this herd is free from pcv and pcv , prrsv, swine in¯uenza virus (siv), porcine respiratory coronavirus (prcv), and a number of other microbiological agents, including mycoplasmas. sows are routinely vaccinated against ppv. the animal experiment was carried out at the isolation facilities of dvivr and the pigs were kept in two groups housed in separate sections of the building. twenty, -week-old colostrum-fed pcv -seronegative piglets born from two sows were randomly divided into two groups, comprising piglets each. the control piglets (group ) were sham-inoculated intranasally with . ml eagle's minimum essential medium (emem), glasgow modi®cation per nare (control group, nos. ± ); the remaining piglets (group ) were inoculated intranasally with . ml virus inoculum per nare, i.e. in total  : tcid pcv (pcv -infected group, nos. ± ). on postinoculation days (pids) and , one-half of the piglets from the control group (nos. ± ) and the pcv -infected group (nos. ± ), were intramuscularly immunised with . ml volume of . mg of klh emulsi®ed in icfa in four sites, both axillas and both hips ( . ml per site). clinical signs and body temperatures were recorded daily. blood samples were collected on pids , , , , , , and from all animals. the piglets were euthanized on pids and for control and pcv -infected animals, respectively, or earlier if found moribund. tissue samples of tonsil, liver, spleen, kidney, lung, thymus, and lymph nodes (mesenteric, bronchial, prescapular and super®cial inguinal) were collected at necropsy for virus isolation, virus titration, cryostat sectioning, quantitative polymerase chain reaction (q-pcr) analysis, and histopathological examination. sera were examined for antibodies against ppv by a blocking elisa (madsen et al., ). an immunoperoxidase monolayer assay (ipma) was carried out for the detection of antibodies against pcv and pcv . in brief, monolayers of pcv-infected pk- cell cultures were ®xed with ethyl alcohol in¯at-bottomed -well microtiter plates. for pcv , persistently infected pk- cells (atcc ccl- , usa) were used and for pcv , pcvfree pk- cells infected with the french pcv isolate (allan et al., ) were used. plates with ®xed pcv-infected cells were stored at À c after addition of % glycerol in phosphate buffered saline (pbs) and washed four times in pbs containing . % tween- (pbst) before use. plates were inoculated with ml of ®ve-fold dilutions at : to : of the serum samples diluted in pbst supplemented with % skimmed milk powder (pbst± % smp) and incubated for min at c. after adsorption, the plates were washed four times in pbst before addition of ml peroxidase conjugated rabbit anti-swine igg (dako p , denmark) diluted : in pbst± % smp followed by incubation for min at c. after the ®nal washing in pbst, ethylcarbazole substrate was added and the plates were left for min at room temperature. speci®c staining of cells indicated the presence of antibodies speci®c to pcv in the serum sample. the titer of a serum sample was determined as the highest dilution containing a detectable level of antibodies. due to cross reactivity between antibodies against pcv and pcv , the interpretation of the results included a comparison of the titers to pcv and pcv . if the titer was highest to pcv , the result indicated an infection with pcv . tissue homogenate suspensions ( %, w/v) in minimum essential medium with eagle's (mem) salts and l-glutamine (gibco brl, cat. no. - , life technologies tm ) containing . mg/ml neomycin, . mg/ml streptomycin supplemented with % foetal calf serum (fcs) were prepared: pool consisted of samples of lymph node tissue (prescapular and super®cial inguinal lymph nodes), pool consisted of lymphoid tissue (spleen, mesenteric and bronchial lymph nodes), pool contained samples of liver, lung, and kidney, whereas pool contained tonsil. pool was sterile ®ltrated before inoculation into cell cultures. the individual pools were examined for pcv by two passages in pcv-free pk- cells grown in mem containing . mg/ml neomycin, . mg/ml streptomycin, and % fcs (pk- medium). the day after cell passage, cultures were incubated with mm glucosamine in hank's salt solution for ± min at c for induction of pcv dna replication (tischer et al., ) . the glucosamine was removed and the monolayers washed with pk- medium. fresh pk- medium was added before incubation for additional or days at c, % co . cell cultures were ®xed in ice-cold absolute ethyl alcohol at c for min and peroxidase stained using a pcv -speci®c monoclonal antibody (mab), f (mcneilly et al., ) diluted : in pbst± % smp, as described for the ipma. in addition, a general virus examination of : mixtures of pools and was carried out by two passages in primary swine kidney cells cultured in emem containing antibiotics and fcs as described for pk- medium. the cell cultures were ®xed and stained for ppv, siv, transmissible gastroenteritis virus (tgev), hemagglutinating encephalomyelitis virus (hev), porcine enterovirus (pev), prcv, classical swine fever virus (csfv), and pcv and pcv using speci®c antisera. all organ pools positive for pcv by virus isolation in pk- cells were titrated in order to determine the infectious titer of the tissue homogenates. infectivity titrations were performed in -fold dilutions in -well microplates adding pk- cells per well in pk- medium. after days of incubation at c, % co , plates were ®xed and stained for pcv as described above. the titers were calculated according to the method of reed and muench ( ) . viral dna levels in serum samples and samples of tonsil, liver, spleen, kidney, lung, and lymph nodes (mesenteric and bronchial) was determined by a q-pcr method developed for the detection of both pcv and pcv . total dna was extracted from either serum or tissue using the qiaamp dna minikit extraction procedure according to the manufacturers instructions (qiagen gmbh, germany), and the concentration of doublestranded dna determined by¯uorometric measurements using picogreen (singer et al., ) . for serum samples, the equivalent of . ml serum was used as template for the pcr. for tissue, the equivalent of ng of double-stranded dna was used as template in the pcr. brie¯y, a forward primer ( h -gatgatctactgagactgtgtga) and a reverse primer ( h - -fam-agagcttctacagctgggaca) conserved between pcv and pcv was used for the pcr together with two probes speci®c for pcv ( h -gcccccc-aggaatggtactcctcxtp- h , where`x' is texas red) and pcv ( h -tcagacc-ccgttggaatggtactcctc-cy - h ). the pcr contained a ®nal concentration of  pcr gold buffer (applied biosystem, denmark), . mm mgcl , . mm dntp, . mm of each primer, . mm of each probe, . u of amp erase (applied biosystems, denmark), and . u of ampli taq gold dna-polymerase (applied biosystems, denmark). all reactions were carried out in triplicates on an abi (abi prism tm sequence detector, applied biosystem, denmark). the cycling conditions were one cycle of ( c for min and c for min) and cycles of ( c for s, c for s and c for s) followed by a melting curve analysis added for speci®city. fluorescence data collection was performed at each annealing step of the pcr with the increase in cy -¯uorescence being proportional to the amount of pcv dna in the sample. for each separate set of pcr reactions, two internal positive controls containing different but known amounts of pcv viral dna template were included. this allowed normalisation for variations in ampli®cation ef®ciency between different sets of pcr reactions. results are presented as mean values of the triplicate reactions. tissue samples of tonsil, liver, spleen, kidney, lung and lymph nodes (mesenteric and bronchial) were collected from each piglet and immediately ®xed by immersion into % paraformaldehyde, c for pathological examination or frozen in isopentane cooled over liquid nitrogen and stored at À c for cryostat sectioning. sections of paraf®n-embedded paraformaldehyde-®xed tissue were stained with hematoxylin and eosin for morphological evaluation. the severity of lesions were scored on a`' to`' scale. selected blocks of tissue were stained with pcv -speci®c polyclonal or monoclonal antibodies as previously described . all histological and immunohistochemical evaluations were performed by individuals who were not aware of the group designation. cryostat sections ( mm) were ®xed with acetone and stained for pcv antigen using the mab f diluted : in pbs without ca and mg (pbs-a) supplemented with % skimmed milk powder for h at c in a humidity chamber. slides were washed in pbs-a and binding of the mab was visualised by incubation for h at c in a humidity chamber with fitc-conjugated goat anti-mouse-ig (jirl - - , usa) diluted : in pbs-a supplemented with % swine serum. pcv positive cryostat sections were scored from`' to`' based on the number of infected cells. none of the uninfected control pigs showed any clinical signs of pmws-related symptoms during the experimental period. the group of pcv -inoculated piglets, however, appeared lethargic for a period of weeks starting on pid . within this period, all piglets experienced mild to more severe oedema in the eye lids. unthriftyness and jaundice were observed for pig no. (pid onwards). especially pig no. appeared very lethargic and suffered from reddened conjunctivae together with swelling and dirt around the eyes. this particular pig also showed dyspnoea on pid . pig no. had persistent fever (determined as body temperature above c) from pid onwards and piglet nos. ± from pid onwards. the remaining pcv -infected piglets had fever occurring intermittently for a single or for two to four consecutive days during the experimental period. pig nos. and showed inappetence on pid and were found dead on pid . pig no. was euthanized in moribund condition on pid . pig no. had to be euthanized on pid suffering from tremor and convulsions. aside from the local reactions to klh/icfa in piglet nos. ± , no gross lesions were seen in the uninfected control pigs at postmortem examination. pig nos. and which died on pid both showed ulceration of the stomach in the oesophageal region and generalised icterus. pronounced thymic and liver atrophy was seen in pig no. euthanized on pid . pig no. , euthanized on pid , did not present any obvious gross lesions. generally, only modestly enlarged lymph nodes were observed within the group of pcv infected pigs. for all pigs, decreasing antibody levels against ppv passively acquired (maternal antibodies) were detected during the experimental period indicating that no infection with ppv occurred. all pcv -inoculated piglets seroconverted to pcv with high titers against pcv compared to pcv . during the experimental period, the remaining two nonimmunostimulated pcv -infected pigs (nos. and ) had reached ipma-titres of against pcv whereas the remaining four immunostimulated pigs (nos. , , , ) had reached titers of (table ) . thus, within the experimental period the serologic response of the immunostimulated pigs appeared increased compared to the non-immunostimulated pigs. on pids and , the four pigs that died during the experiment (nos. ± , and ) had low levels of antibodies against pcv as detected by ipma compared to the remaining pcv -infected pigs (table ) . all control pigs, except pig no. , remained seronegative during the entire experimental period. pig no. seroconverted on pid with an ipma-titer of against pcv . pcv was isolated from all organ pools from the pcv -infected pigs ( table ). the infective titer of different tissues (pools ± as previously described) from each of the pcv -infected pigs are shown in table . tissue pools from pigs ± , and contained the largest quantities of infectious virus. titers ranged from to infectious units per gram of tissue homogenate. tonsil tissue contained a slightly lower amount of infectious virus than the remaining three tissue pools from these pigs, however this may be a result of the sterile ®ltration of the tonsil tissue. in contrast, only low levels of infectious pcv were detected for pigs necropsied at the termination of the experiment (pig nos. ± , , and ) . this observation possibly re¯ects that a larger amount of pcv antigen was present at an earlier stage during infection and that the four pigs most likely died at the peak of infection. no differences in infectious titers were observed between tissue originating from the immunostimulated pigs compared to tissue from the non-immunostimulated pigs. pcv was also isolated from organ pools ± originating from control pig no. which had seroconverted on pid . pcv was not isolated from tissues originating from the remaining control pigs. no virus except pcv was detected by the general virus examination in primary swine kidney cells. high levels of pcv dna were found in serum from the pcv -infected pigs from pid (fig. ) . all four piglets that died prior to termination of the experiment had the highest detected levels of viral dna load in serum indicating a correlation between the viral load and the development of pmws. pig nos. and had reached a maximum of viral dna load on pid and a similar level (above  template copies per ml of serum) was reached by pig nos. and on pid (fig. ) . interestingly, the highest number of pcv template copies detected was observed for the two immunostimulated and as early as on pid . for pig no. , however, the viral dna load rapidly declined as opposed to pig no. where a very high level of viral dna load persisted until this pig was euthanized days later. excluding pig nos. ± , and , immunostimulated pigs (nos. , , , and ) generally possessed higher levels of viral dna in serum than did the non-immunostimulated pigs (nos. and ). the student's t-test used to compare mean levels of viral dna templates detected by the q-pcr for these two groups of pigs on individual sample days did not reveal any statistically signi®cant differences. furthermore, comparison of the results obtained for all non-immunostimulated pigs with the results obtained for all immunostimulated pigs on pids , , , and while the original groups comprising ®ve piglets were still complete, did not reveal any statistical signi®cant differences (student's t-test) in the level of pcv template dna either. none of the examined serum samples was found positive for pcv . however, from a single of the control pigs, no. , pcv viral dna was detected in serum from pid onwards with levels of number of pcv template copies per ml serum. dna sequencing revealed that the pcv isolated from this pig was identical to the osu inoculum indicating a transmission of virus from the pcv -infected group to the control group during the experimental period. serum from the remaining controls were consistently pcv -negative, indicating that the virus did not spread among the remaining control pigs. at termination of the experiment, only a low number of pcv template copies were detected by q-pcr in various tissues from the pcv -infected pigs (table , pig nos. ± , , and ) . at this point, there were no differences in the distribution of pcv dna between tissues originating from immunostimulated pigs compared to non-immunostimulated pigs. for pig nos. ± , and , the load of viral dna per cell was up to higher within tissue samples at the time of death or euthanasia compared to the results for the remaining pigs euthanized at termination of the experiment (table ). the distribution of pcv dna, however, varied between these four individual pigs with samples of tonsil, liver, and lymph nodes containing the highest template copy number. as for the remaining pigs, no apparent effect of the immunostimulation was observed when comparing the q-pcr results obtained for tissue samples from pigs ± , and . the differences in distribution and load of viral dna between these samples likely re¯ect the differences in manifestation of disease within these four pigs. a low copy number of pcv templates per cell were detected by q-pcr in tissues from pig no. . levels similar to the viral dna load within samples from pigs euthanized weeks after infection were observed in lymphoid (spleen and lymph nodes) and lung tissue from this pig. pcv was not detected within tissue samples from the remaining control pigs. the q-pcr did not detect pcv in any of the tissue samples. immunostaining of cryostat sections revealed an abundance of pcv antigen in tissues collected from pigs ± , and which died during the experimental period (table ). for the remaining pcv -infected pigs, lymphoid tissues, i.e. spleen, tonsils, and lymph nodes contained the highest amount of viral antigen (table ). pcv antigen was also found at a low level in lung, spleen, and lymph nodes from control pig no. . pcv was not detected by immunostaining of cryostat sections within tissues from any of the remaining control pigs. À À À a the level of antigen detected in each tissue section was scored from a`' indicating low levels increasing to`' indicating abundant levels of pcv antigen;`±' no pcv antigen detected. b postinoculation day. c pig that died or was euthanized before termination of the experiment on pid . no signi®cant histological lesions were found in the sham-inoculated control pigs (nos. ± ). in contrast, three (nos. ± ) of ®ve pigs in the infected non-immunostimulated group had moderate to severe ( to ) lymphoid depletion in lymphoid organs including thymus, spleen, lymph nodes, and tonsils (fig. ) . this was associated with the presence of copious amounts of pcv antigen in mononuclear phagocytes and syncytial cells. these pigs also had moderate to severe ( to ) hepatic atrophy associated with nonsuppurative cholangiohepatitis. many kupffer cells and mononuclear phagocytes in these atrophic livers contained pcv antigen (fig. ) . the remaining two pigs, nos. and , in this group had mild ( to ) lymphoid depletion in lymph nodes. these pigs also had fig. . hepatic atrophy in a pcv -infected non-immunostimulated pig. immunohistochemical staining of a section of liver which demonstrates the distribution of the capsid structural protein of pcv in the liver of a pmws-affected pig. viral antigen is present in the cytoplasm of kupffer cells and infiltrating macrophages (arrows) as both multiple viral inclusion bodies and as diffusely distributed cytoplasmic product. hepatocytes are largely devoid of viral antigen. mild ( to ) non-suppurative cholangiohepatitis without signi®cant hepatic atrophy. two pigs, nos. and , in the non-immunostimulated group had mild non-suppurative interstitial nephritis, and two pigs, nos. and , had mild ( ) peribronchiolar thickening or hyperplasia of bronchus-associated lymphoid tissue. in the infected immunostimulated group, three pigs (nos. , , and ) had mild ( to ) lymphoid depletion in one lymph node. pig no. also had moderate ( to ) lymphoid depletion in the thymus, spleen, and tonsil. in addition, this piglet (no. ) which showed cns signs clinically had multifocal non-suppurative encephalitis. these lesions did not contain pcv antigen. all immunostimulated pigs had mild ( to ) non-suppurative cholangiohepatitis without hepatic atrophy and three of these pigs (nos. ± ) had mild ( to ) peribronchiolar thickening and/or hyperplasia of bronchus-associated lymphoid tissue. the present study was performed to examine the development of pmws in -week-old spf pcv -seronegative piglets. piglets of this age were used because under ®eld conditions, pmws primarily affects weaned pigs ± weeks of age (allan et al., ; ellis et al., ) , suggesting that infection with pcv occurs when the level of maternal antibodies is low around weaning at ± weeks of age. the results obtained within the present study describes for the ®rst time the reproduction of clinical pmws in colostrum-fed spf pigs experimentally infected with pcv alone irrespective of whether the pigs were immunostimulated or not. four of pigs infected with pcv developed severe pmws characteristic clinical disease and died prior to termination of the experiment at pid . in accordance with ®eld observations (clark, ; harding, ) , the observed clinical signs of pmws varied among individual pigs. a period with persistent fever was observed for each of the four pmws-affected pigs prior to development of clinical disease. the remaining pcv infected pigs experienced transient fever, yet, none of these pigs displayed overt clinical signs of infection. quanti®cation of pcv dna by pcr demonstrated that the four pmws-affected pigs contained extremely high levels of pcv dna in serum. these data suggest a direct association between an increased load of viral dna and clinical pmws in a pcv -infected pig. the highest amount of viral dna detected in serum was observed at pid for two individual pigs. interestingly, one of these pigs developed pmws and died whereas the level of pcv dna detected in serum from the other pig rapidly declined and this pig survived until termination of the experiment. thus, the potential pathogenic effects of an increased level of viral dna needs further investigation. sera from the pmws-affected pigs contained a lower level of pcv -speci®c antibodies compared to the non-diseased pigs. this observation could be explained by the presence of large amounts of pcv antigen binding a substantial amount of virus-speci®c antibodies that were no longer detectable by serologic examinations. furthermore, a dramatic reduction in the number of b lymphocytes observed in pcv -infected pigs with wasting disease syndrome has been reported (shibahara et al., ; segale Âs et al., ) that may also explain the low level of pcv -speci®c antibodies in the pmws-affected piglets. in addition, the elimination of b cells may result in compromised production of antibodies towards other agents and by that immunosupression and inability to respond to concurrent infections. gross and histological lesions in the pigs with clinical pmws were similar to those previously reported in both naturally acquired and experimentally induced disease rosell et al., ) . immunostaining of cryostat sections demonstrated an abundance of pcv antigen in various tissues from the diseased pcv -infected pigs at pids , , and . in contrast, lower amounts of pcv antigen primarily within lymphoid tissues was found weeks after infection in non-diseased pigs. these results were comparable with immunohistochemical staining of tissues. the results of virus isolation were in accordance with the results for immuno¯uorescence staining and pcv was isolated from tissue homogenates from all pcv -infected piglets. a marked difference between diseased and non-diseased pigs was observed with large amount of infectious pcv demonstrated in tissue homogenates from pmws-affected pigs. a similar pattern of pcv distribution was obtained by q-pcr analysis of tissues. five weeks after infection low pcv template copy numbers were detected, whereas high numbers were detected in various tissues from the diseased pigs at pids , , and . thus, in the four pcv -infected pigs that developed pmws extensive viral replication led to high amounts of pcv antigen widely distributed in various tissues. however, since the diseased pigs died or were euthanized ± days before termination of the experiment, there was no data concerning the amount and distribution pattern of pcv antigen at this point in tissues from pigs that did not develop clinical disease. so, the observed difference between diseased and non-diseased pigs regarding virus load may also re¯ect a time dependence. in the present study, development of clinical pmws was demonstrated in both immunostimulated and non-immunostimulated pcv -infected spf pigs for the ®rst time. in contrast to previous studies in neonatal gnotobiotic pigs , immunostimulation of weanling spf pcv -infected pigs had little apparent effect and was not required for the development of clinical pmws. in fact, three out of ®ve nonimmunostimulated pigs compared to one out of ®ve immunostimulated pigs developed severe clinical signs. there were no signi®cant gross pathological differences between non-immunostimulated and immunostimulated pcv -infected pigs although histologic lesions were generally more severe in the non-immunostimulated pigs. five weeks after infection, pcv was isolated from all tissue pools from all pcv -infected pigs with similar titers. furthermore, there were no indications of any immunostimulatory effect upon the distribution of pcv antigen detected by immuno¯uorescence or q-pcr within tissue samples originating from non-immunostimulated pigs compared to immunostimulated pigs. from pids to , there was a tendency for increasing pcv template copy numbers detected by q-pcr in serum from immunostimulated pigs compared to non-immunostimulated pigs but this difference was not statistically signi®cant. three to weeks after infection, the levels of pcv template copy number in serum were also slightly increased in the immunostimulated pigs compared to the non-immunostimulated pigs. again, there were no statistically signi®cant differences in the load of viral dna between the two experimental groups. all pigs infected with pcv seroconverted between pids and , and by pid , all immunostimulated pigs had higher antibody titers than the non-immunostimulated pigs. none of the non-immunostimulated reached this level of antibodies within the experimental period. thus, although immunostimulation was not essential for the development of pmws, it did apparently in¯uence the immune response to the virus. while immunostimulation was non-essential for development of clinical pmws in the present study, it was absolutely essential in the previous study by krakowka et al. ( ) . the immature immunological status of -day-old gnotobiotic pigs used by krakowka et al. ( ) may explain why these pigs were more susceptible to immunomodulation as further stimulation of the more mature immune system of -week-old spf pigs may have little effect on whether they will develop pmws or not upon infection with pcv . furthermore, another difference between the immunostimulation experiment of krakowka et al. ( ) and the present experiment is the fact that the spf piglets were exposed to external factors and other environmental parameters than the gnotobiotic piglets which were held in biologically secure isolators. whether such``additional immune activation'' is critical in the disease process is, however, not known at present. the ®nding of the present study also differs from most previous studies where coinfection with either ppv or prrsv has been required to induce clinical pmws kennedy et al., ; krakowka et al., ) . in that respect, however, the current study support the very recent ®ndings by bolin et al. ( ) and harms et al. ( ) that infection of caesarean-derived colostrum-deprived (cd/cd) pigs with pcv alone may result in development of clinical pmws. this apparent inconsistency between different studies on the requirement for co-infection or co-factors in inducing clinical pmws parallel in many ways the situation from the ®eld. antibodies against pcv are widespread within swine herds in both europe and north america (dulac and afshar, ; hines and lukert, ; tischer et al., ; labarque et al., ; rodriguez-arrioja et al., ) . it seems reasonable to believe that pcv infection is ubiquitous throughout the world. infection of a herd with pcv , however, often results in subclinical infection only (labarque et al., ; rodriguez-arrioja et al., ) . it is therefore critical to identify co-factors that have an in¯uence upon the development of pmws-related problems. differences in immunological status among herds and age of pigs at the time of pcv infection might still be part of the answer. immunological differences are probably more pronounced in the ®eld than under experimental conditions due to larger variability of management conditions and presence of other infectious agents. for example, such differences may explain why countries such as denmark, despite having a high pcv seroprevalence, have not yet experienced severe outbreaks of pmws as reported from other countries (segale Âs et al., ; allan et al., ; ellis et al., ; ladekjñr-mikkelsen et al., ) . as well, there could be as yet unrecognised differences in virulence among pcv isolates that are circulating in various pig populations. introduction of more pathogenic pcv strains could then potentially dramatically increase the rate of pmws-related problems in countries that as yet have not reported pcv -associated disease. another important unexplored co-factor could be genetic variability in pig populations. some pigs may simply be more susceptible to infection with pcv and or may be more permissive for viral replication, and, therefore, may be more at the risk of developing pmws. further examination of material from pmws-affected pigs and pigs without clinical symptoms is necessary to determine whether the q-pcr could serve as diagnostic tool. by qualitative pcr analysis of various tissues from randomly tested diseased pigs with a wide variety of clinical signs and lesions, hamel et al. ( ) detected pcv in more than half the tested individuals. if a certain level of pcv dna is critical for the development of pmws, the q-pcr could discriminate between subclinical and clinical pcv infection and allow a de®nitive diagnosis of pmws. in conclusion, this study for the ®rst time demonstrated the development of clinical pmws by infection of -week-old spf pigs with pcv alone. the effect of immunostimulation of the pcv -infected pigs was found not to be essential for the development of clinical disease. porcine circoviruses: a review pathogenesis of porcine circovirus experimental infections of colostrum deprived piglets and examination of pig foetal material isolation of porcine circovirus-like viruses from piglets with a wasting disease in the united states of america and europe experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv replication postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type porcine circovirus porcine post-weaning multisystemic wasting syndrome in korean pig: detection of porcine circovirus infection by immunohistochemistry and polymerase chain reaction post-weaning multisystemic syndrome porcine circovirus antigens in pk- cell line (atcc ccl- ) and evidence of antibodies in canadian pigs isolation of circovirus from lesions of piglets with postweaning multisystemic wasting syndrome reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome pcr detection and characterization of type- porcine circovirus post-weaning multisystemic wasting syndrome (pmws): preliminary epidemiology and clinical presentation experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus porcine circovirus: a serological survey of swine in the united states. swine health prod reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type alone or in combination with porcine parvovirus viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus and porcine parvovirus activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus- (pcv- ) seroprevalence of porcine circovirus types and in the belgian pig population transplacental infection with pcv- associated with reproductive failure in a gilt detection of antibodies against porcine parvovirus nonstructural protein ns may distinguish between vaccinated and infected pigs experimental transmission of porcine circovirus type (pcv ) in weaned pigs: a sequential study retrospective serological survey of antibodies to porcine circovirus type and type production, characterisation and application of monoclonal antibodies to porcine circovirus characterization of immune response of young pigs to porcine circovirus type infection serum antibodies to porcine circovirus type and type in pigs with and without pmws pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (pmws) in pigs first report of post-weaning multisystemic wasting syndrome in pigs in spain changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting disease (pmws) porcine circovirus induces b lymphocyte depletion in pigs with wasting disease syndrome characterization of picogreen reagent and development of a fluorescence-based solution assay for double-stranded dna quantitation studies on epidemiology and pathogenicity of porcine circovirus replication of porcine circovirus: induction by glucosamine and cell cycle dependence distribution of antibodies to porcine circovirus in swine populations of different farms development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type this work was partly funded by the european union (project qlk -ct- - ) and (canadian) natural sciences and engineering research councilÐcollaborative research opportunities grant - . key: cord- - s b ovi authors: decaro, nicola; campolo, marco; desario, costantina; ricci, dominga; camero, michele; lorusso, eleonora; elia, gabriella; lavazza, antonio; martella, vito; buonavoglia, canio title: virological and molecular characterization of a mammalian orthoreovirus type strain isolated from a dog in italy date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: s b ovi a mammalian orthoreovirus (mrv) strain was isolated from a pup with fatal diarrhea, which had a concurrent infection by canine parvovirus type . the reovirus isolate showed an atypical hemagglutination pattern and a retarded electrophoretic mobility of the s segment, which is characteristic of mrv type (mrv- ). assignment of the isolated virus to mrv- was confirmed by type-specific rt-pcr assays, targeting the s gene, and by subsequent sequence analysis of the pcr product. by phylogeny based on the s gene of several mrvs, the isolate fell into lineage e, along with the murine strain t c / and the bovine strains t c / and t c / . conversely, l sequences were found to segregate regardless of the viral type. a total of fecal samples, nasal and ocular swabs from dogs with diarrhea or nasal/ocular discharge were tested by a nested-pcr assay specific for reoviruses, and no sample was found to contain mrv rna, a finding that is apparently in contrast with the seroprevalence ( . %) observed in dogs. mammalian orthoreovirus (mrv) includes nonenveloped, double-stranded (ds) rna viruses belonging to the genus orthoreovirus within the family reoviridae, which are responsible for either symptomatic or asymptomatic infections in mammals and possess a broad host range (tyler, ) . their genome contains dsrna segments, which are designed as large (l, three segments), medium (m, three segments) or small (s, four segments) on the basis of the electrophoretic mobility (nibert and schiff, ) . three mrv serotypes have been recognized so far, which are distinguishable by means of the capacity of www.elsevier.com/locate/vetmic veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] anti-reovirus sera to neutralize viral infectivity and inhibit hemagglutination (ha) (rosen, ; sabin, ) . neutralization and ha activities are restricted to a single reovirus gene segment, s (weiner and fields, ) , that encodes for s and s s proteins. the s protein, a fibrous trimer located on the outer capsid of the virion (fraser et al., ; furlong et al., ) , is responsible for viral attachment on cellular receptors (lee et al., ; weiner et al., ) , serotype-specific neutralization (bassel-duby et al., ) and hemagglutination (weiner et al., ) . analysis of the s gene of mrvs belonging to different serotypes has shown a strict correlation between sequence similarity and viral serotype (cashdollar et al., ; duncan et al., ; nibert et al., ) . conversely, the other genome segments show no correlation to viral serotype, suggesting that mammalian reoviruses have evolved independently of serotype (breun et al., ; chappel et al., ; goral et al., ; kedl et al., ; leary et al., ) . mammalian reoviruses have a wide geographic distribution and can virtually infect all mammals, including humans (tyler, ) . in carnivores, mrv infections have been sporadically reported, although all the three serotypes have been isolated from dogs and cats (binn et al., ; csiza, ; kokubu et al., ; lou and wenner, ; marshall et al., ; massie and shaw, ; mochizuki and uchizono, ; scott et al., ) . in the present study, the isolation and molecular characterization of a mrv- strain from a dog with diarrhea are reported. rectal swabs from two borzoi greyhounds ( / -a and / -b), belonging to the same litter, were submitted to our laboratory for virological investigations. the pups, months of age, were affected by vomiting and diarrhea, that was bloody in pup / -b. pup / -b died within days from the onset of clinical signs, and it was not possible to perform necropsy. pup / -a underwent a rapid recovery. neither respiratory nor ocular signs were reported by the owner. both the rectal swabs resulted positive to canine parvovirus type (cpv- ) by a real-time pcr assay , showing titers of .  and .  cpv- dna copies per milligram of feces for pups a and b, respectively. both cpv strains were characterized as glu- mutants (buonavoglia et al., ) . the rectal swabs were homogenized in eagle's minimal essential medium (e-mem), treated with antibiotics and inoculated onto freshly trypsinized crandell feline kidney (crfk) cells. the inoculated cells were monitored by an indirect immunofluorescence (if) assay using a cpv- monoclonal antibody. cells inoculated with sample / -a showed no cytopathic effect (cpe), in spite of a strong cpv- specific intranuclear fluorescence, whereas, unexpectedly, in the cells inoculated with sample / -b a cpe was observed, that was characterized by rounding of the infected cells and deterioration of the cell monolayer, in spite of a very weak signal by the if assay for cpv- . in order to prevent cpv- replication on cell cultures, cpv- was selected against by using a monoclonal antibody (a e ). there was no evidence for growth of cpv- in the subsequent passages, as shown by both if and real-time pcr assays. in contrast, the cpe progressively increased, with complete destruction of the inoculated monolayers. the inoculated cells were stained with hematoxylin-eosin (he) for detection of inclusion bodies. negative staining electron microscopy examination was carried out on the cryolysate of the infected cell cultures showing evident cpe. two-fold dilutions in phosphate buffered saline of the isolated virus (cryolysate of the sixth passage on crfk cells), with a titer of . tcid / ml, were subjected to ha assays using -well microtiter plates and % red blood cells (rbc). human ( , a, b and ab), cow, sheep, goat, pig, dog, cat, rabbit and mouse rbc were tested at c room temperature and c. for page, dsrna was extracted from the infected crfk cells using the guanidine thiocyanate/glass milk method (gentsch et al., ) , run in a % polyacrylamide gel at constant current of ma for h and visualized by silver staining as previously described (dolan et al., ) . the rectal swabs from the two dogs and the sixth serial passage of the corresponding inoculated cells were subjected to pcr and rt-pcr assays for the detection of the main viral pathogens of dogs, i.e. canine adenovirus type and type (hu et al., ) , canid herpesvirus type (schulze and baumgartner, ) , canine coronavirus (ccov) (pratelli et al., ) , feline and canine caliciviruses (hashimoto et al., ; marsilio et al., ) and rotaviruses (gouvea et al., ) . rna for the detection of reoviruses was extracted using the guanidine thiocyanate/glass milk method, following the protocol described by gentsch et al. ( ) . for the detection of mrv a nested-pcr assay was performed (leary et al., ) , using primer pairs l -rv /l -rv for rt-pcr and l -rv /l -rv for nested-pcr (table ) . as neither monoclonal antibodies nor polyclonal antisera specific to the three mrv serotypes were available in our laboratory, we developed rt-pcr assays able to differentiate the mrv serotypes, which were used to characterize the mrv strain isolated from sample / -b. in the genbank database, several s sequences of mrv- are available and this allowed us to select primers in highly conserved regions. conversely, type -and type -specific primers were designed on the basis of the s sequence data of a few strains, i.e. the reference strains t l/ and t j/ for mrv- and mrv- , respectively (table ) . the entire s gene was amplified using primers ent-s -r f and ent-s -r r (table ) binding the and ends of the s segment, respectively. the pcr products obtained with primer pairs l -rv /l -rv (l gene, bp) and ent-s -r f/ ent-s -r r (s gene, bp) were cloned and subjected to sequence analysis (genome express, meylan, france). alignments and sequence analysis were performed using the bioedit software package (hall, ) . the nucleotide sequences are available in the ddbj/embl/genbank databases under accession nos. ay (partial l gene) and ay (s gene). the nt sequences of the amplified fragments were aligned with a ent-s -r f d gctattggtcggatggat s type + - ent-s -r r d gatgaaatgccccagtgc À - a leary et al., . primer position is referred to the l sequence of t l/ (accession: nc _ ). b primer position is referred to the s sequence of t l/ (accession: m ). c primer position is referred to the s sequence of t j/ (accession: m ). d primer position is referred to the s sequence of t d/ (accession: m ). selection of mrv reference strains belonging to the three serotypes (table ) . phylogenetic and molecular evolutionary analyses were conducted using mega (kumar et al., ) . parsimony trees were elaborated using a heuristic algorithm and supplying statistical support by bootstrapping over replicates. a total of fecal samples, nasal swabs and ocular swabs were subjected to nested-pcr for detection of mrv (leary et al., ) . the fecal samples were collected from dogs with diarrhea and included samples previously tested positive to ccov by a real-time rt-pcr assay (decaro et al., ) , samples previously tested positive to cpv- by a real-time pcr assay , samples positive both to ccov and cpv- and samples negative to ccov and cpv- . in addition, serum samples collected from dogs of different ages and different geographic origin were tested for antibodies to the mrv isolate by a virus neutralization (vn) assay, using crfk cells and tcid / ml of the virus. at day after the onset of clinical signs, a serum sample was also collected from pup / -a. an mrv strain was isolated on crfk cells from the rectal swab of pup / -b. the cpe was initially characterized by rounding of the infected cells and, after a better adaptation to in vitro growth, by complete destruction of the cell monolayer. at the sixth passage on crfk cells, the isolate reached a titer (fig. ) . by he staining, perinuclear intracytoplasmic inclusions typical of mrv were detected in the infected cells (fig. ) . the mrv strain showed an atypical ha pattern, as it displayed ha activity ( : ) using pig rbc. interestingly, no ha was observed using human type rbc, a pattern that is usually showed by all mammalian reoviruses (tyler, ) . low ha titers ( : ) were obtained using rabbit rbc. unlike other mrv- strains (dermody et al., a; lerner et al., ) , the isolate showed a weak ha activity to bovine rbc and only using high amounts of virus. by page and subsequent silver staining, the dsrna purified from the infected cells showed an electrophoretic pattern typical of mrv, although comigration of the l and l segments was observed (fig. ) . the largest of the s-class gene segments (s ) migrated slowly, which is a characteristic of mrv- strains (hrdy et al., ) . rt-pcr and nested-pcr specific for mrv, carried out on a conserved region of the l gene, yielded products of the expected size ( and bp, respectively) only for specimen / -b and for the corresponding inoculated crfk cells, while no amplification was obtained from sample / -a and the corresponding inoculated cells (fig. ) . by the type-specific rt-pcr assays the isolate was recognized as mrv- (fig. ) , and therefore designated t / canine/italy/decaro/ (t d/ ), according to the conventional system used to identify mrv strains. sequencing of the amplicon generated by primer pair s -r f/s -r r confirmed the characterization obtained by the rt-pcr genotyping assay, and the isolate was definitively assigned to mrv- . comparison of s and partial l nt sequences of strain t d/ with the sequences retrieved from the databases is reported in table . the l fragment displayed the highest nt sequence identity ( %) to the reference strain t d/ . phylogeny showed that the l gene sequences segregate into lineages irrespective of their serotype (fig. a) , confirming the results of a previous study (leary et al., ) . the s gene of strain t d/ revealed the closest nt sequence identity to the murine strain t c / ( %). strain t d/ exhibited % positional identity to reference strain t d/ and % nt identity to the novel human strain t c/ , recently isolated from a child with meningitis (tyler et al., ) . by phylogenetic analysis, a sixth lineage, represented by the type highly divergent t c/ strain, was added to the five lineages previously reported (dermody et al., b) with isolate t d/ falling into lineage e along with the murine strain t c / and the bovine strains t c / and t c / (fig. b) . by nested-pcr, mrv rna was not detected in any of the canine samples analyzed, suggesting that reoviruses are not widespread in the sampled dog population. however, antibodies to mrv were detected in ( . %) out of the sera tested by vn. antibody titers ranging from : to : were detected in dogs, whereas vn antibody titers > : were found in five animals, with the highest titer ( : ) in the recovered pup / -a. the present study represents the first molecular characterization of a reovirus strain isolated from a dog. to date, there are a few reports on the epidemiology of reoviruses in dogs, although all the three mrv serotypes have been isolated in such species (binn et al., ; kokubu et al., ; lou and wenner, ; massie and shaw, ) . mrv- strains have been recovered from dogs with pneumonia (lou and wenner, ) or enteritis (appel, ) , in association with either canine distemper virus or cpv- . mrv- and mrv- have been isolated from dogs with upper respiratory tract disease (binn et al., ) and diarrhea (kokubu et al., ) , respectively. however, to our knowledge, none of the isolates was characterized at the molecular level and no sequence data is available in the databases. as in other mammalians, the pathogenetic role of reoviruses in dogs is still unclear, since attempts to reproduce a specific disease in germ-and disease-free dogs fig. . strain characterization by the type -specific rt-pcr assay targeting the s segment (primer pair s -r f/s -r r, pcr product of bp). m (marker generuler bp dna ladder, mbi fermentas gmbh, germany); t d (strain t d/ ); t a (strain t a/ ); bf (sample / -b, feces); t l (strain t l/ ); t j (strain t j/ ); n (negative fecal sample). resulted in contrasting findings (appel, ; holzinger and griesemer, ) . nevertheless, it has been suggested that mrv infection may act in synergism with other pathogens, aggravating the course of concomitant infections (appel, ) . herewith, we described a double infection by mrv and cpv- in a dog died from severe enteritis. mrv was not detected in the cpv- infected dog that survived, even if mrvspecific antibodies were detectable weeks after the onset of the disease, indicating that the dog had been exposed to mrv- infection as well. strain t d/ displayed an atypical ha pattern, as it was able to agglutinate only pig rbc. unlike other mrv strains (tyler, ) , this isolate did not fig. . maximum parsimony trees based on partial l (a) and s (b) nucleotide sequences of mrv strains. accession numbers of the strains used for phylogeny are reported in table . trees are unrooted and drawn to scale. bootstrap values were calculated and are indicated at each node. agglutinate human type rbc and unlike other mrv- strains (dermody et al., a; lerner et al., ) , ha was weak using bovine rbc. reoviruses possess a wide host range and naturally occurring mrvinfections have been described in cattle, sheep, pigs, horses, dogs, cats, mice, as well as in human and non-human primates (tyler, ) . in mice and in non-human primates, reovirus infections can affect the respiratory, gastrointestinal and nervous systems (tyler, ) . in humans, mammalian reoviruses are mainly responsible for respiratory and/or enteric disease. however, mrv- infection has been associated to a case of meningitis in a child (tyler et al., ) . the ability to induce neurological disease is a characteristic of mrv- and a neurovirulent mrv- strain has been isolated from a cat with ataxia (csiza, ) . because of the apparent lack of species barriers, mrvs may potentially spread from animals to humans and vice versa. from this perspective, further studies would help obtaining a more in-depth understanding of the ecology of reoviruses. virus infections of carnivores identification of attenuating mutations on the reovirus type s double-stranded rna segment with a rapid sequencing technique recovery of reovirus type from an immature dog with respiratory tract disease mammalian reovirus l gene and l core spike protein sequences and whole-genome comparison of reoviruses type lang evidence for evolution of canine parvovirus type in italy the sequence of the s genes of the three serotypes of reovirus sequence diversity within the reovirus s gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif characterization and serotyping of three feline reovirus isolates a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr a sigma region important for serotype reovirus strains sequence diversity in s genes and s translation products of serotype reovirus strains epidemiology of rotavirus electropherotypes determined by a simplified diagnostic technique with rna analysis identification of conserved domains in the cell attachment proteins of the three serotypes of reovirus molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions sigma protein of mammalian reoviruses extends from the surfaces of viral particles identification of group a rotavirus gene types by polymerase chain reaction sequence diversity within the reovirus s gene: reoviruses evolve independently of host species, geographic locale, and date of isolation identification of bovine and porcine rotavirus g types by pcr bioedit: a user-friendly biological sequence alignment and analysis program for windows / /nt genetic analysis of the rna polymerase gene of caliciviruses from dogs and cats effects of reovirus type on germfree and disease-free dogs polymorphism of the migration of double-stranded rna genome segments of reovirus isolates from humans, cattle, and mice detection and differentiation of cav- and cav- by polymerase chain reaction comparative sequence analysis of the reovirus s genes from serotype and serotype field isolates isolation of a reovirus type from dogs with diarrhea mega : integrated software for molecular evolutionary genetics analysis and sequence alignment detection of mammalian reovirus rna by using reverse transcription-pcr: sequence diversity within the l -encoding l gene protein s is the reovirus cell attachment protein hemagglutination with reoviruses natural and experimental infection of dogs with reovirus type : pathogenecity of the strain for other animals virus and virus-like particles in the faeces of cats with and without diarrhea nested pcr for the diagnosis of calicivirus infections in the cat reovirus type in laboratory dogs experimental infections of feline reovirus serotype isolates structure of the reovirus cell-attachment protein: a model for the domain organization of s reoviruses and their replication development of a nested pcr assay for the detection of canine coronavirus serologic groupings of reovirus by hemagglutination inhibition nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies feline reovirus: isolation, characterization, and pathogenicity of a feline reovirus mammalian reoviruses isolation and molecular characterization of a novel type reovirus from a child with meningitis interaction of reovirus with cell surface receptors. i. murine and human lymphocutes have a receptor for the emagglutinin of reovirus type neutralization of reovirus: the gene responsible for the neutralization antigen identification of the gene coding for the hemagglutinin of reovirus we are grateful to dr. michele muscillo (istituto superiore di sanità, rome, italy) for providing the reference mrv strains and dr. colin r. parrish (cornell university, ithaca, ny, usa) for providing monoclonal antibodies to cpv- . key: cord- -xpdtt ej authors: ohshima, t.; kawakami, k.; abe, t.; mochizuki, m. title: a minute virus of canines (mvc: canine bocavirus) isolated from an elderly dog with severe gastroenteritis, and phylogenetic analysis of mvc strains date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: xpdtt ej two of the three adult dogs kept in a family developed severe gastroenteritis. from the feces of one of the affected dogs a minute virus of canines (mvc) was detected by pcr and virus isolation. that this virus had recently infected the dogs was indicated by high anti-mvc antibody titers of their sera. no other virus commonly associated with canine gastrointestinal disease was implicated. as no previous association of mvc infection and disease in aged dogs had been described, further characterization of the isolated virus was performed to determine if it had unique pathogenic or genetic properties. experimental infection of adult dogs did not result in clinical disease and comparison of the viral genome with other mvcs did not reveal any novel elements. the american, japanese and korean mvc strains studied were closely related to bocaviruses of bovine and human origin, and appeared to have evolved uniquely in the dog population after dividing from the common ancestor of bocaviruses. further detailed clinical and virological studies are warranted to define the role of mvcs in disease in adult dogs. canine parvovirus type (cpv- ), canine coronavirus (ccov), canine adenoviruses (cav), and canine distemper virus (cdv) are well-established pathogens of pups as well as adult dogs that possess either no or insufficient immunity. by contrast, another canine virus, minute virus of canines (mvc) (binn et al., ) has been considered to be a ''non-pathogenic orphan virus'' (carmichael, ) . mvc, also known as canine parvovirus type (carmichael, ) or canine bocavirus (manteufel and truyen, ) , and cpv- are unrelated parvovirus species belonging to different genera of the parvoviridae (macartney et al., ; mochizuki et al., ; schwartz et al., ) . however, subsequent experimental studies revealed that mvc is a perinatal pathogen of dogs (carmichael, ; macartney et al., ) . clinical discrimination of the etiology of contagious pneumoenteric diseases of dogs is generally difficult, and at present, diagnosis for mvc infection is available only from particular research laboratories. although only a limited number of studies have been conducted, some features of mvc and its infection have been established. thus, mvc is distributed worldwide among domestic dogs of all ages (carmichael et al., ; hashimoto et al., ; jang et al., ; jä rplid et al., ; mochizuki et al., ; pratelli et al., ; truyen et al., ) . the virus is pathogenic for the fetus and infection of pregnant bitches results in abortion (carmichael et al., ; truyen et al., ) . mvc infection of young pups, typically less than -month old, may be either asymptomatic or cause a milder illness than that caused by cpv- (carmichael et al., ; macartney et al., ) , although two of the three adult dogs kept in a family developed severe gastroenteritis. from the feces of one of the affected dogs a minute virus of canines (mvc) was detected by pcr and virus isolation. that this virus had recently infected the dogs was indicated by high anti-mvc antibody titers of their sera. no other virus commonly associated with canine gastrointestinal disease was implicated. as no previous association of mvc infection and disease in aged dogs had been described, further characterization of the isolated virus was performed to determine if it had unique pathogenic or genetic properties. experimental infection of adult dogs did not result in clinical disease and comparison of the viral genome with other mvcs did not reveal any novel elements. the american, japanese and korean mvc strains studied were closely related to bocaviruses of bovine and human origin, and appeared to have evolved uniquely in the dog population after dividing from the common ancestor of bocaviruses. further detailed clinical and virological studies are warranted to define the role of mvcs in disease in adult dogs. ß elsevier b.v. all rights reserved. more serious disease may occur under certain field conditions (harrison et al., ; jä rplid et al., ; pratelli et al., ) . by contrast, the pathogenic potential of mvc for adult dogs has been considered to be minimal (binn et al., ; carmichael et al., carmichael et al., , hashimoto et al., ; mochizuki et al., ) . genetic analysis has shown that the genomic structure of mvc is similar to those of bovine parvoviruses (bpv) and human bocaviruses (hbov), and the genome size of an american ga strain is nucleotides in length (schwartz et al., ; sun et al., ) . in this context, we had an opportunity to study a new strain of mvc. in a small group of dogs housed together an elderly dog was taken ill with signs of severe gastroenteritis, suspected initially to be due to cpv- . contrary to our expectations, a mvc was the only virus recovered in cell culture from a fecal specimen from the dog. because to our knowledge this is the first time that mvc had been isolated from an old diseased dog, the isolate was subsequently examined for any unique features, focusing especially on its virulence for dogs and its genomic properties. three dogs, one male yorkshire terrier ( years and months old) and two female chihuahuas ( -and -year old), had been kept together for more than year. the dogs had been vaccinated annually with combined canine vaccines that had no ccov component. the yorkshire terrier was taken to a veterinary clinic because of vomiting in november, . on the third day of hospitalization, the dog had bloody diarrhea in addition to vomiting. cpv- was considered as a possible cause but since a fecal specimen was found to be negative by a commercial test kit for cpv- , it was submitted to our diagnostic laboratory for further virological examination. the dog received symptomatic therapy and subsequently recovered. the older chihuahua also had similar signs just before the present case; however, the cause could not be established because no specimen was available for examination. the younger chihuahua did not show any clinical signs throughout the period. serum samples were taken from all three dogs in december, , about month after the incident. the fecal swab specimen obtained from the yorkshire terrier was placed in ml of eagle's minimal essential medium and the extract was clarified by centrifugation at , rpm for min. the resulting supernatant fluid was examined for viruses. for virus isolation, the supernatant was inoculated into both madin-darby canine kidney (mdck) and felis catus whole fetus- (fcwf- ) cell (pedersen et al., ) cultures. the cultures were examined for cytopathic effect (cpe) and were subsequently blindpassaged twice when no cpe was apparent. detection of ccov, cdv, cpv- , canine respiratory coronavirus (crcov), and mvc was attempted by the molecular methods described previously (mochizuki et al., ohshima et al., ; yachi and mochizuki, ) . since the fecal specimen was found to contain either mvc or a related gene fragment by the pcr test, mvc isolation in the mdck cell culture and subsequent specific virus identification were performed by the methods described previously (mochizuki et al., ) . anti-mvc neutralization (nt) antibody in the dog serum samples was determined by the method described previously (mochizuki et al., ) . the serum samples were also examined for antibodies against cav- , cdv, cpv- , canine parainfluenza virus (cpiv), and ccov which are the components of commonly employed combined canine vaccines by the routine methods in our laboratory (see legend to table ). three -month-old spf beagles were used. the dogs had been vaccinated with a canine combined vaccine twice when and weeks old. they did not possess anti-mvc nt antibody before challenge. the present mvc isolate - , passaged times in mdck cell culture, was used as inoculum. each dog received ml of the culture fluid containing tcid of virus by intraoronasal instillation. the clinical condition of each dog was observed for weeks after challenge. rectal swab samples were taken periodically (before inoculation, and on days , , , , and after inoculation) to detect virus excretion by pcr. blood samples were obtained before inoculation and on day after inoculation to determine serum anti-mvc nt titers. in addition to the present isolate - , two previous mvc isolates - and - from japanese dogs (mochizuki et al., ) were used for genome sequencing. genome dna was amplified from the infected-mdck cell culture fluid by the method described previously (mochizuki et al., ; ohshima et al., ) and an almost full-length nucleotide sequence, excepting the -and -terminal palindromes, was obtained for each isolate. the pcr product was purified and sequenced directly with an abi prism bigdye terminator version . cycle sequencing kit on an abi prism genetic analyzer (applied biosystems inc.). the nucleotide sequence was analyzed by the methods described previously (ohshima et al., ) . mvc strains reported thus far were the american ga (af and fj ) and korean hm- (ab ) strains. the nucleotide sequence data of mvc strains - , - , and - first described in the present study are available in the ddbj/embl/genbank databases with accession nos. ab , ab , and ab , respectively. the other parvoviruses used in the phylogeny study are specified in the legend to fig. . a fecal swab was obtained from the affected dog and pcr was carried out on an extract. although no positive pcr amplification was obtained for ccov, cdv, cpv- or crcov, a product specific for mvc was generated (data not shown). subsequently mvc was isolated in mdck cell culture accompanied by the formation of typical intranuclear inclusion bodies that contained mvc-specific antigen (data not shown). this isolate was designated as mvc strain - and was characterized further. no other viral agent was recovered from the specimen in either mdck or fcwf- cell cultures. the serum samples obtained from the three dogs contained antibodies at various titers against cav- , cdv, cpiv, and cpv- , but relatively low or no antibody titer was found against ccov (table ). the titers were not sufficiently high to suggest recent exposure to these viruses. in contrast, the antibody titers against mvc were high enough to indicate a recent infection. none of the experimental dogs showed any clinical signs or shed virus in the feces during the weeks following challenge. however, anti-mvc nt titers of the serum samples taken weeks after challenge were elevated between and indicating that the virus had replicated in vivo. nucleotide sequences composed of and bases were obtained for isolates - and - , and isolate - , respectively, which represented about . % of the mvc genome. in the case of isolate - , one base deletion was found in the untranslated region. the same three open reading frames (orfs) were predicted from the genome sequence of each isolate. the left-hand orf was predicted to encode the non-structural protein ns composed of amino acid (aa) residues. the righthand orf was predicted to encode aa residues, encoding the overlapping capsid proteins vp ( aa residues) and vp ( aa residues). the central orf that overlapped with the ns and vp / orfs was predicted to encode the non-structural protein np and consisted of aa residues. the length of each orf of the present mvc isolate - was the same as those of other mvc isolates including the american ga (schwartz et al., ) and the korean hm- (ohshima et al., ) strains. in addition, neither base insertion nor deletion was detected in the genome sequence of isolate - . among all mvc strains, including the american and korean strains, the predicted amino acid sequence identities ranged from . % to . % for ns , from . % to . % for vp / and from . % to % for np . a noteworthy difference was found in the np identities among mvc strains: complete sequence identity was obtained for those between japanese and american strains, while the sequence identity between korean and japanese/ american strains was . %. two kinds of unrooted phylogenetic tree were made based on the nucleotide sequence alignments of the full genome size data that were available for each parvovirus species. the newly characterized japanese mvc strains together with the previous american and korean mvc strains belonged to the branch of the genus bocavirus with bpv and hbov (fig. ) ; and mvc strains showed a close mutual evolutionary relationship that was distant from different bocavirus species. on the other hand, as shown in fig. , there was a mutual relationship among mvc strains isolated from american, japanese and korean dogs. although the available data may not be sufficient to make a definite conclusion, they suggested that each branch was based on their geographical origin. the genus bocavirus was originally assigned to a small virus group comprising bpv and mvc. subsequently hbov was added as the third member and more recently evidence of porcine bocavirus was found associated with postweaning multisystemic wasting syndrome of pigs (blomströ m et al., ) . characteristically the bocaviruses appear to be pathogenic in very young animals, and mvc is no exception. both experimental and spontaneous field case studies suggest that mvc causes mild to severe pneumonitis and/or enteritis in newborn pups (carmichael et al., ; harrison et al., ; jä rplid et al., ; pratelli et al., ) but is relatively non-pathogenic in older dogs. therefore the association of mvc with an old diseased dog, as described here, is unusual. in addition to the recovery of mvc from the sick dog, the blood samples taken after recovery from the disease were found to possess anti-mvc nt antibody titers between and (table ) . considering the serological data reported previously (carmichael et al., ; hashimoto et al., ; mochizuki et al., ) , higher antibody titers, in the hundreds for example, may be used as grounds for indicating recent exposure to mvc. in this context, mvc was strongly suspected of being associated with the clinical signs of gastroenteritis. in the household, the two older dogs recovered from the illness and in the youngest dog the infection may have taken a subclinical course. this outcome is, however, contrary to the current concept of canine mvc infection (carmichael, (carmichael, , and suggested that the present mvc isolate - might be a novel strain which is pathogenic for adult dogs. subsequently this was not confirmed by the experimental infection of the adolescent spf dogs. in simple terms, the present animal experiment results may reflect the most probable relationship that occurs between mvc and aged dogs in the field: mvc infection of aged dogs results in antibody production but is accompanied only rarely by virus shedding so that mvc has not been found in most field cases. if this is true, the present field case with disease signs and virus shedding is a peculiar clinical experience. as described above, we could not identify any particular pathological characteristics of the present mvc isolate - to explain the present field case of disease. in addition, it had neither insertion nor deletion in the genome sequence and was not a recombinant between other parvoviruses including cpv- . that is, the present mvc isolate - seems to be molecularly very similar to previous mvc strains (schwartz et al., ; ohshima et al., ) . therefore, it is uncertain why the clinical signs suspected of cpv- infection appeared in this case and how mvc played a part in the development of the illness. there is a possibility that some host factors and/or accidental secondary pathogens influenced the disease course, and it may be worthwhile to perform experimental infection of immunosuppressed dogs, for example, to determine if mvc is pathogenic in these conditions. in any event, it would be useful to carry out more field case studies, with mvc included in the list of viruses for routine examination in small animal veterinary clinics. because only a few mvc strains were available (binn et al., ; mochizuki et al., ) , the genetic relation between mvc strains of different origin has not been studied previously. in the present study, the genomes of five mvc strains were compared. all mvcs have diverged from the common ancestor of bocaviruses and have evolved uniquely in the dog population worldwide (fig. ) . a korean mvc is genetically distant from american and japanese mvcs especially when the np genes were compared. mvcs may have evolved further within each geographical region (fig. ) , but the analysis of more mvc isolates would be necessary to confirm such a conclusion. in conclusion, a mvc strain was the only virus recovered from an elderly dog that showed severe signs of gastroenteritis. serological analysis showed that the dogs in the same house had high nt antibodies against mvc, suggesting a close association of mvc with the clinical signs. however the mvc isolate was not pathogenic in aged dogs infected experimentally. it will be necessary to accumulate further field cases to elucidate the clinical significance of mvc as a canine pathogen. phylogenetic analysis of mvc strains including the present mvc isolate suggested a possibility of geographically dependent evolution pattern of mvc. recovery and characterization of a minute virus of canines detection of a novel porcine boca-like virus in the background of porcine circovirus type induced postweaning multisystemic wasting syndrome canine parvovirus type (minute virus of canines) neonatal pup diseases. current status of canine herpesvirus (chv) and minute virus of canines (mvc, canine parvovirus-type , cpv- ) pathogenicity of minute virus of canines (mvc) for the canine fetus minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate a serological survey of minute virus of canines (mvc; canine parvovirus type- ) in dogs in the tokai area of japan seroprevalence of canine calicivirus and canine minute virus in the republic of korea a fatal case of pup infection with minute virus of canines (mvc) fatal disease in nursing puppies associated with minute virus of canines characterization of minute virus of canines (mvc) and its pathogenicity for pups animal bocaviruses: a brief review virologic and serologic identification of minute virus of canines (canine parvovirus type ) from dogs in japan etiologic study of upper respiratory infections of household dogs chronological analysis of canine parvovirus type isolates in japan sequence analysis of an asian isolate of minute virus of canines (canine parvovirus type ) infection studies in kittens, using feline infectious peritonitis virus propagated in cell culture fatal canine parvovirus type- infection in pups from italy the canine minute virus (minute virus of canines) is a distinct parvovirus that is most similar to bovine parvovirus molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses das andere parvovirus: erstbeschreibung des minute virus of canines (canine parvovirus type ) in deutschland survey of dogs in japan for group canine coronavirus infection we are much obliged to oswald jarrett of the university of glasgow, institute of comparative medicine, for valuable discussions in the preparation of the manuscript. key: cord- -nt jcjm authors: garwes, d.j.; lucas, m.h.; higgins, d.a.; pike, b.v.; cartwright, s.f. title: antigenicity of structural components from porcine transmissible gastroenteritis virus date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: nt jcjm pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. similar results were obtained with serum from rabbits inoculated with whole virus and structural components. all three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus. the immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. in each case where the immunoglobulin class was determined, igg was found. one sow that received surface projections also had iga with neutralising activity in her colostrum. in contrast, infection of sows with live whole virus resulted in neutralising antibody of the igg, igm and iga classes. infection of pigs with the coronavirus causing transmissible gastroenteritis (tgev) results in disease that is most severe, frequently fatal, in young animals and of less severity in animals over weeks old. following recovery of the animals from infection, neutralising antibody can be detected in serum and secretions; transfer of this antibody, predominantly iga, in the colostrum and milk from a convalescent dam to her offspring confers reasonable protection against disease in her piglets (bohl et al., b; saif et al., ; stone et al., ) . parenteral administration of inactivated virus stimulates circulating antibody of the igg class saif et al., ; lucas et al., ) but, as little or no specific iga antibody is formed, the low level of igg in the milk affords little protection to newborn piglets during the period that they are most susceptible. work is in progress to investigate how an iga response can be stimulated by inactivated virus but more information is required about the nature of the viral antigen. we have previously demonstrated that inactivated purified tgev is capable of inducing a primary neutralising antibody response in pigs , suggesting that a virus structural element is the antigen responsible for stimulating the neutralising antibody. there is evidence, however, that at least one soluble component from tgev-infected swine testis cells can stimulate neutralising antibody production . following development of techniques to separate and purify the surface profections (sp) from the internal proteins of a subviral particle (svp) (garwes et al., ) it has been possible to determine which of these components are involved in the neutralising antibody response. this report describes the immune response in swine and rabbits inoculated with inactivated tgev and purified subviral components. antigen preparation the fs / isolate of tgev was used after six passes through gnotobiotic piglets aged -- days. a % (w/v) suspension of infected small intestine was clarified by centrifugation at × g for min and was then inoculated onto secondary pig thyroid (apt/ ) cells as monolayer cultures in medium containing galactose . virus was harvested following freezing and thawing the cultures at h after infection and was purified as previously described (garwes and pocock, b) . isolation of purified sp and svp antigen preparations was achieved by differential centrifugation after treatment of purified virus with % nonidet-p (garwes et al., ) . each antigen preparation (whole virus, sp and/or svp) was diluted : with growth medium, formaldehyde was added to . % and the preparations were held at °c for h. following this treatment, no infectivity could be detected in any of the samples. the preparations were stored at °c until used. each preparation of antigen for immunisation studies was derived from -- × pfu (sows) or × l s pfu (rabbits) of tgev. virus used for radio-immune assay was prepared from apt/ cells infected with tgev fs / passaged times through primary pig kidney cells. the medium contained h-uridine (specific activity ci/ mmole; the radiochemical centre, amersham) at pci/ml and the virus was harvested h after infection and purified as described (garwes and pocock, b) . infectivity was titrated by plaque assay. virus neutralisation test sera, colostra and milk samples were tested as previously described (cartwright, ) . purified tgev that had been grown in the presence of h-uridine was mixed with equal volumes of sera or % bovine serum albumin, and incubated at °c for i h. a sample was removed for titration of infectivity and the remainder was layered on to a linear -- % (w/w) sucrose gradient and centrifuged at × g for h. the gradient was fractionated by siphon into aliquots, material that had sedimented to the bottom of the tube was resuspended in distilled water and a sample was removed from each fraction for determination of radioactivity using a toluene-triton x based scintillant. measurement of virus agglutination by antibody was accomplished by incubating # samples of h-uridine labelled tgev with equal volumes of test sera diluted in isotonic saline at °c for h. to each reaction mixture was then added ul of rabbit anti-porcine igg serum (miles laboratories ltd., stoke poges) and incubation continued for h at °c followed by storage overnight at ° c. the samples were passed through cellulose acetate membrane filters of nm mean pore diameter (type eg, millipore (u.k.) ltd., london), the filters were washed with four changes of . ml distilled water and their radioactivity was determined by liquid scintillation. earlier attempts to use membrane filters composed of mixed esters of cellulose (mf filters, millipore (u.k.) ltd., london) were not successful as purified tgev is bound irreversibly to them (unpublished observations). this problem was overcome by using the cellulose acetate membranes to which the virus did not bind. solutions of serum igm and igg and colostral iga were obtained by gel filtration and ion-exchange chromatography (porter, ; vaerman and heremans, ) . antisera were raised in rabbits by injection of precipitin lines made by immunoelectrophoresis of the immunoglobulin solutions against rabbit antiserum to pig serum (goudie et al., ; higgins, ) . sera were made class specific by absorption with glutaraldehyde cross-linked immunoglobulin solutions (avrameas and ternynck, ) . specificity was assessed in immunoelectrophoresis and immunodiffusion tests against serum, colostrum and immunoglobulin solutions. sera and colostral wheys were examined by gel filtration in sephadex g- (pharmacia) in . m tris-hcl, ph . , containing . m naci and mm edta. wheys were also examined by ion-exchange chromatography on deae-cellulose (de- , whatman), using the six step buffer system described by stone et al. ( b) . the eluate obtained with each buffer was treated as a single fraction. presence of igm, igg and iga in the fractions was observed by microimmunodiffusion against specific antisera. fractions were concentrated about five-fold by dialysis against polyvinylpyrrolidone and examined for neutralising antibodies to tgev. the allocation of antibody activity to an immunoglobulin class was made by comparison of the distribution of antibody activity and immunoglobulins in the fractions. twelve pregnant sows were inoculated by the intramammary route: one with live, whole virus, three with formalin-inactivated virus, two with a mixture of purified sp and svp, four with sp alone and two with svp only. in addition one sow was given live virus orally and two sows were kept as uninoculated controls. for the intramammary inoculations, . ml of the antigen preparation was injected through the skin near the teat into three mammary glands of each sow three times. the same glands were inoculated at approximately , and week before farrowing. for the first inoculation, the antigen was emulsified in complete freund's adjuvant. colostrum samples were taken from inoculated and uninoculated glands separately at farrowing and milk samples were taken for several days after that. blood samples were removed before the first inoculation and at the intervals shown in table i . three rabbits were inoculated with tgev antigen: one with inactivated whole virus, one with sp and one with svp. each rabbit received two subcutaneous inoculations of antigen emulsified in complete freund's adjuvant at an interval of weeks, followed by three intramuscular inoculations of aqueous antigen at weekly intervals. one rabbit was kept as an uninoculated control. blood samples were taken from each animal before the first inoculation and at intervals up to months. p/o = orally; i/mam = by the intramammary route. --= not detected. those not tested are indicated by blank spaces. piglets born to the experimental sows were challenged at days of age by oral administration of ml of a suspension of tgev-infected intestine diluted to contain approximately x tcids per ml (equivalent to approximately x lds per ml in piglets reared away from the sows). the suspension was free, as far as it was possible to determine, from moulds, bacteria and other viruses. the results of neutralisation tests for serum and colostrum are shown in table i . after intramammary inoculation of sows, antibody was detected at the time of farrowing in serum and colostrum from all of three sows given whole killed virus, from out of given surface projections with subviral particles, from out of given surface projections only and from neither of the animals given subviral particles alone. neutralising antibody in colostrum only was detected in out of the given purified surface projections. there was little or no difference between antibody titres of colostrum from inoculated and uninoculated glands. the levels of neutralising antibody in milk from sows inoculated with inactivated whole virus or viral components fell to undetectable levels between to days after farrowing. neutralising antibody titres in sera of all sows, including the uninoculated controls, rose during the first weeks after farrowing. this reflects the immune response to infection of the dam by live virus administered to her piglets days after farrowing. the results are shown in table ii . the immune response to infection with live virus (sow no. ) resulted in neutralising antibodies in the three immunoglobulin classes reported previously (ristic and abou-youssef, ; bohl et al., b; salf et al., ; lucas et al., ) although in the example given igg antibody was detected in serum but not in colostrum. samples from sows that received intramammary inoculation contained neutralising antibodies of the igg class in all cases where the specific imunoglobulin could be characterised. no igm antibodies were detected in any sample and iga antibodies were demonstrated in the colostrum from only out of sows that received sp antigen. the only litter that survived challenge was that of sow no. which had been inoculated with sp antigen and had produced neutralising iga antibodies in the colostrum. the other litters experiences - % mortality and all piglets from the control uninoculated sows died. serum samples taken from the rabbit that had been inoculated with inactivated whole tgev showed neutralising antibodies rising to a maximum titre of l/ . neutralising antibodies were detected also in the serum of the rabbit that received sp antigen; the rate of antibody production was slower, however, and the peak titre reached was l/ . no neutralising antibody was found in serum from the rabbits inoculated with svp antigen and the uninoculated animal remained seronegative throughout the course of the experiment. the sedimentation profiles of h-uridine tgev after incubation with bovine serum albumin or with sera from sows that had been inoculated with viral antigen preparations are illustrated in fig. . virus incubated with either bovine albumin or normal pig serum sedimented as a sharp, homogenous band of radioactivity with very little material collecting at the bottom of the gradient. in contrast, however, virus incubated with sera from animals inoculated with viral antigen moved through the gradient as a broad band, suggesting heterogeneity of size and density, with a large proportion of the total radioactivity sedimented at the table i. bottom of the tube. there were no obvious differences between the profiles obtained after treatment of virus with sera that contained neutralising antibodies (sows no. , and ) or with serum from an animal that received svp antigen and which produced no neutralising antibodies (sow no. ). incubation of h-uridine tgev with sera from sows inoculated with viral antigen resulted in the formation of immune complexes that were retained by membrane filters (fig. ) . after incubation with serum from uninoculated animals approximately % of the virus was retained on the filter, probably due to aggregation that occurs in purified tgev preparations. sera from sows inoculated with whole virus, sp antigen or svp antigen preparations produced virus complexes that were retained most efficient samples of h-uridine labelled tgev containing ct/min were incubated for h at °c in dilutions of test sera followed by incubation with rabbit anti-porcine igg serum. after filtration through cellulose acetate membrane filters and repeated washing with isotonic saline, the retained radioactivity was determined by scintillation counting. test sera from: sow no. -" -', sow no. ~ e, sow no. = -', uninoculated sow d d. ly at a serum dilution of / . at higher serum dilutions there was reduced retention, implying that the level of antibodies was limiting. serum dilutions lower than / gave suboptimal retention due, probably, to an excess of antibodies and the formation of smaller complexes. omission of the rabbit anti-porcine igg serum from the test resulted in poorer retention of radioactive virus at all dilutions of experimental serum and a decline in the reproducibility of the test. discussion we report the immune response of sows and rabbits to tgev preparations containing whole virus, isolated surface projections or subviral particles produced by removal of the surface projections and envelope lipid from the virus by detergent treatment. sows receiving whole virus, isolated sp antigen and mixtures of sp and subviral particles produced antibody capable of neutralising virus infectivity; sows that received subviral particles alone, however, did not. similarly rabbits immunised with purified whole virus or surface projections produced neutralising antibody to tgev while administration of subviral particles produced no detectable neutralising activity. these data suggest that neutralising antibody raised during infection of swine with tgev is directed against a structural antigen of the virion and that this antigen is associated with the surface projection, a similar finding to that obtained with other lipid enveloped viruses (webster and laver, ; cartwright et al., ; jennings et al., ; hunsmann et al., ) . the fact that no neutralising antibody was produced in the pigs and rabbits that received subviral particles alone strongly suggests that the structural elements of these particles are not involved in virus neutralisation; conclusive proof of this, however, would necessitate the use of more animals than were available in the present study. the possibility existed that the tgev subviral particles were not antigenic in the animals used. this seemed unlikely as we had previously shown that these structures of nm diameter, readily visualised by electron microscopy, contained all the structural proteins except for the sulphated glycoprotein of the sp (garwes et al., ) . standard serological tests, such as complement fixation and radial immunodiffusion, are not sensitive enough to detect the levels of antibodies present in the experimental sera. the radioimmune assay techniques described above indicated, however, that antibody directed against the surface of the virus was produced in response to inoculation of sows with whole virus or either of the structural moieties. the sedimentation analyses showed that these sera caused an increase in the sedimentation coefficient of the virus, presumably by coating the particles with antibody molecules, and brought about sufficient aggregation to pellet radioactivity to the bottom of the gradient. application of this serum-induced clumping of radiolabelled virus to filtration studies demonstrated that sows receiving svp antigen were capable of producing antibodies to surface components at a similar level to those animals receiving whole virus or sp antigen. the use of this technique to quantitate agglutinating antibody is complicated by the need to incorporate rabbit anti-porcine igg serum into the test. as discussed in the text, purified tgev adsorbs irreversibly to membrane filters containing cellulose nitrate. this necessitated using cellulose acetate filters, for which the smallest pore diameter available is nm. assuming that virus aggregates are approximately spherical and composed of particles -- nm in diameter, the smallest aggregate that sould be retained reproducibly by such filters would need to comprise -- virions. the secondary antiserum to porcine immunoglobulin is required to achieve complexes of this size. the low level of retention of virus incubated with low dilutions of immune sera presumably reflects the solubility of antigen--antibody complexes in the presence of large excesses of either of the two reactants. analysis of the immunoglobulin classes responsible for neutralising activity after inoculation of sows by the intramammary route showed that igg was the predominant antibody species in both serum and colostrum, consistent with previous findings with inactivated tgev lucas et al., ) but differing from the stimulation of iga to ferritin reported by bourne et al. ( ) . whether this discrepancy is due to differences in the types of antigen used is not known. it is of interest that sow no. , which received sp antigen, produced neutralising antibody of the iga class in the colostrum and was the only animal capable of protecting her piglets against challenge with live virus. the possibility that this sow encountered live tgev prior to farrowing and that the experimental inoculation generated a secondary immune response cannot be totally ruled out, since infection with the live virus stimulates igm and iga antibody production (sow no. above; lucas et al., ; stone et al., ) and may result in limited protection of the litter. there was no other reason to believe that prior infection with tgev had occurred, however. all the control sows remained sero-negative throughout the experimental period. the apparent lack of neutralising antibody of the igm class in the pigs that received inactivated viral material may have resulted from the materials used and the route of inoculation; the times at which the serum samples were taken may have been too late, however, to have detected a transitory igm response. most of the sows showed rising neutralising antibody titres after challenge of the piglets, probably in response to cross-infection. those inoculated with sp antigen that had no detectable neutralising antibody at farrowing nevertheless developed titres by to weeks, comparable with those that were sero-positive at farrowing. the two sows inoculated with svp antigen alone, however, as well as having no detectable serum neutralising antibody at farrowing, developed lower titres that were more comparable with those achieved by the uninoculated controls. it has been reported that a soluble antigen from tgev-infected swine testis cells could induce neutralising antibody formation when injected into rabbits. from the data presented above we should anticipate that the soluble antigen concerned is likely to comprise viral sp either free in solution or bound to small fragments of membrane. since the viral sp is characterised mainly by the molecular weight of its component glycopolypeptide and has neither haemagglutinin nor enzyme activity associated with it, the task of recognising the presence of sp in crude tissue extracts is difficult. it should be possible, however, to develop a radioimmune assay using specific antibody to purified components. such a test would facilitate the preparation of potent antigen suspensions and work is in progress to this end. the cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents a. immunology of transmissible grastroenteritis antibody responses in serum, colostrum and milk in swine after infection or vaccination with tge virus the influence of route of vaccination on the systemic and local immune response in the pig surface structure of vesicular stomatitis virus a cytopathic virus causing a transmissible gastroenteritis in swine. ii. biological and serological studies effect of precipitation with methanol on antigenic potency of tge virus the polypeptide structure of transmissible gastroenteritis virus isolation of subviral components from transmissible gastroenteritis virus a simple method for producing antibody to a single selected diffusible antigen. the lancet fractionation of fowl immunoglobulins properties of mouse leukaemia viruses. ix. active and passive immunisation of mice against friend leukaemia with isolated viral gpt~ glycoprotein and its corresponding antiserum the immune response of hamsters to purified haemagglutinins and whole virus vaccines following live influenza virus infections attempts to vaccinate sows against tge the influence of ph on the growth and stability of transmissible gastroenteritis virus in vitro transfer of immunoglobulins igg, iga and igm to lacteal secretions in the parturient sow and their absorption by the neonatal piglets comments on the immunology of transmissible gastroenteritis isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies transmissible gastroenteritis in neonatal pigs: intestinal transfer of colostral immunoglobulins containing specific antibodies a. partial characterisation of the principal soluble antigens associated with the coronavirus of transmissible gastroenteritis by complement fixation and immunodiffusion chromatographic separation of gram quantities of immunoglobulins from porcine colostrum against transmissible gastroenteritis virus immunoglobulin a in the pig. i. preliminary characterisation of normal pig serum iga influenza virus subunit vaccines: immunogenicity and lack of toxicity for rabbits of ether-and detergent-disrupted virus key: cord- -bne cap authors: letellier, c; kerkhofs, p; wellemans, g; vanopdenbosch, e title: detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the ′ untranslated region date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: bne cap a reverse-transcription polymerase chain reaction (rt-pcr) was developed to differentiate the bovine diarrhea virus (bvdv) from other pestiviruses, and to determine the genotype of the bvdv isolates. for this purpose, primer pairs were selected in the ′ untranslated region ( ′utr). the primers b(e) and b( ) were located in highly conserved regions and were pestivirus-specific. two primer pairs named b( )b( ) and b( )b( ) were specific of bvdv genotypes i and ii, respectively. with this technique, an amplification product of the expected size was obtained with either the b( )b( ) or the b( )b( ) primer pairs for the bvdv isolates tested but not for bdv or csfv. for some isolates that were grouped in the genotype ii, sequence analysis of the pcr fragments confirmed their classification into this genotype. bovine viral diarrhea is an important disease of cattle. acute infection is usually mild and often subclinical, and is resolved with the appearance of neutralizing antibodies. transplacental infection of the fetus with a noncytopathic bvdv strain during the first trimester of gestation may lead to persistently infected (p.i.), immunotolerant calves. when these animals are superinfected with an antigenically similar cytopathic strain they may suffer of the fatal mucosal disease (brownlie, ) . another fatal outcome of bvdv infection is associated with severe thrombocytopenia and hemorrhagic lesions. several outbreaks of this syndrome have been reported, essentially in north america but veterinary microbiology ( ) ± cases have also been reported in europe (corapi et al., ; broes et al., ; pellerin et al., ) . bvdv belongs to the pestivirus genus that also comprises border disease virus of sheep (bdv) and classical swine fever virus (csfv). the classification of virus types was made according to the host-species that was infected. pestiviruses are, however, able to cross the species barrier. bvdv can cross-infect cattle, sheep, goats and pigs (carlsson, ; paton et al., ) . bdv is an ovine pathogen that occasionally infects pigs (roehe et al., ; edwards et al., ; vilcek and belak, ) . differentiation between csfv and other pestiviruses can be accomplished by the use of csfv-specific monoclonal antibodies (mabs) but the search for ruminant pestivirus-specific mabs has failed due to a great antigenic diversity (edwards et al., ) . rt-pcr has been proved to be a rapid and sensitive method to detect viral nucleic acids and this technique has been used by several investigators for the detection of pestiviruses using oligonucleotide primers located in conserved regions of the viral genome (lopez et al., ; katz et al., ; ridpath et al., ; wirz et al., ; hofmann et al., ; horner et al., ; tajima et al., ; canal et al., ) . most of the pcr primers that were selected in the utr recognized the greatest number of pestivirus isolates but failed to differentiate bvdv from other pestiviruses vilcek et al., ) . specific detection of csfv by rt-pcr has been described using primers located in the coding part of the genome or by the sequence analysis of the pcr product amplified using primers located in a highly conserved region of the utr wirz et al., ; hofmann et al., ) . the gp , gp , p and p regions did also serve as template for bvdv amplification. however, these rt-pcr tests failed to detect all bvdv isolates (vilcek et al., ; tajima et al., ) . based on the sequence comparison of utr, the bvdv isolates were subdivided in two genotypes. sequence homology within each group was very high (> %) while homology between group i and ii droped near % (pellerin et al., ; ridpath et al., ) . an accurate and fast differentiation between bvdv and another pestivirus is of great importance for the development of control measures, particularly when an outbreak of csfv is suspected. sensitive detection of bvdv contamination in fcs and cell culture would help diagnostic work and would improve the safety of veterinary and human vaccines. the aim of this study was to develop a rt-pcr test that allowed the detection of bvdv and differentiation between both genotypes. published utr sequences were aligned and two pairs of primers were selected. belgium field bvdv isolates were tested by amplification with both sets of primers. sequence analysis of the pcr products confirmed the appartenance of some field isolates to the genotype ii. the bvdv reference strain nadl was received from dr. j.m. aynaud (inra, thiverval, france), the c v strain from dr. straver (cdi, amsterdam), the /han isolate (hewicker-trautwein et al., ) from dr. b. liess and the new york and bdv aveyron (chappuis et al., ) strains were received from prof. e. thiry (university of lie Áge). the viruses were propagated on bovine fetal kidney cells grown in minimum essential medium (mem) supplemented with pestivirus free fetal bovine serum. csfv was kindly provided by dr. f. koenen (cerva). organs (lungs, intestines, brains) were obtained from bvdv-positive cattle with respiratory, digestive or neurological symptoms. these were routine diagnostic samples obtained between and that were assessed as bvdv positive by a direct immunofluorescence test realized on frozen tissue sections. homogenate extracts were then used for rna isolation. leucocytes pellets were prepared from immunotolerant cattle which had previously been detected by an antigen capture elisa kit (rhone me Ârieux). oligonucleotide primers were designed from the utr. the sequence of two pairs of primers, designated b b and b e b , was located in a highly conserved region of bvdv, csfv and bdv. these primers were tested for the amplification of pestivirus cdnas. their sequence, position according to the nadl genome sequence (collett et al., ) and the expected size of the amplified products are presented in table . two pairs of primers were designed from the alignment of sequences published by others (pellerin et al., ) . the sequences were conserved in a bvdv genotype but not between genotype i and ii. the b b pair was tested for the amplification of bvdv type sequences and the b b pair was used for bvdv type amplification. rna was extracted using ml trizol (gibco brl) according to the manufacturer's instructions. the extraction was accomplished using ml of organ homogenate extracts or a pellet of leucocytes corresponding to . ml of heparinized and hemolyzed blood. the rna was resuspended in ml depc-treated water. the reverse transcription was carried out in a volume of ml containing mm tris±hcl (ph . ), mm kcl, mm mgcl , mm dtt, . mm dntp, pmol of the reverse primer b , u m-mlv reverse transcriptase (gibco brl) and ml rna. the cdna was synthesized at c for min and the enzyme was inactivated for min at c. the amplification of cdna by pcr was carried out in a total volume of ml containing mm tris±hcl (ph . ), mm kcl, mm mgcl , . mm dntp, pmol of each primer, ml cdna and . u taq dna polymerase (gibco brl). the reaction was heated in a thermocycle for min at c and then submitted to cycles of amplification. for the b b and b e b primer pairs, the conditions of amplification were min at c, min at c, and min at c. for the b b and b b primer pairs, the conditions were min at c, min at c, and min at c. amplified products were separated by electrophoresis in . % agarose gel in tris±borate edta buffer. after electrophoresis of the b e b amplification products, the agarose gel was soaked two times for min in denaturation solution ( . m nacl, . m naoh), then in neutralization solution ( . m tris±hcl (ph . ), . m nacl). the dna was then transfered to two positively charged nylon membranes (boehringer) and the dna was cross-linked by uv irradiation. the filter was incubated for min at c in dig easy hyb solution (boehringer). then pmol of dig labeled b or b oligonucleotide (eurogentec) was added and the incubation was continued overnight at c. filters were washed and the hybridized probes were detected using dig luminescent detection kit (boehringer) according to the manufacturer's instructions. the pcr products obtained with the b b or b e b primers were purified on centricon filters and were cloned in a pgem-t vector (promega). plasmid dna was purified using qiagen tip columns. a minimum of two clones were sequenced with the sequencing primer m ± , dye terminator cycle sequencing kit (perkin-elmer) and abi prism automated cycle sequencer according to the manufacturer's recommendations. alignment of the sequences was realized using the pileup program included in the gcg software package (devereux et al., ) . distances were calculated by the kimura -parameter method (kimura, ) and used to construct trees using the growtree program of the gcg. sequence analysis was achieved using the belgian embnet node facility. total rna extracted from infected cell cultures was submitted to rt-pcr by using the b e b pestivirus-specific primers. a pcr product of the expected size ( bp) was amplified from bvdv reference strains nadl, new york, c v, /han , bdv aveyron and csfv, and from the bvdv field isolate uvr . the results obtained for bvdv nadl, new york, uvr , bdv aveyron and csfv are shown in fig. (a) . in fig. (b) , the same cdnas were amplified with bvdv type i and type ii-specific primer pairs b b and b b . a fragment of bp was obtained with the former for bvdv nadl and new york, and with the latter for bvdv uvr . this isolate was then used as positive control for the type ii amplifications. no amplification product was obtained for bdv aveyron and csfv. the bvdv strains c v and / han were classified in the genotype i (data not shown). the same results were obtained after southern blot hybridization performed on the b e b amplification products by using the dig-labeled genotype-specific probes. the b e b amplification products shown in fig. (a) were hybridized with b dig probe in fig. (a) and with b dig probe in fig. (b) . with the bvdv type i-specific probe, the amplification products from bvdv nadl and new york were detected. the bvdv type ii-specific probe reacted only with the bvdv uvr isolate. the bdv and csfv amplification fragments did not react with either bvdv-specific probe. a total of bvdv positive samples were tested. rna was extracted from organs or from leukocytes of infected animals. all of the samples contained a pestivirus as demonstrated by a rt-pcr test using the pestivirus-specific primers b b or b e b . the samples were also tested by both genotype-specific pcr and hybridization. the results are presented in table . of a total of organs (lungs, intestines, brains), were assessed as genotype i and nine as genotype ii. two out of immunotolerant cattle were infected with a genotype ii isolate and two out of four laboratory viruses were also classified as genotype ii. as a total, of the isolates tested belonged to the genotype ii. organs positive for infectious bovine rhinotracheitis, bovine parainfluenza type , coronavirus and rotavirus were used as controls. no amplification product was obtained (data not shown). type ii viruses were isolated from to in belgium (table ) . four isolates came from calves with respiratory disease, one from the intestine of a calf. three viruses were isolated from the brain of animals showing neurological symptoms. two genotype ii viruses were isolated from the blood of immunotolerant animals and only one virus was isolated from a case of haemorrhagic syndrome (broes et al., ) . the alignment of nucleotide sequence of a bp dna fragment, flanked by primers b and b is presented in fig. . the sequences of belgian type ii isolates were compared to other genotype ii sequences extracted from a genebank: , isolated from a haemorrhagic syndrome outbreak (pellerin et al., ) , cpa virus isolated from cell culture (harasawa, ) , the ovine pestivirus isolated in the uk (vilcek et al., ) . reference bvdv type i strains nadl and new york and three belgian virus isolated from organs (zvd , zvr ) and from a p.i. calf were also included in the comparison. the sequence alignment confirmed the classification of all the isolates. the sequence of the region corresponding to the oligonucleotides b and b , represented in fig. by a box, showed a high degree of conservation inside both genotypes, and were divergent between the genotypes. a phylogenetic tree was constructed and is presented in fig. . genotype ii viruses were segregated into two groups. the first englobed eight belgian isolates which showed very little genetic divergence. in the other group were classified viruses isolated from cell culture (cpa), from the usa, the uk and two belgian isolates. interestingly, one of them (bse ) was nearly identical to the cpa virus. the zvd , zvr and p.i. viruses were classified in genotype i. the zvd isolate showed a strong homology to the new york strain. rt-pcr tests and sequence analysis of the p and e regions were used by several authors to classify the pestiviruses. using these techniques, four genotypes were identified. the csfv group was restricted to swine, whereas bvdv types i and ii included strains isolated from cattle, sheep, goats and wild ruminants. the bdv genotype comprised strains that were isolated from sheep and pigs (becher et al., (becher et al., , van rijn et al., ; vilcek et al., ) . this classification did not correlate with the hostspecies origin but was supported by serological investigations (dekker et al., ; paton et al., ) . this accurate method cannot however be used for diagnostic work. specific primers located in the utr region were used by others for the diagnosis of bvdv infection by rt-pcr. canal et al. ( ) reported the use of a conserved antisense oligonucleotide and two sense primers, csfv and ruminant-pestivirus-specific, respectively, to distinguish these viruses. another set of primers was designed by sandvik et al. ( ) . pcr fragments from bvdv types i and ii originating from different host-species were successfully amplified. here, we describe a differential system to discriminate between bvdv and other pestiviruses and to determine the genotype of the bvdv isolates, using conserved regions fig. . alignment of nucleotide sequences from the utr of bvdv types i and ii isolates. the sequences are flanked by primers b and b . genotype ii sequences are presented above the horizontal line and genotype i below. sequence of the b and b oligonucleotides is boxed. located in the utr. the sequence of the primer used for the cdna synthesis was conserved between the pestiviruses. this cdna could thus be used in pestivirus-specific but also bvdv-specific pcr. the primers b e and b have already been used by others and were designed to detect all the pestiviruses (vilcek et al., ) . based on the search for genotype-conserved regions, two primer pairs were selected. this allowed us to determine the genotype of the bvdv isolates. the rt-pcr tests were validated with reference bvdv, bdv and csfv strains. with a combination of pcr reactions, the bvdv strains could be differentiated from bdv and csfv and the genotype of bvd viruses could be determined. no cross-reactivity was detected between the type-specific primers. then, the pcr assays were used to characterise field isolates that were previously shown bvdv-positive by other techniques. the rna extraction was performed on organ homogenate extracts or on leukocytes. this excluded a bvdv contamination during cell culture multiplication. among the samples examined, were classified as genotype i and as genotype ii. from the genotype ii viruses that were obtained, five were isolated from organs, two from the blood of p.i. animals and for two other isolates, we were not able to trace the origin of the virus. only one virus was isolated from a case of hemorrhagic syndrome. eight bvdv-positive brains were also tested. three of them harboured genotype ii viruses. our hypothesis is that these were p.i. animals. indeed, the presence of bvdv in the central nervous system (cns) has been described and this seems to be an important location for the persistence of bvdv. primary infection of the cns most likely occurs across the blood±brain barrier (fernandez et al., ; hewicker et al., ; hewicker-trautwein et al., ) . other experiments showed that bvdv was found in the cns of all p.i. animal tested, without causing obvious cellular destruction (wo Èhrmann et al., ) . ten genotype ii and three genotype i isolates were further analyzed by sequencing the amplification dna fragment. based on pairwise alignment, conservation of the sequences inside the genotype ii group was at least %. a minimum of % homology was found inside the genotype i group. when nadl strain was excluded from the comparison, the homology rose to %, suggesting that the belgian isolates tested were closer to genotype ib than ia strains. the homology between genotype i and ii was approximately %. the greatest divergence between genotype ii isolates was between uvd and , which were both isolated from a haemorrhagic syndrome. our results demonstrate that rt-pcr could be performed on rna extracted from organ samples. furthermore, the bvdv genotype could be determined. the same approach has been used by sullivan and akkina ( ) . consensus primers located in the gp region were tested on bovine and ovine isolates, whereas combination of specific primers allowed the differentiation between bvdv type i, type ii and bdv. bvdv has been known not only as a veterinary pathogen but also as a common contaminant in cell culture work and in live vaccines for human, bovine and swine use (harasawa and tomiyama, ; harasawa, ; harasawa and mizusawa, ) . genotype ii bvdv has been described as a contaminant of fbs (ridpath et al., ) , of cell lines (harasawa, ) , ifn for human use (harasawa and sasaki, ) and live virus vaccine for human use (harasawa, ) . the origin of the belgian type ii isolates is not known. the rna was extracted from organs or leukocytes avoiding a possible contamination of the samples by the cell line or fbs. however, we cannot exclude the introduction of these strains via live vaccine for bovine use. epizootical problems due to the circulation of genotype ii viruses in belgium have not yet been encountered. however, these viruses are antigenically different from classical bvdv viruses (ridpath et al., ) . the complete sequence of a genotype ii virus has been published showing that the weak homology with type i strains extended through the complete genome. the gp protein had less than % identity with the type i counterpart (ridpath and bolin, ) . recently, genotype ii strains have been isolated in japan. no cross-neutralization was observed between these type ii and type i isolates (nagai et al., ) . one should thus take account of these viruses for the development of control programs and vaccines against bvdv. further characterization of border disease virus isolates: evidence for the presence of more than three species within the genus pestivirus phylogenic analysis of pestiviruses from domestic and wild ruminants syndrome he Âmorragique chez des bovins infecte Âs par le virus de la diarrhe Âe bovine (bvd/md) differentiation of classical swine fever virus from ruminant pestiviruses by reverse transcription and polymerase chain reaction border disease in sheep caused by transmission of virus from cattle persistently infected with bvdv etudes se Ârologiques et immunologiques re Âalise Âes a Á la suite de l'isolement d'un pestivirus dans un foyer ovina chez des moutons molecular cloning and nucleotide sequence of the pestivirus bovine diarrhea virus severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine diarrhea virus six antigenic groups within the genus pestivirus as identified by cross neutralization assays a comprehensive set of sequence analysis programs for the vax the development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses comparative studies of border disease and closely related virus infections in experimental pigs and sheep viral antigen distribution in the central nervous system of cattle persistently infected with bovine diarrhea virus comparative analysis of the non-coding region of pestivirus rna detected from live virus vaccines evidence of pestivirus rna in human virus vaccine adventitious pestivirus rna in live virus vaccines against bovine and swine diseases demonstration and genotyping of pestivirus rna from mammalian cell lines sequence analysis of the utr of pestivirus rna demonstrated in interferons for human use phylogenetic analysis of pestivirus based on the utr immunohistological detection of bovine viral diarrhoea virus antigen in the central nervous system of persistently infected cattle using monoclonal antibodies infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus variation in neuropathogenicity in sheep fetuses transplacentally infected with non-cytopathogenic and cytopathogenic biotypes of bvdv rapid characterization of new pestivirus strains by direct sequencing of pcr-amplified cdna from the noncoding region comparison of an antigen capture enzyme-linked assay with reverse transcription-polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cdna nested-amplification approach a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences rapid detection of bovine viral diarrhea virus by polymerase chain reaction nucleotide sequence homology to bvdv in the untranslated region of bvdvs from cattle with mucosal disease or persistent infection in japan infection of pigs and cattle with bovine viral diarrhoea virus on a farm in england a proposed division of the pestivirus genus using monoclonal antibodies, supported by cross-neutralisation assays and genetic sequencing identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the noncoding region for detection of bovine viral diarrhea virus segregation of bovine viral diarrhea virus into genotypes the genomic sequence of a virulent bovine viral diarrhea virus (bvdv) from the type genotype: detection of a large genomic insertion in a noncytopathic bvdv characterisation of p sequences from a border disease-like pestivirus isolated from pigs. veterinary microbiol detection and identification of ruminant and porcine pestiviruses by nested amplification of untranslated cdna regions a nested polymerase chain reaction assay to differentiate pestiviruses attempt to discriminate between bovine viral-diarrhea virus strains using polymerase chain reaction subdivision of the pestivirus genus based on envelope glycoprotein e genetic identification of pestivirus strain frijters as a border disease virus from pigs pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis molecular characterization of ovine pestiviruses distribution of bovine diarrhea viral antigens in the central nervous system of cattle with various congenital manifestations detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction the authors thank hans vanderhallen for performing the automated sequencing. key: cord- -kx hfpu authors: mahmood, zana h.; sleman, rizgar r.; uthman, aumaid u. title: isolation and molecular characterization of sul/ / avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: kx hfpu infectious bronchitis virus (ibv) was isolated from trachea and kidney tissues of eight broiler farms in kurdistan region of north iraq from to . the birds were suffering from respiratory and nephropathological symptoms and lesions. a bp hyper mutable spike glycoprotein (s ) gene was amplified and sequenced using conventional rt-pcr. sequence analysis and blast homology search in genbank data base indicate that two of the farms were infected with the / strain, one with an unidentified ibv and five were infected with sul/ / . the birds in the latter five farms were suffering from nephropathogenic lesions, however, the virus was isolated from kidney but not from trachea in these cases. the birds were vaccinated regularly with / or ma vaccine. the deduced amino acid sequence of the isolated and amplified s subunit ( aa) of sul/ / was differed in – % from that of all three vaccine strains ( / , ma , and h ) used in the region. this dissimilarity is most likely the cause of poor efficacy of vaccines used in the region, at least in five of these farms. amino acid sequence comparison and phylogenetic tree analysis with other published ibv genotypes indicate that this newly isolated virus together with other regionally related and recently published isolates from israel (is/ / , is/ ) and egypt (egypt/benisuef/ ) belong to a new genotype. this is the first report of identification and genotyping of ibv isolate in iraq, which indicate the circulation of / along with a new variant (sul/ / ) of ibv in vaccinated broiler farms. avian infectious bronchitis (aib) is an acute and highly contagious viral disease of the respiratory system in chickens. it is of significant economic importance because it results in high mortality and poor weight gain in broilers as well as decreasing egg production and quality in layers. in addition to the respiratory and reproductive system, the virus has been associated with nephritis (dhinakar raj and jones, ; butcher et al., ; cavanagh and naqi, ) . the disease is caused by infectious bronchitis virus (ibv), a member of the group of coronavirus species. cdna representing the entire ibv genome has been cloned and sequenced (boursnell et al., ) , it comprises of approximately . kb, a single stranded rna virus of positive sense, encoding several proteins which are associated with rna replication and transcription. all coronaviruses have four structural proteins, spike protein (s), small membrane envelope protein (e), membrane protein (m), and nucleoprotein (n). the spike protein of ibv undergoes post translational cleavage to form s and s subunits (boursnell et al., ; cavanagh et al., ) . veterinary microbiology ( ) - infectious bronchitis virus (ibv) was isolated from trachea and kidney tissues of eight broiler farms in kurdistan region of north iraq from to . the birds were suffering from respiratory and nephropathological symptoms and lesions. a bp hyper mutable spike glycoprotein (s ) gene was amplified and sequenced using conventional rt-pcr. sequence analysis and blast homology search in genbank data base indicate that two of the farms were infected with the / strain, one with an unidentified ibv and five were infected with sul/ / . the birds in the latter five farms were suffering from nephropathogenic lesions, however, the virus was isolated from kidney but not from trachea in these cases. the birds were vaccinated regularly with / or ma vaccine. the deduced amino acid sequence of the isolated and amplified s subunit ( aa) of sul/ / was differed in - % from that of all three vaccine strains ( / , ma , and h ) used in the region. this dissimilarity is most likely the cause of poor efficacy of vaccines used in the region, at least in five of these farms. amino acid sequence comparison and phylogenetic tree analysis with other published ibv genotypes indicate that this newly isolated virus together with other regionally related and recently published isolates from israel (is/ / , is/ ) and egypt (egypt/benisuef/ ) belong to a new genotype. this is the first report of identification and genotyping of ibv isolate in iraq, which indicate the circulation of / along with a new variant (sul/ / ) of ibv in vaccinated broiler farms. ß elsevier b.v. all rights reserved. serotype-specific determinant of ibv is thought to be located in the hypervariable regions (hvrs) of s glycoprotein (cavanagh et al., ) , and it is the s subunit of the spike protein which induces neutralizing antibody (cavanagh et al., ; koch et al., ) . in addition to the well-known massachusetts (mass) serotype of ibv, many other serotypes, distinct from mass, have been isolated in africa (el houadfi et al., ; abdel-moneim et al., ) , asia (rajeswar et al., ; bochkov et al., ) , australia (ignjatovic et al., ) and europe (capua et al., ; worthington et al., ) . the majority of these strains are endemic for certain geographic regions. three closely related variants of ibv isolates in israel (is/ / and is/ ) and egypt (egypt/beniseuf/ ) have been genotyped by sequencing of the s subunit (meir et al., ; abdel-moneim et al., ) . in spite of the use of three different vaccines (h , ma , attenuated / ) in poultry farms in iraq, outbreaks have been observed with high mortality in broiler farms having nephropathogenic lesions. since outbreaks of ibv still occur in vaccinated flocks and the virus strains isolated are frequently different from serotypes of the vaccine strains used (liu and kong, ; cavanagh et al., ; li et al., ) , continuous identification of the genotype and production of new generations of vaccines are crucial. in order to investigate ibv genotypes in iraq, where the disease is endemic and widely spread in vaccinated and unvaccinated poultry farms mainly associated with kidney damage and urolethiasis, identification and molecular characterization of ibv (sul/ / ) isolated from eight infected broiler farms was conducted and the deduced amino acid sequence of the s subunit of the virus was compared with geographically related isolates. samples: tissue samples from eight suspected ibv outbreaks in sulaimani broiler farms were collected. trachea, lung and kidneys were obtained from to birds in each farm and transferred to the laboratory on ice. the birds suffered from respiratory symptoms and lesions as well as kidney damage (enlargement, congestion, and uroletheasis). the flocks were vaccinated with / or ma vaccine. three different strains of ibv vaccines (h , ceva), (ma , intervet) and ( / , intervet) were used as positive control. serological test: blood samples were collected from birds of each of the eight broiler farms and repeated one week later. the serum was separated; enzyme linked immunosorbent assay (elisa) was conducted for the detection of ibv antibody in clinically suspected farms using ibv antibody test kit (synbiotics, usa). this elisa kit is highly specific in which a titer of (>  ) indicates the present of ibv infection. rna extraction: - mg tissue from trachea, lung and kidney of the birds were homogenized in liquid nitrogen. trizol reagent was used to extract the rna from tissue samples according to the manufacturer's instructions. the rnas were dissolved in ml rnase-dnase free distilled water and directly used for subsequent rt-rcr or stored at (À c). virus isolation: tissue homogenate ( ml) from trachea and kidney of each pcr positive sample was inoculated in the allantoic cavities of spf chicken egg embryos ( - days) and incubated for further days at c and candled daily. after passages, allantoic fluids were collected and rna was extracted for rt-pcr (momayez et al., ) . oligonucleotides: oligonucleotides used in this project are illustrated in table . cdna synthesis: superscript tm ii rt protocol (invitrogen) was used according to the manufacturer's instruction, briefly, ml total rna ( mg/ml) was mixed with ml reverse primer ( pmole/ul), r-n for n gene or r-s for s gene (table ) , ml dntps ( mm each) and rnase free distilled water in a total volume of . ml mixture. the mixture heated to c for min then directly chilled on ice. ml from  first-strand buffer and ml dithiotheritol ( . mm), . ml ( units/ ml) rnase inhibitor were added to the mixture. after incubating the mixture at c for min, ml ( units/ml) superscript tm ii reverse transcriptase was added and further incubated at c for min, and finally, the enzyme was inactivated at c for min. the cdna was stored at c for the following pcr amplifications. detection of the virus by n gene amplification: a primer pair (f-n and r-n ) was used to detect ibv in clinical samples amplifying bp of n gene. this pair of primers was designed specific to a conserved region of nucleocapsid (n) gene to ensure a wide detection range. the pcr amplification reaction was carried out in ml mixture containing . ml of  pcr reaction buffer, . ml of mm dntps, . ml of each of pmol forward table sequences, genome location and the references of primers used in this study. sequences (f-n ) and reverse (r-n ) primers, . ml of red hot taq dna polymerase ( unit/ml) (thermoscientific, uk), ml cdna, the mixture is finalized to ml by the addition of . ml dnase-rnase free distilled water. amplification was performed with a thermo cycler (eppindorf, usa) at c for min initial denaturation step, cycles ( c for min, c for s and c for min) and a final extension at c for min. pcr amplification of s gene: rt-pcr was applied for the first base of s gene which contains the hyper variable regions (hvrs) of s gene (dolz et al., ; cavanagh et al., ) . the amplification reaction was carried out in ml reaction mixture containing . ml of  pcr reaction buffer, ml of mm dntps, . ml of pmol forward (f-s uni +) and reverse (r-xce À) primers, . ml of u/ml thermoprime taq dna polymerase, and ml cdna. amplification was carried out with a thermal profile at c for min initial denaturation then cycles ( c for min, c for s and c for min) amplification with a final extension of c for min. the primer pair (f-col via and r-col via ) (table ) was used to amplify housekeeping gene of chicken collagen via as control for cdna synthesis. nucleotide sequencing: about ml of bp pcr product of amplified s subunit from broiler farms were purified using pcr purification kit (qiagen, germany), and sequenced with two forward (f-s uni + and f-xce +) and one reverse (r-xce À) primers in innovations biochemical laboratory (ibl), vienna-austria. genbank accession numbers: genbank accession number of the nucleotide and amino acid sequence of sul/ / isolate s gene reported in this study is (gq ). genbank accession numbers of ibv sequences used in the analysis are: israel/ / (ay ), is/ (ay ), egypt/beniseuf/ (af ), ir- -ph (ay ), ir- ga (ay ), / (af ), spain/ / (dq ), italy- (aj ), qxibv (af ), h (m ), ma (ay ), m (ay ), connecticut (li ), and australia (ay ). clinical signs and virus isolation: all eight clinically suspected farms were serologically positive, even vaccinated farms had a very high titer of antibody ab to .  , in most of the farms the elisa titer was increased in the second sampling, which indicate that reaction is not due to the vaccination. the ibv-ab-test kit used allows differentiating between vaccinated and nonvaccinated farms, depending on the antibody titer. the virus has been detected in all poultry farms. the mortality in these farms was approximately % in the first days of the onset of the disease and the birds suffered mainly from kidney damage and urolitheasis. in addition to kidney damage, respiratory signs and lesions were found in two broiler farms, where the disease was further complicated with other infections such as airsacculitis due to e. coli infection, in which / was isolated, with a mortality rate of approximately - %. these two farms lucked the hygienic measures and the birds had been vaccinated with / vaccine two weeks before the onset of the disease. the sequences of the isolated viruses from these two vaccinated broiler farms were identical with vaccine strain / . a local isolate sul/ / has been detected in five broiler farms, where the birds were suffering from nephropathologic lesions. on culturing the virus in egg embryos, one third of the embryos died after each passage of / strain. dwarfism has been observed in both / and sul/ / infected embryos. s sequence analysis: the first nucleotides encoding s spike glycoprotein subunit including the hyper variable regions were sequenced. comparative analysis of the nucleotides and the deduced amino acid sequence of s subunit were performed to assess the relation of sulaimani (sul/ / ) isolate with other sequences and to find similarities with vaccine strains h , ma and / . it has been found that sul/ / isolate is - % different from all vaccine strains. however, it only deviates in % from egypt/benisuef/ isolate and % from is/ and israel/ / isolates from israel (table ) . nucleotide sequences of these four isolates are identical with about - % similarity (table ) . only a few restriction enzymes can be used to differentiate between these closely related isolates (fig. ) , these enzymes can be used for restriction fragment length polymorphism (rflp) to identify and differentiate between them. multiple sequence alignment of different strains and isolates have been performed using clustal w version . (www.genome.jp) (fig. ) , which indicate that the regions at the positions - , - , - , - and - of s subunit based on / strain (accession no. af ) are significantly conserved regions in almost all compared sequences. hyper variable regions (hvrs) of selected s amino acid sequences from geographically different regions are located in positions - , - , and - (fig. ) . the phylogenetic tree of the aligned amino acids was also produced using mega beta version . online software (www.megasoftware.net) which shows a typical relatedness between sul/ / isolate and reference ibv strains from israel, but not with isolates from our neighbor country iran (fig. ) . poultry farms in iraq are suffering from huge economic losses caused by avian infectious bronchitis disease. in spite of the use of three different vaccine strains (h , ceva), (ma , intervet) and ( / , intervet) the nephropathogenic type of the disease and even / infection is appearing in vaccinated farms. since iraq suffered from three wars and long time embargos in the last three decades, little has been done to investigate the spread of the disease and the genotype of the causative agent of ibv. a nephropathogenic ibv (sul/ / ) isolate was identified from the tissue of kidney of vaccinated broiler flocks with a mortality rate of approximately % at the time of sampling. sequence analysis of sulaimani isolate s gene revealed its close relatedness only to three field isolates in israel and egypt (table ). since high rate of antiserum immunized with different homologous field genotype could neutralize each other (shimazaki et al., ; wang and huang, ) , which suggest genotypes based on s sequence analysis to some extent correlate with serotypes of ibv strains, therefore isolates from egypt, iraq and israel can be considered as the same novel middle east genotype, and most likely to be the same serotype. in spite of the long border and a large trade relation with iran, especially import of chicken and chicken products since , it is not clear why iraq's isolate (sul/ / ) differs from isolates from iran (ir- -ph, and ir- -gh) in approximately % amino acids. further investigation is necessary to exclude the existence of iranian isolates in other parts of iraq. since diverse coronaviruses have been detected recently in wild birds (hughes et al., ) and it is common among mallard ducks (muradrasoli et al., ) , it is very likely that such identical isolates transmitted between iraq, israel and egypt through wild migrated birds, but not through poultry product trade, which does not existing between these countries. further investigation of ibv in migrated wild birds is necessary to elucidate this measure. the low nucleotide and amino acid similarities between the (h , ma ) massachusetts and / vaccine with sulaimani isolate may account for the occurrence of the ibv outbreak in vaccinated flocks. production of a new vaccine based on the middle east genotype is necessary to fig. . phylogenetic tree of sul/ / isolate and other selected strains and isolates from genbank representing different geographic regions show the relationship among the deduced s amino acid sequence of isolates from the middle east (is/ , israel/ / , and sul/ / ), reference strains from usa (h , ma , m , and connecticut), isolates from europe (spain/ / , and italy- ), isolates from neighbor country iran (ir- -ph, ir- -ga), uk strain ( / ), australian t strain, and qxibv from china. protect the poultry industry in iraq. isolation of / from vaccinated broiler farms may be due to the involvement of the vaccine strain, which has been previously reported (farsang et al., ) . multiple sequence alignment and phylogenetic tree analysis separate the isolates into different groups which are genotypically related to each other. most of the serotypes are different from each other in the first hvr (positions - ) of s spike glycoprotein by more than %. this region seems to be conserved in each geographically isolated virus, which can be used for genotyping of the virus. it is most likely that this region is a serotypespecific determinants of ibv and contains antigenic epitope, which is serologically important for serotyping of ibv. amino acid sequences of european, north american or middle east groups are highly homologous in the first hyper variable region of s subunit of each group, but differ with other groups (fig. ) . this data indicates that in addition to / a new nephropathogenic ibv is circulating in iraq, which belongs to the ibv variant previously isolated in middle east. s gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt molecular analysis of the /b serotype of infectious bronchitis virus in great britain universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction molecular epizootiology of avian infectious bronchitis in russia sequence of the membrane protein gene from avian coronavirus ibv completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus infectious bronchitis virus: classical and variant strains a 'novel' infectious bronchitis strain infecting broiler chickens in italy amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes coronavirus ibv: partial amino terminal sequencing of spike polypeptide s identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m infectious bronchitis variation in the spike protein of the /b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs infectious bronchitis virus: immunopathogenesis of infection in the chicken antigenic and molecular characterization of isolates of the italy infectious bronchitis virus genotype the isolation and characterization of six avian infectious bronchitis virus isolated in morocco molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain genetically diverse coronaviruses in wild bird populations of north england isolation of a variant infectious bronchitis virus in australia that further illustrates diversity among emerging strains antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in south china during identification of a novel nephropathogenic infectious bronchitis virus in israel isolation and identification of infectious bronchitis virus from commercial chickens broadly targeted multiprobe qpcr for detection of coronaviruses: coronavirus is common among mallard ducks (anas platyrhynchos) seroprevalence study of infectious bronchitis among poultry in tamilnadu serological studies of infectious bronchitis vaccines against japanese field isolates of homologous and heterologous genotypes relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus a reverse transcriptasepolymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from this work was carried out with financial support from kurdistan institute for strategic studies and scientific researches, university of sulaimani, and directorate of veterinary in sulaimani, iraq. key: cord- -tudl k g authors: opriessnig, tanja; gauger, phillip c.; faaberg, kay s.; shen, huigang; beach, nathan m.; meng, xiang-jin; wang, chong; halbur, patrick g. title: effect of porcine circovirus type a or b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: tudl k g to determine differences in infection kinetics of two temporally and genetically different type porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type a or b infection, pigs were randomly assigned to one of seven groups: negative controls (n = ); pigs coinfected with a prrsv strain (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with vr- and pcv b (coi- - b; n = ), pigs coinfected with a prrsv strain (nc b) and pcv a (coi- - a; n = ), pigs coinfected with nc b and pcv b (coi- - b; n = ), pigs infected with vr- (n = ), and pigs infected with nc b (n = ). blood samples were collected before inoculation and at day post-inoculation (dpi) , , and and tested for the presence of prrsv antibody and rna, pcv antibody and dna, complete blood counts, and interferon gamma (ifn-γ) levels. regardless of concurrent pcv infection, vr- initially replicated at higher levels and reached peak replication levels at dpi . pigs infected with vr- had significantly higher amounts of viral rna in serum on both dpi and dpi , compared to pigs infected with nc b. the peak of nc b virus replication occurred between dpi and dpi and was associated with a delayed anti-prrsv antibody response in these pigs. pcv coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded, positive-sense rna virus, is characterized by a high mutation rate with the potential of genetically diverse strains evolving over time (forsberg et al., ; hanada et al., ; pirzadeh et al., ; rowland et al., ) . in the past, prrsv isolates have emerged within the swine population with varying degrees of virulence (fang et al., ; han et al., ; nelsen et al., ) possibly due to a high degree of mutation and recombination (yuan et al., (yuan et al., , (yuan et al., , (yuan et al., , . more recently, attention has focused on the occurrence of high mortality in chinese swine herds which veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] to determine differences in infection kinetics of two temporally and genetically different type porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type a or b infection, pigs were randomly assigned to one of seven groups: negative controls (n = ); pigs coinfected with a prrsv strain (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with vr- and pcv b (coi- - b; n = ), pigs coinfected with a prrsv strain (nc b) and pcv a (coi- - a; n = ), pigs coinfected with nc b and pcv b (coi- - b; n = ), pigs infected with vr- (n = ), and pigs infected with nc b (n = ). blood samples were collected before inoculation and at day post-inoculation (dpi) , , and and tested for the presence of prrsv antibody and rna, pcv antibody and dna, complete blood counts, and interferon gamma (ifn-g) levels. regardless of concurrent pcv infection, vr- initially replicated at higher levels and reached peak replication levels at dpi . pigs infected with vr- had significantly higher amounts of viral rna in serum on both dpi and dpi , compared to pigs infected with nc b. the peak of nc b virus replication occurred between dpi and dpi and was associated with a delayed anti-prrsv antibody response in these pigs. pcv coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. ß elsevier b.v. all rights reserved. was associated with novel prrsv isolates and described as porcine high fever disease in tong et al., ; wu et al., ) . the prrsv isolates involved in porcine high fever disease contained unique nucleotide differences compared to other isolates. specifically, a discontinuous, amino acid deletion was identified within the nsp region which was initially suggested to be correlated with the pathogenicity of the virus wu et al., ). however, more recent reports have concluded that this deletion is unrelated to virulence (zhou et al., ) in spite of the high mortality that was initially associated with this prrsv variant tong et al., ) . interestingly, analysis of samples from affected pigs resulted in the identification of prrsv, porcine circovirus type (pcv ) and classical swine fever virus as the most common co-infection pathogens, suggesting that a potential synergistic interaction among these viruses may account for the unusually high mortality (lv et al., ; wu et al., ). pcv is a small, circular, non-enveloped dna virus belonging to the circoviridae family in the genus circovirus. pcv can be further divided into several subtypes of which pcv a and pcv b are prevalent worldwide (patterson and opriessnig, ) . to date, experimental infections comparing pcv a and pcv b in gnotobiotic and conventional pigs have not demonstrated major differences in virulence (beach et al., ; fort et al., ; lager et al., ; opriessnig et al., b) . pcv is the cause of porcine circovirus associated disease (pcvad) with multiple clinical manifestations including respiratory disease . prrsv has become an important component of the porcine respiratory disease complex (prdc) with major economic impact on the swine industry (chae, ) . retrospective studies identified prrsv as the most common cofactor in cases of pcvad (pallaré s et al., ) . experimental coinfection with prrsv and pcv has yielded mixed results. one study completed in reported minimal clinical disease or death loss in conventional pigs coinfected with pcv and prrsv (rovira et al., ) . in contrast, in another study, severe clinical disease and death in of pigs between and days postinfection (dpi) was reported in dually infected, caesarianderived and colostrum-deprived (cdcd) pigs (harms et al., ) . despite differences in severity of clinical presentation, experimental coinfection of pigs with pcv and prrsv has consistently resulted in up-regulation of pcv replication (enhanced viremia and pcv tissue load) and increased severity of prrsv-induced lesions in lung tissues (allan et al., ; harms et al., ; rovira et al., ) . in north america, both prrsv and pcv b have been identified in pcvad outbreaks characterized by excessive mortality suggesting a synergistic relationship between these two viruses gagnon et al., ; horlen et al., ) . the objective of this study was to characterize the infection dynamics and pathogenicity of two different type prrsv isolates in a conventional pig model under the influence of concurrent pcv a or pcv b infection. the severity of clinical disease, macroscopic and microscopic lesions, amount of prrsv and pcv antibodies and nucleic acids in sera, amount of prrsv and pcv antigen associated with lesions, and interferon gamma (ifn-g) concentrations in serum were measured and compared between groups. fifty-three colostrum-fed, crossbred pigs were derived from sows known to be free of pcv , prrsv and mycoplasma hyopneumoniae in two separate batches, pigs in batch (b ) and pigs in batch (b ). in addition, batch (b ) consisted of colostrum-fed crossbred pigs derived from sows free of prrsv and m. hyopneumoniae but seropositive for pcv . b and b pigs were challenged at the same age as b pigs but the experiment was conducted approximately months after b pigs. insufficient numbers of pigs were available from the source herd for singularly prrsv-infected groups to be included with the original experiment. the experimental design and group designations are summarized in table . all pigs were housed under the same conditions and treated in a similar way. all pigs were weaned at three weeks of age and transported to the livestock infectious disease isolation facility at iowa state university, ames, iowa. on the day of arrival, all b pigs were comingled and randomly assigned to one of five rooms each containing or pigs: negative controls (n = ); pigs coinfected with a isolate of prrsv (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with prrsv vr- and pcv b (coi- - b; n = ), pigs coinfected with a isolate of prrsv (nc b) and pcv a (coi- - a; n = ) and pigs coi- - b pcv b nc b prrsv-i- none vr- prrsv-i- none nc b b -prrsv-i- none vr- b -prrsv-i- none nc b a batch and pigs were derived from the same source herd free of prrsv and pcv whereas batch pigs were derived from a different source herd seropositive for pcv . coinfected with prrsv nc b and pcv b (coi- - b; n = ). b pigs were randomly assigned to one of two rooms each containing or pigs which were infected with prrsv vr- (prrsv-i- ) and prrsv nc b (prrsv-i- ), respectively. similarly, b pigs were comingled and randomly assigned to one of two rooms each containing or pigs that were infected with prrsv vr- (b -prrsv-i- ) and prrsv nc b (b -prrsv-i- ), respectively. the pigs from the different batches were kept in different but identical rooms. each room had m of solid concrete floor space, separate ventilation systems and one nipple drinker. inoculation was conducted at approximately days of age. blood samples were collected from all pigs prior to inoculation and at dpi , , and in serum separator tubes ( . ml bd vacutainer, benton dixon, franklin lakes, nj, usa). the blood was centrifuged at  g for min at c and serum was stored at À c until testing. serum samples were analyzed for levels of anti-pcv igg antibody, anti-prrsv-igg antibody, ifn-g, pcv dna, and prrsv rna. in addition, edta tubes ( . ml monoject tm % edta liquid, tyco healthcare group lp, mansfield, ma, usa) were collected at dpi , , , and , stored at room temperature and used within h after collection to determine blood cell counts. all pigs were necropsied on dpi and tissues collected during necropsy were analyzed by immunohistochemistry (ihc) for the presence of pcv and prrsv antigens. the experimental protocol was approved by iowa state university institutional animal care and use committee (iacuc approval # - - -s). prrsv isolate vr- with a rflp pattern - - was recovered in from pig tissues obtained from a sow herd in southwestern iowa affected by severe respiratory disease in - -week-old pigs and high numbers of late term abortions (halbur et al., b; meng et al., ) . the passage virus of the original vr- isolate was used to inoculate pigs in as described previously . serum from the pigs infected with vr- in was used to re-isolate the virus followed by two subsequent passages in marc- cells to produce the virus stock of vr- for this study. prrsv isolate nc b with a rflp pattern - - was isolated in from a clinically affected -week-old pig with systemic pcvad from a group of pigs from north carolina with a history of severe respiratory disease in % of the pigs and approximatley % mortality in the group (gauger et al., ) . the passage virus of the original isolate was used to experimentally infect a set of prrsv-free conventional pigs (data not shown) and the lung tissues from these pigs collected two weeks after infection were used for reisolation of the nc b virus followed by two subsequent passages in marc- cells to produce the nc b virus stock for this study. the two inocula were separated in different aliquots, stored at À c, and virus of the same lot was used for all batches of pigs. on dpi , coi- - a, coi- - b, prrsv-i- , and b -prrsv-i- groups received ml of prrsv isolate vr- at a dose of . median tissue culture infective dose (tcid ) per ml. all pigs in groups coi- - a, coi- - b, prrsv-i- , and b -prrsv-i- received ml of prrsv isolate nc b at a dose of . tcid per ml. inoculation was intranasal by holding the pig in the upright position and slowly dripping ml of the inoculum into each nostril using a ml syringe (fisher scientific, inc.). two different pcv subtypes were used for the inoculation of pigs. pigs in groups coi- - a and coi- - a were inoculated with the pcv a isolate , which was recovered from an iowa farm in (fenaux et al., ) and has been well characterized genetically (fenaux et al., ) and in the conventional specific pathogen free (spf) pig model (opriessnig et al., (opriessnig et al., , a . both pcv a and pcv b viruses were produced as described previously (opriessnig et al., b) and used for inoculation in this study at a titer of . tcid per ml . pigs in groups coi- - b and coi- - b were inoculated with pcv b isolate nc which was isolated in from a pig farm in north carolina (opriessnig et al., b) . both, pcv b nc and prrsv nc b originated from the same tissues. the pcv groups were inoculated intranasally ( ml) and intramuscularly ( ml) with their respective pcv subtype by injecting ml of the inoculum intramuscularly into the right neck area and ml ( . ml per nostril) intranasally by holding the pig in the upright position and slowly dripping . ml of the inoculum into each nostril using a ml syringe (fisher scientific, inc.). edta-treated blood samples were analyzed for white blood cells using a multispecies hematology instrument (hemavet hv fs, drew scientific, inc.). the white blood cell (wbc) count was reported as actual numbers of neutrophils, lymphocytes and total wbc per ml of whole blood. in addition to wbc, a ratio was determined between the total neutrophil count and the total lymphocyte count reported as the n/l ratio. values from negative control pigs were considered as baseline for the infected pigs on each dpi. serum samples from all pigs were also tested for the presence of anti-prrsv antibodies by a commercial prrsv elisa (herdchek prrs virus antibody test kit xr, idexx laboratories inc. westbrook, ma, usa), according to the instructions of the manufacturer. samples were considered positive if the calculated s/p ratio was equal to . or greater. all serum samples were tested for the presence of anti-pcv igg antibodies based on an open reading frame (orf ) elisa (nawagitgul et al., ) . samples were considered positive if the calculated sample-to-positive (s/ p) ratio was equal to . or greater. on dpi , three samples were randomly chosen from each group and room and tested for the presence of swine influenza virus (siv) antibodies by an in house nucleoprotein ns elisa (richt et al., ) and for the presence of antibodies to porcine parvovirus (ppv) by hemagglutination inhibition (hi) assay (mengeling et al., ) . a commercial elisa kit (swine ifn-g; invitrogen, camarillo, ca, usa) was used to detect and quantify ifn-g concentrations in serum according to the instructions of the manufacturer. following prrsv/pcv coinfection, the pigs were monitored daily for respiratory disease (dyspnea, sneezing, coughing, nasal discharge). rectal temperatures and behavioral changes such as lethargy and inappetence/ anorexia were also recorded daily. the observers were aware (not blinded) to the treatment status. rna extraction on serum collected at dpi , , , , and was performed using a qiaamp viral rna mini kit (qiagen, valencia, ca, usa). the agpath-id prrsv multiplex reagent kit (applied biosystems, foster city, ca, usa) was used for the real-time, reverse transcriptase pcr (rt-pcr) on each extracted rna sample. all samples were run in duplicate. each pcr consisted of ml template rna and ml of pcr master mix. the pcr master mix contained . ml of  rt-pcr buffer, . ml  prrsv primer probe mix, . ml  multiplex rt-pcr enzyme mix, . ml of zenorna- internal control rna and . ml nuclease-free water. each reaction included eight progressive : dilutions of a known copy number of prrsv to generate a standard curve for quantification. each plate was run in the sequence detection system (geneamp sequence detection system, applied biosystems) using the agpath-id company specific conditions ( min at c, min at c, followed by cycles of s at c and s at c). samples were considered negative when no signal was observed within the amplification cycles. dna extraction on serum collected on dpi , , , , and days was performed using the qiaamp dna blood mini kit (qiagen, valencia, ca, usa) and subsequently used for detection of pcv dna by quantitative real-time pcr utilizing primers and a probe designed for pcv orf as described (opriessnig et al., ) . the real-time pcr reaction consisted of a ml pcr mixture containing . ml commercially available master mix (taqman universal pcr master mix, applied biosystems by life technologies), . ml dna, ml ( . mm) of each primer, and . ml ( . mm) probe. the reaction was run in a fast real-time pcr system (abi, foster city, ca, usa) under the following conditions: c for min, c for min, followed by cycles of c for s and c for min. all samples were run in duplicate. serial dilutions of a recombinant pcv dna clone were included on each plate to generate a standard curve. viral concentrations were expressed as the dna copy numbers per ml of sample. samples were considered negative when no signal was observed within the amplification cycles. all dna extracts were also tested for presence of pcv a and pcv b dna by utilizing a forward primer ( -gcagggccagaattcaacc- ), a reverse primer ( -ggcggtggacatgatgaga- ), a probe specific for pcv a ( -cal fluor orange -ggggaccaacaaaatctcta-tacccttt-bhq- ), and a probe specific for pcv b ( -quasar -ctcaaacccccgctctgtgccc-bhq- ), which were designed in the pcv orf as described . the multiplex real-time pcr reaction consisted of a total volume of ml containing . ml of the commercially available master mix (applied biosystems), ml dna, . mm of each primer, and . mm of each probe. all samples were run in duplicate. the reactions were carried out under the following conditions: c for min, c for min, followed by cycles of c for s and c for min. the sensitivity and specificity of the real-time pcr reaction was evaluated using known pcv a and pcv b isolates as well as ppv, prrsv, and pcv type (pcv ) isolates. samples were considered negative when no signal was observed within the amplification cycles. open reading frame (orf) of one prrsv rt-pcr positive pig in each group was sequenced on dpi as previously described (gauger et al., ) . on dpi , all pigs were humanely euthanized by intravenous pentobarbital overdose (fatal-plus vortech pharmaceutical, ltd., dearborn, mi, usa). macroscopic lung lesions were estimated based on the percentage of the lung surface affected by pneumonia ranging from to % (halbur et al., b ). the scoring system was based on the approximate volume that each lung lobe contributes to the entire lung: the right cranial lobe, cranial part of the left cranial lobe, and the caudal part of the left cranial lobe contribute % each to the total lung volume, the accessory lobe contributes %, and the right and left caudal lobes contribute . % each (halbur et al., b) . additionally, lymph node size was scored ranging from (normal) to (four times the normal size) (opriessnig et al., ) . lungs were insufflated with fixative as previously described (halbur et al., b) . sections of lymph nodes (tracheobronchial, mesenteric, mediastinal, superficial inguinal, and external iliac), tonsil, thymus, ileum, kidney, colon, spleen, heart, liver, and brain were collected at necropsy and fixed in % neutral-buffered formalin and routinely processed for histological examination. microscopic lesions were evaluated independently by two veterinary pathologists (to, pcg) blinded to the treatment status. sections of lung were scored for the presence and severity of interstitial pneumonia ranging from (normal) to (severe, diffuse) (halbur et al., b) . sections of heart, liver, kidney, ileum, colon and brain were evaluated for the presence of lymphohistiocytic inflammation and scored from (none) to (severe). lymphoid tissues including lymph nodes (trachea-bronchial, mediastinal, mesenteric, external iliac and superficial inguinal), tonsil, spleen and thymus were evaluated for the presence of lymphoid depletion ranging from (normal) to (severe) and histiocytic inflammation and replacement of follicles ranging from (normal) to (severe) (opriessnig et al., ) . . . immunohistochemistry . . . prrsv detection of prrsv-specific antigen was performed by ihc staining on lung sections as previously described (halbur et al., a) . sections were scored for presence of prrsv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. ihc for detection of pcv -specific antigen was performed on sections of lung, lymph nodes (tracheobronchial, mediastinal, mesenteric, superficial inguinal and external iliac), tonsil, spleen, thymus and small intestine using a rabbit polyclonal antiserum (sorden et al., ) . sections were scored for presence and amount of pcv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. if the results obtained by the two pathologists on a certain tissue differed, the mean of the two scores was used. pcv scores ranged from (no antigen) to (more than % of the lymphoid follicles contain cells with pcv -antigen staining) (opriessnig et al., ) . any tissue or tissue pool with detectable staining was given at least a score of . for the purpose of determining prevalence rates, a score of was considered negative and scores of , and were considered positive. for data analysis, jmp software version . . and sas software version . . (both sas institute, cary, nc, usa) were used. summary statistics were calculated for groups to assess the distributional property and data that were not distributed normally (pcr data) were log transformed prior to analysis. as log transformation can only be applied to numbers above , a constant number ( ) was added to each number in the data set prior to log transformation. a linear mixed model with the random effects ''source'' (source a: b and b and source b: b ) and ''batch'' (b , b , b , nested within ''source'') and the fixed effects ''prrsv strain'' (none, vr- , nc b) and ''pcv subtype'' (none, pcv a, pcv b) was used first on all outcomes. from this, it was determined that the random effect ''source'' contributed to the overall variation whereas ''batch'' did not. to decrease the heterogeneity of the animals in the analysis, all data obtained from the second source, b , were removed from the analysis but were provided as supplemental information throughout the ''results'' and tables. the final model to analyze continuous data collected over time (rectal temperatures, blood cell counts, log transformed pcv and prrsv genomic copies, and elisa s/p ratios) was a repeated measures analysis of variance (anova), where prrsv strain, pcv subtype, dpi and their interactions were the fixed effects and pig was the subject of repeated measures. compound symmetry variancecovariance structure was used to model the within pig correlation. a one-way anova was used to analyze crosssectional data (macroscopic and microscopic lung lesions) where prrsv strain, pcv subtype, and their interaction were the fixed effects. differences among the interacting groups (prrsv strain  pcv subtype) in the repeated measures anova or the one-way anova were assessed using tukey's t-test. a p-value of less than . was considered significant. differences in prevalence of prrsv and pcv antigen between groups (ihc staining) were determined by fisher's exact test. mild, transient lethargy and inappetence were observed in all inoculated groups, although coughing or sneezing was not a feature. pigs in all inoculated groups regardless of coinfection status developed a transient to persistent fever ranging from . c to . c between dpi and dpi . the mean rectal temperature time by group interaction after inoculation was significant (p < . ). all six inoculated groups had rectal temperatures significantly higher than the negative controls at dpi and dpi . by dpi , the mean group rectal temperatures in the prrsv-i- , prrsv-i- , coi- - a, coi- - b and coi- - a groups were significantly (p < . ) higher compared to the negative controls. when the effect of ''prrsv strain'' was evaluated across groups, no differences were found. compared to pigs infected with prrsv alone, coinfected pigs had higher mean rectal temperatures at dpi , and . when the effect ''pcv subtype'' was evaluated among coinfected groups, pcv a pigs had significantly (p < . ) higher rectal temperatures on dpi compared to pcv b pigs (data not shown). b pigs (b -prrsv-i- and b -prrsv-i- ) had similar rectal temperatures as b (prrsv-i- and prrsv-i- ) pigs. hematology results are summarized in table . there was an effect of ''prrsv strain'' on white blood cell counts at dpi with pigs infected with nc b having significantly (p = . ) higher levels of white blood cells compared to pigs infected with vr- ( . ae . versus . ae . ). also, there was a significant effect of ''pcv '' (p < . ): pcv -infected pigs had higher levels of white blood cells at dpi and compared to non-pcv -infected pigs ( . ae . versus . ae . and . ae . versus . ae . ). there was no effect of ''prrsv strain'' on numbers of neutrophils; however, there was a significant effect of ''pcv '' on mean group neutrophil counts at dpi , , and with pcv -infected pigs having elevated levels compared to pigs not infected with pcv ( . ae . versus . ae . , . ae . versus . ae . , and . ae . versus . ae . , respectively). differences in mean group lymphocyte counts were only observed on dpi (table ) and there was an effect of prrsv strain (p = . ): pigs infected with nc b had lower levels of lymphocytes compared to pigs infected with vr- ( . ae . versus . ae . ). additionally, pigs coinfected with pcv had higher levels of lymphocytes (p = . ) compared to pigs infected with prrsv alone ( . ae . versus . ae . ) suggesting an effect of ''pcv ''. all pigs in all groups were negative for prrsv-specific antibodies at dpi and negative control pigs remained negative for anti-prrsv antibody throughout the study. prevalence and group mean s/p ratios are summarized in table . overall, there was a significant effect of ''prrsv strain'' and ''pcv '' on the anti-prrsv antibody response at dpi . specifically, pigs infected with vr- had a significantly (p = . ) higher anti-prrsv antibody response compared to those infected with nc b. similarly, coinfected pigs had significantly (p = . ) higher anti-prrsv s/p ratios compared to pigs singularly infected with prrsv. there was no effect of ''pcv subtype'' on the magnitude of the anti-prrsv-antibody response among coinfected groups. all pigs in b and b were negative for pcv -specific anti-igg antibodies at dpi and the negative controls and b pigs remained pcv seronegative throughout the trial. in the pcv coinfected groups, seroconversion was observed at dpi . the prevalence and mean group anti-pcv -igg s/p ratios table mean group leukocyte values ( /ml of whole blood except for ratios) in the different treatment groups on days post-inoculation (dpi) , , , and . data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. hematology a negative controls (n = ) wbc . ae . . ae . are summarized in table . there was no effect of ''prrsv strain'' or ''pcv subtype'' on the magnitude of the anti-pcv -antibody response. b pigs were seropositive for pcv at the time of arrival (mean pcv elisa s/p ratio: . ae . ) and the s/p ratios remained at a similar level for the duration of the study (data not shown). at termination of the study, pigs randomly selected from each batch tested negative for antibodies against ppv, siv h n and siv h n (data not shown). prevalence of ifn-g positive samples and mean group ifn-g concentrations are summarized in table . there was no effect of ''prrsv strain'' or ''pcv '' on the ifn-g levels and no differences were found among groups; however, analysis of an effect of ''pcv subtype'' among coinfected groups revealed that on dpi , pcv b-inoculated pigs had significantly (p = . ) higher levels of ifng compared to pcv a-inoculated pigs ( . ae . pg/ml versus . ae . pg/ml). all pigs were negative for prrsv-rna in serum at dpi and the negative controls remained negative for prrsv rna throughout the study. the prevalence of prrsv rna positive pigs and group mean genomic copy numbers/ml are summarized in table . sequencing of the orf gene of prrsv and comparison with the original inocula confirmed that the correct prrsv isolates were present in the table prevalence of anti-prrsv antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups on days post-inoculation (dpi) , , , and . data presented as prevalence (mean s/p ratio ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. different superscripts (a, b, c) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. prevalence of anti-pcv igg antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups except prrsv-i- and prrsv-i- on day post-inoculation (dpi) , , , and . data presented as prevalence (mean s/p ratio ae se). grey shaded areas indicate the presence of pcv seropositive pigs (s/p ratio > . ) within a treatment group. different superscripts (a, b) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. prevalence of ifn-g and mean group concentration (pg/ml) in the different treatment groups on day post-inoculation (dpi) , , , and . data presented as prevalence (mean log group concentration ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. respective groups and rooms. when results were analyzed for a possible effect of ''prrsv strain'', a significantly (p < . ) higher amount of prrsv rna was detected in pigs infected with vr- at dpi and compared to pigs infected with nc b ( fig. ) . when the pigs infected with prrsv alone were removed from the analysis, coinfected pigs with nc b had significantly higher amounts of prrsv rna in serum compared to pigs infected with vr- on dpi ( . ae . versus . ae . ) and dpi ( . ae . versus . ae . ), respectively. an effect of ''pcv '' or ''pcv subtype'' on prrsv replication was not evident. all pigs were negative for pcv -dna in serum at dpi and the negative controls and b and b pigs remained pcv dna negative throughout the study (data not shown). at dpi , / pcv -inoculated pigs were positive for pcv -dna, and all pcv -inoculated pigs were positive for pcv -dna by dpi and remained positive until dpi . the log group mean pcv dna amounts are summarized in fig. . when results were analyzed for a possible effect of ''prrsv strain'' it was found that there was a significantly higher amount of pcv dna in pigs infected with vr- ( . ae . ) compared to pigs infected with nc b ( . ae . ) on dpi . there was a significant effect of ''pcv subtype'' on dpi ; groups infected with pcv b had significantly higher amounts of pcv dna in serum compared to groups infected with pcv a ( . ae . versus . ae . , respectively). an effect of ''pcv subtype'' was not evident in the later stages of infection. all pigs in the pcv a or pcv b groups were correctly infected with their respective subtype as determined by multiplex real-time pcr (data not shown) and crosscontamination between groups and rooms was not detected. table prevalence of prrsv and group mean log prrsv genomic copies per ml in the different treatment groups on days post-inoculation (dpi) , , and . data presented as prevalence (log prrsv rna ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included into the analysis. group negative controls / ( . ae . ) a / ( . ae . ) a / ( . ae . ) a / ( . ae . ) a coi- - a / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b coi- - b / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b coi- - a / ( . ae . ) b,c / ( . ae . ) b,c / ( . ae . ) b / ( . ae . ) b coi- - b / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b prrsv-i- / ( . ae . ) d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b prrsv-i- / ( . ae . ) c / ( . ae . ) c / ( . ae . ) b / ( . ae . ) b b -prrsv-i- / ( . ae . ) / ( . ae . ) / ( . ae . ) / ( . ae . ) b -prrsv-i- / ( . ae . ) / ( . ae . ) / ( . ae . ) / ( . ae . ) different superscripts (a,b,c,d) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. fig. . log transformed mean prrsv rna genomic copies (aese) in vr- and nc b infected pigs regardless of coinfection status on day postinoculation (dpi) , , , and . significant (p < . ) differences between groups within a dpi are indicated by asterisks. the lines indicate the linear trend for pigs infected with vr- (gray, dashed) or nc b (black, solid). macroscopic lesions were characterized by mild-tomoderate enlargement of lymph nodes (especially tracheobrochiolar lymph nodes and mediastinal lymph nodes) and mottled-tan lungs with varying degrees of the lung surface affected by visible pneumonia lesions. the group mean lung lesion severity scores are summarized in table and were significantly (p < . ) lower for negative controls compared to all coinfected groups. there were no significant differences in lung lesions severity between the negative controls and the pigs infected with prrsv alone. there was an effect of ''pcv '' on the mean group macroscopic lung lesion scores as evidenced by the coinfected pigs having more severe macroscopic lung lesions compared to pigs infected with prrsv alone. however, there was no effect of ''prrsv strain'' or ''pcv subtype'' on the severity of the observed macroscopic lung lesions. lung tissues had multifocal-to-diffuse, mild-to-severe, lymphohistiocytic interstitial pneumonia. the mean microscopic lung lesion scores, which are summarized in table , were significantly (p < . ) lower in the negative controls compared to the four coinfected groups; however, the scores in the negative controls were not significantly lower than observed in the groups singularly infected with prrsv. there was a significant effect of ''pcv '' (p < . ) on microscopic lung lesions but there was no effect of ''prrsv strain'' or ''pcv subtype'' on the severity of the observed microscopic lung lesions. lymphoid lesions were either not observed or were characterized by mild depletion of follicles and minimal granulomatous lymphadenitis in all coinfected groups. significant differences in lymphoid lesion scores were not observed among the groups (data not shown). . . . prrsv all control pigs were negative for prrsv antigen by ihc on sections of lung. the prevalence of prrsv antigen in lung sections was / pigs in the nc b-inoculated group (b : / pigs) compared to / pigs in the vr- -inoculated group (b : / pigs). there were no significant differences in the prevalence rates of prrsv fig. . log transformed group mean pcv dna amounts (aese) in the pcv -prrsv coinfected groups on day post-inoculation (dpi) , , , and . significant (p < . ) differences between groups within a dpi are indicated by different superscripts (a, b). mean group macroscopic (percentage of lung surface affected by lesions) and microscopic (interstitial pneumonia ranging from = normal to = severe, diffuse) lung lesions (mean group amount ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. significant (p < . ) differences between groups are indicated by different superscripts (a, b, c). macroscopic lung lesions ( - %) antigen in lungs between the virus-inoculated groups. the prevalence of prrsv antigen in lungs was independent of ''prrsv strain'' or ''pcv subtype''. all control pigs and all b and b pigs were negative for pcv antigen by ihc. low-to-high amounts of pcv antigen in lung sections were detected in / coi- - a pigs, / coi- - b pigs, / coi- - a pigs and in / coi- - b pigs which corresponds to / pcv a-inoculated pigs and / pcv b-inoculated pigs. moreover, pcv antigen was detected in / vr- -inoculated pigs and in / nc b-inoculated pigs. the prevalence of pcv antigen in lung tissues was independent of ''prrsv strain'' or ''pcv subtype''. in lymphoid tissues, low-tohigh amounts of pcv antigen were detected in / coi- - a pigs, / coi- - b pigs, / coi- - a pigs and in / coi- - b pigs which corresponds to / pigs inoculated with pcv a and / pigs inoculated with pcv b, as well as / pigs inoculated with nc b and / pigs inoculated with vr- . the prevalence of pcv antigen in lymphoid tissues was independent of ''prrsv strain'' or ''pcv subtype''. the objective of this study was to characterize the infection dynamics and pathogenicity of two different type prrsv isolates recovered from pigs in and in a conventional pig model. to mimic field conditions where coinfections frequently occur, the pigs were concurrently infected with either pcv a or pcv b. the effect of each prrsv isolate was also evaluated in singularly inoculated pigs. however, due to limitations in numbers of available pigs from the source herd, the experiments with singularly prrsv-inoculated groups were conducted separately but under the same study conditions, using the same inocula and assays to analyze the samples. the prrsv isolate vr- used in this experiment has been well-characterized in the cdcd and the conventional pig models and is considered a relatively highly pathogenic prrsv isolate from the s (halbur et al., b meng et al., ) . in contrast, nc b represents a more recent prrsv isolated from an outbreak of respiratory disease on a farm characterized by high morbidity and mortality in (gauger et al., ) . the orf - sequence homology between vr- (genbank accession pru and pru ) and nc b (genbank accession hq ) was approximately . %. the orf region demonstrated the least nucleotide and amino acid homology at . % and . %, respectively (gauger et al., ) . in the past, dual infections with prrsv and porcine respiratory coronavirus (prcv) or prrsv and siv were studied using conventional pigs (van reeth et al., ) and gnotobiotic pigs (van reeth and nauwynck, ) and in general disease was found to be more pronounced in dually inoculated pigs. interestingly, in gnotobiotic pigs the effect of the coinfection appeared additive rather than synergistic (van reeth and nauwynck, ) . more recent studies have shown that prrsv modifies the innate immune response, induces immunosuppression and enhances the inflammatory response to prcv in pigs (jung et al., ; renukaradhya et al., ) . in another study, dual infection of specific pathogen-free pigs with prrsv and pseudorabies virus (prv) resulted in more severe clinical signs and increased pneumonia in pigs inoculated with both viruses compared to pigs infected with prrsv or prv alone (shibata et al., ) . it is also well recognized that pcv replication is enhanced by concurrent prrsv infection in both cd and conventional pigs compared to singularly inoculated pigs (allan et al., ; rovira et al., ) . to the authors' knowledge, the pathogenicity of genetically different prrsv isolates in the presence of concurrent viral infection has not been evaluated in vivo. the combination of prrsv and pcv is one of the most common coinfections associated with swine respiratory disease under field conditions (dorr et al., ; pallaré s et al., ) . both prrsv isolates used in the current study were isolated from field cases of high mortality and experimental infection of pigs with prrsv vr- has resulted in severe lesions and high levels of viremia (halbur et al., b . the two pcv isolates were initially recovered from typical field cases of pcvad in iowa and north carolina and have been characterized in the conventional pig model side by side without identifiable differences between pcv subtypes (opriessnig et al., b; sinha et al., ) . in the current study, clinical disease in the treatment groups was characterized by variable numbers of infected pigs experiencing transient, mild lethargy, mild respiratory disease and inappetence. coinfected groups had significantly higher mean rectal temperatures compared to pigs infected with prrsv alone and the negative controls. interestingly, when organized by coinfection status and analyzed by pcv subtype, pigs inoculated with pcv a had significantly higher mean group rectal temperatures compared to pigs inoculated with pcv b on dpi which was associated with an anti-pcv -antibody response in . % ( / ) of the pcv ainoculated pigs on dpi whereas a delayed antibody response was seen in pcv b-inoculated pigs ( . %; / ). it is well documented that pathogenic differences between type prrsv isolates exist (halbur et al., b . the uniqueness of the current study is the utilization of two temporally and genetically different prrsv isolates both from cases of high morbidity and mortality in the field but isolated years apart. in a separate in vitro study comparing phenotypic traits of the two prrs viruses, nc b demonstrated reduced growth characteristics compared to vr- (gauger et al., ) . nc b plaque sizes were slightly smaller than vr- and the peak viral titer demonstrated by nc b was approximately -fold lower than the vr- peak titer. this is in contrast to the in vivo growth characteristics demonstrated in this report. there were clear differences in initial replication between the two prrsv isolates used in this study. the vr- -inoculated pigs had significantly higher levels of prrsv rna in serum on dpi and . moreover, nc b replicated at higher levels at dpi and dpi compared to vr- which was associated with significantly lower levels of lymphocytes at dpi and a significantly lower n/l ratio at dpi . these results suggest that highly pathogenic prrsvs may replicate more efficiently in vivo in contrast to their decreased growth properties in vitro as previously suggested (johnson et al., ; wang et al., ) . this is further supported by the data obtained from the pigs infected with prrsv alone (b and b ) which clearly show an increase in replication in pigs infected with nc b in the later stages of the in vivo study. similar to other pcv -prrsv coinfection studies (allan et al., ; harms et al., ) , macroscopic and microscopic lesions in coinfected groups were enhanced compared to pigs singularly infected with prrsv. recently, it has been shown that pigs infected with vr- had significantly prolonged (until dpi) pcv viremia and shedding in prrsv-pcv coinfected pigs (sinha et al., ) . a similar approach using prrsv nc b, which replicated differently from vr- in the early stages of infection, could potentially offer new insights on viral interactions in pigs. in the current study, pcv b replication was significantly up-regulated shortly after initiation of the study at dpi compared to pcv a. furthermore, the coi- - b group had significantly higher quantities of pcv b in the serum compared to coi- - a (dpi and ) and coi- - b (dpi ) which was associated with a higher prevalence of pcv antigen in tissues ( . % versus . %) indicating a synergistic relationship between prrsv- (vr- ) and pcv . unlike previous studies where the average trial length ranged from to days (allan et al., ; rovira et al., ) , this trial was terminated at dpi to evaluate prrsv-induced lung lesions which tend to be most severe between dpi and dpi . it remains unknown if the observed trend would have resulted in a difference in clinical disease in the later stages of infection. as expected, and similar to a previous study , the pathological lesions associated with pcv were either not present or they were mild; however, pcv antigen was detected in most tissues in coinfected pigs. in this study, pcv naïve pigs were utilized, thus the relevance of the model to actual field situations is limited considering the majority of young pigs have high levels of passively acquired anti-pcv antibodies (opriessnig et al., b) . therefore, the impact of anti-pcv immunity on the pcv infection could not be ascertained in the experiment; however, this was not a major concern as we know from several experiments that pigs with passively derived antibodies, although protected from clinical pcv associated disease, can still be infected with pcv (mckeown et al., ; opriessnig et al., a) . therefore, we believe that a pcv naïve pig model increases the ability to identify trends and associations between prrsv and pcv . in the current study, prrsv-pcv coinfection was administered intranasally on the same day. this model of simultaneous dual inoculation does not fully mimic the population dynamics due to the variability in timing of exposure to these two pathogens within and between herds in field situations. on many conventional farms, endemic exposure and seroconversion to prrsv often occurs earlier than exposure to pcv . infection of pigs with prrsv prior to pcv may contribute to the manifestation of more severe pcv -induced clinical disease and lesions. prrsv is immunosuppressive, primarily infecting porcine alveolar macrophages (drew, ) , which decreases the pig's ability to clear subsequent infections. in contrast, prior prrsv infection may induce an immunostimulatory effect on the host immune response that serves to enhance pcv replication and lesions (krakowka et al., ) . it is possible that amino acid mutations acquired during serial passaging of prrsv on marc- cells could result in attenuation as reported previously (allende et al., ; an et al., ) . while this is also applicable to the current study, we attempted to minimize this risk, by using a relatively low passage of both viruses with a pig passage followed by only two in vitro passages in marc- cells. inoculation was completed two days after weaning and transport of the pigs to the research facility. it is also possible that the stress from weaning, transport, new socialization, and adjusting to a new environment may have affected the ability of the pigs to respond to concurrent prrsv-pcv infection and influenced the level of prrsv replication in the pigs. however, the data obtained from pigs infected with prrsv alone indicate that this was not the case and that the ability of the pigs to develop a humoral immune response was normal. overall, the data indicate no significant differences between the two prrsv isolates based on clinical signs, gross pathology, histology or hematology even though the prrsv isolates we utilized in this study were isolated from geographically separated herds (vr- from iowa and nc b from north carolina) over a period of years. differences in in vivo replication kinetics were identified. vr- initially replicated more quickly and to higher levels and peaked at dpi and the amount of vr- rna steadily declined thereafter. in contrast, pigs infected with nc b had lower levels of prrsv rna in serum initially and this steadily increased through termination of the study at dpi . concurrent pcv viremia was enhanced by prrsv vr- infection but not by concurrent prrsv nc b infection. a higher prevalence of pcv antigen was demonstrated in the lungs of pigs coinfected with vr- ( . %) compared to pigs coinfected with prrsv nc b ( . %). this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens on prrsv kinetics. additional investigations are necessary to further elucidate the specific mechanisms of the pcv -prrsv interaction in pigs. comparative genomic analysis of five pairs of virulent parental/attenuated vaccine strains of prrsv experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv replication mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype novel chimeric porcine circovirus (pcv) with the capsid gene of the emerging pcv b subtype cloned in the genomic backbone of the nonpathogenic pcv is attenuated in vivo and induces protective and cross-protective immunity against pcv b and pcv a subtypes in pigs a review of porcine circovirus -associated syndromes and diseases detection of two porcine circovirus type genotypic groups in united states swine herds epidemiologic assessment of porcine circovirus type coinfection with other pathogens in swine a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus diversity and evolution of a newly emerged north american type porcine arterivirus: analysis of isolates collected between genetic characterization of type porcine circovirus (pcv- ) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of north america and development of a differential pcr-restriction fragment length polymorphism assay to detect and differentiate between infections with pcv- and pcv- a molecular clock dates the common ancestor of european-type porcine reproductive and respiratory syndrome virus at more than years before the emergence of disease porcine circovirus type (pcv ) vaccination of conventional pigs prevents viremia against pcv isolates of different genotypes and geographic origins the emergence of porcine circovirus b genotype (pcv- b) in swine in canada genetic and phenotypic characterization of a united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model replication and expression analysis of prrsv defective rna the origin and evolution of porcine reproductive and respiratory syndrome viruses three cases of porcine respiratory disease complex associated with porcine circovirus type infection experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus a cluster of farms experiencing severe porcine circovirus associated disease: clinical features and association with the pcv b genotype pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (prrsv) are related to viral load in acute infection 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 co-infections activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus- (pcv- ) mortality in pigs given porcine circovirus type subgroup and viruses derived from dna clones an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome effects of porcine circovirus type (pcv ) maternal antibodies on experimental infection of piglets with pcv molecular cloning and nucleotide sequencing of the -terminal genomic rna of the porcine reproductive and respiratory syndrome virus characterization of a high-virulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl size and antigenic comparisons among the structural proteins of selected autonomous parvoviruses modified indirect porcine circovirus (pcv) type -based and recombinant capsid protein (orf )-based enzyme-linked immunosorbent assays for detection of antibodies to pcv porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents 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 effects of the timing of the administration of mycoplasma hyopneumoniae bacterin on the development of lesions associated with porcine circovirus type influence of maternal antibodies on efficacy of porcine circovirus type (pcv ) vaccination to protect pigs from experimental infection with pcv porcine circovirus type (pcv )-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs differences in virulence among porcine circovirus type isolates are unrelated to cluster type a or b and prior infection provides heterologous protection experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with mycoplasma hyopneumoniae and porcine circovirus type effect of vaccination with selective bacterins on conventional pigs infected with type porcine circovirus derivation of porcine circovirus type -negative pigs from positive breeding herds porcine circovirus type (pcv- ) coinfections in us field cases of postweaning multisystemic wasting syndrome (pmws) epidemiology and horizontal transmission of porcine circovirus type (pcv ) genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope gp glycoprotein porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs vaccination of pigs against swine influenza viruses by using an ns -truncated modified live-virus vaccine experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type (pcv ) subtypes pcv a and pcv b by prolonging pcv viremia and shedding development of a polyclonal-antibody-based immunohistochemical method for the detection of type porcine circovirus in formalinfixed, paraffin-embedded tissue emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome proinflammatory cytokines and viral respiratory disease in pigs dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in china porcine circovirus type (pcv ) distribution and replication in tissues and immune cells in early infected pigs complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains heteroclite subgenomic rnas are produced in porcine reproductive and respiratory syndrome virus infection characterization of heteroclite subgenomic rnas associated with prrsv infection recombination between north american strains of porcine reproductive and respiratory syndrome virus the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence the authors thank the iowa livestock health advisory council for funding of this study. key: cord- - u bm fz authors: sánchez-carvajal, j.m.; rodríguez-gómez, i.m.; ruedas-torres, i.; larenas-muñoz, f.; díaz, i.; revilla, c.; mateu, e.; domínguez, j.; martín-valls, g.; barranco, i.; pallarés, f.j.; carrasco, l.; gómez-laguna, j. title: activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of prrsv- infection with the virulent strain lena date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: u bm fz porcine reproductive and respiratory syndrome virus (prrsv) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. pulmonary alveolar macrophages (pams) are the main cells supporting prrsv replication, with cd as the essential receptor for viral infection. although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for prrsv virulent strains. this research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by prrsv- strains of different virulence. conventional pigs were intranasally inoculated with the virulent subtype lena strain or the low virulent subtype strain and euthanised at , , and dpi. lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of il- and ifn-γ in sera compared to -infected pigs. extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in lena-infected pigs. lung viral load and prrsv-n-protein(+) cells were always higher in lena-infected animals. prrsv-n-protein(+) cells were linked to a marked drop of cd (+) macrophages. the number of cd (+) and inos(+) cells gradually increased along prrsv- infection, being more evident in lena-infected pigs. the frequency of cd r (+) and foxp (+) cells peaked late in both prrsv- strains, with a strong correlation between cd r (+) cells and lung injury in lena-infected pigs. these results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response. porcine reproductive and respiratory syndrome virus (prrsv) encompasses two species, betaarterivirus suid and betaarterivirus suid (formerly, prrsv- and prrsv- , respectively) (gorbalenya et al., ) , which present a wide inter-and intraspecies viral diversity (balka et al., ; shi et al., ; stadejek et al., ) . since , different outbreaks characterised by high morbidity and mortality rates, fever, haemorrhages, severe lesions in lung and, eventually, in other organs such as thymus or lymph nodes, have been reported associated with virulent prrsv- strains (canelli et al., ; karniychuk et al., ; morgan et al., morgan et al., , ogno et al., ; sinn et al., ; weesendorp et al., ) . several contradictory results about viraemia, tissue viral load, early virus clearance, low frequencies of prrsv-specific ifn-γ secreting cells or prrsv neutralizing antibodies have been reported after infection with prrsv- virulent strains. however, there is consensus on the fact that some strains are more virulent than others (canelli et al., ; ferrari et al., ; frydas et al., ; geldhof et al., ; morgan et al., ; renson et al., ; stadejek et al., ; weesendorp et al., weesendorp et al., , . prrsv replicates predominantly in the lung, causing a mild to severe interstitial pneumonia which may be complicated to suppurative bronchopneumonia due to the increased lung sensitisation to bacterial infections associated with the damage and impairment of the different pulmonary macrophage subpopulations (pulmonary alveolar macrophages, pams; pulmonary intravascular macrophage, pims; and interstitial macrophages) (brockmeier et al., ; thanawongnuwech et al., ) . pams are the main cellular target of prrsv, although interstitial and intravascular macrophages can be infected too (bordet et al., ; duan et al., ; gómez-laguna et al., ) , with j o u r n a l p r e -p r o o f the nucleocapsid protein n (prrsv-n-protein) as the most abundant viral protein during prrsv infection (rowland et al., ) . pams express high levels of cd scavenger receptor (sánchez et al., ; van gorp et al., ) which is essential to support prrsv internalisation and disassembly interacting with gp and gp viral proteins (burkard et al., ; das et al., ; whitworth et al., ) . a soluble form of cd (scd ), that may be released from tissue macrophages and monocytes, has been identified in plasma as potential biomarker for macrophage activity and inflammation (costa-hurtado et al., ; møller, ; pasternak et al., ) . prrsv is known to modulate the host immune response by inducing changes in the frequencies of immune cell subsets in blood (dwivedi et al., ; ferrari et al., ; morgan et al., ; weesendorp et al., ) and in tissues (gómez-laguna et al., ; rodríguez-gómez et al., ) , leading to an enhanced susceptibility to secondary bacterial infections (karniychuk et al., ; renson et al., ; sinn et al., ). an early decrease in the frequency of monocytes, nk cells or cytotoxic t cells linked to a strong inflammatory response in target organs has been described upon experimental infection with prrsv- virulent strains (ferrari et al., ; morgan et al., ; weesendorp et al., ; . in addition, some studies indicate an early overproduction of pro-inflammatory cytokines, such as ifn-γ, il- β or il- , as the main source of pulmonary injury after infection with virulent prrsv- strains (amarilla et al., ; morgan et al., ; renson et al., ; weesendorp et al., ) . nevertheless, other potential mechanisms, such as an imbalance among pro-and anti-inflammatory responses, might predispose to secondary infections contributing to the onset of the porcine respiratory disease complex (prdc) van gucht et al., ) . in this context, overproduction of nitric oxide (no), mainly triggered by inducible no synthase (inos) (akaike and maeda, ) , or upregulation of cd , as the primary lipopolysaccharide (lps) receptor (zanoni and granucci, ) , could contribute to lung inflammation upon infection with prrsv (chen et al., ; lee and kleiboeker, ; van gucht et al., yan et al., ) . by contrast, the transcription factor forkhead box protein (foxp ), is an essential transcription factor for the development of regulatory t cells (tregs) and hence, a useful marker to detect them. this subset could be involved in supressing the activation of other t-cell populations (käser et al., ) . cd receptor (cd r ), expressed on myeloid cells and b cell subsets (poderoso et al., ) , is an inhibitory surface receptor that might deliver inhibitory signals dampening the activation of cells which express it (vaine and soberman, ) . thus, both immune markers might play an important role inhibiting the production of proinflammatory cytokines (elmore et al., ; nedumpun et al., ; singh et al., ; vaine and soberman, ; wang et al., ) , lessening the exuberant lung injury observed with virulent prrsv- strains. whereas all these markers may play a key role in prrsv virulence, there are scarce studies analysing their role in the context of the lung lesion. therefore, the systemic immune response and immunopathology of lung are evaluated in this study with the goal of exploring the role of selected immune markers in the pro-and anti-inflammatory priming of the lung to secondary bacteria after infection with a virulent prrsv- strain (subtype , lena strain) in comparison with a low virulent prrsv- strain (subtype , strain). j o u r n a l p r e -p r o o f the low virulent strain (subtype prrsv- ) was isolated from the serum of a piglet with pneumonia from a prrsv-positive herd located in spain in . the virulent lena strain (subtype prrsv- ) is considered as the prototype of prrsv- virulent strains. lena strain isolation was performed from lung homogenates obtained from weak born piglets from a prrsv-positive herd from belarus in with a high mortality rate, reproductive failure and respiratory disorders (karniychuk et al., ) . viral stocks were produced from the th passage of each strain on pams, titrated by means of immunoperoxidase monolayer assay and expressed as tissue culture infectious doses (tcid )/ml ( strain: . tcid /ml; lena strain: . tcid /ml). the animals and samples used in this study were part of a project to investigate the pathogenesis of the infection with prrsv- strains of different virulence . briefly, fifty-two -week-old male and female piglets (landrace x large white crossbred) were obtained from a historically prrsv-negative farm. all pigs were negative for porcine circovirus type (pcv ), prrsv and mycoplasma hyopneumoniae by elisa and pcr assays (mattsson et al., ; sibila et al., ) . piglets were blocked by weight and sex and randomly assigned to three different groups and housed in separate pens: lena group (n= ), group (n= ) and control group (n= ). after an acclimation period of seven days, piglets were intranasally inoculated with either the low virulent strain or the virulent lena strain (both used at x tcid /ml, ml/nostril, using the mad nasal™ intranasal mucosal atomization device, teleflex, alcalá de henares, madrid, spain). the control group was mock- commencing day prior to inoculation, piglets were daily monitored to evaluate clinical signs (liveliness, respiratory symptoms and anorexia) and rectal temperature. quantification of clinical signs was performed by applying the following clinical score: liveliness (score , no abnormalities; score , reduced liveliness but with response to external stimuli; score , pig prostration; score , agonic pig); respiratory symptoms (score , no abnormalities; score , mild dyspnoea; score , evident dyspnoea; score , evident dyspnoea with tachypnoea; score , evident dyspnoea, tachypnoea and cyanosis); and anorexia (score , eating without abnormality; score , sporadic frequency of eating; score , no eating). the sum of these scores represented the total clinical score per animal and per day. rectal temperatures > . °c were considered hyperthermia. at necropsy, gross lung lesions were recorded and scored by the same pathologist (halbur et al., ) . afterwards, samples from apical, medial and caudal lobes from the right lung were collected and fixed in % neutral-buffered formalin (fisher scientific ltd., loughborough, uk) for histopathological and immunohistochemical studies. four-micron tissue sections were stained with haematoxylin and eosin and blindly graded by two pathologists for the histopathological evaluation. the severity of histopathological j o u r n a l p r e -p r o o f lesions in the lung was scored as previously described by halbur et al. ( ) : , no microscopic lesions; , mild interstitial pneumonia; , moderate multifocal interstitial pneumonia; , moderate diffuse interstitial pneumonia; and , severe interstitial pneumonia. in addition, a similar score was developed considering the diagnosis of suppurative bronchopneumonia : , no microscopic lesions; , mild bronchopneumonia; , moderate multifocal bronchopneumonia; , moderate diffuse bronchopneumonia; and , severe bronchopneumonia. altogether, the final score included the total of both, the interstitial pneumonia score and the bronchopneumonia score, being points the maximum possible score. rna was isolated from sera using nucleospin ® rna virus (macherey-nagel, düren, germany) according to manufacturer's instructions. for lung, rna was purified from tissue homogenate using a combined procedure with trizol™ (thermo fisher scientific, serial -fold dilutions of or lena orf rt-pcr products with known quantities, ranging from to genomic copies/ml were used as standards to generate a standard curve and, therefore, to determine the prrsv genomic copies in sera and lung. the rt-qpcr efficiency (e) was estimated for each strain by a linear regression model. the e value was calculated from the slope of the standard curve according to equation: also, a set of eight serial -fold dilutions of know tcid /ml (starting from tcid /ml) was included in order to determine a relation between ct-values, genomic copies/ml and tcid /ml. an inter-run calibrator sample with a known number of prrsv copies was introduced in each experiment to self-control inter-run variation. the area under the curve (auc) for viremia and lung viral load was calculated using the trapezoidal approach (greenbaum et al., ) . results of viral load in sera and lung are showed in equivalent tcid (eq tcid ) per ml. results were expressed in pg/ml for ifn-γ, il- and il- , and ng/ml for lbp and j o u r n a l p r e -p r o o f scd . the minimum detectable concentrations were pg/ml for ifn-γ, pg/ml for il- , pg/ml for il- , . ng/ml for lbp and . ng/ml for scd . four-micron sections from lung were dewaxed in xylene and rehydrated in descending grades of alcohol until distilled water. then, endogenous peroxidase inhibition was performed in a % h o solution in methanol for min. epitope demasking, primary antibodies dilutions and blocking of non-specific binding are detailed in table . monoclonal primary antibodies were incubated overnight at ºc in a humid chamber. tris buffered saline (ph . ) were used as wash and diluent buffers, respectively. antibody specificity was verified by exchanging the primary antibody by isotype matched reagents of irrelevant specificity. one negative control which consisted of replacement of the primary antibody by bsa blocking solution was included in each immunohistochemical assay to rule out non-specific bindings. the number of immunolabelled cells was quantified in non-overlapping selected high magnification fields of . mm (olympus bx , olympus iberia sau, l'hospitalet de j o u r n a l p r e -p r o o f llobregat, barcelona, spain) and expressed as the mean of the score for each animal per mm . labelled cells were morphologically identified by differentiating among pams, pims and interstitial macrophages. differences between groups were evaluated for approximate normality of distribution by the d'agostino and pearson omnibus normality test followed by the mann whitney's u non-parametric mean comparisons test. correlation coefficients were assessed by the spearman and pearson tests and were considered relevant with r > . and p < . . data analyses and figures were performed by using graphpad prism . software (graphpad prism software . , inc., san diego, ca, usa) and inkscape . software. a p value lower than . was considered statistically significant and represented as * p ≤ . , ** p ≤ . *** p ≤ . and **** p ≤ . . (table ) . viraemia and lung viral load were determined by rt-qpcr (efficiency of %; slope = . ; detection limit: copy/µl; slope-intercept = . ; and high linearity, r = . ). all animals were negative by rt-qpcr at day and control pigs remained so all throughout the experiment. in sera, four out of five -infected pigs and all lena-infected pigs were prrsv positive as early as dpi. viraemia was always higher in lena-than in -infected pigs from to dpi (p < . at , , dpi; p< . at dpi), reaching the highest viral load at dpi ( . x eq tcid /ml). the auc for viremia (mean) in lena and group were . and . respectively ( fig. a) . the viral load in the lung displayed a similar kinetics to that of serum for both infected groups, reaching the j o u r n a l p r e -p r o o f maximum lung viral loads at dpi in lena group ( . x eq tcid /ml), whereas group peaked at dpi ( . x eq tcid /ml) (fig. b) . by contrast to sera, prrsv- was just detected in lung in two out of five animals in both infected groups at dpi, being positive all infected piglets from dpi onwards. the auc for lung viral load (mean) in lena group was and . for group (fig. b) . in lena infected-group the statistical analysis revealed a positive correlation among viraemia, lung viral load, temperature, clinical signs score and the number of prrsv-n-protein + cells in the lungs (table ) . a correlation among lung viral load and viraemia and prrsv-n-protein + cells was also observed in -infected pigs (r = . , p < . ; and, r = . , p < . , respectively). prrsv-specific antibodies were first detected at dpi in sera from both prrsv- infected groups (non-significant differences in s/p ratios) (data not shown). a significant increase in ifn-γ serum levels was detected after lena infection at and dpi (maximum mean level of ± pg/ml at dpi) compared to control (p< . ) and (p< . ) groups (fig. c ). maximum il- levels in serum of group were observed at dpi (mean of ± pg/ml), whereas pigs belonging to lena group reached the highest il- levels at dpi (mean of ± pg/ml) (fig. d) . il- , lbp or scd were not detected in serum samples from both control and infected groups throughout the study. both viraemia and lung viral load displayed a positive statistical correlation with ifn-γ levels, which in turn were also correlated with temperature and the clinical signs score in lena infected-pigs (table ) . j o u r n a l p r e -p r o o f the labelling of prrsv-n-protein was mainly observed in pams and in a lesser extent in interstitial and intravascular macrophages (figs. a- b) . in lena-infected pigs, clusters of prrsv-n-protein + macrophages were observed within foci of bronchopneumonia at and dpi (fig. b inset) . a progressive increase in the number of prrsv-n-protein + cells was detected throughout the study in both prrsv- -infected groups, reaching a peak at and dpi in lena and -infected piglets, respectively. this increase was significantly higher in lena than in group (p< . at , and dpi) (fig. e , primary axis). no positive cells were detected in control pigs. (table ) . the labelling against cd was mainly observed in the cell membrane and cytoplasm of monocytes, interstitial and intravascular macrophages and, occasionally, in pams (figs. a- b, insets). whereas no changes were observed in the number of cd + cells in the control group along the study, a gradual increase with maximum expression at dpi was detected in both infected groups (fig. e) . lena-infected pigs showed the highest frequency of cd + cells when compared to control animals (p< . ) in association with the presence of suppurative bronchopneumonia (fig. b ). cd + interstitial and intravascular macrophages were observed infiltrating extensive areas of the interstitium, whereas almost no cd + cells were present in the bronchial wall and alveolar lumen. interestingly, the number of cd + cells in lena-infected piglets displayed a strong positive correlation with the concentration of il- in sera (table ) . the granular intracytoplasmic immunostaining of inos was primarily observed in pams and interstitial macrophages in foci of interstitial pneumonia and bronchopneumonia (figs. c- d). the number of inos + cells followed a similar kinetics in both prrsv- infected groups, with a progressive increase from dpi onwards, reaching a significant increase by the end of the study ( dpi) in lena-infected pigs compared to (p< . ) and control groups (p< . ) (fig. f) . cd r labelling was detected in the cytoplasm of intravascular and interstitial macrophages located inside or surrounding bronchopneumonia foci, with occasional j o u r n a l p r e -p r o o f expression in pams and monocytes (figs. a- b, insets). whereas the number of cd r + cells significantly increased in lena-infected pigs at - dpi (p< . at dpi with respect to group; and p< . at dpi and p< . at dpi with respect to control group), this increase was just detected at dpi in -infected pigs (p< . with respect to control group) (fig. e, primary axis) . control animals presented a scarce number of cd r + cells along the study. for lena infected-pigs a strong positive correlation was observed among the frequency of cd r + cells and the microscopic lung lesions (fig. e ) ( table ) . foxp yielded a nuclear immunolabelling in lymphocytes mainly located in areas of atelectasis and interstitial pneumonia (figs. c- d, insets). although two lena-infected pigs exhibited a higher number of foxp + cells at dpi, the kinetics of positive cells for this immune markers showed a gradual increase along the study in both lena-and infected animals, reaching the maximum at dpi (fig. f ). there were no significant differences in the number of foxpp + cells among infected groups. however, a significant increase of foxp + cells was detected at and dpi in lena-infected pigs compared to control animals (p< . ). prrsv plays a pivotal role in prdc, modulating the host immune response and favouring secondary bacterial infections van gucht et al., ) . virulent prrsv- strains cause more severe clinical signs, higher mortality rates as well as marked lung injury with a higher incidence of bronchopneumonia as opposed to low virulent strains (amarilla et al., ; canelli et al., ; frydas et al., ; j o u r n a l p r e -p r o o f gómez-laguna et al., ; morgan et al., ; renson et al., ; rodríguez-gómez et al., ; stadejek et al., ; weesendorp et al., ) . accordingly, we hypothesise that severe pulmonary lesions observed along infection with virulent prrsv- strains might be associated with a higher decrease in the amount of pams as well as an imbalance between anti-and pro-inflammatory responses with different molecules potentially involved in this process. as previously described (renson et al., ; weesendorp et al., ) , severe systemic and respiratory symptoms as well as hyperthermia were observed in animals infected with virulent lena strain, whereas low virulent strain only caused mild clinical signs and a slightly increase of rectal temperature. furthermore, virulent lena strain caused an earlier and stronger onset of lung lesions due to extensive consolidated areas in the apical and medial lobes which were microscopically linked to suppurative bronchopneumonia as well as severe characteristic interstitial pneumonia. on the other hand, prrsv virulence has been associated with higher virus titre and antibody response in vivo (brockmeier et al., ; lu et al., ) although lena virulent strain elicited a quite higher viraemia than the low virulent strain, no differences were observed in the antibody response in the early phase of infection. similar results have been previously reported by others when comparing lena with low virulent strains (renson et al., ; weesendorp et al., ) , and confirm that prrsv- virulence and specific non-neutralizing antibodies are not associated in the acute phase of infection. response when compared with low virulent strains (amarilla et al., ; liu et al., ; levels of il- at dpi and ifn-γ at - dpi were detected in the sera of lena-infected pigs. increased concentration of il- in plasma is associated with both systemic and respiratory symptoms (van reeth and nauwynck, ) and could play a dual role during virus infection: (i) protecting the host from infection and (ii) inducing inflammation and tissue damage when it is overexpressed (liu et al., ) . it is known that ifn-γ is, mostly produced by activated nk cells, nkt cells, γ/δ t cells, cytotoxic t cells and memory t cells (gerner et al., ; mair et al., ) , and participates in regulating the immune and inflammatory responses (van reeth and nauwynck, ) . in fact, an early increase of nkt cells has been associated with viraemia peak in piglets infected with pr cd and inos are involved in lung inflammation after infection with prrsv (chen et al., ; lee and kleiboeker, ; van gucht et al., yan et al., ) upregulation of cd , as lipopolysaccharides (lps) co-receptor, after infection with prrsv sensitises the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to bacterial lps (van gucht et al., ) . for its part, inos is mainly expressed in response to different stimuli, such as cytokines and lps, playing a role in tissue injury upon production of no (chen et al., ; cho and chae, ; vlahos et al., ; yan et al., ) . in this study, an increase in the number of cd + cells after prrsv- infection was observed in association with suppurative j o u r n a l p r e -p r o o f bronchopneumonia, which was more evident in lena-infected piglets at - dpi. this increase was mainly due to cd + monocytes, interstitial and intravascular macrophages infiltrating extensive areas of the interstitium. the influx of cd + monocytes and immature macrophages may be explained by an attempt to replenish the loss of cd + macrophages contributing to clearance of cellular debris and resolution of inflammation, restoring the normal lung function. on the other hand, the increase of cd + cells implies a higher availability of the lps-lbp complex receptor, which is likely to sensitise the lung to future secondary bacterial infections making the onset of prdc easier (van gucht et al., ) . in the case of inos, a significant increase in the number of inos + cells was observed in areas of interstitial pneumonia as well as bronchopneumonia in lena-infected pigs. the induction of inos has been associated with both a direct effect of the viral replication or viral components and an indirect effect mediated by cytokines, such as ifnγ, or by lps (akaike and maeda, ; chen et al., ; lee and kleiboeker, ) . of note, the peak of inos in lena-infected animals appeared in our study just after the peak of prrsv replication in the lung as well as after the peak of serum ifn-γ, being associated with the maximum lung injury and bronchopneumonia lesion. these factors may play a role in the regulation of inos expression along prrsv infection and its role in lung injury development (chen et al., ; lee and kleiboeker, ; yan et al., ) . after a cascade of proinflammatory events, the host is able to trigger off the release of anti-inflammatory or regulatory mediators to restrain the extent of the injury. thus, the role of cd r and foxp was evaluated in the present study. a strong positive correlation was detected among the frequency of cd r + cells and the microscopic score which was mainly associated with a higher severity of typical interstitial pneumonia j o u r n a l p r e -p r o o f and suppurative bronchopneumonia in lena-infected pigs. likewise, an increase in the frequency of foxp + cells between and dpi was triggered by both prrsv- strains when the lung injury was higher. cd r has been involved in reducing the expression of pro-inflammatory cytokines in a wide range of inflammatory diseases (vaine and soberman, ) , nevertheless to the best of the authors' knowledge, the role of cd r in viral diseases of swine is largely unknown. previous studies in a murine model reported that influenza virus infection induced the upregulation of cd r in macrophages, decreasing their responsiveness and increasing the sensitivity to bacterial infection and finally severe lung injury (snelgrove et al., ; vaine and soberman, ) . in contrast, cd /cd r signalling pathway limited type i ifn production during coronavirus infection protecting the host from cytokine storm (vaine and soberman, ) . in addition, foxp has been reported as a potential inhibitor of the cell- the present study dissects the immunopathology of lung injury along an acute infection with prrsv- strains of different virulence, revealing a drop in the number of cd + cells together with an enhancement in the expression of cd and inos as mechanisms involved in the earlier and higher extent of lung lesion in lena-infected piglets. these changes could sensitise the lung to future secondary bacterial infections. in addition, the increase in the number of cd + cells is likely to respond to an attempt to replenish the cd + macrophages subset lost along the infection with both prrsv- strains. on the other hand, the increase in the expression of cd r and foxp represents potential pathways activated to contain the inflammatory response. the authors declare that they have no competing interest. marked microglia activation in the hippocampus and deficits in spatial learning. journal of neuroscience, ( ) temperature (b) for control group (gray circles), -infected group (green triangle) and lena-infected group (red diamonds). "a" indicates a significant difference between the lena and and control groups, "b" a significant difference between the lena and groups and "c" a significant difference between the and control groups. p value lower than . was considered statistically significant and represented as * p ≤ . , ** p ≤ . and *** p ≤ . . days post-inoculation, dpi. (c) at necropsy, lungs were scored and processed for histological and immunohistochemical studies. box plots display the macroscopic lung score for each group (control, gray circles; , green triangles; lena, red for all data, a p value lower than . was considered statistically significant and represented as * p ≤ . , ** p ≤ . , *** p ≤ . and ****p ≤ . . inos -ns: not statistically significant or with r < . . correlation coefficients were considered relevant with r > . and p < . . a comparative study of the local cytokine response in the lungs of pigs experimentally infected with different prrsv- strains: upregulation of il- α in highly pathogenic strain induced lesions a distinct function of regulatory t cells in tissue protection genetic diversity of prrsv in central eastern europe in - : origin and evolution of the virus in the region porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus the presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus comparison of asian porcine high fever disease isolates of porcine reproductive and respiratory syndrome virus to united states isolates for their ability to cause disease and secondary bacterial infection in swine precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function phenotypic characterization of a highly pathogenic italian porcine reproductive and respiratory syndrome virus (prrsv) type subtype isolate in experimentally infected pigs upregulation of pro-inflammatory factors by hp-prrsv infection in microglia: implications for hp-prrsv neuropathogenesis immunohistochemical detection and distribution of inducible nitric oxide synthase in pigs naturally infected with actinobacillus pleuropneumoniae changes in macrophage phenotype after infection of pigs with haemophilus parasuis strains with different levels of virulence genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype i strains the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) respiratory viral infection in neonatal piglets causes inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges phenotypic and functional differentiation of porcine αβ t cells: current knowledge and available tools cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs international committee on taxonomy of viruses (ictv) area under the viraemia curve versus absolute viral load: utility for predicting symptomatic cytomegalovirus infections in kidney transplant patients comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate detection of foxp protein expression in porcine t lymphocytes porcine arterivirus activates the nf-κb pathway through iκb degradation f injury: cytokines, uncontrolled inflammation, and therapeutic implications dynamic changes in inflammatory cytokines in pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus attenuation and immunogenicity of a live high pathogenic prrsv vaccine candidate with a -amino acid deletion in the nsp interleukin- , interleukin- beta, and interferon-gamma levels are linked to prrs virus clearance the porcine innate immune system: an update detection of mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the s rrna gene soluble cd pathology and virus distribution in the lung and lymphoid tissues of pigs experimentally inoculated with three distinct type prrs virus isolates of varying pathogenicity increased pathogenicity of european porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance induction of porcine reproductive and respiratory syndrome virus (prrsv)-specific regulatory t lymphocytes (treg) in the lungs and tracheobronchial lymph nodes of prrsvinfected pigs impact of prrsv strains of different in vivo virulence on the macrophage population of the j o u r n a l p r e -p r o o f thymus development and application of a porcine specific elisa for the quantification of soluble cd analysis of the expression of porcine cd r and cd r l by using newly developed monoclonal antibodies dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype prrsv strain downregulation of antigen-presenting cells in tonsil and lymph nodes of porcine reproductive and respiratory syndrome virusinfected pigs virulent lena strain induced an earlier and stronger downregulation of cd in bronchoalveolar lavage cells the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- activation of the extrinsic apoptotic pathway in the thymus of piglets infected with prrsv- strains of different virulence kinetics of the expression of cd and cd a in the lung and tonsil of pigs after infection with prrsv- strains of different virulence the porcine a antigen is homologous to human cd and related to macrophage differentiation the pd- /pd-l axis and virus infections: a delicate balance molecular epidemiology of prrsv: a phylogenetic perspective use of a polymerase chain reaction assay and an elisa to monitor porcine circovirus type infection in pigs from farms with and without postweaning multisystemic wasting syndrome european genotype of porcine reproductive and respiratory syndrome ( prrsv ) infects monocyte-derived dendritic cells but does not induce treg cells induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus regulatory t cells in respiratory health and diseases. pulmonary medicine emergence of a virulent porcine reproductive and respiratory syndrome virus (prrsv) strain in lower austria a critical function for cd in lung immune homeostasis and the severity of influenza infection pathogenicity of three genetically diverse strains of prrsv type in specific pathogen free pigs the role of pulmonary intravascular macrophages in porcine reproductive and respiratory syndrome virus infection the cd -cd r inhibitory signaling pathway. immune regulation and host-pathogen interactions sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus the combination of prrs virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs porcine reproductive and respiratory syndrome virus infection increases cd expression and lipopolysaccharide-binding protein in the lungs of pigs proinflammatory cytokines and viral respiratory disease in pigs inhibition of nox oxidase activity ameliorates influenza a virus-induced lung inflammation recovery from acute lung injury can be regulated via modulation of regulatory t cells and th cells comparative analysis of immune responses following experimental infection of pigs with european porcine reproductive and respiratory syndrome virus strains of differing virulence lung pathogenicity of european genotype strain porcine reproductive and respiratory syndrome virus (prrsv) differs from that of subtype strains gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus regulation of inos-derived ros generation by hsp and cav- in porcine reproductive and respiratory syndrome virus-infected swine lung injury porcine reproductive and respiratory syndrome virus; cd r , cd receptor ; inos, inducible nitric oxide synthase; foxp , forkhead box protein ; mab, monoclonal antibody; pab citrate ph , microwave heat treatment at w for minutes enzymatic digestion with protease type xiv (sigma-aldrich) at º c for minutes; citrate ph *, autoclave treatment at º c for min we express our appreciation to gema muñoz, alberto alcántara and esmeralda cano for their technical assistance and dr. hans nauwynck for providing us the prrsv- subtype key: cord- -g zhv authors: rowland, raymond r.r; lawson, steven; rossow, kurt; benfield, david a title: lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: g zhv the ability of porcine reproductive and respiratory syndrome virus (prrsv) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to prrsv in utero. in this study, virus replication and prrsv-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at days of gestation with prrsv isolate vr- . eighty-four percent of pigs were born viremic with a mortality of % within days after birth. at approximately days sera from pigs were negative for virus by virus isolation. analysis of virus replication in the tissues of pigs randomly sacrificed between and days showed no evidence of virus in lung and other non-lymphoid organs. however, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. even though replication was at a low level, virus was easily transmitted to sentinel pigs. by days pigs became seronegative and did not transmit virus to sentinel pigs. sacrifice of remaining pigs after days showed no evidence of virus in blood and tissues. this study shows that congenital prrsv-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positivestranded rna virus, prrsv, belonging to the family arteriviridae (wensvoort et al., ; benfield et al., ; nelsen et al., ) . other members of the arterivirus group include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv; for review see plagemann, ) . the arteriviruses, toroviruses, and coronaviruses are members of a single order, nidovirales (cavanagh, ) . arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -co-terminal set of subgenomic mrnas that possesses a common leader and a poly-a tail (snijder and mulenberg, ) . the arteriviruses exhibit several important properties relevant to the study of viral pathogenesis, including cytopathic replication in macrophages, the capacity to establish and maintain an asymptomatic persistent infection, as well as cause severe and fatal disease (plagemann, ) . the outcome following experimental prrsv infection is dependent on the age and reproductive status of the pig, as well as the isolate used for infection (reviewed in rossow, ) . infection of adult pigs usually produces a nonfatal disease, characterized by mild flu-like symptoms, a transient elevation in temperature and inappetance. the reproductive form of prrs occurs following the infection of pregnant gilts or sows. accordingly, virus-induced reproductive failure can present clinically as increased regular and delayed returns to estrus and not-in-pig sows, as well as abortions, mummified fetuses, stillbirths and weak-born pigs. christianson et al., ; mengeling et al., ; benfield et al., ; rossow et al., ) . surviving neonates exhibit the severest form of respiratory disease with mortality often reaching % within weeks after birth. besides interstitial pneumonia, viral lesions in infected neonates can be found in primary and secondary lymphoid organs, including bone marrow, thymus, lymph node, spleen and tonsil, and non-lymphoid organs, such as brain, heart, kidney, and stomach (rossow et al., (rossow et al., , rossow, ; feng et al., ) . the complex pathology following exposure to prrsv in utero represents a unique form of the disease referred to as congenital prrs. another outcome following prrsv infection is persistently infected swine herds, a property that has contributed to the world-wide spread of the disease (albina, ; chung et al., ; duan et al., ; wills et al., ; allende et al., ; bierk et al., ) . it is assumed that persistence is maintained by the ability of prrsv to evade host defenses and establish a long-term asymptomatic infection within individual pigs. even though prrsv-specific antibody appears as early as days post-infection and is followed by serum neutralizing activity and cell-mediated immunity molitor, , ; rowland et al., rowland et al., , persistently infected pigs can continue to shed virus. the mechanistic basis for prrsv persistence is not known. similar to ldv, prrsv may sustain replication in a subpopulation of renewable macrophages, while avoiding host-defenses . another possibility is the restriction of virus replication to immunoprivileged sites, such as the male reproductive tract (christopher-hennings et al., ; sur et al., ) , which is observed during infection with other arteriviruses, such as eav and ldv (holyoak et al., ; anderson et al., ) . in previous work characterizing quasispecies evolution during infection, we proposed that antigenic drift in the ectodomain of gp may lead to the selection of clones that are resistant to neutralizing antibody or change the tropism of the virus for a different population of cells . previous experimental studies designed to understand prrsv persistence have characterized virus replication during prrsv infection of postnatal pigs. these studies indicate that virus is present in the blood for approximately - days after infection; however, in a significant number of animals, virus can be detected in tonsil for up to days and by rt-pcr in semen for days after infection christopher-hennings et al., ; allende et al., ) . more recently wills et al. ( ) reported that six of pigs, infected at days after birth, were positive for the presence of virus at days. however, the ability of these long-term infected pigs to shed virus was not determined. very little is known regarding the course of virus replication in pigs exposed to virus in utero. the purpose of this study was to further understand persistent infection in congenitally infected pigs by characterizing the course of clinical disease, sites of virus replication and the capacity to transmit virus in a group of pigs exposed to prrsv in utero. three pregnant sows at - days gestation were infected with prrsv isolate vr- collins et al., ) passage , which was plaque-purified and propagated on marc- cells, a subclone of the ma- cell line (kim et al., ) . five milliliters of virus, at a concentration of tcid /ml, was divided into two doses and administered into each nare with a cc syringe. the sows were monitored daily for signs of prrsv infection and allowed to farrow naturally. umbilical cord and blood samples were collected from newborn pigs at birth and prior to nursing. blood was then collected from surviving pigs at weekly intervals for the duration of the study. neonates were monitored daily for clinical signs. pigs that succumbed to acute prrsv infection were necropsied and tissues evaluated for pathology and virus replication. at days all surviving pigs were weaned and placed together in a single isolation room in the animal care facility. prrsv-specific antibody in serum was measured using the "old format" herdchek ® prrs elisa (idexx) according to the manufacturer's instructions. this assay was done by personnel at an accredited diagnostic laboratory (animal disease research and diagnostic laboratory at south dakota state university). an s/p ratio greater than . was considered seropositive. prrsv neutralizing activity in serum was measured according to rowland et al. ( ) . serial : dilutions of serum were prepared in culture medium on well plates. an equal volume of the vr- prrsv, at a concentration of tcid /ml, was added to each sample and incubated for h at • c then transferred to a -well plate containing confluent marc- cells. after days the plates were fixed in % acetone and infected cells detected with fitc-labeled anti-nucleocapsid monoclonal antibody, sdow- (nelson et al., ) diluted in pbs with % fbs. neutralizing activity was reported as the log of the highest serum dilution that prevented the replication of prrsv in marc- cells (negative for sdow- staining). virus isolation was performed by incubating dilutions of serum or tissue homogenate on -well plates containing marc- cells. for virus isolation from tissues, approximately g of tissue was homogenized in ml of hanks balanced salt solution and centrifuged at × g for min to remove debris. dilutions ( : and : ) of cleared homogenate or serum were incubated on -well plates of marc- cells for days. plates were then fixed in % acetone and stained with fitc-sdow- . nested rt-pcr amplification of orf , described by christopher-hennings et al. ( ) , was used for the detection of viral rna in serum and tissues. samples were homogenized in an equal volume of lysis buffer ( m guanidinium thiocyanate, mm sodium citrate [ph. ], . % sarkosyl [n-lauryl sarcosine] . m -mercaptoethanol). five hundred microliters of lysate was added to ul of phenol and ul chloroform and mixed. the aqueous phase was removed, the rna precipitated with ethanol and then reconstituted in ul of distilled water. one microliter of rna was reverse transcribed using mulv reverse transcriptase and the outer antisense pcr primer. the outer sense and antisense primers for pcr amplification of a bp product incorporating the entire orf region were -tcgtgttgggtggcagaaaagc- and -gccattcaccacacattcttcc- , respectively. the outer primers were incorporated into a reaction mixture containing cdna, mm mgcl , × pcr buffer and taq polymerase. the product was subjected to a second round of amplification using the nested sense and antisense primers, -ccagatgctggg-taagatcatc- and -cagtgtaacttatcctccctga- , respectively. the second round of amplification incorporated mm mgcl , × pcr buffer and taq polymerase. forty cycles of amplification were performed for each primer pair. all pcr reactions incorporated a • c denaturing step ( s), a • c annealing step ( s), and a • c polymerization step ( s). the reaction products were electrophoresed on an agarose gel and the final bp product detected with ethidium bromide. in situ hybridization for the identification of cells supporting virus replication was done according to lawson et al. ( ) using a modification of the procedure originally described by anderson et al. ( ) . tissues were fixed in % neutral buffered formalin for h at room temperature and embedded in paraffin. thin sections were mounted on denhardt's solution-treated slides and paraffin removed with xylene. after pretreatment, the tissue sections were hybridized with a [ s] dctp labeled random-primed probe complementary to a nucleotide region covering all of orf . slides were hybridized for h at • c, washed, dipped in kodak ntb- autoradiographic emulsion, exposed for approximately days, developed and counterstained with mayer's hematoxylin and eosin y. a positive control included tissues from pigs experimentally infected with prrsv. negative controls included tissues from uninfected pigs and tissues from experimentally infected pigs hybridized with a probe prepared using cdna from ldv orf region. three pregnant sows were infected with vr- at days gestation. clinical signs in sows were minimal, consisting of inappetance in two of the three animals, which began at - days post-inoculation and lasted approximately days. all sows seroconverted and farrowed naturally at days of gestation. outcomes for the piglets from the three infected sows are summarized in table . seven pigs were stillborn and due to the degraded condition of the tissues were not subjected to further study. analysis of virus and antibody in blood and/or umbilical cords from the live neonates showed that at least or % were virus isolation (vi) or rt-pcr positive for prrsv at the time of farrowing indicating that in utero infection was successful. the eight neonates that were identified as prrsv-negative at the time of birth became vi-positive by dpf. neonates as a group exhibited signs consistent with prrsv infection, with pronounced dyspnea being the most common symptom. about half of the pigs exhibited more severe disease signs, including ocular edema and inappetance. microhistology showed prrsv-induced pathological changes in the lungs and other organs of acutely infected symptomatic pigs, including interstitial pneumonia with the infiltration of proteinaceous debris from the interstitial space into the alveoli. lymph nodes were generally enlarged with germinal center hypertrophy, hyperplasia and necrosis. lesions in the brain (encephalitis) and heart (myocarditis) and stomach of some pigs were also observed. there was a % mortality between and dpf. only one pig in the early mortality group was confirmed to have died from a non-prrs-related cause. pigs that survived beyond dpf eventually recovered and showed no external signs of acute infection and there were no prrsv-related deaths after dpf. for the purpose of comparison, we included a litter from a single mock-infected sow. the piglets, born at the same time as the prrsv pigs, appeared normal and showed no mortality within dpf. in the developing fetus, there are two potential sources of virus, which can cause fetal damage. the first source is virus replication within the tissues and organs of the developing fetus. another source is virus replication in the accessory tissues, such as the umbilical cord and placenta. in this study, we were able to obtain umbilical cords of piglets born to infected sows, including umbilical cords from infected newborns and umbilical cords from non-viremic pigs born to infected sows. virus isolation showed all umbilical cords from infected neonates to be positive for prrsv. neonates that were vi or rt-pcr negative for prrsv in serum were also negative for prrsv in umbilical cord tissues. the recovery of virus from umbilical cords presented the possibility that virus was isolated from circulating blood and not from cells supporting virus replication. in situ hybridization, for the detection of cells possessing viral rna, was performed on a single microscopic cross-section of each umbilical cord. we identified prrsv rna-positive cells in of the tissues cross-sections from vi-positive umbilical cords, indicating the presence of infected cells. a representative example of prrsv rna-positive cells in an umbilical cord cross-section is shown in fig. a . positive cells were generally found within the smooth muscle regions of umbilical cord tissues. as a negative control, in situ hybridization was performed on umbilical cord sections from six uninfected pigs. all six were negative for the presence of cells containing viral rna. even though cells supporting virus replication were easily identified in umbilical cords of infected piglets, histology at both gross and microscopic levels revealed no virus-associated lesions. based on the presence of virus and prrsv-specific antibody in pre-suckling serum samples, newborn piglets could be placed into one of the three groups. the first group contained four pigs ( %) that were vi-negative and seronegative. we assume that these neonates were not infected in utero, but were infected immediately prior to farrowing or immediately after farrowing as a result of contact with the dam or infected litter mates. the second group contained ( %) pigs that were vi-positive for prrsv, but seronegative. and finally, the third group was composed of seven ( %) neonates that were both vi-positive and seropositive. each litter contained a mixture of pigs that could be placed in all three groups, indicating that fetuses were infected at different times in the same sow. since pigs are immunocompetent at around days of gestation and there is no passive transfer of antibody from the mother to the fetus (tizard, ) we can conclude that infected ( ) neonates that were born seropositive were infected earlier than those that were vi-positive and seronegative. based on the infection of fetuses at different times in utero, we proposed that the earlier a fetus is infected the greater the probability of early mortality after birth. to test this hypothesis, neonates were divided into two groups. the first group was composed of the piglets that died within dpf and showed symptoms of severe prrs. (one pig succumbed from other than prrsv infection and was eliminated from the analysis.) the second group included the remaining pigs, which recovered and became asymptomatic. in the long-term survivor group only one serum sample was identified as positive for prrsv antibody, as indicated by an elisa s/p ratio greater than . (fig. ) . in pigs that died prior to days, of pigs were seropositive for prrsv. chi-square statistical analysis of these data showed a statistically significant association between the presence of prrsv antibody and mortality prior to dpf (p = . ). it should be noted that in the non-survivor group, of the pigs were vi-negative and seronegative at birth. these two piglets, and two others in the non-survivor group, exhibited clinical features consistent with streptococcus sp. infection, including swollen joints filled with pus, recruitment of pmns into inflammatory lesions, and the presence of streptococci in lung and other tissues. streptococcus suis was isolated from tissues of all four pigs. the presence of virus in the blood was determined by vi, and later when serum samples became vi-negative, by rt-pcr. the analysis of prrsv-specific immune responses included measurements of total anti-prrsv activity by elisa and virus neutralizing activity. these data, collected over a period of days after birth, are graphically summarized in fig. . by dpf, all pigs were vi-positive in serum, with the percentage of vi-positive serum samples declining at about dpf. the decline in the number of vi-positive serum samples was associated with the appearances of peak antibody levels and virus neutralizing activity. the disappearance of virus from the blood also correlated with the recovery of pigs from acute disease. the last vi-and rt-pcr-positive serum samples were obtained at and dpf, respectively. pigs that attained a vi-negative status remained vi-negative throughout the remainder of the study. however, it should be noted that pigs that became pcr negative did not always stay negative. periodically we found serum samples between and dpf, which were pcr-positive, even though the previous and subsequent samples taken from the same animal were negative by pcr. these intermittent pcr-positive results can best be explained by the presence of a small amount of viral rna, but at or near the lower detection limits of the pcr assay (data not shown). there were no positive rt-pcr serum samples after dpf. mean prrsv antibody peaked at approximately dpf with elisa s/p ratios ranging between . and . (n = ), well above the . cut-off that identified a serum sample as seropositive. since pigs were suckling prior to days, we assume that passive antibody from the mother was contributing to the humoral response. after days, antibody gradually declined and by dpf the remaining four pigs had attained a seronegative status (s/p ratio < . ) and remained seronegative throughout the remainder of the study. virus neutralizing activity in serum was first detected in one pig at dpf (neutralizing titer = ). by dpf all pigs showed detectable neutralizing activity. since these measurements were made several weeks after weaning, we presume that virus neutralizing activity was generated by the immune system of the congenitally infected pigs. at their peak, between days and , neutralizing titers ranged between : and : (fig. ) . this relatively low amount of neutralizing activity was not unexpected and is typical of the neutralizing response during infection of older pigs (yoon et al., ; wills et al., ) . interestingly, virus neutralizing activity was detected in sera well beyond days, even though all pigs were seronegative for prrsv by the idexx elisa test (s/p ratios below . ). the source of the virus neutralizing activity in serum was not determined. however, the presence of neutralizing activity in "seronegative" sera can be explained based on the different sensitivities of the assays used to measure antibody and neutralizing activity. for example, we cannot rule out the possibility that pigs possessing an elisa s/p ratio less than . have some prrsv antibody, but at amounts below the confidence limits of the assay. the analysis of virus replication in tissues of congenitally infected pigs focused on three periods of infection, identified by roman numerals ii, iii and iv in fig. . each period was unique in terms of clinical disease signs, viremia, serology and pathology. the first period, indicated by roman numeral ii in fig. , represented the acute, symptomatic stage of infection, which covered the first days after birth. the second period, identified by roman numeral iii focused on a period when pigs were asymptomatic for clinical disease signs and negative in serum for prrsv by vi and largely negative in serum by rt-pcr, but all pigs were still seropositive. and finally, we assessed virus replication in a group of four infected pigs, which had become seronegative (roman numeral iv). during acute infection (roman numeral ii, fig. ) prrsv was easily isolated from all lymphoid and non-lymphoid tissues examined (lung, heart, aorta, kidney, liver, testes, salivary gland, intestine, brain, stomach, thymus, spleen, tonsil, and lymph nodes). in situ hybridization identified cells containing viral rna in the same tissues. fig. b-d shows representative examples of rna-positive cells in lung, salivary gland and kidney. these three tissues represent sources of prrsv shedding by oral-nasal and urinary secretions. also shown are representative examples of virus replication in cells of lymph node, tonsil and thymus (fig. e-g) . in the tonsil, cells supporting virus replication were in close proximity to the tonsilar crypts. these data are consistent with our previous study, identifying multiple sites of virus replication in gnotobiotic piglets at days after experimental infection with vr- (lawson et al., ) . roman numeral iii in fig. shows the next period of infection covered by our study. during this stage, prrsv-specific antibody was declining, but all pigs were seropositive and possessed virus neutralizing activity at a serum dilution of at least : . there were no overt clinical signs of acute prrsv infection and all pigs were vi-negative in serum. virus isolation and rt-pcr were performed on lymphoid and non-lymphoid tissues from nine of the asymptomatic pigs, sacrificed at random between and dpf. vi and rt-pcr results for lung, mandibular lymph node, mesenteric lymph node, tonsil and serum are presented in table . in contrast to the acutely infected pigs, all lung and non-lymphoid tissue samples were negative for virus and viral rna. the absence of detectable amounts of virus in lungs was based on negative results for vi, rt-pcr and in situ hybridization assays of tissue samples from each lung quadrant. virus isolation, rt-pcr and in situ hybridization also failed to identify virus or viral rna in kidney, bladder, heart, aorta, liver, intestines, stomach, testes/ovaries, nasal turbinates, and salivary glands (data not shown). positive vi and rt-pcr results were frequently obtained from homogenates of tonsil (seven of nine pigs) and mandibular lymph nodes (eight of nine pigs; table ). mesenteric, aortic, cervical, tracheal, medial iliac lymph nodes also yielded virus and/or viral rna, but at much lower frequencies (see table for mesenteric lymph node data). virus or viral rna was not detected in spleen or thymus (data not shown). consistent with the vi and rt-pcr results, in situ hybridization identified prrsv rna-positive cells in tonsil and mandibular lymph nodes. prrsv rna-positive cells in a mandibular lymph node at dpf and tonsil at dpf are shown in fig. . only a few rna-positive cells could be identified within a given tissue section. therefore, the locations of rna-positive positive cells were confirmed by performing hybridization of adjacent tissue sections (fig. ) . all pigs sacrificed during this time possessed detectable amounts of virus neutralizing activity. together these data show that prrsv replication during asymptomatic infection is absent from lung and other non-lymphoid tissues and is primarily restricted to tonsil and lymph nodes, even in the presence of humoral immunity. to investigate the significance of this low level of virus replication in asymptomatic pigs, we studied the ability of congenital prrs pigs to transmit virus. age-matched prrsv seronegative sentinel pigs were introduced into the prrsv-infected group at dpf ( sentinels and principals), dpf ( sentinels and principals), dpf ( sentinel, principals) and dpf ( sentinel, principals). sentinel pigs were allowed to commingle with the infected herd for week and then removed to separate isolation rooms for observation and assessment of infection by vi and serology. all sentinel pigs were vi-positive in serum within week after introduction and seroconverted a week later. these results indicate that under natural conditions of normal pig-to-pig contact prrsv is efficiently transmitted to naïve pigs even though virus replication is low. congenital prrs pigs that became seronegative (see roman numeral iv in fig. ) were the subject of the last part of our study. by dpf, all sera from four remaining infected pigs were vi-negative, rt-pcr negative and elisa seronegative for prrsv antibody (s/p ratio less than . ). to determine if prrsv pigs were capable of shedding virus, four age-matched prrsv seronegative sentinel pigs were introduced at dpf and maintained in continual contact with the prrsv pigs for at least days. commingling culminated in the mating of each prrsv pig to an aged-matched sentinel. two litters were obtained from the three prrsv females. the adult female pigs and litters were sacrificed and assessed for prrsv replication. all three female pigs and associated litters were negative for prrsv antibody and viral rna as indicated by negative results for in situ hybridization and by performing rt-pcr in lungs, lymph nodes and tonsils. the single sentinel female pig and litter obtained after mating to the seronegative prrsv boar were also negative for prrsv. since previous work identified long-term virus shedding in semen (christopher-hennings et al., ) , we also performed rt-pcr and in situ hybridization on testes and male accessory reproductive organs of the remaining male pig. there was no evidence of virus replication in any of the male reproductive tissues. the results from this study show that congenital prrsv infection can be divided into distinct stages, unique in terms of serology, virology and clinical disease. the first stage covers the period of in utero exposure to virus. these data demonstrate that not all fetuses are infected at the same time and some fetuses may escape infection altogether. furthermore, the positive association between the presence of antibody at birth with early mortality indicates that the earlier a fetus is infected the more severe the clinical disease (fig. ) . since it takes - weeks to develop antibody, we assume that seropositive fetuses were infected - weeks after infection of the sow or approximately - weeks prior to birth. as a whole, these data show that the placenta is a barrier to prrsv, but this barrier does break down with time. there are at least two sources of virus-related damage that can account for early mortality in the antibody-positive fetuses. the first is the formation of prrsv lesions in fetal organs. however, the target organs in the fetus may be different from those in the postnatally infected pig. for instance, porcine alveolar macrophages, which support virus replication to high levels in postnatal pigs are absent from the fetal lung. in preliminary studies, we found relatively large amounts of virus in the thymus from fetuses obtained - weeks prior to birth (data not shown). feng et al. ( ) observed thymic lesions in congenital prrsv pigs sacrificed at dpf. the possible effect of prrsv infection on fetal/neonatal primary immune organs has obvious implications in the increased susceptibility of congenitally infected pigs to secondary infections. a second source of fetal damage is through the disruption of support tissues, such as placenta and umbilical cord. lager and halbur ( ) reported lesions in umbilical cord, which appeared to be sufficient to cause anoxia in the developing fetus. even though umbilical cord was identified as a sight of virus replication in this study, we were not able to identify prrsv-related lesions. the absence of umbilical cord lesions may reflect the different isolate used in this study. even though immune abnormalities represent a hallmark of prrsv infection, congenital infection did not appear to adversely affect the capacity of surviving pigs to develop an antibody response to prrsv later in life. as a group, the antibody levels in congenital prrs pigs peaked at about days after birth (fig. ) , which is similar to the time-course appearance of antibody in pigs infected several weeks postnatally (yoon et al., ; allende et al., ) . congenital prrs pigs were able to mount a prolonged neutralizing antibody response at levels typically found in experimentally infected older pigs (yoon et al., ) . these results indicate that fetuses exposed to prrsv during late gestation do not become immunotolerant to the virus later in life. a unique aspect of this study was the thorough analysis of virus replication in pig tissues during asymptomatic infection (roman numeral iii, fig. ). this stage of congenital prrsv infection was characterized as the period of time when pigs no longer exhibited clinical signs of acute disease, were vi-negative in serum, but still seropositive. in this study it was represented by a group of nine pigs sacrificed between and days after birth. perhaps, the most interesting finding was the identification of virus in tonsil and mandibular lymph node, but not in lung and other non-lymphoid organs. these results are in agreement with studies identifying tonsil as a source of virus during persistent infection allende et al., ; horter et al., ) , but contradict other studies identifying pulmonary macrophages as the source of virus replication in persistently infected pigs (mengeling et al., ; duan et al., ) . our conclusion that virus is absent from the lungs in persistently infected pigs is based on a thorough analysis of virus replication, including the use of vi, rt-pcr and in situ hybridization. continuous virus replication in regional lymph nodes accounts for the efficient transmission via oral-nasal secretions and in semen during persistent infection (christopher-hennings et al., ) . the basis for an eventual change in organ tropism, from multiple sites of prrsv replication to preferential replication in tonsil and lymph nodes, is not known. in previous work we identified the emergence of a virus sub-population during persistent infection, which possessed a mutation in the ectodomain of orf , the major envelope glycoprotein. this mutation may increase the tropism of prrsv for replication in a macrophage subpopulation that resides in tonsils and lymph nodes. we followed virus and antibody in four congenital prrsv pigs for more than a year. at the time of necropsy these pigs were seronegative for prrsv (s/p elisa ratio less than . ) and showed no evidence of virus replication, as determined by the absence of viral rna in serum and in tissues. in this study we were not able to determine exactly when virus disappeared from these pigs. however, the last rt-pcr positive serum sample was obtained at dpf and sentinel pigs did not become infected when introduced at dpf. the failure of sentinel pigs to become infected after an extended period of intimate contact with the congenital prrsv-infected pigs indicated that prrsv replication was not reactivated. the results from this study show that congenital prrs pigs can shed virus for at least dpf and perhaps for as long at dpf, which support the conclusions of a recent study by wills et al. ( ) . the isolation of virus from lymph nodes at dpf in this study is longer than the isolation of infectious virus at days from pigs infected at month of age (allende et al., ) , but is similar to length of time reported by wills et al. ( ) . because of viral strain differences, it is difficult to assess the impact of congenital infection on virus replication, shedding and persistence by making comparisons with other published studies performed in older pigs. however, in agreement with allende et al. ( ) prrsv replication does not establish a steady-state equilibrium in the manner of other persistent arteriviruses, such as ldv or shfv, but gradually declines over time, with the lymphoid organs as the last vestige of virus replication. the presence of an extended period of ongoing prrsv replication in pigs with substantial anti-prrsv humoral and cell-mediated immune responses suggests that the eventual elimination of the virus from congenital prrs pigs may ultimately reflect the disappearance of prrsv-permissive cells with age. based on our analysis of clinical disease, prrsv-specific antibody and virus replication, congenital prrsv infection can be divided into four distinct stages. stage i covers the period of in utero exposure to virus during which time fetuses are infected at different times and some fetuses remain uninfected. the positive association between the presence of prrsv antibody at birth with early mortality indicates that the earlier a fetus is infected the more severe the clinical disease outcome. stage ii is the period of acute infection. during this time pigs show signs of acute prrsv. in this study virus was isolated from all tissues, and all tissues contained cells supporting virus replication. stage iii is a period when pigs no longer exhibit clinical disease signs, are vi-negative and largely rt-pcr negative in serum and possess peak levels of virus neutralizing activity. even though virus replication is relatively low, persistently infected pigs can efficiently transmit virus to naive pigs. stage iv of congenital prrsv infection is viral clearance. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection lactate dehydrogenase-elevating virus persists in liver, spleen, lymph nodes and testis and results in accumulation of viral rna in germinal centers concomitant with the polyclonal activation of b cells cell-mediated immunity to porcine reproductive and respiratory syndrome virus ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) porcine reproductive and respiratory syndrome diagnostic investigation of chronic porcine reproductive and respiratory syndrome virus in a breeding herd of pigs nidovirales: a new order comprising coronaviridae and arteriviridae pathogenesis of porcine reproductive and respiratory syndrome virus infection in mid gestation sows and fetuses persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars identification of porcine reproductive and respiratory syndrome virus in semen and tissues from vasectomized and nonvasectomized boars persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs virus quantification and identification of cellular targets in the lungs lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with mac- -positive cells days post-infection temporal characterization of transplacental infection of porcine fetuses with porcine reproductive and respiratory syndrome virus diagnosis of porcine reproductive and respiratory syndrome using infected macrophages from live pigs porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of united states and european isolates of porcine reproductive and respiratory syndrome (prrs) virus using monoclonal antibodies lactate dehydrogenase-elevating virus and related viruses lactate dehydrogenase-elevating virus: an ideal persistent virus? porcine reproductive and respiratory syndrome lymph node lesions in neonatal pigs congenitally exposed to porcine reproductive and respiratory syndrome virus fetal microscopic lesions in porcine reproductive and respiratory syndrome virus-induced abortion porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with aminopurine the molecular biology of arteriviruses porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis immunity in the fetus and newborn mystery swine disease in the netherlands: the isolation of lelystad virus porcine reproductive and respiratory syndrome virus: a persistent infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection this work was supported by the usda national research initiative for competitive grants program grants # - - and # - - , national pork producer council grant # , national science foundation grant # osr- , south dakota agricultural experiment station and the south dakota future fund. we thank curt nelson and scott kistler for their excellent assistance in the care and welfare of the animals. key: cord- -rym ik o authors: lemmermeyer, tanja; lamp, benjamin; schneider, rainer; ziebuhr, john; tekes, gergely; thiel, heinz-jürgen title: characterization of monoclonal antibodies against feline coronavirus accessory protein b date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: rym ik o feline coronaviruses (fcovs) encode five accessory proteins termed a, b, c, a and b of unknown function. these proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. in the current study, we produced and characterized b-specific monoclonal antibodies (mabs). a recombinant form of the b protein was expressed as a fusion protein in escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. two hybridoma lines, b and d , were isolated that expressed mabs that recognized b proteins of both fcov serotypes. using an extensive set of n- and c-terminally truncated b proteins expressed in e. coli and a synthetic peptide, the binding sites of mabs b and d were mapped to an -residue region that comprises the only potential n-glycosylation site of the fcov b protein. the two mabs were suitable to detect a -kda protein, which represents the nonglycosylated form of b in fcov-infected cells. we speculate that glycosylation of b is part of the viral evasion strategy to prevent an immune response against this antigenic site. feline coronaviruses (fcovs) are enveloped viruses with a large (approximately kb) single-stranded rna genome of positive polarity. they belong to the family coronaviridae within the order nidovirales (de groot et al., ) . based on phylogenetic analyses, coronaviruses are divided into four genera termed alpha-,beta-, gamma-and deltacoronavirus. feline coronaviruses together with the closely related canine coronavirus (ccov) and porcine transmissible gastroenteritis virus (tgev) have been classified to the species alphacoronavirus in the genus alphacoronavirus. the latter contains a large number of more distantly related alphacoronaviruses, including a number of important medical and veterinary pathogens, such as porcine epidemic diarrhea virus (pedv), human coronavirus e (hcov- e), and human coronavirus nl (hcov-nl ) (de groot et al., ) . the proximal two-thirds of the coronavirus genome comprise orf a and orf b that together encode up to nonstructural proteins (nsps) that form the viral replication/transcription complex but may also be involved in interactions with host cell factors and functions. the remaining part of the genome codes for the structural proteins s (spike), e (envelope), m (membrane) and n (nucleocapsid) (masters, ) and a varying number of so-called accessory proteins with poorly characterized functions. fcovs infect cats and other members of the felidae worldwide. based on serological properties, fcovs are separated into serotypes i and ii (heeney et al., ; kennedy et al., ) . serotype i viruses dominate in the field and account for up to % of fcov infections (hohdatsu et al., ; addie et al., ) , whereas the prevalence of type ii fcovs is much lower. serotype ii fcovs emerged through homologous recombination between serotype i fcovs and ccovs (herrewegh et al., ; terada et al., ) . in addition to a serology-based classification, fcovs may be divided into different biotypes based on their pathogenic potential. the avirulent biotype is generally referred to as feline enteric coronavirus (fecv) and causes inapparent persistent infections of the gut, while the virulent biotype, feline infectious peritonitis virus (fipv), causes a fatal disease called feline infectious peritonitis (fip) (pedersen, ) . according to the "internal mutation theory" fipv evolves from fecv through mutations in approximately - % of the persistently infected cats (vennema et al., ) . so far, the genetic changes responsible for the biotype switch have not been identified, but there is increasing evidence that mutations in the accessory genes and the s gene are involved in the development of fip (vennema et al., ; kennedy et al., ; pedersen, ; chang et al., chang et al., , licitra et al., ; bank-wolf et al., ) . coronavirus genomes comprise a variable number of accessory genes at different positions in the -proximal genome region. with only very few exceptions, homologs of specific accessory genes are only conserved in very closely related viruses (of the same species or sublineage) but not in the more distantly viruses (lai and cavanagh, ) . there is increasing evidence that accessory gene products are important for virulence in the natural host but the precise functions of the vast majority of accessory proteins remain to be investigated. alphacoronaviruses harbor accessory genes at two different positions in their genomes. between the s and e genes, fcovs and the very closely related ccovs possess three orfs ( a- c), while tgev contains only two orfs ( a and b) in this genome region. recently, an additional orf, named orf , was found between the s and e genes in ccov serotype i (lorusso et al., ) . other alphacoronaviruses, such as pedv, hcov- e and hcov-nl , have only one orf . sequence analyses suggest that fcov orf a is homologous to ccov orf a and tgev orf a while the fcov orf c is a homolog of ccov orf c, tgev orf b and orf of all other alphacoronaviruses. downstream of the n gene, all members of the species alphacoronavirus contain a different number of additional accessory genes. thus, tgev contains only one orf (called orf ), which is homologous to orf a of fcovs and ccovs, while the latter two coronaviruses contain yet another orf called b in the -terminal genome region. altogether, the fcov genome encodes five accessory proteins termed a, b, c, a and b (dye and siddell, ; tekes et al., ) . using a reverse genetics approach, haijema et al. ( ) showed that the accessory genes of the fipv strain - are dispensable for viral growth in vitro and recombinant viruses that lack orfs a- c or a and b were unable to induce fip in vivo ( ) . so far, there is no evidence that a- c accessory proteins are produced in infected cells. nevertheless, it has been proposed that c is essential for viral replication in the gut (as is the case for fecv) but dispensable for systemic infections (chang et al., ) . the functions of the fcov-accessory proteins a- c remain to be determined. recently, it was suggested that the accessory protein a represents an interferon antagonist (dedeurwaerder et al., ) , although its expression in infected cells has not been confirmed. among the fcov-accessory proteins, the b protein has been studied most extensively. the b protein has a molecular mass of $ kda, it is secreted from the cell and contains (i) a cterminal kdel-like endoplasmic reticulum (er) retention signal, (ii) an n-terminal signal sequence of amino acids and (iii) a potential n-glycosylation site at aa position (vennema et al., a) . the presence of b-specific antibodies in both fecv-and fipv-infected cats indicates that this protein is produced during natural infections (vennema et al., a,b; herrewegh et al., ; kennedy et al., ) . it was also reported that cell culture adaptation often leads to mutations in the b gene and loss of expression of this protein (herrewegh et al., ) . taken together, there is growing evidence that the accessory genes and their products are involved in fcov persistence and virulence but, up to now, their function is not known. one reason for our limited knowledge on fcov-accessory proteins is the lack of appropriate tools to study these proteins. to our knowledge, specific antibodies against the individual accessory proteins are not available. in this manuscript, we describe the production of fcov b protein-specific mabs. our data show that the mabs recognize the b protein of both serotype i and ii fcovs. moreover, using a panel of n-and c-terminally truncated b proteins and a synthetic peptide, we determined the binding sites of the mabs in b. the data further indicate that the mabs recognize in fcov-infected cells only the nonglycosylated form of b. all animal procedures were performed in strict accordance with the legal regulations of the german animal welfare jurisdiction (tierschutzgesetz). the animal experiment was approved by the giessen regional council (regierungspräsidium) and recorded after approval under reference number a / . crandell rees feline kidney cells (crfk) were purchased from the american type culture collection (attc ccl- ). felis catus whole fetus cells (fcwf- ) were kindly provided by r. j. de groot, university of utrecht, the netherlands. both cell lines were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs), penicillin ( iu ml À ) and streptomycin ( iu ml À ) in % co at c. the type-ii strain fcov - was kindly provided by r. j. de groot, university of utrecht, the netherlands (genbank nc_ ). the recombinant type-i fcov strain recfcov-dstop- b was generated by reverse genetics as described previously (tekes et al., ) . it comprises the type-i fcov strain black with a restored accessory gene b (genbank eu ). the type-ii fcov - and the type-i recfcov-dstop- b were propagated on crfk cells and fcwf- cells, respectively. crfk or fcwf cells were incubated for h with fcov - (multiplicity of infection [moi] of ) or recfcov-dstop- b (moi of . ), respectively. to inhibit n-glycosylation of viral proteins, mg ml À tunicamycin in dimethyl sulfoxide (dmso) were added h post infection (p.i.). at h p.i., the cells were harvested in ripa buffer ( mm nacl, mm tris, % np- , . % na-deoxycholate, . mm pefabloc sc [merck], ph . ) with . % sds for sds-page and western blot analysis. following treatment with tunicamycin, cells were either fixed for immunofluorescence or harvested in ripa buffer for sds-page and western blot analysis at h p.i. deglycosylation of cell lysates was carried out for h with nglycosidase f (pngase f; new england biolabs, ma) according to the manufacturer's instructions. the full-length b genes of fcov - (nucleotides (nts) - ) and recfcov-dstop- b (nts - ) were amplified by reverse transcription polymerase chain reaction (rt-pcr). the amplified product of fcov - was cloned with primers c /c into the ncoi/xhoi sites of a modified pet- a vector (novagen). the resulting plasmid was named pc . and contained a c-terminal  histidine-tag ( b-his). the amplified product of recfcov-dstop- b was cloned with primers c / c into a modified pgex- p- vector (ge healthcare) by digestion with bamhi and xhoi. the resulting plasmid with an n-terminal gst and a c-terminal  his-tag was named pc . (gst- bdstop-his). for the generation of gst- bdss-his (pc . ; c / c ), a deletion mutant of b (amino acid - , without the nterminal signal sequence [dss] ) was cloned into the same modified pgex- p- vector. to determine the antigenic site of the monoclonal antibodies, n-and c-terminally truncated b proteins of fcov - were generated as gst-fusion proteins. to this end, the sequences were amplified by pcr with specific primers and cloned into the modified pgex- p- vector. the resulting plasmids were named pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c / c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ), pc . (aa - ; c /c ) and pc . (aa - ; c /c ). primer sequences are shown in table . the pet- a vector was modified by pcr with primers c and c to insert an ncoi and xhoi restriction site upstream of a bamhi restriction site. furthermore, a  his-tag followed by a stop codon behind the xhoi site was inserted and the existing bamhi site was deleted. in a second pcr with primers c and c , the his-tag was extended with histidines to generate a  his-tag. the pgex- p- vector was modified by pcr with primers c and c to insert a x his-tag downstream of the existing xhoi site. plasmid constructs were verified by sequence analysis. the expression of the recombinant proteins was performed after transformation of the different plasmids into escherichia coli (e. coli) strain rosetta tm (de ) plyss (novagen). an overnight culture was diluted : in fresh lb medium supplemented with ampicillin ( mg ml À ), chloramphenicol ( mg ml À ) and glucose ( %) to a final volume of ml and grown for - h at c to od of . . protein expression was induced by the addition of isopropyl-b-d-thiogalactopyranoside (iptg) to a final concentration of mm. after h of growth at c the cells were harvested by centrifugation and resuspended in mm na hpo , mm nacl and % triton x- . cells were lysed by freeze/thaw treatment ( Â) and sonication on ice. the insoluble protein fraction was separated from soluble proteins by ultracentrifugation ( ,  g for h). to solubilize inclusion bodies the pellet was resuspended in m urea, mm nacl and mm na hpo , ph . or in m guanidinium-hydrochloride (guhcl), mm na hpo , % ethanol, % triton x- and mm nacl, ph . and treated with ultrasonication. after an additional ultracentrifugation at ,  g for min, the solubilized proteins were applied on histrap hp columns (ge healthcare). the purification of the recombinant proteins was performed by metal ion affinity chromatography (imac) with ni + sepharose as recommended in the supplier's protocol. the purified recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and coomassie staining. following electrophoresis the proteins were identified by western blot using anti-his mab. after dialysis overnight against pbs (gst- b-dss-his) or acetone precipitation ( b-his), the proteins were used for the immunization of mice. protein concentrations were determined in bradford assay (bradford, ) . four week old female balb/c mice (charles river breeding laboratories) were injected subcutaneously with mg of purified protein gst- bdss-his in freund's incomplete adjuvant (sigma) on days , and . antibodies binding the recombinant protein were detected in serum samples by western blotting after the third injection. on days , and the mouse with the best immune response was immunized with mg of purified protein b-his without adjuvant. one day after the final antigen application splenocytes were fused with sp / -ag myeloma cells (atcc crl- ) at a ratio of : using polyethylene glycol . (roche) according to standard protocols (köhler et al., ) . the fused cells were cultured in -well plates and hybridoma cells were selected in dmem supplemented with % f (gibco), % f (gibco), % fcs, mm l-glutamine, . % -mercaptoethanol, mm hepes, oxaloacetate-pyruvate-insulin supplement (opi; sigma), and hypoxanthine-aminopterin-thymidine supplement (hat; sigma). for the detection of specific antibodies against the b protein, hybridoma culture supernatants were screened by elisa (engvall and perlmann, ) - days after fusion. to avoid screening for antibodies that react with gst, the fusion protein b-his was used for coating. however coating with b-his resulted in od nm values < . . as an alternative flat-bottomed elisa -well plates (nunc maxisorp) were coated overnight at c with ng purified gst- bdss-his protein in . m na co /nahco (ph . ) per well. the supernatants were also tested against an irrelevant control protein (gst-yfp-his; ng/well) in order to exclude gst-and hisreactive clones. this control protein was expressed in the same vector and purified as described for the gst- bdss-his construct. after washing three times with pbst (pbs containing . % tween- ), the plates were blocked with % fcs in pbst for h at c. then, the plates were washed again and incubated with ml/ well of hybridoma culture supernatants. specific antibodies were detected using goat anti-mouse igg conjugated with horseradish peroxidase (dianova) at dilution : . the elisa was performed with , , , -tetramethylbenzidine (tmb; sigma) and enzyme reactions were terminated with % (v/v) h so solution. the optical density (od values) at nm was measured in a microtiter plate reader (spectra ii, slt). hybridomas with elisa values > . were cloned twice. reactivity was confirmed by elisa and specificity was determined by western blot analysis. to confirm the antigenic sites recognized by the mabs b and d , elisa table primer sequences used to amplify defined fragments of fcov orf b. sequence plates were coated with ng/well of bsa- b-pep (c-rvece-giegfnctwpgfq [centic biotec, germany]). due to problems during the synthesis of the peptide, the internal cysteines were linked by a disulfide bond. therefore, the peptide was incubated with the reducing agent dtt ( mm) for h at room temperature before coating. as negative control, an irrelevant peptide (coupled to bsa), bsa- b-clvg ( mg/well; c-lvgavpkqkrlnvg, biogenes, germany) and, as positive control, the purified protein gst- bdss-his ( ng/well) were coated. the mabs b and d were used at a : dilution, anti-his mab was used at a : dilution. the elisa was performed as described above. the immunoglobulin subclass of both mabs was determined using the commercially available iso-gold rapid mouse-monoclonal isotyping kit (bioassay works) according to the manufacturer's instructions. sds-page was carried out using a % tricine-polyacrylamide gel system (schagger and von jagow, ) . for western blot analysis, the proteins were transferred to a nitrocellulose membrane (pall corporation). residual binding sites were blocked with % skimmed milk in pbst. the membrane was then incubated with monoclonal antibodies of the hybridoma cell culture supernatants (primary antibodies) at a : dilution or sera in pbst ( : dilution). bound antibodies were visualized with hrpconjugated goat anti-mouse or goat anti-cat igg (dianova) and chemiluminescence reagent (western lightning plus-ecl; perki-nelmer). goat anti-cat igg was diluted in pbst with x roti -block (roth) additionally. crfk cells were cultured on sterile cover slips in well plates pretreated with rat tail collagen (sigma). cells were mock or virusinfected and treated with mg ml À tunicamycin h p.i. at h p.i. the cells were fixed with pbs containing % paraformaldehyde for min at c, followed by three pbs washes. cells were permeabilized with triton x- ( % in pbs) for min at room temperature. the cells were rinsed twice with pbs and residual binding sites were blocked with  roti -immunoblock (roth) for min at room temperature. the cells were then incubated with primary antibodies ( : dilution) or sera ( : dilution) in pbs for h at c, followed by three pbs washes. subsequently the cells were incubated with secondary antibodies conjugated to cyanogen- ( : dilution, dianova) in pbs for an additional hour at c. the dna was counterstained with mg ml À , diamidino- -phenylindole (dapi) for min at room temperature. after three washes the cover slips were mounted with mowiol (sigma) containing the anti-fading reagent dabco ( , -diazabicyclo( . . )octane, roth). the immunofluorescent staining was visualized by fluorescence microscopy (axiovert , zeiss). the full-length b protein of fcov - of amino acid (aa) residues was expressed in e. coli with a c-terminal his-tag ( b-his). induction of expression of b-his resulted in insoluble protein aggregates that could be solubilized using m guhcl-containing buffer and purified by ion metal affinity chromatography (imac). the identity of the purified protein was confirmed by sds-page and western blotting using anti-his mab. the protein had an apparent molecular mass of kda as judged by sds-page analysis, which is predicted for this protein, and was recognized by the his-tag-specific antibody (fig. a) . western blot analysis further revealed a protein of approximately kda, suggesting dimerization of the full-length protein. the overall yield for this protein was low. we therefore decided to express b as part of a gst fusion protein. to do this, the region encoding a putative nterminal signal sequence (ss, residues to ) was removed from the b coding sequence and replaced by the gst coding sequence. the resulting construct was termed gst- bdss-his. expression of gst- bdss-his in e. coli resulted again in insoluble protein aggregates. the protein was purified under denaturing conditions using m urea-containing buffer and analyzed by sds-page and western blotting using an anti-his mab. consistent with its calculated molecular mass, the major fraction of the purified protein migrated as a -kda protein in sds polyacrylamide gels. two additional minor bands in the sds-page were specifically recognized by western blotting using the his-tag-specific mab. these bands are consistent with a dimer and a -kda degradation product, respectively, of the gst- bdss-his protein (fig. b) . the total yield of gst- bdss-his was estimated to be times higher compared to b-his. the elution fractions of b-his and gst- bdss-his were pooled, concentrated, desalted and used for immunization ( fig. c and d, respectively) . the fusion protein b-his was used for the immunization of balb/c mice. however, even after four injections over a time period of weeks, no immune reaction against b-his could be detected in sera obtained from immunized animals. we therefore decided to immunize another four balb/c mice using the gst- bdss-his protein. two weeks after the third injection a specific seroconversion was detected. the mouse with the best immune response received an additional boost with b-his. splenocytes isolated from immunized mice were fused with myeloma cells and cell culture supernatants were screened by elisa using the gst- bdss-his as capture antigen. gst-yfp-his was used as a control antigen to identify and exclude clones producing antibodies specific for one of the tags. two supernatants were confirmed to contain antibodies that bind to gst- bdss-his but not the gst-yfp-his control protein. the respective hybridoma cells were subcloned twice and continued to produce b-specific antibodies as determined by elisa. antibodies produced by the two stable hybridoma cell lines ( b [igg a], d [igg ]) were subsequently tested by western blotting. both mabs recognized the recombinant proteins b-his (fig. a) and gst- bdss-his (data not shown). the data also suggest that the formation of the putative b dimer discussed above ( fig. c and text) involves one or more intermolecular disulfide bonds since the higher molecular mass band of > kda was not detectable if -mercaptoethanol was included in the protein sample buffer (fig. b) . to determine whether the two fcov serotype ii (strain - ) b protein-specific mabs recognize epitopes that are conserved among serotype i and ii strains, the mab reactivities against the well-characterized serotype i strain black were analyzed. the b proteins of strains black and - share % amino acid sequence identity. however, strain black contains an internal translation stop codon in orf b, resulting in a c-terminally truncated b protein. we therefore restored the orf b of fcov strain black and expressed the strain black b peptide sequence as part of a recombinant fusion protein (gst- bdstop-his) in e. coli. western blot analysis of lysates obtained from iptg-induced e. coli cells transformed with the appropriate expression plasmid confirmed that the two mabs recognized the recombinant b protein of strain black (data not shown), confirming that the two mabs obtained in this study recognize epitopes that are conserved among the b proteins of representative fcov serotype i and ii strains. to further characterize the newly generated mabs, we sought to determine the binding sites within the b protein. as shown above, both mabs recognized b-his in western blot experiments after sds-page under non-reducing and reducing conditions, suggesting that they recognize a linear epitope on the b protein. first, we produced a set of bacterially expressed segments of b. appropriate coding sequences were cloned into the pc . plasmid and expressed with n-terminal gst and c-terminal histidine tags. mab reactivities against these proteins were tested by western blotting (fig. ) . all proteins containing aa residues - of b (pc . , pc . , pc . , pc . , pc . , pc . ) were detected by the mabs. in contrast, proteins containing residues - (pc . ) or to (pc . ) were not recognized. furthermore, a protein containing residues - of b (pc . ) was recognized while none of the slightly smaller proteins that lacked a few more residues at the carboxyl terminus (pc . , pc . , pc . , pc . ) were recognized. the combined results summarized in fig. led us to conclude that both mabs recognize a peptide segment that includes residues to of the b protein (fig. ) . to further corroborate this hypothesis, a synthetic peptide coupled to bsa (bsa- b-pep, c- rvecegiegfnctwpgfq ) was specificities of b and d were characterized using total lysates of noninduced (À) and iptg-induced (+) e. coli rosetta cells transformed with the appropriate b-his expression plasmid (see materials and methods). proteins were separated by sds-page ( %) under nonreducing conditions and antibody reactivities were analyzed by western blotting using anti- b mabs ( b and d , respectively) and anti-his mab (control) as indicated. (b) the purified protein b-his/e was separated by sds-page ( %) in the absence (À -me) or presence (+ -me) of the reducing agent -mercaptoethanol. western blot analysis was performed using anti- b mab b . arrowheads indicate the recombinant protein b-his. e : elutate fraction obtained with mm imidazole. used in an additional set of experiments. to resolve problems that occurred during peptide synthesis, an intramolecular disulfide bond was introduced. to further characterize the mab binding properties, bsa- b-pep was used as antigen in an elisa that was performed under non-reducing and reducing conditions, respectively. bsa- b-pep was treated with or without dtt and elisa plates were coated with the peptide. bsa- b-clvg and purified gst- bdss-his fusion protein were used as negative and positive control, respectively. as shown in fig. , mabs b and d were confirmed to bind specifically to bsa- b-pep in the absence and presence of dtt. the data provide direct evidence that both mabs recognize a region in the b protein that spans residues to . . . comparative sequence analysis of the peptide region recognized by the mabs as shown above, the epitope(s) recognized by both mabs are located within the amino acid sequence to of the fcov b protein. this part of the b sequence is highly conserved within fcov serotypes i and ii with sequence identities of - %. interestingly, this part of the sequence comprises the only n-glycosylation motif ( nct ) within the b sequence (fig. ) . this motif is conserved in almost all sequenced fcov strains. in order to detect the authentic b protein with our newly generated anti- b mabs, crfk cells were mock-infected or infected with fcov - and analyzed by indirect fig. . determination of the b and d binding sites in the b protein. the scheme shows the full-length b construct and the truncated versions derived from this construct. all proteins were expressed as gst-and his-tag fusion proteins and mab reactivities were tested by western blotting under non-reducing conditions. +: specific binding detected; À: not detected. fig. . reactivities of mabs b and d against bsa- b-pep as determined by elisa. elisa plates were coated with bsa- b-pep in the presence (+) or absence (À) of dtt. as controls, bsa- b-cvlg and gst- bdss-his were used. the data represent three independent experiments; standard deviations are indicated. immunofluorescence assay (ifa) h post infection (p.i.). surprisingly, neither of the anti- b mabs produced a positive signal, although there was a clear reactivity with an antiserum against fcov (data not shown). as the two mabs were shown previously to recognize fcov b fusion proteins in western blot experiments (fig. ) , we used this method to analyze cell lysates obtained at h p.i. both mabs detected a protein with an apparent molecular mass of approximately kda in fcov-infected cells, whereas no signal was detected in mock-infected cells (fig. a) . it has been shown by others that orf b encodes a glycoprotein of kda (vennema et al., a) . in order to investigate whether our mabs detect a glycosylated form of the b protein, pngase f treatment of cell lysates was performed. surprisingly, incubation with pngase f did not affect the migration behavior of the b protein (data not shown), indicating that the protein detected by the mabs represents a nonglycosylated form of the b protein. since the only n-glycosylation motif in b is located at aa position - (nct), we speculated that the n-glycosylation site might be part of the epitope(s) recognized by the mabs. accordingly, the epitope(s) may be masked and thereby inaccessible for our antibodies. in order to test this hypothesis, the effect of an inhibitor of n-linked glycosylation was studied. crfk cells were infected with fcov - or mock-infected and treated with tunicamycin at h p.i. cell lysates were prepared at h p.i. and probed by western blotting. the mabs recognized a single protein with an apparent molecular mass of kda in the absence and presence of tunicamycin (fig. b) . interestingly, the signal intensity obtained with the mabs was much stronger after inhibition of nglycosylation. moreover, after treatment with tunicamycin, a dimer of b was recognized by both mabs which was not detectable in the absence of tunicamycin (fig. b) and disappeared after incubation with -mercaptoethanol (data not shown). these results support the idea that both mabs do not recognize the glycosylated form of fcov b. an anti-fcov serum, which served as positive control, detected the virus-specific proteins n and m (fig. b) . in addition, a protein band with an apparent molecular mass of kda was detected by the anti-fcov serum. this protein may represent the nonglycosylated form of the m protein and/or the b protein. the results outlined above show that the anti- b mabs recognize exclusively the nonglycosylated form of the viral protein in fcov-infected cells. this was particularly striking after treatment of the cells with tunicamycin. in light of this finding, another round of immunofluorescence experiments was performed with the mabs after infection with fcov - and incubation with tunicamycin. as already mentioned, there was no staining after incubation with the mabs in the absence of tunicamycin. however, after tunicamycin treatment, clear signals were obtained in infected cells (fig. ) . at this point, the observed cytoplasmic localization of b cannot be further narrowed down. an anti-fcov serum detected viral proteins in the cytoplasm of infected cells regardless of whether or not they were treated with tunicamycin ( fig. ) . the genome of feline coronavirus (fcov) encodes five accessory proteins termed a- c, a and b. among these, only b has been identified in virus-infected cells (vennema et al., a) . the b protein was also expressed from a plasmid under the control of the t promoter in t polymerase-expressing vaccinia virus-infected cells. the produced b protein was identified with ascites from a fipv-infected cat. fcov b was shown to be a glycoprotein that to some extent may be secreted from infected cells. interestingly, a kdel-like sequence at its c-terminus has been shown to slow down the export of the protein (vennema et al., a) . we are interested in accessory proteins encoded by fcov in particular with respect to potential roles in (i) establishing persistent infections by enteric feline coronavirus in the gut and (ii) the pathogenesis of feline infectious peritonitis (pedersen, ) . since there was already some information on fcov b, we first concentrated on this protein. for further characterization including its unambiguous identification in fcov-infected cells monoclonal antibodies against b protein were generated. using nand c-terminally truncated parts of the b protein expressed by bacteria as well as a synthetic peptide conjugated to bsa, the binding sites of two mabs were determined. according to this analysis, the mabs recognize a stretch of amino acids (aa) between aa position and . interestingly, the only putative nlinked glycosylation site as well as two of the seven cysteines of the fcov b protein are contained within this sequence. this finding led to the question whether the mabs recognize only the nonglycosylated form of b. in fact, both mabs detected in fcov-infected cells only recognized a protein with an apparent molecular weight of kda and not the previously described glycosylated form of kda (vennema et al., a) . the exclusive recognition of the nonglycosylated form was supported by results obtained after tunicamycin treatment of fcov-infected cells. notably, immunofluorescence studies using the mabs led to negative results unless the fcov-infected cells were treated with tunicamycin. the difference to western blot experiments where the nonglycosylated form could be readily shown without inhibition of glycosylation most likely results from different sensitivities of the two methods. masking of epitopes by glycosylation has been shown for many other viral proteins including the hemagglutinin of influenza virus (skehel et al., ) . the authors also used tunicamycin to demonstrate recognition by mabs in the absence of n-linked glycosylation. accordingly, the antigenicity of influenza virus hemagglutinin and of other proteins can be modified by addition of carbohydrate chains. furthermore, since both mabs recognize the same or a nearby epitope in b, it can be assumed that the identified aa stretch represents a highly antigenic site. masking of this region through glycosylation of asparagine may prevent an immune response and could be relevant for establishment of persistent infection by fcov. this could be directly tested by the use of recombinant fcovs, where the n-linked glycosylation site of b is missing. it remains to be seen whether this finding has implications for vaccine development against fcov-induced diseases. another interesting aspect of our studies with fcov b concerns putative intra-and intermolecular disulfide bonds. in this context, it is noteworthy that expression of b in bacteria allowed the detection of disulfide linked dimers, which disappeared after treatment of extracts with a reducing agent. such dimeric forms were also detected in fcov-infected cells, but apparently only after treatment with tunicamycin. thus, it is tempting to speculate that intermolecular disulfide bridges represent an artifact only observed in the absence of glycosylation at asparagine of fcov b. it will be interesting to determine whether one or both cysteines present in our synthetic peptide is/ are involved in intramolecular disulfide bonds. recognition of b by our mabs in the absence and presence of reducing agent may give some indirect evidence in this regard. pilot experiments actually indicate that our mabs recognize the nonreduced form of b from fcov-infected cells better than the reduced one; this result suggests that the native protein contains one or more intramolecular disulfide bridges, which are important for the antigenicity of b. another way to study the relevance of disulfide bonds for the structure of b is the exchange of cysteine codons in different expression systems together with western blots under nonreducing and reducing conditions. however, final proof for the presence of intramolecular disulfide bridges within fcov b will require the determination of its three-dimensional crystal structure. unfortunately, our mabs are not well suited to detect the secreted glycosylated form of b. to further study the synthesis, intracellular localization and secretion of b, we will use our reverse genetics system (tekes et al., (tekes et al., , to obtain recombinant viruses containing the desired changes in the b coding sequence. ultimately, these studies together with the three dimensional structure of b will give more insight into the biological role of the b protein. persistence and transmission of natural type i feline coronavirus infection mutations of c and spike protein genes correlate with the occurrence of feline infectious peritonitis a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral c gene sequence analysis of feline coronaviruses and the circulating virulent/avirulent theory family coronaviridae orf -encoded accessory protein a of feline infectious peritonitis virus as a counteragent against ifn-alpha-induced antiviral response genomic rna sequence of feline coronavirus strain fipv wsu- / enzyme-linked immunosorbent assay (elisa): quantitative assay of immunoglobulin g live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (acinonyx jubatus) the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes feline coronavirus type ii strains - and - originate from a double recombination between feline coronavirus type i and canine coronavirus the prevalence of types i and ii feline coronavirus infections in cats deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis detection of feline coronavirus infection in southern african nondomestic felids evaluation of antibodies against feline coronavirus b protein for diagnosis of feline infectious peritonitis in cats fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines the molecular biology of coronaviruses mutation in spike protein cleavage site and pathogenesis of feline coronavirus gain, preservation, and loss of a group a coronavirus accessory glycoprotein the molecular biology of coronaviruses a review of feline infectious peritonitis virus infection: - tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from to kda a carbohydrate side chain on hemagglutinins of hong kong influenza viruses inhibits recognition by a monoclonal antibody genome organization and reverse genetic analysis of a type i feline coronavirus a reverse genetics approach to study feline infectious peritonitis emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal genomic organization and expression of the end of the canine and feline enteric coronaviruses feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses this study was supported by the "bundesministerium für bildung und forschung" of the german government (zoonosis network, consortium on ecology and pathogenesis of sars, project code ki a-f) as well as by the collaborative research centre : "rna viruses: rna metabolism, host response and pathogenesis". key: cord- - l una authors: kapil, sanjay; richardson, kay l; maag, trisha r; goyal, sagar m title: characterization of bovine coronavirus isolates/from eight different states in the usa date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: l una bovine coronavirus isolates from eight different states of the usa were compared for their antigenic properties and susceptibility to hygromycin b. antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (hi) test using a polyclonal antiserum against the mebus bovine coronavirus isolate. differences were observed on isoelectric focusing among viral proteins with isoelectric points between . – . . most of the bcv isolates were susceptible to hygromycin b ( . mm) whereas a few hygromycin b resistant isolates were also found. diarrhea and respiratory diseases are clinically important and cause high mortality in calves (virtala et al., ) . bovine coronavirus (bcv) is an economically important cause of intestinal and respiratory diseases in calves and has also been implicated in winter dysentery of adult cattle (clark, ) . despite differences in clinical disease, it is believed that all bcv isolates belong to a single serotype (clark, ) . the genome of coronaviruses is the largest among the rna viruses (lai, ) . coronavirus has a high frequency of recombination, rna polymerase lacks proofreading, and defective interfering rnas are frequently generated in coronaviruses (lai, ) . bcv isolates from canada have been divided into three subgroups on the basis of reactivity with monoclonal antibodies (mabs) against spike protein of bcv (michaud and dea, ) . veterinary microbiology ( ) ± the current modified, live bcv vaccine is not clinically effective (thurber et al., ) and lack of clinical efficacy of the vaccine may be due to antigenic differences among the vaccine and field isolates of bcv. minor antigenic and biological differences have been observed among bcv isolates from calf diarrhea and winter dysentery cases from the usa (tsunemitsu and saif, ) and canada (dea et al., ) . to further understand the molecular epidemiology of bcv, bcv isolates from eight states of the usa were compared. we compared antigenic differences on one-way hemagglutination-inhibition (hi) test, isoelectric focusing of bcv proteins, and relative susceptibility of bcv isolates to hygromycin b. bcv isolation from clinical samples containing coronavirus-like particles observed by negative contrast electron microscopy has been described before (kapil et al., ) . briefly, human rectal tumor- cell line (hrt- ) was inoculated with % fecal suspension. before inoculation, the suspension was filtered through a . mm filter (gelman sciences, ann arbor, mi). the confluent monolayers were washed with calcium and magnesium free phosphate buffer saline containing trypsin ( mg/ml). about . ml of filtered fecal suspension was added on to the confluent monolayers and virus allowed to adsorb for an hour. minimum essential medium containing trypsin ( mg/ml) (gibco/ brl, life technologies, gaithersburg, md) and pancreatin ( mg/ml) (sigma, mo) was added. the monolayers were examined daily for ± days for cytopathic effects. we successfully propagated bcv isolates in hrt- cells ( from california, from kansas, from minnesota, from nebraska, from north dakota, from oklahoma, from wisconsin, and from wyoming). all tissue culture propagated bcv isolates were compared at passage . hemagglutination test was performed in v-bottom plates (dynatech laboratories, ma). two-fold dilutions of bcv samples ( ml) were prepared in . m phosphate buffered saline (pbs) containing . % bovine serum albumin (sigma). after addition of ml of % mouse erythrocytes, the plates were incubated at c for min. hemagglutination titers were expressed as the reciprocal of the highest dilution showing complete agglutination. for hi test, cell culture propagated bcv isolates ( from california, from kansas, from minnesota, from nebraska, from wisconsin and from wyoming) and wild-type (original fecal samples but not propagated in cell cultures) bcv isolates ( from california, from nebraska, from wisconsin and from wyoming) were compared by hi test. for of these wild-type samples, cell culture propagated bcv isolates were also tested in hi test. preparation of antiserum and the procedure for hi test have been described before (sato et al., ) . for removing nonspecific hi, ml of anti-bcv antiserum (national veterinary services laboratory, ames, ia) made against mebus isolate, was diluted with ml of pbs, inactivated at c for min, and mixed with ml of % kaolin (fisher scientific, fairlawn, nj). after shaking for h, kaolin was removed by centrifugation of  g for min. clear supernatant was mixed with ml of packed normal mouse erythrocytes to remove nonspecific hemagglutinins. after incubation for h at c, the treated bcv antiserum was separated from mouse erythrocytes by centrifugation. for hi test, four to eight ha units of bcv isolates (in ml) were mixed with ml of treated bcv antiserum. after incubation for an hour at c, % mouse erythrocyte suspension ( ml) was added. plates were reincubated at c for min. the hi activity of bcv antiserum against isolates of bcv was recorded at the reciprocal of the highest dilution of antiserum showing complete inhibition of hemagglutination (sato et al., ) . for each virus isolate, back titration was performed at the same time to assure that four to eight ha units of bcv was used. each sample was repeated three to four times to confirm the results and the test was found to be reproducible. bovine coronavirus purification procedure has been described previously (king and brian, ) . infected hrt- cells were harvested when approximately % of them showed cytopathic effects, such as rounding, detachment and syncytia formation. briefly, after three freeze-thaw cycles, the cells were scraped and pooled. cellular debris was removed by centrifugation at  g for min, and the clear supernatant was filtered through a . mm filter (gelman sciences, mi). polyethylene glycol (peg- ) was added at a final concentration of % (w/v). after overnight incubation at c, the virus precipitate was pelleted at ,  g for min. the pellet was saved and resuspended in tnf buffer (ph . ). the bcv was purified on a sucrose gradient and all sucrose solutions were made in tne. the pellet was layered on a / % (w/w) sucrose gradient and centrifuged at ,  g for h. the virus, at the interphase, was collected and diluted in tne and layered on ± % step gradient ( , , , , and % sucrose, . ml each) and centrifuged at ,  g ( , rpm) overnight. bands were collected, centrifuged at ,  g for h to pellet the virus, and stored at À c. for isoelectric focusing, the gel was made with acrylamide-ampholyte solution ( %, biolyte (bio rad) ph ± ) using ammonium persulfate, riboflavin- h -phosphate, and temed as catalysts for polymerization. after photopolymerization for min on one side, the other side was irradiated for min to eliminate unpolymerized monomer on the gel surface. purified virus was treated with an equal volume of sample buffer ( . m urea, % triton x- , and biolyte %, ph ± at room temperature for h) and then applied (total volume ml) to the gel with disposable templates. samples were allowed to diffuse into the gel for ± min. focusing was done at v for min, then increased to v for min and, to v for h and min. bcv proteins were detected by silver staining (bio-rad). the ief gels on a plastic support were left to dry overnight in a clean area at room temperature. to assess the susceptibility of bcv isolates, hygromycin b was added at a concentration of . mm in the cell culture medium. each sample was propagated in the presence and absence of hygromycin b. after h, the plates were frozen and thawed (three times). the plates were centrifuged and clear supernatant was collected. the amount of bcv in culture supernatant was quantified by a hi test. on hi test, most wild-type bcv samples ( / ) showed very low reactivity (hi titers : , type iii) and only three samples showed high reactivity or inhibition (hi titers : ± : , type i). one sample was inhibited at : , type ii (fig. ) . the hi titers of cell culture propagated bcv isolates ranged from : ± : (fig. ) . when a sample was compared after call culture propagation, in most bcv isolates, except wi- , the hi titers of cell culture propagated bcv were comparatively higher than its wild-type bcv isolate. similar protein bands could be detected in all bcv isolates (ca- , nd- , wi- -sk, wi- , wi- , wi- , wi- , wi- , wi- , wy- , and wy- ) studied by sds-page analysis (data not shown). no protein bands were detected in uninfected hrt- cells processed in a similar manner. the identity of bcv proteins was confirmed by a western blot analysis or radio immunoprecipitation. polyclonal antibody, bovine anti-bcv antiserum, detected all the bcv proteins: spike dimer, spike subunits, nucleoprotein, hemagglutinin-esterase proteins (dimer and subunits), and membrane proteins. the molecular weight of hemagglutinin-esterase protein was the same in all the bcv isolates studied. on the basis of seven wisconsin bcv isolates studied, we concluded that most bands were invariable between different bcv isolates and sharply focused on ief gels. however, bands towards the anodic end of the ief profile (pi . ± . ) showed variation between different isolate and even different fractions of the same isolate. a double-band migrated at pi . in most bcv isolates but in wi- -sk, fraction b, and wi- , fraction , it migrated at pi . . (fig. ) . to demonstrate the differences, all samples were repeated at least three times after the ief protocol was standardized to ensure the reproducibility of the differences between bcv isolates. relative susceptibility of bcv isolates to hygromycin b ( . m) was studied (table ) . a drop in hemagglutination liter by -fold or higher was considered significant and the bcv isolate was considered susceptible to hygromycin b. on the basis of this criteria, four bcv isolates (mn , mn , mn and mn ) were considered resistant to hygromycin b; however, the other bcv isolates were susceptible to hygromycin b. isolates of bcv from calf diarrhea and winter dysentery differ in cytopathology, some isolates are non fusogenic while others are weakly or highly fusogenic (dea et al., ) . cytopathic effects produced by bcv isolates have no correlation with the clinical condition but depends on the presence of trypsin in the medium (dea et al., ) . similarly, no differences were observed in cpe among calf diarrhea and winter dysentery isolates by another group (tsunemitsu and saif, ) . we observed some differences in cytopathic effects among the calf diarrhea isolates; however, passage number, plaque purification, and the presence of trypsin and pancreatin in the cell culture medium greatly affected the cytopathology of bcv isolates. it is possible that some isolates of bcv are fastidious and do not grow on hrt- cell line but will grow in organ cultures (kapil et al., ) . plaque variation has also been observed among bcv isolates and the isolates differ in size and shape of the plaques (kapil et al., ) . it is believed that bcv isolates are antigenically related (clark, ) . bcv isolates do not differ significantly in reactivity on indirect immunofluorescent test (tsunemitsu and saif, ) . either slight or no differences were observed on virus neutralization tests (dea et al., ; tsunemitsu and saif, ) . because spike protein is the major immunogen that elicits antibodies detected by neutralization and indirect fluorescent antibody tests (saif, ) and elicits a bulk of cross reactive antibodies among bcv isolates, spike protein may not be very useful to differentiate bcv isolates. minor antigenic variation has been observed among bcv isolates from neonatal calf diarrhea and winter dysentery cases by a one-way test (dea et al., ) . our results in this study fig. . hemagglutination-inhibition activity of an anti-bovine coronavirus (mebus isolate). antiserum against cell culture propagated bovine coronavirus isolates: on the basis of a one way hi test, bcv isolates were inhibited from : up to : . x-axis denotes the hi titers and the numbers indicate superscripts (log ). y-axis indicates number of bcv isolates inhibited. support and extend the previously reported findings on hi test using a large number ( ) of american isolates collected from eight different states of the usa. none of the previous studies (dea et al., ; tsunemitsu and saif, ) reported hi results on wild-type bcv or the virus in fecal samples. results on wild-type isolates may need cautious interpretation because the presence of colostral iggl and local iga in feces may interfere with results. on the other hand, passaging of bcv also affects hi. passage of bcv in cell culture may also affect the antigenic composition (hussain et al., ) , hemagglutination activity (storz et al., ) , and intestinal replication of bcv (kapil et al., ) . it is important to study all isolates at low passage (up to passage or ) and at the same passage. in the only other american study, samples were studied at different passages (tsunemitsu and saif, ) . we tried to develop a polyclonal antiserum against ca- , a type iii bcv isolate. the type iii antiserum was not high titered and we could not study the two-way hi relationship among bcv isolates. for most bcv isolates, the cell culture passaged virus showed higher hi titers compared to its wild-type virus in fecal samples. when cell culture-propagated and wild-type versions of bcv (wi- ) were compared, they showed hi titers of : and : , respectively. this may be because the hyperimmune serum used in the hi test was produced with a bcv isolate that was highly adapted in cell culture or it could be due to the presence of some antibodies in feces or it may be a unique property of the wi- isolate. typing of wild-type bcv isolates by hi may be useful to study the epidemiology of bcv. hemagglutination and receptor destroying activities have no correlation with the clinical source of bcv isolates (tsunemitsu and saif, ) . a number of studies have been published in which american (hussain et al., ) , french (vautherot and laporte, ) , canadian (michaud and dea, ) , english (el-ghorr et al., ) , and scottish (clark et al., ) isolates of bcv have been antigenically compared using a panel of mabs. all of the previous studies, except the canadian study, have failed to classify the bcv isolates into subgroups. a monoclonal antibody (s / ) neutralizes some bcv isolates (s , scottish isolate, mebus isolate, and pq, a canadian isolate) but does not neutralize other isolates of bcv (english isolate, ck and scottish isolate, s ). however, in a canadian study, quebec isolates, collected between ± , have been classified into three antigenic subgroups (michaud and dea, ) on the basis of reactivity with a panel of mabs. we observed minor differences in the protein profile of bcv isolates by sds-page analysis, western blot analysis and radio immunoprecipitation of bcv isolates (data not shown). similarly, no differences were observed among neonatal calf diarrhea and winter dysentery isolates from quebec by western blot analysis (dea et al., ) . however, minor differences were observed in isoelectric focusing of bcv proteins (fig. ) . different serotypes of infectious bronchitis virus (sadasiv et al., ) and temperature sensitive mutants of mouse hepatitis virus (oleszak et al., ) differ on isoelectric focusing of spike proteins. isoelectric focusing is a tedious procedure and needs a lot of careful standardization in research laboratories. in our studies, a suitable point of application of samples was determined by applying purified bcv at three different places: . , . , and . cm from the cathode. sample application at . cm from the cathode was not suitable because the bands were wavy. application of samples at . cm from the cathode was found to be suitable for ief analysis (fig. ) . we tried three different shapes of the template for the application of bcv protein samples: squares (&), horizontal slits or dashes (-) and vertical slits (|). in our opinion, dashes gave the most clearly visible bands. we observed varying levels of susceptibility of bcv isolates to hygromycin b. the mechanism of action of hygromycin b is not known; however, differential susceptibility of bcv isolates points to direct action of hygromycin b on bcv rna replication. because most isolates of bcv were found to be susceptible to low concentrations of hygromycin b ( . mm) and because hygromycin has no reported toxicity in cattle, hygromycin b may be a possible treatment for scours caused by bcv. moreover, relative susceptibility of bcv isolates to hygromycin b provides an easy epidemiological marker to differentiate bcv isolates, into susceptible and resistant categories and can be easily adopted by diagnostic laboratories trying to differentiate bcv isolates. susceptibility of bcv isolates to hygromycin b can be adopted by most veterinary diagnostic laboratories that have facilities for cell culture. first, bcv is isolated in hrt- in the presence of trypsin and pancreatin (kapil et al., ) . the virus is then propagated in the presence of hygromycin b to access the susceptibility and the titer of the virus is measured by a hi. hemagglutinin-esterase genes of a winter dysentery isolate and two neonatal calf diarrhea isolates from quebec, canada, have been compared to mebus isolate of bcv. no frame shifting, deletion or insertion changes were observed (dea et al., ) . when hemagglutinin-esterase cdnas of highly virulent bovine coronavirus (ly ) and avirulent bovine coronavirus (l ) were compared, four amino acid substitutions occurred (zhang et al., ) . similarly, we have observed only minor changes among two hemagglutinin esterase cdnas sequenced so far (zhang and kapil, unpublished results) . thus, differences observed in hi tests cannot be explained at the level of nucleic acid sequence (dea et al., ) . some wild-type bcv isolates could not be inhibited by the bcv antiserum against the mebus isolate (fig. ) indicating the presence of antigenically different bcv isolates. we speculate that a multivalent vaccine containing different serotypes (types i, ii, and iii) for bcv selected on the basis of hi patterns might offer better protection than the current monovalent modified live virus vaccine that is not clinically effective (deleeuw and tiessink, ) . some of the techniques described in this paper can be useful to diagnostic laboratories. typing of bcv isolates can be easily adopted by most veterinary diagnostic laboratories. bcv isolates can be differentiated by hi and susceptibility or resistance to hygromycin b. a comparison of bovine coronavirus trains using monoclonal antibodies bovine coronavirus comparison of bovine coronavirus isolates associated with neonatal calf diarrhea and winter dysentery in adult dairy cattle in quebec laboratory experiments on oral vaccination of calves against rotavirus or coronavirus induced diarrhea. zbl a serological comparison of bovine coronavirus strains comparison of bovine coronavirus (bcv) antigens: monoclonal antibodies to spike glycoprotein distinguishes between vaccine and wild-type strains plaque variations in clinical isolates of bovine coronavirus factors affecting isolation and propagation of bovine coronavirus in human rectal tumor- cell line excretion and persistence of bovine coronavirus in neonatal calves bovine coronavirus structural proteins transcription, replication, and engineering of coronavirus genes characterization of monoclonal antibodies to bovine enteric coronavirus and antigenic variability among quebec isolates microheterogeneity of s-glycoprotein of mouse hepatitis virus temperature-sensitive mutants pi alteration related to strain variation of infectious bronchitis virus, an avian coronavirus coronavirus immunogens hemagglutination by calf diarrhea coronavirus comparison of hemagglutinating, receptor destroying and acetylesterase activities of avirulent and virulent coronavirus strains field trial evaluation of a reo-coronavirus calf diarrhea vaccine antigenic and biological comparisons of bovine coronaviruses derived from neonatal calf diarrhea and winter dysentery of adult cattle utilization of monoclonal antibodies for antigenic characterization of coronaviruses morbidity from nonrespiratory diseases and mortality in dairy heifers during the first three months of life the hemagglutinin/esterase glycoprotein of bovine coronaviruses: sequence and functional comparisons between virulent and avirulent strains key: cord- -eniovfwy authors: zhao, ye; cheng, jin-long; liu, xiao-yu; zhao, jing; hu, yan-xin; zhang, guo-zhong title: safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: eniovfwy infectious bronchitis (ib) is a highly contagious respiratory and urogenital disease of chickens caused by infectious bronchitis virus (ibv). this disease is of considerable economic importance and is primarily controlled through biosecurity and immunization with live attenuated and inactivated ib vaccines of various serotypes. in the present study, we tested the safety and efficacy of an attenuated predominant chinese qx-like ibv strain. the results revealed that the attenuated strain has a clear decrease in pathogenicity for specific-pathogen-free (spf) chickens compared with the parent strain. strain yn-inoculated birds had clinical signs of varying severity with % mortality, while the attenuated group appeared healthy, with less tissue damage. the attenuated strain also had relatively low tissue replication rates and higher antibody levels. the superior protective efficacy of the attenuated strain was observed when vaccinated birds were challenged with a homologous or heterologous field ibv strain, indicating the potential of the attenuated yn (ayn) as a vaccine. producing a vaccine targeting the abundant serotype in china is essential to reducing the economic impact of ib on the poultry industry. infectious bronchitis virus (ibv) belongs to the order nidovirales, family coronaviridae, and genus gammacoronavirus. infectious bronchitis (ib) is an important worldwide viral disease of poultry. this poses a major economic threat to the poultry industry because of poor weight gain and lost feed efficiency in broilers and reduced numbers and quality of eggs in layers. in addition, some virulent strains cause high mortality in young chickens due to renal disease. while chicks are the most susceptible, ibv affects chickens of all ages. despite a predilection towards the respiratory tract, ibv also infects a wide range of organs, including the kidneys, gastrointestinal tract, and oviduct, causing respiratory disease, interstitial nephritis, and decreased egg production (cavanagh and naqi, ) . clinical signs include coughing, sneezing, tracheal rales, and watery eyes (cavanagh, ; raj and jones, ) . lesions in infected birds include degeneration of renal epithelium, renal interstitial lymphoplasmacytic inflammation, and degeneration and necrosis of the ciliated respiratory epithelium. the main method of protecting chickens from ib is inoculation with both attenuated live and inactivated vaccines. although inactivated vaccines are cheaper and easier to administer, the poultry industry prefers to use live vaccines rather than inactivated ones, as they are more effective (huang and wang, ) . despite widespread vaccination in china and other countries, ib outbreaks still occur sporadically because of little or no cross protection between different ibv serotypes (cook et al., ; liu et al., ). specifically, a new ibv variant has been circulating in china since (wang et al., ) . this virus has been identified as the qx strain and has been primarily associated with various renal pathologies (terregino et al., zsofia et al., . phylogenetic analysis showed that the ibv isolates that clustered with qx were mainly chinese isolates. these results further indicated that ibv isolates that are prevalent in china were significantly evolutionarily distant from mass-type strains. however, mass-type vaccine strains (e.g., h , h , ma , and w ) are mainly ibv vaccine strains typically administered in china today, which may not be able to provide efficient protection against field strains. other vaccine strategies including combination of mass type vaccine with / , conn type etc., were attempted before and could get a broader protection than the mass type vaccine alone (cook et al., (cook et al., , . however, the / and conn type vaccine have not been licensed as commercial vaccines in china nowadays. thus, it will be necessary to rapidly develop new vaccines against the qx-like field strains. it has been shown that the spike glycoprotein (s) of coronavirus is a determinant of tissue tropism and virulence and a tiny change in the s gene may lead to vaccine failure cavanagh et al., ) . thus, until a universal vaccine can be developed, the ongoing determination of epidemic serotypes and production of new vaccines are key factors in controlling infectious bronchitis (jackwood et al., ) . early on, it was recognized that live-attenuated ib vaccines could be developed by reducing virus virulence by multiple serial passages in - -day-old embryonated eggs, and this method is still widely applied today (cook et al., ; gelb and cloud, ) . commercial ib vaccines are developed by multiple passage of a field isolate in specific-pathogen-free (spf) embryonated eggs until the desired blend of non-pathogenicity and immunogenicity has been achieved (bijlenga et al., ; jackwood et al., ) . we have previously isolated and characterized a virulent ibv strain from -day-old vaccinated broiler chickens in the yunnan province of china. this isolate is genetically similar to most of the prevalent strains of ibv found in china, albeit with increased pathogenicity than previously characterized strains (feng et al., ) . to determine its utility as a vaccine, this yn strain was attenuated by serial passages in -day-old spf embryonated chicken eggs. we tested the safety and efficacy of the attenuated strain in this study. the ibv qx-like strain yn (genbank accession no.: jf ) originated from a h vaccinated broiler flock with respiratory and renal disease, with a death rate of % (feng et al., ) . the virus was purified and passaged by inoculating -day-old spf embryonated chicken eggs via the allantoic sac route. inoculated eggs were incubated for h at c and the allantoic fluid harvested for subsequent passages. eggs that died within h of inoculation were discarded. at every th passage, eggs were examined using reverse transcription polymerase chain reaction (rt-pcr) for the presence of virus. the resulting attenuated yn strain was shorted as ayn and was titrated by inoculating -fold serial dilutions with phosphate-buffered saline (pbs) of the virus stocks into the allantoic sac of -day-old spf embryonated eggs. the embryo % infectious doses (eid ) were determined according to the method of (reed and muench, ) . the homologous (yn strain) and heterologous (sd strain) challenged ibv strains were used for determine the protective efficacy of ayn. the heterologous challenged ibv sd strain was isolated from mass-type vaccinated chickens of shandong province in . based on the phylogenetic analysis, it has the highest homology with lx (genbank accession number: ay ), and has a . % similarity with the yn strain. the eid for strain yn and sd was . eid / . ml and . eid / . ml, respectively. one day or -week-old spf chickens were used to determine the safety and efficiency of ayn. all chickens were kept in isolators at china agricultural university throughout the experiment and the animal rearing facilities were approved by the beijing administration committee of laboratory animals under the leadership of the beijing association for science and technology (approval id syxk [jing] - ). all of the animals used in this study were cared for in accordance with established guidelines, and the experimental protocols were performed with the approval of the animal welfare and ethical censor committee at china agricultural university. a total of sixty -week-old spf white leghorn chickens were assigned to groups of chickens each. the chickens were maintained in isolators with positive pressure in air-conditioned rooms. of the three groups, two were inoculated with ml yn or yn attenuated containing . eid by intranasal and intraocular routes. the third group was maintained as a negative control. birds were housed separately in isolators for chickens with consistent conditions, and food and water were provided ad libitum. to determine the pathology produced by the two ibv strains, all birds were observed daily for days post-inoculation (dpi) for clinical signs. clinical signs indicative of infection with ibv strains consisted of hunched posture, depression, reluctance to move, emaciation, diarrhea and soiled vent were given daily clinical scores: for normal, for mild depression, for severe depressed, for paralysis/prostration, and for death. two chicks from each group were sacrificed at , , , and dpi. gross changes were noted and samples of trachea, kidney, lung, proventriculus, duodenum, and bursa of fabricius were collected for virus detection via real-time quantitative reverse transcription polymerase chain reaction (rt-qpcr) and in % neutral formalin for histopathological examination. necropsies were carried out, and external and internal abnormalities were recorded. lesions in the trachea, kidney, lung, proventriculus, duodenum, and bursa of fabricius were scored as for no lesions, for mild, for moderate and for severe lesions. mean lesion scores (mlss) were calculated for each group. blood samples from surviving chicks in each group were collected at the end of the experiment for antibody detection at dpi. tissues collected above were routinely processed, embedded in paraffin wax, and cut into mm sections. sections were stained with hematoxylin and eosin and examined for lesions using light microscopy. the slides were examined by light microscopy and the lesions were scored according to the severity of the observed lesions. absence of injury was classified as -, while mild, moderate and severe were classified as +, ++, and +++ respectively. duplicate tissue sections were prepared for immunohistochemistry (ihc) to detect viral antigen via the following protocol. briefly, mm tissue sections were subjected to antigen retrieval and then incubated in % normal goat serum in pbs for min to block nonspecific binding. slides were further incubated with chicken anti-ibv hyperimmune serum at : dilutions in pbs for h followed by incubation with a horseradish peroxidaseconjugated rabbit chicken igg for h. the reaction was visualized by the addition of , -diaminobenzidine (dab, sigma, st.louis, mo, usa) for min. finally, sections were counterstained with hematoxylin, air dried, and examined by light microscopy. the trachea was removed under strict aseptic conditions. the tracheal rings were cut into semicircles and quarter-circles. they were immersed in % paraformaldehyde and . % glutaraldehyde in . m phosphate buffer, ph . and fixed for h at c. after primary fixation, samples were then washed with pbs, post-fixed in % osmium tetroxide, washed, and dehydrated in an increasing series of ethanol solutions. dehydrated pellets were embedded in an epoxy resin and sections were cut at nm. finally, sections were placed on copper sieves and stained with uranyl acetate and lead citrate. sections were imaged using a jem- tem microscope (jeol, tokyo, japan). tracheal samples were fixed as described above. after dehydration in an increasingly concentrated series of alcohol, samples were critical point-dried (polaron e ) with carbon dioxide and mounted on aluminum stubs. the mounted samples were gold-coated (polaron e ) and imaged using a jeol jsm- lv (jeol, tokyo, japan). total rna was extracted from trachea, kidney, lung, proventriculus, duodenum, and bursa of fabricius using trizol (invitrogen, carlsbad, ca, usa). cdna was obtained via reverse transcription as previously described [ ] . cdna samples were analyzed using sybr green i real-time rt-qpcr to determine viral loads. primers were designed using primer premier . based on the conserved region of n gene of the yn strain (genbank accession no.: jf ). the -ml pcr mixture was composed of mlof power sybr green pcr master mix (applied biosystems, foster, ca, usa), . ml of each primer, ml of template, and ml of doubledistilled water. real-time pcr was performed on a system sds software (applied biosystems) using the following thermal cycles: a min hot start at c, followed by cycles of denaturation at c for s, annealing at c for s and extension at c for s. all qpcr reactions were carried out in triplicate and repeated at least twice. a linear regression was determined plotting the logarithmic values of the number of copies of plasmid dna containing the insert against the cycle threshold (ct) values to convert ct values into copy numbers. the relative n gene expression was analyzed using graphpad prism version . (graphpad software inc., san diego, ca, usa). a commercial enzyme-linked immunosorbent assay (elisa) kit (idexx laboratories, westbrook, me, usa) was used to measure ibv antibody levels according to the manufacturer's instructions. the viral titer was calculated using the formula provided by idexx. ninety -day-old spf chickens were divided into five groups of birds as follows. group ayn-yn and ayn-sd were vaccinated . bars indicate mean ae sd. the significance was considered as follows: significant at p . (*), highly significant at p . (**), and very highly significant at p . (***). intranasally with . eid ayn and challenged intranasally with homologous field strain yn or heterologous field strain sd at a dose of eid /bird at days post vaccination, respectively. group control-yn and control-sd were left unvaccinated and challenged with yn or sd at the same time. group control-nc was maintained as a negative control. all birds were housed separately in isolators with consistent conditions, and food and water were provided ad libitum. following challenge, all birds were observed daily for clinical signs attributable to ib infection for two weeks. two birds from each group were killed humanely at and day postchallenge (dpc) for gross lesions observation and the evaluation of trachea ciliary activity. ten oral swabs from each group were randomly collected at dpc for the detection of virus shedding via rt-pcr. for pcr, ml taq supermix (transgen, beijing, china) and pmol of each primer were added to ng cdna as template in a total of ml reaction volume. pcr was performed at c for min, followed by cycles of denaturation ( c, s), annealing ( c, s), and polymerization ( c, min), and the postpolymerization step was performed at c for min. a pair of primers (forward: -ttttggtgatgacaagatgaa- ; reverse: -cgcattgttcctctcctc- ), which amplify and detect a -bp fragment of the m gene of ibv was used in the procedure. amplified sequences were analyzed by . % agarose gel electrophoresis. for evaluation of tracheal ciliostasis, three sections of the upper, middle and lower part of the upper, middle and lower part of the trachea respectively, nine rings per bird, were analyzed. the rings were placed in a petri dish containing eagle culture medium with % fetal bovine serum. they were then determined by inverted light microscope at a magnification of Â, observing the degree of integrity and preservation of the ciliary movement of the tracheal epithelial cells. a score of was given if the cilia in the complete tracheal section showed movement; a score of was given if the cilia of - % of the tracheal section showed movement; a score of if the cilia of - % of the trachea showed movement; a score of if the cilia of - % of the trachea section showed movement; and a score of if the cilia of less than % of the trachea section showed movement or no movement at all. for each group, the average ciliostasis score was calculated. data were analyzed using unpaired t-test in graphpad prism version . for windows to obtain a statistical analysis of the differences between ayn and yn inoculated groups. in the case of the serology test and ciliary activity inhibition test, collected data were analysed using two-way anova to see whether there was any significant difference between the different groups. multiple comparison between the groups was performed using tukey's multiple comparisons test; the significance was considered as follows: significant at p . (*); highly significant at p . (**); and very highly significant at p . (***). chicks inoculated with the yn strain showed clinical signs as early as dpi persisting until dpi. diseased chicks showed signs of coughing, sneezing, tracheal and bronchiolar rales, listlessness, huddling, ruffled feathers, increased water intake, and slight watery diarrhea. three birds in the yn inoculated group died during the experiment, and the mortality reached % (fig. a) . no obvious clinical signs or deaths attributable to ibv were observed in either the control group or the yn attenuated group (fig. c) . at necropsy, lesions were detected in the respiratory, urinary, and digestive systems in chickens inoculated with yn, including punctate hemorrhages and catarrhal exudates in the throat and trachea (fig. f) . the kidneys were swollen with urate deposits frequently observed in the tubules and ureters (fig. i) . the lungs of several chickens had congestion and edema (fig. l) . digestive tract lesions consisted of thickening of the wall of the proventriculus and duodenum (figs. o and r), in some cases associated with mucosal congestion. the bursas had heavy exudates of mucus and hemorrhage (fig. u ). chickens inoculated with the attenuated strain had only occasional petechiae in the throat (fig. e) and a slight amount of mucus adhered to the bursa (fig. t) . no gross lesions were observed in any birds in the control group (figs. d, g, j, m, p, and s). number of birds with gross lesions and mean lesion scores at necropsy were showed in table . the microscopic lesions were consistent with the gross lesions described above. moderate to severe lesions were predominantly found between and dpi and were more common in the chickens infected with the wild-type than the attenuated strain (fig. ) . inflammatory cell infiltrates and varying degrees of epithelial desquamation of ciliated cells were detected in the trachea ( fig. b and c) , with hemorrhage and mucosa injury in some chickens. foci of necrosis and intense multifocal nephritis, interstitial congestion, with lymphoplasmacytic infiltration and tubular table number of birds with gross lesions and mean lesion scores at necropsy. group the significance was considered as follows: significant at p . (*), highly significant at p . (**), and very highly significant at p . (***). a yn group infected with yn strain; ayn group infected with attenuated yn strain. there were no lesions in birds in the control group. b no. of birds with lesions out of two birds. c mean lesion scores out of two birds. dilation were scattered throughout the kidneys of yn-inoculated chickens ( fig. e and f) . proteinaceous material was detected in the tubules of a number of yn-inoculated chickens. congestion, hemorrhages, erythrocytes and lymphocytes were frequently detected in the bronchial and air capillary lumina of the lungs. a number of yn-inoculated chickens exhibited bronchiectasis ( fig. h and i) . in the proventriculus, lesions included loss of mucosal epithelium with inflammatory cell infiltration ( fig. k and l) . no histologic evidence of damage was detected in the duodenum and bursa (fig. n , o, q, and r). no ibv-related lesions were observed in the control birds ( fig. a , d, g, j, m, and p). histological scores in different organs of spf chicks inoculated with yn or ayn were showed in table . the presence of ibv antigen was widely detected in all ibv target organs. the cytoplasm of epithelial cells of the mucosa and lamina propria of the trachea, air capillaries, renal tubules, proventriculus, duodenum, and bursa of fabricius all had immunoreactivity. more intense immunoreactivity was noted within the yn-inoculated group than in the attenuated group (fig. ) . to further examine ibv replication in the respiratory tract, we observed the virus particles using transmission electron microscopy. as shown in fig. a and b, typical coronavirus particles were noted in the lumina of tracheas inoculated with each of the virus strains. sem examination of yn-infected tracheas revealed severe lesions in the respiratory mucosa caused by replication of the inoculated virus (fig. e, h, and k) . this was evidenced by severe erosion of the respiratory epithelium with associated inflammation. in the yn attenuated group, relatively mild tracheal damage was observed (fig. d, g, and j) . no lesions were observed in the tracheas of control birds (fig. c , f, and i). mean of the severity index from two chicks: -, no change; +, mild; + +, moderate; +++, severe. fig. . immunohistochemical detection of ibv antigens at dpi. black arrows indicate viral antigen immunoreactivity. scale bar = mm. . . . viral genome copy number in tissues no viral dna was detected in tissues from groups before inoculation and in mock-infected chickens. table shows results of viral dna testing from both infected groups. the proportion of positive samples in the group infected with the yn strain was significantly higher than that of the attenuated group. the time-dependent viral load levels in different organs of infected groups are shown in fig. . the viral dna levels in the spleen, lung, and duodenum in the yn group peaked at dpi and fig. . transmission (a-b) and scanning (c-k) electron micrographs of tracheal epithelium at dpi. a, b: virus particles in the tracheal lumen. c, f, i: control epithelium ( ,  . k, and  . k, respectively). d, g, j: respiratory epithelium from yn attenuated infected chickens ( ,  . k, and  . k, respectively). e, h, k: respiratory epithelium from yn infected chickens ( ,  . k, and  . k, respectively). tissue tropisms in -day-old spf chickens. proventriculus lung tracheal duodenum kidney bursa yn attenuated / / / / / / yn / / / / / / subsequently gradually decreased. no virus was detected in the yn attenuated samples from similar time points. the maximum amount of viral dna was detected at dpi in the tracheas of both groups and the proventriculus and bursa from the yn group. viral loads in the kidneys of both groups increased starting at dpi and peaking at and dpi for the yn and attenuated strains, respectively. the proventriculus and lung samples from the yn attenuated group peaked at dpi, and the duodenum and bursa of fabricius samples from the yn attenuated group had peak viral numbers at dpi. all samples except those from the proventriculus contained more viral copies in the yn group. in addition, the number of copies of viral dna in the trachea and kidneys were significantly greater than other organs at the same time. antibody responses were measured using a commercial elisa kit (idexx laboratories). antibodies against ibv were not detected in any groups before inoculation ( dpi) and were never detected in the mock-infected group. chickens in both infected groups fig. . viral loads in samples after yn or yn attenuated strain infection. the significance was considered as follows: significant at p . (*), highly significant at p . (**), and very highly significant at p . (***). fig. . survival percentage (a) and trachea ciliostasis scores (b) in chickens experimentally challenged with ibvyn and sd strains. bars indicate mean ae sd. the significance was considered as follows: significant at p . (*), highly significant at p . (**), and very highly significant at p . (***). seroconverted by dpi and the mean titers induced by the yn and yn attenuated were and , respectively. the differences in the mean titers between birds inoculated with the yn and yn attenuated strains were significantly different (p < . ) (fig. b) . birds in unvaccinated groups which challenged with the yn or sd strain showed clinical signs and death as early as dpc. diseased chicks showed signs of listlessness, huddling, ruffled feathers, increased water intake, and slight watery diarrhea. during the observation period, the mortality in group control-yn and control-sd were % and %, respectively (fig. a) . no obvious clinical signs or deaths attributable to ibv were observed in ayn vaccinated groups and group control-nc. at necropsy, all euthanized chickens showed the slight hemorrhage with serous catarrhal exudate in the trachea and typical kidney lesions characterized by obvious swelling and urate deposition in the tubules and ureters in group control-yn and control-sd. no gross lesions were observed in any birds in ayn vaccinated groups and group control-nc. inhibition of the ciliary activity was measured at and dpc in the trachea. the group control-yn and control-sd showed a maximum average ciliostasis score of , while the average ciliostasis score in group ayn-yn and ayn-sd were below (fig. b) . the difference of ciliostasis was extremely significant between ayn vaccinated groups and unvaccinated groups (p < . ). detected by rt-pcr method, virus shedding rate was % in group control-yn and control-sd at dpc, while the rates were and % in group ayn-yn and ayn-sd, respectively. no virus was detected in the unchallenged group (control-nc). the best protection against challenge is achieved by a vaccine containing homologous strains (gelb et al., ) . as previously demonstrated in some reports, cross-protection is poor between different serotypes and genotypes of ibv strains. consequently, currently available vaccines cannot provide sufficient protection for heterologous challenge gelb et al., ; liu et al., ) . the s gene of ibv has serotype-specific and neutralization-specific epitopes. serotype evolution and the genetic diversity of ibv are monitored by analysis of the s gene. the majority of qx-like ibv isolates present in the field in china had poor similarity in the s part of the spike protein with vaccine strains, indicating the antigenic differences and large evolutionary distances between vaccine strains and ibv field strains in china (liu et al., ; zhao et al., ) . despite the widespread use of live attenuated ibv vaccine (mass serotype), such as strains h , h , and ma , vaccinated chicken flocks usually fail to achieve complete protection against field virulent ibv challenge. the poultry industry has, in recent years, detected an increasing incidence of outbreaks related to qx-like ibv strains of different serotypes in many countries (abro et al., ; terregino et al., ; valastro et al., ; xu et al., ) . optimal vaccines against circulating ibv strains in china require attenuated vaccines designed from local strains in china. a previous ibv isolate (the yn strain) was passaged times through spf chicken embryonated eggs. as a result of this process, the virus becomes more adapted to the embryo, reflected by more efficient replication and higher pathogenicity for the embryo (data not shown). this attenuated yn strain was inoculated via the oral or oculonasal route into spf chickens to compare the tissue tropism and pathogenicity to its parent strain. after infection, birds in the attenuated group showed no common clinical signs as those observed in birds inoculated with the yn strain. however, gross lesions, although much milder and in fewer organs than those in chicks infected with the yn strain, were still detectible at necropsy. histopathology indicated lower pathogenicity of the attenuated strain, which showed moderate inflammatory infiltration and epithelial degeneration. lesions caused by the yn strain were similar to those described in the previous report, which characterized it as a virulent qx-like ibv strain (feng et al., ) . in general, the systemic distribution of yn antigen was demonstrated by ihc staining and was most abundant in the respiratory and urinary systems. the epithelia of the trachea and alimentary tract of the yn-infected group were strongly immunoreactive to ibv antigens compared with the attenuated group. the presence of virus particles in both the infected groups post-inoculation indicates that a significant viral infection was delivered to the experimental spf chickens and suggests that ciliated respiratory tract cells play a significant role in viral replication. these lesions appeared to damage the host cell, leading to the loss of cellular functions and decreasing immunity, increasing the opportunities for secondary or multiple infections (davies et al., ). however, the increased antigen production and cilia loss in the yn strain supports it being more pathogenic than the attenuated strain. in terms of the real-time rt-qpcr examination, both strains were detected in respiratory and non-respiratory tissues, including the kidney, trachea, lungs, proventriculus, duodenum, and bursa of fabricius, indicating viral replication in these organs; however, this was relatively limited in the birds infected with the yn attenuated strain. we also observed that the viral rna levels in the tracheas of birds in both groups declined after the peak, then reached another small peak. loss of ciliated columnar epithelium, and, presumably, the associated viral replication in those cells, is a common lesion with ibv infection (callison et al., ; geerligs et al., ) . the small peak may be related to the recovery of the epithelial cells in the upper-respiratory tract after extensive damage at the early infection period. among the tissues examined by histopathology, the damage was most severe in the trachea and kidney, the primary target organs of ibv (uenaka et al., ) . the viral loads were highest in these two organs. therefore, the higher the viral load, the more severe the histologic damage, and the stronger the positive signal. the yn attenuated strain was able to induce a higher humoral antibody response following inoculation. this suggests that this strain may be useful in vaccination programs under field conditions to reduce the economic losses caused by qx-like ibv infections on commercial layer and broiler farms. information regarding vaccine efficacy against circulating infectious bronchitis virus strains of china will provide valuable knowledge for the poultry industry when considering vaccine types. therefore we invested the efficiency of the yn attenuated strain to determine if it could provide protection against the homologous and heterologous virulent strains. the results indicated that the strain ayn protected spf birds against morbidity and mortality from challenge with the highly virulent strains yn and sd. assessment of immunity to challenge with ibv is most commonly done by removal of trachea at or days after challenge followed by either quantification of ciliostasis cook et al., ) or detection of viral shedding. there was a clear decrease in ciliostasis scores in the yn attenuated vaccinated groups compared with the unvaccinated groups after challenge with homologous and heterologous virulent strains, indicating the yn attenuated strain could protect the respiratory tract efficiently. in addition, virus shedding in the infected chickens is a big challenge to the control of infectious bronchitis virus. it would encourage viral spread among chickens and support virus persistence. thus the reduction of virus excretion should be taken into consideration when choosing the vaccine type and program. in our experiment, a significant decrease was observed in the vaccinated groups after challenge. we also noticed that the virus shedding inhibition in yn challenged group was more significant than the sd challenged group, which indicating that the vaccine strain could provide a better protection against the challenge with homologous strains. the genetic mechanism responsible for loss of pathogenicity is still not well understood. the s gene is responsible for induction of protective immunity, and small differences in s contribute to poor cross protection . it has been shown that a number of amino acid residues in s contribute to ibv attenuation (cavanagh et al., ; liu et al., ) . besides the s subunit, the nucleocapsid protein can also induce protective immune responses in chickens (boots et al., ) . previous studies have shown that amino acid substitutions within the replicase gene may result in attenuation following serial passage in embryonated eggs (armesto et al., ). further genetic investigation will be necessary to determine the key mutations responsible for the attenuation in this sample. moreover, the stability of the vaccine and its tendency to revert should be investigated in the future study before administration in the field to ensure the security and reliability of this vaccine. the attenuated yn strain showed lower replicate ability and decreased pathogenicity than its parent strain, with an efficacious protection against homologous and heterologous field strains, indicating the potential to become an alternative vaccine candidate for controlling ib infections in china. characterization and analysis of the full-length genome of a strain of the european qx-like genotype of infectious bronchitis virus the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review induction of anti-viral immune responses by immunization with recombinant-dna encoded avian coronavirus nucleocapsid protein development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronavirus avian infectious bronchitis virus relationship between sequence variation in the s spike protein of infectious bronchitis virus and the extent of cross-protection in vivo coronavirus ibv: virus retaining spike glycopolypeptide s but not s is unable to induce virusneutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection relationship between sequence variation in the s spike protein of infectious bronchitis virus and the extent of cross-protection in vivo infectious bronchitis variation in the spike protein of the /b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus the long view: years of infectious bronchitis research an assessment of opportunities to dissect host genetic variation in resistance to infectious diseases in livestock virulent avian infectious bronchitis virus, people's republic of china efficacy and safety of an attenuated live qx-like infectious bronchitis virus strain as a vaccine for chickens effect of serial embryo passage of an arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens s gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel development of attenuated vaccines from taiwanese infectious bronchitis virus strains attenuation, safety, and efficacy of an infectious bronchitis virus ga serotype vaccine data from years of molecular typing infectious bronchitis virus field isolates infectious bronchitis virus: s gene characteristics of vaccines used in china and efficacy of vaccination against heterologous strains from china s gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryopassaged, attenuated derivatives complete genome sequence analysis of a predominant infectious bronchitis virus (ibv) strain in china infectious bronchitis virus: immunopathogenesis of infection in the chicken a simple method of estimating fifty per cent endpoints pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the ma and / serotypes intracloacal infection with avian infectious bronchitis virus qxtype infectious bronchitis virus in commercial flocks in the uk isolation and identification of glandular stomach type ibv (qx ibv) in chickens isolation and identification of four infectious bronchitis virus strains in china and analyses of their s glycoprotein gene molecular characterization of an infectious bronchitis virus strain isolated from northern china in comparison of the pathogenicity of qx-like, m and /b infectious bronchitis strains from different pathological conditions this study was supported by beijing agriculture innovation consortium of poultry research system. the authors declare no conflict of interest. key: cord- -vq fh authors: stadejek, tomasz; larsen, lars e.; podgórska, katarzyna; bøtner, anette; botti, sara; dolka, izabella; fabisiak, michał; heegaard, peter m.h.; hjulsager, charlotte k.; huć, tomasz; kvisgaard, lise k.; sapierzyński, rafał; nielsen, jens title: pathogenicity of three genetically diverse strains of prrsv type in specific pathogen free pigs date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: vq fh studies from eastern european countries proved that porcine reproductive and respiratory syndrome virus type (prrsv- ) harbours high genetic diversity and that genetically divergent subtypes – circulate in this area. in the present study, we compared the pathogenicity of two different prrsv- subtype strains and a strain representing prrsv- subtype . four groups of -week-old specific pathogen free pigs were either infected with subtype strain ili , subtype strain or bor , subtype strain , or mock inoculated. the most pronounced clinical signs were observed in pigs infected with bor . pigs from both subtype strain infected groups exhibited significantly elevated mean body temperatures on dpi compared to the other two groups, the difference remaining significant up to dpi for the bor group, only. the pigs in the latter group also displayed significantly highest levels of early viremia together with the most rapid app response. overall, the results indicated that bor strain can be considered a highly pathogenic strain, similarly to subtype strains lena and su -bel, while the virulence of the other subtype strain ili was intermediate between bor and subtype strain. porcine reproductive and respiratory syndrome virus (prrsv) belongs to the arteriviridae family within the order nidovirales (faaberg et al., ) . prrsv is the cause of severe respiratory and reproductive disease in swine worldwide. the virus emerged as a swine pathogen in north america and europe nearly simultaneously in the period - (zimmerman et al., . quickly, nucleotide sequence comparisons of the prototypical isolates lelystad virus and vr- revealed that the european (eu) and north american (na) isolates were only distantly related. later, the eu and na genotypes were officially designated as type (prrsv- ) and type (prrsv- ), respectively (faaberg et al., ) . following the expansion of our understanding of arterivirus evolution and discovery of many new genera in the family of arteriviridae, it was recently proposed to separate prrsv- and prrsv- into two different species (kuhn et al., ) . the results of bioinformatic analysis suggested that prrsv existed at least years back in time (forsberg, ) , and the most recent common ancestor of prrsv- emerged in - , or even earlier (nguyen et al., ) . initially, prrsv- was thought to be genetically homogenous, but studies from italy, lithuania, latvia, belarus and the russian federation established that prrsv- is even more diverse than prrsv- (forsberg et al., ; le gall et al., ; stadejek et al., stadejek et al., , stadejek et al., , stadejek et al., , suarez et al., ) . previously, it has been suggested that the high genetic diversity in prrsv- orf and orf sequences warranted definition of subtypes (stadejek et al., (stadejek et al., , . currently, subtypes (lelystad viruslike), , and are recognized and tentative evidence has been found for potential additional subtypes (stadejek et al., ) . in contrast, multiple genetic clades have been defined in prrsv- but they comprise a degree of sequence diversity found in prrsv- subtype lineages alone (shi et al., ) . the genetic subtyping or clustering the strains of prrsv may be affected by genetic recombination between the strains of the same genotype, and the classification of viruses based on the analysis of small genetic fragments should not be overrated (brar et al., ; franzo et al., ; lu et al., ; martin-valls et al., ; shi et al., ; zhao et al., ) . it has been shown that, in terms of virulence, antigenic characteristics and immunological responses following experimental infection, the belarusian lena subtype strain differed from subtype strains from western europe being generally more pathogenic for pigs (karniychuk et al., ; weesendorp et al., a weesendorp et al., , . later, another subtype belarusian strain su -bel, which was characterized in vitro and in in vivo challenge experiments also proved to be significantly more virulent than western european strains (morgan et al., (morgan et al., , salguero et al., ) . at present, no data are available about the biological characteristics of east european prrsv- strains from other genetic subtypes, e.g. of subtype strains, which compose the second most prevalent cluster of prrsv- , present from lithuania and belarus in the west, to khabarovsk krai in the russian far east in the east (stadejek et al., ) . the aim of the present study was to compare the pathogenicity of different prrsv- strains by inoculation of groups of specific pathogen free pigs. based on orf sequence analysis, two strains from belarus and russian federation and one strain from denmark were classified as subtypes and , respectively. three prrsv- strains isolated in porcine alveolar macrophages (pam) cultures derived from prrs-negative pigs were used in this study. the strain was isolated from a danish pig in (botner et al., ) . the strain ili was isolated in from lung tissue of a russian weaner pig. the strain bor was isolated in from lung tissue of a belarusian pig that died with respiratory disease symptoms. in order to prepare the various inocula, the strains were passaged and titrated in pam cultures. the inoculum of was a th passage (titre of . log tcid /ml). the inoculum of ili was a rd passage (titre of . log tcid /ml). the inoculum of bor was a nd passage (titre of . log tcid /ml). the challenge experiment performed at the bsl animal facilities of dtu national veterinary institute was carried out in accordance with the danish legislation on animal experiments (lbk nr (lbk nr - / / and eu regulations on the use of laboratory animals for research. the pigs used in the experiment were procured from the institute's high health pig production herd, which was tested free from infection with the following pathogens: encephalomyocarditis virus, hepatitis e virus, porcine circovirus type and type viruses, porcine cytomegalovirus, porcine epidemic diarrhoea virus, porcine parvovirus type , porcine respiratory coronavirus, prrsv- and prrsv- , influenza a virus, transmissible gastroenteritis virus, actinobacillus pleuropneumoniae, bordetella bronchiseptica, brachyspira hyodysenteriae, brachyspira pilosicoli and brucella spp., using in-house standard diagnostic methods. twenty-eight -week-old pigs were randomly divided into four groups that were housed separately. after weeks of acclimatization, the pigs (now weeks of age) were inoculated intranasally (i.n.) with ml of virus inoculum in each nostril. group (pigs - ) was inoculated with the strain , group (pigs - ) was inoculated with ili and group (pigs - ) was inoculated with bor . group (pigs - ) was mock-inoculated with ml of eagle's medium in each nostril and served as a prrs negative control group. individual pigs were subjected to daily clinical examination and rectal body temperatures were recorded starting from − day post infection (dpi). in order to obtain a semi-quantitative measure for comparison of clinical disease between the groups, a scoring system developed by mittelholzer et al. ( ) and adapted to prrs experiments was applied. all pigs were evaluated for overall well-being, respiration, eye disorders and appetite. each parameter was scored as (normal condition), (mild disorder), (moderate disorder) or (severe disorder). the scores for individual pigs were added up to a cumulative clinical score (cs) per day. non-stabilized blood samples were collected from the anterior vena cava in ml vacutainers (venoject; terumo europe, leuven, belgium) on − , , , , , , dpi and at euthanasia. serum was isolated (left to coagulate for min and centrifuged at rpm for min at °c) and stored in aliquots at − °c for subsequent real time rt-pcr analysis, or at − °c for antibody and acute phase protein (app) measurements. in parallel to the blood samplings, nasal swab samples were collected on the same days from right nostrils. the swabs were placed in ml pbs (ph . ) and stored at − °c until further analysis with real time rt-pcr. euthanasia of the majority of the pigs was performed on dpi (pigs , , , and ), dpi (pigs , , , , , ) , or dpi (pigs , , , , , ) by intravenous injection of pentobarbiturate ( mg/kg) followed by exsanguination by cutting arteria axillaris. in order to get additional information on the dissemination of virus in individual pigs during the course of infection, pigs representing the various groups (pigs , , , and ) were euthanized on dpi. at necropsy, all pigs were subjected to macroscopic evaluation of respiratory tract lesions, and lung tissue samples were collected for real time rt-pcr and histopathological analysis. lung sections for real time rt-pcr were stored in rnalater according to instruction from the supplier (qiagen) and sections for histopathological evaluation were fixed in % buffered formalin. total rna was extracted from μl serum and nasal swabs in pbs with qiasymphony rna kit automated on qiasymphony sp extraction robot with the protocol ct v , according to instructions provided by the supplier (qiagen, denmark). lung tissue samples stored in rnalater (qiagen) were initially prepared as a % homogenates in rlt buffer (qiagen) containing % β-mercaptoethanol (sigma-aldrich) by homogenization on tissuelyser ii (qiagen, denmark) at hz for min and clarified by centrifugation for min at , rpm. total rna was extracted from μl lung tissue homogenate using rneasy mini kit (qiagen) with the large sample protocol v automated on the qiacube (qiagen) according to instructions from the supplier. the rna was stored at − °c until use. in order to quantify prrsv load in sera, nasal swabs pcr primers and a probe were designed based on orf sequences of , ili and bor . the sequences of the primers and the probe were as follows: fw: ′-ttygggttcachgtcgcag- ′; rev: ′-gaccttcgatarttcgggag- ′; probe: ′-fam-cagagcgcgaacggagaakcgcg-bhq - ′ and were synthesized by eurofins genomics (germany). the pcr reactions contained nm of each primer and nm probe, x qiagen onestep rt-pcr buffer, . mm dntp each nucleotide, μl qiagen onestep rt-pcr enzyme mix and μl rna in a total volume of μl. amplifications were performed on rotor-gene q (qiagen ® ) with the following temperature profile: min at °c for reverse transcription followed by min at °c, and cycles of s at °c, s at °c and s of °c. fluorescent signals were collected during the extension step at each cycle in the green channel and analyzed with rotor-gene q software version . . (qiagen) setting ntc threshold at % and the normalized fluorescence threshold limit at . for cycle threshold (ct) value determination, starting normalization from cycle . samples were tested in duplicates and were considered positive when both replicates had ct below . the efficiency of pcr amplification of all three prrsv strains was between and %. quantification of viral load in experimental samples was performed against a standard curve constructed from a -fold dilution series of . log tcid /ml of the strain. the standard curve had a pcr efficiency of % (r = . ; slope − . ) covering dilution steps corresponding to − . e+ tcid /ml equivalents (equal to . - . log tcid equivalents). antibodies in sera from all pigs were analysed using a prrsv genotype discriminating immunoperoxidase monolayer assay (ipma) (sorensen et al., ) and a genotype discriminating blocking elisa (sorensen et al., ) . for ipma, the serum was initially diluted : and then tested using a fivefold dilution series ( : - : ). the results were expressed as the highest dilution generating a positive signal (titre). in elisa, the serum samples were tested diluted : and the results were expressed as blocking percentage (od%). a sample was considered positive if the od% was below . the concentration of haptoglobin (hp) in serum was determined by a sandwich elisa using an in-house mouse anti-porcine hp monoclonal antibody as catching antibody and a commercial rabbit anti-human hp detection antibody (dako) as previously described (sorensen et al., ) . the serum concentration of c-reactive protein (crp) was analysed by a sandwich elisa using dendrimer-coupled cytidine diphosphocholine (a crp-binding ligand) in the coating layer as described by heegaard et al. ( ) employing polyclonal rabbit antihuman antibodies with cross-reactivity towards porcine crp (dako) followed by peroxidase-conjugated goat anti rabbit antibody for detection (dako). lung sections fixed in % buffered formalin were dehydrated through a graded ethanol and xylene baths and embedded in paraffin wax. sections of - μm were stained with haematoxylin and eosin (he) and microscopic lesions were scored as , no lesion; , mild; , moderate or , severe (table ). the evaluation included interstitial pneumonia infiltration of eosinophils, and hyperplasia of lymphoid follicles. interstitial changes were evaluated at magnification x (objective lens) and x (eyepiece). infiltration of eosinophils was assessed by the average number of cells observed in different highpower fields (hpf), using a magnification of x (objective lens) and x (eyepiece) on the area of about . mm (area of one field of view). hyperplasia of lymphoid follicles in lung parenchyma and balt was observed at magnification of x (objective lens) and x (eyepiece) in one field of view. only visible and activated lymphoid follicles were counted. the details of scoring are presented in table . the microscopic evaluation was performed in a blinded fashion using a standard light microscope olympus bx and cellsens software (olympus). statistical analysis to compare mean viremia, nasal shedding and acute phase proteins concentration between groups was performed at each sampling point using a one-way anova followed by post-hoc tukey's test. if the assumptions of normality or equality of variances were not fulfilled (evaluated by shapiro-wilk and levene's test respectively) non-parametric kruskal-wallis anova test was applied. clinical scores and microscopic lesions scores were compared based on kruskal-wallis anova test. calculations were performed with statistica (statsoft) software. differences were considered statistically significant at p < . . analysis of body temperature was performed using graphpad in stat version . (graphpad software, san diego, ca). student's t-test was used for comparison between means of the infected groups and the control group. no clinical signs were observed in the uninfected control pigs. in groups and , pigs showed mild lethargy on a few days, only (dpi , and , respectively) reflected by cs values of . all pigs in group exhibited varying degrees of clinical signs characterized by mild lethargy, increased respiratory rate, conjunctival hyperaemia and reduced feed-intake. starting from dpi , cumulative cs values of to were observed for an day period with highest scores ( - ) observed in pigs, all infected with bor . between dpi and , cumulative cs in group was significantly higher compared to other groups (p < . ). body temperatures (bt) of all pigs remained within the range of . °c to . °c on dpi - - , and bt did not exceed . °c in any of the controls post inoculation. thus, bt higher than . °c were considered to represent fever. in group , only pig had fever of . °c on dpi. pigs from groups and exhibited significantly elevated mean bt on dpi compared to pigs from group and (p < . ) (fig. ) . hereafter, the mean bt of group was higher than the controls but not statistically significant at any time point. in group , fever with bt ranging between . °c and . °c was recorded in individual pigs for to days in the period from to dpi. two pigs ( and ) in group did not show elevated bt. in group , all pigs had fever of . °c to . °c between dpi and dpi. the duration of fever period was to days for pigs ( and ) and to days for pigs ( , , , ) with the highest bt recorded for the latter pigs. the mean bt for group pigs were significantly higher compared to the controls from dpi to , the highest difference seen on dpi (p < . ) prrsv was detected in serum in all infected pigs at dpi and the viremia persisted until dpi. all pigs from groups and and one pig from group had low level of viremia at dpi (fig. ) . viremia was significantly higher in group infected with bor , at dpi and dpi, when in pigs and it reached about × tcid genomic copy equivalent/ml. on dpi viremia in groups and was significantly higher than in group (p < . ). no viremia was detected in group . prrsv could not be detected in nasal swab samples from any of the control pigs but in most pigs in the infected groups at , and dpi (not shown). at dpi only out of pigs from group had virus positive nasal swab samples compared to and pigs in groups and , respectively. in pig from group virus was detected also at and dpi. there were no significant differences between the groups in the virus detection levels in nasal swabs, the highest observed at and dpi. prrsv was detected in lungs from all inoculated pigs tested with varying ct-values of . ± . (group ), . ± . (group ) and . ± . (group ). however, the differences between the groups were not significant. prrsv could not be detected in lung tissue samples from control animals. all animals seroconverted at - dpi in both the elisa and ipma tests. no differences were seen in the serological profile between the three different strains used for challenge (data not shown). an acute phase protein crp response was observed as early as at dpi, peaking at dpi , in all prrsv infected groups (fig. a) . globally, during the study the mean crp concentration ranged from . μg/ml ( ± . ) to . μg/ml ( ± . ). bor infected pigs showed earliest increase in crp levels. in ili infected pigs, the increase of crp level was slower and peaked at slightly higher level than in bor infected pigs. hp levels ranged from to . mg/ml ( ± . ). an remarkably early response at dpi only in bor infected pigs (p < . ) (fig. b) . no gross lesions of the lungs were observed during necropsy at , , or dpi. microscopically, varying levels of lung lesions were observed in all pigs except one control animal ( table ). the lesions included proliferation of pneumocytes type ii, mononuclear inflammatory cell infiltration (mainly lymphocytes and macrophages) and thickening of alveolar septa. in addition, areas of fibroblast proliferation and fibrosis were observed in lung parenchyma ("honeycomb" pattern). the most affected were lungs of pigs (necropsied at dpi), and (necropsied at dpi), infected with the strain bor , where fibrosis and cellular infiltration were particularly severe, and alveolar lung structure was completely lost. similar severity was observed in pig (necropsied at dpi) infected with the ili strain. fig. . body temperature in pigs infected with prrsv- strain (group ), strain ili (group ) and strain bor (group ), and uninfected control pigs (group ). data are expressed as mean ± sd. statistically significant difference at dpi between groups , and groups , as well as differences between group and remaining groups between dpi - were marked with asterisk (p < . ). fig. . viremia in pigs infected with prrsv- strain (group ), strain ili (group ) and strain bor (group ), and uninfected control pigs (group ). data are expressed as mean ± sd. different superscripts denote significant statistical differences (p < . ). t. stadejek et al. veterinary microbiology ( ) - greater differences existed between the experimental groups in regard to eosinophil infiltration, where again the lungs from the bor infected pigs , and , as well as the ili pig were most affected. eosinophils accumulated around vessels and in the areas with severe fibrosis of the lung parenchyma. occasionally, degranulated eosinophils were also visible. also, a small amount of eosinophils was observed in healthy pigs. however, they were evenly distributed and degranulation was not observed. hyperplasia of lymphoid follicles was higher in bor infected pigs than in and ili infected pigs. the highest score was noted in lungs from bor infected pigs , and , where more than activated lymphoid follicles were observed. in particular, numerous lymphoid follicles were observed in lungs with a high degree of fibrosis and severe cellular infiltration. the most severe interstitial pneumonia, eosinophil infiltration and hyperplasia of lymphoid follicles were observed in the bor infected pigs and that were necropsied at dpi. in the present study, we compared clinical signs, virological parameters, seroconversion, app response and histopathological lesions in pigs infected with either the danish strain from subtype , the russian strain ili from subtype , and the belarusian strain bor from subtype , all belonging to prrsv- . the results indicated that bor strain can be considered a highly virulent strain, similarly to subtype strains lena and su -bel (karniychuk et al., ; morgan et al., ; weesendorp et al., a weesendorp et al., , . the infection with the danish strain did not cause significant clinical signs which is in agreement with the results of earlier studies on prrsv- subtype strains from the netherlands, united kingdom, belgium or korea (frydas et al., ; han et al., han et al., , karniychuk et al., ; morgan et al., ; nielsen and botner, ; weesendorp et al., a) . the mild clinical signs combined with the slightly elevated body temperatures including fever for a few days in pigs infected with the russian ili strain indicate that this is more virulent than , but less virulent than bor , since the pigs infected with the latter strain exhibited more clinical signs, significantly higher body temperatures and more days with fever, together with earlier and higher level of viremia. clinical symptoms of prrsv infection in the field depend on a variety of modifying factors such as differences in genetic susceptibility, environmental factors, immune status, management, virus strain differences or coinfections with other pathogens (zimmerman et al., ) . . concentration of c-reactive protein (a) and haptoglobin (b) in serum from pigs infected with prrsv- strain (group ), strain ili (group ) and strain bor (group ), and uninfected control pigs (group ). data are expressed as mean ± sd. different superscripts denote significant differences (p < . ). t. stadejek et al. veterinary microbiology ( ) - the generally mild clinical disease observed in the present study likely resulted from the use of pigs with a very high sanitary status thus avoiding exaggeration of secondary infections contributing to more overt clinical signs observed in conventional pigs. viremia is one of the measures of prrsv virulence and immediately easier to compare between experiments than clinical signs. in the present study, using quantitative real time rt-pcr, viremia was detected in all challenge groups from dpi to the end of sampling at dpi. at this stage, all pigs infected with and bor were viremic compared to only one pig infected with ili strain. the significantly higher level of viremia detected in pigs infected with bor at and dpi supports the higher virulence of this strain. however, the occurrence of viremia was analysed on predetermined days only, and as such the picture of viremia may not represent its complete dynamics. the analysis of app response performed in the present study provided an important insight into one of the less studied aspects of prrsv infection. apps are part of the systemic acute phase response and important components of the innate immune system. the concentration of app in serum is altered in animals subjected to inflammation, infection or stress. in swine, hp and crp are the main apps. hp is a major antioxidant protective agent mediating removal of free hemoglobin (alayash et al., ) . crp participates in the systemic response to inflammation. its plasma concentration increases during inflammatory states, within hours after tissue injury or infection (black et al., ) . the present work showed that the level of app response can be variable towards different prrsv- strains, likely reflecting the differences in their virulence. similar differences in apps profiles displayed by four prrsv- , subtype strains were recently described by saco et al. ( ) who identified strong correlation between app (in particular hp) concentration in serum and the severity of clinical signs. clinical scores in pigs exposed to eu- prrsv- strain inducing highest hp response, and to ja prrsv- strain inducing highest crp response, where highest (saco et al., ) . in our study, bor infected pigs showed the earliest increase in crp levels but both bor and ili infected pigs had significantly higher levels of crp at dpi compared to pigs infected with the strain. striking differences were observed in hp levels where bor pigs showed an unusual early response at dpi while the response in other groups was absent or minimal (fig. ) . it corresponds well with the previously reported greater and more common responsiveness of crp than hp towards prrsv infection (heegaard et al., ; saco et al., ) . this observation indicates that bor infection is able to stimulate a rapid hp response, possibly by activating neutrophils to release pre-stored hp. such a mechanism has been described for hp (theilgaard-monch et al., ) but not for crp. this finding is another indication that bor is more virulent than ili (and ). it was proposed that the variability in clinical and app response may be related to the different abilities of different prrsv isolates to impact interferon and other cytokines production. in prrsv infections, increased il- levels have been described, while the data on il- β and tnf-α are conflicting (borghetti et al., ; darwich et al., ; weesendorp et al., b) . taking into account that the synthesis of apps is mainly driven by these three pro-inflammatory cytokines, it is expected that more virulent isolates stimulating higher levels of cytokines will also induce stronger acute phase response and more severe clinical symptoms, as in the case of bor , and to a lesser extent ili . results of pathology evaluation were limited in this study as pigs were only necropsied at , , or dpi, rather late in the course of infection at stages when advanced recovery from clinical disease was observed. nevertheless, despite wide range of lesions in each of the experimental groups, bor infected pigs (especially those necropsied at dpi) had generally most evident microscopic lung lesions. as with clinical signs, the lack of gross lesions together with the minor microscopic lesions could result from lack of co-infections in the used pigs of very high sanitary status. the role of eosinophils in lung pathology remains unclear, but the long term consequences of eosinophil activation can result in increased pathological changes in the lungs. these cells produce eosinophilic cationic protein (ecp), which can damage the rna virus, they are also able to present antigens to other cells and capable of phagocytosis and cytotoxicity (giembycz and lindsay, ; gleich et al., ; jacobsen et al., ) . on the other hand, eosinophils may modify inflammation in the lungs through cytokines release and they can also promote fibrosis of the lungs by producing factors participating in transformation of fibroblasts into fibrocytes and myofibrocytes (akuthota et al., ; giembycz and lindsay, ; jacobsen et al., ) . in general, the amount of eosinophils in lung tissues was small but highest in pigs infected with bor strain. in summary, our results suggest that the prrsv- strain bor is more virulent than ili (and ), showing a more rapid and more quickly developing infection, as also supported by the detected early crp and hp responses. the results indicate that the strain bor can be considered a high pathogenic prrsv- virus, similarly to previously evaluated belarusian lena and su -bel (karniychuk et al., ; morgan et al., ) . pathogenicity of bor for conventional pigs, as well as a deep genetic characterization of this strain, remains to be assessed. more efforts should be made in europe and elsewhere to study complete genomes of prrsv- and to study the genetic characteristics of unusually virulent strains, especially from eastern european genetic subtypes. assistance. likewise, bent eriksen and co-workers are acknowledged for taking care of the animals. eosinophils as antigen-presenting cells in allergic upper airway disease haptoglobin: the hemoglobin detoxifier in plasma c-reactive protein cytokine expression, glucocorticoid and growth hormone changes after porcine reproductive and respiratory syndrome virus (prrsv- ) infection in vaccinated and unvaccinated naturally exposed pigs isolation of porcine reproductive and respiratory syndrome (prrs) virus in a danish swine herd and experimentalinfection of pregnant gilts with the virus genomic evolution of porcine reproductive and respiratory syndrome virus (prrsv) isolates revealed by deep sequencing certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology family arteriviridae the genetic diversity of european type prrsv is similar to that of the north american type but is geographically skewed within europe divergence time of porcine reproductive and respiratory syndrome virus subtypes phylodynamic analysis of porcine reproductive and respiratory syndrome virus (prrsv) in italy: action of selective pressures and interactions between different clades different clinical, virological, serological and tissue tropism outcomes of two new and one old belgian type subtype porcine reproductive and respiratory virus (prrsv) isolates pharmacology of the eosinophil biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease pathogenesis of korean type (european genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs comparative pathogenicity of three korean and one lelystad type porcine reproductive and respiratory syndrome virus (pan-european subtype ) isolates in experimentally infected pigs a robust quantitative solid phase immunoassay for the acute phase protein c-reactive protein (crp) based on cytidine '-diphosphocholine coupled dendrimers optimal combinations of acute phase proteins for detecting infectious disease in pigs eosinophils: singularly destructive effector cells or purveyors of immunoregulation pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate molecular variation in the nucleoprotein gene (orf ) of the porcine reproductive and respiratory syndrome virus (prrsv) re-emerging of porcine respiratory and reproductive syndrome virus (lineage ) and increased pathogenicity after genomic recombination with vaccine variant analysis of orf and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes and retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains increased pathogenicity of european porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance pathology and virus distribution in the lung and lymphoid tissues of pigs experimentally inoculated with three distinct type prrs virus isolates of varying pathogenicity a bayesian phylogeographical analysis of type porcine reproductive and respiratory syndrome virus (prrsv) hematological and immunological parameters of / -month old pigs infected with prrs virus c-reactive protein, haptoglobin and pig-major acute phase protein profiles of pigs infected experimentally by different isolates of porcine reproductive and respiratory syndrome virus host-pathogen interactions during porcine reproductive and respiratory syndrome virus infection of piglets molecular epidemiology of prrsv: a phylogenetic perspective evaluation of a blocking elisa for screening of antibodies against porcine reproductive and respiratory syndrome (prrs) virus blocking elisa's for the distinction between antibodies against european and american strains of porcine reproductive and respiratory syndrome virus the porcine acute phase protein response to acute clinical and subclinical experimental infection with streptococcus suis identification of radically different variants of porcine reproductive and respiratory syndrome virus in eastern europe: towards a common ancestor for european and american viruses porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe molecular evolution of prrsv in europe: current state of play phylogenetic relationships of european strains of porcine reproductive and respiratory syndrome virus (prrsv) inferred from dna sequences of putative orf- and orf- genes haptoglobin is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation comparative analysis of immune responses following experimental infection of pigs with european porcine reproductive and respiratory syndrome virus strains of differing virulence phenotypic modulation and cytokine profiles of antigen presenting cells by european subtype and porcine reproductive and respiratory syndrome virus strains in vitro and in vivo lung pathogenicity of european genotype strain porcine reproductive and respiratory syndrome virus (prrsv) differs from that of subtype strains importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china porcine reproductive and respiratory syndrome virus (porcine arterivirus) the research leading to these results has received funding from the european community's seventh framework programme (fp , (fp , - key: cord- -zpf bla authors: holland, robert e; wilson, richard a; holland, margo s; yuzbasiyan-gurkan, vilma; mullaney, thomas p; white, david g title: characterization of eae(+)escherichia coli isolated from healthy and diarrheic calves date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: zpf bla strains of escherichia coli from healthy and diarrheic calves were screened by pcr for the eae (intimin) gene and shiga toxin genes (stx). each eae(+) and eae/stx(+) strain was examined for antimicrobial susceptibility, enterohemolysin activity, and the somatic o antigen was determined. an immunoassay was used to detect shiga toxin antigens for the eae/stx(+)e. coli. significantly more (p = . ) of the healthy calves carried eae(+) and eae/stx(+)e. coli in their feces when compared to strains from diarrheic calves. moreover, shiga toxin antigens were detected significantly more (p = . ) often among the eae/stx(+) strains from healthy calves when compared to eae/stx(+) strains from diarrheic calves. however, significantly more (p = . ) of the eae(+) and eae/stx(+) strains from diarrheic calves were resistant to at least one of the antimicrobials tested, and the strains from diarrheic calves had a significantly (p = . ) higher rate of antimicrobial resistance to at least two different antimicrobial classes. no significant difference (p ≥ . ) was detected among the eae(+) and eae/stx(+) strains from healthy and diarrheic calves for enterohemolysin production. serogroups o-negative, o , o , and o were predominate among both healthy and diarrheic calves. calf strains of escherichia coli possessing the eae gene and having the capacity to induce the attaching and effacing lesion are designated as attaching and effacing e. coli (aeec) (moon et al., ) . intimin, a kda outer membrane protein encoded by the eae gene, mediates adherence of aeec to intestinal epithelial cells; an event critical for the formation of the attaching and effacing lesion (jerse and kaper, ; donnenberg and kaper, ) . thus, the pathophysiologic mechanism by which aeec cause the attaching and effacing lesion in the intestinal tract of calves may follow the three-stage model proposed for human strains of enteropathogenic e. coli (epec) (donnenberg and kaper, ; donnenberg et al., a, b) . the three-stage model for the attaching and effacing lesion consists of localized adherence, signal transduction, and intimate adherence. there is an initial nonintimate attachment of the bacteria to intestinal epithelial cells leading to effacement of the underlying microvilli, followed by intimate attachment, and then the accumulation of cytoskeletal proteins beneath the areas of bacterial attachment. based on expression of intimin, shiga toxins, and the ability to elicit the attaching and effacing lesion, some bovine aeec strains are equivalent to human enterohemorrhagic e. coli (ehec) (levine, ; knutton, ) . thus, two classes of bovine aeec have been described (mainil et al., ) . one class, in addition to carrying the eae gene, carry stx genes; and the second class do not carry stx genes. in calves, the eae gene and intimin have a defined role in causing the attaching and effacing lesion, whereas shiga toxins do not appear to have a role in causing the attaching and effacing lesion (tzipori et al., ; hall et al., ) . further, the importance of shiga toxins in calf diarrhea and systemic disease is not well defined. in humans, shiga toxins are an essential virulence attribute of ehec associated with severe systemic disease such as hemolytic uremic syndrome (karmali, ; tesh and o'brien, ) . in pigs and rabbits, shiga toxins have a role in inducing vascular lesions and have a direct effect on the intestinal epithelium (gyles, ; nataro and kaper, ) . numerous studies have documented the importance of aeec in calf diarrhea (chanter et al., (chanter et al., , sherwood et al., ; hall et al., hall et al., , moxley and francis, ; mohammad et al., mohammad et al., , pospischil et al., ; blanco et al., blanco et al., , schoonderwoerd et al., ; wray et al., ; janke et al., ; synge and hopkins, ; dorn et al., ; mainil et al., ; knutton, ; fisher et al., ; wieler et al., ; saridakis et al., ) . few reports have examined the occurrence of aeec infections concurrently in both healthy and diarrheic calves (mohammad et al., ; wieler et al., ; blanco et al., blanco et al., , . the objectives in this research were to determine the presence of certain virulence properties associated with eae e. coli isolated from healthy and diarrheic calves and to assess the role of aeec in calf diarrheal disease. fecal specimens were obtained from holstein calves after digital stimulation of the rectal mucosa. at the time of fecal collection, calves had diarrhea as determined by visual assessment of the specimen and the calf. on each farm, the diarrheic calves were matched to an approximate number of healthy calves. thus, fecal specimens were collected from calves that did not have diarrhea and appeared healthy. depending on the number of diarrheic calves sampled, to calves (both diarrheic and healthy) were sampled on six farms, and to calves were sampled on six farms. the dairy farms were in michigan. feces were collected into sterile cups, packed on ice packs in chests, and were transported to the laboratory. upon arrival to the laboratory, feces were immediately cultured. at the time of fecal collection, historical and clinical examination data were recorded on each calf. for primary culture, . g of feces was homogenized in . ml of peptone-saline solution. an inoculating loop was used to transfer ml of the fecal/peptone-saline suspension to macconkey (mac) agar plates, and the plates were streaked for isolation of lactose fermenting colonies (lfc). the mac plates were incubated aerobically at c for to h. ten lfc that exhibited e. coli growth characteristics were randomly harvested with sterile toothpicks from the last streaking area of each mac plate. total bacterial dna was extracted and then prepared for pcr analysis. each eae and eae/stx colony from each calf was verified as e. coli by the api e system (biomerieux vitek, hazelwood, mo). ten individual lfc from each calf were suspended in individual ml microcentrifuge tubes (elkay products, shrewsbury, ma) containing ml of lysis buffer ( . % triton x- , mm tris±hcl, . mm edta, ph . ), mixed by vortexing for s, heated at c for min, then centrifuged at ,  g for min. amplification of bacterial dna was performed with ml of bacterial dna in ml of pcr reaction mixture. the pcr reaction mixture contained ml of double distilled water, ml of  pcr buffer, mm mgcl , mm dntp mix, . mm of each primer, and . u of taq dna polymerase (gibco brl, life technologies, inc., grand island, ny). prior to testing each lfc for the eae gene, three sets of published oligonucleotide sequences (gannon et al., ; donnenberg and kaper, ; karch et al., ) were evaluated for detection of eae sequences among well characterized strains of e. coli that have been determined to possess various eae allelic sequences or lacked the eae gene (table ). these strains were of multiple serotypes and were isolated from human and animal sources. based on this study, the ae ±ae oligonucleotide sequences {( h gtg gcg aat act ggc gag act h ) and ( h ccc cat tct ttt tca ccg tcg h )} were chosen to amplify the eae gene (gannon et al., ) . the size of the amplified dna product was bp. the amplification method was c for min for denaturing, c for min for annealing, and c for min for extension for cycles. for amplification of stx genes, the mk ±mk oligonucleotide primers {( h ttt acg ata gac ttc tcg ac h ) and ( h cac ata taa att att tcg ctc h )} were used (karch and meyer, ; schmidt et al., ) . the mk ±mk primer pair was demonstrated to amplify a bp fragment for both stx and stx loci (karch and meyer, ) . the thermocycling program was c for min for denaturation, c for min for annealing, and c for min for extension for cycles. all eae and stx amplifications were performed in a programmable thermal controller (ptc- , mj research, inc., watertown, ma). ml of amplified products were analyzed by gel electrophoresis in . % agarose. products were visualized by ethidium bromide staining followed by uv transillumination. a bp dna ladder (gibcobrl, grand island, ny) was used as the molecular weight marker. for each pcr run, dna from ehec o :h and a calf strain of aeec (serotype :nm) were used as positive controls, and dna from a strain of e. coli and salmonella dublin that lacked eae genes were negative controls. the control strains had been previously evaluated for eae and stx genes by colony blot hybridization and pcr. each eae and eae/stx e. coli was assessed for enterohemolysin activity on % defibrinated washed sheep blood agar plates as described (beutin et al., ) . colonies surrounded by clear zones of hemolysis were defined as exhibiting enterohemolysin activity. serotyping was performed by standard methods at the e. coli reference center, the pennsylvania state university (wilson and francis, ). e. coli strains were grown overnight in tryptic soy broth, then heated to c for h. agglutinations were performed by using preselected dilutions of each of o group antisera in microtitre plates and the positive reactions were confirmed by microtitre titrations against monovalent antisera (glantz, ) . if there was no agglutination in the preliminary screening assay, the bacterial suspension was reheated at c for an additional h and the microtitre agglutination assays were repeated. susceptibility to different classes of antimicrobial agents was performed using standard disk diffusion methods in mueller-hinton agar (bauer et al., ) . results were interpreted according to (national committee for clinical laboratory standards, ) guidelines. the disks containing the following amounts of antibiotics were used: amikacin mg, ampicillin mg, ceftiofur mg, cephalothin mg, chloramphenicol mg, enrofloxacin mg, gentamicin mg, tetracycline mg, trimethoprim/sulfamethoxazole . / . mg, and nalidixic acid mg. to demonstrate shiga toxin antigens, the premier ehec assay (meridian diagnostics inc., cincinnati, oh) was performed on eae/stx e. coli strains known to produce shiga toxins ( table ) . as additional controls, eae only and stx only strains were examined by the enzyme immunoassay. after h growth at c on mac plates, a single colony of each strain was suspended in ml of sample diluent for testing. the immunoassay was performed according to the manufacturer's recommendation. the reaction mixture was read spectrophotometrically at nm. postmortem and microbiologic examinations were performed on of the diarrheic calves. postmortem examinations were performed by attending veterinary pathologists. tissue segments from the duodenum, jejunum, ileum, cecum, spiral colon, transverse colon, descending colon, and rectum were immediately fixed in neutral buffered % formalin. after h in the fixative, tissues were trimmed, routinely processed, and embedded in paraffin. thin sections were cut, mounted on slides, and stained with h & e. slides were examined by light microscopy. intestinal contents or feces from each necropsied calf were examined for cryptosporidium parvum, enterotoxigenic e. coli, salmonella sp., enteric viruses, and bvdv by routine procedures. briefly, c. parvum oocysts were detected by light microscopic examination of concentrated oocysts (garcia et al., ) . g of intestinal contents was dispensed in . ml of peptone-saline and ml was plated onto macconkey and brilliant green agar plates. selenite enrichment broth was inoculated with . ml of the fecal/peptone-saline suspension and was subcultured onto brilliant green agar for detection of salmonella sp. suspect colonies were confirmed as salmonella after differential biochemical and serological testing. strains of e. coli were screened for heatstable enterotoxin type a by pcr using published oligonucleotide sequences (woodward et al., ) . the k fimbrial antigen was detected by agglutination in k monospecific antibody after enhancement on minca isovitalex agar. the presence of enteric viruses was detected by electron microscopy and group a rotavirus antigen was detected by elisa (pathfinder rotavirus, kallestad, chaska, mn). bovine viral diarrhea virus was identified in lysed mononuclear cells or sera by the immunoperoxidase monolayer assay (houe et al., ) . to determine significance of the e. coli properties between healthy and diarrheic calves, the chi-square test of independence was used to evaluate the association between illness and the characteristics of the e. coli strains (snedecor and cochran, ) . based on interviews with calf caretakers and review of individual calf records (when available), of the healthy calves and of the diarrheic calves had received antimicrobial therapy. twenty of these calves had received antimicrobial therapy for their diarrheal disease and calves had been treated for medical problems unrelated to and prior to the onset of diarrhea. eight of the diarrheic calves that carried eae or eae/ stx e. coli had received antimicrobial therapy. gentamicin sulfate, ceftiofur sodium, oxytetracycline (la ), and procaine penicillin were the antimicrobials most often used. the mean age of the healthy calves was . ae . days (range to days) and the mean age of the healthy calves infected with eae e. coli was . ae . days (range to days). the mean age of the diarrheic calves was . ae . days (range to days), whereas, the mean age of the diarrheic calves infected with eae e. coli was . ae . days (range to days). in preliminary experiments, correlation between previously determined eae and stx profiles for select strains (table ) and our pcr results was %. based on these results, the selected primers and the pcr assays were considered definitive for detection of e. coli eae and stx genes. in this study, the premier ehec assay was used to detect shiga toxin antigens associated with eae/stx strains. the assay accurately detected shiga toxin antigens for the eae/stx strains (table ) , no false positives of eae only strains were detected, and of stx only strains were detected. under the conditions used in this study, the premier ehec assay was considered a sensitive assay to detect shiga toxin antigens. overall, of the ( . %) calves carried eae e. coli in their feces. forty of the ( . %) healthy calves and of the diarrheic calves ( . %) carried eae e. coli in their feces. infection with eae e. coli was significantly higher (p . ) in the healthy calves when compared to the diarrheic calves. the stx gene was identified in approximately equal percentages of eae e. coli in healthy and diarrheic calves (table ) . among the eae strains from healthy calves, ( . %) possessed stx genes, whereas, among the eae strains from diarrheic calves, ( . %) possessed the stx gene. when examined in the immunoassay, shiga toxin antigens were detected for all eae/stx ( %) strains from healthy calves and in of ( . %) strains from diarrheic calves. the enterohemolytic phenotype was expressed by . % of the eae and eae/stx strains from healthy calves and by . % of the strains from diarrheic calves. enterohemolysin activity was not restricted to one particular serogroup and was detected in both eae and eae/stx strains (table ) . distribution of eae and eae/stx e. coli among serogroups is shown in table . forty six of the ( . %) eae and eae/stx e. coli strains belonged to different typable serogroups and ( . %) eae e. coli and eae/stx strains were o-negative. serogroups o , o , o , o , and o were detected in both healthy and diarrheic calves. however, strains belonging to serogroups o , o and o accounted for . % and . % of the eae and eae/stx e. coli from healthy and diarrheic calves, respectively. the o-negative strains accounted for . % and . % of the strains from healthy and diarrheic calves, respectively. shiga toxin antigens were detected among e. coli belonging to serogroups , o , o / , o , o , o , o and o from healthy calves, and from serogroups , , , and from diarrheic calves. all strains were sensitive to amikacin, enrofloxacin, and nalidixic acid (table ). significantly more of the eae and eae/stx strains from diarrheic calves, when compared to strains from healthy calves, were resistant to at least one of the antimicrobials tested. moreover, significantly more of the strains from diarrheic calves had multiple antibiotic resistance patterns. four eae/stx strains recovered from diarrheic calves were resistant to chloramphenicol. antimicrobial resistance was not restricted to any one serogroup, nor was resistance associated with enterohemolysin production. most resistance patterns included resistance to tetracycline, ampicillin, and gentamicin (table ) . among the calves that had postmortem examinations, ( . %) had eae or eae/ stx e. coli in their feces. in five calves infected with eae or eae/stx e. coli, and in the tissue sections examined did not have histopathologic evidence of the attaching and effacing lesion, c. parvum, coronavirus, and rotavirus were identified in one calf each. c. parvum and coronavirus were identified in one additional calf. histopathologic lesions consistent with the attaching and effacing phenotype (moon et al., ; hall et al., hall et al., , chanter et al., chanter et al., , moxley and francis, ; pospischil et al., ; janke et al., ) were detected in calves. the lesion was detected in the colon only of six calves and in the ileum and colon of four calves. among these calves, c. parvum was table distribution of eae and eae/stx e. coli among serogroups and relationship to the attaching and effacing lesion detected in five calves, salmonella typhimurium in one calf, and bvdv and c. parvum were detected in one calf. the pathogens screened for by our assays were not detected in feces from of the calves. in this study, significantly more of the healthy calves ( . %) carried eae and eae/ stx e. coli in their feces when compared to diarrheic calves ( . %). however, significantly more eae and eae/stx e. coli that exhibited single and multiple antimicrobial resistances were isolated from diarrheic calves. selection for resistance among the strains from diarrheic calves may have been due to a greater use of antimicrobials among the diarrheic calves. based on limited observations made with calves on one farm, a relation between antimicrobial use and selection for resistance was observed. three of calves developed diarrhea after being treated for respiratory disease. all three calves were subsequently positive for eae (one calf) and eae/stx e. coli (two calves). two of the three strains were resistant to the antimicrobial used to treat the respiratory disease. examination of e. coli from the seven healthy calves did not reveal any eae , eae/stx e. coli or commensal e. coli strains that were resistant to that antimicrobial. in this case, exposure to the particular antimicrobial appeared to select for resistance. however, environmental sources of the resistant strains could not be excluded (linton, ) . our data show that the stx genes were equally distributed among eae e. coli from both healthy ( . %) and diarrheic calves ( . %). furthermore, shiga toxin antigens were detected in more strains from healthy calves ( . %) than in strains from diarrheic calves ( . %). although the immunoassay used detects shiga toxin antigens, the data are consistent with that of others who have demonstrated the prevalence of shiga toxin producing e. coli in the feces of young calves (mainil et al., ; blanco et al., blanco et al., , butler and clarke, ; dorn et al., ; and wieler et al., ) . enterohemolysin, a distinctively different hemolysin from -hemolysin, is synthesized by calf strains of e. coli that produce shiga toxins (beutin et al., (beutin et al., , (beutin et al., , . although the influence of enterohemolysin on calf intestinal disease has not been defined, it has been suggested that enterohemolysins may compliment the effects of shiga toxins (nataro and kaper, ) . furthermore, screening for enterohemolysin has been proposed as a diagnostic marker for shiga toxin producing e. coli (beutin et al., ; wieler et al., ) , since its presence is strongly correlated with shiga toxins. among bovine shiga toxin producing e. coli, enterohemolysin activity has been reported to range from . % to . % (beutin et al., ; wieler et al., ) . our data show that . % and . % of the eae and eae/stx strains from healthy and diarrheic calves, respectively, had enterohemolysin activity. strains of aeec belonging to the same serogroups were often isolated from healthy and diarrheic calves. strains belonging to serogroups o , o , and o accounted for . % of all the typable strains, and for . % and . % of the typable strains from healthy and diarrheic calves, respectively. characterization of aeec by o group only is less powerful than o:h typing since strains of the same o group but possessing different h antigens may have different virulence properties. nevertheless, determination of specific virulence attributes and the association with specific o groups provide valuable epidemiologic information (wieler et al., ) . attaching and effacing e. coli belonging to serogroups o , o , and o are widely distributed among calves and have been demonstrated in natural outbreaks and experimentally (with some strains) to cause diarrhea and dysentery (chanter et al., (chanter et al., , hall et al., hall et al., , schoonderwoerd et al., ; wray et al., ; moxley and francis, ; mainil et al., ; janke et al., ; knutton, ; butler and clarke, ; dorn et al., ) . as calf aeec have a similar pathophysiologic mechanism to human epec and ehec, strains carrying both eae and stx genes, and belonging to serogroups o negative, o , o , o , o , o , o / , o , o , o , o , o and would be of concern to human health. the age at which calves are most susceptible to colonization by eae e. coli and the age at which they are most likely to develop attaching and effacing lesions are not known. based on the distribution of the eae gene among calves of various ages, it appears that an age-associated resistance does not occur during the first week of life as is recognized with etec. furthermore, the mean ages of the healthy ( . days) and diarrheic ( . days) calves infected with eae e. coli would support the notion that calves older than days are more susceptible to infection with eae e. coli. however, this does not mean that the infected calves would develop attaching and effacing lesions. the age of calves infected with eae e. coli and those that had demonstrable attaching and effacing lesions ranged from to days. in natural infections, calves as young as days and as old as months have been described with attaching and effacing lesions (janke et al., ) . antibiotic susceptibility testing by a standardized single disk method close association of verotoxin (shiga-like toxin) production with enterohemolysin production in strains of escherichia coli characterization of hemolytic strains of escherichia coli belonging to classical enteropathogenic o-serogroups entero-hemolysin, a new type of hemolysin produced by some strains of enteropathogenic e. coli (epec) genes coding for shiga-like toxins in bovine verotoxin-producing escherichia coli (vtec) strains belonging to different o : k : h serotypes production of toxins by escherichia coli strains isolated from calves with diarrhoea in galicia enterotoxigenic, verotoxigenic, and necrotoxigenic escherichia coli isolated from cattle in spain diarrhoea and dysentery in calves dysentery in gnotobiotic calves caused by atypical escherichia coli dysentery in calves caused by an atypical strain of escherichia coli construction of an eae deletion mutant of enteropathogenic escherichia coli by using a positive-selection suicide vector enteropathogenic escherichia coli role of the eaea gene in experimental enteropathogenic escherichia coli infection a second chromosomal gene necessary for intimate attachment of enteropathogenic escherichia coli to epithelial cells characteristics of verocytoxin producing escherichia coli associated with intestinal colonization and diarrhea in calves pathogenicity of a bovine attaching effacing escherichia coli isolate lacking shiga-like toxins techniques for the recovery and identification of cryptosporidium oocysts from stool specimens detection and characterization of the eae gene of shiga-like toxin-producing escherichia coli using polymerase chain reaction serotypes of escherichia coli associated with colibacillosis in neonatal animals escherichia coli verotoxins and other cytotoxins dysentery caused by escherichia coli (s - ) in calves: natural and experimental disease attaching and effacing lesions in vivo and adhesion to tissue culture cells of vero-cytoxin-producing escherichia coli belonging to serogroup o and o prevalence of cattle persistently infected with bovine viral diarrhea virus in dairy herds in two counties in central michigan and comparison of prevalence of antibody-positive cattle among herds with different infection and vaccination status attaching and effacing escherichia coli infection as a cause of diarrhea in young calves the eae gene of enteropathogenic escherichia coli encodes a -kilodalton membrane protein, the expression of which is influenced by the eaf plasmid clonal structure and pathogenicity of shiga-like toxin-producing, sorbitol-fermenting escherichia coli o :h single primer pair for amplifying segments of distinct shiga-like toxin genes by polymerase chain reaction infection by verocytotoxin-producing escherichia coli escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent flow of resistance genes in the environment and from animals to man association between the effacing (eae) gene and the shiga-like toxin-encoding genes in escherichia coli isolates from cattle role of verocytogenic escherichia coli in cattle and buffalo calf diarrhoea serotypes of verocytotoxigenic escherichia coli isolated from cattle and buffalo calf diarrhoea attaching and effacing activities of rabbit and human enteropathogenic escherichia coli in pig and rabbit intestines natural and experimental infection with an attaching and effacing strain of escherichia coli in calves performance standards for antimicrobial disk susceptibility tests. approved standard m -a attaching and effacing bacteria in the intestine of calves with diarrhea virulence properties of escherichia coli strains belonging to enteropathogenic (epec) serogroups isolated from calves with diarrhea shiga-like toxin ii-related cytotoxins in citrobacter freundii strains from humans and beef samples colitis in calves: natural and experimental infection with a verotoxin-producing strain of escherichia coli o :nm shiga-like toxin production from escherichia coli associated with calf diarrhea analysis of frequencies in one-way and two-way classifications verotoxigenic escherichia coli o. in scottish calves the pathogenic mechanisms of shiga and shiga-like toxins role of a -megadalton plasmid and shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic escherichia coli o :h in gnotobiotic piglets characterization of shiga-like toxin producing escherichia coli (sltec) isolated from calves with and without diarrhea association of enterohemolysin and nonfermentation of rhamnose and sucrose with shiga-like toxin-genes in escherichia coli from calves shiga toxin-producing escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes fimbriae and enterotoxins associated with escherichia coli serogroups isolated from pigs with colibacillosis detection of entero-and verocyto-toxin genes in escherichia coli from diarrhoeal disease in animals using polymerase chain reaction occurrence of`attaching and effacing' lesions in the small intestine of calves experimentally infected with bovine isolates of verocytotoxic e. coli financial support from the agricultural experiment station, national food safety and toxicology center, and the college of veterinary medicine, michigan state university. key: cord- - dd b di authors: rivera-benitez, josé francisco; de la luz-armendáriz, jazmín; saavedra-montañez, manuel; jasso-escutia, miguel Ángel; sánchez-betancourt, ivan; pérez-torres, armando; reyes-leyva, julio; hernández, jesús; martínez-lara, atalo; ramírez-mendoza, humberto title: co-infection of classic swine h n influenza virus in pigs persistently infected with porcine rubulavirus date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: dd b di porcine rubulavirus (porpv) and swine influenza virus infection causes respiratory disease in pigs. porpv persistent infection could facilitate the establishment of secondary infections. the aim of this study was to analyse the pathogenicity of classic swine h n influenza virus (swh n ) in growing pigs persistently infected with porcine rubulavirus. conventional six-week-old pigs were intranasally inoculated with porpv, swh n , or porpv/swh n . a mock-infected group was included. the co-infection with swh n was at days post-infection (dpi), right after clinical signs of porpv infection had stopped. the pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. in all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. by means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. viral excretion of porpv in nasal and oral fluid was recorded at and dpi, respectively. porpv persisted in several samples from respiratory tissues (rt), secondary lymphoid organs (slo), and bronchoalveolar lavage fluid (balf). for swh n , the viral excretion in nasal fluids was significantly higher in single-infected swh n pigs than in the co-infected group. however, the co-infection group exhibited an increase in the presence of swh n in rt, slo, and balf at two days after co-infection. in conclusion, the results obtained confirm an increase in the clinical signs of infection, and porpv was observed to impact the spread of swh n in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. in the present study, the interaction of swh n infection is demonstrated in pigs persistently infected with porpv. respiratory diseases in pigs are considered a primary health problem and are responsible for great economic losses in the worldwide swine industry (sørensen et al., ) . viruses that cause respiratory disease and pneumonia in growing pigs include the porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), pseudorabies virus (prv), porcine rubulavirus (porpv), porcine circovirus type- (pcv- ), and porcine respiratory coronavirus (prcv). all these viral agents can act individually or through interaction with each other, and associations with other infectious agents of bacterial origin can also occur (choi et al., ; deblanc et al., ; grau-roma and segales, ; kirkland and stephano, ; morin et al., ; rivera-benitez et al., a; segales et al., ) . porpv is the etiological agent of blue-eye disease (bed) in pigs. this disease remains endemic in mexico and has only been diagnosed in that country (escobar-lopez et al., ; stephano et al., ) . the virus genome is composed of single-stranded negative-sense rna. the virus belongs to the family paramyxoviridae (wang et al., ) . in growing pigs, the infection becomes established predominantly in the respiratory tract and the central nervous system. in lungs, interstitial pneumonia has been described, and an increase in respiratory signs was observed in experimental infection (reyes-leyva et al., ; reyes-leyva et al., ; rivera-benitez et al., a; stephano et al., ) . acute swine influenza virus infection causes interstitial pneumonia and bronchiolitis, with cough, dyspnea, fever, and lethargy as clinical manifestations, although recovery is usually rapid. the common subtypes circulating in swine have been characterised as h n , h n , and h n (brookes et al., ; olsen et al., ) . siv has been associated with porcine respiratory disease complex (prdc) in growing or fattening pigs ( - weeks of age), while interactions with mycoplasma hyopneumoniae, prrs, and pcv- (deblanc et al., ; van reeth et al., yazawa et al., have been widely studied. there are no previous studies of experimental or natural co-infection of porpv and siv. however, in the swine farms in the central and western-central regions of mexico, seropositivity (circulating antibodies specific against siv and porpv) in growing pigs is common. these regions are the most important swine production regions in mexico (avalos et al., ; bobadilla et al., ; escobar-lopez et al., ) . primary infection with porpv is common in pigs under field conditions, and it may become persistent (cuevas et al., ; wiman et al., ) . as a consequence, persistently infected pigs have greater susceptibility to secondary infections, and these infections may become exacerbated. the objective of this study was to analyse the pathogenicity of experimental co-infection with porcine rubulavirus and classic swine h n influenza virus in growing pigs. the porpv pac- strain was used (jalisco/ ; genbank access number: ef ) (ramirez-mendoza et al., ) . the viral stock was multiplied in the mdck cell line (madin-darby canine kidney). the pac- strain of porpv has been shown to cause respiratory disease and clinical presentations in experimentally infected growing pigs (rivera-benitez et al., a) . in this study, for coinfection, the a/swine/new jersey/ / (h n ) strain (swh n ) (genbank access number: k -m ) was used. the swh n viral stock was propagated in mdck cell cultures and in the allantoic cavity of -day-old embryonated chicken eggs. the a/swine/new jersey/ / h n strain was isolated from an outbreak of swine influenza in the united states and is considered the classic north american prototype that infects pigs (kendal et al., ) . in both cases, the viral stocks were titrated in cell cultures, and the reed and muench method was used to calculate the titre; the obtained titres were expressed as the % tissue culture infectious dose (tcid ). twenty-four -week-old crossbred conventional pigs were obtained from a porpv-and siv-free commercial farm. when the pigs arrived, their nasal swabs were subjected to real-time rt-pcr to confirm that the pigs were negative for porpv and siv infection. the pigs were housed in experimental isolation units at the departamento de medicina y zootecnia de cerdos of the facultad de medicina veterinaria y zootecnia at universidad nacional autónoma de méxico. all pigs were fed a commercial diet and had access to water ad libitum. after a -day adaptation period, pigs were randomly distributed into groups: porpv/mock (n = ), mock/ swh n (n = ), porpv/swh n (n = ) and mock/mock (n = ). the experimental design described above is summarised in table . all procedures and the experimental protocol were approved by the institutional experimental animal care sub-committee of the universidad nacional autónoma de méxico. the pigs were first evaluated clinically, and the observed clinical signs of respiratory disease were quantified using the loeffen et al. ( ) model. the categories evaluated were as follows: activity (value : active pigs in an alert state, : reduced activity, : apathy), breathing frequency (value : normal, : slightly elevated, : clearly elevated), abdominal breathing (value : normal, : slight abdominal breathing, : abdominal breathing, jerking), and coughing (value : absent; : present). the scores for each observation were recorded for each time point and then arranged on a scale from to (loeffen et al., ) . rectal temperature was measured, and samples were taken both pre-and post-infection. the samples collected were nasal and oral swabs (polyester swabs were placed in ml of antibiotic supplemented culture medium) and blood samples from the jugular vein. the samples (blood and swabs) were collected both pre-and post-infection on day - , , , , , , , , , , and . the pigs of all groups were euthanised at different points during the experiment: and dpi (three pigs in each group) (table ) . during necropsies, all macroscopic alterations of the respiratory tract were recorded, and a series of respiratory tissue (rt) and lymphoid tissue (slo) sections were collected (rt: nasal mucosa, anterior and bronchial trachea, and lung; slo: soft palate tonsil, mediastinal and tracheobronchial lymph nodes). bronchoalveolar lavage fluid (balf) was obtained from each pig using ml of phosphate-buffered saline. the balf samples were then centrifuged (  g/ min/ c), and the cell pellets were homogenised with ml of culture medium. all samples were preserved in liquid nitrogen until use. sections of the cranial lung lobe and mediastinal and tracheobronchial lymph nodes were fixed in % neutral buffered formalin for histopathological and immunohistochemical examination. the formalin-fixed lung samples were embedded in paraffin wax, sectioned, and stained with haematoxylin-eosin. these sections were evaluated by light microscopy for histopathological changes. for analysis, four specific changes were assessed: bronchiolar-associated lymphoid tissue hyperplasia, development of perivascular lymphoplasmacytic infiltration near to respiratory bronchioles, interstitial pneumonia, and increase of alveolar macrophages. lesion severity was scored as follows: , null; mild; , moderate; , marked; and , very marked. the pathological scores were averaged for each group analysed. for the immunohistochemical evaluation, two monoclonal antibodies were used, the first directed to the hn protein of porpv (kindly donated by dr. sandra cuevas-romero) and the second to the ha protein of the influenza virus (mab ; merck millipore, billerica, ma). the procedure was performed according to standardized protocols. immunoreactivity was evidenced using the immunodetector kit (biosb, santa babara, ca). the sera were inactivated and adsorbed to remove nonspecific inhibitors of the haemagglutination. hi was conducted following previously described protocols (oie, ; ramirez et al., ) . the serum samples were placed in a -well u-shaped plate, double serial dilutions were prepared in pbs, and eight haemagglutinating units of virus (porpv or siv) were added to each well. for porpv, the dilutions ranged from : to : , while for swh n , the range was : to : . hi titres ! ( log ) of porpv and ! ( . log ) for swh n were considered as positive. the titre of the haemagglutination-inhibiting antibodies was expressed as the maximum dilution in which the serum was capable of inhibiting the haemagglutinating activity of the virus analysed. the titres were transformed to log values. the fluids collected from the nasal and oral cavities were thawed and centrifuged (  g/ min/ c). the supernatants were inoculated in duplicates in mdck cells at % of confluence. a culture medium supplemented with mg/ml of trypsin-tpck (sigma-aldrich, st. louis, mo) was added, the inoculum were left to adsorb for min at c. afterwards, the inoculum was discarded, and culture medium with % foetal bovine serum was added. the cell cultures were incubated at c for h in a % co atmosphere. tissue samples (rt and slo) were analysed. approximately g of each tissue was homogenised in ml of culture medium, centrifuged and inoculated as described for the swab samples. after h, the cell culture plates were fixed with % paraformaldehyde to perform indirect immunofluorescence assays according to a previously described protocol (rivera-benitez et al., c). rna was extracted from the samples that contained cells (tissues and balf) and from the supernatants of the nasal and oral swabs using the rneasy tm mini kit and qiaamp viral rna tm mini kit (qiagen, dusseldorf, germany), respectively. all procedures were conducted in accordance with the manufacturer's protocol. initial extraction was performed in ml of supernatant from nasal and oral swabs and ml lysates of balf and unfiltered homogenised tissue (approximately mg); elution of total rna was performed in a volume of ml and ml, respectively. the total rna was quantified using spectrophotometry at a wavelength of nm (nanodrop, nd- , wilmington, de). rna integrity was determined by the ratio of the od /od reading, and the inclusion criterion was a ratio greater than . . the rna extracted from the samples was used in the quantification of the n gene of the porpv. real-time rt-pcr quantification was carried out following a previously described procedure (rivera-benitez et al., b) . for swh n , a previously described protocol was used that quantifies a fragment of the ha gene of swine influenza (richt et al., ) . this protocol was conducted in accordance with the established procedure, but with the following modifications: ml of total rna was used, and the amplification program was run for cycles. both taqman hydrolysis probes were marked on their ends with -fam ( -carboxyfluorescein) and on their ends with bhq (black hole quencher tm - ). briefly, a onestep, single-tube qrt-pcr assay was performed using the rna ultrasense tm one-step quantitative rt-pcr system (invitrogen, life technologies, carlsbad, ca). the -ml reaction mixture contained ml of  buffer, ml of enzyme mixture (reverse transcriptase and taq polymerase), . ml of nuclease-free water, nm of each primer, nm of probe, and ml (mean of ng) of total rna. thermal cycling was conducted in a smartcycler realtime pcr thermal cycler (cepheid, inc. usa). the cycling protocol involved an initial incubation at c for min, followed by a denaturation step at c for min, then cycles of c for s and c (for porpv) or c (for swh n ) for s (annealing step). the accumulated fluorescent signal was collected during the annealing step at each cycle. all assays were also performed with a no-template control (ntc), rna extracted from nasal and oral swabs and tissues taken from uninfected pigs. in both cases, tenfold serial dilutions ( À to À ) of the in vitro-transcribed rna were prepared in our laboratory and used to construct a standard curve. the detection limit of quantitative real-time rt-pcr assay was  viral copies/ml (the slope of the line was À . and À . . r was . and . for porpv and swh n , respectively). viral concentration was expressed as viral copy number per millilitre of total rna extracted from samples. spss v. was used for all statistical analyses. the microscopic lesion score was evaluated using a non-parametric test (kruskal-wallis). a student's t-test assuming unequal variance and a significance level of p . was used to compare rectal temperatures and the viral load of porpv and swh n in different samples (nasal and oral swabs, respiratory tissues and slo) between the single-infected groups to the co-infected group. in the porpv/mock and porpv/swh n groups, of the pigs in each group presented nasal secretions and conjunctivitis at dpi. in these same groups, pyrexia (> . c) was registred at and dpi in pigs (fig. a) . the clinical feature in the porpv/mock and porpv/swh n groups consisted of apathy and respiratory distress at dpi. no additional alterations in clinical signs were recorded on the remaining days in the porpv/mock group (except in the rectal temperature). in the co-infection group (porpv/swh n ), an increase of rectal temperatures above . c was observed at dpi of swh n in pigs, but later only one pig had a temperature of . c on dpi. this same pig was observed to have dyspnoea and nasal discharge. in the mock/swh n group, the rectal temperature was increased at dpi of siv inoculation. no significant differences in rectal temperatures were observed between the four groups at different times, and no clinical alterations or increases in rectal temperature manifested in the pigs of the mock/mock group. the clinical score for respiratory signs are shown fig. b. necropsies were performed at and dpi. macroscopic lesions were observed only in one pig in the mock/swh n group, who was found to have mild pneumonia localised in the cranial and diaphragmatic lobes ( dpi-swh n ). none of the other pigs from the analysed groups manifested macroscopic lesions. microscopic lesions were observed and scored (table ). in all infected groups, the most recurrent lesion was hyperplasia of the bronchiolarassociated lymphoid tissue (fig. ) . very marked interstitial pneumonia was observed only in one pig at dpi in the coinfected group (fig. b) . in the other groups, the severity of the interstitial pneumonia was found to be moderate (fig. c,d) . the presence of multinucleated cells in the lumen of the alveoli was a feature that was recurrently observed in / pigs of the porpv/ swh n group and in / pigs of the porpv/mock group (fig. e) . it was also possible to observe the formation of syncytia in the alveolar lumen in infected pigs with porpv (fig. f) . pigs of the mock/mock group presented no related lesions in the lungs (fig. a) . regarding immunohistochemical staining, positive reactivity to porpv was observed in / lung sections of pigs of the porpv/mock group (fig. i,j) . immunoreactivity to influenza was observed in / pigs of the mock/swh n group (fig. k, l) . as for the co-infection group, positive immunoreactivity to porpv was observed in / pigs and for influenza in / pigs. only one pig presented immunoreactivity to both viruses in the the bronchiolar epithelium (fig. g, h) . immunoreactivity to both viruses in mediastinal and tracheobronchial lymph nodes was seen only in two cases. the presence of haemagglutination-inhibiting antibodies against porpv was identified from dpi (porpv/mock group), and there was increased until maximum production was reached at dpi ( . log ). in the porpv/swh n co-infection group, the titre of antibodies remained unchanged at an average of log until the conclusion of the experiment. no antibodies against porpv were observed in the mock/swh n and mock/mock groups (table ) . with respect to the haemagglutination inhibition assay for swh n , antibodies were detected from the moment the pigs arrived in the porpv/swh n group. those antibodies corresponded to passive immunity because the sows are routinely vaccinated against swine influenza virus (subtype h n ). the titres of antibodies for both viruses are shown in the table . viral isolation for porpv and swh n was performed using the nasal and oral fluids and tissue samples. in the nasal swab samples, porpv was isolated at dpi ( / pigs) and up to dpi ( / pigs) in the porpv/swh n group. in the porpv/mock group, porpv was isolated at , and dpi in of pigs analysed. in the oral swabs, it was isolated from day ( / pigs) up to dpi ( / pigs) in the porpv/swh n group. in the porpv/mock group, porpv was isolated at , and dpi ( / pigs). in the mock/ swh n group, swh n -positive samples were detected only in the nasal fluids at ( / pigs) and ( / pigs) dpi. in the coinfection group, no positive samples for swh n were recorded. isolation of porpv and swh n was negative in all tissue samples of the evaluated groups. in the mock/mock group, no positive samples for porpv or swh n were recorded. in the nasal swabs, samples that tested positive for porpv were detected from h post-infection up to dpi (porpv/mock and porpv/swh n groups) (fig. a) , and there were no differences (p > . ) in the mean of viral loads at any time analysed for these two groups. in oral swabs, the excretion of porpv was more prolonged, and positive samples were detected from day up to dpi in the porpv/mock group. in the porpv/swh n group after co-infection with swh n , negative samples were recorded at dpi, and viral load was detected again at and dpi (fig. a) . with respect to balf, positive samples occurred in two pigs euthanised at dpi in porpv/mock group. after co-infection with swh n (in the porpv/swh n group), no positive samples were observed (table ). in slo samples, the assay detected % of positive samples. in samples from the tonsils and the mediastinal and tracheobronchial lymph nodes, the highest viral load was noted at dpi in the porpv/swh n group. in all cases, porpv persisted at % at dpi in the porpv/mock and porpv/ swh n groups (table ). in slo only at dpi were significant differences (p = . ) observed between the porpv/mock and porpv/swh n groups. with regard to tissues from the respiratory tract, positive samples were identified in all of the tissues analysed (i.e., nasal mucosa, anterior trachea, bronchial trachea, and lung), except in the trachea and bronchial trachea of the porpv/ swh n pigs at dpi. in the nasal mucosa, positive samples were detected from to dpi, with the highest viral load recorded on dpi ( . log ) (the porpv/swh n group). in the anterior trachea, the highest viral load was recorded at dpi, whereas positive samples were detected up to dpi (the porpv/ mock group). a similar distribution was observed in the bronchial trachea, with the highest viral load found at dpi. in both sections in the porpv/swh n group, we detected positive samples for swh n in nasal swabs on - dpi after co-infection with siv. in the mock/swh n group, positive tests were detected at and dpi after inoculation with swh n in significantly (p = . ) more pigs than in the co-infected group (fig. b) . for oral swabs, pigs were positive at dpi only in the mock/swh n group. in the balf samples, the highest viral load was recorded at dpi for the porpv/swh n group and dpi for the mock/swh n group (table ). in slo, the presence of swh n was negative in the tonsils of all the groups evaluated. the quantification of swh n was more frequent in the mediastinal and tracheobronchial lymph nodes in the porpv/swh n group. in the mock/ swh n group, only one positive sample was recorded in the tracheobronchial lymph node, at dpi. tissues from the respiratory tract were also analysed for the presence of swh n , but no positive samples were recorded from the nasal mucosa, except in one pig of mock/swh n group at dpi. in the anterior trachea, two samples with an average viral load of . log were detected at dpi (porpv/swh n group). in the mock/ swh n group, positive samples were detected at and dpi. in the bronchial trachea, positive samples were observed in the two groups analysed (porpv/swh n and mock/swh n ). in both tissue samples (anterior and bronchial trachea), the positivity observed at dpi in the co-infection group decreased at dpi, and negative samples were registered. in lung tissue, positive samples were detected more frequently in the co-infection group at and dpi, with viral loads of . and . log , respectively (table ) . no positive tests were recorded in any tissues from pigs from the porpv/mock and mock/mock groups. no significant differences in the viral load of slo and rt were observed between the four groups at two different times of necropsy. the objective of the present study was to evaluate the possible effect of swine influenza virus on growing pigs persistently infected with porcine rubulavirus. in swine farms in the westcentral region of mexico, blue-eye disease has become established as endemic, having reached a seroprevalence of % (escobar-lopez et al., ) . co-infection of porpv with other viral or bacterial agents increases the negative impact on production in this important swine-producing zone. the seroprevalence of siv in the west-central region has been identified at % for the h n swine subtype (avalos et al., ) . under field conditions, infection and co-infection with these two viral agents has been shown to be related to an increase in the number of pigs that experience respiratory disease. no experimental studies have been conducted that would allow us to assess the effects of a secondary infection of siv in pigs previously infected with porpv. in a previous study, we showed that porpv was able to induce a respiratory disease after experimental infection (rivera-benitez et al., a) . in this study, after infection with porpv, clinical observations included nasal secretion, conjunctivitis and decreased activity in the first week. after co-infection with swh n , only one pig presented with dyspnoea and nasal discharge. these results differ from those reported in other models of co-infection with influenza and m. hyopneumoniae (deblanc et al., ; thacker et al., ) and prrs with influenza (van reeth et al., . the findings from these previous studies included acute respiratory disease; these effects can be influenced by both the duration of the co-infection and the virulence of the strains used. the increase of the rectal temperature observed after porpv infection resulted in apathy and a reduction in activity. this increase in temperature lasted for a long period, and it could have been due to an unrelated infection however, this phenomenon was not analysed. in the co-infection group, an increase in rectal temperature was observed in / pigs. in the pigs infected only with swh n , an increase in rectal temperature was noted in / pigs at dpi. other studies of co-infection have reported fever after - dpi (m. hyo-influenza) (deblanc et al., ; thacker et al., ) or - dpi (prrs-influenza) (van reeth et al., ) and - dpi (prrs-influenza) (van reeth et al., ) . these findings indicated that the signs related to co-infection may occur at a subclinical level compared to other similar models. the scores for respiratory signs were low during the single infection phase with porpv. the highest score was recorded after co-infection with table detection of swh n and viral load rna in balf, secondary lymphoid organs (slo), and respiratory tissues (rt) in mock/swh n and porpv/swh n groups. number of positive and viral load of swh n swh n . loeffen et al. ( ) reported similar values in simple influenza infections during an earlier phase ( - dpi). the pigs in the mock/swh n group presented the lowest respiratory signs and rectal temperatures, with no pigs showing a difference in respiration or temperature after experimental infection, a finding that is in accordance with studies that used low-virulence swine influenza virus strains (busquets et al., ) . inspection of the lungs at necropsy revealed mild pneumonia in only one pig of the mock/swh n group. in other experimental infections with siv, marked pneumonia has been observed in the cranial lobes. this depends greatly on the virulence of the strain used (olsen et al., ) . histological evaluation of the lung samples indicated the presence of interstitial pneumonia and hyperplasia of the bronchiolar-associated lymphoid tissue in three infected groups. these results confirm that there is an increase in histological lung lesions after single-infection or co-infection. the increase in the presentation of histological lesions in the lungs has been reported in several experiments examining siv co-infection with other viral and bacterial pathogens (deblanc et al., ; loving et al., ; pol et al., ; thacker et al., yazawa et al., . persistent infection of porpv ( dpi) induces the formation of multinucleated cells and syncytia in the alveolar lumen. this has not been previously described in porpv infection, and this shows that persistent porpv infection generates a chronic disease that is indicated by the presence of microscopic lung lesions. the presence of immunopositivity to both viruses indicates a coinfection, at least in the lung and associated lymph nodes. the antibody response for porpv and swh n was not affected by single-infection or co-infection in all analysed groups. the serological response observed is equal to the normal dynamics previously reported in experimental infection (cuevas et al., ; rivera-benitez et al., a van reeth et al., . the presence of porpv in nasal swabs was detected using real-time rt-pcr from to dpi. a similar situation emerged in the first phase sampling with oral swabs, and later, positive samples were detected up to - dpi in the co-infected group. based on these results, it can be assumed that a reactivation occurred in the viral excretion of porpv, possibly influenced by immunostimulation generated by co-infection with swh n ; however, this is an event that will be studied later by means of immunohistochemical studies. viral isolation for porpv was more frequent in nasal and oral fluids in the first weeks post-infection. viral quantification for porpv was more frequent in oral swab samples. in cases of infection with the mumps virus in humans (a virus that is closely related to porpv), these are the samples chosen for viral quantification studies (boddicker et al., ; krause et al., ; uchida et al., ) . for siv, only samples that tested positive by real-time rt-pcr were recorded in nasal swabs, at and dpi in the two swh n -infected groups (and viral isolation in nasal fluid was only recorded for single-infected swh n group); however, differences in viral load between the mock/swh n group and the co-infection group were observed. in oral swab samples, no positive samples were detected in the porpv/swh n group. only positive samples were detected in oral fluid of pigs in the mock/swh n group. these results indicate that the primary infection with porpv does not cause greater excretion of swh n after co-infection. other models of infection with influenza and m. hyo did not produce increased excretion of siv in pigs previously infected with m. hyo (deblanc et al., ) . the balf samples were collected during the necropsies. in the porpv group, positive samples were detected at dpi. these observations had not been recorded previously at extended time points, and it is interesting to note that this type of sample may be useful for diagnosis of porpv. during the evaluation of swh n , two samples that were positive by real-time rt-pcr were recorded at and dpi in the single-infected and co-infected pigs, respectively. these results are in agreement with those obtained in other experimental infections that used the same influenza sub-type. busquets et al. ( ) reported the presence of influenza h n in bronchoalveolar lavage samples at dpi, and its continuation up to day . in that work, quantification was performed using samples of slo and rt. in the slo, samples that tested positive for porpv were detected on two sampling days. the viral load of porpv in lymphoid organs after co-infection was found to be greater than in the porpv/mock group. the tissue that presented the highest viral load was the soft palate tonsils. previous studies (cuevas et al., ; wiman et al., ) confirmed the persistence of porpv rna in lymphoid tissues from day to day post-infection in experimentally infected pigs. in the samples evaluated for swh n in this study, positive results were noted only in the lymph nodes. there were no positive samples from the tonsils. de vleeschauwer et al. ( ) reported the presence of swh n in tonsils from days to post-infection, and that study emphasised that the route of inoculation (intranasal vs. intratracheal) is an important factor for the distribution of the virus into diverse tissues. the positivity and viral load recorded for swh n in lymphoid organs was more frequent in the co-infected group, which presented with an increase in viral load even in the mediastinal lymph node. in rt, all samples were positive for porpv. in the analysed samples, no significant difference was observed in the viral load for porpv or swh n . in the nasal mucosa, positive samples for porpv were found from days - post-infection, and a higher viral load was observed in the co-infected group. in the case of swh n , only one sample was found to be positive in the mock/swh n group. however, the anterior and bronchial trachea was negative for porpv in the early stage of swh n co-infection. it is probable that after co-infection with swh n , the immune system was reactivated at this site and was able to clear the porpv in the trachea and bronchial trachea, at least in the early stage of infection with siv. with respect to swh n , viral load was found in samples from the trachea and the bronchial trachea on and dpi, predominantly in pigs of the mock/swh n group. previous studies obtained similar results by isolating swh n on days - post-infection (de vleeschauwer et al., ) , revealing a high tropism for this type of tissue. however, in the co-infection group, there was an increase in the presentation of swh n at the early stage of co-infection ( dpi). the increased viral load of swh n in the trachea and bronchial trachea coincides with the decrease in the viral load of porpv. in lung tissue, the distribution of porv was constant and persisted up to dpi. wiman et al. ( ) reported the presence of porv rna in lung tissue days after experimental infection. for swh n , viral rna was detected during the first two days in the co-infected group, and in the single-infected group, at dpi, which is similar to data from previous studies (de vleeschauwer et al., ; weingartl et al., ). an interesting effect was observed in balf and lung tissue: it appears that the pigs of the co-infected group were more susceptible to secondary infection because there was a greater number of a positive sample in this group compared with the single-infected group, thus demonstrating the association between primary porpv infection and subsequent swh n infection. in conclusion, the results obtained confirm infection, seroconversion, excretion, and distribution of porpv and swh n in growing pigs. the observations included an increase in the clinical signs in the co-infected group compared to simple infections. there were no significant differences in other measurements such as rectal temperature, macroand microscopic lesions, and viral loads of both viruses between the analysed groups. however, primary infection with porpv seems to have a positive impact on the spread and viral load of swh n in respiratory and lymphoid tissues in early stages of co-infection, although viral shedding in nasal and oral secretions was not enhanced. in the present study, the interaction of swh n with porpv is demonstrated in persistently infected porpv pigs. under field conditions, preventing porpv infection could also reduce the clinical effect of co-infections with viral or bacterial agents including, as in this case, siv. influenza porcina en méxico dinámica de la producción porcina en méxico de a real-time reverse transcription-pcr assay for detection of mumps virus rna in clinical specimens influenza a (h n ) infection in pigs experimental infection with h n european swine influenza virus protects pigs from an infection with the pandemic h n human influenza virus investigation of t-cell responses and viral mrna persistence in lymph nodes of pigs infected with porcine rubulavirus retrospective analysis of etiologic agents associated with respiratory diseases in pigs comparative pathogenesis of an avian h n and a swine h n influenza virus in pigs pre-infection of pigs with mycoplasma hyopneumoniae modifies outcomes of infection with european swine influenza virus of h n , but not h n , subtype identification of antigenic variants of the porcine rubulavirus in sera of field swine and their seroprevalence detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type , swine influenza virus and aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (pnp) in spain identification and preliminary antigenic analysis of swine influenza-like viruses isolated during an influenza outbreak at fort dix, new jersey paramyxoviruses: rubulavirus, menangle, and nipah virus infections, diseases of swine real-time pcr for mumps diagnosis on clinical specimens-comparison with results of conventional methods of virus detection and nested pcr effect of maternally derived antibodies on the clinical signs and immune response in pigs after primary and secondary infection with an influenza h n virus influenza virus coinfection with bordetella bronchiseptica enhances bacterial colonization and host responses exacerbating pulmonary lesions severe proliferative and necrotizing pneumonia in pigs: a newly recognized disease manual of diagnostic tests and vaccines forterrestrial animals swine influenza, diseases of swine dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract lesions in the reproductive tract of boars experimentally infected with porcine rubula virus hemoaglutinación e inhibición de la hemoaglutinación del paramixovirus porcino a través de la modificación de algunas variables que participan en la prueba detección de viremia en la infección experimental por rubulavirus porcino mecanismos moleculares de la patogenia viral: estudios con el rubulavirus porcino realtime reverse transcription-polymerase chain reaction assays for the detection and differentiation of north american swine influenza viruses respiratory disease in growing pigs after porcine rubulavirus experimental infection efficacy of quantitative rt-pcr for detection of the nucleoprotein gene from different porcine rubulavirus strains persistence of porcine rubulavirus in experimentally infected boars immunohistochemical demonstration of the spread of pneumotropic strain of aujeszky's disease virus in conventional pigs diseases of the respiratory system encephalomyelitis, reproductive failure and corneal opacity (blue eye) in pigs, associated with a paramyxovirus infection interaction between mycoplasma hyopneumoniae and swine influenza virus rapid and sensitive detection of mumps virus rna directly from clinical samples by real-time pcr dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study clinical effects of experimental dual infections with porcine reproductive and respiratory syndrome virus followed by swine influenza virus in conventional and colostrum-deprived pigs virus taxonomy: classification and nomenclature of viruses. ninth report of the international committee on taxonomy of viruses experimental infection of pigs with the human pandemic influenza virus porcine rubulavirus lpmv rna persists in the central nervous system of pigs after recovery from acute infection experimental dual infection of pigs with an h n swine influenza virus (a/sw/ hok/ / ) and mycoplasma hyopneumoniae the authors thank alfredo díaz estrada (departamento de medicina y zootecnia de aves) and josé alfredo sánchez (departamento de patología) of fmvz-unam for their support in sampling and sample processing. this study was partially funded by the following projects: conacyt ac- ,papiit-in - and recursos fiscales inifap . j. f. rivera-benitez received a scholarship (no. ) awarded by the national system of scholarships for graduate studies through the consejo nacional de ciencia y tecnologia (conacyt). the authors affirm that no financial or personal relationships exist that could have inappropriately influenced the content of this manuscript or the opinions expressed. key: cord- -rllxa fj authors: takano, tomomi; kusuhara, hajime; kuroishi, akira; takashina, midori; doki, tomoyoshi; nishinaka, takamichi; hohdatsu, tsutomu title: molecular characterization and pathogenicity of a genogroup gvi feline norovirus date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: rllxa fj norovirus (nov) has been classified into genogroups, gi-gvi. in the present study, we identified novel feline nov (fnov) m - strain. the c-terminal of rna-dependent rna polymerase of the fnov m - strain was highly homologous with giv fnov and giv lion norovirus, whereas vp was highly homologous with gvi canine nov (cnov). based on the results of the simplot analysis, the fnov m - strain may have been produced by recombination between giv. fnov and gvi. cnov. in addition, specific pathogen-free cats inoculated with fnov gene-positive-fecal samples developed diarrhea symptoms, and the viral gene was detected in their feces and blood. norovirus (nov) is an approximately . -kbp single positive-strand rna virus that has been classified into the caliciviridae, genus norovirus (clarke et al., ). there are open reading frames (orfs) in the nov genome. orf encodes ntpase (helicase), vpg and rna-dependent rna polymerase (rdrp), orf encodes the major capsid protein, vp , and orf encodes the minor capsid protein, vp . (clarke et al., ; zheng et al., ) . nov has been classified into genogroups, gi-gvi (clarke et al., ; white, ) , based on the deduced amino acid sequence of the full-length vp region. nov genogroups are further classified into subgenogroups. for example, in giv nov, giv hnov is classified into subgenogroup (giv. ) while giv fnov and giv cnov are classified into subgenogroup (giv. ). gi, gii, and giv (giv. ) have been identified in humans, gii in pigs, giii in ruminants including cattle, giv (giv. ) in cats and lions, gv in mice, and giv(gvi. ) and gvi(gvi. and gvi. ) in dogs (clarke et al., ) . the first nov in carnivores was detected in captive lion cubs with severe enteritis (martella et al., ) . human nov (hnov) is main cause of viral gastroenteritis, and it is also the main cause of epidemic diarrhea especially in infants and the elderly (glass et al., ; harris et al., ; huhti et al., ) . hnov infection occurs in hospitals, schools, and hotels (hutson et al., ) . in cats, feline nov (fnov) was detected in domestic cats in . fnov infection occur in animal shelter. fnov have been detected in - -month-old cats that developed gastroenteritis. however, it remains unclear whether gastroenteritis can be reproduced by inoculating cats with fnov. in the present study, we identified novel fnov m - strain. based on the genome analysis of the full-length vp , this virus was classified into genogroup gvi, which has not previously been reported in cats. this novel gvi fnov was suggested to be produced by recombination between genogroups giv nov and gvi nov. rectal swab samples were collected from cats in an animal shelter in japan. the fnov gene was detected in samples collected from cat no. - , as confirmed by rt-pcr described below, and these samples were subsequently used in this study. the age of cat no. - was unclear because it was a stray (a young, mixed breed cat), but it had a healthy appearance. viral rna was extracted from samples using the high pure viral rna isolation kit (roche, switzerland) following the manufacturer's instructions. cdna was synthesized using an rna preparation from samples (rectal swabs and fecal and serum samples). template rna ( mg), random primers (final concentration of pm), and a dntp mixture (final concentration of mm each) were mixed, and the volume was adjusted to ml with ddh o. this mixture was heated at c for min and rapidly cooled on ice. after mixing ml of the mixture and ml of x primerscript buffer (takara, japan), an rnase inhibitor (final concentration of u; takara, japan) and primescript reverse transcriptase (final concentration of u; takara, japan) were added, and the volume was adjusted to ml with ddh o. the resultant solution was heated at c for min and then reacted at c for min. after heating at c for min, the reaction was rapidly cooled on ice. pcr was performed using the synthesized cdna. two microliters of sample cdna was mixed with ml of quick taq hs dyemix (toyobo, japan), ml of mm primer mix (p d: -gattactccasstgggaytcmac- , p d: -tgacgatttcat-catcmccrta- ) , and ml of distilled water. using a pcr thermal cycler dice (takara, japan), the dna was amplified at c for min, followed by cycles of denaturation at c for s, primer annealing at c for s, and synthesis at c for s, with a final extension at c for min. pcr products ( ml) were electrophoresed with a dna marker on a . % agarose gel. the agarose gel was incubated with midori green dna stain (nippon genetics, japan), and bands of bp (in case of nov) and bp (in case of vesuvius and sapovirus) were visualized using a uv transilluminator. the methods used to detect viral genes other than fnov were described previously (chung et al., ; elschner et al., ; martella et al., ; pinto et al., ; schunck et al., ; takano et al., ) . two microliters of sample cdna was mixed with -ml of -fold primestar buffer (takara, japan), ml of dntp mixture (takara, japan) containing . mm of each dntp, ml of mm primer mix (the primer sequences are shown in tables and ), . ml of primestar hs dna polymerase ( . u/ml; takara, japan), and . ml of distilled water. using a thermal cycler, dna was amplified at c for min, followed by cycles of denaturation at c for s, primer annealing at c for s, and synthesis at c for min, with a final extension at c for min. gel electrophoresis of the pcr products and stain of gels were performed as described above. thirty microliters of pcr products were electrophoresed as described above. singlet bands were excised and transferred to microtubes, and dna was purified using a qiaquick gel extraction kit (qiagen gmbh, germany). the purified dna was subjected to ta-cloning using the mighty ta-cloning reagent set for primestar (takara, japan) following the manufacturer's instructions. the purified plasmid inserting pcr products was sent to sigma-aldrich (japan) for sequencing. sequences of pcr product were determined by sequencing at least clones. the sequences of the virus genomes were determined and phylogenetic trees were analyzed using the mega software (version ). phylogenetic relationships were determined using the neighbor-joining algorithm, and branching order reliability was evaluated by replications of a bootstrap resampling analysis. the constitution of the nov genome was analyzed using simplot (version . . ). table primer sequences used for rt-pcr and sequencing of orf and genes. orientation nucleotide sequence location a length reference forward reverse the animal experiments described below were performed in accordance with the guidelines for animal experiments of kitasato university (the number of approval is - ). spf cats were maintained in a temperature-controlled isolated facility. the fnov gene-positive-rectal swab sample from cat no. - ( ml) and ml of phosphate-buffered saline (pbs) were mixed. this mixture was filtered using a syringe-driven filter unit (sllgh nl, pore size . mm; merck millipore, germany). the filtered sample was administered orally to a two-month-old female spf cat (cat no. - ) . this cat was examined daily for clinical signs, and body temperature and weight were measured for days. when the cat defecated, feces were collected and stored at À c. the collected feces were subjected to rt-pcr using the primer pair of p d/p d. cat no. - -derived fnov gene-positive-fecal samples (sample no. - ) were suspended with pbs to prepare a % (w/v) emulsion, and ml of this emulsion was orally administered to four - -month-old spf cats (cat nos. - , - , - , and - ). these cats were examined daily for clinical signs, and their body temperatures and weights were measured for days. peripheral venous blood was collected every days, and sera were isolated and stored at À c. when the cats defecated, feces were collected and stored at À c. the collected sera and feces were subjected to rt-pcr using the primer pair of p d/p d. rectal swab samples from cat no. - (sample no. ) were subjected to rt-pcr using the primer pair of p d/p d, and pcr products were subjected to sequencing. a clustalw analysis revealed that the pcr products showed high homologies with the -terminal of the orf gene fragments of giv. fnov and giv. lion nov (fig. ) , confirming that the pcr products were derived from nov. full-length vp gene sequencing was attempted in order to genotype fnov (fnov m - strain) detected in sample no. ; however, we could not amplify that of the fnov m - strain. we attempted to grow the fnov m - strain in vivo. we administered sample no. to a -month-old spf cat (cat no. - ), collected fecal samples from the cat daily. cat no. - increased body temperature, and showed low-spirited on days - after the inoculation. fecal samples from cat no. - were tested by rt-pcr using using the primer pair of p d/p d. fnov orf gene remained detectable until day . when the fnov gene-positive pcr products of fecal samples collected on days , , , and were subjected to a sequencing analysis, all were identical with the orf gene of the fnov m - strain. we successfully amplified the orf gene, excluding a part of the n-terminal, in a fecal sample of cat no. - (sample no. - ) collected days after inoculation with sample no. . by referring to the sequence of the amplified orf gene, the amino acid sequence of the rdrp c-terminal was deduced. a phylogenetic tree (fig. ) . we also successfully amplified the full-length orf gene in sample no. - . a phylogenetic tree analysis based on the amino acid sequence of the full-length vp revealed high homologies with gvi. /caninebari- / /ita and gvi. /canine/ fd / /ita were detected. that is, the fnov m - strain was classified into the genogroup gvi cluster, which was different from the genogroup giv fnov (fig. ) . a simplot analysis was performed to compare gene sequences including the recombination breaking point among the fnov m - strain, giv fnov, giv lion norovirus, giv canine nov (cnov), and gvi cnov. orf of the fnov m - strain was classified into the giv. /cu e/usa cluster, whereas orf and orf were classified into the gvi. /bari- /ita and gvi. /fd /ita cluster (fig. ) , suggesting that the fnov m - strain was produced by recombination between cat-derived giv nov and dog-derived gvi nov. fnov m - strain-positive fecal samples were administered to the spf cats. the presence of viruses other than fnov (feline coronavirus, feline calicivirus, feline astrovirus, feline rotavirus, feline kobuvirus, and feline panleukopenia virus) was investigated in the fnov-positive fecal samples collected from cat no. - (sample no. - ). all these viruses were not detect in sample no. - . shedding of fnov into feces started days after the inoculation with the fnov-positive fecal samples in all cats, and the virus was continuously shed until - days after the inoculation (fig. ) . fnov was also shed and days after the inoculation in cat no. - . the induction of viremia by the inoculation with the fnov m - strain was investigated. the fnov gene was detected in the serum of all cats days after the inoculation. the fnov gene was detected in serum of all cats, excluding cat no. - , and days after the inoculation. the fnov gene was detected in the serum of cat no. - on days , , , , , and (table ) . fever developed in cat no. - on day after the inoculation; however, no marked change was noted in the body temperatures of the other cats (fig. ) . in all cats, excluding cat no. - , stools were loose from days to or after the inoculation. in cat no. - , vomiting was noted on day (fig. ) , and the fnov gene was detected in this vomit. body weights increased in of the cats inoculated with the fnov m - strain, while low-spirited and transient weight loss were noted on day in cat no. - , which vomited and persistently defecated loose stools (fig. ) . nov may recombine genomes between orf and orf (bull et al., ; jiang et al., ) . the involvement of recombination in changes in adaptation to the host and pathogenicity of nov has been reported previously (phan et al., ) , and hnov was confirmed to recombine between genotypes (jiang et al., ) ; however, recombination between genogroups is probably rare. based on the results of the simplot analysis, the fnov m - strain (gvi. ) may have been produced by recombination between giv. fnov and gvi. cnov. it currently remains unclear which of these viruses has the same origin. the number of giv nov and gvi nov samples needs to be increased and a gene analysis performed in order to investigate their origins. the high homology of a part of vp between gvi. cnov and oysterderived nov was previously reported (martella et al., ). cat no. , which carried the fnov m - strain, was captured in a region along the sea in which oyster farming is thriving. thus, we cannot rule out that the fnov m - strain was derived from oysters, and needs to be investigated in future studies. no fnov infection experiment has been conducted in cats to date. we administered an fnov gene-positive fecal emulsion to spf cats and monitored them for clinical symptoms. three out of cats inoculated with the fecal emulsion developed diarrhea while one started vomiting. the development of these symptoms was almost consistent with the fnov gene-detecting period, suggesting that fnov is the cause of infectious gastroenteritis in cats. however, it is difficult to demonstrate the pathogenicity of fnov based on these results because only cats were used in this infection experiment. moreover, the presence of unknown pathogens in the fecal emulsion cannot be ruled out. in order to concretely demonstrate that fnov is the cause of infectious gastroenteritis, an infection experiment needs to be conducted that meets the following conditions: ( ) fnov isolated from a fecal emulsion is used to infect animals, ( ) the number of cats is increased, and ( ) a negative control is included in the experiment. furthermore, difficulties are associated with accurately assessing pathogenicity using the fnov m - strain alone. therefore, new strains of fnov need to be isolated from cats in japan and several fnov strains used in infection experiments. - À + + À + À À À À À À - À + À À À À À À À À À - À + + À + À À À À À À - À + + + + À À + + À À +: fnov orf gene positive; À: fnov orf gene negative. the influence of allelic variations in the histo-blood group antigens (hbgas) gene on human nov sensitivity has been reported previously (lindesmith et al., ; tan and jiang, ) . allelic variations in the fut gene encoding flucosyltransferase (fut ) have also been shown to influence nov sensitivity (lindesmith et al., ; tan and jiang, ) . therefore, further studies are needed in order to determine whether fnov infection is restricted by allelic variations in the hbgas and fut genes. viremia has been noted with shedding of the virus in the feces of hnov-infected children (frange et al., ; takanashi et al., ) . viremia was also observed early after infection in gnotobiotic animals inoculated with nov (souza et al., ) . however, viremia was not detected in nov-inoculated chimpanzees even though the virus was shed in their feces (bok et al., ) . transient viremia was noted in all cats with primary infection with the fnov m - strain, and cats clearly showing clinical symptoms were more likely to intermittently develop viremia; however, we could not identify the significance of viremia in fnov m - strain infection. the relationship between viremia and clinical symptoms has not yet been elucidated. in this study, we genotyped the fnov m - strain. when the amino acid sequence of the region from the c-terminal of rdrp to the n-terminal of vp was analyzed, the fnov m - strain was genotyped gvi, which was different from the conventional stain of fnov. fnov was detected with the development of clinical symptoms in spf cats orally inoculated with fecal samples containing the fnov m - strain. these results may be useful for studies on fnov and other giv and gvi nov. chimpanzees as an animal model for human norovirus infection and vaccine development norovirus recombination detection and genetic characterization of feline kobuviruses virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses nested reverse transcriptase-polymerase chain reaction for the detection of group a rotaviruses prevalence and clinical impact of norovirus fecal shedding in children with inherited immune deficiencies norovirus gastroenteritis deaths from norovirus among the elderly, england and wales norovirus gii- causes a more severe gastroenteritis than other noroviruses in young children norovirus disease: changing epidemiology and host susceptibility factor characterization of a novel human calicivirus that may be a naturally occurring recombinant human susceptibility and resistance to norwalk virus infection norovirus in captive lion cub genetic heterogeneity and recombination in canine norovirus enteric disease in dogs naturally infected by a novel canine astrovirus genetic heterogeneity, evolution, and recombination in noroviruses discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the us a simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukemia virus in feces pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup ii. -hs strain of human norovirus detection, genetic characterization, and quantification of norovirus rna from sera of children with gastroenteritis effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle evolution of norovirus norovirus classification and proposed strain nomenclature this work was supported by jsps kakenhi grant number . key: cord- -fm j thy authors: decaro, n.; buonavoglia, c.; barrs, v.r. title: canine parvovirus vaccination and immunisation failures: are we far from disease eradication? date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: fm j thy despite extensive vaccination, canine parvovirus (cpv) remains a leading infectious cause of canine mortality, especially among juveniles. this review provides an update on cpv vaccine types and vaccination protocols. the design of cpv prevention strategies and vaccination programs with a goal of herd immunity has been hampered by deficiencies of studies that model companion animal viral infections and inform an understanding of the basic reproduction number. however, the most important issue in eradication of cpv disease is represented by immunisation failures including: i) the presence of interfering titres of maternally-derived antibodies; ii) the presence of non-responders; and iii) possible reversion to virulence. in contrast, the role of the cpv variants in immunisation failures is widely debated. taking into account the reduced circulation of canine distemper virus and canine adenovirus type in countries where extensive vaccination is carried out, more effort should be made to aim for cpv eradication, including antibody testing to determine the optimal time for vaccinations of pups and adults and homogeneous vaccine coverage of dog population. encompasses all cpv variants and strains descendent from the initial cpv- a global sweep. for the purposes of this review, the three-variant nomenclature will be kept, since this is most used in the current literature. vaccination is the most effective measure to control the spread of the infection in dogs and to prevent the development of clinical cpv infection. therefore, cpv vaccines are considered core vaccines by professional associations with international outreach, such as the vaccination guidelines group of world small animal veterinary association (wsava) and the american animal hospital association (aaha) . a core vaccine is a vaccine that all dogs should receive, regardless of circumstances or geographical location, since it protects animals from severe, life-threatening diseases that have global distribution (day et al., ) . however, despite the intensive vaccination programs that are adopted worldwide, cpv infection represents one of the most frequent infectious disease and cause of death in juvenile dogs even in developed countries . for example, based on data from a recent nationwide survey of veterinary hospitals in australia, the estimated incidence of cpv infection among owned and shelter-housed dogs is . cases per , dogs (kelman et al, b) . very few inactivated cpv vaccines are available on the market, since these formulations have low immunogenicity, and therefore require repeated administration during the primary course and annual boosters. their use is suggested only in exotic animals and pregnant bitches, for which most mlv vaccines are not yet registered (day et al., ) . in a recent study (altman et al., ) , inactivated vaccines were more frequently associated with immunisation failures than mlv vaccines in pups of less than twelve weeks of age, likely as a consequence of a better ability of replicating mlv to override residual maternally-derived antibodies (mda). in contrast, mlv vaccines are widely used, since they induce a strong, long-lasting (usually life-long) immunity by replicating within the host, without producing significant tissue damage or clinical signs. to date, in most countries there are only two cpv types that are included in the cpv mlv vaccine formulations, the original cpv- strain and its variant cpv- b. both mlv cpv vaccine strains are able to cause viraemia and replicate in the intestinal mucosa, albeit at lower titres than field strains, being shed by the faeces of vaccinated dogs for at least - weeks post-vaccination freisl et al, ) . their ability to replicate in the intestinal mucosa has been demonstrated even in dogs with protective circulating antibody titres, which shed low dna titres of vaccine virus in their faeces (freisl et al., ) . cpv mlv vaccines are not recommended by the wsava vaccination guidelines group for use in wildlife, in pups less than - weeks of age, or during pregnancy, due to possible vaccine-associated adverse effects (day et al., ) . however, some recent mlv vaccines have been demonstrated to be innocuous for the both foetuses and for -week-old pups, and are registered for administration during pregnancy and in young pups (n. decaro, personal observation). cpv mlv vaccines are characterised by early onset of immunity (ooi) and long duration of immunity (doi) . some studies have demonstrated that dogs administered mlv vaccines were protected against challenge with virulent cpv as early as days post-j o u r n a l p r e -p r o o f vaccination (schultz and larson, ; n. decaro, unpublished data) . doi after natural cpv infection is considered life-long, but there is evidence of a long-term doi even after cpv vaccination (day et al., ) . dogs vaccinated against cpv, canine distemper virus (cdv) and canine adenovirus (cadv) were protected from disease and/or infection after challenge years from vaccination (schultz et al., ) . most cpv licensed vaccines are registered for -year-interval boosters, but even those claiming a required revaccination interval of or years can be administered at -year intervals (day et al., ; ford et al., ) . all cpv vaccines available on the market are registered for administration through the parenteral route. however, some experimental and/or commercial vaccines have been proposed for intranasal or oral administration to better overcome mda interference (buonavoglia et al., ; martella et al., ; cavalli et al., ) . vaccination is a "customised" action, which should take into account a series of individual factors, including age, breed, life style of the dog, disease prevalence in a particular geographic region, etc. therefore, no standard vaccination policy will cover all possible situations. nevertheless, there are some general protocols that are recommended for use of canine core vaccines that include mlv cpv, cdv and cadv. in client-owned pups, the minimum age to begin the cpv primary vaccination protocol is - weeks, although some vaccines are licensed for use in -week-old pups (day et al., ; ford et al., ) . after the vaccine is given, a - -week interval revaccination protocol is suggested until the age of weeks or even older. in fact, there is some evidence that some pups can still not be immunised at weeks, and that a last vaccination at weeks may be helpful in situations with high incidence of cpv. in dogs older than weeks, which should no longer have interfering mda titres, a single cpv mlv vaccine dose is acceptable (day et al., ; ford j o u r n a l p r e -p r o o f et al., ), although administration of two doses - weeks apart is also considered (ford et al., ) . dogs should receive a booster within year of the primary vaccination course given as a pup (ford et al., ) or at any time point between and weeks of age (day et al., ) , with subsequent revaccinations being given at intervals of years or longer. this vaccination protocol should be followed even if some mlv vaccines against cpv are still licensed for -year or -year boosters and/or claim to induce protection with a primary vaccination series completed at -or -weeks age. however, in some countries practitioners may be legally required to strictly comply with the vaccine registration data and administer -year or -year boosters. more intensive vaccination programs are recommended in the shelter environment, where the virus burden could be high, dogs of unknown vaccination and hygiene status are housed, and the population turnover is high, so that the risk of exposure to cpv is very high (day et al., ; ford et al., ). an alternative to revaccination is to perform antibody testing of dogs that completed the primary vaccination series using external laboratory or in-clinic tests. the gold standard for detection of cpv antibodies is haemagglutination inhibition (hi), which can only be performed in highly specialised laboratories, requiring fresh swine erythrocytes and trained personnel. the cpv hi antibody titre is expressed as the reciprocal of highest serum dilution still inhibiting the haemagglutination activity of a known amount of virus . a more reliable assay for detection of cpv protective antibodies in virus neutralisation (vn), which requires laboratory expertise in cell culture and virus isolation. the vn titre is the reciprocal of the highest serum dilution that completely neutralizes the virus (cavalli et al., ) . in-clinic assays are usually dot-elisa assays licensed to determine the titre of antibodies against canine core vaccines (cadv- , cpv and cdv). the concentration of cpv antibodies in serum samples is defined by the colour intensity of the j o u r n a l p r e -p r o o f dots, which corresponds to the antibody level in the test specimen. results are scored using positive reference dots and a scale (killey et al., ; p. dall'ara, manuscript in preparation) . the presence of cpv antibodies, regardless of titre, in an actively immunized dog over the age of weeks is correlated with protection (day et al., ; decaro et al., ) . antibody testing could be performed at least month after the final primary series vaccination administered at weeks or older and repeated every years in the case of a positive result. "herd immunity" to a pathogen refers to the indirect protection of susceptible members of a population by immune members, due to a lower risk of exposure to an infected individual, and is also influenced by the basic reproduction number (r ) of the pathogen (horzinek, ) . r is defined as the number of new infections generated by the first infectious individual in a wholly susceptible population (metcalf et al., ) . few studies have modelled r for companion animal viral infections, although such an approach would be useful for cpv to better inform prevention strategies. since r is not a fixed parameter and is sensitive to multiple variable host factors such as demography, husbandry and genetics, modelling of r for cpv would need to be done in different settings (woolhouse et al., ) . serological surveillance has provided some insights into herd immunity to cpv among different dog populations. among owned-dog populations (e.g. household pets, dogs in breeding kennels) within the last twenty years, in which most dogs tested have typically been vaccinated, high cpv seroprevalences have been reported in most studies, ranging from to . % (twark and dodds, ; bohm et al., ; mitchell et al., ; riedl et j o u r n a l p r e -p r o o f al., ; killey et al., ; rota et al., ) . by contrast, among shelter-housed dogs, which are usually unvaccinated stray dogs or variably vaccinated guardian-surrendered dogs, lower seroprevalences of to % have been reported (lechner et al., ; litster et al., ; spindel et al., ) . the minimum level of vaccine coverage required to prevent disease outbreaks among owned dog populations is lower than that for shelter-housed dogs because of a lower risk of exposure and transmission. a minimum vaccination coverage of to % has been suggested previously to be adequate to prevent disease outbreaks in owned dog populations (horzinek, ; day et al., ; riedl et al., ) . such estimates may have partially been based on two cpv seroprevalence studies of owned dogs in the uk and us in the s where cpv seroprevalence was % and %, respectively (tennant et al, ; mccaw et al., ) , however, both of these studies were small and seroprevalence was based on a threshold cpv titre ( : ). if all positive antibody titres had been considered protective, in accordance with current wsava guidelines (day, et al., ) , the actual seroprevalence of those dog populations would have been higher. in the absence of interfering titres of mda, attenuated cpv vaccines are strongly immunizing, as demonstrated in a field study of dogs entering two shelters that were vaccinated with or doses of a mlv vaccine; protective antibody titres were present in and % of dogs, respectively, within weeks of vaccination (litster et al, ) . however, when vaccination coverage is not uniform there may be pockets of highly susceptible individuals within an otherwise immune population. geographic "hot-spots" of antibodynegative dogs were identified in one study, which analysed census data and zip code of origin in unvaccinated dogs presenting to shelters in the us. some of these hot-spots were regions with low access to veterinary resources (spindel et al., ) . similarly, kelman et al. in an epidemiological survey conducted in australia, . % of dogs infected with cpv were adults that had received a complete primary vaccination course as a puppy, indicating apparent immunisation failure (ling et al., ) . immunisation failures can be vaccinerelated or host-related. causes of vaccine-related failures include vaccine storage or administration errors, non-compliance with vaccine schedules, and failures in vaccine immunogenicity (decaro et al., a; altman et al., ) . in a survey of australian veterinarians' vaccination protocols, nearly half of respondents did not comply with the recommended guideline to finish primary vaccination at or after weeks of age (kelman et al., ) . host-related factors are associated with age, genetic factors, and impaired health, nutrition or immune status (wiedermann et al., ) . primary immunisation failure, occurring in to % of vaccinated healthy humans, is due to failure to develop detectable antibodies, while secondary immunisation failure is associated with loss of previously acquired protection faster than expected (waning immunity) (wiedermann et al., ) . agerelated immunosenescence, a well-documented phenomenon in humans, is associated with both primary and secondary immunisation failure (grubeck-loebenstein, ). a low cpv seroprevalence of % detected among sick dogs admitted to an intensive care unit may, in part, have been due to secondary immunisation failure (mahon et al., ) . the main causes of immunisation failures are summarised in fig. . one of the main causes of cpv immunisation failures is the presence of interfering titres of mda. in dogs, due to the low permeability of the canine placenta to immunoglobulins, only j o u r n a l p r e -p r o o f - % of mda are transferred during pregnancy. most mda, mainly represented by immunoglobulin g antibodies, are transferred from the bitch to the offspring through ingested colostrum, reaching the small intestine and transported across the intestinal epithelium into the neonatal circulation (winters, ) . passive transfer of cpv mda through milk has been also demonstrated up to at least days after parturition, such that this continued lactogenic immunity may contribute further to protection from cpv infection (decaro et al., ) . mda titres decline exponentially over time, with cpv-specific mda half-lives in serum ranging from . to . days (parrish et al., ; pollock and carmichael, ; mila et al., ) , although they can persist for - weeks (pollock and carmichael, ; buonavoglia et al., ) . maternal immunity represents the first defence of pups against infectious diseases. a correlation between passive immune transfer, in terms of the titre of cpv mda absorbed, and duration of protection against parvovirus infection in weaning puppies was observed (mila et al., ) . in another study, pups with high cpv-specific mda titres were protected against virulent challenge, while pups with intermediate and low or absent cpv-specific mda titres developed mild and severe disease, respectively . mda are able to block active immunization after administration of cpv vaccines (pollock and carmichael, ; buonavoglia et al., ; chappuis, ) . vaccination of puppies with interfering mda titres (hi titres > : ) may result in lack of seroconversion due to neutralisation of vaccine viral antigen by maternal antibodies. since only hi titres ≥ : are considered protective against infection by field strains, there is a period, known as the "window of susceptibility" or "immunity gap", usually lasting - weeks, during which the mda titre falls below that required for protection but is able to neutralise vaccine virus. during this period pups can be infected and sometimes develop disease (pollock and j o u r n a l p r e -p r o o f carmichael, ) . indeed, more recent studies have demonstrated that the cpv variants are able to induce active infection (and disease) even in the presence of mda titres previously considered protective, i.e., : - : . similarly, some vaccines are claimed to confer protection in pups with mda titres previously reported as interfering (n. decaro, personal observation). different strategies have been proposed to overcome mda interference, including i) determination of mda titres; ii) use of high-titre vaccines, and iii) alternative routes of vaccine administration. mda titration at - weeks of age through the hi test, which represents the reference standard for detection or determination of antibodies titres against cpv, can be useful to predict the best time to vaccinate pups, taking into account the curve of mda decline, which is based on the mda half-life. for instance, if a pup displays an hi antibody titre of : at weeks of age, considering an mda median half-life of days, the optimal period for vaccination could be estimated approximately at - weeks, when mda will presumably drop below titres of : - : . although this approach can be cumbersome, requiring serum collection and delivery to a specialised laboratory, future validation of in-clinic tests, currently registered for assessment of post-vaccinal antibody response, for mda titre determination may facilitate routine use of this strategy (p. dall'ara, manuscript in preparation). administering cpv vaccines via alternative routes to the parental one may help limiting vaccine virus neutralisation by mda. experimental vaccines, based on mlv cpv- b, have been administered intranasally and are proven to partially overcome mda interference (buonavoglia et al., ; martella et al., ) . more recently, the oral administration of a commercially available cpv monovalent vaccine was also proven to be effective in overcoming the mda interference (cavalli et al., ) . however, no commercially available cpv vaccine is currently registered for oral administration, so that j o u r n a l p r e -p r o o f administration of cpv vaccines via alternative routes to the parental is considered off-label. in addition, according to the wsava vaccination guidelines, these alternative routes of cpv vaccination are not as effective as parental (subcutaneous or intramuscular) vaccination (day et al., ) another strategy to overcome the mda interference is the use of high-titre vaccines (burtonboy et al., ; buonavoglia et al., ; hoare et al., ; de cramer et al., ) . these vaccines, also commercially available, have the advantage of containing viral titres - logs higher than those of traditional vaccines, such that in the presence of intermediate mda titres not all viral particles are neutralised and are able to infect the vaccinated dog inducing an active immune response (truyen, ) . humoral immunity is the primary mechanism of protective immunity against cpv. the duration of immunity depends on the persistence of memory b and t-cells and longlived plasma-cells, or "memory effector b cells", which synthesise cpv-specific antibodies for years subsequent to the initial challenge or vaccination (shultz, ) . in veterinary medicine, primary vaccine failures that are considered to be genetic non-responders include dogs that repeatedly fail to develop detectable antibodies after both the primary vaccination course (finishing at weeks of age or older) and subsequent revaccination (kennedy et al., b; day et al., ) . a strong genetic factor associated with primary immunisation failure in humans is the major histocompatibility complex, encoded for by the human leukocyte antigen (hla) gene complex. certain hla type ii haplotypes are risk factors for primary immunisation failure to specific vaccines such as hepatitis b or influenza (gelder et al., ) . some canine breeds have been reported to be at higher risk of primary immunisation failure to cpv vaccines, j o u r n a l p r e -p r o o f including rottweilers and doberman pinschers (houston et al., ) . dog leukocyte antigen (dla) type ii haplotype diversity in dogs varies widely between but not within breeds and is restricted in rottweiler as compared to other breeds (kennedy et al., a) . these dogs are assumed to have a higher risk for canine parvovirosis and should be excluded from breeding. however, direct associations between primary vaccine failure and dla haplotype have not been investigated in dogs. while the incidence of genetic non-responders among cpvvaccinated dogs has been estimated at one in dogs, (day et al., ) epidemiological data are lacking, and prospective studies to evaluate the prevalence of primary vaccine failures are needed. reversion of cpv mlv vaccine strains to virulent virus is theoretically possible, but has not been confirmed as a cause of immunisation failure. there is also the chance that the vaccine itself can cause disease after administration. an investigation of dogs that developed severe gastroenteritis shortly after cpv vaccination used a minor groove binder probe quantitative pcr assay to differentiate between vaccine and field strains of cpv (decaro et al., b) . disease was confirmed to be caused by field strains of cpv in over half of the samples ( of ), which contained either field strains alone (n = ) or together with the vaccine strain (n = ). in samples in which vaccine virus was detected but not a field strain, reversion to virulence of cpv mlv vaccine strains was considered unlikely, since co-infections with other pathogens including ccov, cdv and cystoisospora canis were detected. in three pups in which only the cpv mlv vaccine strains were detected, quantities of vaccine virus in faeces were lower than those associated with disease from cpv field strains, ranging from . x to . x dna copies/mg, thus suggesting that the vaccine virus did not have an etiological role in the onset of diarrhoea (decaro et al., b) . although so far errors in manufacturing have not been documented for cpv vaccines, theoretically vaccine design and manufacture may affect the immunogenicity of a particular batch of vaccine. tempesta et al. ( ) reported that an experimental cpv vaccine strain displayed a shorter vp /vp gene, thus leading to the hypothesis that defective particles (non-replicating virus) may hamper the development of a strong immune response. however, large manufacturers marketing their vaccines at the international level are subjected to strict control quality and potency tests that reduce the chance to licence low-quality products (day et al., ) . taking into account that vaccines must be stored at optimal temperatures of - °c, incorrect storage and transportation, i.e., interruption of the cold chain, may potentially inactivate mlv vaccines, especially in warm climate regions (day et al., ) . in some countries, vaccines are transported long distances from the point of manufacture or importation to the veterinary practice, which may hamper the continuation of the cold chain. however, incorrect storage and transportation are unlikely to affect the viability of cpv, due to its environmental resilience, in contrast to cdv, for which the interruption of the cold chain has been involved in apparent vaccination failures (van de bildt et al., ) . the ability of old-type (cpv- based) vaccines to protect against the cpv variants is a topic of debate among parvovirologists. there is concern that the antigenic differences from the currently circulating field strains may decrease the effectiveness of cpv- based vaccines (greenwood et al., ; yule et al., ; pratelli et al., ) . in-vitro cross-neutralisation studies have demonstrated that there is a one-way cross-reactivity between the cpv variants j o u r n a l p r e -p r o o f and the old type cpv- . animals vaccinated with cpv- displayed significant lower virus neutralising (vn) antibody titres against cpv- a, cpv- b and cpv- c with respect to the homologous virus (cavalli et al., ) . in a korean study, the poor cross-reactivity between cpv- and the cpv variants was evident not only by vn that is able to detect protective antibodies, but also by hi, which also detects non-neutralising antibodies (kang et al., ) . accordingly, there are an increasing number of case reports describing the occurrence of parvoviral disease, mainly caused by cpv- c, in dogs "regularly" vaccinated with cpv- . one outbreak occurred in a breeding kennel in italy, affecting dogs aged between months and . years and leading to the death of a -month-old bernese mountain dog pregnant bitch (decaro et al., a) . in that kennel, all adult dogs had not only completed their primary course of vaccinations as pups, but they had also received annual boosters. similarly, a cpv clinical case occurred in a -year-old crossbreed bitch, which had received annual vaccinations using a cpv- vaccine (decaro et al., ) . subsequent studies also reported the occurrence of parvoviral disease in vaccinated dogs, but in most cases the vaccines administered were unknown and the infected dogs had not completed a primary vaccination course as pups. in addition, it could not be ruled out that the few adult animals that had received multiple boosters were non-responders (calderon et al., ; mittal et al., ; woolford et al., ) . in a comprehensive study aiming to assess the distribution of the three cpv variants in italy in a -year period, about a third ( . %) of cpv-infected dogs had been vaccinated, raising doubts regarding the ability of the vaccine to confer protective herd immunity (battilani et al., ) . however, in this study although dogs were categorised as "completely" or "incompletely" vaccinated, these terms were not defined, and vaccination protocols were unknown. since the median age of affected dogs was months, immunisation j o u r n a l p r e -p r o o f failure in many of these cases may have been due to mda interference. similarly, the immunisation failures reported in one australian study were likely caused not by genetic variation of cpv field viruses but by mda interference in the response of pups to vaccination (meers et al., ) . despite the number of reports, sometimes anecdotal, concerning the putative lack of efficacy of traditional vaccines against the cpv variants currently circulating in the field, several studies have demonstrated that currently available vaccines, including those prepared with cpv- , confer a good degree of protection against cpv- a, cpv- b and cpv- c (brunet et al., ; larson and schultz, ; spibey et al., ; siedek et al., ; von reitzenstein et al., ; wilson et al., ; glover et al., ) . most of these studies have been recently reviewed by hernández-blanco and catala-lópez ( ) , showing that cpv- vaccines were generally beneficial in terms of significant reduction of clinical signs and virus shedding caused by subsequent challenge with field isolates. however, in one of these studies, which was the largest study in terms of the number of vaccinates (n = ), leukopenia, diarrhoea and vomiting occurred in some vaccinated pups, although vaccination was carried out in the absence of mda interference since all dogs were specific-pathogen free (von reitzenstein et al., ) . in the same study, the cpv- vaccine was able to prevent faecal shedding of the challenge virus (cpv- c), although viral shedding was evaluated by virus isolation, which has been proven to be poorly sensitive . a recent study by altman et al. ( ) found no strict correlation between immunisation failures and the antigenic cpv type contained in the vaccines, whereas the main risk factor identified was early termination of the primary vaccination course schedule. accordingly, both wsava and aaha claim that all cpv mlv vaccines are expected to provide protection from disease caused by any cpv field variant currently recognised (day et fig. reports schematically the main factors affecting eradication of cpv disease. in countries where dogs' vaccination programmes are extensively carried out, cdv and cadv- circulation has been considerably reduced, so that outbreaks caused by these two viruses are now only sporadically reported (decaro et al. a; maclachlan and dubovi, ; mitchell et al., ) . one almost insurmountable challenge to canine distemper and infectious canine hepatitis eradication is the existence of wildlife reservoirs of infection in wild carnivores (decaro et al., b; beineke et al., ) . importantly, cdv and cadv- outbreaks in wildlife seem to represent spillover events of wide virus circulation in domestic dogs di sabatino et al., dowgier et al., ) , although spillback events from wildlife reservoir hosts to domestic animals occur (kapil and yeary, ). in addition, autochthonous circulation of cadv- in european red foxes was demonstrated, which may represent a potential threat to domestic dogs (balboni et al., ; verin et al., ) . analogously, a diverse range of wildlife in the order carnivora can be infected by cpv, in which subclinical infection appears to be common, or may even be the norm (allison et al., ) . thus, there appear to be significant wildlife reservoirs of cpv, and viral transmission between domestic dogs and wildlife is frequent and bidirectional (allison et al., ) . one recent study analysed the geospatial distribution of wild canids in australia and cpv cases in owned dogs, and found that postcodes in which cpv cases were reported were significantly correlated with the presence of wild canids (van arkel et al., j o u r n a l p r e -p r o o f vaccination against cpv, cdv and cadv- is not performed extensively in many areas of the world, especially in developing countries, such that these areas can represent pockets of infections that can spillover into countries where virus circulation has been reduced (martella et al., ; decaro et al., b decaro et al., , . this, however, does not explain why canine distemper and infectious hepatitis are well controlled by vaccination, while canine parvovirosis still represents a great threat to the canine population. an explanation could be that both maternal immunity towards cpv and window of susceptibility are longerlasting as compared to the situation for cdv and cadv- (griot et al., ; day et al., ; n. decaro, personal observation) . in addition, in contrast to cdv and, to a lesser extent, cadv- , where survival persistence outside the host is relatively short-lived, cpv is resilient, environmentally persistent and able to exist outside the host in favourable environments for months or longer (greene and decaro, ) . cpv is also resistant to commonly used disinfectants (e.g. quaternary ammonium compounds), although a . % sodium hypochlorite solution displayed a good efficacy against the virus (cavalli et al., ) . these factors present another major challenge to halting transmission of cpv, which is mainly indirect through fomites (greene and decaro, ) . the relative impact of environmental reservoirs of cpv on disease transmission is incompletely understood. however, studies that found an association between occurrence of cpv and periods of lower rainfall, suggested that extended dry periods may contribute to environmental persistence and increased risk of exposure (kelman et al., a; rika-heke et al., ) . despite intensive vaccination (at least in developed countries), cpv infection remains a leading cause of death from infectious disease amongst domestic dogs worldwide and at the moment we are far from disease eradication. immunisation failures are uncommon and are j o u r n a l p r e -p r o o f mostly the result of interference from mda in pups under the age of weeks. more research is required to determine the prevalence and genetic causes of non-responders. the most effective tools available for disease prevention are antibody testing to determine the optimal time for vaccination of both pups and adults, and homogeneous coverage of dog populations by vaccination with cpv mlv vaccines in accordance with current global guidelines (day et al., ) . antibody testing to determine the optimal time for vaccinations of pups and adults and homogeneous coverage of dog population are the next logical steps towards achieving disease eradication or at least an improvement of the current situation. none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. are vaccine strain, type or administration protocol risk factors for canine parvovirus vaccine failure? host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species molecular epidemiology of canine adenovirus type and type in free-ranging red foxes (vulpes vulpes) in italy feline panleukopenia: a re-emergent disease molecular epidemiology of canine parvovirus type in italy from to : recurrence of the cpv- b variant cross-species transmission of canine distemper virus-an update serum antibody titres to canine parvovirus, adenovirus and distemper virus in dogs in the uk which had not been vaccinated for at least three years efficacy of vaccination with a canine parvovirus type against a virulent challenge with a cpv type c (glu ) persistenza nei cuccioli degli anticorpi di derivazione materna nei confronti del parvovirus del cane e loro interferenza nella risposta sierologica alla vaccinazione response of pups with maternal derived antibody to modified-live canine parvovirus vaccine intranasal vaccination of pups in the presence of maternally derived antibodies to canine parvovirus (cpv). evaluation of minimal immunizing dose evidence for evolution of canine parvovirus type- in italy performance of high titre attenuated canine parvovirus vaccine in pups with maternally derived antibody molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv c in vaccinated dogs evaluation of the antigenic relationships among canine parvovirus type variants in vitro virucidal activity of sodium hypochlorite against canine parvovirus type oral administration of modified live canine parvovirus type b induces systemic immune response neonatal immunity and immunisation in early age: lessons from veterinary medicine vaccination guidelines group (vgg) of the world small animal veterinary association (wsava), . wsava guidelines for the vaccination of dogs and cats efficacy of vaccination at and weeks in the control of canine parvovirus canine parvovirus-a review of epidemiological and diagnostic aspects, with emphasis on type c canine parvovirus post-vaccination shedding: interference with diagnostic assays and correlation with host immune status evaluation of lactogenic immunity to canine parvovirus in pups maternally-derived antibodies in pups and protection from canine parvovirus infection infectious canine hepatitis: an "old" disease reemerging in italy occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnostic dilemma evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type c canine adenoviruses and herpesvirus severe parvovirus in a -year-old dog that had been repeatedly vaccinated characterisation of canine parvovirus strains isolated from cats with feline panleukopenia molecular characterization of canine minute virus associated with neonatal mortality in a litter of jack russell terrier dogs long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination challenge studies for registration of canine core vaccines: is it time to update the european pharmacopoeia? canine parvovirus infection: which diagnostic test for virus? canine distemper and endangered wildlife: is it time for mandatory vaccination of dogs? vaccine lethal distemper in badgers (meles meles) following epidemic in dogs and wolves a molecular survey for selected viral enteropathogens revealed a limited role of canine circovirus in the development of canine acute gastroenteritis sequential circulation of canine adenoviruses and in captive wild carnivores enteropathogen infections in canine puppies: (co-)occurrence, clinical relevance and risk factors aaha canine vaccination guidelines faecal shedding of canine parvovirus after modified-live vaccination in healthy adult dogs associations between human leukocyte antigens and nonresponsiveness to influenza vaccine canine parvovirus (cpv) type b vaccine protects puppies with maternal antibodies to cpv when challenged with virulent cpv- c virus infectious diseases of the dog and cat comparison of isolates of canine parvovirus by restriction enzyme analysis, and vaccine efficacy against field strains early dna vaccination of puppies against canine distemper in the presence of maternally derived immunity fading immune protection in old age: vaccination in the elderly are licensed canine parvovirus (cpv and cpv b) vaccines able to elicit protection against cpv c subtype in puppies?: a systematic review of controlled clinical trials immunogenicity of a low-passage, hightiter modified live canine parvovirus vaccine in pups with maternally derived antibodies vaccine use and disease prevalence in dogs and cats risk factors associated with parvovirus enteritis in dogs: cases ( - ) prevalence and genetic characterization of canine parvoviruses in korea canine distemper spillover in domestic dogs from urban wildlife socioeconomic, geographic and climatic risk factors for canine parvovirus infection and euthanasia in australia the geographic distribution and financial impact of canine parvovirus in australia canine parvovirus prevention and prevalence: veterinarian perceptions and behaviors canine dla diversity: . disease studies factors influencing the antibody response of dogs vaccinated against rabies long-lived immunity to canine core vaccine antigens in uk dogs as assessed by an in-practice test kit do two current canine parvovirus type and b vaccines provide protection against the new type c variant? prevalence of protective antibody titers for canine distemper virus and canine parvovirus in dogs entering a florida animal shelter risk factors for death from canine parvoviral-related disease in australia prevalence of positive antibody test results for canine parvovirus (cpv) and canine distemper virus (cdv) and response to modified live vaccination against cpv and cdv in dogs entering animal shelters prevalence of serum antibody titers against canine distemper virus and canine parvovirus in dogs hospitalized in an intensive care unit immunogenicity of an intranasally administered modified live canine parvovirus type b vaccine in pups with maternally derived antibodies heterogeneity within the hemagglutinin genes of canine distemper virus (cdv) strains detected in italy fenner's veterinary virology canine distemper epizootic among red foxes serum distemper virus and parvovirus antibody titers among dogs brought to a veterinary hospital for revaccination genetic analysis of canine parvovirus from dogs in australia understanding herd immunity protection against canine parvovirus type infection in puppies by colostrum-derived antibodies duration of serological response to canine parvovirus-type , canine distemper virus, canine adenovirus type and canine parainfluenza virus in client-owned dogs in australia european surveillance of emerging pathogens associated with canine infectious respiratory disease molecular typing of canine parvovirus strains circulating from to in an organized kennel in india reveals the possibility of vaccination failure a novel antigenic variant of canine parvovirus from a vietnamese dog canine parvovirus infections in a colony of dogs natural variation of canine parvovirus rapid antigenic-type replacement and dna sequence evolution of canine parvovirus maternally derived immunity to canine parvovirus infection: transfer, decline and interference with vaccination canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine prevalence of antibodies to canine parvovirus and reaction to vaccination in client-owned, healthy dogs the relationship between the southern oscillation index, rainfall and the occurrence of canine tick paralysis, feline tick paralysis and canine parvovirus in australia serological survey of canine parvovirus antibody titres in breeding kennels in northern italy the new generation of parvovirus vaccines. a comparison study. compendium of continuing education for the practicing veterinarian duration of immunity for canine and feline vaccines: a review age and long-term protective immunity in dogs and cats vaccination with canine parvovirus type (cpv- ) protects against challenge with virulent cpv- b and cpv- c canine parvovirus type vaccine protects against virulent challenge with type c virus evaluation of a community's risk for canine parvovirus and distemper using antibody testing and gis mapping of animal shelter intakes prevalence of antibodies to four major canine viral diseases in dogs in a liverpool hospital population the polymerase chain reaction for the detection of defective interfering canine parvovirus particles evolution of canine parvovirus-a need for new vaccines? clinical use of serum parvovirus and distemper virus antibody titers for determining revaccination strategies in healthy dogs the relationship between reported domestic canine parvovirus cases and wild canid distribution. heliyon , e multicentric molecular and pathologic study on canine adenovirus type in red foxes (vulpes vulpes) in three european countries cross protection of vanguard l -cv vaccine against virulent canine parvovirus- c circulating in the usa limited intrahost diversity and background evolution accompany years of canine parvovirus host adaptation and spread primary vaccine failure to routine vaccines: why and what to do? vaccination of dogs with duramune dappi+lc protects against pathogenic canine parvovirus type c challenge detection of the canine parvovirus c subtype in australian dogs assessing the epidemic potential of rna and dna viruses canine parvovirus vaccine elicits protection from the inflammatory and clinical consequences of the disease key: cord- -ts lyy p authors: pedersen, n.c; elliott, j.b; glasgow, a; poland, a; keel, k title: an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ts lyy p an isolated epizootic of a highly fatal feline calicivirus (fcv) infection, manifested in its severest form by a systemic hemorrhagic-like fever, occurred over a -month period among six cats owned by two different employees and a client of a private veterinary practice. the infection may have started with an unowned shelter kitten that was hospitalized during this same period for a severe atypical upper respiratory infection. the causative agent was isolated from blood and nasal swabs from two cats; the electron microscopic appearance was typical for fcv and capsid gene sequencing showed it to be genetically similar to other less pathogenic field strains. an identical disease syndrome was recreated in laboratory cats through oral inoculation with tissue culture grown virus. during the course of transmission studies in experimental cats, the agent was inadvertently spread by caretakers to an adjoining room containing a group of four normal adult cats. one of the four older cats was found dead and a second was moribund within – h in spite of symptomatic treatment; lesions in these animals were similar to those of the field cats but with the added feature of severe pancreatitis. the mortality in field cats, deliberately infected laboratory cats, and inadvertently infected laboratory cats ranged from – %. this new isolate of calicivirus, named fcv-ari, was neutralized at negligible to low titer by antiserum against the universal fcv-f vaccine strain. cats orally immunized with fcv-f , and then challenge-exposed shortly thereafter with fcv-ari, developed a milder self-limiting form of disease, indicating partial protection. however, all of the field cats, including the three that died, had been previously immunized with parenteral fcv-f vaccine. fcv-ari caused a disease that was reminiscent of rabbit hemorrhagic disease, a highly fatal calicivirus infection of older rabbits. feline calicivirus (fcv) is one of the most common viral pathogens of cats, especially in multi-cat environments such as shelters and catteries. fcv infection and disease occurs in acute and chronic forms (reviewed by studdert, ; reubel et al., ) . the manifesting signs of acute disease depends on the route (oral versus aerosol) and strain of virus. aerosol infection, which is commonly used in laboratory studies, produces a more serious disease than oral infection, with lesions extending further down the respiratory tract. hoover and kahn ( ) described the acute disease produced by ®eld strains of fcv. the disease differed only in severity, with the more virulent strains causing fever, depression, dyspnea, pneumonia, and vesicles/ulcers of the tongue, hard palate and nostrils. lower virulence strains were less likely to affect the lungs, although other signs were similar. transient mild conjunctivitis and rhinitis, has also been associated with acute fcv infection, but unlike pathogens such as feline herpesvirus, upper respiratory involvement is not the major feature of fcv infection. pedersen and colleagues ( ) added the feature of`limping' to the fcv disease syndrome. the lameness appears to be associated with acute viremia and localization of virus and/or immune complexes in the joints (bennett et al., ; dawson et al., ) . following recovery from acute disease, up to one-fourth of cats will shed the virus for a prolonged period of time from their oropharynx (bennett et al., ; harbour et al., ; dawson et al., a, b) . although most fcv carriers are asymptomatic, a small proportion will develop a distinct disease syndrome known as chronic plasmacytic/lymphocytic stomatitis or chronic ulceroproliferative stomatitis (reubel et al., ) . this chronic oral disease is progressive and extremely dif®cult to treat and is perhaps the single most important clinical manifestation of fcv infection today. it is fortunate for cats that fcv is not more virulent. related caliciviruses, such as rabbit hemorrhagic disease virus, have been associated with severe clinical disease and high mortality. although recognized strains of fcv have not been associated with signi®cant acute mortality, the calicivirus genome is highly mutable (johnson, ; pedersen and hawkins, ; geissler et al., ; kreutz et al., ; radford et al., ) and more highly virulent strains could arise at any time. the present report describes the sudden appearance and disappearance of a novel strain of fcv, which induced a severe systemic hemorrhagic-like fever and high mortality. the new strain, named fcv-ari, has weak to negligible cross-reactivity to current vaccine-induced immunity and would pose considerable risk to cats if it were to spread widely among either vaccinated or unvaccinated animals. speci®c pathogen free (spf) cats, of mixed gender, were obtained from the breeding colony of the veterinary nutrition laboratory, school of veterinary medicine, uc davis (compliments of dr. james morris and dr. quinton rogers). cats were from to weeks of age and housed in the experimental animal facilities of the center for companion animal health and cared for under the oversight of the animal resources services, uc davis. complete blood counts, parameter serum chemistries, and various clotting studies were done by either idexx veterinary services, western region, west sacramento, ca, or by the clinical pathology service of the veterinary medical teaching hospital (vmth), school of veterinary medicine, uc davis. gross and/or histopathologic studies were done by either idexx veterinary services or the pathology service of the vmth, uc davis. initial virus isolation were from cats vmth- (ari) and - (ian). swabs of nasal exudate, and herparinized whole blood, were taken from both animals on october . blood ( . ml) was diluted with an equal volume of hank's buffered salt solution (hbss) and centrifuged at Âg for min. the buffy coat was suspended in ml of hbss; the suspended cells were overlayered onto subcon¯uent monolayers of crandell feline kidney (crfk) and felis catus whole fetus- (fcwf- ) cells grown in cm ¯a sks. nasal swabs were placed in ml snap-top tubes containing ml of hbss with  gentamicin and penicillin/streptomycin and allowed to sit for at least h at room temperature. supernatant ( ml) was placed into cm ¯a sks containing subcon¯uent monolayers of crfk or fcwf- cells. cell cultures were observed every h for cytopathic effect (cpe). three -week old spf kittens were infected oronasally with . ml of tissue culturē uid containing approximately  tcid of fcv-ari. one half of the inoculum was instilled up the nostrils and one half placed in back of the throat. complete blood counts and serum chemistries were taken ± h prior to infection and every ± days thereafter. rectal temperatures were recorded starting days prior to infection and daily thereafter. cats were observed for clinical signs of depression, anorexia, limping, subcutaneous swelling and edema, skin lesions, nasal discharge/nasal congestion/ sneezing, diarrhea, and vesicles and/or ulcers of the palate, tongue, gums, cheeks, lips, or skin. for vaccine studies, four ± weeks old spf kittens were immunized oronasally, as described earlier, with ml of tissue culture¯uid containing approximately  tcid of the universal vaccine strain fcv-f (heska subisolate). two sibling kittens were kept strictly isolated and used as unimmunized controls. kittens were monitored for clinical signs of infection as outlined earlier. . . virologic studies . . . fcv strains a number of fcv strains, of vaccine and ®eld origin, were used in this study. the origin and serologic relationships of most of these strains has been previously reported (pedersen and hawkins, ) . vaccine strains, which were genetically identical to each other and to the original fcv-f , were re-isolated from several commercial brands of vaccine (pedersen and hawkins, ) . a nested rt-pcr reaction was used to detect fcv-rna in blood (buffy coat), oral swabs, and feces. forward and reverse primers amplifying a bp region of the capsid gene corresponding to nucleotides ± of the fcv genome were used, following the protocol of radford et al. ( ) . pcr products were run through microcon- columns (millipore, burlington, ma) to remove primers and salts and then submitted to a commercial sequencing laboratory (davis sequencing, davis, ca) for automated sequencing analysis. sequence data were analyzed on a vax computer using the university of wisconsin's genetics computer group (gcg) sequence analysis software package (gcg program manual for the wisconsin package, version , ). a dendrogram (cladogram) was generated by sequence comparison using the cantor-jukes correction, followed by distance calculations using the upgma algorithm. virus neutralizing (vn) antibodies to fcv were measured by a residual virus infectivity assay using constant amounts of virus and antiserum. a diluted tissue culturē uid stock of fcv-f or fcv-ari ( ml), containing approximately tcid , was incubated at c for h with either ml of a : dilution of tissue culture medium (negative control) or a : dilution in tissue culture medium of the cat serum to be tested. ninety-six well microtiter plates containing a monolayer of subcon¯uent crfk cells were used for the titration; each well contained ml of tissue culture¯uid. the virus/antiserum mixture of ml was added to the ®rst well, mixed well, and ml carried serially to each of the next wells in the same manner. this procedure was repeated in triplicate. a similar titration was done using the same virus stock and an equal volume of tissue culture medium alone. the plates were incubated for h and observed under an inverted microscope for typical fcv cpe; the last well containing any detectable cpe was read as endpoint. the vn antibody titer of the serum was calculated from the difference in the average virus titer with and without serum treatment, i.e. if the average virus titer of a sample of virusantiserum was : , and the titer of the corresponding virustissue culture¯uid : , , the difference was six wells ( / ) or a vn titer of : . fcv-ari was infected onto fresh crfk in a cm ¯a sk, and the cultures observed closely for cpe. about h after infection, when cytopathic effect was noticeable but most cells were still attached, the tissue culture medium was decanted and the monolayer overlayered with cold . % paraformaldehyde/ . % glutaraldehyde in . m sorenson's phosphate buffer, ph . . the¯asks were held for several hours at room temperature, and then placed into the refrigerator for several days. post-®xation was in % osmium tetroxide in . m sorenson's phosphate buffer at c. the samples were processed by the electron microscopy service of the dept. of pathology, school of medicine (with special assistance from mr. bob munn). grid staining was with % aqueous uranyl acetate and lead citrate. grids were examined and photographed using a philips em electron microscope. the ®rst tissue analyzed were three full-thickness skin biopsies from the face, nose and ear of the cat named ria. the samples were ®xed in formol-saline and hematoxylin and eosin stained tissue sections prepared by a private laboratory (idexx veterinary services, inc., west sacramento, ca ). subsequent necropsies and gross and microscopic pathologic analyses were done by the veterinary pathology service of the vmth, department of pathology, school of veterinary medicine, university of california, davis, ca . the index case in this outbreak of atypical fcv infection was probably an orphaned, fully vaccinated, -month old, female domestic kitten from a local animal shelter. one of the authors (jbe), noticed that the kitten had a severe upper respiratory infection. therefore, the kitten was brought from the shelter into the clinic on september for hospitalization and treatment. the kitten subsequently developed crusty, ulcerative lesions on her face and oral vesicles on the margins of the tongue and soft palate. the kitten was intensively treated for days before being well enough to be adopted. the kitten intussuscepted week later and died. the second cat in the outbreak was ria, a -year old, spayed female, domestic, strictlyindoors cat owned by a veterinary assistant in the same veterinary clinic. the cat received her last feline panleukopenia-/herpes-/calici-virus (fphcv) immunization on april . the only exposure outside the home was a brief hospitalization for dental prophylaxis on october . ria presented on october with a primary complaint of h of lethargy, anorexia, and fever. physical examination was uninformative except for fever. tests for felv and fiv infections were negative and cbc and blood chemistry analyses were unremarkable. a slight swelling of the dorsal muzzle, extending from one canthus to the other, was observed that afternoon. shortly thereafter, hair over the right side of the muzzle epilated, revealing a mm diameter erythematous patch with a dark necrotic center. ria was sent home for observation, but returned on october still lethargic, anorectic and febrile. additional lesions, with focal crusting, erythema and epilation, were present on the right and left medial canthi, commissures of the mouth, and on the margins of both pinnae. the patient also demonstrated diffuse cutaneous edema of the face and submandibular area and extending down the limbs. biopsies were taken from the muzzle and pinnae lesions and swabs of several sores were collected for routine aerobic bacterial culture. ria was still febrile on october , but did not appear to be in discomfort and was beginning to eat and groom. skin biopsies taken on october revealed multifocal moderate to severe neutrophilic/lymphoplasmacytic perivascular and periadnexal dermatitis, super®cial pyoderma, and extensive ulceration and super®cial dermal necrosis with underlying vasculitis. no infectious organisms were observed in routine or special stained tissues sections and no aerobic bacteria grew in culture. ria was treated with prednisolone and antibiotics; within the next days she was eating well and actively grooming, the cutaneous edema was resolving, and the skin lesions were healing. the third affected cat, indy, was a -month old, domestic, indoor/outdoor cat. he was brought to the hospital on october for castration and his ®nal parenteral fphcv immunization. indy returned to the hospital on october with depression and fever. abnormalities on a standard blood chemistry panel included a mild increase in creatinine phosphokinase (cpk) and total bilirubin. felv and fiv tests were negative and a coronavirus ifa antibody titer was : . indy was treated with iv electrolyte solution and antibiotics. the fever was resolved by october , but ulcerative lesions appeared on the margins of both pinnae on october. the cat made an uneventful recovery. the fourth cat, garth, was a -year old, castrated male, domestic, indoor/outdoor cat and the housemate of indy. the cat had received the recommended series of parenteral fphcv immunizations as a kitten and yearly boosters. garth was seen on october for mild conjunctivitis and squinting and sent home on ophthalmic antibiotic ointment. the cat appeared to make an uneventful recovery, but weeks later the owner noticed focal areas of alopecia and encrustation on the muzzle, base of the right pinna, and bilaterally on both¯anks, compatible with old healing ulcers. the ®fth cat in this focal epidemic was aristotle (ari), a . -year old, neutered male, domestic, indoors-only cat and the son of ria. there were no signi®cant past health problems; he tested negative for felv and fiv infections and had been parenterally vaccinated against rabies and fphcv on april ari presented on october with a history of an acute onset of lethargy and anorexia of days duration. a slight lameness in the right foreleg was noted days prior but had spontaneously resolved. the patient had vomited a bile tinged¯uid with¯ecks of red blood. physical examination revealed fever and a small crusty lesion on the lower lip. ari returned to the hospital on october afebrile and alert, but moderately dehydrated and anorectic. diffuse facial edema with central ulceration were noted, primarily over the muzzle and left pinna; focal crusty and erythematous lesions were also present on the upper and lower lips. intravenous¯uids, metoclopramide and antibiotic therapy were instituted. ari was afebrile on october but anorectic. the facial swelling was mildly increased but the central ulcerated areas seemed to be less exudative. the patient's right forepaw was slightly edematous. ari was still afebrile on october but remained anorectic; the edema of the right forepaw remained unchanged. the patient was transported to the uc davis vmth for additional testing, including blood and nasal swabs for virus isolation and blood for chemistry and coagulation pro®les. blood chemistries revealed moderate elevation of cpk, mild elevation of total bilirubin, and moderate hypoproteinemia. there was evidence of a coagulopathy with moderately increased prothrombin and partial thromboplastin times and a slightly prolonged activated clotting time (act). a fcv was isolated in cell cultures exposed to both blood and nasal exudate. ari was still euthermic on october ; however, he was anorectic and his entire face was edematous. the facial lesions had coalesced to form large irregular crusts on both sides of the muzzle (fig. ) , and the mucus membranes were slightly icteric. a nasoesophogeal feeding tube was positioned and oral hyperalimentation instituted; this therapy that would be continued until his death. ari seemed more responsive on october , although still anorectic and hypocoagulable. the patient appeared comfortable on october ; there was no more vomiting, but the subcutaneous edema of the right foreleg was persisting. ari was transfused with one unit of whole blood and low level heparin therapy was instituted to counteract a suspected disseminated intravascular coagulopathy (dic). dextran was given initially as an iv bolus and then maintained as a continuous infusion. laboratory tests taken in the evening of october indicated a persisting coagulopathy, hypoproteinemia, and hyperbilibubinemia. ari was alert, responsive, and afebrile on the morning of october; however, he was diffusely edematous, had mild watery diarrhea and exhibited a slightly increased respiratory rate and effort. laboratory work revealed a decreased hematocrit, hypoproteinemia and greatly elevated act. the patient was then given a second unit of cross matched whole blood. the respiratory rate continued to increase throughout the day and a thoracocentesis was performed late that evening and a yellowish-transudate withdrawn. ari was alert and responsive on october , but still anorectic. the patient received a third unit of whole blood at midday and more pleural¯uid was removed. on october , ari was quiet, responsive and vocal when handled, but polypneic and totally anorectic. a chest tube was placed to facilitate thoracic drainage and the dextran replaced with hetastarch. ari was still anorectic and lethargic on the morning of october; he was diffusely edematous with small areas of miliary crusting noted in areas of greatest swelling, especially on the distal limbs. despite treatment, ari's condition slowly deteriorated and he died on november from complications of pneumothorax, shock and cardiac arrest. a fcv, genetically indistinguishable from the earlier isolate, was recovered from blood and tissues at postmortem examination. ian, a . -year old, neutered male, domestic, indoors-only cat, also a son of ria, was the sixth cat to be affected. on october , the owner observed that ian was febrile and manifesting signs similar to those shown by his mother. previous clinical history included a negative felv/fiv test on august and routine parenteral vaccination for rabies and fphcv on june . ian became febrile on october and was lethargic and sneezing occassionally. physical examination revealed mild edema of his muzzle with patchy erythematous macules and miliary crusts on margins of both pinnae. ian was transported to uc davis, vmth for consultations with one of the authors (ncp) and a veterinary dermatolgist (dr. peter ihrke). blood and nasal swabs were collected and the cat returned home. a fcv, which was genetically identical to the isolate from ari, was obtained in tissue cultures from both blood and nasal exudate. on october , ian was still febrile at home but was eating and drinking. the facial and pinna swellings were increasing and new crusts were present on his pinnae and around his nares. ian continued to do well at home on october, although the owner reported sneezing and nasal congestion. ian was still sneezing and congested on october and the nasal and ear lesions had begun to coalesce. his appetite and attitude remained very good and he made an uneventful recovery. the seventh cat in the outbreak was emma, a . -year old spayed female, domestic, who was kept strictly indoors. emma tested negative for felv and fiv as a kitten and was last vaccinated for felv and fphcv on june . emma was owned by a second technician who worked at the same practice. emma presented on november with a history of days of lethargy and anorexia. her physical examination was unremarkable except for fever, moderate dehydration and possible abdominal tenderness. the patient was hospitalized and started on intravenous¯uids and antibiotics. presenting lab work revealed a slightly elevated total blood bilirubin and glucose. felv and fiv tests were negative. on november , emma was depressed, anorectic, febrile and hyperirritable. the fever increased by evening and her left hind paw appeared swollen. emma was still anorectic on november but was much more alert; rectal temperatures continued to be elevated and her right hind paw was swollen. emma remained anorectic on november , her hind paws remained swollen and slight swelling was observed on the muzzle. small areas of ecchymosis were noted on the right caudal abdomen. a naso-esophageal feeding tube was inserted and the patient was started on a full strength hyperalimentation solution. the next morning emma became restless and mildly dyspneic, with pronounced decreases in the hematocrit and total plasma proteins. emma was started on vigorous treatment for a developing hypoproteinemia and dic; therapy included intravenous hetastarch, subcutaneous heparin, intravenous antibiotics, and nasoesophageal tube feeding. the patient's respiratory rate continued to increase throughout the day even though her fever abated. a chest tube was placed but only small amounts of a transudate were withdrawn. she became progressively more dypneic and depressed throughout the day and was hypothermic by midnight. results of laboratory tests taken in the evening revealed a moderate hypoproteinemia, hyperbilirubinemia and thrombocytopenia. blood tests taken on november showed an elevated glucose, decreased hematocrit and total protein, and a prolonged act. emma's condition continued to decline throughout the day, with increasing respiratory dif®culty. lasix and dobutamine were administered by intravenous drip and intravenous hetastarch and nasoesophageal tube feeding discontinued. on november , emma's condition continued to decline overnight and the owner elected to have the cat euthanatized. affected cats eight through ( - , - , - and - ) were . ± . years old, neutered male, moderately obese, laboratory cats that were housed two rooms away from kittens that had been experimentally infected with an fcv isolate obtained from the blood of ari (see section . ). the four cats had not been exposed to other agents and were quarantined from other cats through separate ante-rooms, and were cared for by different caretakers. the caretakers took extra precautions in terms of disposable overalls, gloves, boots, and foot baths, as well as separate food and litter sources, to prevent inadvertent infections from the outside. on the morning of november , one of the four cats ( - ) in this group was found dead and the other three animals were depressed and febrile. gross necropsy ®ndings showed a reddened and somewhat swollen pancreas with areas of hemorrhage and saponi®cation in peripancreatic mesenteric fat. several large areas of yellow-tinged subcutaneous edema were observed in the groin and trunks. the lungs appeared grossly congested with many areas of collapse, and there was a small amount of free¯uid in the abdomen and chest cavities. the condition of the remaining three cats rapidly deteriorated, in spite of antibiotic,¯uid and whole blood therapy. cat - was in shock on november and euthanatized. the remaining two cats ( - and - ) were still febrile on november but were becoming rapidly more depressed; a decision was made to euthanatize them rather than to continue symptomatic treatment. blood chemistry panels taken near the time of death from all four animals revealed mild to moderate elevations in blood glucose, moderately decreased serum total proteins, and mild to moderate hyperbilirubinemia. gross necropsy ®ndings in the three latter cats were identical to those observed in the ®rst cat. all three cats had reddened and swollenappearing pancreases with hemorrhage and saponi®cation of peripancreatic fat. in addition to these ®ndings, cat - had an edematous omentum, ulceration of the skin on the phalanges of the right hindleg, sloughing of the claw on second digit of the right hindleg, an ulcer of the skin under the jaw, a yellowish edema under the tongue and icteric appearing nictitating membranes. cat - , in addition to pancreatic lesions, had congestion and consolidation of the right lung lobes, an edematous omentum, and splenomegaly. cat - had identical lesions on the pancreas and surrounding tissues, but also had subcutaneous edema with yellowing of the tissues of the hind limbs and edema of all four paws. crfk and fcwf- cell cultures of blood and pancreatic tissue from all four cats yielded fcv. genetic sequencing of fcv isolates from all four cats showed them to be identical to fcv-ari (data not shown). maximum quarantine was initiated at the time of the secondary outbreak; the virus was only handled under the strictest of quarantine conditions, with special isolation facilities, separate caretakers, complete changes of disposable coveralls, gloves, and foot coverings. no additional cases were observed in either the clinic of origin or in the experimental animal facilities after this time. cytopathic effect, typical of fcv, was evident within h on both crfk and fcwf- cells cultures exposed to blood and nasal secretions of ari. transmission electron micrographs of the cells demonstrated characteristic fcv capsids, ± nm in diameter, within paracrystalline viral arrays in the nuclei (not shown) and parallel linear viral arrays in the cytoplasm (fig. ) . rna was extracted from infected cultures, reverse transcribed, and subjected to pcr. the pcr product was sequenced (table ) and proved to be identical, within the range of strain variation, of a number of vaccine-and ®eld-origin fcvs (fig. ) . the virus isolate was designated fcv-ari. three -week old spf kittens, - , - , and - , were infected oronasally with fcv-ari on october . the kittens were febrile by h post-inoculation. the fever continued unabated for the course of the study, while other signs appeared in rapid progression. evidence of nasal congestion with occasional sneezing and slight ocular discharge were noticeable by the second day. nasal congestion was associated in all three cats with subcutaneous edema over the bridge or side of the nose. in one cat, a shallow ulcer appeared over the site of edema. the feet and lower limbs of the kittens also became edematous. all of the infected cats were anorectic after day and required subcutaneous uid therapy to maintain hydration. cat - was in shock on november and was given ml of fresh whole blood intravenously. he failed to rally and was euthanatized on november . the remaining two cats, - and - , began to show progressive improvement from november onward and were essentially normal by november . signi®cant abnormalities, including hypoproteinemia with hypoalbuminemia and hypoglobulinemia, hyperglucosemia, and hyperbilibubinemia were seen at the peak of illness in cat - on november . hyperbilibubinemia was the only signi®cant ®nding on the remaining two animals. virus was readily isolated from both blood and nasal secretions of the experimentally infected cats. the genetic sequence of pcr products from the two isolates that were tested was identical to fcv-ari isolates obtained from the ®eld cases. detailed necropsy and histopathologic studies were done on ari, four laboratory cats inadvertently infected with fcv-ari, and on four laboratory cats deliberately infected with fcv-ari. gross lesions present on ari included alopecia of the rear limbs, inguinal and perineal regions and the dorsal aspects of the forelimbs, with dozens of ulcers ranging from . ± cm in diameter within alopecic foci. a small vesicle was present on the left carpus and all of the digital pads were ulcerated. small ulcers were also present on the lateral aspects of the gum and medial buccal mucosa. subcutaneous edema was prominent on the lateral body wall, and numerous irregular chalky, white foci were widely disseminated in the subcutaneous fat. the lungs were reddened and atelectatic and ml of a pinkish milky¯uid was present in the pleural cavity. fifty or more miliary grey foci were randomly distributed on the surface of all lung lobes. the cranial mediastinum was thickened and a thrombus was present in the right jugular vein. the abdomen contained about ml of serosanguinous¯uid with ®brin clots and ®brin tags adherent to the surface of the viscera. a . cm . cm ulcer was present in the gastric cardia, and smaller ulcer was also observed on the mucosa of the trigone of the bladder. histopathologic examination showed a severe multifocal epidermal necrosis with ulceration. there was a chronic suppurative necrotizing bronchopneumonia with squamous metaplasia and intralesional fungal hyphae consistent with aspergillus sp. the pulmonary aspergillosis was presumed to be a terminal sequelae of the persistent calicivirus infection, debilitation of the immune system, and prolonged antibiotic therapy. the lung tissue was severely atelectatic, with intraalveolar histiocytic exudate and multifocal vascular thrombosis. a moderately severe and chronic pleuritis was present. tracheobronchial lymph nodes manifested widespread ®brinous sinus thrombi and moderate lymphoid depletion. a severe diffuse and chronic thrombosis was evident in subcutaneous vessels with associated fat necrosis. the spleen showed marked red-pulp histiocytosis. cat - , which was representative of the four laboratory animals that were inadvertently infected with fcv-ari, demonstrated several large areas of subcutaneous yellowish edema, diffuse consolidation of the lungs, swelling and reddening of the pancreas, and numerous chalk-like plaques with surrounding hemorrhage were present in the peripancreatic fat. histopathologic ®ndings included a moderate to severe multifocal pancreatitis with a mixed in¯ammatory in®ltrate, peripancreatic fat necrosis, mild to moderately severe multifocal crypt necrosis in the duodenum and colon, moderately severe and diffuse individual hepatocellular necrosis with mild periportal lymphocytic/ histiocytic in®ltrate, and acute multifocal interstitial pneumonia. gross ®ndings in laboratory cats ( - , - , - , - ) , which were purposefully infected with fcv-ari, were similar, and included patchy to diffuse consolidation of the lungs, small amounts of free pleural¯uid, and areas of subcutaneous edema, especially around the face. intestinal crypt lesions were apparent in all four animals; crypts were lined with large pleomorphic, or¯attened, epithelial cells and necrotic cells and cellular debris often ®lled the crypt lumens. peyer's patches were hyperplastic, and the sinuses of lymph nodes were expanded by numerous foamy macrophages and subcapsular neutrophilic in®ltrate. paracortical lymphoid hyperplasia with abundant apoptotic cells were also evident. the medullary centers of the thymus were often expanded by ®brin and eosinophilic cellular debris. a foci of pancreatic necrosis with in¯ammatory in®ltrate was noted in one animal. pulmonary changes included a mild multifocal atelectasis with a few neutrophils and lymphocytes present in the interstitium of capillaries and small airways. rare in¯ammatory cells were present in the lumen of pulmonary capillaries and bronchioles. multiple randomly situated foci of necrotic hepatocytes, with or without a small number of neutrophils, were observed. marked diffuse edema and epidermal necrosis were present in sections of diseased appearing skin. four -week old spf cats ( - , - , - and - ) were immunized orally with fcv-f and serum collected weeks later. these sera were then tested in a virus neutralization assay against fcv-f and fcv-ari (table ). there was low to negligible cross-reactivity between fcv-f immune sera and fcv-ari. serum collected from cat - weeks following fcv-ari infection was titrated for virus neutralizing activity against a number of vaccine and ®eld strains of fcv (table ) . fcv-ari antiserum neutralized all four vaccine strains (three containing fcv-f ), but failed to neutralize / ®eld isolates. six spf kittens were divided into vaccine-(cats - , - , - and - ) and control-groups (cats - , - ) . the vaccine group was inoculated orally on january with . ml of a tissue culture supernatant containing approximately  table virus neutralizing titer of serum from fcv-ari recovered cat - against several vaccine and ®eld strains of table ). the four recently vaccinated cats, along with two additional unvaccinated animals, were challenge-exposed to fcv-ari on february , which was weeks postvaccination. the route and dosage were identical to the initial experimental infection study. both vaccinated and unvaccinated cats became febrile within h, and the fever persisted for ± days (fig. ) . the febrile responses were slightly lower and about day shorter in duration in vaccinated versus unvaccinated cats. clinical signs were similar in both groups, but noticeably less severe in vaccinates than nonvaccinates. clinical signs included transient anorexia, dehydration, lameness, slight ocular and nasal discharge, mild sneezing, swelling of the face with occasional ulceration, edema of the pinnae with ulceration or crusting of the edges, and variable swelling of the lower limbs. there table the oral shedding (culture/pcr) of feline calicivirus by fcv-f vaccinated cats, following vaccination and after challenge exposure to fcv-ari at week post-immunization a weeks post oral vaccination with fcv-f appeared to be no relationship between whether the cats were still shedding fcv-f vaccine virus at the time of challenge-exposure and subsequent disease severity; cat - , a vaccine virus carrier, was almost as symptomatic as the nonvaccinates. both vaccinated and unvaccinated cats were treated with subcutaneous lactated ringers solution for several days, but all animals made a rapid uncomplicated recovery starting at days ± post-infection. virus shedding, as measured by both tissue culture isolation and pcr, from the oral cavity was observed in both nonvaccinates following fcv-ari challengeexposure and in / vaccinates (table ). virus shedding lasted from ± weeks; no cats were still shedding calicivirus by culture or pcr after week . virus shed at the time of fcv-ari challenge-exposure and earlier was of the f strain, while virus shed after challenge was entirely of the fcv-ari strain as determined by sequence analysis (data not shown). fcv is endemic in most catteries, shelters and large multiple cat households, where up to one-fourth of cats may be orally shedding the virus at any given time (bennett et al., ; harbour et al., ; dawson et al., a, b) . fcv exists in numerous overlapping serotypes but is basically a single species (gillespie and scott, ) . different strains vary somewhat in the main types of disease signs that they cause pedersen et al., ; pedersen and hawkins, ) , but these differences are hard to discern from genetic or serologic comparisons (dawson et al., a, b; pedersen and hawkins, ; geissler et al., ; teewee et al., ) . although original descriptions of fcv infection suggested that it might be a major cause of mortality in young cats, this has not been borne out by subsequent experimental studies. so-called highly virulent strains of fcv, such as fcv- (gillespie and scott, ) , can exist in breeding populations of cats without signi®cant disease; kittens being infected from ± weeks of age when they still have signi®cant levels of passive immunity (johnson and povey, ) . clinical disease can be of acute or chronic form. acute disease, when it is seen, is generally systemic with vesicular involvement of the oral cavity (ulcers on palate, tongue, in¯ammation of gums and oral fauces), arthropathy (limping) and mild interstitial pneumonia pedersen et al., ; pedersen and hawkins, ; teewee et al., ) . upper respiratory disease is relatively uncommon, associated with certain strains much more than others, and manifested as a mild and transient bilateral conjunctivitis and rhinitis (compare fcv- and fcv-cook in pedersen and hawkins, ) . a viremia is detectable during the ®rst week of infection (pedersen et al., ) , and oral shedding occurs for several weeks to months (reviewed by pedersen and hawkins, ) . the source of viral carriage and shedding appears to be the tonsils and surrounding mucosa (dick et al., ) . there seemed to be little doubt that the infection in the ®rst six client-owned cats was nosocomial. however, catteries and shelters are more common sources of infection than veterinary clinics, an observation which may have been borne out by the theorized index case in this outbreak, a -month old female domestic kitten that had been brought into the hospital from a local shelter and treated for a severe atypical viral infection during the period of september to october . the dates that this particular animal was in the clinic both preceded and overlapped the dates when client-owned cats were exposed. this kitten was receiving almost constant medical attention and contamination of hands, clothes, instruments, etc., would have been dif®cult to prevent under the strictest of conditions. the disease caused by fcv-ari in both natural and experimental infections appeared to target blood vessels, as evidenced by the severe edema (sometimes with hemorrhage) in subcutaneous tissues and lungs and local necrosis of skin and adipose tissues. although vasculitis was dif®cult to discern as a distinct lesion in tissues of laboratory infected cats, it was de®nitely a feature of the early skin lesions that were ®rst observed on the cat ria. loss of vascular integrity was also the best explanation for the signi®cant drop in serum proteins, icteric serum (from breakdown of extravasated red blood cells), variable thrombocytopenia, and coagulopathies. elevations in cpk also indicated myonecrosis. the noteworthy feature of the infection was its persistence in the blood of those cases that went on to die; virus was still present in the blood of ari at the time of his death, almost weeks after ®rst clinical presentation. persistence of viremia may also have explained the death of the index kitten after a -week course of illness. the type of disease caused by fcv-ari is highly reminiscent of rabbit hemorrhagic disease, also caused by a calicivirus and ®rst reported in china (liu et al., ) . similarities included the high mortality, acute nature, the tendency to cause more severe disease in older animals, the ease of spread, hepatocyte tropism, and widespread vascular disease. following its apperance in china, rhd spread throughout europe, where it caused severe disease in rabbits but mild to unapparent infections in hares (mitro and krauss, ) . a closely related calicivirus, the european brown hare syndrome virus (ebhs virus) (bascun Äana et al., ) , affects both wild and farmed hares, and was also ®rst reported in the s in europe. both viruses cause severe necrotizing hepatitis and widespread hemorrhaging. the disease caused by the rhdv is particularly severe, with mortalities approaching % in rabbits older than weeks of age (nowotny et al., (nowotny et al., , . the high species speci®city and virulence of rhdv led the australian government to experiment on the virus as a means to control wild rabbits (kovaliski, ) . experiments conducted on an offshore island showed the potential of the virus as a biologic agent, but before a ®nal decision for controlled release could be made, the rhdv appeared in wild rabbits on the mainland. the virus spread rapidly, with over % mortality on wild rabbit populations recorded for one region of south australia (mutze et al., ) . reasons for the high mortality associated with fcv-ari were undetermined. the virus is de®nitely more virulent than any fcv strain yet tested by one of the authors (ncp) or reported in literature. it was noteworthy that the cats that became the most ill were also among the oldest. rhdv causes a mild self-limiting infection in rabbits less than weeks of age, while the mortality approaches % in older animals (mutze et al., ) . inherent resistance factors also seemed to play a role; some individual cats developed mild self-limiting disease, while others were devastated by the infection. the close genetic relationship of ria, ari and ian could have contributed to the severity of their illness. at least one of the cats (ari) was also treated with prednisolone early in the course of his infection, in the belief that the disease was some sort of immune vasculitis. however, another cat (emma) in the initial outbreak, and several laboratory cats, were not treated with prednisolone and had a similar fatal disease course. even though many ®eld strains of fcv do not cross-react with the almost universal fcv-f vaccine strain (pedersen et al., ; knowles et al., ; pedersen and hawkins, ; laruritzen et al., ; hohdatsu et al., ) , none of these strains have been of this virulence and the consequences have therefore been small. antibodies against the universal fcv-f vaccine strain also did not signi®cantly cross-react with fcv-ari, and laboratory cats immunized by the oral route with fcv-f weeks prior to challenge-exposure possessed only a small measure of immunity. two ®eld cats that died of the infection (ari and emma) had been previously immunized for fcv also indicating that current fcv vaccines, given by prescribed parenteral routes, will not protect against fcv-ari. why such a virulent and obviously contagious strain of fcv would appear and disappear so suddenly is unknown. the virus was spread readily from one group of cats to another both within the practice of origin and in the laboratory setting with ease, and it is fortunate that this spread was contained in both areas. unfortunately, highly virulent, vaccine resistant, viruses like fcv-ari may arise again in the future and vigilance is required. detection and differentiation of rabbit hemorrhagic disease and european brown hare syndrome viruses by ampli®cation of vp genomic sequences from fresh and ®xed tissue specimens detection of feline calicivirus antigens in the joints of infected cats typing of feline calicivirus isolates from different clinical groups by virus neutralization tests investigation of vaccine reactions and breakdowns after feline calicivirus vaccination acute arthritis of cats associated with feline calicivirus infection sites of persistence of feline calicivirus genetic and antigenic heterogeneity among feline calicivirus isolates from distinct disease manifestations feline viral infections isolation of feline calicivirus and feline herpesvirus from domestic cats to neutralizing feature of commercially available feline calicivirus (fcv) vaccine immune sera against fcv ®eld isolates experimentally induced feline calicivirus infection: clinical signs and lesions antigenic change in feline calicivirus during presistent infection feline calicivirus infection in kittens born by cats persistently infected with the virus induction of immunity to feline caliciviral disease neutralisation patterns among recent british and north american feline calicivirus isolates from different clinical origins phenotypic and genotypic variation of feline calicivirus during persistent infection of cats monitoring the spread of rabbit hemorrhagic disease virus as a new biological agent for control of wild european rabbits in australia serological analysis of feline calicivirus isolates from the united states and united kingdom a new viral disease in rabbits rabbit hemorrhagic disease: a review with special reference to its epizootiology the initial impact of rabbit hemorrhagic disease on european rabbit populations in south australia zum auftreten der rabbit hemorrhagic disease (rhd) in o È sterreich. i. pathomophologische und virologische untersuchungen mechanisms for persistence of acute and chronic feline calicivirus infections in the face of vaccination a transient febrile limping syndrome of kittens caused by two different strains of feline calicivirus quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats acute and chronic faucitis of domestic cats. a feline calicivirus-induced disease comparison of the primary signs induced by experimental exposure to either a pneumotrophic or a limping strain of feline calicivirus cat # group . b - vaccinate /À c / / À/À À/ /À À/À / À/ À/À - vaccinate / /À / / /À À/À / À/ À/ À/À - vaccinate / / À/ À/ /À / / À/ À/ À/À - vaccinate / / /À /À À/À À/ À/ À/ À/ À/À - nonvaccinate À/À À/À À/À À/À À/À À/À / À/ / À/À - nonvaccinate À/À À/À À/À À/À À/À À/À / À/ À/ À/À a fcv isolates prior to challenge-exposure were uniformly of the fcv-f strain by sequence analysis, while isolates after challenge-exposure were identical to fcv-ari.b challenge-exposed with fcv-ari immediately after oral swabbing. c culture/pcr. fig. . rectal temperatures of previously fcv-f immunized (^ÐÐÐÐ^) and nonimmunized (&ÐÐÐÐ&) cats that were challenge-exposed to fcv-ari. the febrile responses of vaccinates developed more slowly and were somewhat less severe than those of the unvaccinated cats. key: cord- -vy yirek authors: baró, j.; segalés, j.; martínez, j. title: porcine circovirus type (pcv ) enteric disease: an independent condition or part of the systemic disease? date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: vy yirek intestinal disorders in growing and finishing pigs have been associated with several infectious agents, including porcine circovirus type (pcv ). this virus has been mainly related with pcv -systemic disease (pcv -sd); nevertheless, some authors have suggested a possible restricted intestinal infection of this virus associated with enteric clinical signs. this condition has been referred as pcv -enteric disease (pcv -ed). the present study analysed retrospectively, from a pathological point of view, the relation between intestinal disorders and pcv infection in nursery and growing-finishing pigs. among the selected pigs suffering from enteric disease and submitted for necropsy between and , the most prevalent enteric lesions were catarrhal enteritis/colitis ( . %), followed by fibrinous lesions ( . %), granulomatous inflammation ( . %) and other lesions such as haemorrhages or ulceration ( . %). seventy-two pigs ( %) were positive for pcv by in situ hybridization (ish). among positive pigs for pcv ish, animals suffered from pcv -sd and had no lymphoid lesions but low amount of viral nucleic acid in several lymphoid tissues, therefore, these animals did not qualify for pcvd-ed. in conclusion, all animals with enteric disorders that were positive to pcv by ish had evidence of viral systemic infection. these results suggest that pcv -ed is probably a negligible condition and pcv mainly contributes to enteric clinical disorders in relation to pcv -sd occurrence. intestinal disorders in growing-finishing pigs are a common problem in most commercial farms and significantly limit the efficiency and profitability of swine production globally. clinical presentation consists of diarrhoea, poor growth and variable mortality. intestinal disorders in pigs older than four weeks of age can be produced by several infectious agents such as rotaviruses, some coronaviruses, porcine circovirus type (pcv ), escherichia coli, brachyspira spp., salmonella spp., yersinia spp., lawsonia intracellularis, oesophagostomum dentatum and trichuris suis, among others. however, most cases featuring postweaning diarrhoea and colitis are due to concurrent aetiologies (thomson and friendship, ) . pcv is recognized as one of the most important viruses causing severe economic impact in the swine industry worldwide and it has been described to cause different conditions depending on the virus, host immunity, veterinary microbiology ( ) intestinal disorders in growing and finishing pigs have been associated with several infectious agents, including porcine circovirus type (pcv ). this virus has been mainly related with pcv -systemic disease (pcv -sd); nevertheless, some authors have suggested a possible restricted intestinal infection of this virus associated with enteric clinical signs. this condition has been referred as pcv -enteric disease (pcv -ed). the present study analysed retrospectively, from a pathological point of view, the relation between intestinal disorders and pcv infection in nursery and growing-finishing pigs. among the selected pigs suffering from enteric disease and submitted for necropsy between and , the most prevalent enteric lesions were catarrhal enteritis/colitis ( . %), followed by fibrinous lesions ( . %), granulomatous inflammation ( . %) and other lesions such as haemorrhages or ulceration ( . %). seventy-two pigs ( %) were positive for pcv by in situ hybridization (ish). among positive pigs for pcv ish, animals suffered from pcv -sd and had no lymphoid lesions but low amount of viral nucleic acid in several lymphoid tissues, therefore, these animals did not qualify for pcvd-ed. in conclusion, all animals with enteric disorders that were positive to pcv by ish had evidence of viral systemic infection. these results suggest that pcv -ed is probably a negligible condition and pcv mainly contributes to enteric clinical disorders in relation to pcv -sd occurrence. ß elsevier b.v. all rights reserved. co-infections and other environment characteristics (segalé s et al., ; madec et al., ) . these diseases are grouped under the name porcine circovirus diseases (pcvds). pcvds include pcv -systemic disease (pcv -sd, formerly known as postweaning multisystemic wasting syndrome, pmws), porcine dermatitis and nephropathy syndrome (pdns), pcv reproductive disease (pcv -rd), pcv -lung disease (pcv -ld) and pcv -enteric disease (pcv -ed) (segalé s, ). diarrhoea is a feature of some pcvds and has been described in cases of pcv-sd and pcv-ed (kim et al., ; segalé s et al., ; opriessnig et al., ) . pcv -sd occurs in pigs between one and six months of age with the highest number of cases occurring between two and three months (segalé s and cortey, ). it is a multifactorial disease, in which infection by pcv is essential but other factors are usually required for its complete expression (segalé s et al., ; madec et al., ) . from a clinical point of view, there may be a potential diagnostic overlapping between pcv -sd and pcv -ed since diarrhoea can be easily present in cases of the systemic disease (opriessnig et al., ) . however, some authors have suggested that pcv -ed could be a distinct clinical manifestation of pcv- (kim et al., ) . to differentiate between these two conditions, histopathological findings and examination not only of gut but also lymphoid tissues is crucial. pcv-sd is diagnosed when the following are present: (a) clinical signs consisting of weight loss and paleness of skin (respiratory and/or digestive clinical signs may be present as well), (b) moderate to severe lymphocyte depletion with granulomatous inflammation of lymphoid tissues or other organs, and (c) moderate to high amount of pcv in damaged tissues. on the other hand, pcv-ed is diagnosed when (a) pigs suffer from diarrhoea; (b) a granulomatous enteritis and lymphocyte depletion is observed microscopically in peyer's patches, but not in other lymphoid tissues, and (c) a moderate to high amount of pcv is detected in intestinal mucosa or peyer's patches without detection in other lymphoid tissues (segalé s, ) . from a case definition point of view, pcv -ed and pcv -ld share a common characteristic, which is the presence of lesions and pcv antigen/genome in intestines and lung, respectively, in absence of lesions and viral detection in systemic locations (segalé s, ). however, the specific study of pcv -ld in a set of pigs suffering from respiratory disease, indicated that this condition is probably negligible and pcv mainly contributes to respiratory disease in relation to pcv -sd occurrence (ticó et al., ) . this type of assessment has not been yet conducted for pcv -ed. therefore, in order to clarify some points of pcv -ed, the present study aimed to investigate the prevalence of pcv infection in growing-to-finishing pigs with diarrhoea in spain and assess the potential overlap between pcv-ed and pcv -sd. in this study, pigs ( . %) were selected from a total of pigs that were submitted for necropsy between and to the veterinary pathology diagnostic service at the veterinary school of barcelona (spain). animal selection criteria included the following four features: (a) older than four weeks of age (nursery, growing and finishing pigs), (b) clinical digestive signs, (c) availability of intestinal and lymphoid tissues to assess histopathological lesions and to detect pcv dna by in situ hybridization (ish) and (d) presence of microscopic lesions in the intestine. formalin-fixed, paraffin-embedded samples of intestines (portions of jejunum, ileum and colon) and lymphoid organs (mesenteric and inguinal superficial lymph nodes, and tonsil) corresponding to the selected pigs were included in this study. all samples were analysed by the same pathologist. classification of lesions was performed in regards of gross and/or microscopic findings; hence, diagnostic criteria were as follows. catarrhal enteritis or colitis (ce/cc) was consistent with serous or mucous intestinal content in the lumen and moderate to severe lymphoplasmacytic infiltration was observed microscopically within the intestinal mucosa. granulomatous enteritis or colitis (ge/gc) was assessed when multifocal aggregates of macrophages (granulomas), with or without multinucleated giant cells or intracytoplasmic inclusion bodies, and lymphoid depletion were observed in the mucosa and/or peyer's patches. haemorrhagic enteritis (he) was classified when haemorrhagic content was observed in the small intestine. fibrinous enteritis or colitis (fe/fc) consisted of accumulation of fibrin on the mucosa of caecum or colon. ulcerative colitis (uc) was characterized by multifocal to coalescing ulceration of the large intestinal mucosa. mucous-haemorrhagic colitis (mhc) consisted of mucous and haemorrhagic content in the caecal or colonic lumen. lesions were characterized as ''single'' or ''combination'' when one or more lesion types were observed, respectively. diagnosis of pcv -sd was established following internationally accepted criteria previously described (segalé s, ) . regarding the pcv -ed, criteria proposed in the same article were used: ( ) lympho-histiocytic to granulomatous enteritis and lymphocyte depletion with granulomatous inflammation in peyer's patches, ( ) positive ish result for pcv in the intestines, and ( ) absence of pcv genome and histopathologic pcv -sd lesions in other lymphoid tissues (segalé s, ). the presence of pcv in tissues was determined by an ish technique (rosell et al., ) , which was performed on lymph node, tonsil and intestines, using a bp digoxigenin labelled dna probe corresponding to orf of pcv (dig- -cct tcc tca tta ccc tcc tcg cca aca ata aaa taa tca aa- ). quantification of pcv nucleic acid detection was made according to a subjective evaluation of the percentage of positive cells from the totality of the tissue submitted in the slide: -(negative), + (less than %), ++ (between % and %) and +++ (more than %). these samples were analysed in a blinded fashion manner by the same pathologist. all pigs came from different spanish farms, each one with its own particular management, system production and size. from pigs studied, animals ( . %) were necropsied between and (pre-pcv vaccination) and only pigs ( . %) were submitted from to . forty-four animals were among four and eight weeks old (nursery pigs) and fifty-two were older than eight weeks (growing-finishing pigs). a summary of enteric lesions and pcv detection is shown in table , where numbers of pigs with single or combined lesions are given in detail. the majority of pigs displayed a ''single'' lesion ( . %), while only two pigs had a combination of two types of lesions ( . %). the most common lesion was colitis, observed in pigs ( . %), followed by enterocolitis in animals ( . %) and enteritis in pigs ( . %). catarrhal lesions were the most prevalent with pigs affected ( . %), followed by fibrinous lesions observed in animals ( . %), granulomatous inflammation in four ( . %) and other lesions, such as haemorrhages, ulceration or mucohaemorrhagic exudate, in seven pigs ( . %). no lesions compatible with proliferative ileitis were observed. focusing on the age of animals, catarrhal lesions were slightly more frequent in nursery pigs ( . %) compared to growing-finishing animals ( . %). in contrast, fibrinous lesions were more prevalent in the growing-finishing ( . %) than in the nursery period ( , %). only nine animals ( . %) were submitted between - (post-pcv vaccination). in regards of ish positivity for pcv among the intestinal samples, enterocolitis was positive in . % of the animals ( / ) and . % of these cases ( / ) had a diagnosis of pcv -sd. colitis were positive in . % ( / ) and . % ( / ) corresponded to a pcv -sd. enteritis cases were positive to pcv in . % of cases ( / ) and . % of animals ( / ) where diagnosed as a pcv -sd. besides, there was a group of animals where pcv was not detected in the intestine, but yielded positive in lymph nodes and tonsils; this occurred in . % of pigs ( / ); from them, corresponded with a pcv-sd diagnosis. taking only into account the positivity for pcv in the intestinal lesions, from the pigs with intestinal lesions, ( %) were positive for pcv ish and pigs were negative for this virus in intestine and lymphoid tissues (fig. ) . thirty-nine pigs out of the pcv ish positive ( . %) suffered from pcv -sd. the remaining pigs with pcv ish positivity in the intestine had also low or moderate quantity of viral genome in lymph nodes and tonsil. therefore, none of the studied animals qualified for a pcv -ed diagnosis. intestinal disorders are a common health problem in postweaning pigs. however, in the present work, considering the selection criteria, a small percentage of animals were investigated compared to other similar studies performed in pigs with respiratory disorders (ticó et al., ) . several factors could be involved in this low casuistic. first, the necessary presence of gross or microscopic intestinal lesions to be included in the study, as some intestinal disorders such as osmotic or dietary diarrhoeas do not produce lesions. second, most intestinal pcv -sd: porcine circovirus type systemic disease. + ish-pcv was negative in all the tissues analysed. * ish-pcv was negative in the intestine but positive in lymph nodes and tonsil. disorders are usually treated with antibiotics or diet restriction and no animals are submitted for necropsy unless there is no recovery of clinical signs. and third, casuistic of digestive problems in nursery and growingfattening pigs were of lower prevalence than respiratory disorders in the study period. different agents have been involved, including infectious and non-infectious factors, in intestinal disorders in growing-fattening pigs (thomson and friendship, ) . although bacteriological analyses were not performed in the studied cases, from a pathological point of view, intestinal lesions are usually related with specific infectious agents (segalé s and martínez, ). the most prevalent intestinal lesion was catarrhal inflammation of different parts of the intestine. ce is usually associated with colibacillosis, or less frequently with yersiniosis; on the other hand, cc is generally related with the infection of some species of brachyspira. however, other noninfectious factors, such as the composition of diet, could also be involved. another less prevalent lesion was fe/fc which is generally associated with salmonella spp. infections. other type of lesion, such as he and uc or mhc, usually associated with clostridiasis or swine dysentery, appeared in a very small number of cases. surprisingly, only four pigs had granulomatous enteritis (highly suggestive of pcv infection) compared to the high rate of detection of this virus in the intestine or other organs of these animals. these findings suggest that intestinal disorders exclusively attributed to pcv infection in the intestine could be low, but this virus may predispose to opportunistic bacterial overgrowth or dysbacteriosis. pcv has been included in the list of agents involved in the intestinal disorders of these animals (kim et al., ; segalé s et al., ; opriessnig et al., ) . evidence of immunosupression in pcv infected pigs has extensively been reported in regards of several features; for example, the lymphoid depletion observed in infected pigs and the association of pcv -sd with opportunistic pathogens (carrasco et al., ; segalé s and mateu, ; zlotowski et al., ) . another virus associated with immunosuppression is porcine reproductive and respiratory syndrome virus (prrsv) . in the present study, the presence of this virus in lung and lymphoid tissues was analysed by immunohistochemistry in from the pigs, but only were positive (data not shown). in contrast, the present results showed a high prevalence of pcv infection in postweaning pigs with digestive disorders, reinforcing the hypothesis of altered immune response in animals infected by this virus. in this way, only a small percentage of animals corresponded to post-pcv vaccination period. the diagnostic overlapping between pcv -sd and pcv -ed has been previously discussed (opriessnig et al., ; segalé s, ) . kim et al. ( ) proposed that pcv -associated enteritis (equivalent to pcv -ed) should be differentiated from pcv -sd based on clinical and histopathological criteria. however, in the present study, most of pigs showing clinical intestinal disorders with pcv involvement were histopathologically diagnosed as pcv -sd or suffered from subclinical systemic infection of the virus. moreover, the scenario in which pcv was present in intestine but not in lymphoid tissues, as requested to diagnose pcv -ed (segalé s, ), did not occur in any case; so that, pcv -ed as specific clinicopathological entity was absent in the current study. these results coincide with those published by ticó et al. ( ) ; these authors studied the role of pcv associated lung disease (pcv -ld) in cases of respiratory disease of nursery and growing-finishing pigs, but pcv infection exclusively restricted to the lung was not found in any case. all these results support the idea of pcv as a systemic pathogen, rather than virus able to cause a facultative local infection. although, concomitant viral or bacterial agents could be present in the intestinal studied cases (worsening clinical signs), the amount of pcv detected in intestines was never higher than the one detected in lymphoid lesions of the same animal. moreover, in those cases in which pcv -ed was potentially suspected (not diagnosed as pcv -sd), the viral load in intestinal and lymphoid tissues was lower than the amount of virus detected in pcv-sd cases. in fact, obtained findings in pigs which were non-pcv -sd, but pcv -infected, suggest a pcv systemic infection which can be interpreted as: ( ) subclinical pcv- systemic infection, being other pathogens the major contributors of diarrhoea, or ( ) recovery phase from an old pcv -sd infection. the results obtained in this study suggest that pcv is frequent in cases of intestinal disorders in nursery and growing-finishing pigs. from a diagnostic point of view, the present retrospective study shows that the casuistic of enteric disorders cases that could be categorized in the group of pcv -ed was non-existent in contrast to other published data (kim et al., ; opriessnig et al., ) , and pcv mainly contributes to enteric clinical disorders in relation to pcv -sd occurrence. intestinal chlamydial infection concurrent with postweaning multisystemic wasting syndrome in pigs enteritis associated with porcine circovirus in pigs post-weaning multisystemic wasting syndrome and other pcv -related problems in pigs: a -year experience porcine circovirus type associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (pmws) in pigs porcine circovirus type (pcv ) infections: clinical signs, pathology and laboratory diagnosis changes in age at diagnosis of pmws in pigs in spain laboratory diagnosis of enteric disorders immunosuppression as a feature of postweaning multisystemic wasting syndrome porcine circovirus diseases pathological findings associated with naturally acquired porcine circovirus type associated disease digestive system the blurred border between porcine circovirus type -systemic disease and porcine respiratory disease complex porcine reproductive and respiratory syndrome virus (porcine arterivirus) muco-cutaneous candidiasis in two pigs with postweaning multisystemic wasting syndrome the epidemiological data and the paraffin blocks for this study were kindly donated by the servei de diagnòstic de patologia veterinària (sdpv) of the universitat autònoma de barcelona. authors are grateful to the veterinary undergraduate student marta martínez ló pez for her work with the database, and aida neira and blanca pé rez of the sdpv and mó nica pé rez from the centre de recerca en sanitat animal (cresa) for their technical assistance. key: cord- -c ut vn authors: palombieri, andrea; di profio, federica; lanave, gianvito; capozza, paolo; marsilio, fulvio; martella, vito; di martino, barbara title: molecular detection and characterization of carnivore chaphamaparvovirus in dogs date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: c ut vn canine chaphamaparvovirus (cachpv) is a newly recognised parvovirus discovered by metagenomic analysis during an outbreak of diarrhoea in dogs in colorado, usa, in and more recently detected in diarrhoeic dogs in china. whether the virus plays a role as canine pathogen and whether it is distributed elsewhere, in other geographical areas, is not known. we performed a case-control study to investigate the possible association of cachpv with enteritis in dogs. cachpv dna was detected both in the stools of diarrhoeic dogs ( . %, / ) and of healthy animals ( . %, / ). all the cachpv-infected dogs with diarrhea were mixed infected with other enteric viruses such as canine parvovirus (formerly cpv- ), canine bufavirus (cbuv) and canine coronavirus (ccov), whilst none of the asymptomatic cachpv positive animals resulted co-infected. the nearly full-length genome and the partial capsid protein (vp) gene of three canine strains, te/ ovud/ /ita, te/ ovud/ /ita and te/ ovud/ /ita, were reconstructed. upon phylogenetic analyses based on the ns and vp aa sequences, the italian cachpv strains tightly clustered with the american reference viruses. distinctive residues could be mapped to the deduced variable regions of the vp of canine and feline chaphamaparvoviruses, considered as important markers of host range and pathogenicity for parvoviruses. parvoviridae are small (~ nm diameter), non-enveloped, single-stranded and negative-sense dna viruses of . - . kb in length, with the coding region bracketed by terminal repeats that can fold into hairpin-like structures (berns and parrish, ) . they have a large host spectrum, spanning from invertebrates to mammals (pénzes et al., ) . parvoviruses have long been known in dogs, since the identification of canine minute virus, or canine parvovirus (cpv) type (cpv- ; genus bocaparvovirus), in from the faecal samples of healthy dogs (binn et al., ) . cpv- infection is responsible for reproductive disorders and occasionally for respiratory and gastrointestinal signs in young dogs (decaro et al., ) . a second cpv (cpv- ; genus protoparvovirus) was reported in the s in europe and north america in puppies with signs of haemorrhagic gastroenteritis and myocarditis (appel et al., ) . cpv- is currently regarded as the major causative agent of severe gastroenteritis in puppies (decaro and buonavoglia, ) . recent advances in molecular technologies have been allowed the discovery of novel parvoviruses in dogs, including two additional bocaparvoviruses species, cbov- and cbov- identified respectively from healthy and sick dogs respiratory samples (kapoor et al., ) and from the liver of a dog with multiorgan failure (li et al., ) , and the still unclassified carnivore protoparvoviruses likebufaviruses (cbuvs) detected in stool samples of dogs with or without enteric disease and in the nasal and oropharyngeal swabs of animals with respiratory signs. more recently, assessing faecal samples from an unexplained outbreak of diarrhoea in colorado (usa), a novel canine virus has been discovered by metagenomic approach (fahsbender et al., ) . upon sequence analysis of the nearly complete genome of the two strains identified, cachavirus- a strain idexx (genbank accession number mh ) and cachavirus- b strain idexx (mk ), the highest genetic relatedness ( . - . % nt identity) was found to members of the genus chaphamaparvovirus, previously described under an unofficial umbrella term "chapparvovirus" (pénzes et al., ) . on the basis of the international committee on taxonomy of viruses (ictv) j o u r n a l p r e -p r o o f classification criteria for parvoviridae species demarcation, both the canine strains were classified within the species carnivore chaphamaparvovirus (pénzes et al., ) . the genus chaphamaparvovirus includes viruses infecting vertebrate hosts that are more closely related to invertebrate-infecting parvoviruses than to members of the parvovirinae. the discovery of chpvs has compelled a radical rethink on the evolution and genetic relationships among parvoviridae, leading to a recent taxonomical re-classification characterised by the introduction of the novel subfamily hamaparvovirinae that encompasses divergent densoviruses and vertebrateinfecting parvoviruses (pénzes et al., ) . in the last few years, chpvs have been identified in several animal species including bats, rodents, birds, pigs and domestic cats (berker et al., ; reuter et al., ; yang et al., ; palinski et al., ; lima et al., ; de souza et al., ; yinda et al., ; roediger et al., ; william et al., ; . however, information on their epidemiology and/or ability to cause disease in their natural hosts are still limited. preliminary epidemiological data collected by fahsbender et al. ( ) address the question on the possible role of canine chpvs (cachpvs) in the aetiology of dog enteritis. molecular analysis by quantitative pcr (q-pcr) of a large set of enteric samples revealed the presence of cachpvs dna in the faeces of dogs with ( . %) or without ( . %) diarrhoea, although a clear association with enteric signs was not demonstrated. in a more recent study performed in china , the detection rate of cachpvs dna was % and . % in healthy or diarrhoeic dogs, respectively. however, association between cachpvs and gastrointestinal disease was not supported by statistical analysis. in order to gather additional information on the distribution of this novel parvovirus in dogs and to investigate its possible association with enteritis, during the year a surveillance study was initiated by implementing with cachpv-specific assays the diagnostic algorithms of cases of acute gastro-enteritis admitted to the veterinary hospital of the faculty of veterinary medicine, university of teramo (italy). j o u r n a l p r e -p r o o f a case-control study was conducted using two subsets of dogs selected on the basis of the presence of acute gastroenteric signs for clinical cases and the absence of enteritis for controls. a total of rectal swab samples (subset a) was collected from household dogs suffering from gastro-enteric disorders. the inclusion criteria were the presence of mild to severe diarrhoea and age comprised between and months old. rectal specimens (n. ) randomly recruited among healthy dogs during routine visits in and matching with cases for living condition and age were included in the study as control group (subset b). informed consent was obtained from all animal owners. the collected swabs were immersed in ml of viral transport medium consisting of dulbecco's modified eagle's medium (d-mem), and subsequently clarified by centrifuging at g for min. dna and rna were extracted from μl of viral suspension by using the qiaamp cador pathogen mini kit (qiagen s.p.a., milan, italy), following the manufacturer's instructions. all faecal samples were screened for cpv- , cbuv, canine coronavirus (ccov), canine kobuvirus (cakov) and norovirus (nov) by conventional pcr and reverse transcription (rt)-pcr (pratelli, et al., ; buonavoglia et al., ; vennema et al., ; di martino et al., ; martella et al., ) . the presence of cachpv dna was assessed by nested-pcr using diagnostic primer sets cpv_ f/cpv_ r and cpv _ fn/cpv_ rn, following chemical and thermal conditions previously described (fahsbender et al., ) a standard curve was generated using to copies per reaction of cachpv plasmid dna containing the synthetised fragment. viral dna quantification was performed using taqman and type ). the qpcr assay was able to detect > dna copies/ μl of standard dna and . × dna copies/ μl of dna template extracted from clinical samples. in order to gather sequence information from the cachpvs detected in our study, attempts were made j o u r n a l p r e -p r o o f by using diagnostic primer sets (fahsbender et al., ) , cachpv dna was detected in a total of faecal samples, with an overall prevalence of . % ( / on phylogenetic analysis based on the complete ns protein (fig. a) (yinda et al., ) and the chpvs of feline origin . the aa identities within this group was . - . %. the high genetic conservation j o u r n a l p r e -p r o o f among all the cachpvs identified to date (with an overall aa identity of . - . %) was also confirmed in the vp capsid based tree (fig. b) . identities to the feline and bat chpvs were . - . % and . - . %, respectively. in order to further investigate the relationship between the canine and the feline chpvs, the variable regions (vrs) of the cachpv strains b-idexx and te/ ovud/ /ita and those of the feline chpvs /vri/ and idexx- were mapped as described elsewhere (pénzes et al., ) and compared to each other. out of ten vrs (vr-i to vr-x), vr-ii resulted more conserved, whilst the largest differences were located in vr-iii and vr-vi to vr-x (fig. ) . the findings of this study, while providing firm evidence that cachpv is a common component of canine faecal virome, do not allow to make any solid conclusions on the potential role of this novel parvovirus as a primary causative agent of gastrointestinal disease. in our analysis, cachpv dna was detected in animals with enteric signs only in co-infection with other canine viral pathogens. similar results were also obtained in the chinese study, in which two out of the five diarrhoeic although most chpv strains identified to date in the various animal species have been discovered serendipitously in metagenomic studies of enteric virome, evidence for the pathogenic potential of these viruses have been reported. horizontal transmission of the mouse kidney parvovirus (mkpv) (rodent chaphamaparvoviruses species) has been proved to induce inclusion body nephropathy (ibn) and kidney fibrosis in aged immunodeficient mice and, to lesser extent, in immunocompetent mice (roediger et al., ; ge et al., ; lee et al., ) . more recently, a newly identified chpv was identified by qpcr and immunohistochemistry in the heart, intestine, liver, and lung of a dead peafowl suffering of enteritis and pneumonia . sequence analysis of the nearly complete genome of the strains te/ ovud/ /ita and te/ ovud/ /ita and of ~ . -kb long partial vp of the cachpv strain te/ ovud/ /ita revealed a strong sequence conservation with the viruses detected in diarrhoeic dogs in usa (fahsbender et al., ) , either in the ns ( . - . %) or in the vp ( . - . %) encoding genes. upon phylogenetic analysis based on the ns aa sequence, all the strains of canine origin grouped tightly within the species carnivore chaphamaparvovirus . interestingly, this group segregated apart from but close to a chpv identified in stools of a cameroonian fruit bat (yinda et al., ) and to chpvs recently identified in cats with gastroenteritis . the genetic relatedness among j o u r n a l p r e -p r o o f the cachpvs and the strains detected in cats and bats ( . - . %) was consistent with the ranges established by ictv for classification of parvoviruses into the same genus (cut-off > . % aa identity with a ns coverage > . %), whilst it did not match the criteria required to demark the same species (cut-off > . % aa identity) (pénzes et al., ) . the correlation between the canine and feline viruses ( . - . % nt identities in the nearly complete genome) was also confirmed in the vp-based tree. for parvoviruses, the capsid is the major determinant of host range (hueffer et al., ) and subject to antibody-mediated selection (nelson et al., ) . minor genetic changes in these proteins are known to alter the host range and pathogenic potential of parvoviruses (parrish and kawaoka, ) . when comparing the vp sequences of the canine strains b-idexx and te/ ovud/ /ita with those of the feline chpvs /vri/ and idexx- , the highest divergence was observed in the predicted vrs of the capsid (fig. ) , with several aa changes, including residue insertions and deletions, located at vr-iii and vr-vii that have been involved in control of tissue tropism, antibody recognition and receptor attachment of several parvoviruses (halder et al., ; kailasan et al., ) . the marked genetic heterogeneity observed between the cachpvs and the feline strains indicates that at least two distinct groups of chpvs, each characterised by peculiar genetic signatures, circulate in dogs and cats. since some canine viruses can infect cats and vice versa (martella et al., ; matthijnssens et al., ; di martino et al., ; di martino et al., ; diakoudy et al., ) , it will be important to explore whether cross-species transmission of chpvs might occur between the two carnivore species. we gathered epidemiological information of cachpv in a large dog population demonstrating that the circulation of this novel canine parvovirus is not geographically restricted to the american and asiatic continents where the virus was so far detected (fahsbender et al., ; hu et al., ) . this firmly demonstrates that cachpv is a common component of canine enteric virome. the impact of these viruses on canine health remains to establish. in our study, we did not find any possible j o u r n a l p r e -p r o o f association between cachpv and gastro-enteric disease in young dogs. animal experiments or detailed observational studies would be required to address this thoroughly and to investigate whether there may be other possible implications for canine health. all authors declare that there are no financial or other relationships that might lead to a conflict of interest. all authors have seen and approved the manuscript and have contributed significantly to the work. decaro, n., buonavoglia, c., . canine parvovirus -a review of epidemiological and diagnostic aspects, with emphasis on type c. vet. microbiol. , - . j o u r n a l p r e -p r o o f isolation and immunisation studies of a canine parvo-like virus from dogs with haemorrhagic enteritis metagenomic study of the viruses of african straw-coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus parvoviridae recovery and characterization of a minute virus of canines evidence for evolution of canine parvovirus type in italy chapparvoviruses occur in at least three vertebrate classes and have a broad biogeographic distribution viral reproductive pathogens of dogs and cats canine kobuviruses in diarrhoeic dogs in italy a novel feline norovirus in diarrheic cats serological and molecular investigation of -like vesiviruses in cats identification of a novel parvovirus in domestic cats chapparvovirus dna found in % of dogs with diarrhea identification of a new strain of mouse kidney parvovirus associated with inclusion body nephropathy in immunocompromised laboratory mice parvoviruses: structure and infection molecular characterization of cachavirus firstly detected in dogs in china parvovirus host range, cell tropism and evolution crystal structure of the sf helicase from adeno-associated virus type pushing the limits of the scanning mechanism for initiation of translation mega x: molecular evolutionary genetics analysis across computing platforms murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys a novel bocavirus in canine liver virome of a feline outbreak of diarrhea and vomiting includes bocaviruses and a novel chapparvovirus genomic and transcriptional analyses of novel parvoviruses identified from dead peafowl novel parvovirus related to primate bufaviruses in dogs analysis of the capsid protein gene of a feline-like calicivirus isolated from a dog multiple reassortment and interspecies transmission events contribute to the diversity of feline, canine and feline/canine-like human group a rotavirus strains different mechanisms of antibody-mediated neutralization of parvoviruses revealed using the fab fragments of monoclonal antibodies discovery of a novel parvovirinae virus, porcine parvovirus , by metagenomic sequencing of porcine rectal swabs the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses an ancient lineage of highly divergent parvoviruses infects both vertebrate and invertebrate hosts development of a nested pcr assay for the detection of canine coronavirus novel circular single-stranded dna virus from turkey faeces an atypical parvovirus drives chronic tubulointerstitial nephropathy and kidney fibrosis an adeno-associated virus (aav) initiator protein, rep , catalyzes the cleavage and ligation of single-stranded aav ori dna the rule of three, its variants and extensions rational optimization of generic primers used for norwalk-like virus detection by reverse transcriptase polymerase chain reaction distantly related sequences in the alpha-and beta-subunits of atp synthase, myosin, kinases and other atp-requiring enzymes and a common nucleotide binding fold viral diversity of house mice a novel rodent chapparvovirus in feces of wild rats cameroonian fruit bats harbor divergent viruses, including rotavirus h, bastroviruses, and picobirnaviruses using an alternative genetic code a viral phopholipase a is required for parvovirus infectivity funding: this work was financed by grants from università degli studi di teramo.j o u r n a l p r e -p r o o f key: cord- -rrj c authors: moutinho costa, erika; xavier de castro, tatiana; de oliveira bottino, fernanda; nasser cubel garcia, rita de cássia title: molecular characterization of canine coronavirus strains circulating in brazil date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: rrj c to characterize canine coronavirus (ccov) circulating in diarrheic puppies in brazil, fecal samples collected between and were tested. by using rt-pcr to partially amplify the m gene, ccov rna was detected in samples. sequence analysis of the m protein grouped eight strains with ccov-i and another with ccov-ii prototypes. to genotype/subtype the ccov strains and assess the occurrence of single or multiple ccov infections, rt-pcr of the s gene was performed, and / ccov-positive strains amplified with one or two primer pairs. for / samples, single infections were detected as follows: six ccov-i, nine ccov-iia and two ccov-iib. eight samples were positive for more than one genotype/subtype as follows: seven ccov-i/iia and one ccov-i/iib. sequence analysis revealed that the ccov-i and iia strains shared high genetic similarity to each other and to the prototypes. the brazilian strains of ccov-iib displayed an aminoacid insertion that was also described in ccov-iib-ucd- and tgev strains. among the ccov-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. the current study is the first reported molecular characterization of ccov-i, iia and iib strains in brazil. coronaviruses (order nidovirales, family coronaviridae) are enveloped, single-stranded, positive-sense rna viruses with a large genome of - kb. canine coronavirus (ccov) belongs to the genus alphacoronavirus and species alphacoronavirus- along with feline coronavirus (fcov) and transmissible gastroenteritis virus of swine (tgev); these viruses display greater than % sequence identity within the replicase polyprotein (pp ab) gene (adams and carstens, ; carstens, ; pratelli, ) . to date, ccovs can be classified into two genotypes, ccov-i and ccov-ii, according to the genetic identity between these viruses and fcov types i and ii, respectively (pratelli et al., a) . however, a putative recombination between ccov-ii and tgev at the end of the s protein gene gave rise to a new subtype. as a result, the ccov-ii genotype was divided into two subtypes, ccov-iia (classical strains) and iib (tgev-like strains) (decaro et al., (decaro et al., , . infection may occur with a single strain, but the two ccov genotypes are commonly detected simultaneously in the same dog (decaro et al., (decaro et al., , erles and brownlie, ; ntafis et al., ; pratelli et al., a; pratelli, ) since the first reports in (binn et al., ) , ccov infection has been associated with mild cases of diarrhea. clinical signs can range from moderate to severe, depending on whether the infection occurs in combination with other pathogens such as canine parvovirus (cpv), canine distemper virus or canine adenovirus type i (decaro et al., (decaro et al., , a pratelli et al., a pratelli et al., , . in contrast, veterinary microbiology ( ) - to characterize canine coronavirus (ccov) circulating in diarrheic puppies in brazil, fecal samples collected between and were tested. by using rt-pcr to partially amplify the m gene, ccov rna was detected in samples. sequence analysis of the m protein grouped eight strains with ccov-i and another with ccov-ii prototypes. to genotype/subtype the ccov strains and assess the occurrence of single or multiple ccov infections, rt-pcr of the s gene was performed, and / ccov-positive strains amplified with one or two primer pairs. for / samples, single infections were detected as follows: six ccov-i, nine ccov-iia and two ccov-iib. eight samples were positive for more than one genotype/subtype as follows: seven ccov-i/iia and one ccov-i/iib. sequence analysis revealed that the ccov-i and iia strains shared high genetic similarity to each other and to the prototypes. the brazilian strains of ccov-iib displayed an aminoacid insertion that was also described in ccov-iib-ucd- and tgev strains. among the ccov-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. the current study is the first reported molecular characterization of ccov-i, iia and iib strains in brazil. ß elsevier b.v. all rights reserved. highly virulent ccov-ii strains (pantropic variants) that were detected in europe (decaro et al., b ntafis et al., ; zicola et al., ) caused fatal disease in puppies. epidemiological investigations have revealed that ccovs have spread worldwide (decaro et al., erles & brownlie, ; mcelligott et al., ; naylor et al., ; ntafis et al., ; stavisky et al., ) . serological studies conducted in southern brazil revealed the presence of antibodies to this virus in - % of dogs, indicating that ccovs are already widespread in the canine population (castro et al., a; dezengrini et al., ) . nevertheless, no molecular characterization of ccov strains has been reported to date. this study was conducted to characterize, for the first time, the ccov genotypes detected in fecal samples from diarrheic puppies in brazil. fecal samples were collected from a total of privately owned (not kenneled) dogs with diarrhea and examined at veterinary clinics in rio de janeiro from to . dogs were included in the study if the owner reported an increase in the frequency, fluidity or volume of feces (battersby and harvey, ) . information regarding age, breed, vaccination status and clinical findings was obtained from the veterinary records. this trial was licensed by the ethics committee of animal use -ceua-proppi/uff no. / and / . genomic rna was extracted from % fecal suspensions in tris-ca + ( . m, ph . ) using the purelink tm spin column-based kit (invitrogen ). the reverse transcription was performed with random primers (invitrogen ) and the superscript iii enzyme (invitrogen ) by following the manufacturer's instructions. for ccov screening, pcr was performed with the ccov -ccov- ( - ) primer pair, which amplifies a -bp fragment of the gene encoding transmembrane protein m, as previously described (pratelli et al., b) . primer positions refer to the sequence of the ccov-iia insavc strain (d ). differential primers directed to the spike (s) gene were used for ccov genotyping/subtyping as follows: el f/ el r, s f/s r and cepol- /tgsp- (erles and brownlie, ; pratelli et al., a) . primers el f/el r ( - ) were used to amplify a -bp fragment corresponding to the spike gene of the ccov-i elmo/ strain (ay ) (pratelli et al., a) . primer pair s f/s r ( - ) amplified a -bp product corresponding to the spike gene of the ccov-iia insavc strain (d ), whereas primers cepol- and tgsp- amplified a -bp product corresponding to nucleotides (nt) - of the tgev purdue genome (aj . ) (erles and brownlie, ). the pcr assays were performed according to the protocols described by pratelli et al. ( a) and erles and brownlie ( ) . the amplicons obtained after partial amplification of the m and s genes were purified using the gfx tm pcr dna and gel band purification kit (ge healthcare ) and subjected to direct sequencing with the big dye terminator kit and an abi prism dna analyzer (applied biosystems, ca). both strands of each amplicon were sequenced at least twice. sequence editing and multiple alignments were performed with bioedit sequence editor . software. nucleotide similarity with sequences deposited in the genbank database was assessed using the blast tool (http://blast.ncbi.nlm.nih.gov/blast.cgi). sequence analyses were performed with the mega . software program (tamura et al., ) . for the construction of phylogenetic trees, deduced amino-acid sequences were used. bootstrap analysis of replicates was conducted to determine the significance of branching in the constructed tree. the nucleotide sequences generated in this study were deposited in the genbank with the following accession numbers: kf -kf for the m gene, kf -kf for the s gene from ccov-i, kf -kf for the s gene from ccov-iia and kf -kf for the s gene from ccov-iib. fecal samples that tested positive for ccov were also screened for cpv, canine calicivirus (cacv), canine astrovirus (caastv) and group a-rotavirus (rv-a) by either pcr or rt-pcr and sequencing according to the protocols previously described castro et al., ; farkas et al., ; gó mez et al., ; grellet et al., ) . by using pcr to amplify the m gene, ccov rna was detected in out of sampled dogs. the majority of the positive samples ( / ) were from puppies under two months of age. for / strains, the sequences were of sufficient quality for analysis. based on the aa changes found in residues , , , and of the m protein, eight strains were characterized as ccov-i while another as ccov-ii (table ) . alignment of the deduced amino acid (aa) sequences to the sequence of strain insavc (d ) revealed nonsynonymous substitutions. among the aa changes, five were found in all ccov-i strains: (ile/val ! ala), (val ! thr), (ile ! met), (asp ! glu) and (asn ! his). as shown in table , these aa changes were present in the strains from this study and in the reference strains. moreover, six of the brazilian strains analyzed contained non-synonymous substitutions not previously described. aa changes at residues (ile ! thr) and (gly!cys) were found in strain rj / . another two strains (rj / and rj / ) contained a nonsynonymous substitution at residue (ala ! thr), while rj / was substituted at residue (lys ! arg). an aa change at residue (ile ! phe) was found in two ccov-ii strains from (rj / and rj / ) (table ) . a rt-pcr typing approach for the s gene was utilized and / ccov-positive strains showed amplification with one or two primer pairs (table ). in / samples, a single infection was detected: six samples with ccov-i, nine with ccov-iia and two with ccov-iib. eight samples were positive for more than one genotype/subtype: seven samples with ccov-i/iia and one with ccov-i/iib (table ) . among five samples giving negative results for the s gene, two (rj / and / ) were pcr-positive for the m gene but could not be analyzed after sequencing. another three samples (rj / , rj / and rj / ) were characterized as ccov-ii. the results of ccov typing (i and ii) by m and s genes were concordant for / samples from puppies with single infections. characterization of one sample (rj / ) was not possible, as its s gene was amplified by rt-pcr with the ccov-i-specific primers el f/el r, while it was typed as ccov-ii by sequence analysis of its m region. among seven samples that were positive for more than one ccov genotype/subtype by s gene, sequence analysis of the m gene allowed the characterization of only one genotype (table ) . sequence analysis of the brazilian ccov-i strains revealed that they shared . - % nt and . - % aa identity. these strains also shared . - . % nt and . - % aa identity with either elmo/ (ay . ) or / (ay . ) prototypes. amino acid changes were found at residue (lys ! arg) in one sample ( / ) and at (thr ! met) in another six samples (rj / , rj / , rj / , rj / , rj / and rj / ). at the phylogenetic level, the sequenced strains were grouped in the same cluster with either elmo/ or / prototypes (fig. a) . bold indicates amino acid changes of brazilian strains. sequence analysis of the eight brazilian ccov-iia strains showed that they shared . - . % nt and . - % aa identity. these strains also shared . - . % nt and . - . % aa identity with the ccov-iia reference strains insavc (d ), bgf- (ay . ) and cb/ (dq . ). among the aa changes identified, six were found only in samples from this study (rj / , rj / , rj / and rj / ). the sequence of the rj / strain revealed eight aa changes encoded on the -bp fragment that have not been described for other ccov-iia strains (table ) . upon phlylogenetic analysis this strain formed a cluster distinct from the other ccov-iia sequences (fig. b) . sequence analysis of the three brazilian ccov-iib strains showed that they shared . - . % nt and - iib tgev lys phe val val leu val val met leu ile tyr asn leu thr gln thr ser pro asn ucd- leu ------leu ser ---phe leu his asn ile -- - phe -phe -leu ile val -val -glu --his --ser - / phe -phe --ile val ---asp phe leu his ---- / phe -phe --ile val ---glu phe leu his ---- / phe . - . % aa identity. these strains also shared . - . % nt and . - . % aa identity with the ccov-iib reference strains ucd- (af . ) and / (eu . ). the ccov-iib strains from this study exhibited an insertion of three nucleotides at the end of the spike gene, resulting in the addition of an aa at residue five as in the ucd- strain. these samples also showed three non-synonymous substitutions when compared to other ccov-iib strains (table ) . according to the phylogenetic analysis result, the brazilian ccov-iib strains formed a unique clade (fig. c) . mixed infections were detected in / ( %) puppies: cases of ccov/cpv, one of ccov/rv-a, one of ccov/cpv/ rv-a and two of ccov/cpv/caastv. as characterization of the ccov genotype/subtype by s gene rt-pcr was not possible for / samples, they were not included in this analysis (table ) . based on clinical reports, three ccov-i-positive puppies presented mild enteric signs (soft diarrhea), while four ccov-iia-positive puppies showed more severe clinical signs (vomiting, hemorrhagic diarrhea); one died during the hospitalization period. a partial sequence analysis of the s gene showed aa identities of . % to the pantropic strains described in the literature (cb/ and / ). in puppies with dual ccov or mixed infections, the clinical signs varied from mild to fatally severe, as shown in table . among ccov-positive puppies, received either one ( puppies) or three doses ( puppy) of inactivated ccov vaccine. another had not been vaccinated, and for the remaining seven, the vaccination data were not available. in the past decade, several researchers have studied the importance of ccov as an agent of diarrhea in puppies le poder, ; ntafis et al., ; pratelli, ; stavisky et al., ) . in brazil, despite the availability of safe and effective vaccines, cpv is considered the most common viral agent associated with enteritis in puppies (castro et al., b (castro et al., , . however, data regarding ccov infections in puppies have been restricted to serological surveys. the main objective of this study was to conduct a molecular characterization of the ccov strains that circulate in brazil by partially amplifying the m and s genes. the ccov genotypes may be distinguishable using molecular assays that are able to selectively amplify fragments of orf (m gene) and orf (s gene). in this study, the aa changes found in residues , , , and of the m protein could distinguish between ccov-i and ccov-ii strains. these non-synonymous substitutions had already been described in european samples, but the exact consequence of these mutations is not yet clear (erles and brownlie, ; ntafis et al., ; pratelli et al., a) . ccov infection in dogs can occur with a single ccov strain, or two strains may be present simultaneously (decaro et al., ; ntafis et al., ; pratelli et al., a; soma et al., ) . in addition, the ccov-ii genotype has been further divided into two subtypes, including the classical (iia) and the tgev-like (iib) ccovs (decaro et al., ) . although partial amplification of the m gene has been successfully used to detect ccov-i and ii, this approach does not distinguish between single or multiple ccovs in a dog or discriminate between iia and iib strains. to genotype/subtype the ccov strains and to assess the occurrence of single or multiple ccov infections, three different regions of the s gene were amplified. it was shown that the classical ccov-iia was the most predominant type in single and double infections. both ccov genotypes (i and ii) have been reported to be circulating in europe, china and japan, and ccov infections are frequently characterized by the simultaneous presence of both types ntafis et al., ; soma et al., ) . preliminary studies have indicated that multiple ccov or mixed infections may be related to more severe symptoms (decaro et al., ; pratelli et al., a) . in this study, among the ccov-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. it was shown that this one puppy was infected only with a ccov-iia strain that displayed the highest similarity to pantropic strains. as extra-intestinal tissues of this puppy were not available for analysis, it was not possible to confirm infection with a pantropic ccov. in accord with other studies (ntafis et al., ; soma et al., ) , most of the ccov-positive puppies in this study were less than three months of age. although ccov was detected in vaccinated puppies, it should be emphasized that at this age, the vaccination schedule has not been completed. moreover, the value of ccov vaccines in providing adequate immunity is controversial (pratelli et al., b (pratelli et al., , b . in the present study, the ccov-i and iia strains showed high genetic similarity to each other and to the prototypes. further studies are necessary to clarify the importance of the non-synonymous substitutions found only in the brazilian strains. it was shown that the brazilian ccov-iib strains display an aa insertion that has also been described in ccov-iib-ucd- and tgev strains (wesley, ) . this aa insertion has not been found in ccov-iib strains circulating in europe (erles and brownlie, ; ntafis et al., ) , most likely reflecting their geographic origin. in summary, the current study is the first report of a molecular characterization of the ccov-i, iia and iib strains in brazil. the results showed that all three types of ccov circulate among young puppies presenting mild or severe clinical signs of enteritis. the brazilian strains displayed previously undescribed non-synonymous substitutions, but further studies are necessary to clarify the exact consequence of these changes for ccov evolution. the continuous monitoring of ccov will help to elucidate the genetic diversity of these viruses in the brazilian canine population. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses differential diagnosis and treatment of acute diarrhoea in the dog and cat recovery and characterization of a coronavirus from military dogs with diarrhea evidence for evolution of canine parvovirus type in italy ratification vote on taxonomic proposals to the international commitee on taxonomy of viruses canine coronavirus (ccov) in dogs vaccinated and unvaccinated domiciliated in pelotas partial vp sequencing of canine parvovirus (cpv) strains circulating in the state of rio de janeiro, brazil: detection of the new variant cpv- c monitoring of canine parvovirus (cpv) strains detected in vaccinated puppies in brazil canine coronavirus: not only an enteric pathogen canine distemper and related diseases: report of a severe outbreak in a kennel genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs first detection of canine parvovirus type c in pups with haemorrhagic enteritis in spain infectious canine hepatitis: an ''old'' disease reemerging in italy molecular characterization of the virulent canine coronavirus cb/ strain recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs recombinant canine coronaviruses in dogs western european epidemiological survey for parvovirus and coronavirus infections in dogs a pantropic canine coronavirus genetically related to the prototype isolate cb/ soroprevalência das infecções por parvovírus, adenovírus, coronavírus canino e pelo vírus da cinomose em cães de santa maria sequence analysis of divergent canine coronavirus strains present in a uk dog population genetic diversity among sapoviruses rotavirus a genotype p[ ]g : genetic diversity and reassortment events among strains circulating in brazil between prevalence and risk factors of astrovirus infection in puppies from french breeding kennels feline and canine coronaviruses: common genetic and pathobiological features detection and genetic characterization of canine parvoviruses and coronaviruses in southern ireland identification of canine coronavirus strains from feces by s gene nested pcr and molecular characterization of a new australian isolate molecular characterization of a canine coronavirus na/ strain detected in a dog's organs canine coronavirus, greece. molecular analysis and genetic diversity characterization the evolutionary processes of canine coronavirus fatal coronavirus infection in puppies following canine parvovirus b infection development of a nested pcr assay for the detection of canine coronavirus severe enteric disease in animal shelter associated with infection by canine adenovirus type and canine coronavirus identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype efficacy of an inactivated canine coronavirus vaccine in pups two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea safety and efficacy of a modified-live canine coronavirus vaccine in dogs detection and genotyping of canine coronavirus rna in diarrheic dogs in japan cross sectional and longitudinal surveys of canine enteric coronavirus infection in kennelled dogs: a molecular marker for biosecurity me.ga : molecular evolutionary genetics analysis (mega) software version . the s gene of canine coronavirus, strain ucd- , is more closely related to the s gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus fatal outbreaks in dogs associated with pantropic canine coronavirus in france and belgium the authors thank dra. none. key: cord- -rcxjjxw authors: xu, zhichao; lin, ying; zou, chuangchao; peng, peng; wu, yanan; wei, ying; liu, yuan; gong, lang; cao, yongchang; xue, chunyi title: attenuation and characterization of porcine enteric alphacoronavirus strain gds via serial cell passage date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: rcxjjxw porcine enteric alphacoronavirus (peav) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in newborn piglets. in this study, an original, highly virulent peav strain gds was serially passaged in vero cells. the virus titers and sizes of syncytia increased gradually with the cell passages. newborn piglets were orally inoculated with peav p , p and p . compared with p and p , p resulted in only mild clinical signs and intestinal lesions in piglets. the virus shedding in feces and viral antigens in intestinal tract were markedly reduced in p -inoculated piglets. importantly, all p -inoculated newborn piglets survived, indicating that p was an attenuated variant. sequence analysis revealed that the virulent strain gds had four, one, six and eleven amino acid differences in membrane, nucleocapsid, spike and orf ab proteins, respectively, from p . furthermore, more differences in the predicted three-dimensional structure of s protein between gds and p were observed, indicating that these differences might be associated with the pathogenicity of peav. collectively, our research successfully prepared a peav attenuated variant which might serve as a live attenuated vaccine candidate against peav infection. porcine enteric alphacoronavirus (peav) was first detected by our team via genomic analysis of samples collected from a diarrhea-outbreak swine herds routinely vaccinated with porcine epidemic diarrhea virus (pedv) vaccine in a farm in guangdong, china in february (gong et al., ) . this novel swine enteric coronavirus emerged in china at least since august and it has been widely detected in guangdong, china, with a prevalence of . % by a retrospective detection study (zhou et al., ) . recently a peav-like strain cn/ fjwt/ was recently discovered in fujian, china , indicating a prevailing trend in pig farms. since the first report, the complete genome of the peav strain gds was sequenced (gong et al., ) . peav is an enveloped, single-stranded, positive-sense rna virus with a genome of appropriately kb in length (gong et al., ) . the genome organization of peav is similar to that of bat-like hku strains of coronavirus, with an order of: ′ untranslated region (utr), open reading frame a/ b (orf a/ b), spike (s), nonstructural protein (ns ), envelope (e), membrane (m), nucleocapsid (n), nonstructural protein a (ns a), and ′ utr (lau et al., ) . the s protein of coronaviruses (covs), the pivotal surface glycoprotein, is involved in virus attachment and entry, and induction of neutralizing antibodies in vivo (cruz et al., ; woo et al., ) . gds strain of peav has the smallest s protein among all covs (gong et al., ) . the clinical symptoms caused by peav in newborn piglets are similar to that by other porcine enteric pathogens such as pedv and transmissible gastroenteritis virus (tgev), which include vomiting, diarrhea, dehydration, and mortality rate as high as % (gong et al., ; zhou et al., b) . since peav was detected (gong et al., ) , another two swine enteric hku -related covs named sads-cov and seacov were identified in the same region, causing same diarrheal disease as peav strain gds by experimental infection (pan et al., ; xu et al., a; zhou et al., b) . currently, live-attenuated vaccine has been widely used to prevent and control porcine enteric pathogens, such as pedv, with good clinical effect (song et al., ) . live-attenuated vaccine is prepared from less virulent strain. the virulence of pathogenic microorganisms was reduced by various methods (blanco-lobo et al., ; jie et al., ; o'donnell et al., ) , such as genetic manipulation , and cell passage is the most commonly used (jie et al., ) . usually, the virulent parent strain was continuously inoculated cell lines more than generations in vitro, and in this process, some mutations will happen in the viral genome, which might lead to the strain's virulence reduce. these cell-passaged strains are then tested in animals to determine whether they have less virulence and higher immunogenicity before being used to prepare live-attenuated vaccines. of note, several groups have used cell passage to prepare porcine covs attenuated strains, such as pedv (lin et al., ) and tgev (motovski et al., ) , which laid a good foundation for preparation of live-attenuated vaccines. peav is an important enteropathogen in pigs, but currently no effective treatments or vaccines against peav infection are available. to develop a live-attenuated vaccine for peav, we generated candidates via serial cell passage of the parental peav gds strain and evaluated the pathogenicity of peav p , p and p in -day-old newborn piglets. further, we identified genetic changes related to attenuation by performing comparative, complete genomic sequence analysis of virulent and attenuated peav variants. vero cells were obtained from atcc (atcc number: ccl- ) (usa), and cultured in dulbecco' s modified eagle medium (dmem) (hyclone, usa) supplemented with u/ml penicillin, u/ml streptomycin, and % fetal bovine serum (fbs) (bovogen, australia). the maintenance medium for peav propagation was dmem supplemented with μg/ml trypsin (gibco, usa). the isolation and identification of peav gds strain were reported previously by our laboratory (xu et al., a) . serial virus passaging and plaque-purification in vero cells were performed as previously described with some modifications (lin et al., ; xu et al., a) . briefly, vero cells were cultured in -well plates, and washed three times with sterile ph . × phosphate buffer saline (pbs) when % confluent. ten μl of peav gds strain together with ml maintenance medium were added to plate and cultured continuously at ℃ in % co to observe cytopathic effect (cpe). around day post inoculation (d.p.i.), the plates were frozen at - ℃ and thawed twice after cpe was evident in the inoculated cell monolayers. the cells and supernatants were harvested together and used as seed stocks for the next passage. peav gds strain was passaged regularly to a total of passages in vero cells every - days and the p , p and p were plaque-purified. finally, t flasks were used for the p , p and p propagation in vero cells. four-day-old crossbred conventional newborn piglets produced by duroc × landrace × native pigs of guangdong of china were procured from wen' s foodstuffs group co, ltd (guangdong, china). all pigs were maintained in our animal facility with a mixture of skim milk powder (inner mongolia yi li industrial group co., ltd, china) with warm water ad libitum for day before the experimentation. the animal study was approved by the institutional animal care and use committee of the sun yat-sen university (guangdong, china) and animals were treated in accordance with the regulations and guidelines of this committee. kinetics of peav replication in vero cells were performed as previously described with some modifications (lin et al., ) . briefly, vero cell monolayer was inoculated with p , p and p at a moi = . . the virus inoculum was removed at h post inoculation (hpi), washed with ph . × pbs twice and replaced by the maintenance medium. culture supernatant and cell lysates were collected at , , , and hpi, respectively. after freezing and thawing once, the mixtures of culture supernatants and cell lysates were subjected to titration for % tissue culture infectious dose (tcid ) in -well plates and calculated using reed-muench method (reed and muench, ) . thirty-two -day-old conventional newborn piglets were randomly divided into four groups and piglets in each group. prior to inoculation, piglets were confirmed to be negative for the major porcine enteric viruses including pdcov, pedv, tgev, prov, peav, by testing of rectal swabs using specific rt-pcr according to previously described method (xu et al., ; zhou et al., a) . in addition, serum of each piglet was collected and a commercial kit (allright biotechono-logy, china) was applied in order to detect pedv-specific antibody according to the manufacturer' instruction. pedv-specific antibodies were not detected in all sera samples (data not shown). after -day acclimation, piglets in group were orally inoculated with ml of maintenance medium and served as uninfected controls. piglets in group , and were orally challenged with ml of maintenance medium containing a total of × tcid of p , p and p , respectively. to reduce the risk of cross contamination among piglets and piglet death caused by non-experimental factors, the entire animal study period was ended in nine days after challenge. all piglets were observed daily for clinical signs of diarrhea and lethargy. diarrhea severity was scored with the following criteria (chen et al., ) : = normal, = soft (cowpie), = liquid with some solid content, = watery with no solid content. rectal swabs were collected daily from each piglet from d.p.i. to d.p.i., submerged into ml sterile ph . × pbs immediately after collection and stored at - ℃ for virus shedding analysis. two piglets from each group were necropsied at d.p.i.. at necropsy, the fresh jejunums were collected and then formalin-fixed. the formalin-fixed samples were used for histopathology and immunohischemistry analysis. in addition, the mortality of newborn piglets in each group was recorded daily. viral rna shedding in piglet feces after infection with peav was detected as previously described with some modifications (xu et al., a) . briefly, total rna was prepared from the supernatant of rectal swab from each piglet using a rneasy kit (magen, china) according to the manufacturer' instruction, and was treated with dnase i. two μg of total rna was used for cdna synthesis by reverse transcription using rt-pcr kit (takara, dalian). the specific primers for the n gene of peav (sense: ′-ctgactgttgttgaggttac- ′; antisense: ′-tctgcc aaagcttgtttaac- ′), and probe ( ′-fam-tcacagtctcgttctcgc aatca-tarma- ′) were designed with reference to the previous publication (zhou et al., a) and synthesized by invitrogen company (shanghai, china). the real-time pcr assay was carried out with an applied biosystem instrument (life technologies, usa). the pcr was performed in a -μl volume containing μl of cdna, μl of thunderbird probe qpcr mix, . μl × rox reference dye (toyobo, shanghai), . μmol/l of probe, and a . μmol/l of each gene-specific primer. the thermal cycling parameters were as follows: °c for s; cycles of °c for s, °c for s. and the n gene was amplified to generate the standard curve from peav gds strain using the specific primers (sense: ′−ccgctcgagatggcaactgtta attgg- ′; antisense: ′-cgcggatcccgattaataatctcatccac- ′) z. xu, et al. veterinary microbiology ( ) that were designed with reference to the published sequence (genbank, accession no: mf . ), and the pcr products were cloned into the pegfp-n (clontech, usa). the known plasmid concentration was fold serially diluted for generating a standard curve in each plate. the quantity of peav viral rna in tested samples was calculated based on the cycle threshold (ct) values for the standard curve. histological and immunohistochemical staining were performed as previously described with some modifications (xu et al., a) . briefly, tissue samples of jejunum of the piglets from the challenged and control groups were separated, routinely fixed in % formalin for h at room temperature, dehydrated in graded ethanol, embedded in paraffin, cut in -μm sectioned, and mounted onto glass slides. after the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (h&e), the slides were examined and analyzed with conventional light microscopy. five-μm sections of formalin-fixed paraffinembedded tissues were placed onto positively charged glass slides and the slides were air dried for min at °c. the tissue sections were deparaffinized, rinsed and incubated with target retrieval solution (servicebio, china). after being blocked with % bsa (solarbio, china), the sections were incubated with anti-peav m monoclonal antibody (wen's foodstuffs group co., ltd, china) as the primary antibody and the dilution of antibody was : for h at °c. they were then incubated with peroxidase-labeled goat anti-mouse igg secondary antibody (dako, denmark) and the dilution of antibody was : for min at room temperature, and the samples were finally visualized with a , ′-diaminobenzidine (dab) chromogen kit (dako, denmark). hematoxylin was used for counterstaining. tissues of piglets from uninfected control groups were used as negative samples. the immunohistochemistry slides were evaluated according to the evaluation system of histology and immunohistochemistry by jung et al. ( ) . the complete genomes of p , p and p were sequenced by illuminahiseq as previously described with some modifications (malboeuf et al., ) . briefly, total rna was prepared from the peavinfected vero cells using a rneasy kit (magen, china) according to the manufacturer' instruction, and was purified as described above. cdna synthesis was performed by reverse transcription using rt-pcr kit (takara, dalian). the genomic cdna was disrupted and converted to a sticky end by adding the a-base at ′ end of the cdna. the dna containing index sequence was added at either side of sticky end by complementary bases. the target fragment within the scope of a certain length was collected before magnetic beads selection. the sequencing library was built, tested using pcr amplification index sequence, and combined to the chip by bridge pcr. at last, the complete genome was sequenced by iiiumina hiseq. a genome homology analysis and phylogenetic trees were constructed by using the maximum likelihood method with mega software (http://www.megasoftware.net/) based on the whole-genome nucleotide sequences from peav gds , p , p and p together with other covs, like pedv, pdcov and tgev. sequence alignments of n, m, s and orf ab amino acids (aa) of gds , p , p and p were performed using the dnaman. the s protein of cov, the pivotal surface glycoprotein, plays an important role in virus attachment and entry, and induction of neutralizing antibodies in vivo (cruz et al., ; li et al., li et al., , . to further determine the differences of vero-passaged peav strains, the three-dimensional structures of s proteins of gds , p , p and p were predicted in swiss-model using amino acid homology modeling, based on the existing three-dimensional structure of s glycoprotein trimer of human coronavirus nl (pdb code szs) (walls et al., ) in protein data bank. the pymol molecular graphics system (delano scientific; http://www.pymol.org) was used for figures preparation. statistical comparisons were performed using graphpad prism software. the significance of the differences between the growth rate of p and p or p in the tcid was determined by the anova and mann-whitney accordingly. to generate an attenuated peav vaccine candidate, peav gds was passaged regularly to a total of passages in vero cells and the selected passages including p , p and p were characterized by sequencing and analyzing the complete genome (fig. ) . to determine the growth rate of p , p and p in vitro, we inoculated p , p and p at moi = . in vero cells and detected the live virus count at corresponding time point with a tcid assay. interestingly, we observed that the sizes of peav-induced syncytia increased in p and p ( fig. a) . the diameter of p -syncytia was larger than that of p and p ( fig. a ). in addition, the muti-step growth kinetics of peav in vero cells showed similar growth curve trends of p , p and p , but the titers of p were significantly higher than those of p and p at h.p.i. (p < . ) (fig. b) . to evaluate the pathogenicity of vero-passaged peav strains in newborn piglets, we experimentally infected -day-old conventional newborn piglets with p , p and p at a dose of × tcid / head via oral feeding. as shown in fig. a , the newborn piglets inoculated with p , p or p all showed diarrhea from d.p.i. to d.p.i., as compared with the control. but compared to p and p , the degree of diarrhea of the newborn piglets inoculated with p was the lowest, indicating that viral pathogenicity decreased gradually in day-old newborn piglets with cell passage. in addition, we explored the fecal viral shedding in peav-inoculated piglets from d.p.i. to d.p.i. by qrt-pcr. consistent with the clinical signs, the amount of virus rna was lower in p -inoculated newborn piglets, and no peav rna was detected in the negative control piglets during the study (fig. b) . taken together, these results demonstrated that cell passage gradually decreased viral pathogenicity of gds to -day-old newborn piglets. since peav at selected passages caused varying degrees of diarrhea in newborn piglets, we also recorded the mortality of newborn piglets in each group. as shown in fig. , in p -inoculated group, except two piglets that were necropsied at d.p.i., , , , and death (s) were recorded at , , , and d.p.i., respectively. similarly, except two necropsied piglets, piglets in p -inoculated group died from to d.p.i. no piglets died in p -inoculated group and the control group except two piglets that were necropsied at d.p.i.. taken together, these results suggest that peav p strain is low pathogenic to the newborn piglets. to determine the gross pathological and histological changes in piglets infected with peav gds at selected passages, two piglets from each group were necropsied at d.p.i. gross findings were similar in piglets orally inoculated with p and p . the whole intestinal tract, where yellow watery contents accumulated, were transparent, thinwalled, and gas-distended (fig. b&c) . no lesions were observed in the whole intestinal tract of the p -inoculated piglets and the negative control piglets (fig. a&d) . microscopic lesions were also analysed. as shown in fig. f&g , abruption of intestinal villus was observed in p or p -inoculated piglets, whereas the intestines in p -inoculated piglets and the negative control were normal (fig. e&h) . consistent with the histopathological results, peav antigen was detected in the cytoplasm of the villous enterocytes of the p or p -challenged piglets by immunohistochemical analysis, but no antigen observed in p -inoculated and mock piglets (fig. i-l) . taken together, these results indicate that peav p is an attenuated variant. to determine the cause of reduced pathogenicity of vero-passaged peav strains, the complete genomes of p , p and p were sequenced and analyzed. compared to the complete genome of peav gds (mf . ), p possesses point mutations, a -nt deletion and a -nt insertion; p possesses point mutations, a -nt deletion and a -nt insertion; p possesses point mutations, a -nt deletion, a -nt deletion and a -nt insertion, respectively (data not shown), presenting a clue of different pathogenicity of these three strains. in addition, the parental strain gds shares . %, . %, . % nucleotide identity with p , p and p , respectively (data not shown), indicating that differences in viral genome increased gradually with cell passages. in spite of some differences in viral genome of these strains, phylogenetic analysis showed that peav gds , p , p and p were clustered into a large clade (fig. a) . we further analyzed the aa of proteins which might be associated with the pathogenicity of covs, such as m ( results are representative of three independent experiments. data are represented as mean ± sd, n = . *stands for p < . . z. xu, et al. veterinary microbiology ( ) (hou et al., b) . as shown in fig. b , compared to the m protein of peav gds , p possesses aa mutations, p possesses aa mutations, and p possesses aa mutations. compared to the n protein of peav gds , interestingly, only p possesses aa mutation, while no mutations were observed in p and p . compared to the s protein of peav gds , p possesses aa mutation, p possesses aa mutations, while p possesses aa mutations and aa deletions. in addition, compared to the orf ab protein of peav gds , p possesses aa mutations, aa insertions, p possesses aa mutations, aa insertions, p possesses aa mutations, aa deletion and aa insertions. the detailed information of aa changes might help to explain the low pathogenicity of p to the newborn piglets. . . peav p has significant differences in the s protein structure considering the obvious aa mutations and deletions in s proteins of vero-passaged peav strains, we attempted to predict and analyze the three-dimensional structures of s proteins of these peav strains. after analysing the amino acid sequences, the s protein in gds was found to have . % identity with the s protein of human cov nl (walls et al., ) the s protein structures of these strains were predicted by swiss-model, using the s protein of human cov nl as the template (fig. ) . the predicted structure of gds s protein forms a homologous trimer with an expect-value of . , and each monomer is composed of residues in the aa positions - (fig. a&b) . in z. xu, et al. veterinary microbiology ( ) addition, the predicted three-dimensional structures of the s proteins of peav p , p and p have similar overall structures when compared with gds s protein ( fig. c-f) . however the monomer structural overlap of the four s proteins showed that there were three loops and two β strands having significant structural differences in the main chain, especially between p and gds . since the first report of peav in pigs in early february of in guangdong, china (gong et al., ) , this novel swine enteric cov has been widely detected in guangdong, china, with a prevalence of . % and an emergence in china as early as august by a retrospective detection study (zhou et al., ) . although a few studies have demonstrated peav was highly pathogenic to newborn piglets (pan et al., ; xu et al., a; zhou et al., b) , there are no effective treatments or any vaccines against peav infection. in the present study, attenuated peav variants were generated by serial cell passaging and orally inoculated to newborn piglets to evaluate the attenuation in vivo. further, we identified the molecular basis of attenuation by performing comparative, complete genomic sequence analysis of virulent and attenuated peav variants. swine enteropathogenic covs affects all sectors of the pig industry and all cycles of production, which results in significant economic losses. currently, vaccination remains the most effective measure against swine enteropathogenic covs. of note, several groups have used live-attenuated vaccines to prevent and control pedv and tgev in pigs (aynaud et al., ; song et al., ) . as a newly swine enteropathogenic cov, there are no reports about live-attenuated vaccine for peav. pathogenic microorganisms serially passaged in cells (jie et al., ) is one of the most common methods to prepare live-attenuated vaccine. vero cells as a stable african green monkey kidney cell line (rhim et al., ) is commonly used to attenuate covs like pedv (lin et al., ) . in this study, peav gds was passaged regularly to a total of passages in vero cells and the growth rate of p , p and p was determined in vitro. the muti-step growth kinetics of p , p and p in vero cells were similar, but the titers of p were fig. . the survival rate of newborn piglets after infection with peav p , p and p . the mortality of newborn piglets in each group was recorded from to d.p.i.. significantly higher than those of p and p at hpi, similar to observations in attenuated pedv vaccine (lin et al., ) , indicating that p had better adaptation in cells in vitro. usually, adaptation in cell lines coincides with attenuation of the viruses, such as african swine fever virus (asfv) isolate georgioa and pedv isolate pc a, in swine lin et al., ) . the higher fitness of p in vero cells might suggest the changed pathogenicity of peav. for a vaccine candidate, safety is the top concern. it was reported that swine enteric covs like pdcov, pedv and tgev are age-dependent disease (moon et al., ; shibata et al., ; xu et al., b) . similarly, in another study, we found that peav was highly pathogenic to newborn piglets (xu et al., a) , but did not cause vomiting and death in weaned piglets at a medium dose of × tcid within days (data not shown), for which reason we choose newborn piglets to conduct peav pathogenicity study. it was known that peav infection caused typical clinical symptoms characterized by watery diarrhea via oral feeding in newborn piglets (xu et al., a) . in the study, we infected the -day-old newborn piglets with p , p and p via oral feeding. compared with p and p , p caused slight diarrhea in newborn piglets, suggesting that the pathogenicity of peav changed through cell passage. for enteric viruses, viral shedding is often accompanied by diarrhea, so was peav infection (xu et al., a) . thus, fig. . phylogenetic trees constructed on the basis of the complete genome sequences of peav p , p and p or other covs and sequence alignments of peav at selected passages. (a) the dendrogram was constructed using the neighbour-joining method in the mega software package, version (http://www.megasof tware.net). bootstrap resampling with , replicates was performed, and bootstrap values are indicated for each node. reference sequence obtained from genbank is indicated by strain name. the scale bar represents . nucleotide substitutions per site. (b) sequence alignments of the amino acids of m, n s and orf ab proteins were performed using dnaman software. z. xu, et al. veterinary microbiology ( ) we collected fecal swabs from the peav-challenged piglets and detected the fecal shedding by real-time pcr. consistent with the clinical signs, the amount of virus rna was lower in p -inoculated newborn piglets. interestingly and importantly, the presence of peav rna in fecal samples of p inoculated piglets from d.p.i to d.p.i suggested the successful colonization and replication of p in intestinal tract, which is essential to induce mucosal immunity for an enteric pathogen vaccine candidate. it was reported that the lesions were only observed in intestinal tract but not any other organs after peav infection and peav antigen was detected in the villous enterocytes of the peav-challenged piglets (xu et al., a) . compared to p and p , p strain caused slight macroscopic and microscopic lesions in intestinal tract and no peav antigen was detected in the cytoplasm of the villous enterocytes of the p -challenged newborn piglets by immunohistochemical analysis, indicating that the infectiveness of p was reduced. like other swine enteric covs, such as pedv (shibata et al., ) , peav infection caused fatal disease in newborn piglets (xu et al., a) . thus, death after infection is the key indicator to determine the attenuation of the viral strain. compared to p and p , no piglets died in p -inoculated group and the control group during the study, indicating that p is low pathogenic to the newborn piglets. taken together, these results demonstrated that p strain is safe for pigs and effective in colonizing and replicating in intestine, suggesting its future application as a good peav vaccine candidate. compared to p and p strains, the virulence of p was significantly reduced in -day-old newborn piglets. the s protein of covs is the pivotal surface glycoprotein involved in virus attachment and entry, virus attenuation and induction of neutralizing antibodies in vivo (cruz et al., ; lin et al., ; woo et al., ) . in addition, the virulence of a recombinant pedv with inactivation of the endocytosis signal of the s protein was significantly reduced in piglets (hou et al., a) , indicating that the s protein might be associated with the virulence of covs. compared to the s protein of parental strain gds , p possesses aa mutations in aa positions and , which were also found in p and p , indicating that these aa-mutations are insufficient to cause viral attenuation. of note, compared to p and p , p possesses aa deletions and aa mutation in aa positions , , and , respectively, indicating that the absence and mutation of these amino acids might affect the viral attenuation. usually, protein with complete structure has corresponding function, and the changes in the protein's structure might affect its function. overlap of the s protein structures of gds , p , p and p showed that the difference between p and gds was most significant (fig. ) , indicating a potential relationship between these differences of s protein and pathogenicity of peav. it was reported that the s protein is necessary but not sufficient for the virulence of pedv (wang et al., ) . m protein plays an important role in the process of virus assembly by interacting with s and n proteins (de haan et al., ; nguyen and hogue, ) . n protein can encapsulate the viral genome rna to form the nucleocapsid (spaan et al., ; sturman et al., ) . inactivation of the viral ′-o methyltransferase of nsp could help to engineer a live attenuated pedv vaccine (hou et al., a) . in this study, aa changes were also found in m, n and orf ab proteins in p strain as compared to gds (fig. b ). in addition, no additional mutation was observed by sequencing the viruses shed from the challenged piglets (data not shown). therefore, these results suggest that the complete attenuation of a peav strain might be the result of a combination of multiple mutations along the genome, and can occur via multiple molecular mechanisms. whether individual or a combination of genetic changes of peav strains alter viral infectivity, pathogenicity and replication efficiency need further examination by reverse genetics technology. together, all these results confirmed that p generated in this study was low pathogenic to the newborn piglets and might serve as a good peav vaccine candidate. however, there are still several important questions needed to be addressed. for instance, what is the immunogenicity of p strain as a live-attenuated vaccine candidate in pigs? what is the protective effect of p strain in pigs? elucidation of these questions will help us to develop a safe and efficacious live-attenuated vaccine to control peav. in summary, our research successfully generated an attenuated peav variant p . p has the highest titers of virus load, via serial passaging of the parental gds strain. remarkably, inoculation of newborn piglets with p by oral feeding caused the lowest level of clinical symptoms, including slight diarrhea, fecal viral shedding, and pathological changes with a survival rate of % in newborn piglets. the changes in genomic composition and structures found in p by genomic analysis might account for the underlying molecular mechanisms of peav attenuation. cyx, zcx and yl conceived and designed the experiments; zcx, yl, pp, yw, yl performed the experiments; zcx analyzed the data; ycc, cyx, ccz and lg contributed reagents/materials/analysis tools; zcx wrote the paper. cyx checked and finalized the manuscript. all authors read and approved the final manuscript. the animal study was supervised by the institutional animal care and use committee of sun yat-sen university (iacuc dd- - ) and used in accordance with regulation and guidelines of this committee. the authors declare that they have no conflict interest. induction of lactogenic immunity to transmissible gastroenteritis virus of swine using an attenuated coronavirus mutant able to survive in the physicochemical environment of the digestive tract novel approaches for the development of live attenuated influenza vaccines pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -dayold neonatal piglets the gprlqpy motif located at the carboxyterminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus mapping of the coronavirus membrane protein domains involved in interaction with the spike protein a new bat-hku -like coronavirus in swine engineering a live attenuated pedv vaccine candidate via inactivation of the viral ′-o methyltransferase and the endocytosis signal of the spike protein deletion of both the tyrosinebased endocytosis signal and the endoplasmic reticulum retrieval signal in the cytoplasmic tail of spike protein attenuates porcine epidemic diarrhea virus in pigs the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs the progressive adaptation of a georgian isolate of african swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome complete genome sequence of bat coronavirus hku from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies complete genome sequence of a novel swine acute diarrhea syndrome coronavirus cellular entry of the porcine epidemic diarrhea virus attenuation of an original us porcine epidemic diarrhea virus strain pc a via serial cell culture passage complete viral rna genome sequencing of ultra-low copy samples by sequence-independent amplification age-dependent resistance to transmissible gastroenteritis of swine. iii. effects of epithelial cell kinetics on z coronavirus production and on atrophy of intestinal villi experiments to produce an attenuated strain of the transmissible gastroenteritis virus and its use as a live vaccine protein interactions during coronavirus assembly african swine fever virus georgia isolate harboring deletions of mgf and mgf genes is attenuated in swine and confers protection against challenge with virulent parental virus discovery of a novel swine enteric alphacoronavirus (seacov) in southern china a simple method of estimating fifty per cent endpoints biological characteristics and viral susceptibility of an african green monkey kidney cell line (vero) isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr strain coronavirus mrna synthesis involves fusion of non-contiguous sequences isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy the s gene is necessary but not sufficient for the virulence of porcine epidemic diarrhea virus novel variant strain bj c coronavirus genomics and bioinformatics analysis isolation and characterization of a highly pathogenic strain of porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets porcine deltacoronavirus induces tlr , il- , ifn-alpha, ifn-beta and pkr mrna expression in infected peyer's patches in vivo a highly pathogenic strain of porcine deltacoronavirus caused watery diarrhea in newborn piglets suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china development of a taqman-based real-time rt-pcr assay for the detection of sads-cov associated with severe diarrhea disease in pigs key: cord- -a fscyjf authors: smith, joseph a.; wellehan, james f.x.; pogranichniy, roman m.; childress, april l.; landolfi, jennifer a.; terio, karen a. title: identification and isolation of a novel herpesvirus in a captive mob of eastern grey kangaroos (macropus giganteus) date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: a fscyjf a novel herpesvirus was detected in a captive mob of eastern grey kangaroos (macropus giganteus) during diagnostic workup for individuals with ulcerative cloacitis. virus was initially detected in tissues using a consensus herpesvirus pcr. no viral inclusions or particles had been evident in routine histologic or transmission electron microscopic sections of cloacal lesions. virus was isolated from samples and transmission electron microscopy of the resulting isolates confirmed that the virus was morphologically consistent with a herpesvirus. nucleotide sequencing of the pcr product from tissue samples and from the isolates revealed that the virus was in the subfamily gammaherpesvirinae and was distinct from other known herpesviruses. the correlation between the lesions and the novel virus remains unknown. two herpesviruses, both in the subfamily alphaherpesvirinae, have previously been described in macropods and are known to cause systemic clinical disease. this is the first reported gammaherpesvirus within the order marsupialia, and may provide valuable information regarding the evolution and phylogeny of this virus family. based on current herpesvirus nomenclature convention, the authors propose the novel herpesvirus be named macropodid herpesvirus (mahv- ). identification and isolation of a novel herpesvirus in a captive mob of eastern grey kangaroos (macropus giganteus) the family herpesviridae are enveloped, doublestranded dna viruses. two herpesvirus species have been previously described in macropodidae (kangaroos www.elsevier.com/locate/vetmic and wallabies)-macropodid herpesvirus (mahv- ) and macropodid herpesvirus (mahv- ). both viruses have been classified in the subfamily alphaherpesvirinae based on genetic sequencing and biological characteristics (johnson et al., ; johnson and whalley, ; mahony et al., ; davison et al., ) . mahv- was first identified by finnie et al. ( ) in a captive mob of parma wallabies (macropus parma). the viral infection produced fatal systemic disease resulting in severe clinical signs including pyrexia, respiratory signs, conjunctivitis, and anogenital vesicles (finnie et al., ; acland, ) . mahv- was later identified in in captive grey dorcopsis (dorcopsis muelleri luctuosa) and quokkas (setonix brachyurus) . this virus also resulted in fatal systemic disease characterized by conjunctivitis and lesions on the oral and anogenital mucous membranes. latent infection with an unclassified alphaherpesvirus was described in the eastern grey kangaroo (m. giganteus) (guliani et al., ) . herpesvirus infections have also been reported or suspected in other macropod species including tammar wallabies (m. eugenii) and western grey kangaroos (m. fuliginosus), as well as in two species in the family potoroidae-brush-tailed rat kangaroos (bettongia penicillata) and rufous rat kangaroos (aepyprymnus rufescens) (dickson et al., ; callinan and kefford, ; wilks et al., ) . in addition to these natural infections, transient experimental infections with mahv- have been produced in the brushtail possum (trichosurus vulpecula) (zheng et al., ) . a serologic survey revealed that % of wild marsupials tested and % of captive marsupials in australia tested had circulating serum-neutralizing antibodies to a parma wallaby herpesvirus (webber and whalley, ) . however, it is unknown whether the seropositivity reflects exposure to known macropod herpesviruses, previously undescribed herpesviruses, or cross-reaction with antibodies to other antigens unrelated to herpesvirus exposure. in , a novel herpesvirus was found in a captive mob of eastern grey kangaroos while performing diagnostic workups to determine the etiology for ulcerative cloacitis. no clinical signs or evidence of herpesvirus infection were detected in the mob prior to . during the sampling period, the captive mob was composed of individuals consisting of adult breeding males, adult females, and joeys less than years of age ( males, females, and unknown gender). some of the adult females were captive-bred in the united states, some were captive-bred in australia, and some were wild-caught rehabilitated animals. the individuals that originated in australia were imported into the united states between june and december . fifteen of the kangaroos in the mob ( . %; mean age = . years; median age = . years) were evaluated for the presence of herpesvirus. samples collected from live animals included whole blood, cloacal swabs, and cloacal biopsies (table ) . whole blood was collected from the lateral tail vein and placed in glass edta vacutainer blood tubes (becton, dickinson and company, franklin lakes, nj). cloacal swabs were obtained using sterile cotton-tipped applicators. cloacal biopsies were obtained using a sterile punch biopsy. samples collected at necropsy for pcr and viral isolation included - cm sections of cloacal mucosa, liver, spleen, mammary gland, and lymph node. these samples were placed in either sterile vacutainer tubes with no additive (becton, dickinson and company, franklin lakes, nj) or in sterile whirl-pak bags (nasco international, inc., fort atkinson, wi), and either submitted fresh on ice packs or stored frozen. samples for histopathology were placed in % neutral buffered formalin. samples submitted for electron microscopy were placed in karnovsky's fixative. tissues collected at biopsy and necropsy were fixed in % neutral buffered formalin, routinely processed, sectioned at mm, and stained with hematoxylin and eosin (he) for routine light microscopy. for papillomavirus immunohistochemistry, endogenous peroxidase was blocked in deparaffinized and rehydrated sections by immersing sections in hydrogen peroxide ( . %) in methanol for min. slides were rinsed twice in phosphate buffered saline ph . (biogenex, san ramon, ca) for min followed by incubation in a commercially available blocking serum (power block universal blocking reagent, biogenex) for min to prevent non-specific binding. after rinsing in buffer, slides were incubated at room temperature for min with : polyclonal anti-bovine papillomavirus antibody (bpv- , dako, carpinteria, ca). negative serum was used on negative control slides. slides were rinsed in buffer and incubated with the secondary antibody (lsab , dako) at room temperature for min. following an additional buffer rinse, streptavidin peroxidase (dako) was applied. after rinsing, the chromagen, , -diaminobenzidine tetrachloride (dab; biogenex), was applied and allowed to develop for min. slides were rinsed and then counterstained in mayer's hematoxylin (biogenex). for ultrastructural studies, multiple samples containing optimal regions from the cloaca of two kangaroos that were pcr positive for herpesvirus were fixed in karnovsky's fixative or % gluteraldehyde with . % paraformaldehyde, postfixed in % osmium tetroxide, dehydrated, and embedded in epoxy resin. sections were first cut at . mm and stained with toluidine blue and basic fuscin for microscopic evaluation of optimal areas to evaluate by electron microscopy and then sectioned at - nm, stained with uranyl acetate and lead citrate, and examined with a h transmission electron microscope (hitachi high technologies america, inc., pleasanton, ca). dna was extracted from frozen tissue samples using the dneasy kit (qiagen, valencia, ca). nested pcr amplification of a partial sequence of the dnadependent-dna polymerase gene was performed using previously described methods (vandevanter et al., ) . briefly, the first round of amplification utilized forward primers dfa ( -gayttygcna-gyytntaycc- , y = pyrimidine, n = nucleotide) and ilk ( -tcctggacaagcagcarnysg-cnmtnaa- , r = purine, m = a or c) and reverse primer pcr amplification utilized forward primer tgv ( -tgtaactcggtgtayggnttyacngg-ngt- ) and reverse primer iyg ( -cacagagt-ccgtrtcnccrtadat- , d = a, g, or t). the mixtures were amplified with an initial denaturation at virus isolation results marked with ''co'' were negative for herpesvirus, but positive for a coronavirus. b = whole blood, l = liver, s = spleen, mg = mammary gland, ln = lymph node, c = cloaca, . = male, . = female. c for min, followed by cycles of denaturation at c for s, annealing at c for s, dna extension at c for s, and a final extension step at c for min. products were resolved on % agarose gels and bands of the expected size were excised and purified using the qiaquick gel extraction kit (qiagen). to obtain additional sequence, an internal reverse primer was designed (primer roorev tagttctgcctcggagggtgacggt) and used in the second round with dfa. direct sequencing was performed using the big-dye terminator kit (perki-nelmer, branchburg, nj) and analyzed on abi automated dna sequencers at the university of florida center for mammalian genetics dna sequencing facilities. all products were sequenced in both directions. primer sequences were edited out prior to further analyses. sequences were compared with those in genbank (national center for biotechnology information, bethesda, md), embl (cambridge, uk) and the data bank of japan (mishima, shiuoka, japan) using tblastx (altschul et al., ) . predicted homologous - amino acid sequences of herpesviral dna-dependent-dna polymerase were aligned using three methods; clustalw (thompson et al., ) , t-coffee (notredame et al., ) , and muscle (edgar, ) . bayesian analyses of each alignment were performed using mr.bayes . (ronquist and huelsenbeck, ) with gamma distributed rate variation and a proportion of invariant sites, and mixed amino acid substitution models. the first % of , , iterations were discarded as a burn in. maximum likelihood (ml) analyses of each alignment were performed using phylip (phylogeny inference package, version . ) (felsenstein, ) , running each alignment in proml with amino acid substitution models jtt (jones et al., ) , pmb (veerassamy et al., ) , and pam (kosiol and goldman, ) further set with global rearrangements, replications of random input order, less rough, one category of sites, and unrooted. iguanid herpesvirus (genbank accession no. ay ) was designated as the outgroup due to its early divergence from other herpesvirus (wellehan et al., ; . the combination of alignment producing the most likely tree was then used to create data subsets for bootstrap analysis to test the strength of the tree topology ( resamplings) (felsenstein, ) , which was analyzed using the amino acid substitution model producing the most likely tree. a suspension of potoroo kidney cell line (ptk ; atcc, manassas, va, usa) cells was prepared in mem growth medium (sigma chemical co., st. louis, mo, usa) supplemented with % fetal bovine serum (fbs) at a concentration of x cells/ml in a cm cell culture flask. consensus herpesvirus pcr was used to confirm that the cell line was free of herpesviruses prior to inoculation (vandevanter et al., ) . when monolayers of cells were formed, the ptk were infected with a % homogenate of the sample tissue prepared in earl's balanced salt solution and incubated for h, and the inoculum was replaced with % fbs cell culture media and incubated in an incubator at c, % co . the cultures were monitored daily for days for the presence of cytopathic effect. cell cultures were passed to the next flask regardless of whether cytopathic effects were observed or not. supernatant from the flask was checked for the presence of viral material by electron microscopy and for herpesvirus by pcr. eight of the fifteen kangaroos evaluated ( . %; males, females) had gross lesions ( table ) . six of those eight ( . %; males, females) had cloacal lesions ranging from a small focal mucosal ulcer to chronic fibrinonecrotic cloacitis. clinical signs associated with the cloacal lesions included overgrooming and licking of the cloacal region, mild cloacal discharge, and bronzing of the hair around the cloaca. age of affected individuals ranged from . to . years (mean age = . years; median age = . years). the younger kangaroos exhibited smaller, more acute lesions, while the older individuals had more severe, chronic lesions with secondary infections. five of the eight kangaroos ( . %; males, females) with gross lesions had firm, irregular masses palpable within the mammary tissue. age of the female kangaroos with mammary gland masses ranged from . to . years (mean age = . years; median age = . years). all five individuals with mammary masses died during the sampling period. seven of the fifteen kangaroos evaluated ( . %; male, females) died during the sampling period. five of the seven were geriatric females with mammary masses. the remaining two kangaroos ( and ) died from acute head or neck trauma. no other patterns of gross lesions were detected in those individuals presented for necropsy. seven of the fifteen kangaroos died and tissues were available for histological evaluation. antemortem cloacal biopsies were also available from two of these seven animals ( and ). four of seven kangaroos had an ulcerative cloacitis either at biopsy or necropsy. kangaroos , , and did not have any cloacal lesions at necropsy. in affected animals, the cloacal mucosal epithelium was markedly hyperplastic with prominent broad, branching rete ridges and parakeratotic hyperkeratosis of the stratum corneum. within the epithelium there were variable numbers of discrete vesicles containing luminal necrotic cellular debris. adjacent intact epithelium had both intra and intercellular edema. in a few cases, there were regions of ulceration overlain by fibrin, admixed cell-debris, and superficial bacterial colonization. bacteria were confined to the superficial crusts and interpreted as secondary colonizers. in a few cases, there were increased numbers of fibroblasts and small blood vessels in the exposed dermis (granulation tissue). another significant finding in five kangaroos was simple carcinoma of the mammary gland. with the exception of kangaroo , all affected kangaroos had evidence of local lymph node metastasis and kangaroos , and had distant (pulmonary and or hepatic) metastasis. no splenic lesions were noted in any of the examined kangaroos. other histologic findings were considered incidental or age related changes common in this species. there was no specific immunoreactivity in sections of cloaca using antibodies against papillomavirus sp. in kangaroos and . no viral particles were identified by transmission electron microscopy (tem) within any cells or in extracellular spaces in sections of cloaca with hyperplastic, vesicular, and ulcerative lesions from kangaroos and . initial pcr amplification yielded a base pair product (after editing) from all fifteen kangaroos evaluated. details of results can be found in table . nucleotide sequences for all cases were identical. additional sequence for phylogenetic comparison was obtained from spleen and lymph node samples from kangaroo , totaling bp. sequences were submitted to genbank under accession number ef . comparison with other sequences using tblastx revealed that this virus is similar to, but distinct from other herpesviruses present in the available databases. the highest score obtained was with hylobates leucogenys rhadinovirus (genbank accession no. ay ). hylobates leucogenys rhadinovirus is in the subfamily gammaherpesvirinae. bayesian phylogenetic analysis showed the greatest harmonic mean of estimated marginal likelihoods using the muscle alignment (fig. ). the wag model of amino acid substitution was found to be most probable with a posterior probability of . (whelan and goldman, ) , and a posterior probability of . for the rtrev model (dimmic et al., ) . a bayesian tree using the muscle alignment is shown (fig. ) . ml analysis found the most likely tree from the muscle alignment and the pmb model of amino acid substitution. these parameters were used for bootstrap analysis. bootstrap values from ml analysis are shown on the bayesian trees. one significant difference of the ml methods used from the bayesian methods used is the use of a single rate category for sites, which may decrease accuracy. samples from four kangaroos were submitted for virus isolation (table ) . virus was isolated from three of the four kangaroos and the presence of virus was confirmed by tem, pcr, and sequencing of amplified product. a virus with morphology consistent with herpesviruses (an enveloped virus around nm in size) was identified by tem. pcr testing on cell culture and tissue homogenate confirmed the presence of viral dna in the samples tested. cytopathic effects seen in culture included cell rounding, cytoplasmic stranding, and cell nucleus enlargement with some cell destruction visible. a coronavirus was also isolated from three of the kangaroos. herpesvirus was not isolated from any of the samples that were positive for coronavirus. diagnostic testing to evaluate the cause of an ulcerative cloacitis in a captive mob of eastern grey kangaroos resulted in the discovery of a novel herpesvirus. based on sequencing of a pcr product, the novel virus is classified in the subfamily gammaherpesvirinae. all previously described herpesviruses in marsupials have been classified in the subfamily alphaherpesvirinae. the novel herpesvirus does not appear to be associated with the same high degree of mortality associated with mahv- and mahv- . additionally, the clinical signs are not as severe or widespread in the eastern grey kangaroo. the only clinical sign detected in the mob suggestive of herpesvirus infection was the ulcerative cloacitis. however, not all pcr positive animals had cloacitis, and one individual with cloacitis was negative by pcr for herpesvirus in sections of cloaca. additionally, the presence of virus within the affected cloacal regions could not be demonstrated by histopathology or tem. therefore, the potential role of this herpesvirus in the pathogenesis of ulcerative cloacitis remains unclear. in addition, definitive viral cell tropism has yet to be determined. the chronicity of the lesions and the presence of secondary bacterial infections affected the gross and histologic appearance of the lesions. pcr and virus isolation results revealed the presence of virus dna and/or viral particles in a variety of different tissues, including whole blood. pcr-positive sampling sites included areas with gross lesions (e.g. cloaca, mammary tissue), as well as those with no evidence of gross or histologic abnormalities. the lack of correlation further underscores the uncertainty of this virus' pathogenicity. the exact origin of the novel herpesvirus and the reason for the acute onset of ulcerative cloacitis remains unknown. the location of the cloacal lesions is suggestive of venereal transmission. however, the exact modes of transmission and the presence of the novel virus in wild populations of eastern grey kangaroos remain unknown. it is possible that advancing age and concurrent disease caused recrudescence of a latent herpesviral infection in a few individuals. virus particles could then be spread throughout the rest of the mob through direct contact from breeding activity and other interactions. it is possible that the presence of neoplasia in geriatric individuals caused immunosuppression, resulting in a recrudescence of a latent herpesviral infection. it is unknown whether the novel herpesvirus is related to the seemingly high incidence of mammary neoplasia in the mob. gammaherpesviruses have been associated with carcinomas in sealions (lipscomb et al., ) , but further research will be needed to assess the possible association in kangaroos. the herpesvirus sequence in this study is from a conserved region, and these differences together with host species are significant enough to differentiate this from other known herpesvirus species. in another study, analysis of a smaller portion of this region in five strains of human herpesvirus- , strains of human herpesvirus- , and five strains of human herpesvirus- were sequenced. only single base variations were seen within a given species, which did not result in alteration of the amino acid sequence (vandevanter et al., ) . over this shorter region, the novel virus in this study shared identity with only of amino acids when compared to the most homologous sequence, indicating this virus is representative of a new species. current naming conventions for herpesviruses use host family and order of virus discovery (davison et al., ) . there are two previously described herpesviruses from members of the macropodidae, macropodid herpesvirus (mahv- ), and macropodid herpesvirus (mahv- ), both of which are in the subfamily alphaherpesvirinae. the phylogenetic tree demonstrates that the virus in this study significantly clusters with other herpesviruses in the subfamily gammaherpesvirinae (fig. ) . based on naming conventions, this virus should be named macropodid herpesvirus (mahv- ). the topology is generally consistent with that of phylogenetic trees generated from other data sets (wellehan et al., ; . comparative sequence analysis of the herpesviruses of diverse host species should contribute to a further understanding of viral phylogeny and the evolution of this important class of viruses. previous phylogenetic analyses suggest that many elements in the branching patterns of herpesviridae are congruent with branching patterns for the corresponding host species (jackson, , mcgeoch et al., . the branching of the alphaherpesviruses from the betaand gammaherpesviruses has been estimated to have occurred approximately - million years ago, and the branching of the betaherpesviruses from the gammaherpesviruses approximately - million years ago . within the gammaherpesviruses, the divergence of the rhadino-virus/percavirus lineages has been estimated to have occurred - million years ago . looking at host species, the divergence between marsupials and placental mammals has been estimated to have occurred - million years ago (van rheede et al., ) , which would occur temporally after the estimated beta/gamma split but before rhadinovirus/percavirus divergence. this is in agreement with the phylogenetic analysis, and supports coevolution. the availability of more complete data sets for comparison results in greater phylogenetic resolution (flynn et al., ) . the herpesvirus polymerase is the gene for which the most comparative sequence from other herpesviral species is available. additionally, when looking at evolutionary relationships of more distantly related organisms, the continued accrual of mutations resulting in homoplasy can diminish the ability to correctly resolve phylogeny, making a rapidly mutating gene a poor choice. genes for which there is strong negative selection will have fewer nucleotides with a history of multiple changes, making them a better choice for resolving phylogeny over greater distances. genes that are critical for basic organismal functions and are not under heavy immune selection are often highly conserved. therefore, viral polymerases are usually good choices for long-range phylogeny (attoui et al., ; gonzalez et al., ; knopf, ) . it has recently been proposed to split the genus rhadinovirus into three genera; macavirus containing the malignant catarrhal fever viruses, percavirus containing perissodactylid and carnivore gammaherpesviruses, and rhadinovirus containing the remaining viruses from the old genus (mcgeoch et al., ) . while the analysis in this study supports clear divergence of the proposed macavirus from other genera, the division between rhadinovirus and percavirus is not as clear, and the new rhadinovirus appears to be paraphyletic in this analysis. one possible explanation for the differences in this analysis from previous analyses is the inclusion of mahv- and afrotherian gammaherpesviruses in this analysis. mahv- clusters with the rhadinovirus/percavirus/ macavirus lineages, but does not cluster with any one genus, and may be a distinct lineage. other possible explanations for the differences seen are the assessment of multiple alignment algorithms and substitu-tion models for optimality in this study. both nucleotide sequence alignment and amino acid substitution models may have significant effects on results (morrison and ellis, , whelan and goldman, ) . finally, this analysis is of a smaller data set from a single gene which may have resulted in error. the other marsupial herpesviruses, mahv- and mahv- , are members of the genus simplexvirus in the subfamily alphaherpesvirinae (davison et al., ) . they are closely related to eutherian viruses in the genus simplexvirus, and appear to be an example of host switching rather than coevolution lee and smith, ) . alphaherpesvirinae are often capable of infecting a wider range of host cells than beta-and gammaherpesviruses (davison et al., ) , and this may make a host switch more feasible. mahv- may represent a lineage that has evolved in marsupials, may have arisen very close to the basal branching point of the gammaherpesviruses, and may provide insight into the genetic structure of the ancestral herpesviruses. further sequence and analysis of this virus is indicated to clarify. macropodid herpesvirus (mahv- ) is a novel gammaherpesvirus discovered in a captive mob of eastern grey kangaroos. although the clinical signs observed in the mob could not be definitively linked to the novel virus, mahv- in the eastern grey kangaroo does not appear to result in the same severity of clinical signs associated with the previously described alphaherpesviruses in marsupials. this is consistent with a longer established host-virus coevolutionary relationship as seen in other herpesviruses, whereas the oftenfatal disease seen with mahv- and mahv- may suggest a more recent host switch. comparison of sequencing data of a pcr product obtained from the virus to sequencing data from other known herpesviruses classifies the novel virus in the subfamily gammaherpesvirinae. however, the novel virus could not easily be placed into any of the four proposed genera within that subfamily. being the first gammaherpesvirus found in marsupials, mahv- may provide valuable information into the evolution and subsequent phylogenetic classification of herpesviruses. parma wallaby herpesvirus infection gapped blast and psi-blast: a new generation of protein database search programs common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of golden shiner reovirus, grass carp reovirus striped bass reovirus and golden ide reovirus (genus aquareovirus, family reoviridae) mortalities associated with herpesvirus infection in captive macropods family herpesviridae herpesvirus hepatitis in rat kangaroos rtrev: an amino acid substitution matrix for inference of retrovirus and reverse transcriptase phylogeny muscle: multiple sequence alignment with high accuracy and high throughput confidence limits on phylogenies: an approach using bootstrap phylip-phylogeny inference package mortalities in parma wallabies (macropus parma) associated with probable herpesvirus molecular phylogeny of the carnivora (mammalia): assessing the impact of increased sampling on resolving enigmatic relationships a comparative sequence analysis to revise the current taxonomy of the family coronaviridae reactivation of a macropodid herpesvirus from the eastern grey kangaroo (macropus giganteus) following corticosteroid treatment the effect of paralogous lineages on the application of reconciliation analysis by cophylogeny mapping macropodid herpesvirus and : two herpesviruses from australian marsupials differentiated by restriction endonucleases dna composition and hybridisation structure and physical map of the genome of parma wallaby herpesvirus the rapid generation of mutation data matrices from protein sequences evolution of viral dna-dependent dna polymerases different versions of the dayhoff rate matrix analysis of the thymidine kinase genes of macropodid herpesviruses and common metastatic carcinoma of california sea lions (zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus macropodid herpesviruses and occupy unexpected molecular phylogenic positions within the alphaherpesvirinae integrating reptilian herpesviruses into the family herpesviridae on phylogenetic relationships among major lineages of the gammaherpesvirinae topics in herpesvirus genomics and evolution effects of nucleotide sequence alignment on phylogeny estimation: a case study of s rdnas of apicomplexa t-coffee: a novel method for fast and accurate multiple sequence alignment mrbayes : bayesian phylogenetic inference under mixed models clustal w: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position specific gap penalties and weight matrix choice detection and analysis of diverse herpesviral species by consensus primer pcr the platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and therians a transition probability model for amino acid substitutions from blocks widespread occurrence in australian marsupials of neutralizing antibodies to a herpesvirus from a parma wallaby a novel herpesvirus associated with hepatic necrosis in a san esteban chuckwalla (sauromalus varius) a general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach herpesvirus as a cause of fatal disease in australian wallabies experimental infection of possums with macropodid herpesvirus the authors would like to acknowledge the animal care staff of the australia section of the fort wayne children's zoo for their assistance with identification of lesions and with sample collection. we also thank jane chladny and the university of illinois histology laboratory for histologic slide preparation and lou ann miller for preparation of materials for tem, as well as phyllis lockerd, olivia anstaett, and virginie lazar from the purdue university animal disease diagnostic laboratory for technical assistance with sample preparation. key: cord- - qbbgp authors: shibata, isao; tsuda, tomoyuki; mori, masahumi; ono, masaaki; sueyoshi, masuo; uruno, katsuyoshi title: isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: qbbgp this paper describes the isolation of porcine epidemic diarrhea (ped) virus in vero and porcine cell cultures, and the influence of age on disease in experimental infection. ped virus was isolated from the small intestine of piglets inoculated with ped samples and cultured in vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (mm) containing trypsin. in porcine bladder and kidney cell cultures inoculated with isolated ped virus, cytopathic effects (cpe) including cell fusion were detected. specific brilliant fluorescence was observed in the cytoplasm of these cells. two- and -day old, and -, -, - and -week old specific pathogen-free (spf) pigs were orally inoculated with ped virus isolated from an outbreak. all - and -day old pigs inoculated developed severe watery diarrhea from post-inoculation day (pid) and died between pid and . although three of five -week old pigs developed diarrhea on pid – , they eventually recovered. in the -week old group, three of five pigs had mild diarrhea for – days. none of the - and -week old pigs showed any clinical signs. antibodies against ped virus were detected in all surviving pigs by virus neutralization (vn) test and immunofluorescence assay (ifa). therefore, there is an age-dependent resistance to pathogenic ped virus infection in pigs. isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages porcine epidemic diarrhea (ped) virus is a member of the coronaviridae, and is antigenically distinguishable from the other porcine coronaviruses, transmissible gastroenteritis (tge) virus and hemagglutinating encephalomyelitis virus cavanagh, ) . experimental infections with ped virus isolates have indicated that the virus is a primary etiologic agent of diarrhea in pigs (debouck and pensaert, ; debouck et al., ; coussement et al., ) . the principal features of ped are watery diarrhea, dehydration and high mortality in suckling pigs. in , a coronavirus-like particle was ®rst identi®ed during episodes of epizootic diarrhea in pigs in belgium (pensaert and debouck, ) and the uk (wood, ; chasey and cartwright, ) . ped has subsequently been reported in canada (turgeon et al., ) , hungary (horvath and mocsari, ) , germany (pospischil et al., ) and korea (kweon et al., ) . in japan, there was an outbreak of ped-like disease from late to early (takahashi et al., ; kuwahara et al., ) . further outbreaks occurred between late and (sueyoshi et al., ; tsuda, ) . in an acute outbreak of ped in , more than the , suckling pigs died (tsuda, ) . attempts at propagating ped virus in porcine cell or organ cultures have been unsuccessful. the ®rst adaptation of ped virus to vero cell cultures using medium containing trypsin was reported in (hofmann and wyler, ) . in japan, ped virus was ®rst isolated in vero cell culture in the same manner (kusanagi et al., ) . this paper describes the isolation of ped virus not only in vero cells but also in cultures derived from pig bladder and kidney cells by addition of trypsin to the medium. furthermore, to determine the effect of pig age on disease, different age groups of pigs were inoculated with the japanese isolate of ped virus. growth medium (gm) was eagle's minimum essential medium (mem) containing . % tryptose phosphate broth, % inactivated fetal calf serum (fcs) and antibiotics was used for the propagation of cells. maintenance medium (mm) consisted of eagle's mem with . % tryptose phosphate broth, . % yeast extract, and mg/ml trypsin (difco, usa), unless otherwise stated. established cell lines of vero cells derived from african green monkey kidney (rcv ) and ma from fetal macacus rhesus monkey kidney were used. sb and sb cells prepared from the bladder epithelial cells of cesarean-derived colostrumdeprived (cdcd) piglets were used for virus isolation at approximately ± passages. sk cells prepared from the cdcd pig kidney were used at approximately ± passages. the small intestines obtained from four piglets with naturally acquired infection showing severe diarrhea were homogenized with mm. ped virus antigens were detected in the enterocytes of these pigs by immunohistochemistry. the % pooled homogenates were centrifuged and the resulting supernatants were passed through a nm membrane ®lter. six -day old cdcd piglets were orally inoculated with ml of the homogenized samples and sacri®ced on post-inoculation days (pid) ± . the small intestines collected from these infected pigs were treated as described above and the pooled homogenates were used as virus stock. the virus stock was stored at À c until experimental inoculation of pigs. the virus stock was approximately . ld / ml for a -day old pig. for virus isolation, three -day old cdcd piglets were inoculated with the virus stock and sacri®ced on pid (second passage in pig). homogenized samples were prepared for virus isolation according to the method described previously (hofmann and wyler, ). after removal of gm, con¯uent cell cultures in collagen-coated -well tissue culture plates (iwaki glass, japan) were washed once with mem. then, the cells were inoculated with . ml per well of the clari®ed homogenate. after adsorption at c for h, the inoculum was removed and the monolayers were washed three times with ml of mem. the cell cultures were fed with ml per well of mm. control cultures were mock-inoculated with the same volume of mm instead of viral inoculum. if no cytopathic effect (cpe) was detected within days, ®ve blind passages were performed using supernatant¯uids of cell culture in the same manner as the original samples. twenty eight speci®c pathogen-free (spf) pigs in different age groups (six at days old, two at days old, ®ve each at , , and weeks old) were obtained from an spf pig herd. each group of pigs was housed separately in a containment room and fed a commercial milk or solid diet. the ambient room temperature was maintained at ± c for -and -day old pigs and ± c for the other pigs. two-day old pigs were divided into uninoculated control or infection groups. two-day old and all other pigs were orally inoculated with ml of -and -fold diluted virus stock, respectively. blood samples were collected weekly from each pig. clinical signs were recorded twice a day until slaughter. surviving pigs were euthanized and necropsied at post-inoculation week (piw) or . vero cell cultures on coverslips were ®xed in acetone for min, about h after virus inoculation. they were then stained with the experimentally inoculated pig serum at c for min. after incubation, they were washed with phosphate buffered saline (pbs) three times and stained with protein a conjugated with¯uorescein isothiocyanate (zymed, usa) at c for min. after washing, the cells were mounted with buffered glycerol and examined under a¯uorescence microscope. the vn test was carried out by the microtiter method using vero cells. sera were heated at c for min before use. serial two-fold dilutions of serum were mixed with an equal volume of z p strain virus suspension containing tcid / . ml. the mixture were incubated at c for h and . ml of virus±serum mixture was inoculated into each of the two wells. following adsorption at c for h, the inocula were removed and the monolayers were washed three times with mem. then, . ml of mm containing mg/ml trypsin was added to each well and the cultures were incubated at c for days. the antibody titer was expressed as the reciprocal of the highest serum dilution inhibiting cpe in at least one of the two wells. the avidin±biotin (ab) technique was used for the detection of ped virus antigen in tissues. an abc kit (vector laboratories, usa) was used to examine formalin-®xed, paraf®n wax-embedded sections of the gastrointestinal tract. sections were counterstained with methyl green. rabbit antiserum against ped virus (tsuda, ) was used as primary antibody. histopathological examination was performed according to routine procedures. in brief, tissue samples were ®xed in % neutral phosphate-buffered formalin. thin sections of paraf®n-embedded samples were stained by hematoxylin and eosin. as shown in table , cytopathic agents were isolated in vero, sk and sb cell cultures inoculated with second passage intestinal samples. at ®rst passage, cpe was inapparent vero À a sk À À sb À sb À À À ma À À À because of the cytotoxic effect of the inoculum. on passage in vero cell culture, cpe characterized by cell fusion and syncytial formation was detected microscopically after an incubation period of ± days. cpe progressed more slowly in sk and sb cell cultures than in vero cell culture. the number of cells which were rounded, detached and fused were increased after ± days of incubation (fig. ) . although syncytial formation was not apparent under microscope in non-stained cultured cells, fused cells were obvious in sb cell culture samples stained with wright giemsa's solution (fig. ) . vero cells inoculated with viruses isolated in sb or sk cells showed the same cpe with syncytial formation. by ifa, speci®c brilliant¯uorescence were observed in the cytoplasm of these cells (fig. ) and the isolates were identi®ed as ped virus. nō uorescing cells were observed in uninoculated control cells. the isolated ped virus from pig no. in vero cell culture was clone-puri®ed three times by plaque selection and designated z p . cpe was not detected microscopically in sb and ma cells at the ®fth passage. all -and -day old pigs inoculated with ped virus developed severe watery diarrhea from pid and died between pid and (table ). ped virus antigen was detected by the ab technique in the small intestine of dead pigs. vomiting was occasionally seen on pid ± . non-inoculated control -day old pigs kept under the same conditions remained healthy. three of ®ve -week old pigs developed diarrhea on pid ± which lasted ± days. although all these pigs were slightly depressed and anorectic after inoculation, they recovered after week. ped virus antigen was detected by the ab technique in the jejunum and ileum of pig no. which was euthanized on pid . in the -week old group, three of ®ve pigs had mild diarrhea which started on pid ± and continued for ± days. in this group, other clinical signs such as depression and anorexia were not observed. all of -and -week old pigs showed no clinical signs including diarrhea throughout the experiment. all the pigs in the -, -, -and -week old groups survived. before infection, all of the pigs were negative for antibody against ped virus by both vn test and ifa. the antibodies were ®rst detected on piw or and they peaked between piw and on vn test and on piw on ifa (fig. ) . ifa titers were higher than those of vn antibody. all of the non-inoculated control pigs were negative for both antibodies throughout the study. in the present study, ped virus was successfully propagated not only in vero cells but also in sb and sk cell cultures. hofmann and wyler ( ) ®rst isolated and adapted ped virus to serial propagation in vero cells, which are derived from african green monkeys, by adding trypsin to the mm. however, attempts at isolating ped virus in porcine cell cultures in the presence or absence of trypsin have been unsuccessful until now. previous reports suggested that porcine cell cultures were damaged by trypsin, which then might not be able to support virus replication (hofmann and wyler, ) . in the present study, collagen-coated plates were used; cells cultured on collagen-coated plates are more resistant to trypsin (data not shown). this might be the main reason for the successful isolation of ped virus in porcine cell cultures in the present study. this method may be suitable for cell culture in medium containing trypsin or other proteinases. ped virus was isolated in sb cells but not in sb cells. although ped virus propagated in sb cells showed cpe in sb cells, the titer in these cells was lower than in sb cells (data not shown); the cells had been derived from the bladder of different pigs and were used at the same passage numbers. the reason for the difference in susceptibility for viral propagation between these cells is not known. upon experimental infection, -and -day old pigs inoculated with ped virus developed severe diarrhea and died. the -and -week old pigs showed mild diarrhea after inoculation and survived. the -and -week old pigs did not show any clinical signs, but developed antibody against ped virus. coussement et al. ( ) described that -or -day old cesarian-derived piglets infected oronasally with the cv strain of ped virus showed severe diarrhea after an incubation period of ± h. debouck and pensaert ( ) reported that pigs between and days old experimentally inoculated with ped virus developed diarrhea and some pigs younger than days old died. these results are similar to those of the present study. in several outbreaks, ped has caused diarrhea in pigs of all ages (pensaert and debouck, ; takahashi et al., ) . in the present study, however, severe diarrhea was only observed in -and -day old pigs. the difference in the clinical signs between ®eld cases and the present examination may re¯ect differences in the infectious dose, the susceptibility of pigs, the environmental conditions or the virulence of the strains. the majority of herd outbreaks of ped occur during the colder months, especially between january and april (tsuda, ) . in tge virus infection, (shimizu et al., ; shimizu and shimizu, ) showed that a high ambient temperature resulted in increased disease resistance, while a low ambient temperature or temperature changes caused a dramatic enhancement in clinical signs. in the present study, ± -week old pigs were kept at ± c throughout the study. consequently, a stable comfortable ambient temperature might induce resistance to clinical disease after ped virus infection. classi®cation and nomenclature of virus. fifth report of the international committee on taxonomy of viruses virus-like particles associated with porcine epidemic diarrhea pathology of experimental cv coronavirus enteritis in piglets experimental infection of pigs with a new porcine enteric coronavirus cv the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv propagation of the virus of porcine epidemic diarrhea in cell culture ultrastructural changes in the small intestinal epithelium of suckling pigs affected with a transmissible gastroenteritis (tge)-like disease isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate passage in piglets of a coronavirus associated with porcine epidemic diarrhea isolation of porcine epidemic diarrhea virus (pedv) in korea a new coronavirus-like particle associated with diarrhea in swine an immunoelectron microscopic and immuno¯uorescent study on the antigenic relationship between the coronavirus-like agent, cv , and several coronaviruses light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic diarrhoea virus (evd): comparison with transmissible gastroenteritis (tge) virus and porcine rotavirus infection effects of ambient temperatures on clinical and immune responses of pigs infected with transmissible gastroenteritis virus effects of ambient temperatures on induction of transmissible gastroenteritis in feeder pigs an immunohistochemical investigation of porcine epidemic diarrhoea an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan porcine epidemic diarrhea: its diagnosis and control coronavirus-like particles associated with diarrhea in baby pigs in quebec an apparently new syndrome of porcine epidemic diarrhoea key: cord- - lh y j authors: chen, jianing; cui, yaru; wang, zemei; liu, guangliang title: identification and characterization of pedv infection in rat crypt epithelial cells date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: lh y j porcine epidemic diarrhea (ped) is a devastating enteric disease to the world's swine production. porcine epidemic diarrhea virus (pedv), as the ped causative agent, has been commonly propagated and investigated in vero cells, as well as in ipec-j , a porcine epithelial cell-jejunum . however, vero cells, which are defective in interferon production, cannot represent the host response in enteric cells while pedv replicates poorly in ipec-j cells. in this study, we observed that rat crypt epithelial cells (iec- ) were highly susceptible to different subtypes of pedv. the replication kinetics of pedv in iec- cells is similar to that in vero cells, but it is much higher than in ipec-j cells. besides that, pedv infection in iec- cells can induce the production of inflammatory cytokines and interferon, especially the type iii ifns. collectively, our findings suggest that iec- is an ideal cell line for pedv replication and immune response studies. the swine enteric coronaviruses, a family of the most significant problematic pathogens to the world's swine industry, include porcine epidemic diarrhea virus (pedv), porcine transmissible gastroenteritis virus (tgev), porcine deltacoronavirus (pdcov), and swine acute diarrhea syndrome coronavirus (sads-cov) (qiuhong et al., ) . among these four viruses, pedv has been recognized as the most devastating pathogen, causing up to % mortality in piglets younger than one week old. since its first discovery in the s, pedv has then subsequently identified nearly worldwide (wood, ) . later in the s, variant pedv strains were emerged and killed hundreds of millions of suckling piglets (jung and saif, ; wang et al., ) . pedv is an enveloped, single-stranded, positive-sense rna virus, which targets the small intestine, especially the ileum (lee, ) . pedv infection in enterocytes can exhibit acute necrosis, leading to marked villous atrophy in the small intestine (ducatelle et al., ) . consequently, malabsorption, caused by the massive loss of absorptive enterocytes, contributes to diarrhea and even death of newborn piglets. the cell line, which is permissive for pedv replication and possesses the intestine function and responses, may facilitate the studies about pedv. vero cells are commonly used for pedv propagation. however, this cell line is characterized by lacking the production of interferon (desmyter et al., ) . consequently, it is valuable in vaccine production but defective in immune studies. ipec-j is a non-transformed porcine columnar epithelial cell line and used in the investigations of pedv lin et al., ) . however, it is semi-permissive to pedv infection and defective in the production of several cytokines (brosnahan and j o u r n a l p r e -p r o o f brown, ) . these drawbacks of the two cell lines restrict further understandings of pedv. therefore, it is of great importance to screen proper cell lines for pedv investigations. in this study, we identified rat crypt epithelial cells (iec- ) was highly susceptible to a different subtype of pedv, which could be an ideal cell model for pedv studies. the rat small intestine epithelium iec- cells were purchased from american type culture collection (atcc) and cultured in dulbecco's modified eagle's medium (dmem) (sigma, germany) supplemented with . unit/ml bovine insulin and % fetal bovine serum (gemini, usa). african green monkey epithelial vero cells were cultured in dmem medium supplemented with % fetal bovine serum. the intestinal porcine epithelial cell line j (ipec-j ) cells were cultured in dmem/f (sigma, germany) supplemented with % fetal bovine serum, μg/ml insulin/selenium/transferrin (life, usa) and ng/ml epidermal growth factors (egf) (life, usa). the pedv ljx /gs/ strain was isolated and conserved in our laboratory. the pedv cv strain were isolated from attenuated vaccine and maintained in our laboratory. iec- cells were seeded in -well plates. when grown to % confluency, cells were infected with pedv at a moi= . . after hours, the cells were fixed with % j o u r n a l p r e -p r o o f paraformaldehyde for min and then permeabilized with . % triton x- for min at room temperature. after blocking with % skim milk for h, cells were incubated with mouse anti-pedv-nucleocapsid monoclonal antibody ( : dilution) for h, followed by incubating with the alexa fluor goat anti-mouse igg antibody ( : dilution) (thermo, usa) for h. the cell nuclei were stained by dapi (beyotime, china). the cells were then examined under a fluorescence microscope (te u; nikon) with a video documentation system. to assess the host response against pedv, the three cell lines were infected with pedv at a moi= . . the supernatant was collected at , , , , and hours postinfection and used for viral titration. at hours post-infection, the cells were also lysed with trizol (takara, japan) for rna extraction. confluent monolayers of iec- cells in -well plates were inoculated with pedv and incubated for h at room temperature on rocker. after three washes, the cells were overlaid with . % low melting point agarose (sigma, germany) in dmem containing % fbs and then incubated at ℃ for around h. to visualize plaques, cells were stained with % crystal violet in ethanol. vero cells were seeded into -well plates and cultured for - % confluence. the cell monolayers were washed three times with pbs. serial -fold dilutions of the collected j o u r n a l p r e -p r o o f supernatant were made, and l of each dilution were inoculated to five wells of the well plate. the plates were incubated at • c and supplied with % co . and then, the cultures were checked under a light microscope for cytopathology every day for days. the dilution of the supernatant was titrated by tcid assay and calculated based on the reed-muench method. for the detection of host gene expression level, the real-time qpcr was performed with unique aptamer qpcr sybr green master mix (novogen, china) in the bio-rad cfx system. the reactions were incubated at °c for s, followed by cycles at °c for s and °c for s. all reactions were run in triplicate using the primer sets listed in table . the -∆∆ct method was employed to measure the expression level of target genes. the student's t-test was used for the examination of statistical significance between matched groups. an unadjusted p value of less than . was considered significant; a p value of less than . was considered highly significant. iec- cell line, originally derived from the rat small intestinal crypt, was commonly used in the nutritional investigations (quaroni et al., ) . to investigate if it is susceptible to pedv, the iec- cells were infected with two different subtypes of pedv at a moi= . and observed for cytopathic effect (cpe) under a microscope. the pedv j o u r n a l p r e -p r o o f vaccine strain cv belongs to subtype g , while the field strain ljx isolated by our group belongs to subtype g (guo et al., ) . as shown in fig. a , typical cpe became visible at hours post-infection. the morphology of pedv-infected cells became enlarged fusiform at early stage. lately, they were wrinkled and detached. however, the mock-infected cells remained normal during the whole process. ifa was then performed to confirm its infection by using a mab against pedv nucleocapsid protein (yang et al., ) . the results showed that strong pedv-n positive signals were observed in iec- cells infected with either ljx strain or cv strain (fig. b) , indicating iec- cells were successfully infected by pedv. then the total rna was extracted and analyzed by rt-pcr. the results demonstrated that the pedv-specific bands were amplified from virusinoculated cells (fig. c) . finally, the western blotting analysis showed that the pedv nucleocapsid protein was detected in cells infected with pedv (fig. d) . we also attempted to identify whether the iec- were susceptible to tgev but no positive signal were obtained (data not shown). taken together, the above data illustrated that iec- cells were susceptible to pedv but not tgev. vero and ipec-j cell lines were commonly used for pedv investigation. to test and compare the replicative capacity of pedv in different cell lines, the pedv replication kinetics were measured in these three cell lines. these cells were seeded in the -well plate and inoculated with pedv ljx strain or cv strain at a moi= . . the supernatant was collected every hours and used for viral titration. the results showed that the pedv ljx strain replicated poorly in ipec-j cells with viral titers lower than j o u r n a l p r e -p r o o f tcid /ml. pedv ljx strain exhibited a robust and similar growth curve in vero and iec- cells, showing higher than tcid /ml viral titers. pedv cv strain reached its most elevated viral titers in both vero and iec- cells but poorly replicated in ipec-j cells (fig. e) . the plaque assay is usually employed for virus purification and titration. it is of great importance to virus related studies. we also tested whether iec- could be used to conduct plaque assay. the data showed that the infection of both ljx and cv strains resulted to the formation of plaque (fig. f) . these results further showed that iec- cells supported pedv infection and replication. pedv infection in the porcine small intestine is characterized by inflammatory cytokines and interferon (jung and saif, ) . the type iii interferon is considered to play a critical role in enteric immunity. therefore, we examined whether pedv infection could induce the typical enteric immunity in iec- cells compared to its infection in ipec-j and vero cells. pedv ljx was a field strain and was used to infect the three cell lines. after hours post-infection, the rna was extracted for rt-qpcr analysis. the results showed that both type i and type iii interferon responses were significantly activated in pedv infected iec- and ipec-j cells while the type i interferon were absent in vero cells ( fig. a, b, d, e ). due to the lack of ifn-λ gene of green monkey in the ncbi or ensembl database, we failed to detect its gene expression level. although the ifn-λ gene of green monkey was strongly upregulated in pedv infected vero cells, its ct value was about , while the ct value to gapdh was about in pedv infected cells. in mock j o u r n a l p r e -p r o o f cells, the ct values of about for ifn-λ and for gapdh. for type ii interferon, pedv infection led to a significant upregulation in iec- cells but not in ipec-j and vero cells (fig. c) . pedv infection also enhanced the expression of il- , il- , il- , and tnfα in iec- cells except for il- a (fig. f, g, h, i, j ). all these results demonstrated that iec- cells were able to generate a robust immune response against pedv infection. although vero and ipec-j were commonly used in pedv studies, they were defective in immune response and viral replication. there were also other cell lines identified permissive for pedv infection recently. pedv can also infect hek t, ip - i cells, and primary bovine mesenchymal cells (jung et al., ; wang et al., ; zhang et al., ) . however, hek t is not an intestinal epithelium cell line, while ipi- i is semi-permissive to pedv infection. the primary bovine mesenchymal cells cannot culture for a long time. the rat small intestinal crypt epithelium cell line iec- is non-transformed. and it was often used as a necrotizing enterocolitis model system and nutritional studies (braga-neto et al., ; ruthig and meckling-gill, ; yan et al., ) . the uptake, metabolism, and transport processes are observed in iec- cells (said et al., ; sanderson and he, ) . therefore, iec- is recognized as an ideal model for intestine relating studies. however, it is rarely used in viral studies. only modified vaccinia virus ankara was reported to multiply in iec- cells (okeke et al., ) . in this study, we tested the susceptibility of iec- cells to pedv infection and found it was highly susceptible. the growth curve of pedv in iec- was similar to it in vero cells with the viral titers higher than tcid /ml. it was also found that iec- could mimic the antiviral immune response in the small intestine. pedv infection in the small intestine is characterized by the induction of proinflammatory responses (huan et al., ) . in our study, il- b, il- , il- , and tnfα were upregulated by pedv infection in iec- cells. only tnfα, il- a and il- were found increasingly expressed in ipec-j cells. il- was only activated by pedv infection in iec- cells. besides that, interferon is recognized to play critical roles in the innate immune response. the type iii interferon is significantly essential in maintaining intestinal homeostasis, especially to pedv (ingle et al., ; zhang et al., ) . in pedv infected iec- cells, the expression of type i and type iii interferon was drastically upregulated, while type ii interferon was also increasingly expressed. vero cells are defective in interferon production. the ct values of all genes were higher than while it was about for gapdh. although ifnλ was highly upregulated in vero cells, its ct value was also higher than . in ipec-j cells, the production of ifnα, ifnλ , and ifn-λ was increased but not as high as in iec- cells. all these data suggested that pedv infection in iec- cells could induce strong antiviral responses. in summary, our studies identified the rat small intestinal crypt epithelium iec- cells were highly susceptible to pedv. pedv could efficiently replicate in iec- cells as it does in vero cells. besides that, pedv infection activated potent inflammatory cytokines and j o u r n a l p r e -p r o o f interferon, primarily type iii interferon in iec- cells, which highly simulated its infection in the small intestine. all these data above suggested that iec- cells can be used as an excellent cell model for pedv studies. protective effects of alanyl-glutamine supplementation against nelfinavir-induced epithelial impairment in iec- cells and in mouse intestinal mucosa porcine ipec-j intestinal epithelial cells in microbiological investigations porcine endemic diarrhea virus infection regulates long noncoding rna expression defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains glycyrrhizin inhibits porcine epidemic diarrhea virus infection and attenuates the proinflammatory responses by inhibition of high mobility group box- protein distinct effects of type i and iii interferons on enteric viruses porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis replicative capacity of porcine deltacoronavirus and porcine epidemic diarrhea virus in primary bovine mesenchymal cells porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus differential protein analysis of ipec-j cells infected with porcine epidemic diarrhea virus pandemic and classical strains elucidates the pathogenesis of infection modified vaccinia virus ankara multiplies in rat iec- cells and limited production of mature virions occurs in other mammalian cell lines emerging and re-emerging coronaviruses in pigs. current opinion in virology epithelioid cell cultures from rat small intestine. characterization by morphologic and immunologic criteria both (n- ) and (n- ) fatty acids stimulate wound healing in the rat intestinal epithelial cell line, iec- intracellular regulation of intestinal folate uptake: studies with cultured iec- epithelial cells nucleotide uptake and metabolism by intestinal epithelial cells porcine epidemic diarrhea in china susceptibility of porcine ipi- i intestinal epithelial cells to infection with swine enteric coronaviruses an apparently new syndrome of porcine epidemic diarrhoea. the veterinary record fish oil-derived lipid emulsion induces rip -dependent and caspase -licensed necroptosis in iec- cells through overproduction of reactive oxygen species generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid characterization of porcine epidemic diarrhea virus infectivity in human embryonic kidney cells type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling. key: cord- -agm zbcx authors: kennedy, melissa; boedeker, nancy; gibbs, pam; kania, stephen title: deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: agm zbcx a population of persian cats experienced an epidemic of feline infectious peritonitis (fip) over years. twelve cases of fip occurred in litters born during this period. cats contracting fip were all genetically related through the sire. feline coronavirus (fcov) genomic rna was detected consistently in this study in biologic samples from adult cats, kittens suffering from fip, and their siblings. analysis of viral a/ b open reading frame (orfs) were analyzed and revealed two distinct virus variants circulating in the population, one with an intact a orf and one with two major deletions in the a orf. the b orfs were intact and similar among all virus isolates, although point mutations resulting in amino acid changes were present. the sire was determined to be infected with both variants, and was persistently virus-infected. we speculate the deletion variant arose from the non-deletion variant during viral replication in this population, possibly in the sire. feline infectious peritonitis (fip) is a serious disease of domestic and wild felidae. it is an important disease of cats in multi-cat households, catteries, and shelters (scott et al., ; wolf, ) . this disease can manifest as an effusive peritonitis and/or pleuritis with a short course ending in death; or it may present as a more insidious disease with veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] granulomatous lesions affecting multiple organs that also progresses to death (pedersen, ; hoskins, ; pedersen, ) . the etiologic agent of fip is feline coronavirus (fcov) . in environments where large numbers of cats are housed closely together, - % of the animals may be seropositive to the virus (pedersen, ) . despite these numbers, fip occurs sporadically -fatal disease is an uncommon manifestation of infection with fcov. virus factors are important to disease development, as virus strains vary in virulence (pedersen, ) . virulent fcov is theorized to arise from mutation of the infecting fcov during replication in the intestinal tract of infected cats (poland et al., ; vennema et al., ) . the b open reading frame (orf), the -most gene, has been speculated to have a role in virulence, as deletions in this region lead to decreased virulence (vennema et al., ; herrewegh et al., ; vennema et al., ) . despite this data, the specific virus and host factors involved in the production of lethal disease are not known. the occurrence of fip in feline populations is rarely higher than % (hoskins, ) . we investigated a colony of adult persian cats that experienced cases of fip in their kittens over years. we detected fcov genetic material in the feces of the adult cats as well as tissue and fluid sample from the fip victims. the a/ b orf from the fcov in this population was characterized to determine if mutations in this region were occurring. a persian cattery consisting of adult cats was selected for study. all animals in the population were negative for feline leukemia virus and feline lentivirus. they were routinely vaccinated for feline herpesvirus, calicivirus, panleukopenia virus, and rabies. twelve cases of fip occurred in six litters over years ( ) ( ) ( ) . diagnosis was based on clinical signs, cbc and blood chemistry, coronavirus serology, histopathology, and pcr on plasma or effusion. this latter parameter was found to correlate with histopathology for diagnosis of fip (kennedy et al., ) . fecal samples, whole blood, tissue and ascites from two clinical cases were collected at the time of illness. in addition, fecal and whole blood samples were collected from seven adult cats and unaffected kittens in the household at the same time point. total rna was extracted from the specimens using trizol ls according to the manufacturer's directions for reverse transcription and nested polymerase chain reaction (gibco brl, baltimore, md). primers encompassed the a/ b orfs, the -most orfs of the genome (kennedy et al., ) . reverse transcription was done with moloney murine leukemia virus reverse transcriptase according to the manufacturer's recommendations with the downstream external primer (gibco brl, baltimore, md). nested polymerase chain reaction was done using extaq polymerase (intergen, purchase, ny) as described previously (kennedy et al., ) . products were analyzed on a % agarose gel. amplification products were cloned into pcr . using the ta cloning system (invitrogen, carlsbad, ca). cloned cdna was sequenced by molecular biology resources service (university of tennessee, knoxville, tn) and analyzed using gcg software (university of wisconsin, madison, wi). a minimum of two clones from two separate pcr reactions was used for sequencing and analysis. we detected fcov in a purebred population of cats that had experienced a high rate of fip over a -year period, with cases of fip in six litters. the fcov infecting the population was analyzed, focusing on the a/ b orf. this genomic region was chosen for analysis because of its speculated role in virulence (vennema et al., ; herrewegh et al., ; vennema et al., ) . amplification of fcov rna was successful in samples from nine cats. seven of these were resident adults and included the sire and queens of affected offspring. two were from kittens that died from fip. two distinct virus variants were found, one with an intact a orf and one with two deletions in the a orf (fig. ) . the deletions encompassed nucleotides - and nucleotides - of the a gene. four additional nucleotides (tctt) were present in all of the deletion mutants at the position corresponding to nucleotide of the undeleted virus. the protein predicted from the nucleotide sequence contains different amino acids in the one-half of the predicted protein due to the altered reading frame caused by the second deletion (fig. ) . it is not known if the a protein predicted from the nucleotide sequence is expressed in the deletion mutant virus. the remainder of the a/ b orf was highly conserved among all isolates, including the region encoding the b orf (fig. ) . interestingly, amino acid residues and of the predicted b protein were histidine and lysine. this is contrary to the findings of vennema et al. ( ) that fip viruses contained tyrosine and lysine at these positions whereas putative avirulent fcov contained histidine and arginine. both virus variants were identified in one cat, the sire ''dan'', as sequence analysis of clones from a single pcr from this animal revealed the presence of the a deletion mutant as well as the intact isolate (dan and in fig. ). this cat was pcr-positive for virus in multiple samples and may be persistently infected with fcov. he was also the sire of all the fip victims in the first months of the study. litters resulting from the breeding of this sire to his daughters from previous litters experienced morbidity and mortality of - % within the litter (n ¼ ). during the last months of the fip outbreak, a new sire was used and bred to the original sire's daughters. morbidity and mortality has since decreased to - % within litters (n ¼ ), with the last case occurring in spring of . amplification of fcov a/ b orf from the most recent cases of fip (n ¼ ) have not been successful. fig. shows the phylogenic relationship among the virus isolates resulting from nucleotide sequence alignments. the two distinct variants are clearly delineated. the deletion mutants show a very close similarity to one another and form a closely related cluster. we have characterized the a/ b orf of fcov variants in a population experiencing an epidemic of fip. one variant had an intact a/ b orf, while another had two major deletions in the a orf. the latter group appears to be very closely related, and probably arose from a single mutant strain. the first deletion resulted in no change in the reading frame, but the second deletion led to an alteration of the reading frame by one nucleotide leading to an altered amino acid sequence in the predicted protein. we speculate that these deletions arose from ''looping out'' of rna regions due to the predicted secondary structure of the single stranded rna genome in this region (data not shown). this may result in the viral rna polymerase ''skipping'' certain regions during transcription. it is not known if the a protein is expressed in the mutant virus. the first nucleotides of the cdna sequence, which corresponded to nucleotides - of the a gene and included the start codon, corresponded to the upstream internal primer. some of these nucleotides must be present in the viral template in order for hybridization of primer to template to occur but it is not known how many of nucleotides - are present in the virus. thus, it is unclear if this start codon is present. if it is, translation of the a orf in the deletion mutant would result in a predicted protein that is nearly half the size of the native protein ( amino acids in deletion mutant versus amino acids residues in the native virus). the sire of the majority of fip kittens was dually infected with both virus variants as revealed by sequence analysis of cloned a/ b genes from this cat. other cats in this study may have also been dually infected with both virus variants, however, only one virus variant was identified by pcr in each of the remaining cats tested. this may be due to a quantitative difference in the amount of each virus variant in each cat tested. both variants were circulating as every cat tested was infected with one of the virus variants. some infected with the intact a/ b orf variant were ill and some infected with the deletion mutant were ill. however, as both variants were circulating and thus, every cat was exposed to both variants, there may be a causal relationship between the mutation that occurred and the increased incidence of disease seen in this population. the variation in disease may be related to host factors, such as generation of an effective immune response, rather than solely to the virus itself. alternatively, mutations in other genomic regions may be responsible for the variation in virulence observed with these isolates. host factors may play an important role in the virulence of fcov. increased incidence of fip in purebred cats, as well as in cheetahs, which are relatively genetically homologous, is known to occur (o'brien et al., ; foley and pedersen, ) . in this investigation, all of the fip victims were genetically related through the sire. those related to him through both the queen and sire had the highest morbidity while those related to him through the queen or sire only had lower morbidity. this would support the belief that susceptibility to fip following infection with fcov has a host genetic component. genetic detection targeting the a/ b orf of fcov was not successful in all fip victims. the deletions we have characterized in the a orf occurred near or within the binding site of the internal upstream primer. as the virus has persisted in this population, we postulate that this primer-binding site may have been lost due to the occurrence of additional deletions in the a viral gene. mutation of fcov in some cats may lead to changes in virulence ultimately resulting in fip. point mutations, recombination, and deletions have been observed (poland et al., ; herrewegh et al., ) . mutations are more likely to occur if virus is not cleared from a host population and viral replication continues at a significant level . it is not known if two virus variants entered this population or if one variant is a mutant of the other. the latter may be more likely, as the cats in this population have remained virus-infected for an extensive period increasing the likelihood of genetic mutation. deletions have been noted in the b orf from previous studies (vennema et al., ) . this is the first report of a deletion occurring in the a orf in a natural infection of a cat population. other virus mutations in addition to those which we have characterized may have occurred in the fcov from this population and may correlate directly with virulence. further analysis of additional genomic regions of these fcovs is required to completely explain the increased occurrence of fip in this population. the inheritance of susceptibility to feline infectious peritonitis in purebred catteries the molecular genetics of feline coronavirus: comparative sequence analysis of the orf a/ b transcription unit of different biotypes perspectives on feline coronavirus evolution veterinary clinics of north america: small animal practice correlation of genomic detection of feline coronavirus with various diagnostic assays for feline infectious peritonitis genetic basis for species vulnerability in the cheetah virologic and immunologic aspects of feline infectious peritonitis virus infection an overview of feline enteric coronavirus and infectious peritonitis virus infections two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus evaluation of the safety and efficacy of primucell-fip vaccine genomic organization and expression of the -end of the canine and feline enteric coronaviruses feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses the impact of feline infectious peritonitis on catteries we gratefully acknowledge and thank morris animal foundation for its financial, technical, and administrative assistance in funding and managing the research through which this information was discovered. we would also like to thank dr. michael kiningham for his assistance in this investigation. key: cord- -tigj fhc authors: cleveland, christopher a.; denicola, anthony; dubey, j.p.; hill, dolores e.; berghaus, roy d.; yabsley, michael j. title: survey for selected pathogens in wild pigs (sus scrofa) from guam, marianna islands, usa date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: tigj fhc pigs (sus scrofa) were introduced to guam in the ’s and are now present in high densities throughout the island. wild pigs are reservoirs for pathogens of concern to domestic animals and humans. exposure to porcine parvovirus, transmissible gastroenteritis, and leptospira interrogans has been documented in domestic swine but data from wild pigs are lacking. the close proximity of humans, domestic animals, and wild pigs, combined with the liberal hunting of wild pigs, results in frequent opportunities for pathogen transmission. from february–march , blood, tissue and ectoparasite samples were collected from wild pigs. serologic testing found exposure to brucella spp. ( %), toxoplasma gondii ( %), porcine reproductive and respiratory syndrome (prrs) virus ( %), porcine circovirus type ( %), pseudorabies virus ( %), actinobacillus pleuropneumoniae ( %), lawsonia intracellularis ( %), and porcine parvovirus ( %). eleven ( %) samples had low titers ( : ) to leptospira interrogans serovars bratislava (n = ), icterohaemorrhagiae (n = ), pomona (n = ), and hardjo (n = ). kidney samples from nine pigs with leptospira antibodies were negative for leptospira antigens. numerous pigs had metastrongylus lungworms and three had stephanurus dentatus. lice (hematopinus suis) and ticks (amblyomma breviscutatum) were also detected. no antibodies to influenza a viruses were detected. in contrast to the previous domestic swine survey, we found evidence of numerous pathogens in wild pigs including new reports of pseudorabies virus, prrs virus, brucella, and leptospira in pigs on guam. these findings highlight that domestic swine-wild pig interactions should be prevented and precautions are needed when handling wild pigs to minimize the risk of pathogen transmission. wild pigs are nearly globally distributed and are hosts for many parasites and bacterial and viral pathogens, some of which are transmissible to agricultural animals, wildlife, and humans (barrios-garcia and ballari, ; bevins et al., ) . numerous studies have investigated the prevalence and distribution of various pathogens in wild pigs and the risk they pose for pathogen transmission in the united states and europe, but there are relatively few reports from southeast asia (baroch et al., ; hill et al., ; pedersen et al., pedersen et al., , pepin et al., ) . wild pig populations are increasing dramatically on several pacific islands, yet data on pathogen exposure are limited or absent. wild pigs were introduced to guam, an unincorporated territory of the united states located in the marianna island chain, in the late 's and despite current liberal hunting regulations, populations continue to increase (conry, ) . this population increase has led to severe ecological damage and agricultural losses. the only report of swine pathogens on guam was limited to domestic pigs (dugies et al., ) . the lack of data on pathogens in wild pigs is a concern as increased swine populations have led to increased pig-human interactions. recent reports of leptospirosis in residents and tourists is one concern and although rodents tend be considered the most common reservoir of leptospira, there is evidence of leptospira in domestic swine on guam (dugies et al., ) , and wild pigs in numerous countries have antibodies to leptospires (jansen et al., ; corn et al., ) . in , there was an effort to decrease wild pig populations within fenced areas of two military bases on guam to minimize the ecological damage caused by the pigs. samples collected from these animals were used to conduct a comprehensive surveillance project on pathogen exposure of wild pigs on guam. andersen air force base (aafb) is a ha military installation in northern guam ( . °n, . °e) . forested portions of the base contain high quality native habitat, some of which is included in the guam national wildlife refuge. the naval base guam naval munitions site (nbg nms) is centrally located on the island and covers approximately acres ( . °n . °e). removal of wild pigs was conducted via strategic sharp-shooting techniques conducted by well-trained shooters using suppressed . caliber rifles mounted with scopes. during removal, shooters only fired if the situation met the following criteria: ) there was certainty that the animal would be dispatched and not escape, ) if other animals were nearby, every animal had a high probability of being dispatched, and ) it was safe to dispatch the animal. the shooting methods followed the american veterinary medical association's guidelines for humane euthanasia of animals (avma, ) . pigs were aged based on tooth eruption patterns and wear. animal and sample collection procedures were reviewed and approved by uga's institutional animal care and use committee (a - ). immediately after euthanasia, blood samples were collected via cardiocentesis and placed into ethylenediaminetetraacetic acid (edta) and plain tubes (greiner bio-one, monroe, nc). clotted blood was centrifuged at g for min and serum was removed and frozen at − °c until diagnostic testing. whole blood was also frozen at − °c until testing. representative ectoparasites were collected and preserved in % ethanol. information on pathogens we screened pigs for as well as diagnostic assays and diagnostic laboratories used are listed in table . most pigs were serologically tested for all of the pathogens listed; however, due to limited sample volume, some pigs were only tested for selected pathogens. small sections of lung were fixed in formalin and processed for routine histology. small sections of kidneys were also fixed in formalin and if antibodies to leptospira were detected, they were tested for leptospira antigens by immunohistochemistry (ihc). if any gross lesions were noted during necropsy, they were also collected in formalin for histologic examination. pigs were not systematically examined for internal parasites, but if any were seen they were collected in formalin for identification. body weights were compared between male and female swine using a two-sample t-test. prevalence of pathogen exposures was compared between males and females and between adult and juvenile swine using fisher's exact test. all testing assumed a two-sided alternative hypothesis and p < . was considered statistically significant. analyses were performed using commercially available statistical software (stata version . , statacorp lp, college station, tx). from the two sites, a total of wild pigs were sampled including six ( . %) juveniles and ( . %) adults; ( . %) were males and ( . %) were females. weight (kilograms) was recorded for individuals: juveniles and adults. all juveniles weighed < . kgs and the mean ± sd weight of the adult males ( . ± . ) was significantly greater than that of the adult females ( . ± . ; p < . ). all pigs were positive for at least one of the pathogens included in the study. exposure to or infection with influenza a virus (iav), brucella, porcine epidemic diarrhea virus, babesia spp., transmissible gastroenteritis/porcine respiratory coronavirus and trichinella spp. was rare or absent (table ). based on antibody testing, a high percentage of pigs were exposed to actinobacillus pleuropneumonia (app; %), lawsonia intracellularis ( %), and porcine parvovirus ( %). exposure to multiple serotypes of app was also common ( table ). based on mat testing, antibodies to four leptospira serovars were detected in pigs (tables and ) , with some individuals having antibodies to more than one serovar. kidney samples from nine pigs with leptospira antibodies were negative for leptospira antigens by ihc. juvenile pigs had a significantly higher prevalence of antibodies to porcine circovirus type ( / [ %]) compared with adults ( / ; p = . , respectively). there was no difference in prevalence between males and females for any pathogen. only pigs were examined for ectoparasites; lice (hematopinus suis) and ticks (amblyomma breviscutatum) were found on ( %) and seven ( %) pigs, respectively. lung samples from pigs were examined histologically and eight ( %) were positive for metastrongylus lungworms. stephanurus dentatus were found in abdominal lesions from three pigs and one pig had a liver abscess with intralesional nematode larvae which could not be identified. our results indicate that wild pigs on guam are exposed to multiple pathogens of zoonotic and agricultural importance. guam has many free-range pig operations which have an increased risk of domestic pigwild pig interactions. previous work on guam was restricted to domestic pigs and in contrast to our data, domestic pigs were exposed to relatively few pathogens (duguies et al., ) . clinically, several diseases were noted, but the only pathogen detected by serologic testing was parvovirus ( / , %). although domestic pigs were not serologically tested for app or l. intracellularis, porcine pleuropneumonia and proliferative enteritis were noted. domestic pigs tested negative for antibodies to pseudorabies virus (n = ), porcine reproductive respiratory syndrome virus (n = ), brucella spp. (n = ), leptospira spp. (n = ), swine influenza virus (n = ), transmissible gastroenteritis virus (n = ), mycoplasma hyopneumoniae (n = ), and trichinella spp. (n = ) while we found serologic evidence of the first four pathogens in wild pigs (duguies et al., ) . porcine pleuropneumonia, caused by app, is a highly infectious disease that is economically important for domestic swine. although known to occur on guam among domestic pigs, prevalence was unknown (duguies et al., ; bossé et al., ) . clinical disease and mortality resulting from infection with one or more app serotypes can vary geographically; however, serotype has been consistently associated with morbidity and mortality in domestic pigs (baroch et al., ) . over half of the wild pigs from guam had antibodies for serotypes - - - . reproductive disease in domestic pigs due to porcine parvovirus (ppv) has been reported on guam (duguies et al., ; ruiz-fons et al., ) . in addition to reproductive failure due to ppv infection, coinfection with porcine circovirus type (pcv ) in domestic pigs can result in mortalities (ellis et al., ) . we report in wild pigs the first evidence of pcv on guam; surveillance and vaccination for these pathogens may be warranted. l. intracellularis has been documented worldwide in domestic and wild pigs and is the etiologic agent of proliferative enteropathy (chiriboga et al., ) . the high prevalence of these three pathogens in wild pigs, indicates that transmission to domestic pigs is a risk. exposure to leptospira interrogans and toxoplasma gondii, two important zoonoses, was common in wild pigs on guam. leptospirosis is a growing concern in guam ( mason et al., ) . while exposure to several serotypes was detected, prevalence is likely underestimated because antigens from serotypes readily available for testing in our us laboratory were used rather than those likely present in guam/other pacific nations. regardless, exposure of pigs does not implicate them in human cases due to the diverse reservoir-range, but suggests that proper ppe should be utilized when in contact with pigs. domestic pigs have not been previously tested for t. gondii but evidence of t. gondii was noted in goats (duguies et al., ) . this parasite has a wide host range and can pose a risk to native avian fauna that do not have an evolutionary history with the parasite (work et al., ) . in addition, t. gondii is zoonotic; due to the popularity of wild pig hunting for sport and population management, hunters in guam should properly butcher and cook wild pig meat (hill et al., ; conry ) . several pathogens were not detected or were rare in wild pigs. no evidence of exposure to trichinella spp., iav, or coronaviruses associated with enteric and respiratory disease was found; one pig had antibodies to porcine epidemic diarrhea virus. previously, trichinella was absent from domestic pigs, but because of the zoonotic potential of this parasite, surveillance and education campaigns to ensure pork is thoroughly cooked (which would also kill any t. gondii present) should continue. swine influenza, a zoonosis, is relatively common in domestic pigs worldwide, but exposure of wild pigs is not ubiquitous and varies geographically (smith et al., ) . while prevalence was high in a study in southern china (luo et al., ) , in korea, the us and spain, seroprevalence was generally low (< %) and varied seasonally; it is possible that our sample size was insufficient to detect exposure in guam (hall et al., ; corn et al., ; feng et al., ) . enteric and respiratory coronaviruses, which have been reported in several asian countries, are of significant concern due to high morbidity and mortality in naïve pig populations (song and park, ) . porcine epidemic disease virus is clinically similar to transmissible gastroenteritis; both are coronaviruses that can lead to acute diarrhea in all age groups of swine, often resulting in poor body condition or mortalities (lee, ) . one wild pig had antibodies to brucella; the species could not be distinguished based on serologic testing. serology indicates if brucella is present in a population, but culture and/or advanced molecular assays are required to confirm species (leiser et al., ) . brucellosis, a zoonotic disease, has been documented in wild pig populations globally (leiser et al., ) . lice (h. suis) were common on guam, similar to studies on domestic and wild pigs worldwide (girişgin et al., ) . h. suis can transmit swine poxvirus and classical swine fever virus but there is no evidence that these viruses are present on guam. amblyomma breviscutatum were found on several pigs but little is known about this tick species; it is believed to have been historically present on guam and other islands in greater micronesia (vander velde and vander velde, ) . currently, there are no data on pathogens associated with a. breviscutatum. our testing method for helminth parasites was limited due to time and cleveland et al. veterinary microbiology ( ) - importation restrictions; we only examined small sections of lung tissue (or lesions if noted). confirmed infections with metastrongylus lungworms and s. dentatus were relatively common, and likely underrecognized because of testing method. both metastrongylus lungworms and s. dentatus, previously reported in domestic pigs on guam, can impact domestic pig health (duguies et al., ) . our study highlights the importance of surveillance efforts for pathogens transmitted by wild pigs on guam as many of the pathogens circulating in wild pigs can cause disease in domestic swine. we detected serologic evidence for multiple zoonotic pathogens, necessitating simple yet effective preventative measures. wearing appropriate ppe, practicing good hygiene, washing all utensils and surfaces that have come into contact with butchered wild pigs, and thoroughly cooking meat prior to consumption may assist in preventing infection. management of wild pigs is often controversial as they are seen as a food source for hunters or in some cases may be considered native species. the wild pig populations on guam are not native, harbor pathogens of importance to domestic swine and people, and very importantly, cause major damage to the environment leading to economic losses for farmers and extreme habitat destruction for native species of wildlife. guidelines for the euthanasia of animals, edition. avma, schaumburn, il exposure of feral swine (sus scrofa) in the united states to selected pathogens impact of wild boar (sus scrofa) in its introduced and native range: a review consequences associated with the recent range expansion of nonnative feral swine actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection detection of lawsonia intracellularis in faeces of swine from the main producing regions in brazil management of feral and exotic game species on guam survey of selected diseases in wild swine in texas pathogen exposure in feral swine populations geographically associated with high densities of transitional swine premises and commercial swine production animal health survey for guam coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome influenza a subtype h viruses in feral swine occurrence of haematopinus suis linnaeus, (insecta, anopluridae) on a wild boar (sus scrofa) influenza exposure in united states feral swine populations surveillance of feral swine for trichinella spp. and toxoplasma gondii in the usa and host-related factors associated with infection leptospirosis in urban wild boars porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus feral swine brucellosis in the united states and prospective genomic techniques for disease epidemiology exposure to swine h and h and avian h and h influenza a viruses among feral swine in southern china leptospira interrogans antibodies in feral pigs from new south wales identification of brucella suis from feral swine in selected states in the usa widespread detection of antibodies to leptospira in feral swine in the united states contact heterogeneities in feral swine: implications for disease management and future research seroprevalence of six reproductive pathogens in european wild boar (sus scrofa) from spain: the effect on wild boar female reproductive performance diversity of piroplasms detected in blood-fed and questing ticks from several states in the united states origins and evolutionary genomics of the swine-origin h n influenza a epidemic porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines known and potential ticks and tick-borne pathogens of micronesia fatal toxoplasmosis in free-ranging endangered 'alala from hawaii primary financial support for this project came from the us department of the navy (n - -r- and n - -r- ). additional financial support was provided by the wildlife management agencies of the southeastern cooperative wildlife disease study member states through the federal aid to wildlife restoration act ( stat. ) and by the us department of the interior cooperative agreement g ac . the authors thank the personnel at the diagnostic laboratories for diagnostic support and r.l. poulson for comments on this manuscript. key: cord- -r d oaez authors: rottier, peter j.m. title: the molecular dynamics of feline coronaviruses date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: r d oaez feline coronaviruses are widespread and come in different flavors. there are two main serotypes both of which occur in two pathotypes, the avirulent enteric viruses and the virulent, usually fatal peritonitis viruses, the latter in turn occurring either in a ‘wet’ or exudative form or in a ‘dry’ or proliferative form. in this paper a concise overview is given of the molecular features of these viruses. special attention is given to the genetic dynamics of the viruses as these now allow us to begin to understand the origin of the different phenotypes, in particular the genesis of virulence during persistent infection. as discussed, the surprising new insights obtained over the last few years call for a critical reevaluation of strategies for protection. the utrecht group has been accumulating data on feline coronaviruses for more than two decades; marian c. horzinek started the work on feline infectious peritonitis in , and with raoul de groot, harry vennema, herman egberink and bart haagmans our group has made major contributions on this topic. arnold herrewegh's dissertation, based on five papers, is the last coherent addition to the subject (herrewegh, ) . coronaviruses are positive-stranded rna viruses, recently accommodated in the order nidovirales. the family coronaviridae includes the genera coronavirus and torovirus, whereas the arteriviridae contains but a single genus (arterivirus) so far. the viruses have been classified in the order nidovirales mainly on the basis of two features: their genome organization and their replication strategy (for review, see de vries et al., ) . the organization of the genomes of the corona-, toro-, and arteriviruses is quite similar: they have a large open reading frame at the '-end of their genome, which encodes the polymerase (fig. ). it is synthesized as a precursor protein, which is in part generated by ribosomal frameshifting and which is processed by proteolytic cleavages to generate the functional proteins. further downstream from the polymerase gene there is a collection of open reading frames, amongst which the genes for the structural proteins are located, as well as genes of largely unknown function. there is some variation in the genetic makeup of coronaviruses, e.g., with respect to the hemagglutinin/esterase (he) gene, which is lacking in feline viruses. the genes for the structural proteins are obviously present in all viruses, but again there are differences: thus toroviruses lack an e protein, which for coronaviruses is essential in the assembly process. the other common feature is the replication strategy, more specifically: the way these viruses make their proteins. the polymerase protein is translated from the genome, the incoming viral rna. the other open reading frames are translated from subgenomic messenger rnas that are generated by a specific mechanism of discontinuous transcription. each mrna of this collection is responsible for one protein by translation of the '-most open reading frame. coronaviruses can be grouped into three clusters on the basis of genetic comparisons (table ) . we find the feline coronaviruses in group , together with e.g., porcine transmissible gastroenteritis virus and canine coronavirus. the most prominent member of group is mouse hepatitis virus, also the coronavirus type species. infectious bronchitis virus and its many variants constitute group . electron microscopically these viruses present a characteristic picture, with a`corona' surrounding the enveloped particle, a halo of very typical surface projections. within the envelope resides the nucleocapsid, which harbors the plus-stranded huge rna molecule of some kb, the longest mature mrna known to occur in nature. the rna is packaged by one type of protein, the nucleocapsid (n) protein, which is surrounded in turn by an envelope containing three membrane proteins. the spike (s) protein, is the most obvious protein in that it forms the above-mentioned`corona'. another envelope constituent is the membrane protein (m), which is most abundant. finally there is the small envelope protein (e), which has been discovered only recently and which occurs only in very low numbers in virions. in feline coronaviruses the number has not yet been determined but in the related tgev it was estimated to occur at a rate of about molecules per particle (godet et al., ) coronaviruses do not bud from the plasma membrane but are assembled within the cell, at intracellular membranes. these have been identified as the membranes from the intermediate compartment, a complex situated between the endoplasmic reticulum and the golgi apparatus. this is how it happens: the nucleocapsid, the ribonucleoprotein structure composed of the rna and the nucleocapsid protein is synthesized in the cytoplasm. it subsequently encounters the membrane proteins which after their synthesis in the endoplasmic reticulum have traveled to the intermediate compartment. in this compartment the membrane proteins accumulate and interact with the nucleocapsid. subsequent budding gives rise to viral particles which are moved to and through the golgi complex by vesicular transport, undergoing various modifications while on their way. from the golgi complex they are transported (again by vesicles) to the plasma membrane and exocytosed. many properties of the membrane proteins are known. the s-protein is a homotrimer of a glycoprotein of some kda. the m-protein is a triple-spanning membrane protein ± a typical nidovirales protein found also in toro-and arteriviruses in that configuration. finally there is the largely hydrophobic kda e-protein. as we have shown, only the last approximately residues of this polypeptide are exposed at the cytoplasmic side of cellular membranes; these residues end up in the interior of the virus particle after assembly. we have extensively studied the coronaviral envelope proteins, amongst others by coexpressing their genes in different combinations. using the vaccinia virus/t polymerase expression system with plasmids coding for the respective proteins we asked the question what would emerge from these cells. when we coexpressed all three membrane proteins, we encountered particles in the medium, which were morphologically indistinguishale from coronavirions (vennema et al., ) . we have done the experiment for mouse hepatitis virus and repeated it with feline coronaviruses, with the same result the particles produced by coexpression are indistinguishable from authentic virions. the surprise was great when we studied the various combinations and found that, with only the m and e envelope proteins particles were still formed, while nothing happened when the proteins were expressed separately. this finding means that while the m protein alone is unable to assemble coexpression of the e protein allows the formation of a curvature in the membrane, and eventually budding of vesicles with virion size. if the s protein is present, it is co-incorporated. we know that the s protein interacts with the m protein whereby it is apparently drawn into these particles. the conclusion of this experiment is that the assembly of the coronavirus envelope is independent of the nucleocapsid, the m and e proteins being essential, while the spike protein is also dispensable. these findings are interesting not only for fundamental studies on virus assembly, but also for applied research. feline coronaviruses can be grouped on the basis of different criteria, one of them being virulence for the host. this has led to the definition of biotypes, or pathotypes as we may want to call them. we have on the one hand avirulent strains which cause usually mild or even subclinical enteric infections. they are mostly referred to as feline enteric coronaviruses (fecv). on the other hand we have the virulent strains which cause feline infectious peritonitis (fip), and we then talk about fip viruses (fipv). for a general review, see de groot and horzinek ( ) . another distinction is made on the basis of serology, which has allowed the definition of two serotypes. the epidemiology of coronavirus infections can be summarized as follows. all viruses are transmitted by the oro-faecal route, the viruses being widespread in all cat populations known. if you look in catteries or community shelters where cats are in crowded situations, seropositivity is high; but even many single-cat households appear to be seropositive. on the other hand fip is a rare consequence of the infection, occurring in only ± % of the infected animals, mainly in kittens, but sometimes also in old cats. the signs of the infection leading to fip are chronic undulating fever, anorexia, general malaise, ocular and neurological disorders. very characteristic is the abdominal extension which is caused by the formation of ascitic fluid in the`wet' or exudative form of fip; there is also a`dry' or proliferative form where only little if any exudate is found. the pathogenesis of fip is clearly immune-mediated. the key pathogenic event appears to be the infection of cells of the monocyte/macrophage lineage. as shown recently by bart haagmans there is bystander apoptosis of activated t-cells mediated by some soluble factor, while the t-cells are not replicating the virus (haagmans et al., ) . also antibody enhancement is a very well-known phenomenon, with early death as a consequence under experimental conditions. the enhanced infection of macrophages is probably mediated by antibodies complexed with virions and taken up via a fc receptor. what is the origin of the coronaviruses causing fip? this question has kept us busy for many years. what is the relation of fipv with fecv, so to say. how do fip-inducing coronaviruses arise? a quote from a recent paper of harry vennema (vennema et al., ) is appropriate here: fipv is derived by mutation from endemic enteric coronaviruses and the paper provides strong indications for that. there were already indications from the work of arnold herrewegh and others before, but this paper most convincingly shows that fipv is a variant, a mutant of fecv arising in an infected animal. vennema and coworkers came to this conclusion on the basis of extensive sequence analyses and sequence comparisons. they compared sequences of fipv viruses from different geographic areas with those of fecv viruses from these same areas and concluded that the viruses isolated from fip cases are most similar to the coronaviruses from the area where this particular case of fip occurred. this important conclusion also has implications e.g., for protection. another pathogenetic indication obtained was that virulence, the sudden appearance of an fipv biotype, appears to correlate with deletions in the c and b genes. what the c gene actually does is unknown; in essence the same is true for the b gene product, though we know that this is a small glycoprotein that is released into the medium from infected cells in culture. we have suggested that it might have a signalling function, acting as a`virokine'. the two feline coronavirus serotypes can be distinguished on the basis of their relationship to canine coronavirus. ccv specific sera neutralize type ii viruses but fail to neutralize, or neutralize only poorly those of type i. the viruses also have different in vitro characteristics: type ii strains grow well in tissue culture, type i strains grow poorly (pedersen et al., ) . as a consequence more is known about type ii viruses than about type i viruses. in the field, however, type i strains are predominant; in europe for instance, type ii strains have hardly been found, if ever, and also in the us they have been detected only incidentally. in japan they are reported to constitute ± % of the viruses. what is the origin of the serotype ii, what is the molecular basis for the serotypes? it has become clear from sequence comparisons by vennema et al. ( ) , motokawa et al. ( ) and herrewegh et al. ( ) that the type ii strains are the result of double recombination events between fecv type i strains and ccv (see abstract from harry vennema in this issue). recombinations have been found to occur somewhere in the e and the m gene, the second cross-over point being found in orf . since in the various cases of type ii viruses analyzed the same picture has been found (where the spike protein of ccv has been acquired by a type i virus) it is understandable why type ii viruses are ccv related serologically. the fact that several genetically different fecv type i/ccv recombinant strains have been recognized indicates that they have been independently generated, and this double recombination is apparently not a rare event. for many years there have been indications that feline coronaviruses can persist in their natural host. epidemiological studies have indicated that and there is of course niels pedersen's classical experiment where he infected a cat with a sublethal dose of fipv, put the cat in isolation, waited for months and then superinfected it with felv. the immunosuppression caused by the retrovirus made the coronavirus reappear, it was activated to replicate, and eventually the disease picture of fip was seen (pedersen and floyd, ) . more recently, using our sensitive rt-pcr methods (herrewegh et al., a, b) , we designed studies in a closed cat facility in hannover, germany, which confirmed and extended these observations. this was the laboratory cat facility of the medical school, and its history was well documented. it had been fecv-free for many years when, in , feline coronavirus has been inadvertently introduced and from that time on there were yearly cases of ftp; a total of some % of the cats succumbed to the disease. when we came to analyze the situation, no fip cases had been observed over the last years. irrespective of this fact, the virus was really still around: of the cats studied, were seropositive by immunofluorescence; % of these cats were rt-pcr positive in faeces and/or serum and/or plasma. after months we re-examined five rt-pcr positives and found that four of them were still positive. . these data strongly suggested virus persistence in this cattery. the question was then asked whether the continuous presence of the virus was due to recurrent infections between animals or to its extended presence in individual cats. consequently, two pcrpositive cats were placed in strict isolation and their faeces collected. after months, one cat (#h ) was sacrificed, and the collected samples were tested by rt-pcr. the results shown in fig. clearly show that this animal had remained rt-pcr positive for the entire observation period. the long bars in fig. indicate that the samples were positive by direct pcr, the short bars indicate that a nested pcr was required to show a positive result. in essence the same was true for the cat that had been maintained in isolation (cat #h ). we saw that the infection waned and some samples were negative even using a nested pcr. later on positive results were recorded for up to months. this finding demonstrates that the virus can really persist in isolated cats for a long time. from cat #h , the animal that had been sacrificed, a number of tissues were examined for the presence of virus in different tissues. by pcr, viral (genomic) rna was found in many organs. we then employed a pcr designed to detect only mrna, to fig. . chronic shedding of feline coronavirus as monitored by rt-pcr on feces. amplification reactions were targeted to the ' nontranslated region of the viral genome. cats h and h were placed in isolation at day . the numbers of days that the cats were in isolation is on the horizontal axis. the rt-pcr results are expressed by bars above (i.e. positive in rt-pcr) or below (i.e. negative) the horizontal axis. long and short bars represent single and nested rt-pcr. check for replication occurring in these tissues, and there oniy the lower part of the gastro-intestinal tract was found positive. immunohistochemistry performed by bart haagmans evidenced single, scattered antigen-positive cells in various areas in the ileum, close to peyer's patches. the conclusion from these experiments is that feline coronaviruses may persist in the lower intestinal tract where the virus continues to replicate at low levels. we used the samples collected in this cattery also for sequencing, to learn more about the evolution of the viruses, about their genetic changes. the b gene is quite a stable gene (herrewegh et al., c) and was taken for comparison. we found that feline coronaviruses in this colony formed a distinct dade . when we compared them with viruses from other sources and with laboratory strains, they were quite distinct. the viruses from the hannover cattery form one dade, as do viruses originating from another cattery. most probably the viruses in the hannover cattery originated from a single founder infection. the most interesting finding came when we compared the viruses in their ' end of the s gene, known to be highly variable. it appeared that each cat harbors its own distinct feline coronavirus quasispecies. conceivably, the persisting virus confers to its host resistance against superinfection by the closely related feline coronaviruses, which were infecting the other cats. this is an important piece of information when we think about protection, especially the fact that there is resistance against superinfection. we have seen that the feline coronaviruses are harbored in the lower intestinal tract. the question remains whether they are restricted to the enteric epithelium or whether they can cross it. the idea was that`feline enteric coronaviruses' are indeed restricted in tropism, while`fip viruses' would cross the epithelium, infect macrophages and go systemically. epithelial cells are specialized cells; they possess an apical membrane facing e.g. the gut lumen, and a basolateral membrane keeping contact with the neighboring cells and facing e.g., the submucosa;`tight junctions' form a barrier between both the compartments and make sure that different proteins and lipids can be kept separately in them. these cells are specialized in the sorting of proteins and lipids, in the transport of immunoglobulins by transcytosis etc. to study their functions, these cells are grown on filter membranes, whereby the basolateral and apical membranes are separately accessible, e.g., for a virus infection. when the monolayer is complete, there is no transport of medium from the upper part into the lower part of the filter device. using this system, john rossen from our group studied the infection and release behavior of fipv, fecv, ccv, mhv, and tgev. we knew from earlier experiments that tgev is secreted into the apical medium, and by inference into the gut lumen if one considers the intestinal tract, with progression of the infection through apical virus uptake. in contrast, mhv is released into the basolateral medium, and in the mouse, this virus causes a systemic infection, giving rise to hepatitis amongst other symptoms. thus, its basolateral release would be in line with the systemic infection (rossen et al., a (rossen et al., , b, . when examining fipv, fecv, and ccv we found virus release into the basolateral medium in all cases, which would mean that the direction of release is toward the blood stream. so we will probably need to modify our preconception that these viruses are restricted to the intestine. it needs to be examined, of course, if the same holds true for the natural situation. there have been many attempts to protect cats against fip, using various approaches. the result of all these studies is that generally there is no protection when an antibody response to the spike protein is induced there is rather an enhancement of the infection, with an`early death' phenomenon. all these attempts although fruitless in the practical sense have gathered a body of information that allows a number of better focused questions to be asked. one important question is: do we really want to protect against the fipv infection? since fip-inducing coronavirus mutants are generated within the persistently infected animal the objective would rather be to prevent fecv infetion and persistence. when fipv is generated in an animal and causes disease, it is probably shed to some extent, but other cats usually do not get infected. so the transmission is either not very efficient, or most of these cats are resistant to superinfection due to an infection with a related coronavirus variant. usually one sees catteries with one or two isolated cases but the virulent virus obviously does not spread very efficiently. so who is the enemy? the enemy is fecv. what we should want to prevent is the establishment of an fecv infection, because it will eventually be the source of an fipv strain. what we would want to achieve is to protect cats against an infection with fecv. it is highly unlikely that any vaccination strategy will result in sterile immunity. it is unlikely that any vaccine will prevent an infection to occur. there will probably always be a few cells that become infected. what one would want to achieve then is to have an immune system in place that can either terminate or contain that infection immediately. replication of the infecting fecv must be kept to a minimum, such that the viral load and the eplication determinants for the stochastic appearance of fipv mutants are as low as possible. what are the correlates of protection? cellular immunity is probably most important, and there are many indications that this is where the solution must be sought. the vaccinological objective can be summed up in one sentence: novel vaccines aimed at protection against fip should induce a vigorous t-cell response, above all against type i viruses. feline infectious peritonitis the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses tgev coronavirus orf encodes a membrane protein that is incorporated into virions apoptosis and t-cell depletion during feline infectious peritonitis molecular genetics, persistence and evolution of fcov persistence and evolution of feline coronavirus in a closed cat-breeding colony detection of feline coronavirus rna in feces, tissues and body fluids of naturally infected cats by reverse transcriptase pcr polymerase chain reaction (pcr) for the diagnosis of naturally occurring feline coronavirus infections the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes feline coronavirus type ii strains fecv - and fipv - originate from a double recombination between feline coronavirus type i and canine coronavirus comparison of the amino acid sequence and phylogenetic analysis of the peplomer integral membrane and nucleocapsid proteins of feline canine and porcine coronaviruses pathogenic differences between various feline coronavirus isolates experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd , and fipv-ucd a murine and a porcine coronavirus are released from opposite surfaces of the same epithelial cells coronavirus infection of polarized epithelial cells mhv-a enters polarized murine epithelial cells through the apical surface but is released basolaterally nucleocapsid-independent assembly of coronavirus-like particles by coexpression of viral envelope proteins a comparison of the genomes of fecvs and fipvs and what they tell us about the relationships between feline coronaviruses and their evolution key: cord- - p ld ur authors: suzuki, kazuhiko; matsui, yusuke; miura, yasuo; sentsui, hiroshi title: equine coronavirus induces apoptosis in cultured cells date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: p ld ur equine coronavirus (ecov) was first isolated from a diarrheic foal and was found genetically similar to group ii coronaviruses. however, its pathological characteristics were not adequately investigated. in our preliminary in vitro investigation, ecov-induced cell death was observed in bovine kidney-derived mdbk cells. based on this finding, we investigated whether the ecov-induced cpe was apoptosis. following ecov infection, mdbk cells showed morphological changes such as cell rounding and detachment from the culture surface. moreover, syncytium formation was observed as the other type of cytopathic effect in ecov infection. morphologic and biochemical features of apoptosis, such as nuclear fragmentation and dna ladder formation, were also detected in ecov-infected cells. moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases – caspase- / – and initiator caspases – caspase- and caspase- – that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ecov-infected mdbk cells. therefore, it was suggested that ecov can induce apoptosis in mdbk cells via a caspase-dependent pathway. apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. although the pathological characteristics of ecov are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ecov infection. apoptosis is a physiologically important process that eliminates damaged cells and is defined by typical changes in cellular morphology and biochemical features, including dna fragmentation, cytoplasm vacuolization, chromatin condensation and margination, and cellular breakdown into apoptotic bodies (kerr et al., ) . apoptosis can be triggered by diverse intracellular and extracellular signals, including virus infections. many viruses have the ability to induce apoptosis, which is one of the cytolytic properties of viral infections, and show the phenotype of cytopathic effect (cpe) in vitro; this is particularly www.elsevier.com/locate/vetmic observed in rna viruses, including coronaviridae (collins, ; eleouet et al., ; liu et al., liu et al., , liu and zhang, ; ruggieri et al., ) . the coronaviridae family is a group of enveloped viruses with a large, positive-stranded rna genome and comprises two genera-coronavirus and torovirus. coronaviruses are classified into three groups (i-iii) based on serological cross-reactivity and phylogenetic analysis (gonzalez et al., ) . equine coronavirus (ecov) was first identified in the feces of a diarrheic foal. when the amino acid and genome sequences of ecov were compared with those of other avian and mammalian coronaviruses, ecov was found to be relatively similar to group ii coronaviruses such as murine hepatitis virus (mhv) and human coronavirus (hcov) oc and particularly to bovine coronaviruses (bcovs) (guy et al., ; zhang et al., ) . although there have been reports on suspected cases of spontaneous ecov infection showing severe diarrhea, investigations on the pathogenicity of ecov infection have not been conducted until now. in in vitro cultures, human rectal adenocarcinoma (hrt- ) cells were used for propagation of ecov, but this cell line could not induce a distinctive cpe (guy et al., ) . in our preliminary investigation, we found that mdbk cells (a bovine kidney-derived cell line) infected with ecov showed more distinctive morphological changes. in this study, to elucidate the mechanisms of cell injury by ecov, we investigated whether the ecov-induced cpe was apoptosis by using cytological change, dna fragmentation, and caspase activity as the indices. ecov nc was kindly provided by the equine research institute of the japan racing association. mdbk cells were cultivated in eagle's minimum essential medium (emem) (nissui pharmaceutical co., ltd., tokyo, japan) supplemented with % fetal bovine serum and . % tryptose phosphate broth. subconfluent cells infected with ecv at tcid were harvested and collected at , , , , , , , , and h after treatment (hat). mock-infected cells were used as controls. ecov-infected and mock-infected mdbk cells were collected at the predetermined time points after infection, fixed in methanol for min, stained with giemsa's solution for min, and examined microscopically. the apoptotic dna ladder detection kit (che-micon international inc., ca, usa) was used for dna extraction from cells. briefly, ecov-infected mdbk cells collected at the predetermined time points after infection were lysed by the addition of ml of te lysis buffer and ml of enzyme a (rnase a) and incubated at c for min. then, ml of enzyme b (proteinase k) was added, and the lysate was further incubated at c for min. dna was precipitated using ethanol. after washing with % ethanol, the pellet was resuspended in dna suspension buffer. for detecting the dna ladder, the extracted dna samples were run on a % agarose gel in tris-acetic acid-edta buffer. after electrophoresis, the gel was stained with ethidium bromide (nippon gene co. ltd., toyama, japan), visualized with a uv light transilluminator, and photographed. caspase- / activity was measured by apo-one homogeneous caspase- / assay (promega corporation, wi, usa), according to the manufacturer's instructions. in brief, subconfluent mdbk cells seeded in -well plates were infected and harvested at the various predetermined times post-infection. ecovinfected and mock-infected cells were treated with equal volumes of the homogeneous caspase- / reagent and incubated for h. then, their fluorescence was measured using a fluorescence microplate reader. individual caspase- / activities were measured as the difference in fluorescence between ecov-infected cells and mock-infected cells at each time point. caspase- and caspase- activities were measured using a caspase-glo assay kit (promega corporation, wi, usa), according to the manufacturer's instructions. in brief, subconfluent mdbk cells seeded in -well plates were infected and harvested at the various predetermined times post-infection. ecovinfected and mock-infected cells were treated with equal volumes of the caspase-glo or caspase-glo reagent and incubated for min. then, their luminescence was measured using a luminometer. individual caspase- and caspase- activities were measured as the difference in fluorescence between ecov-infected cells and mock-infected cells at each time point. the ecov-infected mdbk cells gradually developed the cpe, including cell rounding and detachment, at and after hat. when stained with giemsa's solution, syncytium formation was also observed as a variant of the cpe (fig. a) . in addition to syncytium formation, chromatin fragmentation was observed in many ecov-infected cells at and after hat ( fig. b and c) . dna fragmentation started at hat, and the ''ladder'' pattern was apparent at and hat (fig. ) . we measured caspase- / activity based on the cleavage of the selective fluorescent substrate. the results showed that caspase- / activity was significantly increased in ecov-infected cells at and after hat, and the maximum increase was observed at hat ( . -fold increase in activity when compared with that of mock-infected cells) (fig. ) . further, the activities of caspase- and caspase- , which are representative initiator caspases in the death receptor-mediated and mitochondrial apoptotic pathways, respectively, were also measured based on the cleavage of the luminogenic substrates. caspase- and caspase- activities were increased in ecov-infected cells at and after hat, and the maximal increases were observed at hat ( . -fold and . -fold increases in activity, respectively, when compared with those of mock-infected cells). apoptosis represents an important antivirus defense mechanism of the host cell, and viruses have evolved strategies to counteract and regulate apoptosis in order to maximize the production of virus progeny and promote the spread of virus progeny to neighboring cells. ecov was isolated from the feces of a diarrheic foal, and investigations revealed that it was genetically similar to bcov (guy et al., ) . however, no other study has examined ecov after this. therefore, as the first step to clarify the biological characteristics of ecov, we investigated whether ecov could induce apoptosis. we characterized the apoptotic cell death of ecovinfected mdbk cells. at and after hat, there was a gradual increase in the number of infected cells that showed the morphological changes of cell rounding and detachment from the culture surface. in addition, chromatin fragmentation was observed at and hat. moreover, dna ladder formation was observed at and after hat and was most prominent at hat. ecv-infected cells also showed syncytium formation prior to apoptosis induction. syncytium formation has been previously reported in some coronaviruses (kusanagi et al., ; lavi et al., ; li and cavanagh, ) . in a study on group ii coronaviruses, mhv- -infected macrophages from balb/c mice showed syncytium formation and apoptosis, but the cell populations that showed the phenomena were different (belyavsky et al., ) . in contrast, infectious bronchitis virus (ibv)infected vero cells showed syncytium formation followed by caspase-dependent apoptosis (liu et al., ) . these findings indicate that since ibv belongs to group iii coronaviruses, ecov and ibv are considerably separated on the phylogenetic tree based on genome sequences but are similar with respect to biological behavior . ibv is known as a prototype virus of the coronaviridae family. therefore, it is conceivable that ecov has genetically evolved into a group ii coronavirus but has conserved the functions of the coronaviridae family. fig. . agarose gel electrophoresis of dna from ecv-infected cells. dna fragmentation was observed at and after hai, but was prominent at and hat. m: bp dna marker (takara bio inc., shiga, japan), - : , , , , , , , , and hat, respectively. caspases are cysteine proteases that play fundamental roles in the apoptotic responses of cells to different stimuli. in particular, caspase- , caspase- , and caspase- are classified as effecter caspases that are activated by the respective initiator caspases in the death receptor-mediated and mitochondrial apoptotic pathways and can be considered as key enzymes involved in apoptosis since they are indispensable for chromatin condensation and dna fragmentation. in the death receptor-mediated apoptotic pathway, caspase- is the initiator caspase that is activated by the interaction between external apoptotic factors and cell surface molecules such as fas or fas ligand via adaptor proteins such as fas-associated death domain (fadd) (cohen, ) . on the other hand, in the mitochondrial apoptotic pathway, caspase- is the initiator caspase whose activity is regulated by the expression balance between bcl- and bax via cytochrome c/apaf- complex formation (cohen, ; strasser et al., ) . in ecov-infected mdbk cells, caspase- / activity was increased at and after hat, and caspase- and caspase- activities were increased at and after hat. these results indicate that ecov can induce caspasedependent apoptosis in mdbk cells via both the death receptor-mediated and mitochondrial apoptotic pathways. however, the activation of the initiator caspase- can also be related to the mitochondrial apoptotic pathway via cleavage of bid (a member of the proapoptotic bcl- family) and translocation of the truncated bid to mitochondria . in some coronaviruses, both initiator caspases are activated in the course of apoptosis; the fas-signaling pathway in which both initiator caspases are activated via cleavage and translocation of bid was suggested as the mechanism of apoptosis, particularly in mhv infection (eleouet et al., ; liu et al., ; liu and zhang, ) . therefore, it was speculated that ecov also induces apoptosis via a caspase-dependent pathway that may be activated by an interaction between virus protein and cell surface signaling factors with participation of both the death receptor-mediated and mitochondrial apoptotic pathways. however, further investigations that will focus on the kinetics of signal transduction factors are necessary to clarify the detailed mechanisms of ecov-induced apoptosis. in conclusion, this report showed that ecovinduced apoptosis via caspase-dependent pathway. this finding indicated that mdbk cells can be use for propagation and establishment of serological diagnosis system for ecov. however, because the findings from bovine-derived cell line may not faithfully reflect on the in vivo situation, further investigation using equine-derived cell line will be needed to clarify the pathobiological characters of ecov. coronavirus mhv- -induced apoptosis in macrophages caspases: the executioners of apoptosis induction of apoptosis in mrc- , diploid human fetal lung cells after infection with human coronavirus oc transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase- and - during tgev-induced apoptosis a comparative sequence analysis to revise the current taxonomy of the family coronaviridae characterization of a coronavirus isolated from a diarrheic foal apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus coronavirus ibv-induced membrane fusion occurs at near-neutral ph murine coronavirus-induced oligodendrocyte apoptosis is mediated through the activation of the fas signaling pathway induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis canine coronavirus induces apoptosis in cultured cells apoptosis signaling bid: a novel bh domain-only death agonist genomic characterization of equine coronavirus key: cord- -g y au a authors: mitchell, judy a.; brooks, harriet w.; szladovits, balázs; erles, kerstin; gibbons, rachel; shields, shelly; brownlie, joe title: tropism and pathological findings associated with canine respiratory coronavirus (crcov) date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: g y au a canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. tropism and pathological findings associated with canine respiratory coronavirus (crcov) a b s t r a c t canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. ß elsevier b.v. all rights reserved. since its initial discovery in (erles et al., ) ; canine respiratory coronavirus (crcov) is now considered to be a significant cird pathogen, most frequently detected in dogs with mild respiratory clinical signs during the early stages of cird onset (erles et al., ) . although crcov has been found worldwide (decaro et al., ; erles and brownlie, ; kaneshima et al., ; priestnall et al., priestnall et al., , yachi and mochizuki, ; knesl et al., ) ; little is known regarding its pathogenesis, tissue tropism or virulence differences among global isolates in the canine host. it is postulated that crcov plays an important role during the early stages of cird by predisposing the dog to more severe clinical disease from secondary infections. through the use of an in vitro tracheal explant culture system, a moderate reduction in ciliary function and a down-regulation of pro-inflammatory cytokine mrna levels (tnf-a, il- and the chemokine il- ) was observed in response to crcov exposure (priestnall et al., ) . such alterations in the mucociliary and innate immune systems could be linked to increased susceptibility to secondary infection and is consistent with the proposed role for crcov in cird. however, the limitation of this in vitro model precludes the understanding of the clinical relevance and pathogenesis of a crcov infection in the dog. furthermore, given the global presence of this virus, insight into crcov pathogenesis among isolates originating from geographically distinct locations would be valuable to determine the need for a global vaccine. recently our group has collected findings from a preliminary in vivo challenge study of crcov. in that study we demonstrated that young dogs were susceptible to experimental infection with both crcov isolates, which gave rise to clinical signs of respiratory disease consistent with naturally occurring infection. crcov was detected in the oropharynx of infected dogs and spread rapidly to sentinel dogs which also displayed clinical signs of disease (mitchell et al., unpublished data) . here we extend this study to gain a better understanding of crcov pathogenesis in vivo. analyses specifically focused on the histopathological changes in the canine upper and lower respiratory tissues, virulence differences among crcov isolates derived from cird cases representing wide geographical locations; uk and usa [mo, ne, ut and mi] , and the demonstration of koch's postulates. the information obtained from this study vastly enhances our understanding of crcov pathogenicity and its involvement in the cird complex. five crcov isolates originating from geographically distinct regions of the uk and usa were used in this study (table ) . crcov isolates uk , np , np , and np were propagated in culture on human rectal tumour cells (hrt- g; american type culture collection cell line, manassas, va, usa), and used at tcid /ml for intranasal challenge. crcov lu was not was not propagated or expanded in vitro, instead . mm filtered viral fluids obtained from the tissues of a crcov infected dog in the usa were used to challenge animals in to . prior to challenge each of the crcov virus and control hrt- g cell culture fluids were satisfactorily tested for sterility and canine extraneous agents (including canine distemper virus, measles, canine adenovirus- , canine parainfluenza virus, canine rotavirus, rabies, canine parvovirus and canine enteric coronavirus). thirty-six, - -week old specific pathogen free (spf) purpose bred beagle dogs were used in this study. all dogs were demonstrated as crcov negative and seronegative for crcov and b. bronchiseptica prior to the study. dogs were housed in temperature controlled isolation rooms with dedicated shower in and out procedures, disinfection and sterilisation of all items prior to entry and a pasteurised diet were used to maintain bio-security. colony dogs are screened quarterly to determine the spf status. throughout the study dogs from the same treatment group were housed in pairs to minimise stress. dogs were randomly divided into six treatment groups (t -t ) (table ) , each consisting of six dogs. dogs in groups t -t inclusive were challenged with the crcov isolates as described in table . dogs in t were mock challenged with uninfected hrt- g cell culture supernatant to serve as negative controls. intranasal inoculations took place over two consecutive days (study day zero and one). on each challenge day dogs were sedated prior to intranasal administration with . ml of virus or control material ( . ml/nostril). dogs were monitored throughout the trial for clinical signs of disease. on study days , , and , as detailed in table , dogs were humanely euthanatized within each treatment group as determined by randomisation completed prior to the start of the trial, and necropsies were performed immediately. gross pathological changes were documented throughout each necropsy and the lungs of each animal were photographed. all experiments involving animals were carried out at a contract research organisation, in compliance with national legislation, and subject to local ethical review. general health observations were performed on each dog, twice daily, and scored for general appearance, breathing, sneezing, coughing, and ocular and nasal discharge as detailed in table . body temperatures were recorded twice daily via implanted microchips, and dogs were weighed on study days À , À and on the day of euthanasia. appetite was recorded according to the quantity of food eaten per room. oropharyngeal viral swabs for rt-pcr analysis (sterilin, uk) and virus isolation (dacron swabs, puritan, in ml virus transport medium (pah)) were collected on study days À (pre challenge) , , , , , , , , and from all dogs on the study. rectal swabs were collected from each dog at necropsy. after sampling, viral swabs were frozen and stored at À c until processed. swab tips for rt-pcr analyses were immersed in ml of rpmi medium (sigma, dorset, uk) and mixed prior to analysis. samples from the following tissues were harvested at necropsy and stored at either À c or fixed in % buffered formalin: nasal cavity (included ciliated area), nasal tonsil, palatine tonsil, trachea, apical lung lobe, diaphragmatic lung lobe, and bronchial lymph node. each tissue sample was taken using a new set of sterile instruments. lung lavage fluid samples were collected in dmem cell culture medium and stored at À c. formalin-fixed tissues were processed and stained for histology and were examined by a veterinary pathologist as described in section . . on study days À , , , and , serum and edta whole blood samples were collected for serological and haematological analysis as described below. swabs and tissue samples were tested for the presence of crcov by rt-pcr and virus isolation. prior to inoculation on to hrt- g cells, swab and lung lavage samples were clarified by centrifugation, and filtered ( . mm filter). similarly, approximately g of each tissue sample was homogenised in . ml of virus transport medium (vtm), clarified by centrifugation and filtered ( . mm filter). filtered samples were inoculated onto confluent t or t monolayers of hrt- g cells. inoculated cultures were maintained in dmem supplemented with final concentrations of mm l-glutamine, . mg/ml gentamicin, and % fbs. inoculation media was also supplemented with trypsin. trypsin concentrations were determined for each batch (circa mg/ml) based of cell toxicity. cell culture fluids were sampled weekly for weeks to test for the presence of crcov by immunofluorescence assay (ifa) (section . . ). rna was extracted using the rneasy mini kit (qiagen, crawley, uk) as recommended by the manufacturer from ml of the swab fluid or a . cm piece of tissue. rna was transcribed into cdna using random hexameres (ge healthcare, little chalfont, uk) and improm ii reverse transcriptase (promega, southampton, uk) according to the manufacturer's protocol. all samples were tested for the presence of the house keeping gene glyceraldehyde- -phosphate dehydrogenase (gapdh) by pcr as described previously (grone et al., ) . results were expressed as a positive or negative outcome to ensure successful nucleic acid extraction. samples were analysed for crcov using the nested spike gene pcr as described previously (erles et al., ) . formalin-fixed tissues were processed for histology and sections were stained using haematoxylin and eosin. the histological sections were examined by a veterinary pathologist who was blinded to the study groups. each tissue was ascribed a histological and cilia score as described in table . a single overall histological ''weighted'' score was assigned to each dog, in which scores assigned to the lower respiratory tract (trachea, lungs, bronchial lymph nodes) were considered more noteworthy than those in the upper respiratory tract (nares, tonsils). this weighting of the overall score was used to take into account that histological changes (especially inflammatory reactions or lymphoid hyperplasia) in the upper respiratory tract are not uncommon, especially in young animals, and unless considerable, are considered part of the normal defences of the upper respiratory tract. conversely, such changes in the lower respiratory tract are more likely to be clinically significant. thus moderate to marked lymphoid hyperplasia or neutrophilic inflammation of the palatine tonsil (score ) would have less effect on the overall histology score for that individual dog than would a similar grading of lymphoplasmacytic or neutrophilic aggregation in the tracheal mucosa or lungs. paraffin-embedded formalin-fixed tissues ( mm) were mounted on superfrost plus microscope slides (menzel-glä ser, braunschweig, germany). slides were heated at c for h, deparaffinised and dehydrated. endogenous peroxidase was blocked with % h o for min then slides were washed in dh o, then incubated in prewarmed ( c) protease xiv . % (sigma) in tbs for min. slides were rinsed in dh o then incubated with blocking serum ( % normal goat serum [vector laboratories, peterborough] in tbs). staining for crcov was performed as described previously (priestnall et al., ) . positive cells were identified microscopically by the presence of staining. haematological analysis of edta blood samples was performed using a cell-dyn automated haematology machine. a cell differential count of the white blood cells was performed on blood smears stained with a hematek automated stainer using modified wright's stain, by a board certified veterinary clinical pathologist blinded to the treatment groupings. the results for day À were analysed for normality and reference intervals were estimated (n = ). total neutrophil, band neutrophil, lymphocyte and monocyte concentrations were calculated using the automated total wbc concentration, and the observed percentages of each leucocyte type determined by the differential count. the results within each treatment group for days , and were compared with each other and with those at day À to evaluate trends or significant differences. the paired t-test or wilcoxon signed ranks test with repeat measures were used if distribution was normal or non-normal, respectively. statistical significance was set at p = . . hrt- g cells were cultured to confluency in well plates. monolayers were inoculated with a usa crcov isolate, incubated for days, and fixed with % acetone. canine serum samples were diluted : in pbs supplemented with % bovine serum albumin (w/v) (steris corporation, mentor, oh, usa) and . % sodium azide (mallinkrodt chemicals, hazelwood, mo, usa) followed by two-fold serial dilutions to : . diluted serum was dispensed at ml/well onto the fixed cells, incubated for h, and then washed twice with water. bound antibody was detected with ml/well of fluorescein isothiocyanate-labelled secondary antibody (rabbit anti-dog igg) (sigma-aldrich, jerusalem, israel) diluted : in pbs supplemented with % bovine serum albumin (w/v) and . % sodium azide (w/v), incubated and washed as before. endpoint crcov titres were observed using fluorescence microscopy and defined as the inverse of the last dilution of serum exhibiting definite crcov fluorescence. in instances where no virus-specific fluorescence at the : dilution was observed, dogs were considered seronegative or non-exposed to crcov. dog sera collected on study day were tested for serum neutralising antibody titres against uk, and usa crcov isolates. hrt- g cells were grown to confluency in a well plate. cells were rinsed once in serum-free dmem and then pre-treated for hour with ml/well of serum-free dmem containing c mg/ ml trypsin. heat inactivated serum samples were diluted -fold, : through : , in dmem containing % fbs. a further : dilution of the serum was made in - tcid of virus per ml, to obtain final serum dilutions of : - : . virus:serum mixtures were incubated for h at room temperature. cells were inoculated in quadruplicate with ml/well of the virus: serum mixture, incubated for - days, and fixed with % acetone. virus growth was detected by ifa as described previously (section . . ). the fifty percent neutralisation endpoint for each serum sample was calculated by the statistical methods of spearman-karber. oropharyngeal amies swabs (sterilin, uk) and lung lavage fluid samples were collected from all dogs at necropsy in order to screen for the presence of bacterial pathogens. briefly each swab was plated onto  chocolate agar,  macconkey agar, and  blood agar (  aerobic and  anaerobic) and submitted to the clinical services division at the royal veterinary college for analysis. samples were also plated onto mycoplasma experience agar (mycoplasma experience ltd. surrey, uk) and incubated at c with % co for days. dogs from all six treatment groups were healthy prior to challenge. dogs in t (negative control) remained healthy for the duration of the study, the only exception being one dog which had some serous ocular discharge prior to challenge, and throughout the study. all dogs completed the study on the pre-assigned day. following challenge, a number of dogs from t to t inclusive displayed mild clinical signs of respiratory disease which included nasal discharge, sneezing and coughing. table shows the total scores for each observation by treatment group. there were no significant changes in body temperature all dogs had normal or fair appetites and gained weight over the course of the study (data not shown). no consistent differences between dogs treated with different strains of the virus were observed, although respiratory signs were recorded most frequently and most severely in one dog from t and one from t (table ). it is worth noting that in both instances these dogs were euthanized on study day and displayed the clinical signs of respiratory disease recorded throughout the -day study period, although the scores were highest in these two dogs on study days - . oropharyngeal swabs were analysed for crcov by both rt-pcr and virus isolation (fig. ) . all dogs from all six treatment groups were negative for crcov on study day À (pre-challenge) using both techniques. all dogs in t (negative control) remained negative for the duration of the study. shedding of crcov in t -t was consistently detected for up to days post challenge by both rt-pcr and vi. all rectal swabs, collected at necropsy, were positive for the internal pcr control gapdh. all rectal swabs collected from dogs in t (negative control) were negative for crcov by both rt-pcr and vi for the duration of the study, as was the case for dogs in groups t , t and t . in t , two dogs euthanized on study day , and one dog euthanized on study day were positive for crcov, as was one t dog euthanized on study day . the remaining dogs in these groups were negative. all rectal swabs were negative for crcov by vi (data not shown). tissues collected at necropsy were tested for the presence of crcov by rt-pcr and vi (fig. ) . crcov was detected in at least five of the eight tissues [diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, bronchial lymph node, nasal and palatine tonsil and lung lavage fluid] from groups t -t by rt-pcr. in t crcov was detected in all the tissues tested. vi was successful in seven of eight tissue types from groups t -t including the diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, nasal and palatine tonsil as well as the lung lavage fluid. only bronchial lymph nodes were negative for crcov by vi. overall, crcov was detected most frequently from the trachea, followed by the lung lavage fluid, nasal cavity and nasal tonsil, in all five treatment groups. histological examination was carried out on the palatine tonsil, external nares, nasal tonsil, trachea, apical and diaphragmatic lung lobes, and bronchial lymph node. the most significant findings were seen in the trachea and nares where inflammation, with notable changes in the length and distribution of the cilia were observed as described below and shown in figs. and . in t the overall weighted histology score was consistently minimal (average . ), with minimal changes in all the tissues examined. in t the weighted score indicated minimal to modest abnormalities (average . ), including inflammatory aggregates in the nares and inflammation in the trachea which was associated with loss of cilia from the mucosal surface. one dog in particular on day showed marked changes (scoring or ) in all of the tissues examined, with the exception of the diaphragmatic lung lobe and nasal tonsil where only minimal to modest changes were observed. in t and t the overall weighted score showed modest to marked histological abnormalities with average scores of and . , respectively. in the nares modest to marked inflammation was seen, which in t trailed off in the later stages of the trial period, whilst inflammation in the trachea of dogs from both groups was mild but with moderate shortening and irregularity of the cilia. in t , with the exception of two dogs (one each on days and ), marked histological changes in the nares were observed throughout the trial period. in addition marked changes were also observed in the trachea and cilia. one dog on day also had marked changes in the bronchial lymph node and diaphragmatic lung lobe (data not shown). the overall weighted scores indicated consistently marked abnormal histological changes in all dogs in this group with an average score of . . in t modest to marked inflammation was observed in the trachea of dogs in t which was reflected in the condition of the cilia, whilst only modest changes were observed in the nares. on study day marked changes were also seen in the bronchial lymph nodes. the overall weighted score showed consistently marked abnormal histological changes (average . ), with the exception of one of the dogs at the first time point which had a score of . in t -t inclusive histological abnormalities in the lungs, although not marked, were variable and tended to be lymphoid aggregates adjacent to airways or blood vessels, an observation which was not seen in the broth control which yielded consistently mild scores. in all groups, including the broth control, the palatine tonsils had medium to high histological scores (data not shown). coronavirus antigen-positive cells were detected within the epithelium of the trachea and bronchioles of the infected dogs (fig. ) . positive staining was present in the cytoplasm of ciliated columnar epithelial and goblet cells. positive cells were widespread but often found in focal clusters, and often associated with small aggregates on the luminal surface of the trachea. positive cells were surrounded by areas of vacuolation within the epithelium, possibly representing a cytopathic effect of the virus. seroconversion to crcov was measured by ifa. all dogs were seronegative (ifa < ) for crcov on study days À , and , and all dogs in the t control group remained seronegative throughout the study whilst all dogs in the crcov challenge groups seroconverted by study day . crcov serum cross-neutralisation antibodies from serum collected on study day were tested against uk and usa crcov isolates (table ). all serum samples tested from dogs in crcov treatment groups were shown to be serum neutralisation positive against all the crcov isolates tested. serum collected from t control dogs showed no neutralising activity against any of the crcov strains. the mean concentrations of lymphocytes, neutrophils and monocytes, for each group on study days À , , and were determined (data not shown). in t , and t no statistical differences in the lymphocyte, neutrophil or monocyte concentrations were detected. in t , t , t and t increases in total white blood cell concentrations were observed as a result of lymphocytosis which was significant in t (p = . ) on day and t (p = . ) on day . in t a marked lymphocytosis was seen on day . in t there was moderate lymphocytosis accompanied by significant changes in monocyte concentrations which peaked on day [>day (p = . ), >day (p = . )]. in contrast there was a significant decrease in neutrophil concentration on day in t , t and t (p = . , p = . and p = . , respectively) compared to day À , which in t resulted in of the dogs becoming neutropenic. across the groups seven dogs had rare to occasional toxic neutrophils exhibiting foamy cytoplasm, mild cytoplasmic basophilia or rare dohle bodies, and detectable levels of band neutrophils. in group t , all dogs had detectable levels of band neutrophils on day , which was a significant increase when compared to day À (p = . ). on day one dog in each of t , t , and t had band concentrations higher than the estimated upper reference limit; however this did not result in statistically significant changes at a group level and no other significant changes could be detected. oropharyngeal amies swabs collected from all dogs on day À and at euthanasia yielded growth of normal respiratory flora. a majority of dogs also yielded scanty growth of either streptococcus canis or staphylococcus intermedius. in the vast majority of dogs the lungs were sterile for bacterial growth, with the exception of the growth of mycoplasma spp. in four dogs, one from each treatment group t -t inclusive (data not shown). previous publications have collectively demonstrated the global distribution of crcov and its association with respiratory disease in dogs under field conditions (erles et al., ; decaro et al., ; kaneshima et al., ; priestnall et al., priestnall et al., , yachi and mochizuki, ; knesl et al., ) . this is the first publication to report experimental infection of dogs using crcov, and the comparison of different crcov isolates from wide geographic origins. each crcov isolate was derived from dogs with respiratory disease in mo, mi, ut and ne in the usa, and one isolate from london, uk. this study builds upon a preliminary experimental challenge study (mitchell et al., unpublished data) . in that study we demonstrated rapid shed-spread of crcov from experimentally infected dogs to sentinel dogs within days of exposure. peak viral loads were detected in oropharyngeal swabs at and days post infection in experimentally infected, and sentinel dogs, respectively; with shedding lasting for - days in both groups. both inoculated and sentinel dogs displayed clinical signs of mild respiratory disease, and seroconverted to the virus. in the current study prolific shedding of crcov from the oropharynx of dogs in all treatment groups (detected by rt-pcr and vi from oral swabs) was seen by day . in a majority of dogs viral shedding ceased after day , although crcov was detected up to days post infection by rt-pcr in one dog. small differences in the duration of viral shedding between the two studies are most likely to be explained by differences in the age of the dogs used in each of the studies (preliminary study: - weeks old, current study: - weeks old). despite differences, both studies clearly illustrate that crcov is a quick-hit respiratory pathogen, and supports field data in which the rapid spread of crcov throughout a large rehoming centre was observed (erles et al., ) . consistent with observations made during naturally occurring infection, dogs in this study also displayed clinical signs of mild respiratory disease following viral challenge (nasal discharge, sneezing, and coughing); whilst the control group remained healthy. there appeared to be no profound difference in the clinical observations made between groups of dogs challenged with the different strains. two dogs however (one each in t and t ) showed more severe and prolonged clinical signs compared to the others. this included increased respiratory noise, and more frequent and prolonged coughing and sneezing. the reason for this was unclear. disease in these two dogs did not appear to be associated with any secondary bacterial infections in the lung, nor with notably different histopathological changes when table study day cross neutralisation titres. two dogs remained on the study at day in each treatment group. samples with sn values of were considered negative at the : starting dilution. serum samples collected at earlier time points were negative for crcov antibody. treatment group (isolate) lu lu lu lu lu np np np np np uk t (lu ) t (uk ) t (np ) compared to other challenge dogs in the same group (data not shown). given its multifactorial aetiology; modelling cird under controlled conditions, to mimic disease as it appears in the field is difficult to achieve. when studied in isolation dogs infected with other viruses involved in the cird complex, such as cpiv and cav- , also cause only mild respiratory disease such as a dry cough and nasal discharge (appel and binn, a,b; castleman, ; buonavoglia and martella, ) . more severe clinical signs, representative of the disease complex, are likely to occur when other viruses or bacterial respiratory pathogens are also present, and when environmental stresses such as those encountered in rehoming facilities are experienced (appel and binn, a,b; buonavoglia and martella, ) . overall there appeared to be little difference in tissue tropism, when comparing the different crcov isolates investigated. crcov was detected in at least five of the eight necropsy samples examined (lung lavage, diaphragmatic lung lobes, trachea, nasal tonsils and nasal cavity) in dogs from all five challenge groups. crucially, isolation of crcov was achieved from six of the seven tissues collected (the bronchial lymph node remained negative) as well as the lung lavage fluid. re-isolation of virus from experimentally infected dogs displaying clinical signs of disease, signifies a causal relationship between crcov and respiratory disease, which until now has been best demonstrated through epidemiological surveys (erles et al., (erles et al., , decaro et al., ; kaneshima et al., ; priestnall et al., priestnall et al., , knesl et al., ) . in all treatment groups crcov was detected most frequently in the trachea, nasal tonsil and lung lavage fluid, and these same tissues also exhibited the highest viral titres (data not shown). this is consistent with previous findings from a small cohort of naturally infected dogs in which the highest viral loads, detected by quantitative rt-pcr, were seen in the trachea and nasal tonsil . importantly, histological analyses were consistent with our molecular and virological findings. ihc revealed coronavirus positive staining in the cytoplasm of ciliated epithelial and goblet cells of the trachea and bronchioles of crcov infected dogs. this pattern of staining is consistent with previous work which identified coronavirus antigenpositive epithelial cells within the trachea of dogs naturally infected with crcov (ellis et al., ; priestnall, ) , and in crcov infected tracheal sections maintained in culture (priestnall et al., ; priestnall, ) . furthermore, histopathological analysis showed a clear association between exposure to crcov and inflammation in the nares and trachea, with loss or damage to cilia in the latter. these changes were particularly marked in dogs belonging to t and t , which may indicate a higher degree of pathogenicity for those crcov strains; although, with the exception of one dog in t this was not apparent from the clinical signs. ciliary clearance is a key strategy for the removal of pathogens from the respiratory tract. reductions in the efficiency of ciliary clearance would potentiate infection with secondary respiratory pathogens, leading to increased severity and duration of disease. in addition to the trachea, the nasal tonsil may also represent an important site for crcov infection and pathogenicity; given the consistency and duration of crcov isolation and detection in this tissue. preliminary ihc analysis has shown that crcov infection in the nasal tonsil is associated with the epithelium, and with large mononuclear cells which have the appearance of macrophages (priestnall, ) . a clear association of crcov with macrophages is yet to be confirmed, but this could present another interesting area given the importance of macrophages in the pathogenesis of disease associated with other coronaviruses, such as feline infectious peritonitis virus (fipv), severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus e (hcov- e) (desforges et al., ; rottier et al., ; shieh et al., ) . the detection and re-isolation of crcov from the lungs indicates the virus can also establish an infection in the lower airways. gross and histopathological analysis showed that both the control and challenged dogs had focal reddening of the lungs at necropsy, however no significant histological abnormalities were associated specifically with this reddening, and such reddening is assumed to be an artefact of barbiturate euthanasia (lopez, ) . nonetheless, in most cases there were microscopic differences between the lungs of these dogs, which could not be appreciated grossly, and therefore the gross appearance of the lungs was not considered an accurate predictor of histopathological changes in these experimental conditions. pulmonary inflammation in crcov challenge dogs was associated with lymphoid aggregates adjacent to the airways or blood vessels. the significance of this is uncertain, but notably these aggregates were not a striking feature of the control dogs. histological changes in the lungs and bronchial lymph nodes of dogs in t tended to be more consistent and mild; whilst scores for dogs in crcov challenge groups, although not marked, were more variable throughout the study. histological changes in the lymphoid tissues of most dogs in the study (t -t inclusive) included hyperplasia. lymphoid hyperplasia is not uncommon in dogs at this age, particularly in the palatine tonsil. acute inflammatory responses were evident; however in some dogs this may have been associated with the presence of fragments of foreign material (e.g. hair) in the tonsillar crypts. where marked lymphoid hyperplasia was seen, this was reflected in the histological scores, which were more variable in the challenge dogs compared to those in the broth control animals throughout the trial period. the close genetic relationship between crcov and bovine coronavirus (bcov) (erles et al., , raised the question as to whether crcov may have an extended tropism which involves the gastrointestinal tract, as seen with some bcov strains (park et al., ) . the molecular detection of crcov in the rectal swabs of some dogs from t is interesting, and consistent with findings from a number of previous reports of dogs naturally infected with crcov (decaro et al., ; yachi and mochizuki, ; mitchell et al., ) . dogs in the t challenge group were unique in that they were the only dogs to be challenged with virus that had not been grown in vitro. the inability to detect crcov from dogs in other challenge groups may therefore be a result of cell culture adaptation; alternatively, this may be a strain specific phenomenon. at present there is no conclusive evidence that crcov displays a true dual tropism for respiratory and enteric tissues. the failure to isolate crcov from enteric samples collected during the peak oropharyngeal shedding period, suggests that crcov may pass through the canine gut as a bystander, without infection. nevertheless, enteric shedding could have implications for managing the spread of disease, and therefore further investigation is warranted. in addition to the histological changes observed in the tissues, significant changes in the leukogram can also be detected in association with crcov infection. this is summarised by a statistically and also clinically relevant lymphocytosis between days and ; most clearly seen in groups t , t , and t . this observation is not unexpected due to the viral antigenic stimulation, and the lymphocytic reactions found in various tissues. what was somewhat less expected is the high number of dogs with decreased neutrophil concentrations, including a number which developed neutropenia, mild left shift and toxicity, best seen in groups t , t , t , and t . these changes suggest an acute inflammatory reaction with a high demand for neutrophils and accelerated production in the marrow. transient neutropenia is not uncommon during infections with some viruses; however, there is currently limited data relating to the leukogram profile following infections with other beta coronaviruses. in one report detailing the experimental infection of cows with bcov, significant reductions in neutrophil concentrations were observed at days post infection, followed by neutrophilia at days post infection (traven et al., ) . in sars coronavirus infected patients the picture is mixed. neutropenia has been reported in some cases, however, in most cases high blood neutrophil concentrations were observed, and this is often associated with a poor clinical outcome manocha et al., ; wong et al., ) . neutrophil infiltration of tissues infected with different coronaviruses such as sars, mhv and rat cov has also been described (bhatt and jacoby, ; iacono et al., ; ding et al., ) ; however, their role in viral clearance and possible immune pathology is largely unknown. considering the presence of mostly lymphoid aggregates in crcov infected tissues in this study, the observed changes are intriguing, and a direct effect of crcov on the myeloid series in the bone marrow cannot be ruled out. seroconversion to crcov and the acquisition of neutralising antibodies to heterologous strains from distinct geographical locations within the usa and uk occurred in all challenge dogs remaining on the study at day . this degree of serological cross reactivity is not unexpected given the high degree of amino acid similarity seen in the spike protein of different isolates published to date. whilst the correlation between neutralising titres and protection in vivo is yet to be determined, this finding supports published data which demonstrated that the presence of crcov specific antibodies in dogs significantly decreased the risk of developing respiratory disease upon entry to the kennel (erles et al., ) . these findings support a possible role for vaccinating against crcov as part of a preventative strategy for respiratory disease in kennelled dogs. moreover, based on the high degree of cross neutralisation and high degree of amino acid identity among the crcov spike proteins described to date, it is anticipated that a single crcov isolate as a vaccine antigen will be sufficient to provide protection against crcov induced respiratory disease. in summary, this is the first study to describe the development of a model for studying crcov pathogenesis; and to fully demonstrate koch's postulates through the successful re-isolation of crcov from experimentally infected dogs with clinical signs of respiratory disease. isolation of crcov was achieved from a wide variety of respiratory and mucosal associated lymphoid tissues, lung lavage fluids and swabs collected over the -week period, thus providing clear evidence of tropism for the canine respiratory tract, accompanied by respiratory shedding. moreover, we have shown crcov infection is associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia alongside inflammatory responses. significant effects on the leukogram, in the form of clinically relevant lymphocytosis and neutrophil changes were also documented. strong serological and cross neutralising reactivity between heterologous crcov isolates demonstrates antigenic homogeneity among crcov from the two continents. this study provides vital evidence supporting a role for crcov in the cird complex. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. canine infectious tracheobronchitis short review: kennel cough canine infectious tracheobronchitis short review: kennel cough sv- -like parainfluenza virus in dogs bordetella and mycoplasma respiratory infections in dogs and cats pathogenesis of canine bordetellosis experimental infection of adult axenic rats with parker's rat coronavirus canine respiratory viruses bronchiolitis obliterans and pneumonia induced in young dogs by experimental adenovirus infection mycoplasmas associated with canine infectious respiratory disease serological and molecular evidence that canine respiratory coronavirus is circulating in italy activation of human monocytes after infection by human coronavirus e the clinical pathology of severe acute respiratory syndrome (sars): a report from association of canine adenovirus (toronto a / ) with an outbreak of laryngotracheitis (''kennel cough''): a preliminary report detection of coronavirus in cases of tracheobronchitis in dogs: a retrospective study from to investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex detection of a group coronavirus in dogs with canine infectious respiratory disease longitudinal study of viruses associated with canine infectious respiratory disease isolation and sequence analysis of canine respiratory coronavirus canine glyceraldehyde- -phosphate dehydrogenase complementary dna: polymerase chain reaction amplification, cloning, partial sequence analysis, and use as loading control in ribonuclease protection assays both spike and background genes contribute to murine coronavirus neurovirulence the prevalence of a group coronavirus in dogs in japan canine tracheobronchitis: isolation and characterization of the agent with experimental reproduction of the disease role of bordetella bronchiseptica in infectious tracheobronchitis in dogs the seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in new zealand a major outbreak of severe acute respiratory syndrome in hong kong respiratory system, thoracic cavity and pleura in thomson's special veterinary pathology severe acute respiratory distress syndrome (sars): a critical care perspective development of a quantitative real-time pcr for the detection of canine respiratory coronavirus dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves the role of a novel coronavirus in canine infectious respiratory disease. royal veterinary college serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus quantification of mrna encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (crcov) acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis survey of dogs in japan for group canine coronavirus infection this study was funded by pfizer animal health. the authors would like to thank m. ikeh for technical assistance. rvc manuscript id number: pid_ . key: cord- -o oauxex authors: fritzen, juliana t.t.; oliveira, marcos v.; lorenzetti, elis; miyabe, flávia m.; viziack, mariana p.; rodrigues, carlos a.; ayres, henderson; alfieri, alice f.; alfieri, amauri a. title: longitudinal surveillance of rotavirus a genotypes circulating in a high milk yield dairy cattle herd after the introduction of a rotavirus vaccine date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: o oauxex worldwide, neonatal diarrhea is one of the most important health issues affecting dairy calves, and rotavirus a (rva) is one of its primary causes. among the measures to mitigate the risk of diarrhea outbreaks, cow vaccination stands out as one of the most important. however, the immune pressure resulting from routine vaccination may be able to select specific g and p genotypes in rva field strains. this study aimed to determine the frequency and intensity of neonatal diarrhea and the incidence of rva and attempted to monitor the g and p genotypes present in the rva strains circulating in a high milk yield cattle herd vaccinated with rva g p[ ] strain. fecal samples (n = ) from holstein heifer calves between – days old that were born from rva-vaccinated cows were collected at different time points, regardless of the presence or absence of diarrhea. the presence of rva in fecal samples was determined by the polyacrylamide gel electrophoresis (page) technique and confirmed by reverse transcription polymerase chain reaction (rt-pcr). g and p amplicons from rva-positive fecal samples from calves of different ages and collections were subjected to nucleotide sequencing. the proportion of the calves and fecal samples that were positive for rva were . % ( / ) and . % ( / ), respectively. using sequence analysis, all rva field strains presented genotype g p[ ]. the protection of g p[ ] vaccination is clear, as this genotype was not detected in this study, and it is known that vaccination against rva reduces the incidence of diarrhea independent of genotype involved. this result demonstrates the importance of epidemiological monitoring of rva genotypes circulating in vaccinated dairy cattle herds to the early detection of new potential pathogenic rva strains. neonatal calf diarrhea (ncd) is the most common cause of calf morbidity and mortality, which can exceed % in certain instances (uetake, ) . the factors that influence the etiology of this syndrome include nutritional and sanitary management, immunological aspects, and infectious agents, such as bacteria, viruses, and protozoa (coura et al., ) . bovine rotavirus a (rva) is one of the most prevalent viral agents associated with ncd in dairy and beef cattle herds worldwide (al mawly et al., ; coura et al., ) . the non-enveloped virion of rva is composed of a triple-layered capsid that surrounds dsrna segments that encode structural (vp -vp , vp , and vp ) and nonstructural (nsp -nsp / ) proteins (greenberg and estes, ). based on their antigenic characteristics and sequence analyses of the vp gene, viruses within the rotavirus genus are primarily classified into distinct groups/species (ictv, ; matthijnssens et al., ; mihalov-kovacs et al., ) ; a newly-proposed species j has been described in the bat . the vp and vp genes encode structural proteins responsible for the induction of the immune protective response and are used for the binary classification of rva strains in terms of their g and p genotypes, respectively. currently, g and p genotypes are recognized by the rotavirus classification working group (rcwg, ) . calves aged to weeks have antibody levels from passive immunity decreasing to a level that is still sufficiently high to block responses to vaccines but is not sufficiently high to combat infection. thus, this period is considered a window of opportunity for the infection of microorganisms and, consequently, the occurrence of disease (chase et al., ) . therefore, the implementation of a cow vaccination program is an important strategy to protect the calves from rva infection and consequent neonatal diarrhea. the commercial vaccines that are available in brazil contain inactivated rva and other infectious agents that are implicated in ncd. there were different rva brazilian vaccine formulations: i) the bivalent vaccine g p[ ] and g p[ ]; ii) and monovalent g p[ ] genotype, which is the most prevalent genotype worldwide (papp et al., ) as well as in brazil (medeiros, ) . the aim of this study was to determine the frequency and intensity of neonatal diarrhea and the incidence of rva and to identify the rva g and p genotypes circulating in dairy calves born from cows that are regularly vaccinated with the rva g p[ ] strain in a high milk yield dairy cattle herd. this study was conducted in a brazilian dairy farm that is operated as a closed dairy cattle herd with holstein cows in lactation. the cows were managed using appropriate nutritional and health practices. this high milk yield dairy cattle herd produces an average of liters/ day/cow. the calves are reared in individual pens on elevated floors. the colostrum intake began shortly after birth and has taken place with adequate frequency and quantity for all newborn calves. sixty or days before calving, the cows were vaccinated against ncd. the commercial vaccine used in this study contained inactivated rotavirus a (uk-compton strain -g p[ ]), inactivated bovine coronavirus (mebus strain), and e. coli (k ) adesine f . the vaccination was performed according to the manufacturer's instructions. the fecal samples from heifer calves born from rva-vaccinated cows were collected at different time points ( , , , , , , , , , and days after birth), regardless of the presence or absence of diarrhea. the samples were collected between march and may , and a total of fecal samples were stored at - °c until analysis. at the time of collection, all of the fecal samples were classified according to their consistency using the following scale: , normal; , pasty; , soft; , watery. a score of or was considered non-diarrheic, and scores of or were considered diarrheic. the fecal samples from the same animal that were collected at different time points were processed at the same time to avoid any bias. the nucleic acid extraction was performed using a combination of the phenol/chloroform/isoamyl alcohol ( : : ) and silica/guanidinium isothiocyanate extraction methods according to alfieri et al. ( ) . the presence of rva dsrna in fecal samples was evaluated using polyacrylamide gel electrophoresis (page) (pereira et al., ) followed by silver staining (herring et al., ) . fecal samples with doubtful page results, as bands with low intensity or in anomalous positions, extra bands or undefined electropherotype were confirmed by reverse transcription polymerase chain reaction (rt-pcr) assay. the presence of rva in the fecal samples that were determined to be positive for rva via page was confirmed by rt-pcr using consensus primers to amplify a bp fragment from the vp gene (gouvea et al., ) and a bp fragment from the vp (vp *) gene (gentsch et al., ; martella et al., ) . the rt-pcr products were analyzed using electrophoresis with % agarose gels stained with ethidium bromide and observed under ultraviolet (uv) light. ten rt-pcr products of good quality from the rva-positive fecal samples from calves of different ages in the range of to days old and collections, including beginning, middle, and end of the experiment, were selected for sequence analysis. the rt-pcr products were purified using an illustra gfx pcr dna and gel band purification kit (ge ® , buckinghamshire, uk), quantified using a qubit ® fluorometer (invitrogen ® life technologies, eugene, or, usa), and sequenced using an abi genetic analyzer sequencer with a bigdye terminator v . cycle sequencing kit (applied biosystems ® , foster city, ca, usa). the phred and cap software packages were used for the nucleotide (nt) quality analysis and contig assembly of the rva sequences, respectively (http://asparagin.cenargen.embrapa.br). sequence similarity searches were performed using the basic local alignment search tool (blast) software (http://blast.ncbi.nlm.nih.gov/), and the genotype identification was performed using the rotac . tool (http://rotac. regatools.be/). phylogenetic trees were obtained using the neighbor-joining method with kimura -parameter model in mega v software. the bootstrapping probabilities were calculated using replicates. the bioedit software, version . . . , was used for the construction of the nt sequence identity matrix. the gene sequences described in the present study have been deposited in the genbank database under accession numbers: mh and mh . seventy-six ( . %) of the heifer calves were rva-positive according to at least of the fecal samples evaluated, and of these, ( . %) calves were determined to have excreted rva on more than one occasion. worldwide, the percentage of calves that are rva-positive varies based on the experimental design used in each study. in scotland and northern england (snodgrass et al., ) and new zealand (al mawly et al., ) , both using commercial enzyme-linked immunosorbent assay (elisa), rates of rva infection of . % ( / ) and . % ( / , ), respectively, were reported. in brazil, also using the page technique, coura et al. ( ) reported that . % ( / ) of dairy heifer calves that were - days old excreted rva during a longitudinal study, and in a transversal study only of ( . %) of dairy calves - days old were found to be rva-positive (alfieri et al., ) . the percentage of rva-positive fecal samples obtained during this study was . % ( / ). other longitudinal studies that have been conducted in brazil to determine the level of rva infection in calves born from vaccinated dairy cows had percentages of rva-positive diarrheic fecal samples that were . % ( / ) (coura et al., ) and . % ( / ) (rocha et al., ) . the distribution of the total samples and the rva-positive fecal samples for each fecal consistency score and time point at sample collection are shown in table . the frequency of diarrhea in calves during the experiment, shown in the table, was not high. fecal samples with scores of and (n = ) represented . % of the total samples, while samples with scores of and , indicating the presence of diarrhea, comprised . % (n = ) and . % (n = ) of the total samples, respectively. in a similar study conducted in brazil on rvaunvaccinated dairy cattle herds, the proportion of diarrheic fecal samples ( . %; / ) within the total number of samples was greater than that in this study (coura et al., ) . previous studies have indicated that the presence of rva infection in feces from diarrheic calves is significantly higher than that in nondiarrheic feces (al mawly et al., ; bartels et al., ; chitambar et al., ) . forty-eight of the ( . %) diarrheic fecal samples evaluated were rva-positive. on average, the frequency of rva-positive samples obtained from diarrheic feces in calves worldwide ranges between . and . % (alfieri et al., ; badaracco et al., ; barreiros et al., ; brito et al., ; buzinaro et al., ; caruzo et al., ; chitambar et al., ; falcone et al., ; malik et al., ; silva et al., ) . in this study, dsrna to from the rva was identified in . % ( / ) of the fecal samples with scores of or and in . % ( / ) and . % ( / ) of the fecal samples with scores of and , respectively. although rva was more frequent in diarrheic calves, the overall frequency of rva diagnosis was lower than that observed in unvaccinated herds (rocha et al., ) . the distribution of the fecal samples according to the age of the calves reveals that during the first and fourth weeks, the frequency of diagnosis of rva was . % ( / ) and . % ( / ), respectively. however, in the second and third weeks, the frequency of rva-positive fecal samples was higher, with the rate for the second week reaching . %. this finding suggests the occurrence of a decrease in passive immunity, corroborating the findings of previous studies that suggest that passive immunity protects calves during their first week and that their own natural resistance is not initiated until weeks of age (alfieri et al., ) ; suggesting the high susceptibility of these animals to disease during the period between the first and fourth week. meganck et al. ( ) and coura et al. ( ) reported that diarrhea caused by rva is most frequent in -to -week-old calves, and alfieri et al. ( ) suggested that animals from dairy cattle herds were most susceptible to rva infection at to weeks of age. in order to reduce the occurrence of neonatal diarrhea, the dairy farm utilizes vaccination with a commercial vaccine containing the rva uk-compton strain with the genotype g p[ ] . this vaccine is frequently used in dairy and beef cattle herds both from brazil and other countries around the world mainly in countries of the northern hemisphere such as canada, the usa, and the european union. to determine the g and p genotypes of rva strains present in the fecal samples collected during this study, rva-positive fecal samples were selected from calves of different ages and collections for nt sequence analysis of the vp and vp amplicons. the analysis enabled the identification of the genotypes, which were g p [ ] in all of the wildtype rva strains with a high ( . to %) nt similarity between them. viruses with the g and p[ ] genotypes are not included in the rva vaccine used by the dairy farm evaluated in this study. the sequence obtained of vp gene of rva had the highest ( %) nt similarity with that of the prototype b strain (genbank accession number: x (xu et al., ) ), which belongs to g lineage iv (fig. a) , and the vp gene had the highest similarity ( . %) to that from the turkish e tr strain (genbank accession number: fj (unpublished data)) of p[ ] lineage iii (fig. b) . a putative vaccine breakthrough associated with heterotypic rva infection in newborn calves was described in turkey, where the emergence of an rva strain with the genotype g p[ ] was reported in a cattle herd vaccinated with the g p[ ] rva strain, which demonstrated a failure of heterologous protection (karayel et al., ) . the occurrence of emerging heterologous rva genotypes is a phenomenon has been previously observed in brazilian dairy and beef cattle herds after the administration of a rv vaccine (medeiros et al., ; rocha et al., ) . similar occurrences have also been described in humans in brazil and other countries around the world (banyai et al., ; cowley et al., ; doro et al., ; khandoker et al., ; santos et al., ) . however, depending on other factors, such as the type of cattle (beef or dairy herds), the method of cow and calf management (intensive or extensive), and the inclusion of vaccination for neonatal diarrhea in the health program, different results can be found between studies. in france, kaplon et al. ( ) evaluated beef cattle herds subject to extensive management and found no differences in the frequencies of diarrhea and rva-positive fecal samples between vaccinated and unvaccinated herds; the majority of the genotyped rva-positive diarrheic fecal samples were found to have the same genotype (g p[ ]) as that of the vaccine strain. among the measures used to mitigate the risk of neonatal diarrhea outbreaks, the vaccination of cow herds appears to be the most important. in humans, the world health organization recommends the inclusion of the rva vaccination in all national immunization programs (who, ) . several studies have demonstrated that vaccination does not totally eliminate the risk of infection, but it certainly reduces both the frequency and severity of the episodes of neonatal diarrhea caused by rva infection in calves (kaplon et al., ; parreno et al., ) . considering that the immune pressure exerted by routine vaccination may have the potential to select strains containing specific g and p genotypes, it is important to monitor the genotypes present in the rva strains that are circulating among and within vaccinated cattle herds (cashman et al., ) . in summary, in this longitudinal study evaluating fecal samples ( calves), the low occurrence of rva in diarrheic fecal samples from calves born from regularly rva g p[ ]-vaccinated cows was determined. in addition, the genotype g p [ ] , present in the rva vaccine strain used on the farm, was not identified in any rva field strains identified in calves in this study. additionally, the presence of the rva g p[ ] genotype was verified, which was different from the vaccine strain, reinforcing the vaccine protection. the presence of rva in vaccinated herds may occurs due to immune pressure, individual factors, management procedures, nutrition, and environmental variations. the constant monitoring of circulating rva strains in neonatal diarrhea in dairy cattle herds is important to identify the emergence of new rva strains, highlighting those with higher virulence and/or zoonotic potential. risk factors for neonatal calf diarrhoea and enteropathogen shedding in new zealand dairy farms frequency of group a rotavirus in diarrhoeic calves in brazilian cattle herds bovine rotavirus strains circulating in beef and dairy herds in argentina from systematic review of regional and temporal trends in global rotavirus strain diversity in the pre rotavirus vaccine era: insights for understanding the impact of rotavirus vaccination programs candidate new rotavirus species in schreiber's bats, serbia g and p genotypes of group a rotavirus from diarrhoeic calves born to cows vaccinated against the ncdv (p[ ],g ) rotavirus strain prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young dutch dairy calves characterization of mixed infections with different strains of bovine rotavirus in an outbreak of diarrhea in dairy herds in goiás, brazil ocorrência dos genótipos g e p de rotavirus do grupo aem bezerros de rebanhos de corte no estado de são paulo molecular characterization of g and p-types bovine rotavirus strains from goias, brazil: high frequency of mixed ptype infections changing profile of the bovine rotavirus g population in the south of ireland from neonatal immune development in the calf and its impact on vaccine response molecular characterization of unusual bovine group a rotavirus g p[ ] strains identified in western india: emergence of p[ ] genotype longitudinal study of salmonella spp., diarrheagenic escherichia coli, rotavirus, and coronavirus isolated from healthy and diarrheic calves in a brazilian dairy herd novel g p[ ] rotavirus strain, northern territory review of global rotavirus strain prevalence data from six years post vaccine licensure surveillance: is there evidence of strain selection from vaccine pressure? determination of bovine rotavirus g and p serotypes in italy by pcr identification of group a rotavirus gene types by polymerase chain reaction polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens rotaviruses: from pathogenesis to vaccination rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels international committee on taxonomy of viruses impact of rotavirus vaccine on rotavirus genotypes and caliciviruses circulating in french cattle putative vaccine breakthrough event associated with heterotypic rotavirus infection in newborn calves molecular epidemiology of rotavirus gastroenteritis in japan during - : characterization of re-emerging g p[ ] after rotavirus vaccine introduction frequency of group a rotavirus with mixed g and p genotypes in bovines: predominance of g genotype and its emergence in combination with g /g types identification of a novel vp genotype carried by a serotype g porcine rotavirus strain vp -sequence-based cutoff values as a criterion for rotavirus species demarcation bovine neonatal diarrhea: epidemiology and molecular characterization of g (vp ) and p (vp ) genotypes of rotavirus a, brazil phylogenetic analysis of a g p[ ] bovine rotavirus strain isolated in a neonatal diarrhea outbreak in a beef cattle herd vaccinated with g p[ ] and g p[ ] genotypes advances in prevention and therapy of neonatal dairy calf diarrhoea: a systematical review with emphasis on colostrum management and fluid therapy candidate new rotavirus species in sheltered dogs review of group a rotavirus strains reported in swine and cattle modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus electrophoretic study of the genome of human rotaviruses from rio de janeiro, são paulo and para, brazil rotavirus classification working group. newly assigned genotypes longitudinal study of bovine rotavirus group a in newborn calves from vaccinated and unvaccinated dairy herds rotavirus genotypes circulating in brazil before and after the national rotavirus vaccine program: a review molecular characterization of group a bovine rotavirus in southeastern and central-western brazil aetiology of diarrhoea in young calves newborn calf welfare: a review focusing on mortality rates world health organisation. rotavirus vaccines: an update sequence of the gene encoding the major neutralization antigen (vp ) of serotype rotavirus the authors thank the following brazilian institutes for financial support: the national council of scientific and technological development (cnpq), the brazilian federal agency for support and evaluation of graduate education (capes), and the araucária foundation (fap/pr). alfieri, a.a. and alfieri, a.f are recipients of cnpq fellowships. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord- -b no yrv authors: davies, robert l; maccorquodale, roslyn; caffrey, bridget title: diversity of avian pasteurella multocida strains based on capsular pcr typing and variation of the ompa and omph outer membrane proteins date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: b no yrv one hundred avian pasteurella multocida isolates recovered from cases of fowl cholera and related infections in england and wales over a -year period were characterised by capsular pcr typing and analysis of outer membrane protein (omp) profiles. sixty-eight percent of the strains were of capsular type a, % were type f, % were type d, % were type b and % were untypable. nineteen distinct omp profiles (omp-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (ompa) and porin (omph) proteins. fifty-six percent of the isolates were represented by omp-types, whereas % of the isolates were associated with four omp-types. the extensive molecular mass heterogeneity of the ompa and omph proteins supports previous findings that avian p. multocida strains are very diverse. furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. the high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of p. multocida are opportunistic pathogens of relatively low virulence. strains of capsular types b, d and f, as well as the untypable isolates, were associated exclusively with specific omp-types and represent distinct and widely disseminated clonal groups. these observations support the view that avian strains of p. multocida have a clonal population structure. based on previous studies, the molecular mass heterogeneity of the ompa and omph proteins might provide a selective advantage to p. multocida by generating antigenic variation. pasteurella multocida is the aetiological agent of fowl cholera, a widely distributed and economically important disease of poultry, particularly chickens, turkeys, ducks and geese rimler and glisson, ) . the organism is also responsible for disease in wild birds, commercially raised game birds and caged birds . four capsular serogroups are recognised among avian strains of p. multocida, namely a, b, d and f rimler, , ; rimler and rhoades, ) . strains of serogroup a are recognised as the primary cause of fowl cholera, whereas isolates of serogroups b, d and f are less frequently associated with disease rimler, , ; wilson et al., ) . in addition, some avian strains of p. multocida are non-encapsulated and are not serogroupable wilson et al., ) . sixteen somatic serotypes ( - ) are also recognised in p. multocida , a and most of these have been demonstrated in avian capsular serogroup a strains . there is considerable evidence, based on a wide range of molecular studies (snipes et al., ; carpenter et al., ; christiansen et al., ; wilson et al., wilson et al., , blackall et al., blackall et al., , gunawardana et al., ; petersen et al., ) , that avian strains of p. multocida are extremely diverse. in particular, a study of the population genetics of australian strains using multilocus enzyme electrophoresis (mlee) identified electrophoretic types among only field isolates (blackall et al., ) . based on dna-dna hybridisation and sugar fermentation patterns p. multocida has been subdivided into three subspecies, subsp. multocida, subsp. gallicida, and subsp. septica (mutters et al., ) and all of these have been isolated from birds (snipes et al., ; hirsh et al., ; fegan et al., ) . however, conflicting results from ribotyping and s rrna sequence data (petersen et al., ) suggest that the precise phylogenetic relationships of strains representing each of these subspecies is complex and has yet to be satisfactorily resolved. control of fowl cholera is primarily by good management practice and vaccination in areas where the disease is endemic (rimler and glisson, ) . both whole-cell bacterins and live vaccines composed of attenuated strains are currently available but neither is entirely satisfactory. bacterins only induce serotype-specific protection, whereas live vaccines sometimes cause disease (bierer and derieux, ; schlink and olson, ; prantner et al., ) and there is increasing interest in the development of subunit vaccines (kasten et al., ; luo et al., ) . outer membrane antigens that might be considered as potential vaccine candidates include the heat-modifiable or ompa and the porin or omph proteins mittal, , ; luo et al., luo et al., , . both of these proteins are expressed in high copy number, are surface exposed and immunogenic (hancock, ; tagawa et al., ; yi and murphy, ; zeng et al., ; neary et al., ) . the omph protein has been shown to be heterogeneous in strains of p. multocida representing somatic serotypes - (luo et al., ) and there is evidence that anti-omph antibodies are protective in chickens (luo et al., ) and mice . there is less information available about the ompa protein of p. multocida, but this protein also exhibits variation in other bacterial species (duim et al., ; webb and cripps, ) . the aim of the study was to investigate capsular and outer membrane protein (omp) diversity among avian p. multocida strains isolated from diseased poultry in england and wales. in particular, heterogeneity of the ompa and omph proteins was examined and used as the basis for an omp classification scheme. since ompa and omph are important surface-exposed components of the outer membrane, analysis of their diversity in avian p. multocida strains will contribute to our understanding of host-pathogen interactions in fowl cholera, including the role of these proteins in immune evasion, and to the development of improved vaccines against this pathogen. one hundred avian field isolates of p. multocida were investigated. these were obtained from regional laboratories of the veterinary laboratories agency (vla) and originated from widespread geographic locations within england and wales over a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the isolates were recovered predominantly from cases of fowl cholera and related acute disease conditions such as septicaemia and pneumonia. however, some isolates were associated with chronic conditions such as conjunctivitis, sinusitis, swollen head, arthritis, etc. properties of the isolates and details of the clinical symptoms of the birds of origin are provided in table . the capsular reference strains x (a), m (b), p (d), p (e) and p (f) were kindly provided by dr. r. rimler, national animal disease center, ames, ia. the isolates were stored at À c in % (v/v) glycerol in brain heart infusion broth (bhib). from À c stock cultures, bacteria were streaked onto blood agar (brain heart infusion agar containing % (v/v) defibrinated sheep's blood) and incubated overnight at c. for preparation of dna, a few colonies were inoculated into ml volumes of bhib and grown overnight at c at rpm. for preparation of outer membranes, . ml of overnight growth in bhib was inoculated into ml volumes of bhib in l erlenmeyer flasks and incubated for h at c at rpm. cells from . ml of overnight cultures were harvested by centrifugation for min at ;  g and washed once in sterile, distilled h o. dna was prepared with the instagene matrix (bio-rad) according to the manufacturers' instructions and stored at À c. the capsular types were determined by multiplex capsular pcr typing with the capsule-specific primer pairs (capa, capb, capd, cape and capf) described by townsend et al. ( ) . isolates that were negative for all five capsular types were confirmed as p. multocida with a p. multocida-specific primer set (kmt t and kmt sp ) (townsend et al., ) in separate pcr reactions (see section ) and classified as untypable. all primers were synthesised by sigma-genosys (cambridge, uk) and the capsular gene fragments were amplified with a taqdna polymerase kit (boehringer mannheim) according to the manufacturers instructions. pcrs were carried out in a geneamp pcr system (applied biosystems) thermal cycler using the following amplification parameters: denaturation at c for s, annealing at c for s and extension at c for min. thirty cycles were performed and a final elongation step of c for min was used. production of pcr amplicons of the expected size was confirmed by electrophoresis in % agarose gels. pooled pcr amplicons of capsular type a, b, d, e and f reference strains were used as standards in each gel. omps were prepared by sarkosyl extraction as previously described (davies et al., ; davies and donachie, ) . protein concentrations were determined by the modified lowry procedure (markwell et al., ) and adjusted to . mg/ml. omps were separated by sds-page in % (w/v) resolving gels (hoefer se electrophoresis apparatus) using the sds discontinuous system of laemmli (laemmli, ) as previously described (davies et al., ; davies and donachie, ) . unless otherwise stated all samples were heated at c for min prior to electrophoresis. twenty micrograms of protein were loaded per lane and the proteins were visualised by staining with coomassie brilliant blue. protein molecular mass standards (pharmacia) consisted of phosphorylase b ( kda), bovine serum albumin ( kda), ovalbumin ( kda), carbonic anhydrase ( kda), trypsin inhibitor ( . kda) and a-lactalbumin ( . kda). the molecular masses of individual proteins were calculated with the labworks tm image acquisition and analysis computer software. the capsular types of the avian p. multocida isolates were determined by capsular pcr typing and typical results are shown in fig. . the distribution of capsular types among the isolates is summarised in table . sixty-eight ( %) isolates were of capsular type a, ( %) were of type f, five ( %) were of type d, four ( %) were of type b and nine ( %) isolates were untypable. capsular type e was not detected among the population sampled. the p. multocida-specific primers were omitted from the capsular primer mixture because they interfered with the capsule-specific primers and resulted in a reduction of capsular pcr product (i.e. reduced band intensity). therefore, isolates that were negative for capsular typing were confirmed as untypable p. multocida in separate pcr assays with the p. multocida-specific primers (townsend et al., ) . microscopic examination of the untypable isolates after indian ink staining indicated that they were non-encapsulated. the stability of the omp profiles was examined by comparing the profiles of two isolates after repeated subculture and at different stages of the growth cycle. the profiles of these isolates were identical after , , and rounds of subculture on blood agar and after , , and h of growth in bhib (results not shown). the omp profiles of the isolates were analysed by sds-page and provisionally assigned to omp-types based on profile similarity (described below). isolates assigned to the same omp-type were subsequently rerun on up to three or four occasions such that isolates of the same omp-type were directly compared on the same gel. an omp classification scheme was devised-based, firstly, on molecular mass variation of the two major proteins, ompa and omph (omp-type , , etc.), and, secondly, on variation of minor protein patterns (omp-type . , . , etc.). the ompa and omph proteins have overlapping molecular mass ranges ( - kda) and were distinguished on the basis of their different behaviours in sds-page gels after heat-treatment. the omph porin protein is tightly associated with peptidoglycan and is not released unless heated at a temperature of approximately c or higher (rosenbusch, ) . therefore, the omph protein does not migrate into the gel unless heated at c or higher prior to sds-page. in contrast, the ompa protein is not associated with peptidoglycan and freely migrates into the gel after heat-treatment at temperatures below c prior to sds-page. however, the ompa protein undergoes a characteristic conformational change when heated at c that results in an increase in its apparent molecular mass in sds-page gels (beher et al., ) . therefore, to identify ompa and omph, one isolate representing each omp-type was subjected to heat-treatment at , , , , and c prior to sds-page. the results for two isolates of omp-types . and . are shown in fig. . the ompa (a) and omph (h) proteins for each omp-type are indicated in fig. and the results are described below. the isolates consisted of major omp groups that were classified as omp-types - based on variation of ompa and omph (described above). based on variation of minor proteins isolates of omp-types , and could be further subdivided into omptypes . - . , . and . , and . - . , respectively. profiles representing each of these omp-types (with the exception of omp-types . and . ) are shown in fig. . the molecular mass of ompa (a) varied from . to . kda and that of omph (h) varied from . to . kda. the distribution of omp-types among the avian isolates is shown in table . isolates of omp-types . ( %), . ( %), . ( %) and . ( %) were the most numerous and accounted for % of the total. a smaller number of isolates, ranging from to , were associated with each of the other omp-types but these accounted for % of the total number of isolates. there was a strong correlation between certain capsular types and specific omp-types (table ). the frequently occurring capsular type a was associated with isolates representing of the omp-types. in contrast, capsular type b was associated exclusively with the four isolates of omp-type . ; these isolates originated from four different regional laboratories. capsular type d was associated with / isolates of omptype . and with the four isolates of omp-type . ; three of the four isolates of omptype . originated from different regional laboratories and the single omp-type . isolate came from a fourth laboratory. capsular type f was associated with the two isolates of omp-type . , with / isolates of omp-type . and with / isolates of omp-type . . all seven of the isolates representing omp-types . - . were untypable, as were / isolates of omp-type . . the seven untypable/omp-type isolates originated from six different regional laboratories; the two omp-type . isolates also came from different laboratories. overall, the majority of omp-types were represented by a single capsular type, but isolates of omp-types . and . were associated with capsular types a and f, and isolates of omp-type . were either untypable or possessed capsular type d. there are a number of difficulties associated with conventional capsular serotyping of p. multocida (chengappa et al., ; rhoades, , ) . however, townsend et al. ( ) described an alternative and highly specific multiplex capsular pcr assay that is based on nucleotide sequence variation within the five capsular biosynthetic loci. this pcr-based capsular typing method was used in the present study and found to be a reliable and rapid method for capsular typing large numbers of p. multocida isolates. reference strains were used as internal standards and no cases of ambiguity occurred. the observed incidence of capsular serotypes in our sample was very similar to that described in the study of isolates by rhoades and rimler ( ) . in the latter investigation, capsular types a, f, b and d were associated with , , and % of isolates, respectively, whereas % of strains were untypable. the significantly higher incidence of serotype a strains with respect to isolates of serotypes b, d and f in this and previous studies wilson et al., ) suggests that the various serotypes differ in their virulence characteristics. although virulence studies have shown that strains of serotypes b, d and f are potentially pathogenic rimler, , b) , there is very little information about the comparative virulence of strains representing the different serotypes. the omp profiles of the avian p. multocida isolates were very diverse. the isolates could be classified into distinct omp-types based on variation of ompa and omph and, to a lesser extent, of the minor proteins. fifty-six percent of the isolates were represented by omp-types, whereas % of the isolates were associated with four omp-types. the high degree of heterogeneity observed in the omp profiles, and of ompa and omph in particular, was not unexpected because previous studies have shown that avian p. multocida strains are extremely diverse (snipes et al., ; christiansen et al., ; wilson et al., wilson et al., , blackall et al., blackall et al., , petersen et al., ) . in particular, blackall et al. ( ) identified electrophoretic types among only p. multocida isolates from australian poultry by mlee. in a previous study of mannheimia haemolytica (davies and donachie, ) , strains were sub-divided into three distinct groups based on their omp profiles and these were subsequently shown to represent phylogenetically distinct lineages by mlee (davies et al., ) . however, no such demarcation was apparent among the omp profiles of the avian p. multocida isolates. omp patterns have been shown to be closely associated with electrophoretic types and clones identified by mlee in other species (achtman et al., ; musser et al., musser et al., , achtman and pluschke, ; kapur et al., ; davies et al., ) . the exclusive association of isolates of the less common capsular types with specific omp-types provided evidence that omp-types mark individual clonal groups of p. multocida (achtman and pluschke, ) . for example, isolates of omp-type . were associated with capsular type f, isolates of omp-type were untypable, isolates of omp-type . were associated with capsular type b and isolates of omp-type . were associated with capsular type d (table ) . furthermore, almost all of the isolates representing each of these groups originated from a different regional laboratory. this is significant because a characteristic feature of clonal bacterial populations is that strains representing the same clone originate from widespread geographic origins (selander and musser, ) . the association of capsular types b and d and certain untypable isolates, with specific omptypes is also important because it demonstrates for the first time that strains of these uncommon capsular types, together with untypable isolates, probably represent specific clones of p. multocida. in contrast, dziva et al. ( ) were unable to demonstrate a relationship between rapd patterns and capsular serogroups in their study of zimbabwean isolates of p. multocida (dziva et al., ) . isolates of omp-types . and . were associated with capsular types a and f. this observation is probably due to the close relationships of these two capsular types (townsend et al., ) . in many pathogenic bacterial species, the majority of cases of infectious disease are often caused by a small proportion of the total number of extant clones (selander and musser, ) . in this respect, avian p. multocida strains differ from many other pathogens because the majority of cases of disease were associated with a relatively large number of omp-types/clones. a possible reason for this is that the isolates were recovered from a diverse range of lesions and tissues and were associated with different types of infection ranging from pneumonia and septicaemia to sinusitis, conjunctivitis and swollen head. high levels of diversity were also observed among eschericha coli strains isolated from chickens with swollen-head syndrome and from birds with colibacillosis (white et al., ) . it was suggested that the large number of clonal genotypes associated with these avian diseases was due either to the opportunistic nature of the infections or to the widespread occurrence of unknown virulence factors (white et al., ; whittam, ) . swollen-head syndrome associated with e. coli is thought to be the result of a secondary infection subsequent to an initial viral infection caused by paramyxovirus, coronavirus, or pneumovirus. the high level of diversity observed among avian p. multocida isolates, together with the wide range of clinical symptoms and tissues of origin, similarly suggests that a high proportion of the isolates might represent opportunistic pathogens of relatively low virulence. in particular, isolates associated with conjunctivitis, sinusitis and swollen head could potentially be secondary pathogens following initial viral infection. confirmation of this hypothesis will require the comparison of bacterial isolates from diseased birds with the normal avian flora. the ompa and omph proteins of avian isolates of p. multocida were shown to be heterogeneous since numerous molecular mass variants were identified (fig. ) . however, the omph protein ( . - . kda) is clearly more heterogeneous than the ompa protein ( . - . kda). comparative nucleotide sequence analysis of the omph proteins representing the somatic serotypes of p. multocida has shown that the molecular mass heterogeneity of this protein is due to variation in the number of amino acids ( - ) in the protein (luo et al., ) . however, most of this variation is confined to two discrete hypervariable regions (amino acids - and - ) which are thought to correspond to external surface-exposed loops (luo et al., ) . similar heterogeneity occurs in the corresponding p (omph) and p (ompa) proteins of haemophilus influenzae, and has also been shown to be due to differences in the size of hypervariable surface-exposed loop regions (forbes et al., ; sikkema and murphy, ; duim et al., ; webb and cripps, ) . these surface-exposed loops are thought to interact with the host immune system and, by undergoing antigenic variation, provide the bacterium with an important defence mechanism murphy, , ; neary et al., ) . furthering knowledge of the molecular basis of this diversity in p. multocida will lead to a better understanding of the role of these proteins in avian disease and contribute to the development of improved vaccines. in summary, this investigation of capsule and omp variation has confirmed the view that avian p. multocida isolates are very diverse. a possible explanation for the high level of strain diversity observed in the study is that many of the isolates were associated with chronic infections, were recovered from a wide range of lesions and tissues, and represent opportunistic pathogens. the association of certain capsular types with specific omp-types suggests that omp profiles mark individual clones of p. multocida. in particular, isolates of the uncommon capsular types b and d and certain untypable isolates, represent distinct clonal groups. the ompa and omph proteins exhibit extensive molecular mass heterogeneity that might provide a selective advantage to the pathogen by generating antigenic variation. clonal analysis of descent and virulence among selected escherichia coli six widespread bacterial clones among escherichia coli k isolates major heat-modifiable outer membrane protein in gram-negative bacteria: comparison with the ompa protein of escherichia coli immunologic response to turkey poults of various ages to an avirulent pasteurella multocida vaccine in the drinking water characterisation of pasteurella multocida isolated from fowl cholera outbreaks on turkey farms population structure and diversity of avian isolates of pasteurella multocida from australia molecular epidemiology of pasteurella multocida in turkeys identification of type d pasteurella multocida by counterimmunoelectrophoresis transmission of pasteurella multocida on california turkey premises in - intra-specific diversity and host specificity within pasteurella haemolytica based on variation of capsular polysaccharide, lipopolysaccharide and outer-membrane proteins outer membrane protein and lipopolysaccharide variation in pasteurella haemolytica a under different growth conditions evolutionary genetics of pasteurella haemolytica isolates recovered from cattle and sheep molecular variation in the major outer membrane protein p gene of nonencapsulated haemophilus influenzae during chronic infections random amplification of polymorphic dna and phenotypic typing of zimbabwean isolates of pasteurella multocida phenotypic characterisation of pasteurella multocida isolates from australian poultry variation in length and sequence of porin (ompp ) alleles of non-capsulate haemophilus influenzae molecular characterisation of avian pasteurella multocida isolates from australia and vietnam by rep-pcr and pfge bacterial outer membranes: evolving concepts serum resistance is correlated with encapsulation of avian strains of pasteurella multocida characteristics of pasteurella multocida isolated from waterfowl and associated avian species in california outer membrane protein patterns mark clones of escherichia coli o and o strains that cause avian septicemia pasteurella multocida produces a protein with homology to the p outer membrane protein of haemophilus influenzae cleavage of structural proteins during the assembly of the head of bacteriophage t cloning and characterization of the major outer membrane protein gene (omph) of pasteurella multocida x- sequence analysis of pasteurella multocida major outer membrane protein (omph) and application of synthetic peptides in vaccination of chickens against homologous strain challenge a modification of the lowry procedure to simplify protein determination in membrane and lipoprotein samples a population genetic framework for the study of invasive diseases caused by serotype b strains of haemophilus influenzae clonal population structure of encapsulated haemophilus influenzae reclassification of the genus pasteurella trevisan on the basis of deoxyribonucleic acid homology, with proposals for the new species pasteurella dagmatis, pasteurella canis, pasteurella stomatis, pasteurella anatis, and pasteurella langaa antibodies to loop of the p porin protein of nontypeable haemophilus influenzae are bactericidal against multiple strains genetic diversity of pasteurella multocida fowl cholera isolates as demonstrated by ribotyping and s rrna and partial atpd sequence comparisons the pathogenesis of pasteurella multocida serotype a: , infection in turkeys: a comparison of two vaccine strains and a field isolate capsular groups of pasteurella multocida isolated from avian hosts virulence of avian capsular serogroup b pasteurella multocida for turkey poults fowl cholera somatic serotypes of pasteurella multocida strains isolated from avian hosts ( - ) virulence and toxigenicity of capsular serogroup d pasteurella multocida strains isolated from avian hosts fowl cholera serogroup f, a new capsule serogroup of pasteurella multocida pasteurella multocida characterization of the major envelope protein from escherichia coli. regular arrangement on the peptidoglycan and unusual dodecyl sulphate binding vaccination of turkey breeder hens and toms for fowl cholera with cu strain population genetics of bacterial pathogenesis molecular analysis of the p porin protein of nontypeable haemophilus influenzae use of an rrna probe and restriction endonuclease analysis to fingerprint pasteurella multocida isolated from turkeys and wildlife characterization of a heat-modifiable outer membrane protein of haemophilus somnus genetic organization of pasteurella multocida cap loci and development of a multiplex capsular pcr typing system characterization of an outer membrane protein of pasteurella multocida belonging to the ompa family role of outer membrane protein h (omph)-and ompa-specific monoclonal antibodies from hybridoma tumors in protection of mice against pasteurella multocida the kda major outer-membrane protein of pasteurella multocida capsular serotype d secondary structure and molecular analysis of interstrain variability in the p outer-membrane protein of non-typable haemophilus influenzae isolated from diverse anatomical sites genetic relationships among strains of avian escherichia coli associated with swollen-head syndrome genetic population structure and pathogenicity in enteric bacteria comparison of dna fingerprinting and serotyping for identification of avian pasteurella multocida isolates pasteurella multocida isolated from wild birds of north america: a serotype and dna fingerprint study of isolates from to mapping of a strain-specific bactericidal epitope to the surface-exposed loop on the p porin protein of non-typeable haemophilus influenzae importance of an immunodominant surface-exposed loop on outer membrane protein p of nontypeable haemophilus influenzae molecular cloning of the pasteurella haemolytica poma gene and identification of bovine antibodies against poma surface domains this study was supported by a wellcome trust university award to r.l. davies ( /z/ /z). we are extremely grateful to staff of the veterinary laboratories agency (vla) for the provision of isolates, including those at bury st. edmunds for making available strains of their p. multocida collection. key: cord- -btdbv tk authors: xu, xingang; zhang, honglei; zhang, qi; huang, yong; dong, jie; liang, yabing; liu, hung-jen; tong, dewen title: porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: btdbv tk porcine epidemic diarrhea (ped) is an acute and highly contagious enteric disease of swine caused by porcine epidemic diarrhea virus (pedv). the porcine intestinal epithelial cell is the pedv target cell. in this study, we established a porcine intestinal epithelial cell (iec) line which can stably express pedv n protein. we also investigate the subcellular localization and function of pedv n protein by examining its effects on cell growth, cycle progression, interleukin- (il- ) expression, and survival. the results show that the pedv n protein localizes in the endoplasmic reticulum (er), inhibits the iec growth and prolongs s-phase cell cycle. the s-phase is prolonged which is associated with a decrease of cyclin a transcription level and an increase of cyclin a degradation. the iec expressing pedv n protein can express higher levels of il- than control cells. further studies show that pedv n protein induces er stress and activates nf-κb, which is responsible for the up-regulation of il- and bcl- expression. this is the first report to demonstrate that pedv n protein can induce cell cycle prolongation at the s-phase, er stress and up-regulation interleukin- expression. these findings provide novel information on the function of the pedv n protein and are likely to be very useful in understanding the molecular mechanisms responsible for pedv pathogenesis. porcine epidemic diarrhea (ped) is an acute and highly contagious enteric disease in swine characterized by severe enteritis, vomiting, and watery diarrhea and has high mortality in piglets (ducatelle et al., ) . ped is one of the most important causes of economic loss in many swine-raising countries. this is mainly due to its high prevalence compared to the rare incidence of transmissible gastroenteritis (tge) and the asymptomatic characteristics of the rotavirus (rv) infections (carvajal et al., ) . the causative agent of ped is the porcine epidemic diarrhea virus (pedv), which belongs to the family coronaviridae and was first reported in (pensaert and de bouck, ) . pedv is an enveloped virus possessing a single-stranded positive-sense rna genome approximately kb in size with a cap and a polyadenylated tail. the genome comprises a untranslated region (utr), a utr, and at least seven open reading frames (orfs) that encode structural proteins porcine epidemic diarrhea (ped) is an acute and highly contagious enteric disease of swine caused by porcine epidemic diarrhea virus (pedv). the porcine intestinal epithelial cell is the pedv target cell. in this study, we established a porcine intestinal epithelial cell (iec) line which can stably express pedv n protein. we also investigate the subcellular localization and function of pedv n protein by examining its effects on cell growth, cycle progression, interleukin- (il- ) expression, and survival. the results show that the pedv n protein localizes in the endoplasmic reticulum (er), inhibits the iec growth and prolongs s-phase cell cycle. the s-phase is prolonged which is associated with a decrease of cyclin a transcription level and an increase of cyclin a degradation. the iec expressing pedv n protein can express higher levels of il- than control cells. further studies show that pedv n protein induces er stress and activates nf-kb, which is responsible for the upregulation of il- and bcl- expression. this is the first report to demonstrate that pedv n protein can induce cell cycle prolongation at the s-phase, er stress and up-regulation interleukin- expression. these findings provide novel information on the function of the pedv n protein and are likely to be very useful in understanding the molecular mechanisms responsible for pedv pathogenesis. ß elsevier b.v. all rights reserved. [spike (s, , envelope (e, kda), membrane (m, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and nucleocapsid (n, kda)] and three non-structural proteins (replicases a, b, and orf ); these are arranged on the genome in the order -replicase ( a/ b)-s-orf -e-m-n- (song and park, ; kocherhans et al., ; yeo et al., ) . pedv n protein binds to virion rna and provides a structural basis for the helical nucleocapsid. also, it can be used as the target for the accurate and early diagnosis of pedv infection (song and park, ) . it has been suggested that n protein epitopes may be important for induction of cell-mediated immunity (curtis et al., ; saif, ) . to date, no data exists on the subcellular localization of pedv n proteins and its effects on cell growth and cell cycle progression. porcine intestinal epithelial cells (iecs) are the cells targeted by pedv and the epithelial cells in the gut serve as a physical barrier which restricts the movement of components and passage of potentially harmful microorganisms between the lumen and underlying mucosa (schierack et al., ) . in the present study, we demonstrate for the first time that pedv n protein induces endoplasmic reticulum stress and up-regulates nf-kb, bcl- , and interleukin . in addition to above findings, we also uncovered that pedv n protein prolongs the s phase of stage cell cycle. the results have potentially important implications for understanding the molecular mechanisms of pathogenesis for this economically important porcine disease. the pegfp-n eukaryotic expression vector was purchased from clontech (usa) and escherichia coli dh a used for cloning were purchased from tiangen biotech (china). in this study, the pedv shaanxi strain was isolated from intestinal tract contents of pedv infected piglets in shaanxi province of china and n gene of pedv was amplified as described previously (honglei et al., ) . the established swine intestinal epithelial cell lines (iec), which were kindly provided by prof. yan-ming zhang, college of veterinary medicine, northwest a&f university, were cultured as described previously (jing et al., ) . briefly, iec cells were grown in dulbecco's modified eagle medium (dmem) (gibco brl, gaithersburg, md, usa) supplemented with % heat-inactivated new born calf serum (gibco brl), iu of penicillin and mg of streptomycin per ml, at c in a % co atmosphere incubator. mouse monoclonal antibodies against cyclin a, grp , b-actin were purchased from santa cruz biotechnology (santa cruz, inc., ca, usa). mouse anti-gfp monoclonal antibody was purchased from millipore (millipore, temecula, ca, usa). horseradish peroxidase (hrp)-conjugated secondary antibody was purchased from pierce (pierce, rockford, il, usa). the mg proteasome inhibitor was purchased from calbiochem (calbiochem, san diego, ca, usa) and the nuclear staining dye hoechst and er-tracker tm red probe were obtained from invitrogen (invitrogen, carlsbad, ca, usa). the primers used to amplify n gene of pedv were as follows: forward primer (pedv-xhoi), -ccgctcga-gatggcttctgtcagctttca- ( - of cv strain) and reverse primer (pedv-hind iii), -cccaagctt atttcctgtatcgaagat- ( - of cv strain). the restriction sites are underlined. the primers were designed according to the archived pedv cv strain nucleotide sequence (genbank: af . ). the amplified product was cloned into the corresponding sites in the pegfp-n expression vector. the recombinant plasmid was identified by enzyme digestion and dna sequencing. the recombinant plasmid was named as pegfp-n. iec cells were seeded into -well dishes h before being transfected (up to - % confluence). cells were transfected with pegfp-n and pegfp-n control vector using lipofectamine (invitrogen) and maintained (up to - % confluence) in selection media containing mg/ml g for two weeks. when all control cells had evidence of death in the presence of the selection agents, cultures transfected with pegfp-n and pegfp-n were propagated for two further weeks in medium containing mg/ml g . the resulting stably transfected cell lines expressing either gfp or gfp-n fusion proteins were used for subsequent analysis. the stable cell lines expressing gfp-n protein and gfp were seeded respectively into -well dishes at a suitable concentration of cells each well. after incubation at c with % co for h, the culture medium was replaced with fresh medium containing mm mg , and then incubated in a co incubator at c for h. after h incubation, the cells were washed with phosphatebuffered saline (pbs) for twice and incubated with hoechst at c for min. images were viewed after cells washed with pbs for twice by fluorescence microscope (model te , nikon, japan). to examine the expression and subcellular localization of pedv n protein, the cells expressing gfp-n protein or control cells (the cells expressing gfp and untransfected iec cells) were grown on glass bottom dishes ( mm) and washed with hank's balanced salt solution (hbss) and incubated with hoechst at c for min, and then washed twice with hbss. cells were then incubated with er-tracker red probe (invitrogen) at c for min and washed with hbss for twice. images were viewed by laser confocal scanning microscopy (model lsm meta, zeiss, germany). cells were harvested and treated with lysis buffer, equivalent amounts of proteins were loaded and electrophoresed on % sds-page and transferred to pvdf membranes. the membranes were blocked with % nonfat dry milk and then incubated with indicated primary antibodies over night at c, followed by hrp-conjugated secondary antibodies. the signal was detected using enhanced chemiluminescence (ecl) reagents (pierce, rockford, il, usa). the mtt cell proliferation assay was performed to determine the growth properties of cells expressing pedv n protein and control cells as described previously (li, ) . data are presented as a percentage of the control, and results are the mean ae sd of three independent experiments performed in duplicate. the cell cycle was measured by using propidium iodide staining as described previously (xu et al., ; tang et al., ) . briefly, approximately  cells of the stable cell lines and control cells were treated with trypsin, washed with phosphate-buffered saline (pbs) for twice, resuspended in % ethanol and fixed at c for days. after washing with pbs, cell were resuspended in pbs containing mg/ml of rnase a and mg/ml of propidium iodide (pi) and incubated at c for min in the dark. the nuclear dna content was examined by a coulter epics xl flow cytometer (beckman coulter, usa). the procedures of real-time quantitative pcr were described previously (xu et al., ) . briefly, total rna was extracted from cells using trizol agent (invitrogen, california, usa), and mg each rna sample was reversetranscribed using first-strand cdna synthesis kit (invitrogen, california, usa). the expression of genes was quantified using bio-rad iq real time pcr system by means of a quantitative real-time pcr assay (qrt-pcr). the primers for qrt-pcr in this study were shown in table . reactions were carried out in ml volume containing  sybr premix ex taq tm ii (takara, dalian, china), sense and anti-sense primers ( . mm) and target cdna ( ng). the cycling conditions were c for min, followed by cycles of c for s, c for s. a negative control was included in each run and the specificity of amplification reaction was checked by melting curve (tm value) analysis. the individual samples were normalized for genome equivalents using the respective ct value for the porcine b-actin housekeeping gene. the relative quantification of gene expression was analyzed by the two-ddct method as described previously (livak and schmittgen, ) . to determine the alteration of nf-kb activity by gfp-n and gfp proteins in the established cell lines, the level of nf-kb activity was measured using the nf-kb p transam kit (active motif) according to the manufacturer's instructions. briefly, cells nuclear extraction was prepared by using the nuclear extract kit (keygen, nanjing, china) and protein concentrations were measured using the bca protein assay reagent (pierce, rockford, il, usa). lysates ( mg total proteins) were incubated in elisa wells coated with the oligo-nucleotide motif recognized by active p , then detected using a specific antibody against p , followed by a horseradish peroxidase (hrp)-conjugated secondary antibody. the colorimetric reaction was measured at od nm. the stable pedv n protein expressing cells and the control cells were seeded in -well plates at a density of  cells/ml in dmem supplemented with % calf serum and cultured for h. as suggested previously, mg that was discovered to block il- expression was added after h (matsuo et al., ). the culture medium was then collected and centrifuged in a microcentrifuge at  g for min to remove debris, the supernatants were then frozen at À c until analyzed. the concentrations of il- were measured using a swine il- elisa kit according to the manufacturer's instructions (invitrogen). all data were means ae sd from three independent experiments in triplicate. results were analyzed by student's t-test. p value less than . was considered to be statistically significant. western blot analyses show that the cells transfected with pegfp-n -n and pegfp-n plasmid expressed table sequences of primer pairs used for qrt-pcr. forward primer ( - ) reverse primer ( - ) product (bp) accession no. (fig. ) . since the molecular mass of gfp is known to be approximately kda, this is in good agreement with the predicted size of the n protein, viz. approximately - kda. however, no signal was detected from the negative iec control cells. the degradation characteristic of n protein was investigated using fluorescence microscope. the results show that gfp-n proteins were expressed in greater amounts in the cell lines treated with mg than untreated cells, while gfp protein expression has no change in the untreated cells and cells treated with mg (fig. ) . the results show that pedv n protein degradation was mainly via a proteasome pathway rather than a lysosomal one, and that the n protein degradation can be inhibited by mg proteasome inhibitor. the subcellular localization of n protein was investigated by confocal fluorescence microscopy. the results show that gfp-n proteins are localized in the endoplasmic reticulum (er), while the gfp protein was distributed throughout the whole cell (fig. ) . compared with pegfp-n transfected and untransfected control cells, the n protein expressing cells divided much more slowly, leading to a significantly decreased cell number after a certain period of time. cell proliferation of n expressing cells measured by the mtt assay was decreased by approximately % over a time course of h (fig. ) . to investigate whether the growth inhibition of pedv n expressing cells was due to arrest of the cell cycle at a particular phase or phases of the cell cycle progression, flow cytometric analysis was performed based on the dna content in nuclei stained with pi. the proportions of g /g phase, s-phase and g /m phases for the control cells were %, . %, and . %, respectively. for iecs expressing gfp, the proportions of the phases were g /g : . %, s-phase: . %, and g /m: . %. on the other hand, for gfp-nexpressing iec stable cells, the proportions were g /g : . %, s-phase: . %, and g /m: . % (fig. a) . further quantitative analysis of the histograms was performed to determine the percentage of cells in each of the g /g , s, and g /m phases (fig. b) . the g /g phase cells show a n dna content and the g /m phase cells show a n dna content. these data suggest that the pedv n protein prolongs the s-phase cell cycle and that prevents gfp-n-expressing cells from entering the g /m phase. the results show that, relative to control cells, pedv n protein expression causes a significant increase in the proportion of cells in the s-phase accompanied by a decrease in the proportion of cells in the g /g phase. taken together, these results strongly suggest that n protein causes the inhibition of cell growth by prolongation the s-phase cell cycle. cyclin a is a key regulator of the cell cycle progression from the s phase to the g /m phase. to investigate the mechanism of n-induced s phase cell cycle prolongation, we first examined the cyclin a protein levels in transfected and control cells using western blot assay. as shown in fig. a , the cyclin a expression level was significantly down-regulated in cell lines that expressed n protein compared with control cells. this indicates that it is the expression of n, rather than gfp, that prolongs the s phase cell cycle progression. to further support these findings, quantitative real-time rt-pcr was employed. the results show that cyclin a mrna levels in the gfp-n-expressing cell lines were significantly lower than in control cells (fig. b ). this suggests that s-phase prolongation induced by pedv n protein is associated with cyclin a protein degradation and a decrease in cyclin a transcription. to analyze the expression of glucose regulated protein (grp ), we chose a well characterized er chaperone protein that is a marker of er stress (hong, ; lee, ; li et al., ) . we examined the grp protein levels in transfected and control cells by western blot assay. the grp expression level was significantly upregulated in cell lines that expressed n protein compared with control cells (fig. a) . moreover, the grp transcription level detected using real-time pcr assay was also significantly increased in gfp-n-expressing cell line compared with controls (fig. b) . analysis of the activity of nf-kb in gfp-n-expressing cell lines demonstrates that nf-kb was significantly activated compared with control cells (fig. ) . together, these analyses show that expression of pedv n protein results in the up-regulation of grp and nf-kb, which suggests the n protein has a role in er stress activation. secretion of il- in the supernatant liquid from untransfected and transfected cells was examined using elisa assay. as shown in fig. a , gfp-n expressing cells were found to express higher levels of il- compared to control gfp expressing cells and untransfected cells. after treatment with mg , the level of il- in the supernatants from control cells is significantly decreased. in contrast, there was no such change in supernatants from the gfp-n expressing cells. an investigation of the transcriptional levels of il- using real-time quantitative pcr found that the mrna levels of il- in the gfp-n expressing cells is also higher than in the gfp transfected and untransfected cells (fig. b) . these results suggest that pedv n up-regulates il- expression in iecs. il- expression is regulated by activation of nf-kb and, in turn, the activation of nf-kb is associated with er stress during viral infection (hoffmann et al., ; waris et al., ) . thus, these results suggest that pedv n expression results in er stress and nf-kb activation which is responsible for the up-regulation of il- . it is well-known that the anti-apoptotic molecule bcl- is tightly regulated by the transcription factor nf-kb fig. . cell proliferation assays of the stable n protein expressing cells. the mtt assay was used to measure proliferation of  cells from iec cell lines over time. each data set represents the mean ae sd of six replicates. (fahy et al., ) . also, bcl- as an anti-apoptotic molecule which is associated with cell survival (batsi et al., ; ricca et al., ; seo et al., ) . a quantitative real-time rt-pcr was employed and the results show that bcl- expression in the gfp-n expressing cells is higher than in the control cells (fig. ) . the results suggest that pedv n protein may play a very important role in protecting the host cells from functional damage or apoptosis. recent years, many studies were focus on the gene sequence analysis of pedv. however, the subcellular localization and function of the pedv n protein is still unclear. also, the function of this protein is yet to be determined, particularly with regard to its effect on host cell physiological changes. recently, the n protein function of other coronaviruses also has been studied, especially in the n protein of transmissible gastroenteritis virus (tgev) has extensively studied. the n protein of tgev is an rna chaperone and a naked plasmid dna encoding the n protein of tgev can elicit both humoral and cell-mediated immune (cmi) responses (liu et al., ; zú ñ iga et al., ) . previous study suggested a correlation among the replication cycle of sars-cov, subcellular localization of n, induction of apoptosis, and the subsequent activation of caspases leading to cleavage of the n protein of sars-cov (diemer et al., ) . in this study, we constructed a eukaryotic expression vector and generated stably expressing cell lines of pedv n in fusion with the gfp protein that allowed the analysis of many of these properties. colocalization studies clearly showed that gfp-n localized in the er. it has been reported that the n proteins of mhv and ibv localized in the nucleolus of a small number of infected cells (wurm et al., ) . tgev n protein was not observed in the nucleolus of infected st cell lines (calvo, ) . in the case of our study, pedv n protein was not observed in the nucleolus of the cells by confocal microscopy. viruses manipulate progression through the cell cycle and alter checkpoint signaling in order to provide a favorable environment for their own replication (liu et al., ; chulu et al., ) . recently, it was reported that the sars-cov n protein induced cell cycle arrest in the s phase through down-regulation of the s phase gene products expression (surjit, ) . one of the most critical phases during cell cycle progression is the s phase, since it can provide a cellular environment that is beneficial for viral replication. in this study, our findings show that the n protein of pedv is able to inhibit the cell proliferation and prolongs the s-phase cell cycle. cyclin a is very important in cells from the s phase to g /m phase. in this study, the results of a western blot analysis show that cyclin a protein levels in pedv n protein-expressing cell lines are significantly lower than in control cell lines. furthermore, we also find that pedv n protein significantly inhibits the transcription of cyclin a. these results show that the pedv n protein plays an important role, not only in the cyclin a protein expression levels, but also in the transcription of cyclin a. our observations show that the pedv n protein is likely to be responsible for inducing er stress. we have shown that pedv n protein localizes in the er and is able to induce er stress. this was indicated by the significant upregulation of the molecule chaperon grp , a typical marker of er stress. under conditions of er stress, mammalian cells accelerate the retrograde export of proteins from the er to the cytosol for ubiquitylation and proteasome-mediated degradation (friedlander et al., ; rao and bredesen, ; schrö der and kaufman, ) . thus, we speculate that the pedv n-expressing cells accelerate the retrograde export of cyclin a proteins from the er to the cytosol for ubiquitylation and proteasomemediated degradation. the er has essential roles in multiple cellular processes that are required for normal cellular functions and cell survival (anelli and sitia, ) . viruses use the er as an integral part of their replication strategy and must therefore contend with the er stress response and downstream consequences of er stress signaling. this includes initiation of an inflammatory response through activation of nf-kb (todd et al., ; waris et al., ; zhang and kaufman, ) . il- as a pro-inflammatory neutrophil chemotactic factor plays an important role in the promotion of cell survival signaling and antagonizes the anti-viral activities of interferon. our results show that pedv n is able to up-regulate the expression of il- in iec cells. pedv n protein induces er stress and significantly activates nf-kb, which consequently leads to promotion of il- transcription. further research suggested that il- production in control cells treated with mg was significantly lower than in untreated cells. however, il- production from the gfp-n expressing cells treated with mg was not changed significantly compared with the untreated cells. taken together, our findings suggest that mg is capable of inhibiting il- production in control cells and pedv n protein seems to antagonize the function of mg . in addition, nf-kb is a transcription factor that controls the expression of a variety of genes involved, not only in innate and adaptive immunity, but also in cell survival (geng et al., ; li and verma, ; wietek and o'neill, ) . as is known, bcl- is an anti-apoptotic molecule which is associated with cell survival (batsi et al., ; ricca et al., ; seo et al., ) . previous study suggests that the expression of bcl- is regulated by the nf-kb (fahy et al., ) . in this study, the anti-apoptotic molecule bcl- was significantly elevated in pedv n protein expressing cells. therefore, pedv n protein may play an important role in protecting the host cells from morphological and functional damage or apoptosis. in conclusion, pedv n protein is localized in the er. it inhibits iec growth and prolongs the s-phase cell cycle. sphase prolongation is associated with an increase in cyclin a degradation and a decrease in cyclin a transcription level. the pedv n protein is able to up-regulate il- expression in iecs. the up-regulation of il- and bcl- expression is ascribed to the er stress response and activation of nf-kb induced by pedv n. thus, the data suggest that pedv n likely plays an important role in the inflammatory response and in persistent pedv infection. this study has uncovered some novel features of the function of the pedv n protein which are likely to be very useful in understanding the molecular mechanisms of pedv pathogenesis. xingang xu and honglei zhang performed the majority of experiments and involved in manuscript preparation, qi zhang and yong huang participated in editing of the manuscript. jie dong and yabing liang participated part of the experiments. dewen tong and hung-jen liu conceived of the study, participate in its design and coordination, and revised the manuscript. all authors read and approved the final manuscript. there is no conflict of interest of any authors in relation to the submission. protein quality control in the early secretory pathway bcl- blocks -methoxyestradiol induced leukemia cell apoptosis by a p kip -dependent g /s cell cycle arrest in conjunction with nf-kb activation phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies avian reovirus p regulates g /m cell cycle arrest and shutoff cellular translation involves activation of p -dependent pathways heterologous gene expression from transmissible gastroenteritis virus replicon particles cell type-specific cleavage of nucleocapsid protein by effector caspases during sars coronavirus infection in vivo morphogenesis of a new porcine enteric coronavirus, cv targeting bcl- overexpression in various human malignancies through nf-kb inhibition by the proteasome inhibitor bortezomib a regulatory link between er-associated protein degradation and the unfolded-protein response phosphorylation of nf-kb p at ser controls its commd -dependent ubiquitination and target gene-specific proteasomal elimination multiple control of interleukin- gene expression transcriptional regulation of the grp promoter by endoplasmic reticulum stress: role of tfii-i and its tyrosine phosphorylation rt-pcr identification of porcine epidemic diarrhea virus and sequence analysis of its m, n and e gene the isolation and identification of neonatal swine intestinal epithelial cells completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence the er chaperone and signaling regulator grp /bip as a monitor of endoplasmic reticulum stress the unfolded protein response regulator grp /bip is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells nf-kb regulation in the immune system swainsonine activates mitochondria-mediated apoptotic pathway in human lung cancer a cells and retards the growth of lung cancer xenografts retardation of cell growth by avian reovirus p through the activation of p pathway dna mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus analysis of relative gene expression data using real-time quantitative pcr and the Àddct method proteasome inhibitor mg inhibits angiogenesis in pancreatic cancer by blocking nf-kb activity a new coronavirus-like particle associated with diarrhea in swine misfolded proteins, endoplasmic reticulum stress and neurodegeneration bcl- over-expression enhances nf-kappab activity and induces mmp- transcription in human mcf (adr) breast-cancer cells coronavirus immunogens characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine er stress and the unfolded protein response inhibitory heterotrimeric gtp-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of bcl- via nf-kb in h human lung cancer cells the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines classic swine fever virus ns protein leads to the induction of cell cycle arrest at s-phase and endoplasmic reticulum stress the endoplasmic reticulum stress response in immunity and autoimmunity endoplasmic reticulum (er) stress: hepatitis c virus induces an er-nucleus signal transduction pathway and activates nf-kappab and stat- diversity and regulation in the nf-kb system localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin- expression cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus chinju coronavirus nucleocapsid protein is an rna chaperone from endoplasmic-reticulum stress to the inflammatory response this work was supported by grants from the basic research and operating expenses of northwest a&f university (grant nos. qn and z ), the international science and technology cooperation fund of northwest a&f university (grant no. a ), and the ministry of education, taiwan, r.o.c. under the atu plan. key: cord- - p gnpgf authors: zang, yue; tian, ye; li, yungang; xue, ruixue; hu, liping; zhang, dong; sun, shengfu; wang, guisheng; chen, jing; lan, zouran; lin, shaoli; jiang, shijin title: recombinant lactobacillus acidophilus expressing s( ) and s( ) domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: p gnpgf porcine epidemic diarrhea (ped) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (pedv), which is characterized by a high mortality rate in piglets. since , a remarkable growth in ped outbreaks occurred in many pig farms in china, landing a heavy blow on the pig industry. in order to develop a new effective vaccine for the current pedv, oral vaccines were generated by transferring eukaryotic expression recombinant plasmids carrying the s( ) and s( ) (antigenic sites of the s protein) epitopes of pedv into a swine-origin lactobacillus acidophilus (l. acidophilus). after oral immunization of the balb/c mice, higher levels of anti-pedv specific igg and siga antibodies and cellular immune responses were detected in mice orally administered with the recombinant l. acidophilus-s( ) compared to the l. acidophilus-s( ). furthermore, l. acidophilus-s( ) was used to inoculate the pregnant sows orally and the results showed that the recombinant l. acidophilus-s( ) could elicit a specific systemic and mucosal immune response. in summary, our study demonstrated that oral immunization with l. acidophilus-s( ) could improve the humoral and mucosal immune levels in sows and would be a promising candidate vaccine against pedv infection in piglets. porcine epidemic diarrhea (ped), caused by ped virus (pedv), is an acute enteric infectious disease characterized by severe vomiting, diarrhea, and dehydration (debouck and pensaert, ; pensaert and de bouck, ) . the spike (s) protein of pedv is a type transmembrane envelope glycoprotein, which is responsible for the virus invading host cells through membrane fusion and mediating the production of neutralizing antibodies in infected hosts (lee et al., ) . based on the homology of the coronavirus s protein, it can be divided into two domains: s ( - aa) and s ( - aa) (sun et al., ; follis et al., ) . there are four neutralizing epitope domains in the pedv s protein: coe ( - aa), ss ( - aa), and ss ( - aa) are located in the s domain, and c ( - aa) is in the s domain (chang et al., ; cruz et al., ; j o u r n a l p r e -p r o o f et al., ) . s is responsible for binding receptors and s is responsible for membrane fusion (gallagher et al., ; song et al., ; oh et al., ) . since , outbreaks of ped have significantly increased in china, and the mortality rate of suckling piglets is as high as %- %, which seriously hinders the healthy development of the pig industry (zhang et al., ) . due to immature immune system development in newborn piglets, vaccination with traditional vaccines does not produce protection in time, resulting in immune failure zhao et al., ) . to prevent intestinal infectious diseases in newborn piglets, an ideal strategy is for maternal antibodies to be produced in immunized pregnant sows and passively transferred to suckling piglets via colostrum . gut-associated lymphoid tissues (galt) are distributed in the pig intestine. the immunity induced at mucosal sites in the pregnant sow and passively transferred to suckling piglets via colostrum and milk (lactogenic immunity) is crucial for immediate protection of neonates against enteric infections. as recognized in previous studies of passive immunity to transmissible gastroenteritis virus (tgev), sows that recovered from tgev infection protected their exposed litters against tgev. this protection was associated with high levels of antibodies in the milk (lactogenic immunity), but not in the serum of the sows bohl and saif, ; saif et al., ; saif, ) . oral immunization can induce more iga-plasmablasts and t cells in the intestine. in addition to localized intestinal tracts, some of these cells migrate to the breast via the "intestinal-mammary-siga axis" to produce more siga and cytokinesin milk (song et al., ) . the presence of gastric acids and proteases in the gastrointestinal tract of animals affects the effectiveness of oral vaccines. in recent years, many scholars have studied how to improve the effect of oral immunization, and the application of antigen-presenting carriers is an effective strategy. as an important predominant bacterium in intestine, lactobacillus acidophilus (l. acidophilus) can not only effectively antagonize pathogenic bacteria in the digestive tract and maintain the intestinal microecological balance, but also be used as an antigen-presenting carrier in j o u r n a l p r e -p r o o f practical production. l. acidophilus is the candidate with the greatest potential for oral vaccines with a series of advantages (yu et al., ) . in a previous report, tetanus toxin was successfully constructed on a lactobacillus expression vector to be expressed as an immune antigen to induce the immune system to produce specific igg and siga antibodies in mice inoculated by nasal cavity (grangette et al., ) . in china, scientists have compared the effectiveness of lactobacillus carrier vaccines on piglets by using both oral and injection immunization routes. the results showed that oral administration of the vp gene of foot-and-mouth disease virus using lactobacillus as a carrier could produce a higher antibody titer than injection, and induce humoral and cellular immunity (li et al., ) . in this study, eukaryotic recombinant expression plasmids prc/cmv -s -rep. and prc/cmv -s -rep. carrying the s and s epitopes of pedv and replication gene rep. of l. acidophilus were constructed and transformed into swine-origin l. acidophilus. subsequently, balb/c mice and pregnant sows were orally immunized with the recombinant l. acidophilus. the results showed that the recombinant l. acidophilus vaccine prc/cmv -s -rep. could induce higher levels of humoral and mucosal immune response. the plasmid prc/cmv (sigma, germany) is a . kb vector designed for high-level stable (zhang et al., ) . the pcr products were identified and purified by gel electrophoresis. purified s and s genes and plasmid prc/cmv were subjected to a hind iii and not i double digestion separately and ligated by t dna ligase (tiangen, beijing, china). the recombinants prc/cmv -s and prc/cmv -s were sequenced and analyzed. the plasmid of pgem-t-rep. (shandong provincial center for animal disease control and prevention, china) carrying replication gene rep. of l. acidophilus (li et al., ) and two recombinants were then subjected to a not i and apa i double digestion separately and ligated by t dna ligase. the resulting recombinants prc/cmv -s -rep. and prc/cmv -s -rep. were then sequenced and analyzed. for detection of the displayed s and s of recombinant plasmids, ifa was used as described previously (xue et al., ) . in brief, the bhk- cells were transfected with the recombinant plasmids prc/cmv -s -rep. and prc/cmv -s -rep. , as well as the negative control plasmid prc/cmv with vigofect transfection reagent (vigorous biotechnology, beijing, china), respectively. after incubation for h, the ifa was conducted with the swine pedv-positive serum al., ) . in brief, l. acidophilus sw colony was inoculated into ml of mrs broth (hopebiol, qingdao, china) for static cultivation at °c with % co for h and then further cultured for - h until the same situation was obtained (od . - . ). cells (od . - . , incubation for - . h) were chilled on ice for min and washed twice with ice-cold epwb ( mm sodium, mm mgcl , ph . ). a total of μl of plasmid dna (prc/cmv -s -rep. or prc/cmv -s -rep. , ng/μl) was mixed with μl of the ice-cold cell suspension in a . cm cuvette on ice for min. bio-rad gene pulser (bio-rad laboratories, richmond, ca) was used for electroporation. meanwhile, the non-electroporated bacterial cells were set as a negative control. positive clones were confirmed with primers ls (f and r) and ls (f and r) by nucleotide sequencing. to detect the stability of the two recombinant plasmids in l. acidophilus sw , the recombinant l. acidophilus-prc/cmv -s -rep. (l. acidophilus-s ) and l. acidophilus-prc/cmv -s -rep. (l. acidophilus-s ) were passaged five times in mrs broth without any antibiotics, and all the generations were detected by pcr with primers ls and ls respectively. spedv fragment concluding the coe ( - aa) and c ( - aa) epitopes, based on the nucleotide sequence of spike protein of the strain (genbank acc. no. jq ), was synthesized after codon-optimization by genscript (piscataway, nj, usa). spedv was cloned into the pet- a (qiagen, germany) plasmid dna vector, and then positive recombinant plasmids were transformed into escherichia coli (e. coli) bl (tiangen, beijing, china) for protein expression. briefly, the positive bl colony containing recombinant expression vector pet- a-spedv was cultured in lb broth containing μg/ml of kanamycin for - h at °c until the od reached . . iptg was then added to a final concentration of mmol/l to induce the expression at °c for h. recombinant protein was purified as described previously (xue et al., ) . after sonication, the lysate was centrifuged at rpm for min. the supernatant and precipitate were collected and j o u r n a l p r e -p r o o f submitted to sds-page and western blot as described previously (zhang et al., ) . sds-page was conducted on pre-made % polyacrylamide mini-gel run in a mini-protean electrophoresis system (bio-rad, ca, usa). the expressed soluble protein was purified according to the procedure of the his·bind® purification kit (novagen, usa) and determined according to the manufacturer's instructions of the bca protein assay kit (beyotime, shanghai, china). western blotting was conducted with swine pedv-positive serum as the first antibody and hrp-conjugated goat anti-swine igg antibody (biodee, beijing, china) as the second antibody. to detect the igg and siga antibodies specific to spedv epitope, indirect elisa methods were developed referring to published protocols with minor modifications (liu et al., ) . briefly, the optimal concentrations of serum and coating antigen were determined by a checkerboard titration; -well microtiter plates coated with two-fold diluted spedv protein antigen (from μg/well to . μg/well, μl/well) were incubated at °c for h and then °c overnight in carbonate buffer ( mm nahco , mm na co , ph . ). after washing, the wells were incubated with two-fold diluted mouse/pig pedv positive sera and negative sera (from : to : , μl/well) at °c for h. a total of μl hrp-conjugated goat anti-mouse (pig) igg (iga)-specific antibodies (biodee, beijing, china) was added and incubated at °c for h after washing with pbst. o-phenylenediamine dihydrochloride substrate ( μl/well) (biodee, beijing, china) was added and further incubated for min. the reaction was terminated with stop solution ( m h so , μl/well) and the optical density (od) was read at nm. the optimum sera titer and concentration of coating antigen were established with the chessboard test. a total of six-week-old female balb/c mice (experimental animal center of shandong university, china) were randomly separated into groups, and ones in every group. the first group was orally immunized with cfu recombinant l. acidophilus-prc/cmv -s -rep. (l. acidophilus-s ) ( μl of the suspension). the second group was orally immunized with cfu j o u r n a l p r e -p r o o f recombinant l. acidophilus-prc/cmv -s -rep. (l. acidophilus-s ) ( μl of the suspension). the third group were subcutaneously immunized with μl pedv commercial inactivated vaccine (lanzhou pharmaceutical factory of biology, china). the last group was orally administered with μl sterile phosphate buffer saline (pbs). the induction of antigen specific serum igg and siga levels were measured by elisa. the assay procedures were the same as described in the indirect elisa section. all mice were boosted at weeks post first immunization by the same strategy. sera for the detection of pedv specific antibody were collected via tail-bleeding at , , , , , and days post immunization (dpi) and stored at − °c until used. fecal samples used for detecting siga antibodies were collected in the same periods and treated according to methods described previously with slight modification (liu et al., ) . cytokines ifn-γ and il- levels were measured by elisa according to the manufacturer's instructions (r&d systems, shanghai, china) . data were acquired on an automated elisa plate reader at od nm immediately. the most effective vaccination group was used to test protective efficacy in the pig vaccination. pedv-seronegative, crossbred, pregnant sows were obtained from a local farm and all the sows were confirmed to be negative for pedv, from both pathogenic and serological tests by pcr (vipotion, guangzhou, china) and elisa (boyang, guangzhou, china), respectively. nine sows were randomly divided into three groups ( ones in every group) and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. the first group was orally dosed with ml of l. acidophilus-s ( × cfu/ml). the control groups of sows were orally dosed with ml of pbs and subcutaneously immunized with an equal volume of pedv commercial inactivated vaccine. all pigs were first immunized at dpi and received booster immunization at dpi under the same conditions. sera samples were collected at , , , , , and dpi. farrowing was induced at days after the last vaccination. colostrum samples were collected on the day of farrowing and treated as described previously (adams and marteau, ) . the levels of anti-pedv specific igg antibody in the serum and siga antibody in the colostrum were determined by indirect elisa. piglets were allowed to suckle their dams and their sera samples collected on the th day after birth were tested by anti-pedv igg elisa. all of the data were analyzed a paired samples t-test in spss software. comparison of antibody titers and cytokine levels at each time point were conducted by a paired t-test and the values are presented as mean ± standard deviation (sd), with p < . and p < . considered as statistically significant and highly significant, respectively. all animal experiments were carried out in accordance with guidelines issued by the shandong agricultural university animal care and use committee (approval number, sdaua- - ). the prc/cmv -s and prc/cmv -s plasmids were obtained by amplifying and sub-cloning the s and s fragments into the prc/cmv vector, and the replication gene rep. of l. acidophilus from pgem-t-rep. was then subcloned into prc/cmv -s and prc/cmv -s , resulting in prc/cmv -s -rep. and prc/cmv -s -rep. . the expression levels of recombinants were assessed in vitro by ifa. the results showed that positive fluorescence signals were observed in the cytoplasm under confocal microscope, while no fluorescence was observed in the mock cells, indicating that the s and s epitopes were expressed successfully in bhk- cells ( figure a ). recombinant plasmids prc/cmv -s -rep. and prc/cmv -s -rep. were transformed into swine-origin l. acidophilus sw by electroporation assay. to determine whether the bacterial j o u r n a l p r e -p r o o f strains were carrying the recombinant plasmids after electroporation assay, the specific primers of s and s genes were used for colony identification. in order to test if prc/cmv -s -rep. and prc/cmv -s -rep. could be carried in recombinant l. acidophilus steadily, the positive l. acidophilus were cultured for five generations and analyzed through pcr, and the genes were detected as expected (data not shown). the bp and bp bands were observed by agarose gel electrophoresis and the results showed that recombinant plasmids from all five passages were carried in recombinant l. acidophilus steadily without any antibiotics in the media. to establish spedv-based indirect elisa, the protein was expressed and purified in bl (de ) figure , the expression of the spedv epitope protein was assessed via sds-page; a band with the expected molecular mass of . kda was observed upon staining with coomassie brilliant blue (figure e ). the spedv protein was purified successfully with the his·bind purification kit (figure f) , and the concentration of the purified protein was . mg/ml referring to bca protein assay. the purified spedv protein was further identified by western blot using pedv positive swine serum (figure g) . a checkerboard titration was used to determine the optimal dilutions of antigen and serum. the optimal antigen concentration and serum sample dilution were set at . μg/well and : , respectively. the levels of anti-pedv igg antibodies in mice induced by the recombinant l. acidophilus were determined by indirect elisa. compared to the pbs mock group, from the th day, significant levels of anti-pedv igg antibody (p < . ) were induced in mice that were orally administered l. acidophilus-s and l. acidophilus-s groups, and subcutaneously immunized with commercial inactivated vaccine group (figure a) . moreover, the igg antibody levels of the commercial inactivated vaccine group were obviously higher (p < . ) than those of oral vaccine groups between dpi to dpi, and the igg antibody level of the l. acidophilus-s group were higher (p < . ) than that of the l. acidophilus-s group. notably, compared to the pbs mock group and commercial inactivated vaccine group, the mucosal siga levels increased significantly (p < . ) after first immunization with pedv l. acidophilus-s and l. acidophilus-s vaccines ( figure b ). the pedv specific siga level of the l. acidophilus-s group was higher (p < . ) than that of the l. acidophilus-s group after dpi, suggesting the l. acidophilus-s vaccine could efficiently induce mucosal immunity in mice. these results indicated that the oral recombinant l. acidophilus vaccines induced both humoral and mucosal immunity, and the oral recombinant vaccines induced much greater mucosal immunity and significantly less humoral immunity than that of the commercial inactivated vaccine (figure ). the serum ifn-γ and il- levels of the three vaccine immunized groups began to increase after immunization and reached the peak at dpi (table and table ). the serum ifn-γ level of the three vaccine groups was obviously higher than that of the pbs mock group (p < . ) between dpi and dpi (table , figure s ), while the il- level of the two oral vaccine groups was obviously higher than that of the pbs mock group (p < . ) between dpi and dpi (table ) . though the serum ifn-γ and il- levels of the l. acidophilus-s group were higher than those of the l. acidophilus-s group during the whole test period, the differences between the two orally immunized groups were not significant (p > . ). the serum ifn-γ and il- levels of the commercial inactivated vaccine group was obviously higher than those of the oral vaccine groups between dpi to dpi (p < . ), but the differences between the two oral vaccine groups and the inactivated vaccine group were not significant (p > . ) at dpi to dpi. to determine the immunogenicity of the recombinant l. acidophilus-s , pregnant sows were used for the program and the humoral immune responses were examined by indirect elisa. the l. acidophilus-s and commercial inactivated vaccine groups both showed growth in specific serum igg after the first and second vaccination (figure ) , whereas no increasing trend was noticed in the control sow (p < . ). furthermore, in contrast to the l. acidophilus-s group, the commercial inactivated vaccine group demonstrated obviously higher serum igg levels at dpi (p < . ). piglets that regularly suckle the immune mother receive colostrum/milk antibody, a process that transfers passive immunity to the piglets (makadiya et al., ) . therefore, we tested colostrum samples collected from the sows on the day of farrowing for the presence of pedv specific siga antibodies. the levels of siga antibodies were obviously higher in colostrum of the recombinant l. acidophilus-s group compared to the control and commercial inactivated vaccine groups (p < . ) ( figure a ). moreover, serum of piglets collected on day after birth had also high pedv specific igg in the recombinant l. acidophilus-s group, while no specific antibodies were found in the control and commercial inactivated vaccine groups (p < . ) ( figure b ). to date, the most effective measure to control viral infectious diseases still depends on vaccination in china. ped is a highly infectious disease with intestinal tissue tropism. immunization with non-oral vaccines mainly stimulates the body to produce igg. igg in serum cannot neutralize the free pedv particles in the intestine and cannot prevent the invasion of pedv in the intestine (song and park, ) . oral vaccines cannot only stimulate the body to produce igg, but also stimulate the intestinal mucosa to produce siga, protecting pigs from infection (liu et al., ) . however, if a live-attenuated pedv vaccine is directly orally immunized, it will be damaged after entering the gastrointestinal tract. therefore, there are broad prospects for development of a live vector oral vaccine which can effectively stimulate mucosal immunity by simulating the natural infection route of pedv. lactobacillus is a normal intestinal bacterium and has adjuvant properties. it can combine immune adjuvant and target antigen to produce antigens and regulate the body's immune system for a long time to continuously stimulate the body's mucosal surface lymphocytes to produce specific antibodies (yu et al., ) . as is well known, the most important features of lactobacillus are its non-toxic side effects and safety in clinical use, and the entire surface of most lactobacillus is covered with a single crystal protein material. this structure can secrete proteins, so can be used as a secretory expression vector. lactobacillus-based vaccines will receive increasing attention. the mechanism by which the lactobacillus vaccine induces an immune response is unknown. in short, based on the outstanding advantages of lactobacillus itself, it will become an attractive engineering bacterium and represents a safe, emerging, and promising oral vaccine carrier (song et al., ; shi et al., ; yu et al., ; song et al., ) . in previous studies, a genetically engineered lactobacillus casei (l. casei) oral vaccine in china, outbreaks of ped have caused great economic losses to the swine industry in recent years zhang et al., ) . it is speculated that genetic mutations lead to less protective effect of the traditional vaccine. g group of pedv is the main predominant in china, and ss and coe domains in s protein displayed -aa or -aa mutations in most field strains of g group compared with traditional strain cv (zhang et al., ) . in this study, s and s genes were amplified from pedv ch-sdbz- - strain, which was located in g group. we developed two oral vaccines encoding s and s epitope domains of pedv spike protein, delivered by live l. acidophilus. the mice immunization results suggested that oral l. acidophilus vaccines j o u r n a l p r e -p r o o f were able to induce pedv specific humoral antibodies. moreover, pedv specific siga levels of oral l. acidophilus vaccine groups were significantly higher than the commercial inactivated vaccine group after dpi, and the l. acidophilus-s vaccine efficiently induced mucosal immunity in mice compared to the l. acidophilus-s group (figure ) . the serum levels of ifn-γ and il- indicated that our two oral l. acidophilus vaccines might mainly enhance a th -type immune response to stimulate the cellular immune responses. although mice are not susceptible to pedv infection, good immune responses of the oral l. acidophilus vaccine in pigs were also observed in this study. sows vaccinated two times with l. acidophilus-s had higher igg antibody levels in the serum as compared to the control sows ( figure ) . also, obviously higher levels of siga antibodies were found in the colostrum of the orally vaccinated sows. furthermore, maternal transferring of antibody was demonstrated, as only the serum of suckling piglets had higher levels of pedv specific igg. all data indicated that the oral recombinant l. acidophilus-s delivering the pedv specific antigens could act as a novel mucosal vaccine formulation and provide a useful strategy to induce efficient immune responses against pedv infection. in this study, the oral vaccines were designed as only s or s protein was expressed in recombinant l. acidophilus. in previous reseach, lc-expressed n protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by lc-expressing s region (liu et al., ). an oral vaccine using l. acidophilus to co-express the s and n proteins of pedv should be developed to yield better immune efficacy. in summary, an oral vaccine strategy using l. acidophilus to deliver s and s epitopes of pedv spike protein was explored to develop an anti-pedv vaccine for oral administration in this study. we demonstrated that the genetically engineered prc/cmv -s -rep. could efficiently induce mucosal, humoral, and cellular immune responses against pedv, suggesting a promising vaccine strategy. the authors declare that they have no competing interests. antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus passive immunity in transmissible gastroenteritis of swine: immunoglobulin characteristics of antibodies in milk after inoculating virus by different routes identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus experimental infection of pigs with a new porcine enteric coronavirus, cv furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry coronavirus spike proteins in viral entry and pathogenesis mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant actobacillus plantarum oral immunization against pedv with recombinant lactobacillus casei expressing dendritic cell-targeting peptide fusing coe protein of pedv in piglets an improved method for the electrotransformation of lactic acid bacteria: a comparative survey heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea immune responses generated by lactobacillus as a carrier in dna immunization against foot-and-mouth disease virus molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in south china development of an indirect elisa for the detection of duck circovirus infection in duck flocks comparison of the immune responses induced by oral immunization of mice with lactobacillus casei-expressing porcine parvovirus vp and vp fused to escherichia coli heat-labile enterotoxin b subunit protein high-level mucosal and systemic immune responses induced by oral administration with lactobacillus-expressed porcine j o u r n a l p r e -p r o o f epidemic diarrhea virus (pedv) oral recombinant lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus s domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein a new coronavirus-like particle associated with diarrhea in swine isolation of porcine immunoglobulins anddetermination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies enteric viral infections of pigs and strategies for induction of mucosal immunity immunoprotection against influenza virus h n by the oral administration of recombinant lactobacillus plantarumnc expressing hemagglutinin in balb/c mice chromosomal insertions in the lactobacillus caseiupp gene that are useful for vaccine expression oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr strain porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines oral vaccine of recombinant lactococcus lactisexpressing the vp protein of duck hepatitis a virus type induces mucosal and systemic immune responses adjuvant effects of l. acidophilus lw on immune responses to the foot-and-mouth disease virus dna vaccine in mice spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus coe antigen: a promising vaccine strategy against pedv efficacy and immunogenicity of a live l. acidophilus expressing sad epitope of transmissible gastroenteritis virus as an oral vaccine faeg in conjunction with a heat-labile enterotoxin a (ltak ) and heat-labile enterotoxin b (ltb) of enterotoxigenic escherichia coli as an oral adjuvant in mice mucosal lactobacillus vectored vaccines identification of a conserved neutralizing linear b-cell epitope in the vp proteins of duck hepatitis a virus type and two distinct genotypes of porcine epidemic diarrhoea virus in vaccinated pig flocks in shandong province of china complete genome sequence of a vero cell-adapted isolate of porcine epidemic diarrhea virus in eastern china this study was funded by the provincial natural science foundation of shandong, china (zr bc ). different lowercase letters denote differences among groups (p< . ) between the assays for the same immune time. different uppercase letters denote significant differences among groups (p< . ) between the assays for the same immune time.j o u r n a l p r e -p r o o f different lowercase letters denote differences among groups (p< . ) between the assays for the same immune time. different uppercase letters denote significant differences among groups (p< . ) between the assays for the same immune time. key: cord- - odc un authors: hasslung, f.; wallgren, p.; ladekjær-hansen, a.-s.; bøtner, a.; nielsen, j.; wattrang, e.; allan, g.m.; mcneilly, f.; ellis, j.; timmusk, s.; belák, k.; segall, t.; melin, l.; berg, m.; fossum, c. title: experimental reproduction of postweaning multisystemic wasting syndrome (pmws) in pigs in sweden and denmark with a swedish isolate of porcine circovirus type date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: odc un an experimental model using -day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type (pcv ) and porcine parvovirus (ppv) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (pmws). a swedish isolate of pcv (s-pcv ) retrieved in from a healthy pig has been used in this model to reproduce pmws in pigs from northern ireland. this virus has been present in the swedish pig population for at least a decade without causing any known pmws disease problems, despite its potential pathogenicity. the reasons for this are unknown, but could be related to genetics, absence of triggers for pcv upregulation (infectious agent and/or management forms) within swedish pig husbandry. in order to confirm the pathogenicity of s-pcv , swedish and danish pigs were experimentally infected with this isolate according to the established model. swedish pigs were also infected with a reference isolate of pcv (pcv - ) to compare the severity of disease caused by the two isolates in swedish pigs. both danish and swedish pigs developed pmws after the experimental infection with s-pcv . antibodies to pcv developed later and reached lower levels in serum from pigs infected with s-pcv than in pigs inoculated with pcv - . in general, pigs infected with s-pcv showed more severe clinical signs of disease than pigs infected with pcv - , but pigs from all pcv -inoculated groups displayed gross and histological lesions consistent with pmws. all pigs inoculated with ppv, alone or in combination with pcv , displayed interleukin- responses in serum while only pigs infected with ppv in combination with pcv showed interferon-α in serum on repeated occasions. thus, the pathogenicity of s-pcv was confirmed and a role for cytokines in the etiology of pmws was indicated. porcine circovirus type (pcv ) is now accepted as the causal agent of postweaning multisystemic wasting syndrome (pmws). however, it is also recognised that other additional infectious (allan et al., ; allan et al., a; krakowka et al., ) or non-infectious factors (rose et al., ) are necessary for the full clinical expression of the disease. pmws was first observed in high health herds in canada in ellis et al., ) , and has since then rapidly become a major problem in many pig-producing countries throughout the world. retrospective studies have demonstrated that a pcv virus has been present in pigs for many years prior to the recognition of pmws without being associated with any specific disease syndrome (rodriguez-arrioja et al., ; walker et al., ) . the global epizootic spread of pmws since suggests that the pcv virus in pigs may have mutated to a more pathogenic form or that another agent in combination with pcv is necessary for the development of pmws. alternatively, it has also been suggested that the susceptibility of the host to pcv associated clinical disease has, in some way, changed due to alterations in the pig industry. efforts to identify new pathogenic genotypes of pcv (de boisseson et al., ) or new common co-infecting microorganism have, to date, failed and epidemiological studies including numerous aspects of husbandry forms have not yet revealed any particular factor(s) that predispose for pmws (larochelle et al., ; pogranichniy et al., ; rose et al., ) . dual infection with pcv and porcine parvovirus (ppv) or porcine reproductive and respiratory syndrome virus (prrsv), as well as immunomodulators, have been used to successfully reproduce pmws experimentally in pigs (for review see allan et al., ) and one of the most reproducible infection models involves co-infection with pcv and ppv in -day-old snatch-farrowed colostrum-deprived (sfcd) piglets (allan et al., ) . this model was recently used in northern ireland to demonstrate the potential pathogenicity of a swedish isolate of pcv from (allan et al., ) . the swedish pcv (s-pcv ) was isolated from a clinically healthy pig, which was raised in a spf-herd that seroconverted to pcv at that time (wattrang et al., ) . sweden remained free from pmws until december , and as of october , only farms have been diagnosed as affected by the disease. it has been suggested that differences in pig husbandry practices, animal genetics and/or viral pathogenesis could have contributed to relative freedom of swedish pigs from disease. the present experimental infection with s-pcv and ppv was conducted using swedish and danish pigs. to allow comparison between various pcv isolates, one group of swedish pigs was also infected with a reference isolate of pcv (imp. ). clinical manifestations of disease, histological lesions, levels of virus antigen in affected tissues and development of antibodies to pcv were recorded and the il- and interferon-a responses were determined in serum obtained from swedish pigs. an experimental model, using a dual infection with porcine circovirus type (pcv ) and porcine parvovirus (ppv) for induction of pmws was used as previously described (allan et al., ) . two isolates of pcv were used: pcv - (pcv stoon) isolated from an outbreak of pmws in a high health herd in canada and s-pcv isolated from a lymph node collected in from a clinically healthy pig reared in a swedish spf-herd (allan et al., ; wattrang et al., ) . for coinfection, ppv (isolate pool ) recovered from pmws-affected pigs was used (allan et al., b; krakowka et al., ) . all viruses were propagated in a pcv -free cell line (pk/ a) as previously described (allan et al., ) . the experimental infections were performed in two sets, one using swedish pigs and the other using danish pigs. in the first set, swedish pigs were snatched-farrowed (sf) and hand-reared on colostrum and milk substitutes (snatch-farrowed colostrumdeprived (sfcd)), whereas the danish pigs were caesarean-derived colostrum-deprived (cdcd). the swedish pigs were obtained from a conventional piglet-producing herd. to date, the farm has not reported any pcv -associated disease problems and the herd is free from infections with salmonella spp, sarcoptes scabei, serpulina hyodysenteriae and toxin producing strains of pasteurella multocida. the sows were vaccinated against escherichia coli, erysipelothrix rhusiopatiae and ppv. the piglets, originating from five litters (crossed hampshire, yorkshire and swedish landrace) designated a-e. the piglets were transported within h after birth to the animal department at the national veterinary institute (nvi), uppsala, sweden, and distributed into four groups that were housed in separate rooms with individual ventilation in a clean but not sterile environment. during the first days of life, the piglets were hand reared on bovine colostrum substitute (calf volostrum, volac international ltd., uk) and subsequently on commercial pig milk substitute (piggi milk, manufacturer no. , uk) and pellets (primary elite, primary diets ltd., uk). all animals were treated with intramuscular injections of antibiotics (nuflor, florfenicol g/ ml . ml/pig, day) for the first days, and thereafter orally (baytril, enrofloxacin mg/ml . ml/pig, day) once daily throughout the experimental period. pigs showing severe signs of disease were euthanised and of the initially piglets were included in the experiment, which was approved by the ethical committee for animal experiments, uppsala, sweden. in the second set, eight danish piglets (nos. - ) danish landrace-yorkshire crossbreds were derived from two sows originating from the spf-herd at the danish institute for food and veterinary research (dfvf), kalvehave, denmark. this herd is free from pcv and pcv , prrsv, swine influenza virus (siv), porcine respiratory coronavirus (prcv), as well as mycoplasms. the sows were vaccinated against ppv. the experimental infection of the cdcd piglets was carried out at the isolation facilities of dfvf and the pigs were housed in a separate section of the building. in all other aspects, rearing of the pigs including colostrum and milk substitutes used and antibiotic treatments was the same as described for the swedish sfcd pigs. on the rd day of life, all piglets were inoculated with . ml per nare of cell lysates from indicated cell culture. pigs in swedish group (n = ) were mock inoculated with cell culture supernatant from uninfected pk/ a cells; pigs in swedish group (n = ) were inoculated with ppv . tcid alone; pigs in swedish group (n = ) were inoculated with a mixture of pcv - . tcid and ppv . tcid ; and pigs in swedish group (n = ), as well as the danish pigs in group (n = ), were inoculated with s-pcv . tcid and ppv . tcid . the health status of all individual pigs was assessed twice daily and pigs developing signs of severe disease were euthanised and necropsied (see below). for swedish pigs, weights and rectal temperatures were measured twice every week. blood samples were collected from vena cava cranialis in evacuated test tubes prior to infection, and on days , , and (groups - ) or , , , and post infection (group ). at blood sampling, individual faecal samples were collected from pigs in groups - , and samples of faeces were collected from the floor of each of these groups daily. at the end of the trial, weeks post infection, all remaining pigs were sacrificed, autopsied and gross lesions recorded. general bacteriological examination of samples from spleen, liver and lung tissues was performed by routine methods at the bacteriological departments at nvi and dfvf, respectively. tissue samples were collected from lymph nodes and tonsils, lungs, heart, liver, kidneys, spleen, small intestine and any other tissues showing gross pathological changes. tissues were fixed in % paraformaldehyde or buffered formalin for histopathological analysis or rapidly frozen in isopropanol on dry ice and stored in liquid nitrogen for immunohistochemical staining. sections of fixed, paraffin-embedded tissue were stained with hematoxylin and eosin for morphological evaluation. the severity of lesions was scored as none visible (À), mild (+), moderate (++) or severe (+++). blocks of tissue from spleen, liver and lymph node were stained with pcv -specific polyclonal or monoclonal antibodies (mcneilly et al., ) as previously described. the presence of pcv was determined on cryostat sections and scored as negative (À) or containing minimal (+) up to abundant (+++++) amount of pcv antigen. levels of pcv dna were determined in serum samples collected from piglets in group by quantitative pcr (q-pcr) as described elsewhere (ladekjaer-mikkelsen et al., ) . results were presented as mean values of duplicate reactions. two sets of pcr methods for detection of pcv were designed. one that detected both pcv isolates used in this study, and one that discriminates between pcv - and s-pcv . for the first set, pcr primers were selected based on sequence alignments of the genomes of several pcv isolates, and two primers, cag caa gaa gaa tgg aag and tat gtg gtt tcc ggg tct were selected for the initial pcr product. for the discriminative pcr, primers were chosen according to sequence differences within the orf regions, aag taa tca ata gtt cta being specific for s-pcv and aag taa tca ata gtg gag being specific for pcv - . these primers were used in combination with an orf full-length primer. the specificity of both pcr methods was verified using purified viral dna from each isolate as control. samples of dna were purified from splenic tissue of experimental pigs according to standard protocols, using proteinase-k digestion, phenol-chisam extraction and ethanol precipitation. the full-length dna sequence of the s-pcv isolate used in the experimental infection was compared to the sequence of pcv from the original lymph node preserved in liquid nitrogen since . both genomes were amplified by pcr using orf r ( aaa ggatcc tca gta att tat ttc ata ) and orf r ( ttt aag ctt cca tga cgt atc caa gga gg ) primers. the pcr products were subsequently ligated into the t-vector (pgem-t easy vector system i, promega) according to the manufacturer's instructions. sequence analysis of the pcr products was performed by standard procedures used at the department of animal breeding and genetics, section of disease genetics, at the swedish university of agricultural sciences, uppsala, and at uppsala genome centre, uppsala university, sweden. alignments were made using dnastar and pairwise comparison of nucleotide and amino acid sequence identities was performed using megalign software version . (dnastar, wisconsin). the nucleotides were numbered in analogy with meehan et al., . antibodies to pcv were detected in an immunoperoxidase monolayer assay (ipma) as described elsewhere (allan et al., b; ladekjaer-mikkelsen et al., ) . sera were examined for antibodies to ppv by blocking elisa (madsen et al., ) or by a competetive elisa (svanovir ppv-ab, svanova biotech, uppsala, sweden). serum ifn-a was determined in samples from the piglets in groups - by a dissociation-enhanced lanthanide fluoroimmunoassay (delfia), as described earlier (artursson et al., ) . the delfia, which is based on two mabs directed to porcine ifn-a, had a lower detection limit of . u ifn-a/ml. presence of il- was determined in serum samples diluted : by elisa (biosource, camarillo, ca) according to the manufacturers description. the sensitivity of the elisa is pg/ml which corresponded to an absorbance value (a nm) of approximately . . among the swedish pigs, animals in total belonging to all four experimental groups developed transient diarrhoea within the first days post infection (dpi). five of these pigs were sacrificed within the first dpi due to moribund conditions. from three of these animals, enrofloxacin and florphenicol resistant klebsiella pneumoniae were isolated from liver, intestine, spleen and peritoneal cavity. from one pig, pseudomonas aeruginosa intermediate sensitive to enrofloxacin and florphenicol resistant were isolated from lungs, intestine, liver and spleen. among pigs in group , no pathogenic bacteria were detected at necropsy, except for lung tissue of piglet from which non-haemolytic e. coli was isolated. apart from these early manifestations of disease, no pigs in groups (mock) or (ppv) developed signs of clinical disease during the experimental period. at necropsy, only three pigs in the control groups (groups and ) displayed minor lesions as specified in table . in group (pcv - and ppv), one pig (d ) developed congestive heart failure and was found dead on day pi. this pig also had overall enlarged lymph nodes, some with subcapsular haemorrhages, and patchy kidneys due to haemorrhages. another pig (d ) was sacrificed due to weakness and bleeding from the nose. this pig displayed severe haemorrhages in the ventral neck region, enlarged axillary lymph nodes and was slightly jaundiced. in addition, three pigs were slightly weak during the last days of the experiment. gross lesions indicating pmws were found in all pigs at necropsy (table ) . in group (s-pcv and ppv), two pigs developed clinical signs indicating pmws. one pig (d ) was found listless on day post infection, and was sacrificed the following day in moribund condition. necropsy demonstrated severe jaundice, overall enlarged lymph nodes and liver, and haemorrhages in lungs, liver and kidneys. the other pig (d ) became weak and lethargic dpi and was subsequently sacrificed. necropsy revealed haemorrhagic lesions in the kidneys and haematuria, as well as jaundice and overall enlarged lymph nodes with subcapsular haemorrhages. no other animals in group showed clinical signs consistent with pmws, but at necropsy, all pigs showed varying degrees of gross lesions consistent with pmws (table ) . in group (s-pcv and ppv), three pigs (nos. , and ) either died or were euthanised during the experimental period. piglet appeared pale and lethargic dpi, experienced severe respiratory distress, and was euthanised dpi. piglet was found dead on dpi. prior to this, it had suffered from respiratory distress and appeared lethargic with intermittently observed tremors. it was also pale and anorexic - days prior to dying. pig was euthanised dpi in a moribund condition after showing pronounced respiratory distress since almost weeks. in addition, this pig showed inappetence, appeared pale and lethargic and suffered from tremor attacks days prior to euthanasia. the remaining five piglets (nos. , , , and ) showed mild clinical symptoms primarily characterized by periods of respiratory distress and lethargic appearance intermittently during the entire experimental period. all pigs in group displayed gross lesions consistent with pmws at necropsy (table ) . histological lesions found in organs tested from all experimentally infected pigs are summarized in table . in brief, all but one of the pigs in group , infected with ppv alone, had mild to moderate lesions mainly affecting myocardial tissue and kidneys. in groups , and , a majority of the pigs displayed mild to severe lesions consistent with pmws in most of the tissues tested. among the uninfected control pigs (group ), three animals displayed mild histological lesions at necropsy dpi. immunohistochemical staining of cryostat sections revealed that all pigs in group , and were positive for pcv in two or more tissues tested (table ) . furthermore, discriminative pcr analysis confirmed that pigs in group were infected with pcv - only, whereas pigs in group were only infected with s-pcv . no pcv antigen was detected in cryostat sections of liver, spleen or lymph node from any of the pigs in the control groups or . quantitative pcr performed on serum samples obtained from the danish pigs infected with s-pcv and ppv (group ) demonstrated pcv dna from days pi and onwards (fig. ) . the levels of pcv dna increased during the first weeks reaching levels of - template copies per ml serum. at dpi, however, the amount of pcv dna had declined slightly in four of the five pigs remaining in the experiment. the results are given as percent of affected pigs of those tested in the group for each lesion and average severity of lesions in affected pigs scored as (+) mild, (++) moderate or (+++) severe; nt = not tested. sequence comparison of the genome of s-pcv used in the experimental infection and pcv retrieved from the original lymph node revealed differences in five nucleotides at position , , , and . one of these exchanges caused a substitution of amino acids (no. ) located in orf . in orf , only one exchange of nucleotides (no. ) was found, not leading to any change in amino acid sequence. in addition, an intronic nucleotide deletion between orf and orf (no. ) was found. thus, repeated passages in cell culture had only caused minor changes in the genome of s-pcv . none of the pigs had antibodies to pcv or ppv prior to infection. all pigs in group remained seronegative to both pcv and ppv, and pigs in group remained seronegative to pcv throughout the experimental period. all pigs in groups , , and had seroconverted to ppv week pi, and remained positive for ppv at the following sampling occassions (data not shown). the development of antibodies to pcv in the pigs dually infected with ppv and pcv is shown in table . in brief, the two pigs in group (s-pcv + ppv) that developed clinical pmws, died or were sacrificed before serum antibodies appeared. three of the other pigs in group displayed low levels of antibodies to pcv , whereas one (c ) developed high titers of antibodies. in group , all animals but two had seroconverted to pcv on dpi, and on dpi, all the remaining pigs were seropositive, whereas piglet died before seroconversion occurred. of the five piglets surviving the weeks of experimental period, piglets , and only reached low antibody titers to pcv . in general, pigs in group (pcv - + ppv) developed a higher titer of antibodies to pcv than those infected with s-pcv and ppv. no such difference between these two groups was apparent for the antibody response to ppv (data not shown). table immunohistochemical staining for pcv antigen in cryostat sections obtained from liver, spleen and lymph nodes (ly node) of pigs experimentally infected with pcv - and ppv (no. ) or s-pcv and ppv in sweden (no. ) amounts of pcv antigen were scored as not detectable (À), or minimal (+/À) to abundant (+++++). all serum samples collected before infection ( dpi) of the piglets in groups - were negative for ifn-a (fig. ) . one week later, however, pigs from all experimental groups, including the mock-inoculated controls, displayed ifn-a in serum. thereafter, only pigs infected with both pcv and ppv had serum ifn-a, and some of these pigs still had detectable levels of ifn-a upon termination of the experiment. serological testing of the swedish pigs revealed that most of the samples contained less than pg il- per ml serum. however, comparison of absorbance values obtained by the elisa (fig. ) indicated a low il- response at day pi which occurred in the majority of pigs exposed to ppv, alone or in table development of antibodies to pcv in serum of pigs experimentally infected with pcv - and ppv (no. ) or the serum levels of antibodies are given as reciprocal titers, determined in an immunoperoxidase monolayer assay. combination with pcv . in groups and , elevated levels of il- were also detected in serum collected dpi. pmws could be reproduced in both swedish and danish pigs with a swedish isolate of pcv in combination with ppv, using a previously established model for experimental infection (allan et al., ) . the swedish pcv used (s-pcv ) was isolated from a pig raised in a spf-herd at the time of herd seroconversion to pcv more than years ago (wattrang et al., ) . at that time, transient reproductive problems were noticed in the herd, but later no effects of the infection could be observed. the present study thus confirms that a strain of pcv virus can persist in a pig population without causing clinical pmws, and still be pathogenic under certain conditions. this potential of s-pcv has previously been demonstrated in the same experimental model using pigs derived from an spf unit in northern ireland (allan et al., ) , and the present study shows that swedish pigs are susceptible to experimental reproduction of pmws. moreover, it was confirmed that the pathogenicity of s-pcv is indistinguishable from that of the reference strain pcv - in this experimental model. pcv - was initially recovered from a diseased pig in canada. at the time of this study, pmws had not been diagnosed in sweden, whereas in denmark, the disease spread rapidly after it was first discovered in (http://www.dfvf.dk). pmws was diagnosed in sweden in late and so far the spread of disease is moderate with only few animals showing clinical disease in affected farms. it is to date not possible to predict the final outcome of pcv -related diseases in sweden, but it seems that the prevalence and mortality within each herd is comparably low, approximately % mortality and runts after weaning. thus, the situation is more similar to what has been reported from north america than that reported from the rest of europe (harding, ) . generally, postweaning mortality in sweden is low ( . % of weaned pigs in , http://www.qgenetics.com) and the country is free from many porcine pathogens such as prrsv, adv and enteric corona virus. also, rearing of pigs according to the ''swedish model'' includes a relatively high weaning age ( - weeks), batch-wise rearing from birth to slaughter, and a reduction of transportation and/or mixing of pigs from different litters. all of these factors may contribute to the relative resistance of the swedish pig industry to pcv -related diseases, although it is now clear that swedish pigs can develop pwms both experimentally and naturally. notably, the first pmws outbreaks in sweden occurred in herds where high production intensity had resulted in deviations from this ''swedish model''. the clinical and pathological evidence of disease in s-pcv and ppv co-infected animals was similar in the swedish and danish experiments presented herein. to further evaluate the pathogenicity of s-pcv , one group of swedish pigs was infected with a reference strain of pcv (imp. , stoon) in combination with ppv. no pigs in this group died from pmws, and no animal showed clinical signs of disease related to pmws during the experimental period. at necropsy, however, all pigs from this group displayed gross lesions consistent with pmws. the combined manifestation of clinical disease and pathological lesions caused by the two isolates indicates that the pathogenicity of the swedish isolate is equal to or even higher than that of pcv - . in two previous studies applying the same experimental model, however, the proportions of pigs developing clinical pmws was somewhat larger, both concerning pcv - and s-pcv (allan et al., ; allan et al., ; allan et al., ) . this discrepancy could reflect a diminished pathogenicity of pcv - , possibly caused by several in vitro passages of the virus, but variations in the experimental conditions cannot be excluded. to rule out that s-pcv had undergone major changes during the isolation procedure, a comparison of the genomes of s-pcv before and after repeated passages in cell culture was performed. this revealed only minor changes, indicating that the virus used in the experimental infection was very similar to the swedish field isolate from . as previously described, this isolate differs from pcv - in the nucleotide sequence of orf (allan et al., ) causing eight amino acid substitutions. nucleotide sequence analysis revealed several differences between s-pcv used in the experimental infection and pcv isolated in december from the first natural cases of pmws in sweden (data not shown). the occurrence of ifn-a in serum samples collected on day from pigs in groups - indicates that some pigs experienced infections unrelated to the experimental infections. this was also evident from early deaths among pigs in all experimental groups, which in most cases were related to navel infections, and could be expected for pigs snatched-farrowed in a conventional farm. on later sampling occasions, however, ifn-a could only be detected in sera from pigs in groups and that had been inoculated with pcv . in contrast, elevated serum il- was indicated in pigs infected with ppvalone or in combination with pcv . this suggests that ppv induces a systemic response of this cytokine, which might be of importance for the development of pmws in the experimental co-infection with pcv . in natural cases of pmws, an over-expression of il- mrna has been found in pbmc (sipos et al., ) and thymocytes (darwich et al., b) , and pbmc from pmws affected pigs preferentially produced il- and ifn-g at in vitro re-exposure to pcv (darwich et al., a) . if an altered cytokine profile is caused by progressing pmws or induced by other factors that might predispose for pmws, remains however to be determined. interestingly, prrsv infections, often considered as a co-factor for development of pmws, induced increased expression of il- mrna in pbmc, lung tissue and bronchoalveolar cells (chung and chae, ; johnsen et al., ; suradhat and thanawongnuwech, ) . thus, if il- expression is important for pmws development, the prrsv free status of sweden may well explain the lower incidence of pmws recorded within swedish herds. the impact of ppv infections in the pathogenesis of pmws was recently demonstrated under field conditions where pmws was induced in segregated earlyweaned - week old pigs co-infected with ppv and pcv (opriessnig et al., ) , which further justifies the current experimental model. it should, however, be noted that the permanent treatment with broadspectrum antibiotics severely affected the normal bacterial flora of the gut. pen floor samples revealed less than cfu coliforms per gram faeces (data not shown) compared to - in samples from nontreated pigs of the same age category (melin et al., ) . the importance of a normal microflora for the development of both mucosal and systemic immunity has been described for several species, including gnotobiotic piglets (tlaskalova-hogenova et al., ) . in addition to the antibiotic treatment, piglets were handfed with cow-milk based colostrum substitutes during the first days of life to avoid passive transfer of porcine immunoglobulins. this treatment might further affect the function of the gut associated lymphoid tissue of the experimental pigs. since large amounts of pcv are secreted through faeces during pmws, the gut and surrounding tissues are likely to be involved in the replication of pcv as well as of ppv. thus, several non-physiological effects are caused by the present experimental model, which have to be considered for a better understanding of co-factors that contribute to the development of pmws. nevertheless, the results presented here clearly show that a virus strain that persisted for years in a spf-herd without causing clinical pmws was able to induce this disease under experimental conditions in pigs of different origin. further studies concerning the effects of husbandry forms as well as genetics of the animals and microorganisms are needed to elucidate why the spread of pmws differs between regions. isolation of porcine circovirus-like viruses from pigs with a wasting disease in the usa and europe experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv replication a sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation reproduction of post weaning multisystemic wasting syndrome in pigs experimentally inoculated with a swedish porcine circovirus isolate pmws: experimental model and co-infections interferon-alpha production and tissue localization of interferon-alpha/beta producing cells after intradermal administration of aujeszky's disease virus-infected cells in pigs expression of interleukin- and interleukin- in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (prrsv) cytokine profiles of peripheral blood mononuclear cells from pigs with postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens cytokine mrna expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome molecular characterization of porcine circovirus type isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome porcine circovirus- and concurrent infections in the field the clinical expression and emergence of porcine circovirus cytokine mrna profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of ifn-gamma and il- after viral clearance viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus and porcine parvovirus reproduction of postweaning multisystemic wasting syndrome (pmws) in immunostimulated and non-immunostimulated -week-old piglets experimentally infected with porcine circovirus type (pcv ) comparative serologic and virologic study of commercial swine herds with and without postweaning multisystemic wasting syndrome detection of antibodies against porcine parvovirus nonstructural protein ns may distinguish between vaccinated and infected pigs production, characterisation and applications of monoclonal antibodies to porcine circovirus characterization of novel circovirus dnas associated with wasting syndromes in pigs weaning of piglets. effects of an exposure to a pathogenic strain of escherichia coli effect of porcine parvovirus vaccination on the development of pmws in segregated early weaned pigs coinfected with type porcine circovirus and porcine parvovirus case-control study on the association of porcine circovirus type and other swine viral pathogens with postweaning multisystemic wasting syndrome retrospective study on porcine circovirus type infection in pigs from to in spain risk factors for porcine post-weaning multisystemic wasting syndrome (pmws) in french farrow-to-finish herds systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome (pmws) as determined by semiquantitative rt-pcr and flow cytometric intracellular cytokine detection upregulation of interleukin- gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type exudative epidermitis and porcine circovirus- infection in a swedish spf-herd this work was supported by formas, agrifun-gen and the programme for biology of infection at the veterinary faculty, swedish university of agricultural sciences. financial support was also provided by the european union (project qlk -ct- - ), danish research agency (no. - - ) and (canadian) natural sciences and engineering research council-collaborative research opportunities grant - . the authors also wish to thank barbro högberg and per carlsson for invaluable help with the animals, as well as lisbeth fuxler and maria persson for excellent laboratory work. key: cord- -f i lq w authors: bachofen, claudia; braun, ueli; hilbe, monika; ehrensperger, felix; stalder, hanspeter; peterhans, ernst title: clinical appearance and pathology of cattle persistently infected with bovine viral diarrhoea virus of different genetic subgroups date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: f i lq w bovine viral diarrhoea (bvd) is an economically important cattle disease with a world-wide distribution that is caused by bvd virus, a pestivirus of the flaviviridae family. bvd viruses are genetically highly variable. they are classified into two genetic species (bvdv- and - ) that are further divided into numerous subgroups, particularly for bvdv- . the complexity of these viruses is also reflected in their interaction with the host animals. infections are either transient or persistent and can cause a wide spectrum of clinical signs, from no or very mild disease to severe forms, reminiscent of viral haemorrhagic fevers. in this work, we have analysed the clinical signs and the pathology of bvd viral infections in a cattle population where different subgroups of bvdv- genotype viruses are endemic. in addition, we have examined potential virulence properties of bvdv- subgroups during persistent infection by comparing the viral subgroups present in clinical cases with those detected in persistently infected (pi) animals sampled for epidemiological criteria, irrespective of their health condition. furthermore, the clinical and postmortem findings were compared with respect to genetic characteristics of the viruses isolated from these animals. our results indicate that the bvdv positive animals fall roughly into two categories, depending on the primary organ affected and the age, with lung-centred pathology occurring mainly in young animals and mucosal pathology predominantly in older animals. furthermore, we found a markedly higher proportion of representatives of the bvdv- e subgroup in stillborn calves and aborted foetuses originating from epidemically unrelated cattle herds, suggesting that bvdv- e may play a special role in prenatal and perinatal losses. bovine viral diarrhoea (bvd) is an economically important cattle disease with a worldwide distribution that is caused by bvd virus, a pestivirus of the flaviviridae family. bvd viruses are genetically highly variable. they are classified into two genetic species (bvdv- and - ) that are further divided into numerous subgroups, particularly for bvdv- . the complexity of these viruses is also reflected in their interaction with the host animals. infections are either transient or persistent and can cause a wide spectrum of clinical signs, from no or very mild disease to severe forms, reminiscent of viral haemorrhagic fevers. in this work, we have analysed the clinical signs and the pathology of bvd viral infections in a cattle population where different subgroups of bvdv- genotype viruses are endemic. in addition, we have examined potential virulence properties of bvdv- subgroups during persistent infection by comparing the viral subgroups present in clinical cases with those detected in persistently infected (pi) animals sampled for epidemiological criteria, irrespective of their health condition. furthermore, the clinical and postmortem findings were compared with respect to genetic characteristics of the viruses isolated from these animals. our results indicate that the bvdv positive animals fall roughly into two categories, depending on the primary organ affected and the age, with lung-centred pathology occurring mainly in young animals and mucosal pathology predominantly in older animals. furthermore, we found a markedly higher proportion of representatives of the bvdv- e subgroup in stillborn calves and aborted foetuses originating from epidemically unrelated cattle herds, suggesting that bvdv- e may play a special role in prenatal and perinatal losses. ß elsevier b.v. all rights reserved. growth in cultured cells. the two biotypes also differ with respect to their effects in infected animals. only the ncp biotype can establish a persistent infection of the foetus if the dam is transiently infected between approximately days and of its development. even though such foetuses may develop normally, they remain infected for life and shed large quantities of virus, thereby assuring the persistence of bvdv in the cattle population even in the absence of animals susceptible to transient infection. different from other persistent viral infections in man and animals, bvdv persistently infected (pi) cattle are immunotolerant to the infecting viral strain (reviewed in peterhans et al., ) . the clinical consequences of infection with bvd virus are as diverse as the genetic and antigenic properties of these viruses. disease can result from different pathogenetical mechanisms, depending on the different types of infection. most transient infections may take an inapparent or mild course, associated with low-grade fever, diarrhoea and coughing. rarely, however, acutely infected animals may suffer from high grade fever and bleeding in internal organs. this often fatal form of acute infection was reported in outbreaks in the late s and early s, caused by highly virulent bvd virus- strains (ridpath et al., (ridpath et al., , . although generally considered to be of low virulence, bvdv- , too, may cause similar severe disease signs in acutely infected animals, albeit only rarely (liebler-tenorio et al., ; ridpath et al., ) . in the case of persistent infections, clinical signs in pi animals can be differentiated pathogenetically into mucosal disease (md) and non-md cases. the onset of md is associated with the appearance of the cytopathic biotype of bvdv that arises as a result of mutations and/or recombination events from the persisting ncp virus (kü mmerer et al., ; tautz et al., ; becher et al., ) . this lethal disease is characterised by mucosal lesions, destruction of the lymphoid tissue in the gastrointestinal tract (liebler et al., ) and untreatable diarrhoea. the onset of the clinical signs is often acute but a more chronic form, called late onset md, is also described (liebler-tenorio et al., ; fritzemeier et al., ) . the clinical signs in pi animals that have not (yet) developed md can encompass a wide spectrum of symptoms, ranging from normal health to subclinical disorders, ill-thrift and growth retardation. chronic or recurrent intestinal and/or pulmonary symptoms are frequently observed but occasionally dermatological, neurological or haematological disorders are the only signs of a persistent infection (braun et al., ; taylor et al., ) . in many cases the clinical and necropsy findings do not allow a clear differentiation between md and non-md cases. the isolation of both biotypes, ncp and cp, proves a pi animal to suffer from md. possible reasons for the variable clinical signs seen in bvdv infections have been analysed repeatedly and in some cases an impact of the infecting virus strain was proven (baule et al., (baule et al., , fulton et al., ; jones et al., ; ridpath et al., ) . however, these studies focused on transient infections. moreover, the analyses were restricted to single outbreaks of identical or very closely related viral strains that affected several animals within one herd or in different herds simultaneously, or to experimental infections. hence, the observations may be representative for a given viral strain rather than for an entire subgroup of bvdv. in contrast to transient infections, disease associations between individual bvdv strains or subgroups have not been investigated in persistent infection. bvd is endemic in switzerland, with roughly % of cattle seropositive and . % persistently infected, with seemingly no change in prevalence over a time period of some years (bü rki et al., ; homberger et al., ; rü fenacht et al., ; stalder et al., ) . in this work, we have analysed pi animals referred by private veterinary practitioners to the clinical and pathology departments of the veterinary hospital of the university of zü rich. due to the endemic nature of bvd, these animals may represent the most severe clinical forms of persistent infection, comparable to the tip of the iceberg. we have recorded and applied a scoring protocol for the clinical and postmortem findings of the diseased animals and analysed possible correlations between different organ manifestations and the subgroups of persisting bvdv. to obtain an insight into the possibility of virulence of bvdv in persistent infection, we have analysed the viral subgroups present in diseased pi animals as compared to subgroups present in animals diagnosed bvdv pi independently of the clinical status. at the department of farm animals of the university of zurich, all calves less than months of age and chronically diseased animals are routinely tested for the presence of bvdv. we have retrospectively analysed all clinical reports of pi animals shown at the clinic for ruminants from first of january to st of december (group a, table ). in addition, the prospective cases from the years and were included (group b, table ) , yielding clinical case reports. all animals had undergone the same thorough clinical examination following a standardised protocol as described previously (braun et al., ) . haematological, biochemical, bacteriological and parasitological analyses were performed as described by braun et al. ( ) . the bvdv status of animals of groups a and b was determined intra vitam by immunohistochemistry (ihc) of skin biopsies ( cases) and by antigen-capture elisa of blood leukocytes ( cases) (strasser et al., ) or of skin biopsies ( cases) (idexx herdcheck bvdv ag/serum plus, idexx europe b.v., ne schiphol-rijk, the netherlands). some animals were repeatedly tested by multiple methods. ten animals of groups a and b were only diagnosed after being euthanised. the bvdv status of all animals was confirmed postmortem by ihc of organ samples. all samples revealed the staining pattern typical for persistent infection, as described previously by hilbe et al. ( ) . as a result of the unfavourable prognosis, all animals were euthanised within - days after the initial clinical investigation and sent to the institute of veterinary pathology for necropsy. a necropsy report was available of of the cases from groups a and b. in addition, we included in our study necropsy reports of animals that had been euthanised or had perished and been submitted by private veterinarians for necropsy to the institute of veterinary pathology where they were tested bvd viruspositive by ihc of organ samples (group c, table ). the reasons for the submission of these carcasses for postmortem analysis were variable and encompassed examination of herd problems, clarification of ambiguous clinical disease signs or suspicion of inherited disease such as spinal muscle atrophy of swiss brown calves. no information on intra vitam examinations and their results were available of the animals of group c. all in all, we analysed necropsy reports (groups a, b, c), of which contained complete data sets (groups a, b, c. ). in cases, postmortem reports were only available for single organs or body parts (group c. , table ). the cases analysed were extended by cases of bvdv infected neonates (stillbirths and calves younger than days) and aborted foetuses, tested positive by ihc (staining pattern typical for persistent infection (group d, table )). descriptive information from the standardised clinical and necropsy reports was transformed into ordinal data (e.g. general health condition, degree of lung alteration) or nominal data (e.g. gender) with the help of a specially created evaluation protocol. the evaluation protocol applied was created in collaboration with clinicians and pathologists and is based on clinical scoring protocols used previously (braun et al., (braun et al., , . for the majority of findings we used a simple -level grading: nothing abnormal detected = , mild = , moderate = , severe alteration = . the evaluation of all reports was performed by the same person and without any information on the bvdv subgroup isolated from the corresponding pi animal. the scoring was based on the graduation of the clinical and postmortem findings made by the various clinicians and pathologists who had initially recorded the cases. grading the findings is an essential part of the standardised protocols used for initial clinical examination and necropsy. in those cases where the organ was only partially affected (e.g. an abscess in a single lung lobe) we considered that the severity and spread of the lesion would serve to assess the general impairment of the organ. to compare the mucosal alterations between different pi animals, we used a standardised mucosal (mc) index calculated for each animal. this index considers the number of affected sections of the alimentary tract (from nose to large intestines = max. organs; affected = any pathological change from the normal tissue integrity) and the severity of the alterations ( = no alteration, = mild, = moderate, = severe alteration). with respect to the multiple dispersed lesions typically seen in md cases (liebler-tenorio et al., ) the calculation places more weight on the number of affected sections than on the severity of the alterations. the mc index may range from to . swiss bvdv strains, isolated between and were sequenced and phylogenetically analysed by stalder et al. ( ) . these viruses originated from animals detected to be pi as a result of a cross-sectional epidemiological study to determine the prevalence of bvdv in switzerland (rü fenacht et al., ) . in addition, viruses were derived from herd analyses performed in order to assess diagnostic tests or to apply molecular epidemiology. the bvdv isolates of the clinical cases described here were isolated, sequenced and phylogenetically analysed for a study published previously (bachofen et al., ) . for all statistical calculations the ncss/pass software (kaysville, ut, usa) was used. as the data were not normally distributed, we used the non-parametric oneway anova test (kruskal-wallis) to compare medians of three or more groups of patients. if this test revealed a significant over-all difference, pair-wise comparisons were performed using the wilcoxon rank-sum test. to compare proportions we applied the chi-square or fisher's exact test. the probability level was set to %. the displayed pvalues represent the results of the two-tailed hypothesis testing. we tested serum of pi animals from the prospective cases for the existence of cytopathic bvdv virus as a hallmark for the onset of md. the sera were diluted seven times in tenfold steps in cell culture medium (earle's minimal essential medium (mem)) enriched with % foetal bovine serum (fbs). mem was purchased from seromed (biochrom, munich, germany) and fbs from sigma or oxoid gmbh (wesel, germany). fbs was free of bvdv and antibody to bvdv as tested by virus isolation and serum neutralization test, respectively. each serum dilution step was distributed to six wells ( ml) of a -well microtiter plate, seeded with primary bovine turbinate cells. after days of incubation at c and % co , ml of supernatant was transferred to a fresh -well microtiter plate, pre-seeded with bovine turbinate cells. after addition of ml of fresh mem, the microtiter plate was incubated as before and the passaging procedure repeated once more. after each passage, the cells were fixed and stained for viral protein as described by adler et al. ( ) . the cells were then microscopically controlled for the presence of a cytopathic effect. in order to investigate differences between bvdv subgroups regarding virulence during persistent infection, clinical recordings and necropsy reports of pi animals infected with viruses of different bvdv- subgroups were analysed. we included patients from the clinic for ruminants of the department of farm animals (university zü rich) and additional necropsy reports of animals referred by private veterinarians directly to the institute of veterinary pathology (university zü rich) for postmortem analysis. as shown in table , the cases could be separated into four groups: retrospective cases (group a, n = ) and prospective cases (group b, n = ) with both a clinical and a necropsy report, cases directly referred for necropsy by private practitioners without clinical report (group c, n = ) and postmortem analyses of abortions and neonates (animals days of age) without clinical report (group d, n = ). of the cases of groups a, b and c (abortions and neonates not included since the gender is often not stated) % were females, while for the animals with a clinical report (groups a and b, n = ) the proportion of females was %. animals of five different breeds and diverse crossbreeds were analysed. the majority of cases (n = ) were of the swiss brown ( %), holstein-frisian ( %) and swiss fleckvieh ( %) breeds. these numbers reflect the over-all breed proportions of the patient population of the clinic. single cases of rhä tisches grauvieh, dexter, limousin and beef crossbreeds were also represented. the median age of the animals (n = ) was months, with a range from days to years. animals referred to the clinic (groups a and b, n = ) were significantly older (median = months) than those submitted directly for necropsy (group c, n = , median age = months). of the animals with a clinical record (n = ), % had an age of months or over and four animals were older than years ( , , and months). the cases originated from of the swiss cantons. most of the cases (n = ) had a history of recurrent or untreatable diarrhoea ( %), pneumonia ( %) or both conditions together ( %). neurological symptoms ( %) were often anamnestically described in necropsy reports of young calves, directly referred for postmortem examination (group c). other anamnestic signs were abortion, lameness due to claw problems, anaemia and ill-thrift. in % of the cases (n = ) the anamnestic report indicated concomitant health problems in the herd. growth retardation was indicated in % of the cases. the most frequently observed clinical and postmortem manifestations are shown in fig. . nearly all animals showed unspecific signs like loss of appetite ( %), reduction of the general condition ( %) and ruminal hypoperistalsis ( %), followed by intestinal disorders like diarrhoea ( %) and the resulting dehydration ( %). respiratory sounds of different degree were present in % of the cases. mucosal erosions as a more specific manifestation were found in the oral cavity and on the muzzle of % and % of the animals, respectively (fig. a) . for the determination of the most frequent postmortem findings, only cases with a complete necropsy report were included, whereas partial necropsies (e.g. single organs or body parts) were excluded (group c. ). group d was excluded, because the abortions were often in a condition that would no longer allow clear postmortem diagnosis or consisted only of foetal body parts. the most frequent necropsy findings differed between animals with clinical reports (groups a and b) and those referred directly for necropsy (group c. ) (fig. b) . in the latter, these were pathological changes in the lung, while alterations in one or more mucosal organs (from muzzle to large intestines) were the primary findings in the clinical cases. generally, except for the lung, a higher proportion of cases referred to the clinic (groups a and b) showed macroscopic lesions than animals directly referred for a postmortem examination (group c. ). haematological data were available from of the cases with a clinical report (groups a and b). the most frequently observed changes were elevated bilirubin levels ( %) and monocytosis ( %), neutrophilia (in %) and leukocytosis (in % of the animals). decreased leukocyte counts were found in % of the cases. alterations in erythrocyte parameters were frequent and were displayed as polycythemia ( % of the animals), decreased cellular haemoglobin content ( %) and a decreased erythrocyte volume ( %). for groups a and b, intra vitam parasitological and bacteriological analyses were not performed routinely but rather upon clinical suspicion. however, with the exception of gastrointestinal nematodes that were present in % of the cases analysed, parasites and bacteria such as liver fluke, lung worm, salmonella, campylobacter and enterotoxic escherichia coli were only occasionally confirmed ( - % of analysed cases). furthermore, all animals analysed for the presence of ovine herpesvirus- , the causative agent of malignant catarrhal fever in cattle, were negative. four and five animals were analysed for the presence of rota-and coronavirus, respectively. for each virus, one animal tested positive. bovine herpesvirus- that can cause symptoms similar to bvd/md is eradicated in switzerland and can be excluded as contributing factor to the symptoms observed. for all groups, postmortem table ). (b) the most frequently observed necropsy findings from pi animals submitted for postmortem examination from the clinic (black bars) or directly from private practitioners (grey bars). only necropsies of entire carcasses were included. the animal groups indicated refer to table . a one or multiple pathological alteration(s) of the mucosa of the whole alimentary tract, including nose and muzzle. b one or multiple pathological alteration(s) of the mucosa of the gastrointestinal tract. c macroscopic alteration of the mediastinal or mesenterial lymph nodes. parasitological and bacteriological analyses were performed sporadically, depending on demand and suspicion and were not included to the study. in many cases, mucosal lesions were observed at multiple locations of the gastrointestinal tract. to be able to compare the general degree of mucosal lesions between groups of animals, it was necessary to condense the multiple mucosal alterations to one parameter. we used the number of affected organs and the severity of the single alterations to calculate an index for each animal, referred to as mucosal (mc) index (see section for details). the highest value that is theoretically possible (= all analysed organs are severely affected) is . the maximal and minimal mc index values observed from the cases analysed (complete necropsy reports only: groups a, b and c. ) was and , respectively, with an arithmetic mean of . . because intestinal and pulmonary symptoms were the most frequent findings observed in the bvdv infected animals, we investigated whether these findings were correlated. interestingly, these organ manifestations tended to exclude each other. as shown in fig. a , animals with a high mucosal index had significantly less severe lung alterations than those with a low mucosal index. moreover, animals with severe pulmonary manifestations were significantly younger than those with less severe pathological changes in the lungs (fig. b ). by contrast, the mucosal index was significantly higher in older animals than in young ones (fig. c) . linear regression analysis (spearman rank) demonstrated a weak (r = . ) but statistically significant (p = . ) correlation between the mc index and the age of the animals. the dichotomy between lung and mucosal lesions pointed to roughly two groups of bvdv infected patients. in order to investigate if this correlated with the presence or absence of mucosal disease, we tested serum samples of pi animals of group b for the presence of cytopathic (cp) virus by passaging different dilutions of the sera three times on cultured bovine turbinate cells. this analysis was restricted to animals, since blood samples were only available from the prospective cases (group b) and young calves with maternal antibodies had to be excluded due to inhibition of virus isolation. the results are shown in table . with one exception ( - ), the cp biotype was only present in sera of animals with an mc index of ! . , suggesting that these patients had suffered from md. no cp biotype was observed in animals with an index of . . therefore, we decided to set the threshold between mdand non-md cases to . . extrapolating this finding to the (table ) . mucosal-indices of all cases, % of the animals ( / ) had suffered from md. of the cases analysed in this work (groups a, b, c and d), the partial genomic sequence of the persisting virus was known from cases (bachofen et al., ) . for the cases of group b, viral rna was isolated from blood, from the other cases from frozen tissue samples. phylogenetic subgroup assessment was performed based of the untranslated region and in some cases additionally on the n pro coding region. the subgroups present are shown in fig. b . the majority of the animals harboured viruses of the subgroups bvdv- e ( %) and h ( %), followed by k ( %) and b ( %). from one single animal we isolated the bvd- x virus as described previously (bachofen et al., ) . these are (with exception of x) the same subgroups as described by stalder et al. ( ) (fig. a) . the subgroup e was most prominent in the latter ( %) while h was the most frequent subgroup in clinical cases ( %) (fig. ) . however, statistical analysis revealed no significant differences of the proportions of bvdv subgroups present in the diseased pi's and animals detected persistently infected independent of their health condition. cases with a complete necropsy report and a known bvdv sequence could be included in this part of the study. this number is composed of animals of group a, of group b and the cases of group c. . the single animal harbouring bvdv x was excluded from the analysis, resulting in pi animals. of these, % had harboured viruses belonging to subgroup b, % to e and h each, and % to k (fig. ) . we observed no statistically significant differences in the associations of viruses of a given subgroup with mucosal or pulmonary alterations or suspected md cases (fig. ) , nor did we see differences with regard to different age groups, breeds or other clinical, postmortem or haematological findings (data not shown). however, there was a tendency for h viruses to be more frequently isolated from animals with mucosal problems, presumable md cases (mucosal index ! ), and thus older animals, while e was the predominant subgroup of bvd virus in patients with pulmonary alterations and thus younger animals (fig. ) . to broaden the age range of the patients, we additionally analysed the proportions of the different bvdv- subgroups in the cases of group d (abortions, stillbirths and neonates). interestingly, we observed a clear predominance of representatives of subgroup e bvd viruses in these animals (fig. ) . the subgroup proportions in group d were significantly different from those observed in animals with mucosal lesions and suspected md cases (mc index ! ). the spectrum and severity of clinical signs caused by bvdv infection are highly variable. factors contributing to this complexity include two types of infection with entirely different involvement of the immune system (immune response in transient versus immunotolerance in persistent infection), two different biotypes of virus (noncytopathic versus cytopathic) and the diverse genetic these animals were chosen due to availability of serum and absence of maternal antibodies. in persistent infection, in contrast, analysis of a potential impact of the viral strain on the clinical picture is, for a number of reasons, more difficult. among them is the existence of disease signs associated with persistent infection as such, and the occurrence of md as a form of infection associated with the emergence of a cytopathic biotype of bvdv in these immunotolerant animals. furthermore, some of the signs of persistent infection unrelated to md may be of chronic nature, such as growth retardation and general ill-thrift. however, as shown by pluriparous pi cows and pi bulls detected among the candidates for artificial insemination stations, clinical signs of persistent infection can be absent in some animals. experimental generation of a sufficient number of pi animals with different strains of bvdv would be laborious and require long-term follow-up. in addition, many different factors such as time point of maternal infection, genetic background of the animals and environmental factors, e.g. the presence of various other cattle pathogens, may modulate the outcome. moreover, the epidemiological situation -endemic versus non-endemic -may influence the clinical appearance of infections. it is not surprising, therefore, that questions such as the impact of bvdv genetic properties on the health status of a pi animal, or possible associations between disease signs and a given subgroup of bvdv during this type of infection, have to date not been addressed. we have investigated these questions in an endemic situation that was virtually undisturbed by vaccination or other control measures, both of which might influence the outcome of infection. in order to obtain information on the pathogenic potential of bvdv in persistent infection, we analysed clinical and necropsy findings of diseased pi animals and compared the bvdv subgroups present in these animals those isolated from animals diagnosed as pi independently of the health status. since we included prospective as well as retrospective cases examined over a period of years, the long time range and the fact that different clinicians and pathologists examined the animals might influence the comparability of the raw data. however, the protocol applied to both the clinical and pathological investigations did not change over this time. in addition, the fact that the initial examinations were performed by many different clinicians and pathologists decreases the risk of a systematic bias. moreover, the transformation of nominal into numerical data was done by one person, using a standardised protocol. to obtain a larger number of cases, we analysed different groups of patients. while the retro-and prospective cases from the ruminants' clinic (groups a and b) showed similar demographic parameters and postmortem findings (data not shown), we observed significant differences to the animals of group c. animals of groups a and b were significantly older and generally more severely diseased than the cases directly referred for postmortem analysis (group c) (fig. b) . this might be due to the fact that, generally, the more valuable and thus primarily older animals with complex or unsuccessfully pre-treated health problems are referred for a stay at the clinic. the high proportion of females in groups a and b simply reflects the fact that most of these animals originate from dairy farms. interestingly, also the main postmortem findings differed between groups a and b versus group c, with mucosal alterations playing a prominent role in groups a and b and pulmonary lesions in group c (fig. b) . keeping in mind the age difference between the groups, we tested the statistical correlation between lung and mucosal manifestations and the age of the animals. mucosal lesions were more frequent in older and lung lesions in younger animals (fig. ) ; that held also true when the two animal groups (a + b and c) were tested separately (data not shown). the reason for the correlation between age and the postmortem finding may be related to the higher risk of md onset with increasing age. md is correlated to the appearance of the cp virus that arises as a result of mutations or recombination during viral rna replication from the persisting ncp virus (deregt and loewen, ; neill and ridpath, ; lackner et al., ) . statistically, a longer time period of viral replication may increase the risk of generating mutations leading to a cp biotype. due to the retrospective nature of the study, we were not in a position to fulfill the virological criterion for diagnosing md by showing the presence of cytopathic and non- fig. . proportions of the four main swiss bvdv subgroups isolated from different groups of pi animals. a only complete necropsy reports were included: cases of group a, of group b and cases of group c (table ) = . however, one single animal harboured a virus that did not belong to any of the four subgroups analysed and was therefore not included (n = ). b cases of group d (table ). cytopathic bvdv in all cases. to overcome this problem we combined a virological determination of md of selected cases (namely the isolation of both biotypes) with a standardised pathological investigation ( table ) . the latter was achieved by creating a ''mucosal (mc) index'', comparable to other clinical or pathological indices, e.g. the psoriasis area severity index (pasi) in human dermatology. typically, md leads to mucosal lesions at multiple locations of the digestive tract. therefore, the mc index places more weight on the number of affected organs than on the severity of single lesions. this approach also minimizes individual differences in rating the severity of lesions and favours completeness of the necropsy reports. the results of the comparison of the biotype analysis with the mc index (table ) support the validity of the concept. taking all bvd viral sequences of groups a, b, c and d together, we did not observe statistically significant differences to the bvdv subgroup distribution previously described by stalder et al., (fig. ) . with the exception of the single ''orphan'' bvd x strain, we found the same four bvd- subgroups in the diseased animals and in the pi animals sampled in the course of an epidemiological survey. although sampled in the same time period, the two large groups of pi animals are not matched with respect to breed, age or gender. on the one hand, using unmatched hosts, only major differences in tropism and virulence would show up. the results of our study argue against such major differences between entire subgroups. on the other hand, this observation does not exclude that individual strains may differ in virulence and tropism, as shown for transient infections. a different aspect concerns possible bvdv subgroup affiliations with the two main clinical and pathological patterns observed, i.e. lung-versus gastrointestinal tractcentred. in this context, the possibility that these patterns might be influenced by the presence (or absence) of other pathogens needs to be considered. such pathogens could include other viruses, bacteria and parasites. viruses known to induce similar clinical signs and pathology, i.e. bovine herpesvirus- and ovine herpesvirus- , can be excluded. the former is eradicated in switzerland, and all pi animals suspected of suffering from malignant catarrhal fever tested negative for this lethal form of infection caused by ovine herpes virus- (ackermann, ) . where data were available, we recorded additional bacteriological and parasitological examinations. however, in most cases the suspected infections were not confirmed. we did not find a statistically significant association between a given bvd viral subgroup and the two major disease manifestations (fig. ) , nor to any other clinical or mortem finding (data not shown). however, we observed a tendency for viruses of subgroup h to be more frequently isolated from animals with mucosal lesions, while e was the predominant subgroup in animals with lung alterations (fig. ) . regarding the age dependency of the mucosal and pulmonary manifestations (fig. ) , the observed subgroup tendencies may well correlate to different age groups of pi animals rather than reflect a selective organ tropism. indeed, % of the animals younger than months ( months = median age of all animals analysed) harboured bvdv- e, while this proportion was only % in older animals (! months). since all pi animals are infected as foetuses, animals infected with bvdv- e may die younger than animals infected with other subgroups and are therefore less frequently observed among older animals. this is supported by the observation of a surprisingly high proportion of bvdv- e strains in abortions, stillbirths and neonates (fig. ) , which clearly encourages additional investigations. we can exclude that a e live vaccine may be responsible, since, as indicated above, vaccines are used only very infrequently, and a e vaccine is not licensed in switzerland. moreover, we can also exclude that a particular strain of e may cause these abortions because the sequence data indicate that genetically different strains were present in these animals (bachofen et al., ) . in summary, our study showed that, in an endemic situation, clinical cases of bvdv pi animals fall roughly in two distinct categories, with lung-centred pathology occurring mainly in young animals and mucosal pathology mainly in older animals. moreover, even though we did not find evidence for one bvdv subgroup being generally more virulent during persistent infection, the epidemiologically unrelated concentration of e subgroup bvd viruses in stillborn calves and aborted foetuses provides preliminary evidence that viruses of this subgroup may play a special role in prenatal and perinatal losses due to bvd virus. virus in sheep's skin macrophages infected with cytopathic bovine viral diarrhea virus release a factor(s) capable of priming uninfected macrophages for activationinduced apoptosis co-existence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation genetic heterogeneity of bovine viral diarrhoea viruses isolated in southern africa pathogenesis of primary respiratory disease induced by isolates from a new genetic cluster of bovine viral diarrhea virus type i phylogenetic analysis of pestiviruses from domestic and wild ruminants rna recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease genetic and antigenic characterization of novel pestivirus genotypes: implications for classification bovine virus diarrhea mucosal disease: clinical findings in calves and heifers clinical findings and treatment of cattle with botulism clinical findings in cattle with traumatic pericarditis kasuistischer beitrag zur mucosal disease bovine viral diarrhea virus: biotypes and disease genetic heterogeneity of bovine viral diarrhoea virus in italy the development of early vs. late onset mucosal disease is a consequence of two different pathogenic mechanisms bovine viral diarrhea virus (bvdv) b: predominant bvdv subtype in calves with respiratory disease immunohistochemical diagnosis of persistent infection with bovine viral diarrhea virus (bvdv) on skin biopsies zur epizootologischen bedeutung der sö mmerung bei der mucosal disease/virusdiarrhö e des rindes the extended genetic diversity of bvdv- : typing of bvdv isolates from france phylogenetic analysis of bovine pestiviruses: testing the evolution of clinical symptoms bovine viral diarrhea virus strain oregon: a novel mechanism for processing of ns - based on point mutations temporal modulation of an autoprotease is crucial for replication and pathogenicity of an rna virus experimental mucosal disease in cattle: changes of lymphocyte subpopulations in peyer's patches and in lymphoid nodules of large intestine comparative investigation of tissue alterations and distribution of bvd-viral antigen in cattle with early onset versus late onset mucosal disease recombination with a cellular mrna encoding a novel dnaj protein results in biotype conversion in genotype bovine viral diarrhea viruses identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities how the bovine viral diarrhea virus outwits the immune system phylogenetic, antigenic and clinical characterization of type bvdv from north america impact of variation in acute virulence of bvdv strains on design of better vaccine efficacy challenge models multiple outbreaks of severe acute bvdv in north america occurring between and linked to the same bvdv strain prevalence of cattle infected with bovine viral diarrhoea virus in switzerland genetic heterogeneity of pestiviruses of ruminants in switzerland detection of bovine virus diarrhea virus (bvdv) in peripheral blood, cell cultures and tissue using a monoclonal antigen-capture elisa pathogenesis of mucosal disease, a deadly disease of cattle caused by a pestivirus performance, survival, necropsy, and virological findings from calves persistently infected with the bovine viral diarrhea virus originating from a single saskatchewan beef herd genetic diversity of international bovine viral diarrhoea virus (bvdv) isolates: identification of a new bvdv- genetic group bovine viral diarrhoea virus genotype can be separated into at least eleven genetic groups sequencing and comparative analysis of a pig bovine viral diarrhea virus genome this work was supported by the swiss national science foundation and a grant from vetsuisse. we thank matthias schweizer and reto zanoni for helpful discussions, the team of the diagnostic unit for technical help, marcus doherr for statistical support and ruth parham for linguistic assistance. key: cord- -a h u x authors: cho, yong-il; han, jae-ik; wang, chong; cooper, vickie; schwartz, kent; engelken, terry; yoon, kyoung-jin title: case–control study of microbiological etiology associated with calf diarrhea date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: a h u x calf diarrhea is a major economic burden for the us cattle industry. a variety of infectious agents are implicated in calf diarrhea and co-infection of multiple pathogens is not uncommon in diarrheic calves. a case–control study was conducted to assess infectious etiologies associated with calf diarrhea in midwest cattle farms. a total of and fecal samples were obtained from diarrheic and healthy calves, respectively, from cattle farms. samples were tested by a panel of multiplex pcr assays for enteric pathogens: bovine rotavirus group a (brv-a), bovine coronavirus (bcov), bovine viral diarrhea virus (bvdv), bovine enterovirus (bev), bovine norovirus (bnov), nebovirus, bovine torovirus (btov) salmonella spp. (salmonella), escherichia coli (e. coli) k (+), clostridium perfringens with β toxin gene and cryptosporidium parvum (c. parvum). the association between diarrhea and detection of each pathogen was analyzed using a multivariate logistic regression model. more than a half of the fecal samples from the diarrheic calves had multiple pathogens. statistically, brv-a, bcov, bnov, nebovirus, salmonella, e. coli k (+), and c. parvum were significantly associated with calf diarrhea (p < . ). among them, c. parvum and brv-a were considered to be the most common enteric pathogens for calf diarrhea with high detection frequency ( . % and . %) and strong odds ratio ( and . ). unexpectedly bnov (or = . ) and nebovirus (or = . ) were identified with high frequency in diarrheic calves, suggesting these viruses may have a significant contribution to calf diarrhea. calf diarrhea is a major cause of economic loss with high morbidity and mortality in the cattle industry worldwide (bartels et al., ; de la fuente et al., ; kelling et al., ; uhde et al., ; united, ) . many factors are known to contribute to calf diarrhea. historically, calf diarrhea has been commonly attributed to bovine rotavirus group a (brv-a), bovine coronavirus (bcov), bovine viral diarrhea virus (bvdv), salmonella spp. (salmonella), escherichia coli (e. coli) k + , and clostridium perfringens (c. perfringens) type c and cryptosporidium parvum (c. parvum) (acha et al., ; reynolds et al., ; saif and smith, ; snodgrass et al., ) . the specific etiology of many field cases of calf diarrhea still remain undiagnosed (milnes et al., ) . recently, bovine norovirus (bnov), nebovirus, bovine enterovirus (bev) and veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] calf diarrhea is a major economic burden for the us cattle industry. a variety of infectious agents are implicated in calf diarrhea and co-infection of multiple pathogens is not uncommon in diarrheic calves. a case-control study was conducted to assess infectious etiologies associated with calf diarrhea in midwest cattle farms. a total of and fecal samples were obtained from diarrheic and healthy calves, respectively, from cattle farms. samples were tested by a panel of multiplex pcr assays for enteric pathogens: bovine rotavirus group a (brv-a), bovine coronavirus (bcov), bovine viral diarrhea virus (bvdv), bovine enterovirus (bev), bovine norovirus (bnov), nebovirus, bovine torovirus (btov) salmonella spp. (salmonella), escherichia coli (e. coli) k + , clostridium perfringens with b toxin gene and cryptosporidium parvum (c. parvum). the association between diarrhea and detection of each pathogen was analyzed using a multivariate logistic regression model. more than a half of the fecal samples from the diarrheic calves had multiple pathogens. statistically, brv-a, bcov, bnov, nebovirus, salmonella, e. coli k + , and c. parvum were significantly associated with calf diarrhea (p < . ). among them, c. parvum and brv-a were considered to be the most common enteric pathogens for calf diarrhea with high detection frequency ( . % and . %) and strong odds ratio ( and . ). unexpectedly bnov (or = . ) and nebovirus (or = . ) were identified with high frequency in diarrheic calves, suggesting these viruses may have a significant contribution to calf diarrhea. ß elsevier b.v. all rights reserved. bovine torovirus (btov) have been identified as potential causes of calf diarrhea (blas-machado et al., ; haschek et al., ; hoet et al., a; kaplon et al., ; otto et al., ; park et al., park et al., , a . some of these agents (i.e., bnov, bev and btov) have also been found in feces from clinically healthy calves (haschek et al., ; jimenez-clavero et al., ; mijovski et al., ; shanks et al., ) and many of previous epidemiological studies for bnov and btov have been focused only on diarrheic calves (hoet et al., b; milnes et al., ; park et al., park et al., , b . their role in calf diarrhea still remains to be evaluated. various laboratory methods have been applied for the detection of infectious agents in feces. historically, virus isolation, electron microscopy, enzyme-linked immunosorbent assay, latex agglutination test, bacterial culture, direct microscopy of fecal smear (acid-fast stain), and/or fecal flotation have been commonly used to test fecal samples for enteric pathogens . these procedures are reliable; however, they are time-consuming and require specialized knowledge. recently, nucleic acid based tests, such as polymerase chain reaction (pcr) assays, have become popular for rapid and sensitive detection of infectious agents cho et al., ) . multiplex real-time pcr panels have been proven to be a useful diagnostic tool for concurrent detection of several target enteric pathogens with high sensitivity and specificity cho et al., ) , which decreases bias in diagnostic outcome due to testing method. the following case-control study was conducted to: (a) assess the prevalence of infectious agents consisting of common [brv-a, bcov, bvdv, salmonella, e. coli k + , c. perfringens with b toxin gene (cpt b) and c. parvum] and emerging enteric pathogens (bnov, nebovirus, bev and btov) in fecal samples from healthy and diarrheic calves in the midwest by using a panel of pcr assays; and (b) determine their association with diarrhea as well as investigate their potential interactions in expression of disease. all fecal samples used in the study were originated from clinically diarrheic and healthy calves during year - . a total of fecal samples from diarrheic calves were procured from submissions to the iowa state university veterinary diagnostic laboratory (isuvdl) and used as cases. the samples were from cattle farms with the most of the samples ( %) originated in the midwest [iowa ( %), minnesota ( %), wisconsin ( %), missouri ( %), ohio ( %), illinois ( %), south dakota ( %) and nebraska ( %)]. no more than samples were randomly selected from the same farm if a large number of samples were submitted. a vast majority of the samples tested were from sick animals before treatment begun according to referring veterinarians. approximately % and % of the samples were from dairy and beef breeds, respectively. the remaining . % of the samples were submitted without breed identification. physical appearance of first of the fecal samples was recorded as 'watery' or 'semi-solid' upon receiving as fresh samples were available to the investigators before freezing. a total of fecal samples were collected from clinically healthy (i.e., no diarrhea) calves in different beef or dairy farms which were evenly distributed across the state of iowa and used as controls. these farms were pre-selected to be part of other field-based study in which on-going health monitoring was required including use of any medication. samples were collected twice from each farm at approximately -week intervals with continuous monitoring of health status to ensure lack of diarrhea among animals on each farm. at each time of sample collection, calves were randomly selected for sampling. most of the source farms were similar in overall farm management, including vaccination and medication, and nutritional status. most ( . %) of the calves tested were less than months old in age. two third of the control calves were less than months of age while % of the case calves were less than months of age. only and cases were submitted from a -month-old diarrheic calf and clinically healthy yearlings or older cattle, respectively. all fecal samples were examined for different microorganisms (i.e., brv-a, bcov, bvdv, bev, bnov, btov, nebovirus, salmonella, e. coli k + , c. parvum and cpt b) using a panel of polymerase chain reaction (pcr) based assays. all except bev have been reported as pathogens implicated in calf diarrhea. before pcr testing, each fecal sample was suspended in . m phosphate-buffered saline (ph . ) to make % fecal homogenates and then centrifuged for min at  g for clarification as previously described . the supernatant was then used for viral and bacterial nucleic acid extraction using magmax tm total nucleic acid isolation kit (applied biosystems, austin, tx) according to the manufacturer's instruction. the extraction procedure was performed using kingfisher magnetic particle processor (thermo fisher scientific inc., waltham, ma). all extracts were stored at À c until tested. probe-based real-time pcr (rtpcr) assays for all pathogens except btov and nebovirus were performed in a duplex or singleplex pcr format with path-id tm multiplex one-step rt-pcr kit (applied biosystems, austin, tx) and agpath-id tm one-step rt-pcr kit (applied biosystems, austin, tx), respectively. for btov, a sybr green rtpcr assay was used with quantitest tm sybr green pcr kit (qiagen, valencia, ca). for rtpcr set-up, ml of template and ml of the reaction mixture for the duplex pcrs (table , real-time pcr set , , and ) and ml of template and ml of the reaction mixture for singleplex pcrs (table , real-time pcr set and ) were used. all reaction mixtures contained nm of each primer, nm of the probe except btov, rt-pcr buffer, rt-pcr enzyme mix, and nuclease-free water. the volume of each reagent added to a reaction mixture was as per manufacturer's instruction. the sequence information of primers and probes used for specific detection of each pathogen is summarized in table . amplification of the targeted genomic region was conducted using abi fast real-time pcr system (applied biosystems, austin, tx). cycling conditions of the probe-based rtpcrs were as follows: (a) reverse transcription (rt) for min at c ( c for singleplex); (b) activation of dna polymerase at c for min ( min for singleplex); and (c) cycles of denaturation at c for s and annealing/extension at c for s ( s for singleplex). the rt step was applied only for viral targets. running conditions of the sybr green rtpcr for btov were: (a) rt step for min at c; and (b) cycles of denaturation at c and annealing/extension at c for . after cycle reaction, the melting curve analysis was performed. samples with cycle threshold (ct) for any given targets were considered positive for those pathogens. for detection of nebovirus, a gel-based nested rt-pcr was used as previously described (jor et al., ) . the pcr was conducted using onestep rt-pcr kit (qiagen, valencia, ca) with qiagen rnase inhibitor and hotstartaq dna polymerase kit (qiagen, valencia, ca) for rt-pcr and nested pcr, respectively, according to the manufacturer's instruction. cycling conditions of the rt-pcr were: (a) rt step at c for min; (b) dna polymerase activation step at c for min; (c) cycles of denaturation at c for s, annealing at c for s and extension at c for min; and (d) followed by a final cycle at c for min. cycling conditions of the nested pcr were: (a) activation step at c for min; and (b) cycles of denaturation at c for s, annealing at c for s and extension at c for min; and (c) followed by a final cycle at c for min. table oligonucleotide sequence of primers and probe used in pcr to detect each target enteric pathogen. target pathogen [primer/probe sequence ( - )] detection frequencies of selected enteric pathogens (i.e., brv-a, bcov and c. parvum) in calf diarrhea cases submitted to isuvdl from to were compared based on laboratory methods used. before , antigencapturing elisa and fecal smear direct microscopy (acidfast stain) tests were used to detect brv-a/bcov and c. parvum in feces, respectively. since then, a bovine enteric panel (bep) consisting of multiplex rtpcr tests was implemented for simultaneous detection of brv-a, bcov, and c. parvum in feces. all diagnostic data were retrieved from the isuvdl laboratory information management system. the pcr results on each of the fecal samples were recorded as either positive or negative for each pathogen and categorized under disease status (i.e., diarrheic versus non-diarrheic) of each animal. the association between diarrhea and detection of each pathogen was determined using a multivariate logistic regression model. the probability of concurrent detection among pathogens was also analyzed in the same manner. the final model was built with stepwise selection using firth's penalized likelihood method due to quasi-complete separation of the data. odds ratios (ors) with % confidence intervals were calculated to assess the likelihood of association. the association between the severity of diarrhea (i.e., watery versus semi-solid) and the presence of each pathogen was also analyzed using multivariate logistic regression model with stepwise model selection. since bnov and bcov were detected in feces from both diarrheic and healthy calves at a relatively high frequency, ct values of feces for bnov and bcov were analyzed by the non-parametric wilcoxon rank-sum test to evaluate the quantitative difference in virus shedding between diarrheic and healthy calves. all statistical analyses were conducted by using sas . (sas institute, cary, nc). for all analyses, a value of p < . was considered significant. a total of the fecal samples from diarrheic calves and fecal samples from healthy calves were tested for putative enteric pathogens. pcr testing revealed that . % and . % of the diarrheic and normal fecal samples, respectively, were positive for at least one of these infectious agents. as summarized in table , bnov ( . %), c. parvum ( . %), bcov ( . %), brv-a ( . %), nebovirus ( . %) and salmonella ( . %) were commonly detected in feces from the diarrheic calves, while bvdv, btov, e. coli k + and bev were found at a much lower frequency ( . - %). bnov ( . %) and bcov ( . %) were also detected in the feces from healthy calves but at a lower frequency than that in diarrheic feces. while nebovirus ( . %) and bvdv ( . %) were infrequently detected in the feces from healthy calves, c. parvum, brv-a, e. coli k + and btov were detected only in the feces from diarrheic calves. in contrast, bev ( . %) was much more frequently detected in the feces from healthy calves than those from diarrheic calves. c. perfringens with b toxin gene (i.e., c. perfringens type b or c) was not detected in any of the feces examined in this study. although bnov and bcov were detected in feces from both diarrheic and healthy calves, the detection frequency and fecal shedding quantity of the viruses were significantly higher in the feces from diarrheic calves except for one healthy calf feces which showed the lowest ct value ( . ) for bcov (fig. ) , as compared to those in the feces from healthy calves. the median (mean ae se) ct values of feces from diarrheic calves positive for bnov and bcov were . ( . ae . ) and . ( . ae . ) respectively, whereas those from healthy calves positive for bnov and bcov were . ( . ae . ) and . ( . ae . ) respectively. with respect to age distribution, many of fecal samples from diarrheic calves positive for bnov, c. parvum, bcov, brv-a, nebovirus, salmonella, btov, and e. coli k + were from calves at to weeks of age (fig. ) . in particular, calves at - weeks of age were the most commonly positive for these pathogens. as summarized in table , the presence of c. parvum, e. coli k + , salmonella, brv-a, nebovirus, bcov and bnov in feces were significantly associated with calf diarrhea (p < . ). among these pathogens, c. parvum, e. coli k + , salmonella, brv-a and nebovirus showed a stronger association with diarrhea (or > . ). in contrast, detection of bev was inversely correlated with diarrhea (or = . ); therefore, bev was not included in further statistical analyses. no statistically significant association between the presence of btov in feces and diarrhea was observed in this study even though the virus was detected only in the feces from diarrheic calves, probably due to a low frequency of detection ( table ). the ors could not be calculated for bvdv and cpt b because of either extremely low frequency of detection or no detection; hence, statistical significance could not be determined. bovine rotavirus group a was the only pathogen significantly (p = . ) associated with liquid form of diarrheic feces (table ) . while % of the diarrheic fecal samples had more than enteric pathogen detected, only % of the fecal samples from healthy calves had multiple pathogens (fig. ) . in the diarrheic fecal samples, the presence of different pathogens ( %) was the most commonly seen and % of the samples even had up to different pathogens concurrently. the probability of detecting certain agents together is summarized in table . bovine norovirus, bcov, salmonella, and c. parvum were commonly detected in feces which were also positive for brv-a. nebovirus was commonly detected in feces also positive for bcov, c. parvum or btov. bnov presence was significantly correlated with c. parvum presence in addition to brv-a. while many of the pathogens were concurrently detected with more than other pathogens, btov and salmonella were identified only with nebovirus and brv-a, respectively. the concurrent presence of btov and nebovirus was much stronger ] as compared to other mixed infections. statistically significant synergistic interaction between pathogens for causing the diarrhea or exacerbating the severity of diarrhea was not observed. when the pathogens were sorted based on their taxonomical property (i.e., virus, bacteria and protozoa) and compared for their detection frequency between diarrheic and healthy calves, virus only ( . %) or virus/c. parvum co-infection ( . %) was the most commonly observed in the diarrheic calves. in comparison, virus only ( . %) was common in the healthy calves (table ) . bnov and bcov were the pathogens that were the most commonly detected in the feces from healthy calves. the mean detection frequency of brv-a, bcov and c. parvum in diarrhea cases during year - were . %, . % and . %, respectively, when antigen-capturing elisas and direct microscopy (acid-fast stain) were the main laboratory methods for detection of these pathogens at isuvdl. after implementation of a pcr panel for the major calf diarrhea pathogens, the mean detection frequency of brv-a, bcov and c. parvum were . %, . % and . %, respectively, during year - (table ) . in this study, we investigated the prevalence of calf enteric pathogens consisting of common (brv-a, bcov, bvdv, salmonella, e. coli k + , cpt b and c. parvum) and emerging pathogens (bnov, nebovirus, bev and btov) and then evaluated two aspects; their clinical significance in calf diarrhea and co-infection between them. not unexpectedly, % of diarrheic calves tested were positive for at least one of the target enteric pathogens, suggesting that the infectious factor is still a major cause of calf diarrhea. more than % of the diarrheic calves tested were concurrently infected with more than one pathogen. coinfection with pathogens was the most common finding ( %) with up to pathogens detected in % of the fecal samples from diarrheic calves. the majority of diarrheic cases were identified among -to -week-old calves and concentrated among calves at - weeks of age, which is similar to previous reports by other investigators (bartels et al., ; de la fuente et al., ; mcdonough et al., ) . high frequency of co-infection by multiple pathogens in young animals emphasizes that interventions for calf diarrhea should be focused on husbandry and management strategies, including assurance of colostrum intake, hygiene, reduction of population density, or modified components of the sandhills calving system (larson and tyler, ) . twenty percent of the diarrheic calves were negative for all of the pathogens in this study. while low sensitivity of the test might be accounted for the negative result, the role of non-infectious factors (e.g., cold weather, impaired uptake of colostrum, or poor sanitation) in calf diarrhea cannot be discounted. in addition, the possibility of other pathogens (e.g., rotavirus b or c; coccidia; c. perfringens type a, d or e; and other pathogroups of e. coli) or previously unrecognized agent(s) involved in diarrhea remains to be further studied. viral infections ( . %) or combination of viruses and c. parvum ( . %) were the most commonly detected etiology in feces from diarrheic calves, which is similar to previous reports on calf diarrhea (de la fuente et al., ; garcia et al., ; mcdonough et al., ) . in contrast, the proportion of bacteria-positive samples was relatively small. of three target bacterial pathogens, salmonella ( %) was the most commonly detected in the diarrhea feces examined. interestingly, none of the fecal samples from both diarrheic and healthy calves was positive for cpt b which is contained in either c. perfringens type b or c. this was an unexpected observation since c. perfringens type c has been postulated as the main type causing calf diarrhea. although the pcr results were not confirmed by anaerobic bacterial culture, it should be noted that our observation is in agreement with previous reports by other investigators describing no ferrarezi et al., ; sting, ) or very low detection of cpt b (gurjar et al., ) in diarrheic calves, suggesting that c. perfringens type c is rarely involved in outbreaks of calf diarrhea or is simply an opportunistic bacterium causing acute enterotoxemia under certain favorable conditions. as it was suggested that all types of c. perfringens should be considered as a calf diarrhea etiology (ferrarezi et al., ) , involvement of other types of c. perfringens in diarrhea cases may be necessary. c. parvum was frequently ( . %) detected in calf diarrhea cases, which is in agreement with previous reports (bartels et al., ; izzo et al., ; mcdonough et al., ; uhde et al., ) . it may imply the difficulty with c. parvum control in the field due to autoinfection, environment resistance of oocysts and lack of effective treatment and vaccine (joachim et al., ) . preventative measures for c. parvum in cow-calf operations should be focused on keeping good herd sanitation and sick animals segregated from healthy ones (trotz-williams et al., ) . co-infection with viruses ( . %), particularly brv-a (or = . ), bnov (or = . ) and nebovirus (or = . ), was much more common than with bacteria in our study. while co-infection of brv-a and c. parvum in diarrheic calves has been frequently reported (bartels et al., ; bjorkman et al., ; de la fuente et al., ; garcia et al., ; uhde et al., ) , common association of nebovirus and c. parvum in diarrheic animals is a new observation. it has been reported that viral infections, such as porcine circovirus type and human immunodeficiency virus, can increase the susceptibility of pigs and humans, respectively, to c. parvum (nunez et al., ; putignani and menichella, ) , suggesting that immunosuppressive viruses can predispose animals or humans to c. parvum. in the absence of effective treatment options for c. parvum, it may be prudent to rely on management practices and specific aids in prevention of viral infections to reduce clinical problems with c. parvum infections. bovine rotavirus a was found solely in many of the diarrhea cases ( . %) and positively correlated with the severity (i.e., liquid feces) of diarrhea (or = . ). this observation is similar to reports of human rotavirus infection being highly associated with acute watery diarrhea (olesen et al., ; wilhelmi et al., ) . a high correlation between brv-a detection and diarrhea (or = . ) and a wide range of association with other pathogens (bnov, bcov, salmonella and c. parvum) may be an evidence that brv-a is a primary major bovine enteric pathogen of calf diarrhea, which is also in agreement with previous reports describing the primary role of brv-a in neonatal calf diarrhea (bartels et al., ; garcia et al., ; uhde et al., ) . our and others' observations raise concerns regarding vaccination practices on farms and the efficacy of current licensed brv-a vaccines since vaccination has been a main tool for prevention of brv-a associated diarrhea in neonates. implementation of a regular vaccination program for brv-a can be easily achieved through enhancing the awareness of the high frequency of rotavirus-associated calf diarrhea in the field, but continuing efficacy of brv-a vaccines may require frequent surveillance and further characterization of rotaviruses circulating in the field. surveillance is warranted since antigenic variation of rotavirus due to frequent mutation and recombination is of a great concern for emerging a variant or new serotype (martella et al., ) . new and emerging viruses with pathogenic potential for calf diarrhea (i.e., bnov, nebovirus and btov) were also studied together with historically well-known major enteric pathogens (i.e., brv-a, bcov, bvdv, c. parvum, salmonella and e. coli k + and cpt b). the most noteworthy observations from our study were the significant association of bnov (or = . ) and nebovirus (or = . ) with calf diarrhea and their frequent detection ( . % and . %, respectively) in calf diarrhea cases, suggesting that bovine caliciviruses may play a more significant role in calf diarrhea than what was believed. it is an unexpected observation that nebovirus was detected in diarrheic animals at a much higher rate than what was previously reported from france (kaplon et al., ) . a high frequency of bnov detection is, on the other hand, not a surprise since many other investigators have previously reported a high prevalence of bnov infection in the studied bovine populations di bartolo et al., ; jor et al., ; kaplon et al., ; mijovski et al., ; park et al., ; reuter et al., ; van der poel et al., ; wise et al., ; yilmaz et al., ) . clinical significance of bnov infection has not been clear in the field because the virus has also been found in clinically healthy calves (jor et al., ; mijovski et al., ) as also shown in our study. recently an animal study has demonstrated that bnov is pathogenic to naïve calves (otto et al., ) . in our study, which is the first case-control study evaluating bnov as bovine enteric pathogen for calf diarrhea, a significant quantitative difference in the virus amount between fecal samples from diarrheic and healthy calves was detected, suggesting that disease progression may depend upon the initial exposure dose of the virus or factors contributing to bnov replication to a high titer. while such a difference in replication ability did not appear to be due to unique genetic profile of bnov's polymerase gene (data not shown), further study remains to redefine the pathogenicity of bovine caliciviruses and to determine the correlation between virus amount and the pathogenicity and identify contributing factors. unlike bovine caliciviruses, it was difficult to judge the role that btov may play in calf diarrhea because the virus was detected in a relatively small number of the fecal samples examined ( . %). such a detection frequency of btov in our study is similar to what was previously reported from korea ( . %) and austria ( . %) but different from that reported in usa ( . %) and japan ( %) (duckmanton et al., ; haschek et al., ; kirisawa et al., ; park et al., b) . although a statistically significant association between btov and diarrhea could not be demonstrated due to a low prevalence, it must be pointed out that the virus was detected only in feces from diarrheic calves. a survey on a larger number of animals, longitudinal cohort study or animal challenge study would be necessary to determine the clinical significance of btov for calf diarrhea. bovine coronavirus is historically believed to be a major bovine enteric pathogen causing calf diarrhea, corroborated by pathologic studies (boileau and kapil, ) . however, such a role has been challenged as some epidemiological studies could not demonstrate a statistically significant association between bcov infection and calf diarrhea (bartels et al., ; bjorkman et al., ; uhde et al., ) . a recent cohort study on dutch cattle farms even suggested potential opportunistic nature of bcov infection with previous history of diarrhea (bartels et al., ) . in our study, bcov was found to be significantly associated with calf diarrhea although its association strength with calf diarrhea was relatively weak (or = . ) as compared to pathogens historically known to be causes of calf diarrhea such as brv, c. parvum, e. coli k + and salmonella. as reported by other investigators, the virus was also detected in some of the fecal samples ( . %) from healthy calves. while this was initially suspected to be due to fecal shedding of a vaccine virus (theil and mccloskey, ; thiel et al., ) , brv-a was not detected concurrently in those bcov-positive samples, noting that commercial vaccines for calf scouring contain both bcov and brv-a in a live form. co-infection or other factors may contribute to diarrhea in association with bcov infection as levels of bcov in feces from diarrheic calves were significantly higher than those in feces from healthy calves. such a quantitative difference may be a useful criterion in determining the clinical significance of bcov detection during diagnostic investigation. bovine enterovirus is commonly present in gastrointestinal track in cattle and highly prevalent in high-density cattle farms ley et al., ) . the virus is also known to be stable in the environment . most of bev infections are subclinical, although gastroenteritis and reproductive disease associated with bev infection have been reported (blas-machado et al., . in our study, detection of bev did not demonstrate a statistically significant association with calf diarrhea (or = . ). in fact bev was more commonly detected in feces from healthy calves, which supports asymptomatic infection of bev in bovine gastrointestinal track ley et al., ) . accurate and rapid diagnosis of pathogens of bovine enteric disease is important for quick and appropriate interventions in the field to mitigate losses (mcguirk, ) . the detection frequency of brv-a, bcov and c. parvum was increased by . - . times after implementing the bep, pcr-based testing in isu-vdl. such an increase in incidence and/or prevalence is more likely attributed to higher sensitivity and specificity of bep than conventional tests and accurately reflects actual epidemiology of these pathogens in the field, while there was neither apparent increase of sample submissions nor change in personnel or fees associated with calf diarrhea testing at the lab were made during the study period. interestingly, the detection frequency of c. parvum in diarrhea cases increased by . times (i.e., from . % to . %) after implementation of bep, raising awareness of the epidemiological and clinical significance of c. parvum in the field. this observation is an example of the bias of test sensitivity on interpretation of infection prevalence or disease prevalence, which, in turn, can misguide veterinary practitioners or producers on disease intervention or animal management on farm. continuous and frequent evaluation of the performance of diagnostic tests in context of impact on the animal (infection versus disease) is highly desired to minimize misclassification of data (david et al., ) . our study was not an age-matched case-control study, which could introduce a bias into frequency of certain pathogen detection. besides, use of diagnostic submissions for the study could also bias the study outcome as sick animals may have handled differently before samples were taken from the animals. nonetheless, age distribution between cases and controls was similar. many other factors, such as sex, breed, sampling season and farm management, were similar between case and control groups. use of a pcr-based panel for all targeted agents may have reduced potential bias due to section of different tests for different pathogens. therefore, observed detection frequency and association strength between certain pathogens and calf diarrhea would be decent assessment. in conclusion, co-infection of multiple pathogens is common in calf diarrhea cases although clinical significance/role of each pathogen in diarrhea may vary and remains to be further studied for some pathogens. c. parvum and brv-a appear to be the primary enteric pathogens significantly contributing to calf diarrhea under conditions presented in the study. frequent detection of bovine caliciviruses, such as bnov and nebovirus, in feces from diarrheic calves raises the need to pay attention to these viruses with respect to the management of enteric disease on farm. use of a pcrbased testing panel (e.g., multiplex real-time pcrs) covering a wide range of known and potential pathogens with defined sensitivity and specificity is strongly recommended for monitoring/surveillance of populations for diseases, particularly when dealing with multifactorial diseases such as calf diarrhea or bovine respiratory disease complex. such a screening test for multiple pathogens would be useful for not only studying the host-agent ecology, disease expression and dynamics in a population but also developing an effective intervention strategy for disease control or prevention. in addition, further characterization of pathogens with high rate of mutation on the on-going basis may be necessary to keep a vaccine-based intervention strategy effective. ctagtaaccaggctgatgtcaatacc bcov-rev: ggcggaaacctagtcggaata bcov-probe: (fam/mgb) cggctgacattctcgatc brv-fwd : tcaacatggatgtcctgtactcct brv-fwd : tcaacatggatgtcctgtattcct brv-fwd : tcaacatggatgtcctttattcct brv-rev : tcctccagtttggaactcatt brv-rev : tcccccagtttggaattcatt brv-rev : ccctccagtttggaattcatt brv-probe : (vic/mgb) tcaaaaactcttaaagatgctag brv-probe : (vic/mgb) real-time pcr (set ) bev-fwd: gccgtgaatgctgctaatcc bev-rev: gtagtctgttccgccycyract bev-probe: (fam/bhq ) cgcacaatccagtgttgctacgtcgtaac cy /bhq ) real-time pcr (set ) bnov-fwd: cgctccatgttygcbtgg bnov-rev: tcagtcatcttcatttacaaaatc bnov-probe: (fam/zen/iabkfq) sybr green real-time pcr (set ) btov-fwd: ttactggytattgggcmyt btov-rev: aaaggrgtgcagtgwagctt before bep studies on calf diarrhoea in mozambique: prevalence of bacterial pathogens real-time multiplex pcr assays for reliable detection of clostridium perfringens toxin genes in animal isolates prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young dutch dairy calves cryptosporidium parvum and giardia intestinalis in calf diarrhoea in sweden fatal ulcerative and hemorrhagic typhlocolitis in a pregnant heifer associated with natural bovine enterovirus type- infection pathogenesis of a bovine enterovirus- isolate in experimentally infected calves bovine coronavirus associated syndromes detection and molecular characterization of bovine norovirus among bovine diarrhea cases in us midwest development of a panel of multiplex real-time polymerase chain reaction assays for simultaneous detection of major agents causing calf diarrhea in feces activeepi companion textbook proportional morbidity rates of enteropathogens among diarrheic dairy calves in central spain cryptosporidium and concurrent infections with other major enterophatogens in to -day-old diarrheic dairy calves in central spain a pilot survey of bovine norovirus in northern italy detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms genotyping of clostridium perfringens isolated from calves with neonatal diarrhea rotavirus and concurrent infections with other enteropathogens in neonatal diarrheic dairy calves in spain real-time multiplex pcr assay for rapid detection and toxintyping of clostridium perfringens toxin producing strains in feces of dairy cattle real-time pcr for quantification of giardia and cryptosporidium in environmental water samples and sewage detection of bovine torovirus in neonatal calf diarrhoea in lower austria and styria (austria) detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves development of universal sybr green real-time rt-pcr for the rapid detection and quantitation of bovine and porcine toroviruses prevalence of major enteric pathogens in australian dairy calves with diarrhoea survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription-pcr comparison of viability assays for cryptosporidium parvum oocysts after disinfection sybr green based real-time rt-pcr assay for detection and genotype prediction of bovine noroviruses and assessment of clinical significance in norway possible novel nebovirus genotype in cattle effect of infection with bovine viral diarrhea virus alone, bovine rotavirus alone, or concurrent infection with both on enteric disease in gnotobiotic neonatal calves detection of bovine torovirus in fecal specimens of calves with diarrhea in japan reducing calf losses in beef herds bovine enteroviruses as indicators of fecal contamination detection of bovine viral diarrhea virus by taqman reverse transcription polymerase chain reaction zoonotic aspects of rotaviruses enteric pathogens in intensively reared veal calves disease management of dairy calves and heifers detection and molecular characterisation of noroviruses and sapoviruses in asymptomatic swine and cattle in slovenian farms retrospective study of noroviruses in samples of diarrhoea from cattle, using the veterinary laboratories agency's farmfile database real-time pcr method for salmonella spp. targeting the stn gene coinfection by cryptosporidium parvum and porcine circovirus type in weaned pigs etiology of diarrhea in young children in denmark: a case-control study infection of calves with bovine norovirus giii. strain jena virus: an experimental model to study the pathogenesis of norovirus infection molecular epidemiology of bovine noroviruses in south korea molecular detection and characterization of unclassified bovine enteric caliciviruses in south korea molecular epidemiology of bovine toroviruses circulating in south korea global distribution, public health and clinical impact of the protozoan pathogen cryptosporidium detection of genotype and bovine noroviruses in hungary microbiology of calf diarrhoea in southern britain enteric viral infections of calves and passive immunity quantitative pcr for detection and enumeration of genetic markers of bovine fecal pollution aetiology of diarrhoea in young calves detection of beta and major toxin genes by pcr in clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia rotavirus shedding in feces of gnotobiotic calves orally inoculated with a commercial rotavirus-coronavirus vaccine how long are viruses detected by pcr in blood and nasal swabs of calves vaccinated with infectious vaccines? calf-level risk factors for neonatal diarrhea and shedding of cryptosporidium parvum in ontario dairy calves prevalence of four enteropathogens in the faeces of young diarrhoeic dairy calves in switzerland epidemiology of norwalk-like virus infections in cattle in the netherlands rapid detection of escherichia coli virulence factor genes using multiplex real-time taqman pcr assays viruses causing gastroenteritis molecular characterization of noroviruses detected in diarrheic stools of michigan and wisconsin dairy calves: circulation of two distinct subgroups sensitive multiplex real-time reverse transcription-pcr assay for the detection of human and animal noroviruses in clinical and environmental samples first report on the phylogeny of bovine norovirus in turkey the authors would like to thank jessica boor and jacqueline thomas for their excellent assistance in sample collection from vdl submissions. the authors are grateful to drs. annette o'connor and grant dewell for their critical review of the manuscript. the study was supported in part by funding from calf scouring fund, usda cooperative state research, education, and extension service (award no. - - ), the national research foundation on behalf of the korean ministry of education, science and technology (award no. - ) and vdl r&d fund. key: cord- -jb u xn authors: gerdts, volker; zakhartchouk, alexander title: vaccines for porcine epidemic diarrhea virus and other swine coronaviruses date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: jb u xn the recent introduction of the porcine epidemic diarrhea virus (pedv) into the north american swine herd has highlighted again the need for effective vaccines for swine coronaviruses. while vaccines for transmissible gastroenteritis virus (tgev) have been available to producers around the world for a long time, effective vaccines for pedv and deltacoronaviruses were only recently developed or are still in development. here, we review existing vaccine technologies for swine coronaviruses and highlight promising technologies which may help to control these important viruses in the future. coronaviruses were first described in the mid- s and subsequently isolated from a number of species including man, mice, swine and chicken. these viruses share a common morphological characteristic, a fringe of club-shaped projections, - nm long, around a pleomorphic - nm viral particle, having a resemblance to a solar corona (masters, ) . coronaviruses infect humans and various animal species, causing respiratory, gastrointestinal and neurological diseases as well as hepatitis. prominent examples include the severe acute respiratory syndrome virus (sars-cov), middle-eastern respiratory syndrome virus (mers-cov) and the feline infectious peritonitis virus (fipv), to name a few. swine coronaviruses can be divided into respiratory (prcov) and enteropathogenic coronaviruses such as transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov). the latter have similar epidemiological, clinical and pathological features. the family coronaviridae is currently divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. tgev and pedv belong to the alphacoronavirus genus, whereas pdcov belongs to genus of deltacoronaviruses. coronaviruses are enveloped, single-stranded, positive-sense rna viruses with the largest rna genome of approximately kb reported to date. the genomic rna includes and untranslated regions (utr), and it is capped and polyadenylated. open reading frame (orf) a and orf ab occupy the two-thirds of the genome and encode two replicase polyproteins (pp a and pp ab). expression of pp ab protein requires a ribosomal frameshift during translation of the genomic rna. produced polyproteins are proteolytically cleaved into nonstructural proteins, nsp through nsp by the proteinase activity of nsp and nsp . the -proximal one-third of the genome encodes four structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. some betacoronaviruses have an additional membrane protein, hemagglutinin esterase (he). interspersed between these genes are genes encoding accessory proteins. the number of these genes varies between different coronaviruses. for instance, tgev has accessory genes, pdcov has , whereas pedv has only one (fig. ) . the viral rna genome is packaged by the n protein into a helical nucleocapsid. in addition to the structural role, the n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, up-regulates interleukine- expression and antagonizes type i interferon production (ding et al., ; xu et al., b) . the s protein, which forms peplomers on the virion surface, mediates binding to host receptors and membrane fusion. it can be divided into s and s domains. in some coronaviruses the s protein is processed into s and s fragments by cellular proteases or trypsin (belouzard et al., ; wicht et al., ) . the s protein is a major target for virus neutralizing antibodies (chang et al., ; reguera et al., ) . the m protein is the most abundant virion component and also contains conserved linear b-cell epitopes (zhang et al., ) . the e protein is responsible for the assembly of virion, and it causes endoplasmic reticulum stress and interleukin- expression up-regulation (xu et al., a) . accessory genes are dispensable for virus growth in vitro, but they may play an important role in the virus survival in the infected host. indeed, the product of the tgev accessory gene orf reduces the expression of genes involved in the antiviral defense of the immune system, e.g. the interferon response, and inflammation (cruz et al., ) . the orf protein of pedv functions as an ion channel, and it is thought to be related with virulence of pedv (song et al., ; wang et al., ) . one of the pedv non-structural proteins, nsp , was shown to be a type i interferon suppressor (zhang et al., a) . interestingly, pdcov lacks the nsp gene. coronaviruses target predominantly type i and ii pneumocytes (prcov) or villous-and crypt enterocytes in the intestine (tgev, pedv and pdcov). pedv also infection goblet cells in the small intestine (jung and saif, ) . in addition, infection of alveolar macrophages and lamina propria macrophages has been shown for some but not all swine coronaviruses (laude et al., ; park and shin, ) . entrance of the virus into the target cells is mediated by a series of receptor ligand interactions including heparin sulfate (huan et al., ) and aminopeptidase n (apn) (chen et al., ; li et al., ) . importantly, the expression levels of aminopeptidase n appear to correlate with the level of infection, at least for pedv. the higher the expression levels the more severe is the infection (li et al., ; zhang and yoo, ) . as a result, it may be perceivable that piglets born with lower apn levels in the brush border may be more resistant to pedv than piglets with higher levels. enteric infections with tgev and pedv are characterized by severe diarrhea, vomiting and dehydration with high morbidity and mortality especially in piglets less than two weeks of age. in contrast, infections with respiratory coronaviruses cause very mild and transient disease in pigs of all ages, which often get unnoticed by the producer. unless complicated by concurrent infections, prcov infections are only short lasting with temporary phases of coughing and respiratory distress. however, prcov can become a more significant problem during co-infections with other pathogens such as the porcine reproductive and respiratory syndrome virus prrsv (jung et al., ) . infection of enterocytes with pedv results in villous atrophy which can lead to malabsorption, diarrhea and anorexia. within - h post infection, vomiting may occur, which typically does not last longer than - days post infection. diarrhea can be found within to h post infection depending on the dose of the virus and the age of the piglets. diarrhea typically lasts for about - days, but can last longer, and results in severe weight loss that often cannot be made up during the normal production cycle. viral shedding is highest between days - , but can last for days to weeks post infection. surviving piglets start to recover around - days post infection, typically around the same time when proliferation of the crypt epithelium and regeneration of the villi occurs. similarly, tgev infects villous enterocytes and causes disease that clinically is indistinguishable from pedv. mortality rates are highest in young piglets, often reaching about %. in contrast, infection with pdcov causes milder infections in piglets between and weeks of age. diarrhea, vomiting and anorexia can be found in infected animals. in general, infected animals display much milder signs compared to infections with pedv and tgev. the innate immune response to enteric coronaviruses in pigs is characterized by a rapid antiviral response in the intestine, including the release of interferons, nuclear factor kb and other antiviral molecules (chattha et al., ; jung and saif, ; sang et al., ) . pigs can produce three types of interferons (sang et al., ) : type i interferons include well known interferons such as interferon a and b (ifn-a/b) and in pigs are encoded by as many as different genes. the only type ii interferon in pigs is ifn-ɣ. type iii interferons include ifn-l (interleukin ; il- ), ifn-l (il- a), ifn-l (il- b) and ifn-l (kotenko et al., ; park et al., ; prokunina-olsson et al., ; sheppard et al., ; zhang and yoo, ) . their functions are unknown in pigs. especially type i and iii interferons are used by the host to counteract viral infections. in response, most viruses including pedv and tgev have developed strategies to evade and interfere with the interferon response. several viral proteins, including structural and non-structural proteins have been identified for pedv and tgev that can suppress the interferon response. for an excellent review on the evasion of immunity by porcine coronaviruses please see (zhang and yoo, ) . the adaptive immune response to swine enteric coronaviruses is based on secretory antibodies and cytotoxic t cells. these include secretory iga antibodies (siga) that are produced by antibodysecreting cells in the lamina propria of the mucosal tissues and systemic antibodies such as igg and igm are found in serum and interstitial tissues and some isotypes can be transsudated across the mucosal epithelium into the lumen (chattha et al., ; horton and vidarsson, ) . the cellular response to swine coronaviruses is characterized by t helper cells that are supporting the production of antibodies and cytotoxic t cells that are targeting virus infected epithelial cells. in pigs, these are predominantly ɣɗ- cells, most of which can be found within the intraepithelial layer (bonneville et al., ) . the majority of t cell epitopes are located in the spike and nucleoprotein of coronaviruses (channappanavar et al., ; saif, ; sestak et al., ) . additionally, cd t cell epitopes have been found in the membrane protein of the human sars-cov (yang et al., ) in neonatal piglets, the main mechanism of protection is mediated by lactogenic immunity. during lactation, siga, igg and igm are passively transferred to the piglet via colostrum and milk (bohl and saif, ; saif and bohl, , ; salmon et al., ) . colostrum contains predominantly igg, which is transudated from sow serum and absorbed by the piglet within the first - h of life. secretory iga is predominantly found in milk, after transitioning from colostrum to milk around - days of age (langel et al., ) . siga is produced by antibody secreting cells in the mammary gland, and it was shown many years ago by bohl and saif (bohl et al., a ) that these cells migrate from the gut to the mammary gland at the end of pregnancy. this was confirmed by others in a variety of species and chemokines such as ccl and others have been found responsible for recruiting these antibody secreting cells to the mammary gland (bourges et al., ; lazarus et al., ; meurens et al., meurens et al., , wilson and butcher, ) . thus, in order to enhance the level of maternal immunity the oral route seems to be the most obvious choice for vaccinating the sow. indeed, most vaccines for enteric coronaviruses are designed to induce lactogenic immunity by vaccinating the sow, however, most of them are administered via systemic injection. in the absence of effective vaccines for pedv, many producers are currently using a lock-down of the barn combined with feeding back infectious live virus to pregnant sows. however, the duration of immunity often does not extent more than a few years (table ) , depending on the type of vaccine being used with live vaccines typically providing longer lasting immunity. even after feed-back, immunity starts to wane after a relatively short period of time, often even less than a few months. for an excellent review of the role of lactogenic immunity for pedv see (langel et al., ) . in addition to antibodies, the colostrum also contains innate effector molecules such as defensins and antimicrobial peptides, interleukins and cytokines hlavova et al., ; mair et al., ; nechvatalova et al., ; salmon et al., ) . the level of cross-protection is somewhat unclear for coronaviruses. for pedv, goede et al. reported that -day old piglets born to sows that had been infected with a mild strain of pedv seven months previously, were protected against infection with a more virulent strain of pedv (goede et al., ) . in this experiment the sows were challenged with a more virulent pedv virus at day of gestation, and orally re-challenged when the piglets were three days old. none of the sows displayed significant clinical symptoms. the piglets were orally challenged with ml of mucus scrapings of the more virulent pedv. while mortality and morbidity rates varied significantly amongst the piglets in each group, the overall morbidity and mortality was reduced in piglets born to sows that had been pre-exposed to pedv. in the s, tgev was responsible for severe economic losses around the globe. several vaccine technologies were developed and commercialised. by administration to sows, the importance of lactogenic immunity was established (bohl et al., b; saif and bohl, ) . however, with a disappearance of the disease in many parts of the world, fewer vaccines are now commercially available in north america and europe (table ). most current commercial tgev vaccines are live attenuated vaccines that are given to the sow during gestation in order to provide lactogenic immunity to the newborn piglet. these vaccines are often available as bi-or trivalent vaccines combined with rotavirus, pedv and/or escherichia coli. experimental vaccines include novel dna vaccines, vectored vaccines and recombinant vaccines (table ) . for example, the porcine adenovirus was used to deliver the tgev spike protein (tuboly and nagy, ) . yuan et al. used the swine pox virus to express the a epitope of the spike protein (yuan et al., ) . dna plasmids were generated for both pedv and tgev for the development of a dna vaccine (meng et al., ) . recombinant proteins (spike and nucleocapsid) have been extensively evaluated as recombinant vaccine following expression in bacteria, yeast and plants. many of these are being assessed for their potential to mucosal immunity after oral administration. oral delivery has not been demonstrated. the first vaccine for pedv in the us was developed by harrisvaccines tm in iowa, and conditionally licensed in . the vaccine, initially called iped vaccine, was based on a truncated version of the pedv spike gene produced in the sirravax℠ rna particle technology platform , which is based on the a pvek replicon vector derived from the venezuelan equine encephalitis virus. the technology is a propagation defective, single cycle rna particle technology that is believed to target dendritic cells. a longer version of the spike genes was codon optimized and used in the second-generation vaccine called iped plus, which now is commercially available as porcine epidemic diarrhea vaccine, rna (ped rna). the vaccine induced immunity in young pigs after two doses given intramuscularly in a threeweek interval. vaccinated weaned pigs when challenged with homogenized gut tissue from a clinical isolate displayed significantly reduced severity of clinical signs (diarrhea) and reduced viral shedding for the first h (mogler et al., a) . vaccine efficacy was further tested in naïve sows. after three vaccinations at , , and weeks pre-farrowing piglets from both groups were challenged between and days of age with tcid (pedv/co/ ). average litter mortality in the control group was %, while average mortality in the vaccinated groups was % (crawford et al., ) . similar results were found in sows previously exposed to pedv. after oral challenge of piglets within the first week average litter mortality was reduced from % in the control group to % in the vaccinated group. finally, greiner et al. ( ) evaluated the vaccine in sows that had been previously exposed to pedv. vaccination induced higher titers against the s protein in the colostrum of vaccinated sows and reduced overall pig mortality by % (greiner et al., ) . a second vaccine for pedv in the us was developed by zoetis and made commercially available in under a conditional license. the vaccine consists of an inactivated whole virus formulated with an adjuvant. the vaccine was tested in pedv negative sows, which were vaccinated twice, weeks apart, with the vaccine (n = ) or an adjuvant placebo (n = ). the vaccine was safe and immunogenic and induced neutralizing antibodies to both the whole virus and the spike protein . the vaccine was also tested under field conditions in a pedv positive commercial herd. sows (n = ) were vaccinated at -and weeks pre-farrowing, control sows (n = ) received placebo. vaccination resulted in reduction of pre-weaning mortality due to pedv from . % in piglets born to control sows versus . % in piglets born to vaccinated sows. vaccination resulted in about times higher neutralization antibody titers compared to the control group, and an additional . pigs per sows survived . a third vaccine was recently developed by the vaccine and infectious disease organization-intervac in canada. the vaccine is based on inactivated virus formulated with an adjuvant. when administered to seronegative sows and weeks prior to farrowing, high levels of neutralizing antibodies against pedv were found in colostrum and milk, as well as in the serum of piglets born to vaccinated sows. piglets were orally infected at days of life with pfu of pedv isolate co . it was found that % of all piglets (n = ) born to vaccinated sows survived the infection and showed significantly reduced clinical symptoms, weight loss and viral shedding. in contrast, all piglets from unvaccinated sows displayed severe clinical symptoms including weight loss and dehydration, and % of these piglets died within days post infection. these results were confirmed in two additional clinical trials. a large field trial involving > sows was performed in three commercial units in saskatchewan, canada to assess the vaccine in different genetics, health statuses and management systems. the vaccine demonstrated to be completely safe to use; no adverse events including injection site reactions and reproductive complications were observed. vaccine efficacy was evaluated in % of these animals by transporting the pregnant sows a week before farrowing to the vido-intervac high containment facility. following oral challenge at days of age, survival was significantly higher in piglets born to vaccinated sows than those from control sows (berube et al., ) . the assessment of duration of immunity is currently ongoing. in addition, an affinity tagged pedv s protein was expressed in the hek system to be used as subunit vaccine. when administered to pregnant sows the vaccine partially protected newborn piglets against infection with pedv (makadiya et al., ) . since early s, pedv outbreaks have been reported in several asian countries, including china, japan, thailand, taiwan, the philippines, south korea and vietnam. in october , a largescale outbreak of severe pedv was reported in china (wang et al., ) . in late , pedv outbreaks occurred in japan, south korea and taiwan . phylogenetic analysis of pedv fulllength genomic sequences reveals that pedv can be genetically divided into groups: g (classical) and g (field epidemic or pandemic). each group can be further divided into two subgroups: a and b, a and b, respectively. it is also possible that the low to moderate effectiveness of current pedv vaccines are due to genetic differences between vaccine and field epidemic strains (kim et al., ) . for disease control, an inactivated bivalent tgev and pedv vaccine was introduced in china in . in march , a trivalent attenuated vaccine (pedv, tgev and porcine rotavirus) was also approved. all these vaccines were based on the classical cv (g -a) strain that can be grown to high titers in green monkey kidney vero cells. there are no data published on the efficacy of these vaccines. however, de arriba et al. (de arriba et al., ) orally inoculated -day-old conventionally reared piglets with two different doses of the attenuated cv- strain and challenged with the same virulent pedv strain three weeks later. the vaccinated pigs were partially protected against the challenge, and % of the low dose-and % of the high dose-exposed pigs did not shed virus after challenge. since winter of , china experienced severe ped outbreaks with devastating damage to the swine industry. these outbreaks can be explained by re-emergence of new pedv strains. to answer industry demand, chinese researchers have developed a bivalent (pedv and tgev) attenuated vaccine that contained the pedv strain zj (g -b) and a bivalent attenuated vaccine based on the aj strain (g -b). inactivated bivalent vaccine based on the g b strain has also been developed. these vaccines are currently under clinical evaluation (wang et al., ) . in japan, pedv strain p- (g -a) was attenuated after passages in vero cells (sato et al., ) . subsequently, this strain has been employed as an intramuscular (im) live attenuated vaccine (p- v) in japan and south korea. in addition, two south korean virulent pedv strains (sm - and dr- ) were attenuated by serial cell culture passages. the attenuated sm - strain has been used as an im live or killed vaccine, whereas dr- was used as an oral live vaccine. this oral vaccine was registered and commercialised in the philippines in . in south korea, the multiple dose vaccination program ( or im vaccinations in the following order: live-killed-killed or live-live-killed-killed, respectively) at -or -week intervals starting before farrowing is commonly recommended in pregnant sows (lee, ) . according to a south korean study, the administration of commercial vaccines increased the survival rate of piglets challenged with a virulent wild-type pedv from . % to over %. however, all vaccines did not significantly reduce morbidity rate and virus shedding (lee, ) . also, song et al. (song et al., ) reported % reduction of the mortality rate of the suckling piglets born to the sows that were im vaccinated with dr- pedv vaccine. despite of nationwide use of commercially available vaccines, south korea experienced a devastated ped epidemic in - . pedv g -b strains were responsible for recent severe ped epidemics in asian countries and north america. considering this fact, south korean researchers (baek et al., ) tested an inactivated vaccine based on serially cultured g -b strain kor/ knu- / . pregnant sows were immunized im with the inactivated adjuvanted vaccine at and weeks prior to farrowing. six-day old piglets were challenged with the homologous virulent virus. piglets born to vaccinated sows had reduced morbidity, mortality and quickly recovered daily weight gain. further studies are needed to evaluate efficacy of this vaccine in -or -day old piglets under field conditions. all commercially available vaccines that are in use in asia are traditional live attenuated or killed vaccines. however, researchers are working on the next-generation vaccines for pedv (table ) . for instance, hou et al. (hou et al., ) expressed the pedv n protein on the surface of lactobacillus. oral and intranasal inoculations of recombinant l. casei into pregnant sow and mice resulted in high levels of n-specific serum igg and mucosal iga. similarly, liu et al. (liu et al., ) expressed s and n protein in recombinant l. casei and reported enhanced mucosal and systemic immune responses after oral immunization of mice. meng et al. (meng et al., ) evaluated the immunogenicity of recombinant dna plasmids expressing s genes from pedv and tgev in mice. the results showed that the recombinant dna plasmids increased the proliferation of t lymphocytes and the number of cd + and cd + t lymphocytes. in addition, the dna vaccines induced a high level of ifn-g in the immunized mice. at days post-immunization, the recombinant dna plasmids bearing full-length s genes of tgev and pedv stimulated high levels of virus-neutralizing antibodies. zhang et al. (zhang et al., b) have also constructed bivalent dna vaccine co-expressing s genes of tgev and pedv. attenuated salmonella typhimurium was used for oral delivery this vaccine into pigs. vaccinated pigs developed tgev and pedvspecific cellular and humoral immune responses; however, challenge experiment was not conducted in this study. there have also been reports on expressing recombinant pedv s protein fragments in plants (bae et al., ) and in a cell line (oh et al., ) . feeding mice with transgenic tobacco plants that express the s protein fragment containing the pedv neutralizing epitope induced pedv-specific antibody and cell-mediated immune responses. in another study, oh et al. (oh et al., ) established porcine cell line stably expressing s fragment of the pedv spike protein. pregnant sows were immunized im times with the purified and adjuvanted protein , and weeks prior to farrowing. the sows developed pedv-specific neutralizing antibody response in serum and colostrum. one -to -day-old piglet was selected randomly from each farrowing sow for challenge with virulent pedv. the piglets born to vaccinated sows showed reduced morbidity, mortality and virus shedding. however, a low number of challenged piglets hamper the author's conclusion about efficient protection of neonatal piglets. with the disappearance of tgev around the world, the need for tgev vaccines has dropped over the last few years. for north america and europe, only two major international animal health companies continue to offer tgev vaccines. in contrast, many parts of asia including china and korea are still dealing with tgev outbreaks, and different types of vaccines are still available. the introduction of pedv into the north american herd in - , however, has reversed this picture and has highlighted the global need for effective vaccines. coronaviruses represent an important group of animal pathogens that can have devastating impacts in a variety of species. for an industry to rely on biosecurity alone seems somewhat risky and, in case of an emerging disease, can lead to major economic losses, as witnessed in north america over the last two years. indeed, the phylogeny of coronaviruses demonstrates a great deal of diversity in antigenic variants, which may lead to limited cross-protection against infection with different strains. thus, it is important to continue to survey novel pedv variants that may emerge locally or globally through antigenic drift (point mutations) or antigenic shift (recombination events). effective prevention and control of pedv and other coronaviruses can only be achieved through the use of vaccines. an ideal vaccine prevents mortality and clinical disease in newborn piglets, the age group most seriously affected by the disease, as well as viral shedding. as lactogenic immunity is a key mechanism of protection, efforts to enhance the levels of antibodies in the milk through formulation (adjuvants) and delivery (mucosal) are critical. however, time is also of essence when dealing with a new strain, as traditional manufacturing vaccine methods like virus isolation, inactivation or attenuation can be time-consuming. therefore, research on next-generation vaccines such as rna particle, dna, sub-unit and viral-vectored approaches is critical for the prevention of future outbreaks of emerging coronavirus diseases. induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen efficacy of an inactivated genotype b porcine epidemic diarrhea virus vaccine in neonatal piglets colostral antibodymediated and cell-mediated immunity contributes to innate and antigenspecific immunity in piglets mechanisms of coronavirus cell entry mediated by the viral spike protein development of a novel vaccine for porcine epidemic diarrhea virus passive immunity in transmissible gastroenteritis of swine: immunoglobulin characteristics of antibodies in milk after inoculating virus by different routes immunology of transmissible gastroenteritis antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus gammadelta t cell effector functions: a blend of innate programming and acquired plasticity new insights into the dual recruitment of iga+ b cells in the developing mammary gland identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus t cell-mediated immune response to respiratory coronaviruses strategies for design and application of enteric viral vaccines p , a murine membrane protein expressed on mast cells and some macrophages, is mouse cd /aminopeptidase n protective efficacy of a replicon particle vaccine in both naïve and previously exposed gilts against porcine epidemic diarrhea virus coronavirus gene counteracts host defenses and modulates virus virulence porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk safety and antibody response of pigs to an experimental porcine epidemic diarrhea virus (pedv) vaccine, killed virus previous infection of sows with a mild strain of porcine epidemic diarrhea virus confers protection against infection with a severe strain evaluation of a ped vaccine on piglet mortality and sow immunity the phenotype and activation status of t and nk cells in porcine colostrum suggest these are central/effector memory cells antibodies and their receptors: different potential roles in mucosal defense surface-displayed porcine epidemic diarrhea viral (pedv) antigens on lactic acid bacteria porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis 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 co-infections genetic characterization of porcine epidemic diarrhea virus in korea from ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts replication of transmissible gastroenteritis coronavirus (tgev) in swine alveolar macrophages a common mucosal chemokine (mucosae-associated epithelial chemokine/ccl ) selectively attracts iga plasmablasts outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine aminopeptidase n is a functional receptor for the pedv coronavirus high-level mucosal and systemic immune responses induced by oral administration with lactobacillusexpressed porcine epidemic diarrhea virus (pedv) s region combined with lactobacillus-expressed n protein the porcine innate immune system: an update s domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen ) fields virology evaluation on the efficacy and immunogenicity of recombinant dna plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus expression of teck/ccl and mec/ccl chemokines and their respective receptors ccr and ccr in porcine mucosal tissues expression of mucosal chemokines teck/ccl and mec/ccl during fetal development of the ovine mucosal immune system vaccination of pedv-naïve dams with replicon rna particle vaccine protects suckling piglets from challenge development of an alphavirus rna particle vaccine against porcine epidemic diarrhea virus transfer of humoral and cell-mediated immunity via colostrum in pigs immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein porcine epidemic diarrhea virus infects and replicates in porcine alveolar macrophages il- is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus field efficacy of an experimental porcine epidemic diarrhea (ped) vaccine administered to pregnant sows structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows animal coronavirus vaccines: lessons for sars humoral and cellular factors of maternal immunity in swine differential expression and activity of the porcine type i interferon family molecular evolution of the porcine type i interferon family: subtype-specific expression and antiviral activity mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo active immunity and t-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins il- , il- and their class ii cytokine receptor il- r differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr strain construction and characterization of recombinant porcine adenovirus serotype expressing the transmissible gastroenteritis virus spike gene pedv orf encodes an ion channel protein and regulates virus production porcine epidemic diarrhea virus variants with high pathogenicity porcine epidemic diarrhea in china proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture ccl controls immunoglobulin (ig)a plasma cell accumulation in the lactating mammary gland and iga antibody transfer to the neonate porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin- expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin- expression long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp construction of a bivalent dna vaccine co-expressing s genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated salmonella typhimurium mucosal and systemic isotypespecific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus the authors declare no conflict of interest. key: cord- -foifc ch authors: zhou, ying shun; zhang, yi; wang, hong ning; fan, wen qiao; yang, xin; zhang, an yun; zeng, fan ya; zhang, zhi kun; cao, hai peng; zeng, cheng title: establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: foifc ch infectious bronchitis virus (ibv) strain h was successfully rescued as infectious clone by reverse genetics. thirteen . – . kb fragments contiguously spanning the virus genome were amplified and cloned into pmd -t. transcription grade complete length cdna was acquired by a modified “no see’m” ligation strategy, which employed restriction enzyme bsa i and bsmb i and ligated more than two fragments in one t ligase reaction. the full-length genomic cdna was transcribed and its transcript was transfected by electroporation into bhk- together with the transcript of nucleocapsid gene. at h post transfection, the medium to culture the transfected bhk- cells was harvested and inoculated into -days old spf embryonated chicken eggs (ece) to replicate the rescued virus. after passage of the virus in ece five times, the rescued h virus (r-h ) was successfully recovered. r-h was subsequently identified to possess the introduced silent mutation site in its genome. some biological characteristics of r-h such as growth curve, eid and ha titers, were tested and all of them were very similar to its parent strain h . in addition, both r-h and h induced a comparable titer of ha inhibition (hi) antibody in immunized chickens and also provided up to % of immune protection to the chickens that were challenged with mass ibv strain. the present study demonstrated that construction of infectious clone from ibv vaccine strain h is possible and ibv-h can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach. avian infectious bronchitis virus (ibv), the causative agent of infectious bronchitis (cavanagh, ; cavanagh et al., ) , is a gammacoronavirus in the family coronaviridae (casais et al., ; saif et al., ; tidona and darai, ) . ibv is worldwide distributed and difficult to control because of the existence of multiple serotypes and variants of the virus (cavanagh, ) . ibv is an enveloped, unsegmented, positive sense ssrna virus and has a genome of approximately . kb in length (casais et al., (casais et al., , hodgson et al., ) . similar to other coronaviruses, the two thirds of the ibv genome encodes two polyproteins, pp a and pp ab, and the latter is an extension product of pp a as a result of a À frameshifting (almazan et al., ; brierley et al., ) . the remaining one third of the genome encodes the structural proteins and group-specific orfs, including spike glycoprotein (s), envelope protein (e), membrane veterinary microbiology ( ) infectious bronchitis virus (ibv) strain h was successfully rescued as infectious clone by reverse genetics. thirteen . - . kb fragments contiguously spanning the virus genome were amplified and cloned into pmd -t. transcription grade complete length cdna was acquired by a modified ''no see'm'' ligation strategy, which employed restriction enzyme bsa i and bsmb i and ligated more than two fragments in one t ligase reaction. the full-length genomic cdna was transcribed and its transcript was transfected by electroporation into bhk- together with the transcript of nucleocapsid gene. at h post transfection, the medium to culture the transfected bhk- cells was harvested and inoculated into -days old spf embryonated chicken eggs (ece) to replicate the rescued virus. after passage of the virus in ece five times, the rescued h virus (r-h ) was successfully recovered. r-h was subsequently identified to possess the introduced silent mutation site in its genome. some biological characteristics of r-h such as growth curve, eid and ha titers, were tested and all of them were very similar to its parent strain h . in addition, both r-h and h induced a comparable titer of ha inhibition (hi) antibody in immunized chickens and also provided up to % of immune protection to the chickens that were challenged with mass ibv strain. the present study demonstrated that construction of infectious clone from ibv vaccine strain h is possible and ibv-h can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach. ß elsevier b.v. all rights reserved. protein (m) and nucleocapsid protein (n), which are essential for replication of the virus (armesto et al., ; fang et al., ; youn et al., ) . in addition, ibv also encodes a set of accessory proteins of unknown function that may be absent in some strains and not essential for virus replication in vitro (armesto et al., ; casais et al., ; hodgson et al., ; youn et al., ) . the reverse genetic system for ibv was firstly established using vaccinia vector (casais et al., ) . subsequently, a more elegant protocol to obtain viral infectious rna was developed. in this system, full-length viral cdna was assembled in vitro by orderly ligating viral genomic cdna fragments and directly used as dna template for reverse transcription of viral infectious rna. the new technique was successfully applied to the studies on the role of accessory genes in viral replication (youn et al., ) , virulence determinant of beaudette strain (fang et al., ) and the relationship between s gene and tissue tropism of the virus casais et al., casais et al., , casais et al., , youn et al., ) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were based on vero cell-adapted beaudette strain, which was considered to be poorly immunogenic and never used as a vaccine strain (geilhausen et al., ) . therefore, application of the reverse genetics techniques to other strains including vaccine strains could improve the technique on modification of viral genome and provide a powerful tool for novel vaccine development. h , an attenuated live vaccine strain of massachusetts (mass) serotype, was originally obtained by serial passage of strain h that was isolated in the netherlands in in embryonated chicken eggs up to the th passage (bijlenga et al., ) . in recent years, h was considered to be one of the safest vaccine strains and used worldwide as a primary vaccine in broilers, breeders, and future layers. the complete genome of h was sequenced in our previous study (zhang et al., ) . in this study, we describe the in vitro assembly and recovery of an infectious clone of ibv-h , the biological and immune characteristics of the rescued h virus (r-h ), and the potential to use r as a candidate of vaccine vector in the future vaccine development. the ibv strains, h and mass , obtained from china institute of veterinary drug control (ivdc), were propagated in the allantoic cavities of the -day-old specific pathogen-free (spf) embryonated chicken eggs (ece), and the allantoic fluid was harvested h post inoculation. bhk- cells were maintained in dmem containing % fbs, u/ml penicillin and mg/ml streptomycin. viral rna was extracted from h -infected allantoic fluid with trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's directions. reverse transcription was performed with superscript iii (invitrogen). the ibv-h sequence (genbank, accession number fj ) was used for primer design and nucleotide sequencing. each dna fragment was amplified from cdna templates by pcr using kod plus polymerase (toyobo, japan). pcr primer pairs used to amplify genomic regions are listed in table . pcr amplification of cdna fragments was performed in the following conditions: denaturation at c for min, cycles of c for s, c for s, and c for - min depending on the size of the products and final extension at c for min. the pcr products were isolated from agarose gels and ta-cloned into pmd -t vector (takara, japan), according to the manufacturer's directions. the entire nucleocapsid (n) orf, including the -utr, was amplified by rt-pcr from total rna extracted from h -infected allantoic fluid, and the rt-pcr product was also cloned into pmd -t vector. in order to transcribe rna by using t rna polymerase, t promoter sequence was incorporated to end primers of f and n- fragments. in addition, a silent nucleotide change of a to t at position , nt was incorporated in to f f primer as a molecular marker. to determine the consensus clone, two to four independent clones of each amplicon were sequenced by sangon biological engineering technology & services co., ltd. each amplicon that represents the consensus sequence of ibv-h was then released from cloning vector by restriction enzyme digestion and recovered for construction of the full-length ibv genome in vitro. briefly, the plasmid carrying f amplicon was digested with sal i and nci i, and treated with calf intestine alkaline phosphatase (ciap: takara), before digesting with the bsmb i; the plasmid with f amplicon was digested with xho i, ciap treated and digested with bsmbi; the plasmids carrying amplicons f and f were digested with nci i and bsmb i; and the other plasmids inserted amplicon f , f , f , f , f , f , f , f and f , were digested with bsai. all the digested plasmids were separated on . % agarose gels and the bands corresponding to each amplicon were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc., valencia, ca). the cdna amplicons were ligated orderly with t dna ligase as following: ( ) ligation of the amplicons into four fragments (f + f + f , f + f + f , f + f + f , and f + f + f + f ) in equal mole ratio and recovering the appropriately sized four fragments from % agarose gels and ( ) ligation of the four fragments into full-length ibv genome (fig. ) . the final ligation product was extracted with phenol/chloroform/isoamyl alcohol ( : : ), precipitated with ethanol, and detected by electrophoresis on . % agarose gel. full-length transcript was generated in vitro using mmessage mmachine t kit (ambion, austin, tx) according to the manufacturer's instructions with certain modifications. briefly, ml of transcription reaction with a : ratio of gtp to cap analog was sequentially incubated at c for min. a similar reaction was performed for the n transcripts, which were generated by using a linearized pmd -t-n- containing the ibv n gene and the -utr region as templates. a : ratio of gtp to cap analog was used for the transcription of ibv n gene (fang et al., ; youn et al., ) . in the same time, bhk- cells were grown on monolayers to % confluence, treated with trypsin, washed with cold depc-treated pbs twice, and resuspended in depc-treated pbs at a concentration of cells/ml. rna transcripts were added to ml of bhk- cell suspension in microfuge tubes on ice, gently pipetted and transferred to electroporation cuvettes. three consecutive electrical pulses of v at af were given, using the gene pulser xcell tm electroporation system (bio-rad). the transfected bhk- cells were incubated at c for h in dmem supplemented with % fbs (invitrogen). after h, the virus containing medium was collected and inoculated into the -day-old spf embryonated chicken eggs. after passages in ece, allantoic fluid was harvested according to the previous description (cavanagh, ) . to differentiate the rescued viruses from the parental viruses, viral rnas were extracted from allantoic fluid (r-h and parental h ) as described previously. rt-pcr was performed with selected primer pairs ( -aatataagacagagcacaag- , -ctgtcatacaaagcagcactaca- ) to amplify the fragment consisting of the silent nucleotide change of a to t at position , nt. the pcr fragment was cloned and sequenced to check the silent mutation site. to determine % egg infection dose (eid ) of the r-h , serial -fold dilutions ( À - À ) of the amplified virus were inoculated into the -day-old spf ece. for each dilution, . ml of virus solution was injected into each egg and four eggs were used for each dilution. the parental strain h was used as a positive control. the eid calculation was based on reed and muench ( ) method. r-h virus hemagglutination antigen was prepared by using the filtrate from a clostridium welchii to treat the -fold concentration. ha antigens were doubly diluted in a range of : - : and titrated with . % chicken erythrocytes. parental h , spf allantoic fluid (free ibv) and pbs were used as controls. to determine the growth kinetics of the rescued virus r-h , the viral titre (eid ) were tested. in this procedure, the parental strain h was a postive control. briefly, . ml of virus . eid titre of r-h and h were inoculated into the allantoic cavities of -days old embryonated eggs, and the allantoic fluid of six eggs from each group were harvested at the time points of , , , , and h post inoculation and pooled for the determination eid , were carried out as described by the office of international des epizooties. all the assays were run in triplicates and the eid of each virus were calculated according to standard curve. . . immunization characteristics of r-h as a vaccine vector candidate . . . immunization of chickens with r-h to determine the immunization characteristics of r-h virus, chickens of -day-old were randomly divided into three groups and raised separately. r-h virus at . eid /ml (group ) was injected intramuscularly to both quadriceps; h virus at . eid /ml (group ) was injected in the same way; and . ml pbs (group ) was used as control injection. all groups were boosted with an equivalent dose on days post the initial inoculation. serum samples were taken weekly from the three groups of immunized chickens on , , , , and -day posthatch for haemagglutination inhibition (hi) assay. the hi tests were carried out as described by the office of international des epizooties (oie, ) . the maximum dilution of each serum sample that caused ha inhibition (hi) was recorded as the endpoint of dilution and the hi titers were determined as the geometric mean titer (gmt) of log . the immunized chickens were challenged with eid of ibv strain mass in . ml by the nasal ocular route on the th day after the hatching. chickens were examined daily for weeks for the clinical symptoms such as coughing, sneezing, dyspnea or death. dead chickens were necropsied to confirm that the death was due to ibv infection. the chickens in each group were euthanized on days post challenge. kidney and lung tissues were collected individually from either the dead or euthanized chickens and virus in these tissues was detected by rt-pcr. bp, bp, bp, bp, bp, bp, bp, respectively assembly of the fragments into four / full-length cdna fragments (fa: f + f + f ( . kb), fb: f + f + f ( kb), fc: f + f + f ( kb), and fd: f + f + f + f ( . kb)), then assembly of the four / full-length cdna fragments into a full-length cdna clone, and in vitro transcription of the full-length transcripts. thirteen . - . kb fragments spanning the entire ibv genome, designating f -f , were obtained by rt-pcr and all the fragments do have the same size as the predicated from h sequence. to facilitate the assembly of the fulllength cdna in vitro, each fragment has been ranged between two restriction enzymes in h -ibv genome, e.g., bsmbi or bsai, either at the or ends of each fragment (fig. ) . the amplified pcr fragments were recovered from agarose gel and all of them were successfully cloned into pmd -t cloning vector. determined by sequencing, the consensus clones including those that contains the correctly introduced sequences such as t promoter at -end of f and poly(a) at -end of f were chosen to construct the full-length cdna clone. the cdna fragments were prepared by digestion of the corresponding plasmids with assigned restriction enzymes such as bsmbi and bsai and recovery from agarose gel. the full-length cdna was assembled by orderly ligation of the purified fragments in vitro, which in turn was used as the template for in vitro transcription. coronavirus n gene transcripts were shown to enhance the recovery of the rescued virus from the in vitro-synthesized full-length transcripts (casais et al., ; youn et al., ; yount et al., yount et al., , yount et al., , , therefore, n-gene orf was also prepared for transcription in vitro (fig. a ) . the full-length transcript together with the n transcript was electroporated into bhk- cells and cytopathic effects (cpe) including cell clustering and falling-off were observed on two days post transfection. to amplify the rescued virus, the supernatant from transfected cell culture was harvested and inoculated into allantoic cavities of embryonated chicken eggs for replication of the rescued virus. after passages in ece, typical embryo lesions such as curling, stunting and dwarfing were observed, which indicated the successful rescue of h virus from infectious rna. finally, the rescued virus was designated as ibv r-h . the recovery of r-h from the ligated cdna was confirmed by rt-pcr amplifying and sequencing the genomic region that contained molecular marker to distinguish r-h from h . as expected, a nucleotide change at , nt was identified in r-h genome. the virus titer and growth curve of r-h were also estimated and compared with the parental strain h . the results showed that ( ) r-h was pathogenic to chicken embryos and the eid titer was comparable to its parental virus h (fig. a) ; ( ) the tested ha titers of r-h was almost equal to that of h (fig. b) ; and ( ) like the parental strain h , r-h could replicate in ece cells and both viruses had very similar growth pattern (fig. c) . the above experimental data indicated that the rescued virus shared several essential properties with its parental virus h . . . serum hi antibody assay in chicken after immunizing the h and r-h to estimate the immunogenicity of the rescued virus, an important factor for using it to develop vaccine, r-h and h viruses were immunized to two groups of chickens and the induced serum antibody was titrated. the dynamic changes of hi antibody titer following the inoculation of r-h and h were observed (table ) and no statistic difference of hi antibody titer was found between r-h and h groups. however, a significant difference was fig. . biological characteristic of the r-h . (a) the eid of the r-h ,the parental strain h was the positive control, spf allantoic fluid (free ibv) and pbs were the negative controls. (b) ha titer of the r-h , parental strain h was the positive control, spf allantoic fluid (free ibv) and pbs were the negative controls. (c) comparison of the replication kinetics of r-h and h in ece cell. the rescued virus h (r-h and parent h ( . ml of eid ) were inoculated into the allantoic cavities of -day old embryonated eggs, and the allantoic fluid of six eggs from each group was harvested at the time points of , , , , and h post inoculation and pooled for the determination of eid in ece. detected between pbs and virus inoculated groups (p < . ). three groups of chickens were challenged with the virulent strain mass to test if the rescued virus could protect the immunized animals from ibv infection. on the th day post challenge, chickens started to show clinical signs of infection or death. the data shown affected chickens in each group after challenge, such as mortality rate and protection rate, were listed in table . the chickens injected with pbs alone were not protected from ibv infection and developed cough, nasal discharge and dyspnea. the death rate in the group injected with pbs reached to %, but it was only % for both groups and . to evaluate the level of protection, shielding virus in the collected lung and kidney samples were estimated by rt-pcr. the results showed that % of the chickens in groups and had virus shielding in their lungs and/or kidney in compared with % in control group (pbs). the results suggested that r-h and h both provide protection to immunized chickens from virulent ibv challenge. through different methodologies, vaccinia virus vectors or bac as cloning systems and the in vitro assembly strategy, reverse genetics systems of ibv and other coronaviruses (tgev, hcov e, mhv-a , and sars-cov) have been constructed (almazá n et al., ; casais et al., ; yount et al., yount et al., , . for the purposes of studying the mechanisms of pathogenesis, or building transfer vectors, so far, casais et al. ( ) , youn et al. ( ) and fang et al. ( ) established the reverse genetic system for vero cell-adapted beaudette strain. after that, this reverse genetic system for vero cell-adapted beaudette strain are used to research the pathogenicity, tissue tropism and functionality of accessory gene of the ibv (armesto et al., (armesto et al., , britton et al., ; casais et al., ; hodgson et al., ) . previous reverse genetic system set for coronavirus was assembly of a genomic length cdna from to contiguous cdna subclones in size - kb (fang et al., ; youn et al., ) . although this segmentation strategy can decrease the number of cdna fragments and is easier to link to full-lengthen cdna, it is difficult to get the longer pcr product with correct sequence and clone these cdna fragments. in addition, the recombined plasmid containing larger fragments may be harmful to host bacteria, resulting some of them could not replicate in escherichia coli host cells. in order to solve these problems, certain modifications for assembling of ibv genomic rna in vitro were made to improve the efficiency of generating full-length cdna. in this study, two improvements for assembly of full-length viral genome were applied. at first, the ibv genome was cloned as thirteen . - . kb fragments. the shortened length of cdna fragments made them easier to clone, with less sequence mutation in each fragment and more stable in bacteria. secondly, three or four fragments were ligated in one t dna ligase reaction to reduce the number of recovery times which was negative correlated to dna recovery rate from agarose gels. the genome of the avain coronavirus was established to provide a tool not only for the mechanism studies but also for the development of new vaccines (matthijs et al., ; ziebuhr, ) . in this study, we were established a reverse genetics system based on h strain of ibv, a worldwide used attenuated live vaccine strain of massachusetts (mass) serotype. in china, h is always considered as the first vaccine selection. compared with the vero cell-adapted beaudette strain, h can be developed as the vaccine vector to express other antigens since it has a stronger immunogenicity than that of beaudette strain, although the latter was a widely used model of ibv on reverse genetics study and never considered as a vaccine strain (geilhausen et al., ) . according to the present data, the biological and immune data within a column with different letters differ significantly (p < . ); the same letters means no significance. protective effects in -week-old spf chickens immunized with r-h and h against challenge of ibv mass strain. characteristics of r-h including the eid and ha titer, growth curve and protection of immunized animals from challenge were all very similar to its parental strain h . the same situation was found in other covs such as ibv beaudette strain (fang et al., ) , mhv (yount et al., ) , tgev (yount et al., ) , ndv (hu et al., ) and avian influenza virus (hoffmann et al., ; jackson et al., ) . it might imply that h was a good strain for the development of engineering avian vaccines. in addition, the . kb genome of h as a vaccine vector can be recombined with larger pathogenic antigen gene compared with kb newcastle disease virus vaccine vector, which was a vector used presently to express different pathogenic antigen genes as vaccine to protect the immunized animals from correspondence pathogen's challenge (dinapoli et al., ; nakaya et al., ; veits et al., ) . it would make the procedure of constructing reverse genetics system labor intensive if a continuous cell line could not be used in infectious rna transfection and rescued virus replication. in our study, the continuous cell line, bhk- , was applied to generating modified virus after electroporation, indicating that bhk- can be utilized to rescue virus from infectious rna although bhk- was never used to replicate h virus before. at present, most ibv strains replicate in primary cells like ck and cek and do not adapt to continuous cell lines (casais et al., ) . therefore, in vitro assembly strategy is applicable to ibv strains that were not adapted to the commonly used continuous cell lines through the established approach in the present study. it is interesting to find that the rescued virus r-h and h viruses cannot efficiently replicate in bhk and make the cells emerge obvious cpe, but r-h do rescue from bhk cell. in next step, the s gene of h may be modified by reverse genetics to find out bhk adapted h that may reveal the information on cell tropism of the virus. in conclusion, the present study demonstrated that the rescued virus r-h has similar biological and immunogenic characteristics with its parental strain h and it would be possible to develop novel avian vaccine through reverse genetics approach by modification or recombination of the present vaccine virus h . engineering the largest rna virus genome as an infectious bacterial artificial chromosome the nucleoprotein is required for efficient coronavirus genome replication the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity a recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the / serotype development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot genes and of infectious bronchitis virus are accessory protein genes gene of the avian coronavirus infectious bronchitis virus is not essential for replication recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism reverse genetics system for the avian coronavirus infectious bronchitis virus coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides nidovirales: a new order comprising coronaviridae and arteriviridae coronavirus avian infectious bronchitis virus longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp ) is lethal to the coronavirus infectious bronchitis virus in cultured cells the pathogenesis of virulent and avirulent avian infectious bronchitis virus neither the rna nor the proteins of open reading frames a and b of the coronavirus infectious bronchitis virus are essential for replication eightplasmid system for rapid generation of influenza virus vaccines a vaccine candidate of attenuated genotype vii newcastle disease virus generated by reverse genetics a new influenza virus virulence determinant: the ns protein four cterminal residues modulate pathogenicity effect of ibv-h vaccination in broilers on colibacillosis susceptibility after infection with a virulent massachusetts-type ibv strain recombinant newcastle disease virus as a vaccine vector manual of diagnostic tests and vaccines for terrestrial animals a simple method of estimating fifty per cent endpoints coronaviruses of domestic livestock and poultry: interspecies transmission, pathogenesis, and immunity the springer index of viruses newcastle disease virus expressing h hemagglutinin gene protects chickens against newcastle disease and avian influenza in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the a open reading frame is not essential for replication strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain h the coronavirus replicase key: cord- - drhoz authors: tabynov, kairat; sansyzbay, abylay; tulemissova, zhanara; tabynov, kaissar; dhakal, santosh; samoltyrova, aigul; renukaradhya, gourapura j.; mambetaliyev, muratbay title: inactivated porcine reproductive and respiratory syndrome virus vaccine adjuvanted with montanide™ gel st elicits virus-specific cross-protective inter-genotypic response in piglets date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: drhoz the efficacy of a novel bei-inactivated porcine reproductive and respiratory syndrome virus (prrsv) candidate vaccine in pigs, developed at ribsp republic of kazakhstan and delivered with an adjuvant montanide™ gel st (d/kv/adj) was compared with a commercial killed prrsv vaccine (nvdc-jxa , c/kv/adj) used widely in swine herds of the republic of kazakhstan. clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [elisa and virus neutralizing (vn) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time pcr and cell culture assay) were assessed in vaccinated and both genotype and prrsv challenged pigs. our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, kazakh strains of prrsv type and type genotypes. in contrast, clinical protection, absence of viremia and lung lesions in d/kv/adj vaccinated pigs was associated with generation of vn antibodies in both homologous vaccine strain lkz/ (prrsv type ) and a heterogeneous type prrsv strain (cm/ ) challenged pigs. thus, our data indicated the induction of cross-protective vn antibodies by d/kv/adj vaccine, and importantly demonstrated that an inactivated prrsv vaccine could also induce cross-protective response across the viral genotype. porcine reproductive and respiratory syndrome (prrs) is an economically devastating disease in pigs that causes an estimated direct loss of greater than $ million annually to the us pork industry (chand et al., ; holtkamp and kliebenstein, ) . the causative agent, prrs virus (prrsv), belongs to the family arteriviridae, order nidovirales, and it causes respiratory problems in pigs of all ages and reproductive failure in sows, including still births and mummification, birth of weak piglets and high preweaning mortality (kimman et al., ; rossow et al., ) . genetic and antigenic analyses have revealed two distinct prrsv genotypes, european (type ) and north american (type ). marked genetic and antigenic differences of up to % are observed between the type and type prrsv genotypes (kim et al., ; nelsen et al., ) . in addition, within the genotypes up to % genetic variation for type i and % for type ii prrsv do exist (kim et al., ; zimmerman et al., ) . this implies complexity of the prrsv and difficulties to develop protective vaccines (li et al., ) . vaccination is the only viable strategy practiced to control the clinical prrs disease and transmission in pigs. two types of prrsv vaccines are commercially available, modified live virus (mlv) vaccine and a killed virus (kv) vaccine (charerntantanakul, ) . prrs mlv vaccine has been shown to provide protective efficacy against prrsv infection in the field; however, it provides limited protection against heterologous viruses. additionally, prrs mlv vaccine has the intrinsic risk of reversion to a virulent strain (kwang et al., ) . though prrs kv vaccine is safe to use in pigs, it elicits insufficient immunity in pigs (lee et al., ) . overall, commercially available prrs kv vaccine does not induce strong required immune response, and thus failed to protect pigs against viremia to homologous as well as heterologous viral challenges (nilubol et al., ; scortti et al., ; zuckermann et al., ) . interestingly, experimental studies showed that it is possible to induce production of virus neutralizing (vn) antibody response in naïve pigs by immunization with inactivated prrsv (misinzo et al., ; renukaradhya et al., a) . passive transfer studies demonstrated that the prrs vn antibody response is responsible for viral clearance from the blood and lungs, and reduce transplacental transmission of the virus, thus played a significant role in protective immune response against prrs (lopez and osorio, ; osorio et al., ) . recent research efforts aimed to improve protective immune response to prrs kv have been focused on utilizing potent vaccine adjuvants, which help to reduce viremia, lung lesions and clinical disease in homologous and heterogeneous prrsv challenged pigs. several types of vaccine adjuvants have been investigated for their ability to potentiate the immune response to prrs vaccines (charerntantanakul, ; li et al., ) , for example polymeric adjuvant montanide tm gel (deville et al., ; parker et al., ; tabynov et al., ) . previous work in our laboratory demonstrated that montanide tm gel potentiates the immune response of a candidate inactivated prrs vaccine , but at that time the efficacy was not compared with the commercial vaccine. therefore, we developed a binary ethylenimine (bei)-inactivated experimental prrsv vaccine containing the prrsv strain, arterivirus/lkz/ (lkz/ ) (type ), and coadministered intramuscularly with montanide tm gel st adjuvant. the vaccine virus was isolated in the territory of the republic of kazakhstan in . further, to elucidate efficacy of our candidate kv prrs vaccine, vaccinated piglets were challenged with the lkz/ (parental type vaccine strain) or kostanay-cm/ (type heterogeneous strain) of prrsv. prrsv strain lkz/ (national repository of especially dangerous diseases of the republic of kazakhstan, registration number m- - /d) was propagated in marc- cells to use in our experimental vaccine preparation. virus infected cell culture supernatant at fifth passage was harvested after h of infection and centrifuged at rpm for min, and filtered through . mm filter. virus titers were determined using a confluent monolayer of marc- cells cultured for h in -well tissue culture plates, and the viral titer was expressed in tcid /ml (reed and muench, ) . inactivation of prrsv with bei was performed as described (bahnemann, ) with some modifications (azanbekova et al., ) . briefly, bei . m stock solution was prepared by cyclization of -bromoethylamine in . m naoh for h at c and stored at c. virus was inactivated by incubation with mm bei for h at c, and the unutilized bei was neutralized by incubation with . mm sodium thiosulphate at c overnight. to confirm complete inactivation, the inactivated virus suspension was inoculated onto marc- cells cultured in cm tissue culture flasks in ml medium. the cells were cultivated for week at c, during which time they were passaged three times and the absence of cpe was confirmed. furthermore, ml of inactivated prrsv was injected into three month-old pigs and serum samples collected every week for weeks was assayed for viremia by real-time pcr as described in section . . after confirmation of viral inactivation, the viral antigen was mixed with a final concentration of % (w/w) gel st polymeric adjuvant (montanide tm , seppic, france) by manual shaking for min. the prrsv vaccines used in this study were: ( ) our candidate kv vaccine mixed with a polymeric adjuvant (d/kv/adj; lkz/ ; ribsp, gvardeiskiy, kazakhstan; batch ); and ( ) a commercial nvdc-jxa kv vaccine containing oil based adjuvant (c/kv/adj; guangdong dahuanong animal health products co., ltd., jinan, china; batch ). the challenge prrsv strains include, prrsv lkz/ (type ; isolated from lugovskoi konniy zavod (lkz) farm, zhambylskaya, oblast, kazakhstan) (orynbayev et al., ) and cm/ (type ; isolated from a private farm in zatobol village, kostanayskiy rayon, kazakhstan) (tabynov et al., ) , were obtained from the depository of especially dangerous infectious diseases, ribsp. the challenge virus strains lkz/ and cm/ were propagated in marc- cells, and confirmed as type and type viruses by ez-prrsv tm mpx . real time rt-pcr using target-specific reagents kit for the rapid identification & differentiation of north american and european prrs viral rna (tetracore, md, usa). fifty large white breed pigs weighing $ kg each (average months-old) were purchased from a specific-pathogen-free herd with certified records showing free from prrsv, porcine parvovirus, porcine respiratory coronavirus, aujeszky's disease, classical swine fever virus, transmissible gastroenteritis virus, swine influenza virus and mycoplasma hyopneumoniae. the negative prrsv status of the animals was confirmed by serology using a commercial elisa kit after arrival of the animals to our facility. pigs were randomly assigned into six treatment groups: groups and , mock-vaccinated negative control groups (received ml final concentration of % gel adjuvant); groups and , vaccinated three times ( , , days post-first vaccination [dpfv]) with a volume of ml d/kv/adj containing gel adjuvant. groups and were vaccinated twice ( and dpfv) with ml of commercial c/kv/adj vaccine containing oil adjuvant as per the manufacturer's recommendations. the virus titers present in the d/kv/adj and c/kv/adj vaccines before inactivation were . tcid /ml and . tcid /ml, respectively. at dpfv, the negative control groups - (n = pigs/group) and groups - (n = pigs/group) were challenged intranasally ( . tcid ) and intramuscularly ( . tcid ) with lkz/ or cm/ prrsv (table ) . serum samples were collected from pigs at , , , , , , , , , , , , , and dpfv and stored at À c until used in assays. pigs were euthanized at dpfv using the mixture of ketamine and xylazine as previously described and performed necropsy. pigs were housed in our bsl isolation animal facility at the research institute for biological safety problems science committee, ministry of education and science of the republic of kazakhstan (ribsp). animals had free access to filtered water and unmedicated sterilized feed. pigs were initially kept for acclimation period for days before started vaccination. this study was carried out in compliance with national and international laws and guidelines on animal handling. the animal use protocol was approved by the committee on the ethics of animal experiments at the ribsp (permit number: / ). serum samples collected from pigs were aliquoted and stored at À c until used in the assays. elisa for prrsv antibodies was performed using a commercial kit (bionote, inc., hwaseong, korea) according to the manufacturer's instructions. the optical density was measured at nm using the multiskan plus microplate reader (labsystems, vantaa, finland). the presence or absence of prrsv antibodies was determined by calculating the sample to positive (s/p) ratio. samples were considered positive for prrsv antibodies if the s/p ratio was > . . prrsv vn antibody titers in serum samples collected at , , , , , , , , , , , , , and dpfv were analyzed by indirect immunofluorescence assay (ifa) as previously described (christopher-hennings et al., ) . briefly, samples were subjected to uv-treatment for min to inactivate any prrsv and subjected to heat inactivation at c for min to inactivate the complement function. two-fold dilutions of test samples prepared in serum free dmem ( ml/well) were incubated with ml of prrsv (lkz/ or cm/ ) tcid per well for h at c, subsequently ml of the suspension was transferred into well microtiter plate containing confluent monolayer of marc- cells and incubated for h at c. further, ml/well of dmem containing % fetal bovine serum was added to each well and incubated for h at c in a co incubator. the contents of the wells were removed from the plate and the cell monolayer was fixed with % acetone in water for min and the plates were allowed to dry in fumehood for min. cells were rehydrated with ml/well of pbs for - min. cpe in both the plates meant for virus titration (see paragraph . ) and vn titer were examined after treatment with ml/well of mouse anti-pprsv nucleocapsid protein specific mab (sdow- ) ( : ) followed by alexa- conjugated anti-mouse igg (h + l) secondary antibody ( : ). after each treatment plates were incubated at c for h, and washed times in between the treatments and observed under an inverted fluorescent microscope after mounting the cell monolayer with glycerol-pbs in : ratio ( ml/well). the vn antibody titer was determined to be the reciprocal dilution ratio of the sample at which > % inhibition of prrsv-induced immunofluorescence was observed. rectal temperature was measured daily from to days postchallenge (dpc) and the threshold for fever was defined as . c. respiratory disorders were scored as: = normal; = mild dyspnea and/or tachypnea if the animal was stressed by handling for s; = mild dyspnea and/or tachypnea at rest; = moderate dyspnea and/or tachypnea when stressed; = moderate dyspnea and/or tachypnea at rest; = severe dyspnea and/or tachypnea when stressed; = severe tachypnea and/or dyspnea at rest. during necropsy the lungs were excised and macroscopic lung lesions were scored to estimate the percentage of the lungs affected by pneumonia as described (halbur et al., ) . one gram of lung tissue was collected from each pig into a ml conical tube containing ml of dmem, minced, and homogenized using the ika t ultra turrax high speed homogenizer (cole-parmer, il, usa) for one min on ice. the clarified supernatant was collected by centrifugation at g for min, aliquoted and stored at À c until assayed. marc- cells were seeded onto -well plates h before infection. prrsv supernatants were serially -fold diluted (from À to À ) and ml of each dilution were plated in six replicate wells. cell culture media without prrsv was used as a control. cells were incubated at c for h in a % co incubator, fixed and immunostained as described above ( . . prrsv vn titer assay). the titers were calculated as described previously (reed and muench, ) and expressed as tcid /ml. prrsv rna was extracted from serum samples and lung homogenate using magmax tm - virus isolation kit (ambion/ applied biosystems, carlsbad, california, usa) as per the manufacturer's instructions. real-time rt-pcr was performed using the ez-prrsv tm mpx . assay (tetracore ; rockville, md, usa), which covers two target regions of prrsv type and type genes using specific primers and probes. in the assay, -fam ( carboxyfluorescein) was used as a reporter dye for detection of type and type prrsv; and the alternative reporter dye cy was used for detection of extraction/inhibition control. the reaction volume per well included . ml of ez-prrsv tm mpx . reagent (includes buffer, primer and probes), . ml of enzyme blend and ml of the extracted serum sample. each pcr run included two positive controls for type and type viruses and one negative amplification control. negative and positive controls per well contained . ml of ez-prrsv tm mpx . reagent, . ml of enzyme blend, . ml of inhibition control and ml of the positive ( . ml of type and prrsv) or negative control (  te buffer). plates were briefly mixed by shaking on a vortex mixer ( s), centrifuged, and loaded into the thermal cycler ( fast real-time pcr system; applied biosystems , foster city, ca, usa). the thermal cycling conditions were as follows: one cycle at c for min, one cycle at c for min, cycles at c for s and c for s. samples with c t values < for either genotype prrsv were considered positive. quantification of samples was expressed in terms of the number of rna copies per ml for serum and copies per gram of lung tissues. these estimates were based on linear extrapolation of the cycle threshold values against a standard curve generated by serial dilutions of known amounts of in vitro transcript rna product (  to  copies per ml). all data were expressed as the mean ae standard error mean (sem) value of - pigs in each group. the differences in body temperatures, lung pathology scores, humoral responses and viremia between groups were assessed by one-way anova followed by tukey's multiple comparisons test. p-values < . were considered significant. all statistical analysis was performed using graphpad prism software, version . (graphpad software inc., san diego, ca, usa). all animals remained in good health after vaccination with d/ kv/adj or c/kv/adj vaccine and prior to viral challenge. any local or systemic vaccine side effects were absent pre-and postchallenge and none of the experimental pigs died during the entire study period. the body temperature of the animals in every group fluctuated within the normal physiological range after vaccination, but not statistically significant (data not shown). after pigs were challenged with lkz/ or cm/ , the body temperature in the d/kv/adj-vaccinated pig groups remained within the normal range ( . - . c) during the entire study period ( days). while the body temperature of the animals in the mock-vaccinated control and c/kv/adj-vaccinated pig groups were increased > . c on days - and days - post-challenge, respectively (fig. a ). there were significant differences in mean body temperature between the mock-vaccinated, c/kv/adj-vaccinated and d/kv/ adj-vaccinated pigs following the viral challenge. the magnitude of increase in body temperature was correlated with the severity of clinical disease, i.e., lower the body temperature lower the overall severity of clinical disease. severe respiratory disorders were observed after challenge with cm/ and lkz/ at and dpc, respectively, with significantly higher respiratory disease scores in the mock-vaccinated and c/kv/adj-vaccinated animals than the d/ kv/adj-vaccinated pigs (fig. b) . the bionote elisa demonstrated that all pigs were seronegative for prrsv at dpfv and remained low until dpfv ( fig. a-c) . the animals in the d/kv/adj-and c/kv/adj-vaccinated groups became prrsv seropositive as follows: at dpfv ( % and %), ( % and %), ( %), ( % and %) and ( % and %), respectively (fig. d-h) . this result indicated that the d/kv/adj vaccine induced greater prrsv specific antibody production than the commercial c/kv/adj-vaccine in pigs. all the c/kv/adj-vaccinated pigs ( %) became seropositive at dpc ( fig. i-k) . at dpc and , all the c/kv/adj vaccinated groups showed significantly higher s/p ratios compared to the control groups (p < . ). at dpc and , the s/p ratios of the d/ kv/adj-vaccinated groups were significantly higher than the c/kv/ adj-vaccinated groups (p < . ; fig. l -o). there was no significant difference between the s/p ratios of the c/kv/adjvaccinated and mock-vaccinated pig groups at dpc and ( fig. m-o) . additionally, the antibody titers of the control groups and remained negative ( fig. a-o) . serum prrs vn antibody titers in pigs were analyzed by the standard serum neutralization assay, and our results showed neither the d/kv/adj nor c/kv/adj vaccine induced vn antibodies to lkz/ (type ) or cm/ (type ) after the first or second vaccination. in the d/kv/adj-vaccinated pigs, vn titers against lkz/ were detected at dpfv (fig. a) . the vn titers in the d/ kv/adj-vaccinated groups were significantly higher compared to the c/kv/adj-vaccinated pigs at dpc and (p < . to p < . ) (fig. a) . a rapid vn antibody response against lkz/ virus was observed in the d/kv/adj vaccinated pig group indicating that the vaccine induced better protective memory immune response. to assess broad vn antibody activity, we used heterologous prrsv strain (type ) cm/ in vn assay and detected vn titers at dpfv in pig groups and vaccinated with d/kv/adj (fig. b) . the vn titers of these animals were significantly higher than those of c/kv/adj-vaccinated pigs (group ) at dpc - (p < . to p < . ) (fig. b) . the vn titers against the challenge virus strain of the pig group vaccinated with d/kv/adj and challenged with cm/ strain were similar to those of group animals challenged with the homologous virus strain lkz/ . this indicates that our experimental pigs received prrsv vaccination (strain lkz/ ) followed by a heterologous cm/ strain challenge generated antibodies with a broader neutralizing spectrum. significant difference in the macroscopic lung lesions scores were observed between pig groups and during necropsy at dpc . in a similar trend to the body temperature, less clinicallyaffected d/kv/adj-vaccinated pigs had significantly fewer lung lesions scores (p < . ) compared to severely clinically-affected mock-vaccinated and c/kv/adj-vaccinated pigs (fig. a ). all the mock-vaccinated and c/kv/adj-vaccinated pigs challenged with lkz/ or cm/ prrsv had gross lung lesions in the cranial, middle and accessory lobes, which resulted in failure of the lungs to collapse during necropsy and lung parenchyma remained firm and rubbery. no obvious lung lesions were observed in d/kv/adjvaccinated pig groups, indicating that this vaccine-adjuvant formulation enabled pigs to tolerate challenge with type and type prrsv. further, prrsv rna copy numbers in the lungs at necropsy ( dpc) was measured using the commercial quantitative pcr (qpcr) kit. in pig groups that were immunized with d/kv/adj, prrsv rna copies were undetectable in / animals (< rna copies per gram of lung tissue). the viral loads of the mock-vaccinated and c/ kv/adj-vaccinated pig groups were up to three logs ( ) above prrsv threshold detection limits; however, the viral loads of these groups were not significantly different (fig. b ). all the serum samples collected at dpfv , , , , , and were negative for prrsv type rna copies (data not shown). viremia after challenge was assessed by both qpcr and viral titers on marc- cells (fig. ) . prrsv rna could not be detected in the serum samples obtained from the pigs vaccinated with d/ . the viral rna copy numbers in the lung homogenates were quantified by qrt-pcr and its limit of detection was rna copies/ml. data is shown as mean ae sem viral rna copy numbers of or pigs in each group; ****p < . ; analyzed by two-way anova followed by tukey's multiple comparisons test. kv/adj at any time point throughout the period post-challenge, except in group pigs observed .  copies at dpc following challenge with the type viral strain cm/ . contrastingly, serum collected from pigs vaccinated with mock and c/kv/ adj had viremia with the viral copy numbers from .  to .  copies (fig. a) . the serum samples were also analyzed of virus titration using the marc- cells (fig. b) . prrsv was not detected in the serum of pig groups and collected at dpfv , , , , , and , confirming that the virus was properly inactivated (data not shown). the mock-vaccinated pig groups challenged with lkz/ or cm/ exhibited the highest mean viral titer of . and . log tcid /ml at dpc in serum samples, respectively. all the animals vaccinated with c/kv/adj remained viremic until dpc . in contrast, d/kv/adj vaccinated pigs did not show viremia at all the dpcs by both rt-pcr and viral isolation methods (fig. ) . inactivated (killed) and mlv prrsv vaccines are commercially available and licensed for use in many countries to control reproductive and respiratory forms of prrs (murtaugh and genzow, ) . however, each vaccine type possesses its own strengths and limitations. due to safety issues inactivated vaccines are preferred over attenuated vaccines; however, efficacy of current inactivated prrsv vaccines is questionable (renukaradhya et al., a; scortti et al., ; vanhee et al., ; zuckermann et al., ) . inactivated prrsv vaccine under field situations showed to elicit protective immune response under certain conditions, which was depending on the strain of infecting field virus. by employing a controlled inactivation procedure and using a suitable adjuvant, an inactivated prrsv vaccine could be developed that systematically induces prrsv specific vn antibody response after two vaccinations in naïve piglets (nilubol et al., ) . serum vn antibodies have been identified as a key component of protective immunity against prrsv infection (osorio et al., ) . researchers have previously showed that an inactivated vaccine could induce vn antibodies resulting in strong to partial virological protection in challenged pigs (misinzo et al., ; renukaradhya et al., a) . experiments of passive transfer of vn antibodies in pigs prior to prrsv infection showed vn antibodies could fully protect pigs against viremia and reproductive failure (lopez and osorio, ; osorio et al., ) . however, others observed only low to moderate degree of protection against heterologous strains of virus when pigs were immunized with inactivated prrsv vaccines (labarque et al., ; lager et al., ) . but intranasal delivery of poly(lactic-coglycolic) acid nanoparticle-entrapped uv-killed prrsv vaccine adjuvanted with soluble mycobacterium tuberculosis derived whole-cell lysate showed the induction of cross-protective immune response in pigs (binjawadagi et al., a (binjawadagi et al., , b . at present, it is generally accepted that there is a need for new and safe vaccines that protect against homologous and heterologous/heterogeneous prrsv infections. in this study, our objective was to assess the efficacy of a candidate bei-inactivated prrsv vaccine (d/kv/adj), which contains a recently isolated prrsv field strain (lkz/ ) in kazakhstan, against a homologous (type ) and heterogeneous (type ) viral challenges. a commercial killed prrsv vaccine (c/kv/adj, strain nvdc-jxa , type ) was selected as a reference vaccine, which has been in use for immunization of pigs against prrs in most of the pig farms in kazakhstan. the efficacy of our vaccine candidate was assessed by evaluating its ability to reduce viremia and prevent clinical disease, including lung lesions in vaccinated pigs challenged with a homologous or heterogeneous prrsv. elisa antibody analysis demonstrated that the commercial vaccine induced prrsv-specific antibodies; however, only % of vaccinated pigs became seropositive prior to challenge. after challenge by day all the c/kv/adj vaccinated pigs became seropositive. however, we did not observe vn titers to lkz/ or cm/ in the serum pre-challenge, and only a slightly elevated neutralizing antibody response was detected post-challenge in c/ kv/adj-vaccinated pigs. these results are consistent with other published studies (scortti et al., ; zuckermann et al., ) , wherein vaccination with inactivated prrsv candidate vaccines slightly improve vn antibody response post-challenge. moreover, comparable to mock-vaccinated pigs, c/kv/adj vaccinated animals became ill post-challenge and manifested characteristic clinical signs of prrs (severe respiratory disorders with high scores), with simultaneous detection of viremia. noticeable macroscopic lung lesions were observed at necropsy in all the mock-and c/kv/adjvaccinated pigs challenged with lkz/ (type ) or cm/ (type . data in (a) indicates the mean prrsv ae sem rna copy numbers of or pigs in each group, and data in (b) were expressed as the geometric mean ae sem log tcid /ml of or pigs in each group. serum samples of pigs were collected up to dpc ( dpfa) challenged with prrsv (lkz/ or cm/ ). the limit of detection by the assay kit was rna copies per ml. the limit of detection for the virus titration assay was . tcid /ml. ) virus, and detected high challenged viral rna copy numbers in the lungs. thus, we conclude that the commercial c/kv/adj vaccination in pigs does not protect against clinical disease, viremia and lung lesions against field variants of kazakh strains of prrsv. these results are in line with earlier studies, such as vaccination against prrs in the field provides variable degrees of protection, and reports of clinical outbreaks of prrs in vaccinated pigs have led to doubts about efficacy of current commercial prrsv vaccines (geldhof et al., ; thanawongnuwech and suradhat, ) . in contrast, elisa results demonstrated that the d/kv/adjvaccinated pigs produced prrsv-specific antibodies more rapidly than the c/kv/adj and were % seropositive. after the third immunization, vn antibodies were detected in all the animals vaccinated with d/kv/adj prior to challenge, albeit at low levels. interestingly, the vn titer did not reduce after challenge in any pig vaccinated with d/kv/adj, and the animals had consistently high vn titers against both homologous and heterogeneous prrsv. this data is in agreement with an earlier study, which showed that high vn titers are required at the time of challenge to offer full protection against high dose of virus used to infect animals (geldhof et al., ) . the detectable levels of replicating prrsv was absent post-challenge in d/kv/adj vaccinated pigs, which could be attributed to early appearance (pre-challenge) of vn antibodies targeting the putative neutralizing epitopes on the prrsv gp and m proteins (yang et al., ) , but it needs further investigation. inactivated vaccines predominantly elicit th response (spellberg and edwards, ), but we cannot exclude the possibility of th -th -balanced response in d/kv/adjvaccinated pigs, which also requires further research. in the lkz/ (type ) and cm/ (type ) prrsv strains challenged pigs vaccinated with d/kv/adj, we observed a strong association between the induction of virus-specific neutralizing antibody response and the absence of viremia, lung lesions and clinical disease; indicating that vn antibodies may have contributed significantly to cross-protection. similar results were also observed in our previous study with this candidate vaccine, but challenged with a heterologous nadc- (type ) strain of virus . these findings demonstrated that our candidate killed prrsv vaccine has a safety profile, and it is efficacious when administered three times at days , and to large-white breed of pigs aged months. the results of this study is interesting as the candidate killed prrsv vaccine provided strong heterogeneous protection, because even modified live prrsv vaccines have been shown ineffective against inter-genotypic virus and provided incomplete protection against reinfections and heterologous viruses (botner et al., ; kimman et al., ; martelli et al., ; renukaradhya et al., a,b) . the reasons beyond better protective response by our candidate vaccine could be attributed to vaccination of pigs three times with montanide tm seppic adjuvant compared to others, wherein in many vaccine trials killed or subunit prrsv vaccines were vaccinated twice using relatively weaker adjuvant than montanide adjuvant (renukaradhya et al., a) . furthermore, at this moment due to lack of genome sequence data of both the vaccine and challenge viruses, it is difficult to draw any meaningful conclusions on this interesting result. in this study, although our candidate bei-inactivated prrsv vaccine adjuvanted with montanide tm gel st provided protection against homologous (type ) and heterogeneous (type ) strains of virus, this does not confirm that this vaccine formulation would cross-protect against other heterogeneous and heterologous field strains of viruses; which needs further vaccine trials. due to lack of sequence data of vaccine and challenge viruses used in this study, it is likely that though the commercial vaccine virus and the challenge virus strain are type viruses, the level of genetic variability is not known. however, to our knowledge, this is the first report demonstrating that an inactivated prrsv vaccine could also provide cross-protective response against a different viral genotype in pigs, which offers new perspectives for the development of effective and safe prrsv vaccines. the authors declare no potential or actual conflict of interest. selection of effective activation agents for porcine reproductive and respiratory syndrome virus inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs appearance of acute prrs-like symptoms in sow herds after vaccination with a modified live prrs vaccine pathogenesis of porcine reproductive and respiratory syndrome virus adjuvants for porcine reproductive and respiratory syndrome virus vaccines detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars load reduction in live prrs vaccines using oil and polymer adjuvants comparison of the efficacy of autogenous inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus prrs costs industry $ million annually pork checkoff study effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology antibody and cellular immune responses of swine following immunisation with plasmid dna encoding the prrs virus orf's , , and impact of genetic diversity of european-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challenge-exposed with an antigenically distinct prrsv isolate protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein genomic analysis of two chinese strains of porcine reproductive and respiratory syndrome viruses with different virulence montanide tm gel st adjuvant enhances prrs modified live vaccine efficacy by regulating porcine humoral and cellular immune responses role of neutralizing antibodies in prrsv protective immunity efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to a heterologous european (italian cluster) field strain: clinical protection and cellmediated immunity efficacy of an inactivated prrsv vaccine: induction of virus-neutralizing antibodies and partial virological protection upon challenge immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents the effect of a killed porcine reproductive and respiratory syndrome virus (prrsv) vaccine treatment on virus shedding in previously prrsv infected pigs identification of porcine reproductive and respiratory syndrome virus of north american genotype in republic of kazakhstan (in russian) passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity adjuvant formulation for veterinary vaccines: montanide tm gel safety profile a simple method of estimating fifty per cent endpoints inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv type /type immunity in infectious diseases isolation of the european genotype porcine reproductive and respiratory syndrome virus (prrsv) in kazakhstan the pathogenicity of swan derived h n virus in birds and mammals and its gene analysis an influenza viral vector brucella abortus vaccine induces good cross-protection against brucella melitensis infection in pregnant heifers bei-inactivated prrs vaccine adjuvanted with montanidetm gel st elicits virus specific cross-protective immune responses in piglets development of an experimental inactivated prrsv vaccine that induces virusneutralizing antibodies categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp and gp proteins and susceptibility to neutralization porcine reproductive and respiratory syndrome virus (porcine arterivirus). diseases of swine assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge key: cord- -zlr nwc authors: burimuah, vitus; sylverken, augustina; owusu, michael; el-duah, philip; yeboah, richmond; lamptey, jones; frimpong, yaw oppong; agbenyega, olivia; folitse, raphael; tasiame, william; emikpe, benjamin; owiredu, eddie-williams; oppong, samuel; adu-sarkodie, yaw; drosten, christian title: sero-prevalence, cross-species infection and serological determinants of prevalence of bovine coronavirus in cattle, sheep and goats in ghana date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: zlr nwc cattle, goats and sheep are dominant livestock species in sub-saharan africa, with sometimes limited information on the prevalence of major infectious diseases. restrictions due to notifiable epizootics complicate the exchange of samples in surveillance studies and suggest that laboratory capacities should be established domestically. bovine coronavirus (bcov) causes mainly enteric disease in cattle. spillover to small ruminants is possible. here we established bcov serology based on a recombinant immunofluorescence assay for cattle, goats and sheep, and studied the seroprevalence of bcov in these species in four different locations in the greater accra, volta, upper east, and northern provinces of ghana. the whole sampling and testing was organized and conducted by a veterinary school in kumasi, ashanti region of ghana. among sampled sheep (n = ), goats (n = ), and cattle (n = ), the seroprevalence rates were . %, . % and . %. for cattle, seroprevalence was significantly higher on larger farms ( . % vs . %, comparing farms with > or < animals; p = . ). highest prevalence was seen in the northern province with dry climate, but no significant trend following the north-south gradient of sampling sites was detected. our study identifies a considerable seroprevalence for bcov in ghana and provides further support for the spillover of bcov to small ruminants in settings with mixed husbandry and limited separation between species. cattle, goats and sheep are among the major livestock species in ghana. the present numbers in based on the food and agriculture organization (fao) animal production database are estimated to range around . , . , and . million cattle, goats and sheep in the country, respectively. among livestock, only chicken outnumber these species ( million). while disease surveillance is in place, there are knowledge gaps concerning the laboratory-based prevalence of some major livestock diseases. among these is bovine coronavirus (bcov) that affects cattle and other livestock species including horses and camels. bcov (yang and leibowitz, ; oma et al., ; pfefferle et al., ) . while different strains may have some antigenic variability, all strains elicit cross-reactive seropositivity and thus form a single serotype (clark, ; el-ghorr et al., ) . the virus is an important livestock pathogen causing effects on animal welfare as well as the economy (lathrop et al., a) . it causes diarrhea and respiratory disease in calves, as well as winter dysentery in adult cattle (boileau and kapil, ; ksiazek et al., ) . transmission of bcov is mainly through respiratory or fecal-oral routes (clark, ) , infecting the respiratory (nasal, tracheal, and lung) and intestinal (villi and crypts of the ileum and colon) epithelial cells (park et al., ) . when infected with bcov, within-herd transmission is generally rapid and infected animals display diverse clinical signs including diarrhea with or without blood, fever, and respiratory signs, which range from none to severe (clark, ; boileau and kapil, ) . in many african countries including ghana, livestock species live in close contact and animals serve diverse purposes such as transportation, draught power, fuel, clothing and as a source of meat and milk. husbandry practices do not involve the same standards of species separation and hygiene as in other parts of the world. close and sustained interaction between different animals as well as between animals and humans pose a risk of interspecies spillover of pathogens. bcov is characteristically a cattle virus. however, reports indicate bcov infections also occur in small ruminants. previous studies in australia (pass et al., ) , new zealand (durham et al., ) , chile (reinhardt et al., ) , and scotland (snodgrass et al., ) reported bcov infection in small ruminants. eisa and mohamed, also detected bcov antigens in goats (eisa and mohamed, ) whereas tråvén et al., detected bcov antibodies in sheep (tråvén et al., ) . recently, gumusova et al., have also detected bcov antibodies in goats (gumusova et al., ) . studies regarding the prevalence of bcov and its associated risk factors are however limited in africa, and none have been conducted in ghana. this study evaluated the sero-prevalence of bcov infection and assessed its associated risk factors among cattle, sheep, and goats in ghana. this study employed a cross-sectional design and was conducted between january to december in five districts in four regions of ghana. ghana is located in the west of africa, sharing borders with togo to the east, cote d'ivoire to the west, burkina faso to the north and the gulf of guinea, to the south and lies on latitude . and longitude - . . ghana has a tropical climate with an average annual temperature of about °c and the annual rainfall of . mm/ ″. agriculture dominates the economy of ghana and extensive farming practices in the country increase the livestock-wildlife-human interface this study was approved by the wildlife division of the ghana forestry commission (approval number: ao ). a total of animals aged ≥ months, comprising cattle, sheep and goats were included in the study. animals aged < months were excluded due to the possibility of detecting maternal antibodies. sampling was done using a simple two-stage cluster sampling technique. the regional veterinary officers of the ministry of food and agriculture (mofa), ghana in the selected regions were contacted for information on animal populations in their respective regions prior to the study. the list provided served as the sampling frame. prior to the study, a survey was carried out and an inclusion criteria of animal population (cattle, sheep and goats) ≥ for districts to be eligible for selection for the study was upheld. as a result, five ( ) districts that fulfilled the criteria were randomly selected. secondly, farms within these districts with herd size ≥ animals were randomly selected. all cattle, sheep, and goats within the selected farms were included in the study. if a district meets the first criteria, but the individual farms fail to meet the second criteria, farms which were very close to each other were pooled together. sites included were: bongo district in the upper east; savelugu and wale wale in northern; ada west in greater accra, and north tongu in volta. the map with the locations for sampling in shown in fig. . a validated questionnaire was used to obtain data on possible risk factors of bcov. data collected include: age, sex, dietary changes, parturition, and lactation status of female animals. additionally, the body score, presence of ectoparasites, and signs of infections such as fever, diarrhoea, respiratory distress, neurological disorder, and icterus were also assessed. ten milliliters ( ml) of blood was collected through jugular puncture from each animal after disinfection of the site with % using vacutainer tubes (becton dickinson, nj, usa) with needles ( gauge). in the field, the blood was allowed to clot before transportation to the veterinary laboratory in the district. at the district laboratory, the samples were spun for min at rpm to obtain the sera. the sera were transferred into three separate aliquots in cryotubes for each animal. the tubes were subsequently placed in liquid nitrogen to minimize antibody degradation. these processes were undertaken under sterile conditions. upon obtaining representative samples per district, the frozen samples were transported in a cold-chain to the kumasi centre for collaborative research (kccr) for long term storage at − °c prior to laboratory analysis. sample collection and preparation in each district and transportation to the kccr lab took an average of - days. during laboratory analysis, frozen sera were thawed at room temperature, vortexed and aliquots of μl of each sample were prepared. the aliquots were incubated at °c for min in warm water prior to recombinant immunofluorescence assay (ifa) as previously described (hoye et al., ; el-duah et al., ; reusken et al., a) . briefly, vero b cells were co-transfected with pcg plasmids bearing human coronavirus oc spike proteins. after overnight transfection, cells were harvested by treatment with trypsin to detach them in a cell culture incubator at °c and re-suspended in dulbecco's modified eagle medium (dmem) (invitrogen, usa) in % foetal calf serum (fcs). aliquots of cells were pelleted at x g for min and washed twice with ml phosphate buffered saline (pbs). fifty microliters ( μl) of cell pellets were spotted on well slides by dispensing and immediately aspirating, allowing s interval between spotting. the cells were fixed using ice cold acetone/methanol ( : ), dried at ambient temperature, and kept at °c after drying for min. to conduct the assay, μl of protein-free blocking solution (roti®-block, carl roth, karlsruhe, germany) was first added to each of the spotted fields on the slide and incubated at room temperature in a humid box for min, followed by rinsing with x tween-free pbs. after inactivation at °c for min, sera to be tested were diluted : in a x concentration of the protein-free blocking solution. subsequently, μl of the diluted sera was applied on each of the spotted area and incubated at °c for h in a humid chamber, followed by rinsing with . % tween in x pbs. secondary antibody detection was done by the alexa fluorescent reporter-conjugated goat anti-bovine, donkey anti-sheep, and donkey anti-goat igg antibodies for cattle, sheep, and goat bcov igg respectively. test evaluation was done by microscopic examination under a fluorescent microscope and a positive outcome was determined by bright green cytoplasmic fluorescence as shown in fig. . data were presented as frequencies (percentages) and chi square test was used to test for association where applicable. univariate and multivariate logistic regression analysis were performed to determine the possible factors associated with bcov sero-positivity for cattle, sheep, and goats. a p-value < . was considered statistically significant. all statistical analyses were performed using ibm statistical package for the social sciences (spss) software (spss inc., chicago, il, usa), and graphpad prism version . (graphpad software, inc., la jolla, california usa). the proportions of sheep, goat, and cattle were . %, . %, and . % respectively. there were more adults than weaners ( . % vs . %) and more female animals ( . %) compared to male animals ( . %), with . % of the females being lactating females at the time of the study. majority of the animals had high rectal temperature ( . %) and . % had physical signs of disease of which . % was diarrhea, . % was respiratory distress, and . % of the animals were icteric. none of the animals had neurological disorders. additionally, a higher proportion of the animals had ectoparasites ( . %), were thin ( . %) and have not had any dietary change ( . %) ( table ) . the sero-prevalence of bcov in the entire animal population was . %. upon stratification by sheep, goats, and cattle, the prevalence was . %, . % and . %, respectively. cattles had significantly higher prevalence of bcov compared to sheep and goats. among the entire animal population, sero-positivity of bcov was significantly associated with farms with ≥ animals ( . % vs . %, p < . ). upon stratification by type of animal, this effect seemed to be explained by cattle ( . % vs . %, p = . ) but not sheep and goats that are normally kept in smaller groups ( table ). the sero-prevalence of bcov was highest in the northern region followed by the volta region (table ) . even though our sampling sites formed a north-south gradient, there was no latitude-dependent trend in seroprevalence. there was no statistically significant association between the possible risks factors assessed and bcov sero-positivity among all animals with the exception of dietary change, where a significantly lower odd of bcov was observed among cattle with recent dietary change [or = . , % ci ( . - . ), p = . ] ( (table ) . bcov is a ubiquitous infection and bcov-specific antibodies have been detected in cattle populations in numerous countries (hasoksuz et al., ; kapil et al., ; lathrop et al., b; yavru et al., ) . bcov shares recent common ancestry with human coronavirus oc (hcov-oc ) (vijgen et al., ) and the two are serologically closely related to the extent that hcov-oc is often used as a proxy in serological testing as previously described where specific proteins of bcov were not available for serological testing or when national legislation restricts the use of certain livestock pathogens (reusken et al., b; meyer et al., ) . in most african countries such as ghana, diverse farm animals live in close contact that poses a risk of cross-species infection. indeed, we found seropositivity against bcov not only among cattle ( . %) but also among sheep and goats at . % and . % prevalence rates, respectively. this prevalence was predominant among ontario feedlots, respectively based on enzyme linked immunosorbent assay (elisa) method. bidokhti et al. ( ); hasoksuz et al. ( ) , and yildirim et al. ( ) also reported a sero-prevalence of - %, . %, and . %, respectively among cattle. the discrepancies in the prevalence rates compared to that of this present study could be attributed, at least in part, to differences in geographical location, different management systems, source population size, method employed for bcov antibody detection, and the samples size used in the different investigations. in addition, the higher prevalence rate among cattle could be due to the fact that a higher proportion of the animals were cattle as well as the tropism of bcov to cattle few studies have been conducted to evaluate the prevalence of bcov among small ruminants. the most recent study was conducted by gumusova et al. in in turkey. in their study, they evaluated the sero-prevalence of bcov in goats by employing commercially available competitive elisa kits and reported a bcov sero-prevalence of . % (gumusova et al., ) . in a previous study, eisa and mohamed reported detection of bcov antigens in goats (eisa and mohamed, ) . additionally, tråvén et al., in a study among sheep from flocks in different parts of sweden, reported that % of the sheep were positive for bcov antibodies (tråvén et al., ) . prior to these recent studies, there had been reports of bcov infection in small ruminants in australia (pass et al., ) , new zealand (durham et al., ) , chile (reinhardt et al., ) , and scotland (snodgrass et al., ) . our finding in sheep and goats, thus, provides update information of spillover of bcov from cattle. granted the contagious nature of bcov, it is imperative that factors that influence exposure and the determinants of bcov infection be identified which would assist in the development of apt control and preventive measures against bcov and other infectious diseases. though there was no statistically significant association between the possible risks factors assessed and bcov sero-positivity, we found bcov sero-positivity to be significantly associated with farms with higher cattle density. this finding is in harmony with studies by beaudeau et al. ( ) ; hägglund et al. ( ) , and ohlson et al. ( ) who reported that large herd size is a risk factor for bcov infections in dairy cattle. this may be due to poor biosecurity especially among farms with larger herd size in ghana, and also due to the close contact between animals in these farms which could potentiate the transmission of bcov compared to farms with small herd size (beaudeau et al., ; ohlson et al., ) . infectious diseases surveillance can be greatly enhanced by research studies, as often there is close collaboration between governmental and academic institutions. restrictions in the movement of samples to prevent the spread of notifiable livestock diseases create a demand for domestic laboratory capacities. through this study we hope to demonstrate the value of capacity building in field-based research. sığırlarda coronavirus enfeksiyonunun epidemiyolojisi. ankara Üniv vet fak derg spatial patterns of bovine corona virus and bovine respiratory syncytial virus in the swedish beef cattle population reduced likelihood of bovine coronavirus and bovine respiratory syncytial virus infection on organic compared to conventional dairy farms bovine coronavirus associated syndromes bovine coronavirus rotavirus and coronavirus associated diarrhoea in domestic animals role of enteric pathogens in enteritis in lambs, goat kids and children and their zoonotic importance potential intermediate hosts for coronavirus transmission: no evidence of clade c coronaviruses in domestic livestock from ghana a serological comparison of bovine coronavirus strains first report of bovine rotavirus and bovine coronavirus seroprevalance in goats in turkey dynamics of virus infections involved in the bovine respiratory disease complex in swedish dairy herds detection of respiratory and enteric shedding of bovine coronaviruses in cattle in an ohio feedlot detection of respiratory and enteric shedding of bovine coronaviruses in cattle in northwestern turkey surveillance of wild birds for avian influenza virus excretion and persistence of bovine coronavirus in neonatal calves a novel coronavirus associated with severe acute respiratory syndrome association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle antibodies against mers coronavirus in dromedaries the relationship between the occurrence of undifferentiated bovine respiratory disease and titer changes to bovine coronavirus and bovine viral diarrhea virus in ontario feedlots risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds bovine coronavirus in naturally and experimentally exposed calves; viral shedding and the potential for transmission dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves intestinal coronavirus-like particles in sheep with diarrhoea distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus e in bats diagnosis of coronavirus in sheep in valdidia province middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study virus infections in cattle and sheep in scotland - serum antibodies to bovine coronavirus in swedish sheep evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc the structure and functions of coronavirus genomic ′ and ′ ends bovine coronavirus (bocv) infection in calves with diarrhoea and their dams seroprevalence of the rotavirus and corona virus infections in cattle this work was supported by deutsche forschungsgemeinschaft under a grant to y. a. s. and c. d. (dr / - ). key: cord- -a vblbbz authors: decaro, nicola; buonavoglia, canio title: canine parvovirus—a review of epidemiological and diagnostic aspects, with emphasis on type c date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: a vblbbz canine parvovirus type (cpv- ) emerged in late s causing severe epizootics in kennels and dog shelters worldwide. soon after its emergence, cpv- underwent genetic evolution giving rise consecutively to two antigenic variants, cpv- a and cpv- b that replaced progressively the original type. in , a new antigenic variant, cpv- c, was detected in italy and rapidly spread to several countries. in comparison to the original type cpv- , the antigenic variants display increased pathogenicity in dogs and extended host range, being able to infect and cause disease in cats. epidemiological survey indicate that the newest type cpv- c is becoming prevalent in different geographic regions and is often associated to severe disease in adult dogs and also in dogs that have completed the vaccination protocols. however, the primary cause of failure of cpv vaccination is interference by maternally derived immunity. diagnosis of cpv infection by traditional methods has been shown to be poorly sensitive, especially in the late stages of infections. new diagnostic approaches based on molecular methods have been developed for sensitive detection of cpv in clinical samples and rapid characterisation of the viral type. continuous surveillance will help assess whether there is a real need to update currently available vaccines and diagnostic tests. canine parvovirus type (cpv- ) emerged in late s causing severe epizootics in kennels and dog shelters worldwide. soon after its emergence, cpv- underwent genetic evolution giving rise consecutively to two antigenic variants, cpv- a and cpv- b that replaced progressively the original type. in , a new antigenic variant, cpv- c, was detected in italy and rapidly spread to several countries. in comparison to the original type cpv- , the antigenic variants display increased pathogenicity in dogs and extended host range, being able to infect and cause disease in cats. epidemiological survey indicate that the newest type cpv- c is becoming prevalent in different geographic regions and is often associated to severe disease in adult dogs and also in dogs that have completed the vaccination protocols. however, the primary cause of failure of cpv vaccination is interference by maternally derived immunity. diagnosis of cpv infection by traditional methods has been shown to be poorly sensitive, especially in the late stages of infections. new diagnostic approaches based on molecular methods have been developed for sensitive detection of cpv in clinical samples and rapid characterisation of the viral type. continuous surveillance will help assess whether there is a real need to update currently available vaccines and diagnostic tests. ß elsevier b.v. all rights reserved. . an overview on canine parvovirus the family parvoviridae comprises two subfamilies, parvovirinae and densovirinae, infecting vertebrates and insects, respectively. currently, five genera are included in the subfamily parvovirinae, namely parvovirus, erythrovirus, dependovirus, amdovirus and bocavirus. canine parvovirus (cpv) belongs to genus parvovirus and has been included in the unique species feline panleukopenia virus together with feline panleukopenia virus (fpv), mink enteritis virus (mev) and raccoon parvovirus (rpv) (tattersall et al., ) . cpv is genetically and antigenically unrelated to canine minute virus (cnmv), formerly known as canine parvovirus type (cpv- ), which is responsible for neonatal death in dogs and is now included in the genus bocavirus together with bovine parvovirus and human bocavirus (tattersall et al., ) . parvoviruses are small (diameter of nm), nonenveloped viruses infecting vertebrates and insects. the parvovirus virion consists of a spherical capsid, which is composed by three proteins and contains a linear, singlestrand dna molecule (muzyczka and berns, ) . by means of x-ray crystallography, the parvovirus capsid has been found to be formed by copies of a combination of vp , vp and vp . vp contains the full-length vp sequence plus an additional n-terminal domain. vp , the most abundant structural protein, accounts for % of the viral capsid, representing the major determinant of host range and virus-host interactions, and is cleaved to vp by host proteases. the capsid protein subunits are made up of a core eight-stranded, antiparallel b-barrel domain with loop insertions between the strands forming large spike-like protrusions at or surrounding the icosahedral threefold axes. other characteristic features of the parvoviral capsid include a -Å canyon-like depression about the fivefold axes and a dimple-like depression at the icosahedral twofold axes. the parvoviral genome consists of a $ -nucleotide dna molecule containing two large open reading frames (orfs) and smaller or overlapping genes, encoding for two nonstructural (ns and ns ) and two structural (vp and vp ) proteins through alternative splicing of the same mrnas. virus replication takes place in the cell nuclei and requires rapidly dividing cells of fetuses and newborns or of hematopoietic and intestinal tissues of young and adult animals. their replication in vivo is rarely associated with the appearance of nuclear inclusion bodies, whereas the cytopathic effect induced in vitro is not always evident. all parvoviruses are highly stable in the environment, as they are extremely resistant to ph and temperature changes and to treatment with lipid solvents, trypsin and most disinfectants. virions can be inactivated by formalin, sodium hypochlorite, beta propiolactone, hydroxylamine, oxidizing agents and ultraviolet irradiation. several parvoviruses are able to agglutinate erythrocytes of different mammal and bird species, and some diagnostic tests for parvovirus infections rely on this hemagglutination activity (muzyczka and berns, (truyen, ) . the original viral strain, designated as cpv- to distinguish the novel virus from the previouslyknown cpv- , caused severe, fatal epizootics of hemorrhagic gastroenteritis and subacute myocarditis in kennels and shelters worldwide (truyen, ) . there are at least six or seven amino acid (aa) changes between fpv and cpv- (table ) , mostly accumulated in the vp domain interacting with the host-cell transferrin receptor (tfr). these changes may have contributed to the gain of affinity for canine tfr observed during the shift from fpv or fpv-like parvovirus to cpv- (shackelton et al., ) . another important difference between these two carnivore parvoviruses is that cpv evolves much more rapidly than fpv (battilani et al., ; decaro et al., c decaro et al., , b , showing genomic substitution rates similar to those of rna viruses, with values of about À substitutions per site per year (shackelton et al., ) . active virus circulation and initial vaccination programmes helped develop herd immunity in canine populations, which greatly reduced mortality and further spreading of the virus. however, host-immunity pressure may have also contributed to the progressive emergence of cpv- antigenic variants. in the s, two antigenic variants, distinguishable using monoclonal antibodies (mabs), emerged within few years and were termed cpv types a (cpv- a) and b (cpv- b). currently, the antigenic variants have completely replaced the original type , which is still used in most commercial vaccines, and are variously distributed in the canine population worldwide. cpv- a and cpv- b differ from the original strain cpv- in five or six aa residues of the vp capsid protein (table ). in contrast, only two residues differentiated cpv- a from cpv- b, i.e., n d and i v (truyen, ) . residue is situated in epitope a, over the threefold spike of the capsid, and a role of antigenic escape has been assigned to the same residue of the vp of the parvovirus minute virus of mice. at residue , old type a cpvs displayed an isoleucine residue differentiating this variant from cpv / b strains both exhibiting v . with the subsequent introduction of the change i v, the recent cpv- a strains differ from cpv- b/ c only at position of the vp protein (table ) (martella et al., ; decaro et al., b) . other mutations occurring during the last decades and being fixed in type a/ b cpvs include s a and t a. residue is located in the middle of epitope b and substitutions at this position may be responsible for changes in antigenicity of cpv variants (truyen, ) . change t a has been reported worldwide (kapil et al., ; kang et al., ; decaro et al., b) , but its implications are not clearly understood. the majority of these changes affects the vp region comprised between residues and , which forms the gh loop located between the bg and bh strands and is affected by the greatest variability among parvoviruses due to its exposure on the capsid surface. cpv recognizes as target tissues for viral replication the intestinal crypts and the lymphoid organs, but the virus can spread to all tissues (pollock, ) , including the brain (elia et al., ; decaro et al., a) . after penetration through the oronasal route, the virus replicates in gastroenteric associated lymphoid tissues and is disseminated by infected leukocytes to the germinal epithelium of the crypts of the small intestine, causing diarrhea. infection of leukocytes, mainly circulating and tissue-associated lymphocytes, induces acute lymphopenia (often associated with neutropenia) (pollock, ) . in - -week-old seronegative pups, cpv is also able to replicate in cardiac cells inducing a fatal myocarditis. however, this form is no longer observed as almost all young pups are protected by maternally derived antibodies (mda) (greene and decaro, in press ). the most characteristic clinical form induced by cpv is represented by hemorrhagic enteritis, the extent of which is often dependent on the mda titers of the infected pups at the moment of infection. clinical signs occur after an incubation period of - days and consist of anorexia, depression, vomiting and mucoid or bloody diarrhea, frequently dehydration and fever. leukopenia is a constant finding, with white blood cell (wbc) counts dropping below - cells/ml of blood. however, total wbc counts may be even within normal ranges due to concomitant virus-induced lymphopenia, and neutrophilia consequent to infections by opportunistic bacteria. concurrent pulmonary infections may lead to the onset of respiratory distress. subclinical and inapparent infections are frequently detected, mainly in pups with intermediate mda titers and in adult dogs (decaro et al., a) . the mortality rates can be high (up to %) in pups, but are usually less than % in adult dogs. hemorrhagic enteritis of the small intestine and enlargement of mesenteric lymph nodes and peyer's patches are the main gross lesions observed in dogs that table amino acid variations in the vp protein of feline and canine parvoviruses. a aa residue codon affected by snps used to design type-specific probes differentiating cpv- from cpv- a/ b/ c. c codon affected by snps used to design type-specific probes differentiating cpv- a from cpv- b and cpv- b from cpv- c. die as a consequence of cpv infection. histopathologically, the small intestine is affected by multifocal crypt necrosis and intranuclear inclusion bodies, whereas extensive depletion of lymphocytes is seen in peyer's patches, lymph nodes, spleen and thymus (greene and decaro, in press ). prophylaxis of cpv infection relies mainly on extensive vaccination. since inactivated vaccines are able to induce only short-term immunity, modified live virus (mlv) vaccines are widely used. these vaccines, prepared by using either the original type cpv- or its variant cpv- b, are highly effective, being able to protect dogs against parvoviral disease as well as infection, and almost completely safe, as postvaccinal reactions are very rarely observed. a recent study showed that most dogs developing parvovirus-like diarrhea after vaccination were infected by the field virus alone or with the attenuated vaccine virus (decaro et al., b) . the primary causes of failure of cpv vaccination are interfering levels of mda that are transmitted by bitches to their offspring through colostrum and, at a lesser extent, milk. thus, in order to avoid the interference with active immunisation, vaccines should be administered to pups only after waning of mda (greene and decaro, in press ). different strategies have been proposed to overcome the mda interference, including high-titer vaccines (burtonboy et al., ) and intranasal vaccination . the vaccination guidelines group of the world small animal veterinary association also recommends delaying finish of primary cpv vaccination course to - weeks of age to ensure protection even in pups with longlasting mda. in addition, there are some concerns about the complete efficacy of type -based vaccines against the antigenic variants (kapil et al., ; decaro et al., a decaro et al., , a . in , a new antigenic type of cpv was detected in italy . the fecal samples of two dogs with hemorrhagic enteritis tested positive for cpv by hemagglutination (ha) and both strains were characterised as cpv- b on the basis of their reactivity to monoclonal antibodies (mabs) and the results of genotyping polymerase chain reaction (pcr). however, by sequence analysis of a large fragment of the vp gene, the two cpv- b strains were found to contain two unexpected amino acid variations, s a and d e, with respect to classical type b cpvs. while the change at residue is shared by most type a and b cpvs circulating currently (truyen, ) , the substitution d e, due to a single nucleotide change at the third codon position, had not been observed previously and is situated in epitope a, over the three-fold spike of the capsid. subsequently, viruses sharing this unusual mutation, firstly named glu mutant, were detected at high frequency in italy (decaro et al., b (decaro et al., , a and were found to have a worldwide distribution (nakamura et al., ; decaro et al., c decaro et al., , a decaro et al., , b hong et al., ; kapil et al., ; perez et al., ; vieira et al., ; touihri et al., ; nandi et al., ; ntafis et al., ; filipov et al., ) . the widespread distribution of the glu- mutant posed a taxonomical issue as this mutant was proposed as a new variant cpv- c (decaro et al., b) . however, the same designation had been previously assigned to cpv strains detected in domestic and wild felids in southern asia. those mutant strains displayed the novel substitution irrespective of the antigenic type ( a or b), so that had been referred to as cpv- a(c) or cpv- b(c) on the basis of the aa encountered at residue (asn or asp) of the vp protein (ikeda et al., ) . the mutation at residue was associated to the further adaptation of the cpv variants to the feline host (ikeda et al., ) , as indicated by the appearance of the same change in a cpv mutant after consecutive passages in feline cells in vitro. although this change has been recently reported in a dog in korea (kang et al., ) , it was absent from all cpv variant strains sequenced in several studies conducted in different parts of the world (meers et al., ; ohshima et al., ; decaro et al., b) , thus restricting circulation of those mutants to south-east asia. in contrast, the glu- mutant has spread very efficiently to the canine population in italy and has been reported in other parts of world. moreover, although mutation g d is located in the middle of antigenic site b and controls the binding of many antibodies to the capsid, the changed e has been previously taken into account for classification of the variants a and b (truyen, ) . on the basis of the above considerations, the glu- mutant has been tentatively designated as true antigenic variant c, whereas the asp- cpvs should be regarded as mutants of cpv- a and cpv- b (decaro et al., b) . the recent development of real-time pcr assays based on minor groove binder (mgb) probe technology has led to a considerable simplification of the diagnostic approaches for cpv characterisation. type-specific mgb probe assays were employed to characterise cpv-positive samples collected in italy during the decade - , finding ( . %) type a, ( . %) type b and ( . %) type c cpvs. the retrospective analysis has revealed that cpv- c was not present in italy before . the frequency of the variants underwent a rapid fluctuation during years - , with cpv- c replacing very rapidly cpv- b, that has been detected more rarely in italy in the last years (decaro et al., b) . more recently, the new variant c was detected in several countries. in an epidemiological survey for cpv, nakamura et al. ( ) found a cpv- c strain in vietnam. in , such a variant was isolated from an outbreak of fatal gastroenteritis occurred in a breeding kennel of basset hounds located in catalonia, spain (decaro et al., c) . the results of two different european epidemiological surveys (decaro et al., a (decaro et al., , b showed that cpv- c is now predominant in italy, germany and spain and is also widely distributed in portugal, france and belgium, where cpv- b or cpv- a are more frequently detected (table ) . sporadic isolation of cpv- c has been obtained also from the united kingdom, greece and bulgaria, where there was a higher frequency of cpv- a/ b detection (decaro et al., a; ntafis et al., ; filipov et al., ) . the oldest cpv- c strain was isolated in (decaro et al., a) , thus providing evidence that this variant had been circulating in germany for years before its first detection in italy in . vp sequence analysis of cpv strains obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the united kingdom over a -year period revealed that % of viruses were cpv- a, and % cpv- b, with no type c found (clegg et al., ) . outside europe, all three variants are well represented in tunisia (touihri et al., ) . type b and c isolates predominate in north america (hong et al., ; kapil et al., ) , whereas cpv- c is more widespread in south america (perez et al., ; calderon et al., ) with the exception of brazil, where all circulating strains were characterised as cpv- a (castro et al., ) . cpv- a is the predominant variant in asia (kang et al., ; ohshima et al., ; nandi et al., ) and australia (meers et al., ) , although few cpv- c strains have been detected in india (table ) . there are at least six or seven aa changes between fpv and cpv- , and at least five or six aa changes between the variants cpv- a/b and the original type (table ) . these few aa differences in the vp sequences of fpv, cpv- and cpv- a/b account for important antigenic and biological changes (e.g., the host range shift), making cpv an important model to study virus evolution (hueffer and parrish, ) . in vitro, while fpv replicates efficiently only in feline cell lines, cpv can infect with the same efficiency cells of canine and feline origin. in vivo, fpv replicates in dogs in the thymus and bone marrow without being shed in the feces, and the original canine virus, cpv- , does not replicate at all in cats. conversely, both the type a and b variants have re-gained the ability to replicate in vivo in the feline host (truyen, ) . studies on the interactions of fpv and cpv with their cellular receptor, the transferrin receptor type , have revealed that fpv specifically binds the feline tfr, whereas cpv- and its variants can bind both the feline and the canine tfrs. interestingly, the antigenic variants of cpv- bind the canine and feline tfrs less efficiently than the original type- (palermo et al., ) . cases of feline panleukopenia caused by cpv- a or b in wild and domestic felids have been reported in different parts of the world (truyen, ) . as for the new variant cpv- c, a first case was reported in a cat in italy, but nothing was known about the clinical conditions, hematological parameters and outcome of the infection (battilani et al., ) . more recently, two cases of cpv- c infection in cats were observed in the same country (decaro et al., a (decaro et al., , a . in both cases, a mild form of disease was evident with no or moderate modifications of hematological parameters; neurological disease occurred in one cat, but this was associated to a concurrent intracranial abscess (decaro et al., a) . another biological consequence of the accumulation of aa changes in the vp protein is the increased pathogenicity of the cpv- variants (carmichael, ) . in comparison to the original type , the antigenic variants a and b have been reported to be shed in the feces at much higher titers and to cause a more severe disease. in addition, a lower virus dose seems to be required for efficient infection. experimental infection of dogs with different levels of mda showed that cpv- b is shed at very high titers in the feces of the infected dogs and that even pups with mda hemagglutination-inhibition titers of : may become infected (decaro et al., a) . the pathogenicity of cpv- c has been investigated in two litters of german shepherds with natural infection (decaro et al., b) . all pups displayed clinical signs of parvovirosis (mucoid or watery diarrhea and transient leukopenia), but none showed either hemorrhagic diarrhea or vomiting and all recovered in a few days. by real-time pcr, prolonged shedding (up to days) of cpv- c was observed. however, the benign course of the cpv- c infection is in contrast with recent findings that are indicative of a more severe disease induced by this mutant. spibey et al. ( ) have carried out an experimental infection of beagle dogs with a field isolate of cpv- c. this experimental challenge was carried out principally to confirm the ability of an existing type -based vaccine to prevent clinical signs and shedding. however, in this context it is of interest that all six of the infected control dogs became severely ill and displayed leukopenia from day post-infection; three of them had to be euthanized and the remaining three recovered but required a supportive therapy. to date, whether there is a greater infection or virulence by one variant over the others is not well established either in experimental systems or on the basis of field observations. indeed, it is reasonable to think that all three variants have a similar pathogenetic potential, which can increase depending on particular field conditions (age, immunological status, stressing factors, etc.). commonly, cpv infects - -week-old pups that are prone to acquire the virus in concomitance with the wane of mda (greene and decaro, in press) . adults are thought to be resistant to cpv infection due to the age-reduced susceptibility and presence of specific immunity induced by vaccination or previous (often subclinical) infections. although cpv infection is generally restricted to young animals, it has recently become an issue also in adult dogs. apart from personal observations (kapil et al., ) , there are some scientific reports of the occurrence of parvovirosis in adult dogs, mostly associated to cpv- c infection decaro et al., a decaro et al., , a . in one episode, a two-year-old mixed breed dog was severely affected as a consequence of infection with a strain which was mischaracterised as cpv- b by means of mabs . however, molecular characterisation by pcr-rflp and sequence analysis of the vp protein gene was able to correctly type the virus as 'true' cpv- c (martella, unpublished data) . another cpv- c outbreak occurred in a breeding kennel in italy, affecting dogs aged between months and . years and leading to the death of a month-old bernese mountain pregnant bitch (decaro et al., a) . a case of disease induced by this variant was described even in a -year-old dog displaying fever ( . c), accelerated pulse and respiratory rates, abdominal pain, vomiting, hemorrhagic diarrhea and dramatic lymphopenia. however, this dog recovered progressively despite the severity of the disease (decaro et al., a) . in most cpv- c outbreaks reported in adult dogs so far, the animals had undergone the full vaccination schedules including a booster vaccination on yearly basis (decaro et al., a (decaro et al., , a . in cpv- variant outbreaks involving younger dogs, the vaccination protocol scheduled for the first year of age had been completed (kapil et al., ; perez et al., ; calderon et al., ; ntafis et al., ; castro et al., ; filipov et al., ) . protection elicited by cpv- based vaccines against the field variants still represents a ''vexata quaestio'' as the current opinions are highly divergent. many authors have suggested that the old type- based vaccines are still protective against the variants currently circulating in the field. on the opposite side, other researchers believe that the immunity induced by cpv- vaccines is effective against the homologous (vaccine) virus but significantly lower against the variants, thus allowing an aggressive strain to cause infection and even mortality in dogs ''regularly'' vaccinated. it has been shown that there is a one-way cross-reactivity between the antigenic variants and the original cpv- . sera raised against cpv- displayed lower virus neutralizing (vn) titers against the heterologous virus (cpv- b) in comparison to those obtained when sera raised against cpv- b were run against the original cpv- . similar experiments were carried out for evaluation of serological crossreactivity between cpv- and its antigenic variants in sera of dogs and rabbits (cavalli et al., ) . animals administered cpv- showed significant lower vn antibody titers against the heterologous viruses with respect to the homologous virus. these findings were confirmed by cross-hemagglutination inhibition studies conducted in dogs (ohshima et al., ) and guinea pigs (kang et al., ) . despite this measurable difference in strain recognition, other studies have shown that 'old-type' vaccines are able to protect dogs against next challenge with cpv- c in experimental conditions (spibey et al., ; siedek et al., ) . however, in those experiments the challenge was carried out in controlled conditions few weeks after vaccination, when antibodies commonly reach the highest levels (decaro et al., a) . nothing is know about the protection induced by the original type against cpv- c (as well as against the other variants) after a long period interval between vaccination and challenge, when the type- antibody titers could be not high enough to efficiently prevent infection and disease caused by a field strain. in addition, in one study dogs vaccinated with cpv- were clinically protected from challenge with virulent variant virus, but shed the challenge virus, thus proving to be susceptible to infection (siedek et al., ) . taking into account the concern that the antigenic differences between the original type and its variants may decrease the effectiveness of the cpv- based vaccines, a more extensive use of vaccines prepared with the strains circulating in the field (type b-based) has been suggested martella et al., ; cavalli et al., ; decaro et al., a decaro et al., , a ohshima et al., ; calderon et al., ; ntafis et al., ; castro et al., ) . nevertheless, even considering all case reports of vaccination failures, there is no definitive evidence for the absence of cross-protection from clinical disease between old and variant cpvs. thus, the primary cause of widespread virus circulation despite extensive vaccination is still represented either by interference of mda in vaccinated pups or by poor efficiency of immune system in old dogs. as a consequence of the emergence of the cpv- variants in the feline population, a similar debate has arisen about the real efficacy of traditional fpv-based vaccines against the infections caused by cpv- a, cpv- b and cpv- c. in a recent study (gamoh et al., ) , an fpvbased vaccine was able to cross-protect against the subsequent infection with a virulent cpv- b strain. however, in this study only two vaccinated cats were used that were challenged in a very short period after the administration of the second vaccine dose. therefore, even acknowledging that additional studies are required to address this issue, the development of multivalent vaccines containing fpv in combination with a cpv variant strain should be seriously considered (decaro et al., a (decaro et al., , a . a rapid diagnosis of cpv infection is especially important in kennels and shelters in order to isolate infected dogs and prevent secondary infections of susceptible contact animals. clinical diagnosis is indecisive and several other viral pathogens may cause diarrhea in dogs, such as coronaviruses, adenoviruses, morbilliviruses, rotaviruses, reoviruses, noroviruses (greene and decaro, in press ). thus, a suspect clinical case should always be confirmed by laboratory tests. several methods have been developed for the laboratory diagnosis of cpv infection, which is usually carried out on the feces (or intestinal contents if the animal is dead) of affected dogs. in the late stages of infection, edta-blood samples have been proven to be more useful for the diagnosis as cpv viremia is exceptionally long lasting (decaro et al., a,b) . tests detecting viral antigens by means of antibodybased methods are suitable for cpv diagnosis in the veterinary practice and represent the only tests available in the field (greene and decaro, in press ). however, their sensitivity, like other traditional diagnostic methods, has been proven inferior to molecular assays. an immunochromatographic (ic) test was compared to molecular techniques, showing that the relative sensitivity of the test did not exceed % with respect to the nucleic acid-based methods, whereas the specificity was %. the poor sensitivity of the ic test was associated with the low amounts of virus shed in the feces during the late stages of infection and/or the early presence of high cpv antibody titers in the gut lumen that may sequestrate most viral particles . a more recent study compared the performances of three different commercially available, antibody-based tests for rapid detection of cpv antigens with pcr and immunoelectron microscopy, confirming the high specificity and low sensitivity of the antigen-detection kits (schmitz et al., ) . taking advantage on the close genetic and antigenic relationship among carnivore parvoviruses, in-house test systems developed for cpv diagnosis are able to detect also fpv (neuerer et al., ) . surprisingly, some concerns have been expressed about the ability of in-hospital rapid parvovirus tests to recognise efficiently the new variant cpv- c. those concerns took into account the circumstantial evidence that the increase in rapid test failure paralleled the emergence and spreading of cpv- c (kapil et al., ) . almost simultaneously, it was recommended to test the mabs contained in the in-house assays against the additional mutations detected in strains currently circulating (hong et al., ) . however, by ic testing of specimens representative of the three cpv variants including samples containing the recently identified cpv- c, the detection rate of this variant was similar to those of cpv- a/cpv- b, thus dissipating any previous concerns about the hypothesized but never demonstrated less efficiency of the test in detecting the new variant (decaro et al., b) . alternative techniques, such as hemagglutination (ha) and virus isolation (vi), can be carried out only in specialized laboratories. fresh erythrocytes are required for ha testing causing problems related to the management and housing of donor pigs. other species' erythrocytes, e.g., cat or rhesus monkey red cells, may be used, but they are either difficult to obtain in the quantities required, or are expensive. nevertheless, for a clear reading of the ha test, good quality erythrocytes should be ensured since the test is affected by an altered coefficient of erythrocyte sedimentation which may occur in case of stress or disease of the donor pig . furthermore, cpv- strains lacking ha activity have been reported . nevertheless, the ha test carried out in a well plate format allows rapid processing of many samples. results are read after only h. vi requires the availability of cell cultures that can be propagated only in laboratories with specialized personnel and a cell culture capability. moreover, vi is time-consuming; it requires a long incubation period ( - days) and additional testing by immunofluorescence or ha in order to detect viral antigens. the main disadvantage of ha and vi, however, is the low sensitivity, most likely due to antibodies in the intestinal lumen of the infected dogs which may bind virions and prevent both ha and viral attachment to cell receptors . it has been demonstrated in natural (decaro et al., b) and experimental (decaro et al., a) infections, that cpv is detectable by ha and/ or vi only for few days post-infection, whereas those techniques may give false-negative results despite high amounts of viral dna detected by real-time pcr analysis. a simpler ha protocol, designated as slide agglutination test, has been also proposed to detect all cpv variants in fecal and intestinal samples, but this test does not seem to overcome the limitations of the classical methods (marulappa and kapil, ) . electron microscopy identification of parvovirus-like morphology is poorly sensitive, although it is possible to concentrate viral particles by means of specific antibodies in the immunoelectron microscopy technique. viral antigens can be also detected by means of immunohistochemistry carried out on intestinal, brain or tongue sections (greene and decaro, in press ). while molecular methods are generally not affected by the host immune response, they are generally timeconsuming, labour-intensive, and need the expertise of specialists. a nucleic acid hybridization assay was available since early s (remond et al., ) . subsequently, several pcr assays were developed that displayed increased sensitivity and specificity in comparison with traditional methods . a loop-mediated isothermal amplification assay has been also proposed in recent years as an alternative to pcr-based methods (cho et al., ) . however, none of these nucleic-acid detection methods were designed to be quantitative, although they are time consuming and contain a certain risk of carryover contamination, especially when a high sample throughput is required. a real-time pcr assay based on the taqman technology was developed for rapid, specific and sensitive detection of cpv dna (decaro et al., d) . real-time pcr has several advantages over conventional pcr, allowing a large increase in throughput and enabling simultaneous processing of several samples. real-time pcr is run in a -well format, and many of the steps in the assay are automated. because of the inexpensive and quick method used for dna preparation, based on boiling of fecal homogenates, the total time requested for analysis of - samples was about and h for conventional and real-time pcr, respectively (decaro et al., d) . the high sensitivity and reproducibility of the real-time pcr assay may allow for identification of dogs shedding cpv at low titers in their feces, helping to adopt adequate measures of prophylaxis to prevent cpv infection, especially in kennels and shelters, where this virus is often responsible for dramatic epizootics. the established taqman assay was successfully employed in pathogenesis studies carried out during natural (decaro et al., b) and experimental (decaro et al., a) infections and was also used to evaluate virus distribution in different tissues (decaro et al., c) . a taqman real-time rt-pcr assay developed for detection of cpv rna transcripts demonstrated the presence of replicating virus in cns (elia et al., ) . a sybr greenbased real-time pcr assay was proposed as an alternative method to the taqman technology, displaying the same detection limit ( copies of viral dna) . with the exception of discrimination between vaccine and field viruses, virus strain determination may have a limited interest for practitioners, taking into account that prognosis and treatment of cpv enteritis (mainly supportive) disregard the involved variant. however, virus characterisation is of particular interest under an epidemiological point of view. the methods used for characterisation of the cpv strains can be performed only in highly specialised laboratories. while diagnosis of cpv infection is easily feasible using innovative techniques, such as pcr and real-time pcr, the identification of the viral type is sometimes indecisive and more than one method is often required for the definitive prediction of the antigen specificity. for several decades, the only method available for characterisation of cpv strains has been hemagglutination inhibition (hi) using mabs. a panel of four mabs (a e , b a , c d and b e ) was developed for antigenic characterisation of fpv, cpv- , cpv- a and cpv- b, through assessing the hi reactivity. types a and b differ for a lack of reactivity of mab b a to cpv- b; however, this mab is not able to recognise even cpv- c. recently, a mab was developed which can differentiate the new variant c from type b cpvs (nakamura et al., ) . however, mab analysis can be applied only on fecal samples with optimal ha activity or with cpv isolates obtained in cell cultures. only specimens with ha titers! : can be characterised, but several samples with high cpv dna titers test negative or poorly positive by ha (decaro et al., d; desario et al., ) . moreover, only few ha-negative samples are successfully amplified on cell cultures in order to increase their ha titer. a genotyping pcr system was developed by pereira et al. ( ) . due to the limited number of nucleotide variations between types a and b, the type b-specific primers were selected taking advantage of two single nucleotide polymorphisms (snps), a t and g a, that determine the replacement of asparagine by aspartic acid at position and of isoleucine by valine at position , in types a and b, respectively (table ) . each primer was selected to have one such mutation at the very end, as nucleotide mismatches that occur at the end of a primer are highly detrimental to primer extension and strongly decrease pcr amplification. however, these mismatches, albeit present at the very end of the primers, were not sufficient to completely prevent the amplification of the other cpv types (martella, personal observation) . moreover, currently circulating type a strains have the mutation i v, due to the nucleotide change g a (martella et al., ) . therefore, the pcrbased genotyping system developed by pereira and colleagues (pereira et al., ) is no longer able to discriminate between type a and type b strains, as almost all the novel type a strains ( -val) will go mischaracterized as type b. finally, by the type-specific pcr assays, type c cpvs are not detectable, since the substitution d e is due to a change (t ! a) in the third codon position, at nucleotide , so that this mutant is erroneously recognised as type b by this pcr strategy . to address this point, a pcr-rflp assay using enzyme mboii was developed which was proven to be useful for discrimination between types b and c . however, rflp analysis is not able to differentiate cpv- b from cpv- a, since both types remain undigested after mboii digestion. in addition, cpv- a strains have been detected in hungary that were erroneously recognized as cpv- c by this method due to a constant point mutation in the vp gene introducing an mboii restriction site (demeter et al., ) . therefore, only sequence analysis could predict definitively the viral type (type a or b) for samples negative by ha and not digested or giving misleading results by pcr-rflp, leading to a remarkable increase of costs and time losses. sequence analysis can give ample information for cpv typing since the fragment amplified by conventional pcr using primer pair for/ rev encodes for at least one informative aa (residue ) of the vp protein, thus allowing differentiation between cpv- a, cpv- b and cpv- c, but not between cpv- (old type) and cpv- a. in the effort to speed up routine analysis, two real-time pcr assays, based on the mgb probe technology, were designed in order to recognize snps (table ) existing between types a/ b (a t) and between types b/ c (t a) at codon of the vp protein (decaro et al., c (decaro et al., , b . both assays were found to be highly sensitive and reproducible. in comparison with traditional typing methods, the mgb probe assays are more labour-and timesaving. in fact, the -well format and the automation of some steps allow for a simultaneous processing of several samples and ensure a direct characterisation of the cpv variants within a couple of hours, considering that the assays can be run in parallel in the same plate. recently, cpv- c strains uncharacterised by the mgb probe assays have been reported (decaro et al., b) . such strains displayed a non-coding mutation in the probe binding region which prevented the correct hybridization of the type-specific probe. these findings highlight the need to update the cpv-typing methods based on snps as additional mutations may hinder the correct strain characterisation, reinforcing previous suggestions to keep using diagnostic molecular tools targeting more conserved regions to avoid false-negative results (decaro et al., b) . accordingly, the untypeable samples were found to be cpv positive using a taqman assay able to detect all cpv types and flpv as well (decaro et al., d (decaro et al., , b . similar concerns have been recently expressed by american scientists that detected type c strains with the change a g, which is located near the critical codon (kapil et al., ) . albeit present within the target region of the mgb assay, that mutation should not prevent the correct strain characterisation being not included in the primer/probe binding sites. indeed, a us strain displaying the change a g (hong et al., ) was submitted to the b/ c assay, being correctly recognised by the type c-specific probe (decaro et al., b) . diagnosis of cpv infection may be ambiguous when carried out on fecal samples from dogs presenting with diarrhea few days after vaccination. in fact, the modifiedlive virus contained in the vaccines is able to replicate in the intestinal mucosa of vaccinated dogs, despite the unnatural route of administration (intramuscular or subcutaneous instead of oronasal), and to be shed in the feces, albeit at low titers and for a shorter time period with respect to field strains (decaro et al., a) . in such a circumstance, the detection of cpv- or its nucleic acid by conventional assays (ict, ha, vi or pcr) in the feces of vaccinated dogs could provide false-positive results, leading to a misdiagnosis of the disease probably caused by other enteric pathogens of dogs. moreover, it would be important to rule out vaccine-induced disease due to regaining of virulence of the vaccine virus. cpv vaccines are exceptionally safe and cases of vaccine-induced disease have been only anecdotally reported but never clearly demonstrated. however, considered that reversion to virulence of cpv vaccines can theoretically occur, virus strain determination is required at least when gastroenteritis occurs few days after cpv vaccination. cpv characterisation using traditional techniques is often inconclusive, since a simultaneous infection by the type -based vaccine and wild-type virus may mislead the results of hi with mabs, pcr-rflp and sequence analysis. to address this point, a pcr-based approach has been proposed by senda et al. ( ) , taking advantage of two snps, a t and c g, that determine the replacement of methionine by leucine at position and of alanine by glycine at position , in old and wild-type strains, respectively (table ) . however, in our experience, such mutations were not sufficient to prevent completely amplification of the old-type virus (vaccine) (personal observation). moreover, samples containing both vaccine and wild-type strains are amplified successfully, so that the pcr-based strategy will not be able to detect the simultaneous presence of the two viruses in the feces. the mgb probe assays developed for the rapid characterisation of the cpv strains (decaro et al., c (decaro et al., , b do not discriminate between the old type cpv- and type a. even sequence analysis of the vp protein gene can be inconclusive or misleading as, unless sequencing of several clones is carried out, pcr may selectively, or more efficiently, amplify either of the viruses, so that the other one remains undetected by subsequent sequence analysis. to overcome the limitation of existing methods, an mgb probe assay (decaro et al., a) was established which is able to discriminate between type (vaccine) and variant (field) strains on the basis of the snp t c responsible for the change from isoleucine to threonine at vp residue (table ) . thus, all samples collected from vaccinated dogs and characterised as cpv- a should be tested by the novel mgb probe assay in order to assess whether they are true type a (field) strains or vaccine (old type) virus. few companies in europe have licensed type b vaccines. although at the moment such vaccines are not used widely, some problems may arise when analysing samples collected from dogs administered a type b vaccine. using the type /variants mgb probe assay (decaro et al., b) , type b vaccine viruses shed in the feces would be recognized by the probe specific for the cpv variants, being characterised as field strains instead of vaccine strains. similarly, the type a/ b assay is not able to distinguish between type b vaccinal and field strains, since the same nucleotides are encountered in the binding region of the type b-specific probe (decaro et al., b) . consequently, additional two mgb probe assays were designed to precisely differentiate cpv- b strains that circulate in the field from those contained in commercially available vaccines (decaro et al., d) . to the authors' knowledge, the developed assays are able to detect all vaccine strains available at least in the european market. if additional vaccines containing different cpv strains are licensed in the future, further tests should be established to ensure vaccine strain determination. the established assays can be useful to address controversies arising between dog owners, veterinarians and vaccine companies when diarrhea occurs within few days after cpv vaccine administration. the molecular assays discriminating between vaccine and field viruses were successfully applied to fecal samples collected from dogs displaying acute gastroenteritis shortly after cpv vaccination (decaro et al., b) . the results showed that, in most cases, a cpv field strain or a different dog pathogen was being incubated at the moment of vaccination, thus ruling out any reversion to virulence of the vaccine viruses. while fpv since its first identification in has not undergone significant changes in the antigenic and biological properties (decaro et al., c) , cpv has progressively evolved under partly positive selection (shackelton et al., ; decaro et al., b) , showing genomic substitution rates similar to those of rna viruses, with values of about À substitutions per site per year (shackelton et al., ) . such an evolution has lead to the emergence of antigenic variants that seem to be more virulent than the original type. in contrast, despite some anecdotal reports claiming a higher pathogenicity of cpv- c, there is no evidence for a different virulence between the variants. thus, to what extent antigenic variants identified by mouse monoclonal antibodies represent clinically relevant immunological events is still to be determined. at the same time, concerns about the real efficacy of cpv- vaccines against the antigenic variants are mainly based on in vitro cross-neutralization assays that might be even negligible in vivo. indeed, apart from well-documented case reports, experimental data and field observations seem to suggest that the great majority of infections still occur in the puppies about the time when mda wane and the animals become susceptible to any strain of virus. in conclusion, continuous evolution of cpv through accumulation of point mutations in the viral genome presents two different, but equally important, implications: (i) the emergence of new antigenic variants displaying different biological and antigenic properties may require an extensive revision of the vaccination programmes and an update of the viral strains contained in the commercially available products; (ii) the sophisticate molecular methods able to detect and characterise the cpv strains, that are based on perfect matching between viral dna and assay oligonucleotides, may be affected by the onset of point mutations and should be therefore updated as well. thus, a continuous epidemiological surveillance is needed to detect new cpv variants potentially escaping the host immune system and detection methods. analysis of the evolution of feline parvovirus (fpv) evidence for evolution of canine parvovirus type- in italy performance of high titre attenuated canine parvovirus vaccine in pups with maternally derived antibody molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv c in vaccinated dogs an annotated historical account of canine parvovirus monitoring of canine parvovirus (cpv) strains detected in vaccinated puppies in brazil characterization of a canine parvovirus strain isolated from an adult dog evaluation of the antigenic relationships among canine parvovirus type variants detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification molecular epidemiology and phylogeny reveal complex spatial dynamics in areas where canine parvovirus is endemic maternally-derived antibodies in pups and protection from canine parvovirus infection clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant new approaches for the molecular characterization of canine parvovirus type strains a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs a minor groove binder probe real-time pcr assay for discrimination between type -based vaccines and field strains of canine parvovirus characterisation of the canine parvovirus type variants using minor groove binder probe technology first detection of canine parvovirus type c in pups with haemorrhagic enteritis in spain diagnostic tools based on minor groove binder probe technology for rapid identification of vaccinal and field strains of canine parvovirus type b molecular epidemiology of canine parvovirus occurrence of severe gastroenteritis in pups after canine parvovirus vaccine administration: a clinical and laboratory diagnostic dilemma tissue distribution of the antigenic variants of canine parvovirus type in dogs evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type c specific identification of feline panleukopenia virus and its rapid differentiation from canine parvoviruses using minor groove binder probes genetic analysis of feline panleukopenia viruses from cats with gastroenteritis severe parvovirus in a -yearold dog that had been repeatedly vaccinated genetic analysis of canine parvovirus type c characterisation of canine parvovirus strains isolated from cats with feline panleukopenia detection of canine parvovirus type c by a commercially available in-house rapid test canine parvovirus type c infection in a kitten associated with intracranial abscess and convulsions western european epidemiological survey for parvovirus and coronavirus infections in dogs misleading results of the mboii-based identification of type a canine parvovirus strains from hungary reacting as type c strains canine parvovirus infection: which diagnostic test for virus? detection of infectious canine parvovirus type by mrna real-time rt-pcr canine parvovirus epidemiology in bulgaria efficacy of an inactivated feline panleucopenia virus vaccine against a canine parvovirus isolated from a domestic cat canine viral enteritis occurrence of canine parvovirus type c in the united states parvovirus host range, cell tropism and evolution predominance of canine parvovirus (cpv) in unvaccinated cat populations and emergence of new antigenic types of cpvs in cats prevalence and genetic characterization of canine parvoviruses in korea canine parvovirus types c and b circulating in north american dogs in and development of a sybr green based real-time pcr assay for detection and quantitation of canine parvovirus in faecal samples immunogenicity of an intranasally administered modified live canine parvovirus type b vaccine in pups with maternally derived antibodies evolution of cpv- and implication for antigenic/genetic characterization simple tests for rapid detection of canine parvovirus antigen and canine parvovirus-specific antibodies genetic analysis of canine parvovirus from dogs in australia parvoviridae: the viruses and their replication a novel antigenic variant of canine parvovirus from a vietnamese dog occurrence of canine parvovirus type c in the dogs with haemorrhagic enteritis in india comparison of different in-house test systems to detect parvovirus in faeces of cats characterization of canine parvovirus variants circulating in greece chronological analysis of canine parvovirus type isolates in japan purified feline and canine transferrin receptors reveal complex interactions with the capsids of canine and feline parvoviruses that correspond to their host ranges molecular characterisation of canine parvovirus in brazil by polymerase chain reaction assay first detection of canine parvovirus type c in south america experimental canine parvovirus infection in dogs canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine partial dna cloning and sequencing of a canine parvovirus vaccine strain: application of nucleic acid hybridization to the diagnosis of canine parvovirus disease comparison of three rapid commercial canine parvovirus antigen detection tests with electron microscopy and polymerase chain reaction detection by pcr of wild-type canine parvovirus which contaminates dog vaccines high rate of viral evolution associated with the emergence of carnivore parvovirus vaccination with canine parvovirus type (cpv- ) protects against challenge with virulent cpv- b and cpv- c canine parvovirus type vaccine protects against virulent challenge with type c virus family parvoviridae molecular characterization of canine parvovirus- variants circulating in dogs in tunisia evolution of canine parvovirus-a need for new vaccines? canine parvovirus c infection in central portugal this work is funded by grants from university of bari, italy, ricerca di ateneo , project ''epidemiologia molecolare del parvovirus del cane nei carnivori domestici e selvatici'' and contribution to prin , project ''aspetti molecolari ed immunologici delle infezioni da parvovirus nei carnivori''. key: cord- -w iqeayr authors: gallien, sarah; moro, angélique; lediguerher, gérald; catinot, virginie; paboeuf, frédéric; bigault, lionel; gauger, phillip c.; pozzi, nathalie; berri, mustapha; authié, edith; rose, nicolas; grasland, béatrice title: limited shedding of an s-indel strain of porcine epidemic diarrhea virus (pedv) in semen and questions regarding the infectivity of the detected virus date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: w iqeayr pedv is mainly transmitted by the oro-fecal route although pedv shedding in semen has already been shown for an s-non-indel pedv strain infection. the aim of this study was to determine if pedv can be shed in semen from spf (specific pathogens free) boars infected by a french s-indel pedv strain (pedv/fr/ / ) and in case of positive semen to determine the infectivity of that semen. both infected boars had diarrhea after inoculation and shed virus in feces. pedv genome was also detected by rt-qpcr in the sperm-rich fraction of semen ( . × ( ) and . × ( ) genomic copies/ml) from the two boars infected with the s-indel pedv strain but only once at dpi. in addition, pedv rna in peyer’s patches and in mesenteric lymph nodes was also present for the two inoculated boars. the pedv positive semen (s-non-indel and s-indel) sampled during a previous trial and in this boar trial were inoculated to six spf weaned pigs. the inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at dpi. but, pedv could be detected in intestinal tissues such as duodenum, jejunum and jejunum peyer’s patches by rt-qpcr except for one pig. even if pedv genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected pedv. porcine epidemic diarrhea (ped) was described for the first time in europe in the s (jung and saif, ; song and park, ) . this disease is characterized by a severe, profuse watery diarrhea with or without vomiting and dehydration and is caused by an etiologic agent called porcine epidemic diarrhea virus (pedv), a positive-sense, singlestranded rna enveloped coronavirus of -kb (jung and saif, ) . after several outbreaks in the 's, ped has been persisting in europe with sporadic cases until the late 's (jung and saif, ) . outbreaks of ped have also been described in asia in the s and from (jung and saif, ; wang et al., ) . currently, two genotypes are circulating in several regions in the world, namely the s-non-indel strains and the s-indel strains, showing insertions-deletions in the s segment of the s gene (jung and saif, ; vlasova et al., ) . from clinical reports, these two types of strains seemed to be different in terms of morbidity and mortality with the s-non-indel pedv strains associated with more severe clinical cases and higher case-fatality rate compared to the others (vlasova et al., ; wang et al., ) . in contrast to s-non-indel pedv strains, exclusively reported from america and asia, s-indel pedv strains have also been identified in europe since (efsa, ; stadler et al., ; vlasova et al., ) . pedv is transmitted mainly by the oro-fecal route, but also contact with contaminated equipment, vehicles used for pig transport or the staff (bowman et al., ; jung and saif, ; lowe et al., ) . airborne transmission of the virus was also shown for s-non-indel strains (alonso et al., ) . pedv transmission through contaminated milk from dam to piglets can also occur (sun et al., ) but vertical transmission of pedv through semen has never been shown. s-non-indel pedv strain shedding has recently been evidenced in the different fractions of semen (seminal and sperm-rich fractions) and in gelatin plug of specific pathogen free (spf) boars experimentally inoculated (gallien et al., b) . however, to the best of our knowledge, the presence of pedv rna in semen of boars infected by an s-indel pedv strain has not been studied so far. thus, the aim of this study was to determine if pedv can be shed in semen from spf boars infected by an s-indel french pedv strain (pedv/fr/ / ). subsequently, the infectivity of quantitative rt-pcr (rt-qpcr) positive semen was evaluated in an experimental infection of spf weaned piglets. two experimental trials were carried out in the air-filtered level biosecurity facilities of the french agency for food, environmental and occupational health & safety (anses) in accordance with the european and the french regulations on animal welfare. protocols used were approved by the ethics committee registered under number # by the french ministry of . the first trial (trial ) was performed to evaluate pedv shedding in semen from large white spf inoculated boars. two boars (boars i and i aged ½ and ½ years-old) were housed in the same room but in two separate pens. the two other boars (boars c and c ), of similar age to boars i and i , were used as negative controls and housed within the biosafety level- , air filtrated anses spf herd. this experiment lasted for days post-inoculation (dpi). at dpi, the inoculated boars were euthanized including anesthesia (zoletil®, virbac, carros, france, mg/kg) followed by bleeding before necropsy. the boars c and c were not necropsied. the second trial (trial ) was carried out for days to assess the infectivity of rt-qpcr pedv-positive semen. the positive semen fractions from two experiments were used and were collected from trial with the s-indel pedv strain and from a previously described trial involving an s-non-indel strain (gallien et al., b) . six spf weaned pigs of three week-old were housed in the same room in two different pens separated by a plastic partition. two pigs of the same age housed in another room were used as controls. at dpi, pigs were euthanized after anesthesia as already described and necropsied. inoculation procedures the two boars of the trial received orally ml of a homogenate of the pedv french s-indel strain pedv/fr/ / (genbank number: kr ) titrating viral genome copies/ml (equivalent to ≈ tcid /ml). the pedv french s-indel strain was amplified in a threeweek-old spf weaned pig inoculated with a homogenate of jejunum collected from ped-affected pigs belonging to a french farm in . the inoculum was prepared from the jejunum of this spf pedv-inoculated pig by homogenization in dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa) ( % w/v). the homogenate was centrifuged at , ×g for min at °c and the supernatant filtered through a . μm filter. next-generation sequencing (ngs) was performed on the inoculum to obtain the pedv complete genome sequence and to ensure the absence of other viral rna sequences. the absence of porcine circovirus type (pcv ) and porcine reproductive and respiratory syndrome virus (prssv) in the inoculum was assessed by the absence of seroconversion against pcv and prssv in the inoculated boars at the end of the trial. the six spf weaned pigs of trial were orally inoculated with ml of contaminated semen: pigs # , # , # , # with the s-non-indel and pigs # , # with the s-indel (table ). the semen samples inoculated were stored at − °c until inoculation of the piglet. the semen were defrosted on ice just before the inoculation and reconstituted in cold dulbecco's phosphate-buffered saline to avoid thermal shock. for the two trials, clinical signs (lethargy, outward appearance, behavior, breathing, diarrhea and vomiting) were recorded daily. pedv shedding in fecal samples was evaluated daily the first week after inoculation, and then three times a week until dpi for trial and for trial , at . dpi, twice a day from to dpi, once at and dpi and then three times a week until the end of the trial. pedv shedding was also assessed in semen for trial . semen was collected before inoculation and every day the first week post-inoculation and then twice a week until the end of the trial. the belly and the sheath of the boars were cleaned with cleaning wipes before sampling in order to avoid any contaminations of the semen with positive pedv-feces/aerosols. a swab of the prepuce was also done before each collection to rule out the possibility of external contamination of semen. semen was collected manually without sexual stimulation with support of a collection dummy. the gelatin plug was also collected at the end of the semen ejaculate. blood samples were also collected before inoculation and at the end of trials for the two trials, and at dpi for trial and once a week until the end of the trial for trial to assess seroconversion and viremia by rt-qpcr ( fig. ) . during necropsy, organs of the digestive tract (duodenum, jejunum, ileum, colon, peyer's patches (jejunum and ileum)), spleen, liver, mesenteric and inguinal lymph nodes, lungs, were collected and stored in rna later tissue storage reagent (sigma-aldrich, saint louis, mo, usa) for the two trials. organs of the reproductive tract were also collected for the trial (vas deferens (right and left), testicles (right and left: apical pole, distal pole, median axis), epididymis (right and left: head, body, tail), prostate, cowper's glands (right and left), seminal vesicles (right and left: apical and distal pole) and spermatic cords (right and left)) and stored in the same tissue storage reagent. macroscopic lesions were also evaluated on necropsy for the two trials. all samples collected during the two trials were stored at − °c. fresh semen samples were centrifuged at ×g for min at °c (pal et al., ) . this centrifugation step allowed separating the seminal fraction from the sperm-rich fraction of the semen. these two fractions and the gelatin plug were stored at − °c. blood samples were centrifuged at ×g for min at °c and then sera were stored at − °c until use. one gram of feces (or ml of feces in case of liquid feces) was homogenized in ml of dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa). the fecal homogenates were then centrifuged at , ×g for min at °c. tissues were homogenized in dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa) at % v/w using a bead mill (retsc, haan, germany). these homogenates were centrifuged at , ×g for min at °c and the supernatants were stored at − °c. total rnas were extracted from the fecal and tissue homogenates, from the preputial swabs and from sera using the qiagen rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. five μl of eluted rna were used as templates for pedv rt-qpcr. rna extraction controls were performed for every five samples to check for any pedv contamination by replacing sample with rnase free water. rna extracted from feces with the rneasy mini kit was diluted : to avoid any pcr inhibition. total rnas were extracted from the two semen fractions (seminal and sperm-rich fractions) and the gelatin plug using trizol® assay (thermo fisher scientific, waltham, ma, usa) (gallien et al., b) . the number of pedv genome copies was assessed by real-time rt-pcr using power sybr® green rna-to-ct ™ -step kit (thermo fisher scientific, waltham, united states of america) on an applied biosystems® real time pcr system (thermo fisher scientific, waltham, united states of america) as already described (gallien et al., b; kim et al., ) . the primers used were designed from the conserved regions of the pedv nucleocapsid gene for universal detection of both strains (forward, ′-cgcaaagactgaacccactaa- ′; reverse, ′-ttgcctctgttgttacttggagat- ′). for each pcr run, a positive control containing pedv rna extract from a pedv cell culture supernatant was included. two negative controls were also included on the plate, the rna samples were replaced by rnase free water. one negative control was placed close to the positive control and the second one at the end of the plate. all samples were processed in duplicate. sera were tested for pedv antibodies using a commercial elisa test, id screen® pedv indirect (id vet, grabels, france). the elisa test is validated if the mean value of the positive control optical density (od) is greater than . and if the ratio of the mean values of the positive and negative controls is greater than . for each sample, the s/p (sample-to-positive) ratio was calculated. samples with s/p ratios equal to or higher than % were considered positive for pedv antibodies (fig. ) . both inoculated boars demonstrated clinical signs. they were lethargic as soon as dpi and until dpi. a reduction of food intake was also observed for the two inoculated boars at dpi and until dpi. diarrhea was also observed at dpi. none of the control boars demonstrated clinical signs during the trial. the inoculated boars were pedv seronegative before inoculation and became seropositive at ± dpi (s/p% i = . % and s/p% i = . %). only boar i was still positive for pedv antibodies at the end of the trial (s/p% i = . %). the two control boars were seronegative before and at the middle of the trial. all boars were rt-qpcr negative for pedv in feces prior to inoculation. the two control boars remained pedv negative in feces until the end of the trial. however, pedv rna was first detected in the feces of boar i at dpi ( . × genome copies/ml of feces). at dpi, boars i and i showed . × and . × genome copies/ml of feces, respectively. the viral genome was continuously detected until dpi. maximum shedding was detected between . and dpi for the two boars ( . × genome copies/ml for boar i at . dpi and . × genome copies/ml for boar i at dpi). pedv rna was then sporadically detected for the two boars (at dpi for boar i and at and dpi for boar i ) (fig. ) . no pedv nucleic acid was detected in the seminal or the sperm-rich fractions of semen and in the gelatin plug collected from boars i , i , c and c before inoculation. the two control boars remained negative for pedv rna in all fractions of semen and in the gelatin plug throughout the trial. pedv rna was detected for both inoculated boars at dpi in the sperm-rich fraction of semen with . × and . × genome copies/ml for boars i and i , respectively. no pedv rna was detected by rt-qpcr thereafter in this fraction. the inoculated boars remained rt-qpcr negative for the seminal fraction and the gelatin plug throughout the trial. all swabs collected from the sheath tested negative for pedv rna, even when the virus was detected in the spermrich fraction ( dpi). pedv rnas were detected in the jejunum peyer's patches ( . × and . × genome copies/ml for boars i and i respectively), in the ileum peyer's patches ( . × and . × genome copies/ml for boars i and i respectively) and in the mesenteric lymph nodes ( . × and . × genome copies/ml for boars i and i respectively). pedv rna was not detected from the organs of the reproductive tract. no lesions were observed in the gastrointestinal or reproductive tract. none of the inoculated piglets showed clinical signs throughout the trial. no seroconversion of these pigs was observed at dpi. no pedv nucleic acid was detected in feces of pigs # , # , # , # , # and # during the trial except for pig # at dpi ( . × genome copies/ ml). some digestive tissue samples were found positive for pedv in rt-qpcr for pigs # , # , # , # and # at dpi (table ) but no lesion was observed in the gastrointestinal tract. oral inoculation of mature boars by an s-indel french pedv strain demonstrated less severe clinical signs compared to those observed in boars inoculated with a s-non-indel us pedv strain (gallien et al., b) . however, the infection induced profuse diarrhea and affectd growth performances similarly to weaned pigs inoculated with this s-indel french pedv strain (gallien et al., a) . the duration of virus fecal shedding determined in our study ( days) was shorter compared to weaned pigs inoculated with an s-indel pedv strain ( - days) (leidenberger et al., ; lohse et al., ) or for boars inoculated with an s-non-indel pedv strain ( - days) (gallien et al., b) . in this experimental challenge, the presence of pedv rna in the fig. . fecal pedv shedding detected from boar i and i (log(pedv-genome copies/ml)) in trial . pedv genome loads (genome copies/g) detected in the tissues collected at necropsy from spf weaned pigs inoculated with pedv rt-qpcr positive semen in trial . sperm-rich fraction of semen of both inoculated boars was detected at dpi only, which is much more limited than in the sperm-rich fraction of semen from boars inoculated with an s-non-indel pedv strain (gallien et al., b) . in the previous study, pedv rna could be detected for a longer period in the sperm-rich fraction of semen: i.e. transient detections of pedv rna during three distinct periods comprised between and days. pedv rna was also detected in the seminal fraction of semen and in gelatin plug in s-non-indel pedv inoculated boars contrasting to the observations from the present study. this difference regarding pedv rna detection in semen could be linked to the viral strain used for inoculation and its virulence. clinical signs induced with s-non-indel pedv were more severe compared to clinical signs observed in s-indel pedv challenged boars (gallien et al., b) . viral shedding in the sperm-rich fraction of semen has already been reported for prrsv which belongs to the same order as pedv, the nidovirales order, and differences in terms of shedding in semen between genotype and highly virulent genotype prrsv strains have already been shown (christopher-hennings et al., ; prieto and castro, ) . prrsv genotype strains could be detected during longer periods in semen compared to genotype strains. hence, the duration of detection of highly virulent genotype prrsv strains in semen was reported between to days (christopher-hennings et al., ) while the presence of genotype prrsv strains in semen could be observed more sporadically: only at one point after infection, at dpi (prieto et al., ) or during shorter periods comprised between and days (swenson et al., ) . detection of s-indel pedv rna in semen appeared sporadically after the occurrence of clinical signs and detection in feces in contrast to what we observed for an s-non-indel pedv strain. moreover, no s-indel pedv rna was detected in semen after cessation of clinical signs and fecal shedding in contrast to what was observed with the s-non-indel strain (gallien et al., b) . these data suggest that the risk of introduction of pedv shedding boars via imports should be more predictable from clinical exams or fecal shedding assessment for s-indel strains than for s-non-indel. absence of detection of pedv rna in the genital tract has been shown in boars inoculated with s-indel pedv strain as well as in boars inoculated with an s-non-indel strain at dpi (gallien et al., b) . mc carty and al., showed that pedv rna could be detected in penis and testicle at days post-infection (mccarty et al., ) . that presence was noticed at infection peak when a viremia could be noticed too. that could explain the absence in reproductive tract of pedv we noticed at days post infection, a very late date compare to the infection peak. during the second trial, no shedding was detected in feces of spf weaned pigs inoculated with s-indel and s-non-indel rt-qpcr pedv positive semen except for pig # at dpi. the presence of low pedv genomic loads could be noticed in different intestinal tissues. however, clinical signs and seroconversion were absent in all inoculted piglets. the results of the present study neither confirms nor denies if the rt-qpcr pedv positive semen contained infectious virus. additional trials with longer periods of observation, sequential slaughters or the use of a more sensitive model such as neonatal piglets should be conducted in order to determine if the pedv rna detected in the tissues were caused by pedv infection. the use of neonatal piglets could have been in fact a most sensitive model to evaluate pedv infectivity but to realize that kind of trial in our experimental facility, we should have infected two sows with their suckling piglets and for material reasons (place, cost, animal availability…), this option could not be selected. moreover, with these results, it is impossible to speculate whether pedv-positive semen could infect sows via natural insemination. in fact, it has already been shown for other porcine viral pathogens that contaminated semen could be infectious, but could not infect sows or gilts through artificial insemination. weaned pigs inoculated with pcv positive semen, for example, presented a viremia and anti-pcv antibodies but the same pcv positive semen could not infect sows through artificial insemination (madson et al., ) . the amount of virus contained in semen could explain the impossibility of viral transmission through artificial insemination (grasland et al., ; madson et al., ). an impact of the amount of virus contained in semen on the capacity of the transmission of viral pathogens has also been shown for prrsv. the prrsv can be transmitted through semen but only when it is present in this matrix at a given concentration. under experimental conditions, prrsv transmission was successful when semen contained × tcid /ml of virus i.e. on average genome copies/ml. however, when the amount of virus contained in semen was approximately × tcid /ml ( genome copies/ml on average), transmission through semen was limited. when the amounts were even lower ( × tcid /ml, genome copies/ml on average), transmission was no longer effective (prieto and castro, ) . a possible impact of the amount of genomic load of pedv contained in semen on the capacity of the transmission of pedv might be suspected in the present case. to conclude, a very transient shedding of pedv in semen was observed in case of infection by an s-indel pedv strain conversely to what was observed with s-non-indel pedv strain. however the infectivity of the virus present in pedv s-indel or s-non-indel positive semen was not evidenced. evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars collection and review of updated scientific epidemiological data on porcine epidemic diarrhoea better horizontal transmission of a us non-indel strain compared with a french indel strain of porcine epidemic diarrhoea virus evidence of porcine epidemic diarrhea virus (pedv) shedding in semen from infected specific pathogenfree boars evaluation of the transmission of porcine circovirus type (pcv- ) genogroups a and b with semen from infected specific-pathogen-free boars porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis multiplex realtime rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus virulence of current german pedv strains in suckling pigs and investigation of protective effects of maternally derived antibodies experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain role of transportation in spread of porcine epidemic diarrhea virus infection, united states infectivity of porcine circovirus type dna in semen from experimentallyinfected boars case report describing the clinical course of porcine epidemic diarrhea in a commercial boar stud and return of the stud to service after whole-herd inoculation with porcine epidemic diarrhea development and validation of a duplex real-time pcr assay for the simultaneous detection and quantification of porcine circovirus type and an internal control on porcine semen samples porcine reproductive and respiratory syndrome virus infection in the boar: a review semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (prrs) virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines emergence of porcine epidemic diarrhea virus in southern germany outbreak of porcine epidemic diarrhea in suckling piglets excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars distinct characteristics and complex evolution of pedv strains porcine epidemic diarrhea virus variants with high pathogenicity new variant of porcine epidemic diarrhea virus the authors are grateful to anses, inra and lncr for their financial support. we also would like to thank phillip gauger from iowa state university who provided the us non-indel strain and all the team members at the experimental laboratory for their contribution. all the authors designed and carried out the experiments. sg analyzed the data and wrote the manuscript. bg, nr and mb supervised the project. all the co-authors revised the manuscript. all authors read and approved the final manuscript. the authors declare that they have no conflict of interests. key: cord- -g udfu a authors: decaro, nicola; cirone, francesco; mari, viviana; nava, donatella; tinelli, antonella; elia, gabriella; di sarno, alessandra; martella, vito; colaianni, maria loredana; aprea, giuseppe; tempesta, maria; buonavoglia, canio title: characterisation of bubaline coronavirus strains associated with gastroenteritis in water buffalo (bubalus bubalis) calves date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: g udfu a recently, a coronavirus strain ( / - ) was isolated from water buffalo (bubalus bubalis) and the virus which displayed a strict genetic and biological relatedness with bovine coronavirus (bcov) was referred to as bubaline coronavirus (bucov). here, we report the characterisation of four bucovs strains identified in the faeces or intestinal contents of water buffalo calves with acute gastroenteritis. single bucov infections were detected in all but one cases from which two clostridia species were also isolated. sequence and phylogenetic analyses of the ′ end of the spike-protein gene showed that three bucovs were closely related to the prototype strain / - , whereas the fourth isolate ( / -c) displayed a higher genetic identity to recent bcov reference strains. three strains adapted to the in vitro grow on human rectal tumour cells were also evaluated for their ability to replicate in a bovine cell line (madin darby bovine kidney) and to cause haemagglutination of chicken erythrocytes and all displayed biological properties similar to those already described for the prototype bucov. the present report shows that albeit genetically heterogeneous, the different bucov strains possess a common biological pattern which is different from most bcov and bcov-like isolates. coronaviruses (covs) are enveloped, positive-stranded rna viruses that cause respiratory and/or enteric disease in mammals and birds (enjuanes et al., ) . with the largest known genomic rna, covs are prone to dramatic shifts of the tissue tropism or host range through accumulation of point mutations or deletions/insertions or recombination events affecting the structural and nonstructural proteins (decaro and buonavoglia, ) . currently, covs are classified into three different genetic and antigenic groups with group being further divided into subgroups a and b. subgroup a consists of mouse hepatitis virus, rat sialodacryadenitis virus, human coronavirus (hcov) hku (woo et al., ) and several bovine-like covs, including bovine coronavirus (bcov), porcine haemagglutinating encephalomyelitis virus (phev), hcov-oc , human enteric coronavirus (hecov) (enjuanes et al., ) , and the newly recognized equine coronavirus (ecov) (guy et al., ) and canine respiratory coronavirus (crcov) (erles et al., ; decaro et al., ) . there are multiple genetic and antigenic evidences that several subgroup a covs, such as hcov-oc , hecov- , phev and crcov, have arisen as consequence of trans-species infections caused by bcov (zhang et al., ; vijgen et al., vijgen et al., , erles et al., veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] recently, a coronavirus strain ( / - ) was isolated from water buffalo (bubalus bubalis) and the virus which displayed a strict genetic and biological relatedness with bovine coronavirus (bcov) was referred to as bubaline coronavirus (bucov). here, we report the characterisation of four bucovs strains identified in the faeces or intestinal contents of water buffalo calves with acute gastroenteritis. single bucov infections were detected in all but one cases from which two clostridia species were also isolated. sequence and phylogenetic analyses of the end of the spike-protein gene showed that three bucovs were closely related to the prototype strain / - , whereas the fourth isolate ( / -c) displayed a higher genetic identity to recent bcov reference strains. three strains adapted to the in vitro grow on human rectal tumour cells were also evaluated for their ability to replicate in a bovine cell line (madin darby bovine kidney) and to cause haemagglutination of chicken erythrocytes and all displayed biological properties similar to those already described for the prototype bucov. the present report shows that albeit genetically heterogeneous, the different bucov strains possess a common biological pattern which is different from most bcov and bcov-like isolates. ß elsevier b.v. all rights reserved. ; lorusso et al., ) (de groot et al., ) . recently, another bovine-like cov, which was referred to as bubaline coronavirus (bucov), was isolated from water buffalo (bubalus bubalis) calves with fatal gastroenteritis in italy (decaro et al., d) . the virus was characterised at the genetic level, displaying intact structural and non-structural proteins with respect to bcov, whereas biologically it presented unique characteristics including the inability to grow on bovine derived cell cultures and to agglutinate chicken erythrocytes. here, we report the clinical history and virus characterisation of four outbreaks of gastroenteric disease caused by bucov in italian buffalo herds. the first outbreak was registered in july, in a buffalo herd of the caserta province (campania region) and involved - -day-old calves with the occurrence of depression, anorexia and haemorrhagic diarrhoea. the affected calves recovered progressively after treatment with oral rehydrants and antibiotics (sulfadimidin + trimetoprim). the laboratory analyses were carried out on faecal samples collected from six diarrhoeic calves ( / -a-f). signs of enteritis and tympanism occurred in february, in - -day-old calves of a buffalo herd of the province of latina (lazio region). in this case, the mortality rates did not exceed %. necropsy carried out on a dead buffalo calf ( / ) revealed the presence of severe gastroenteritis, with enlargement of the mesenteric lymph nodes and gall bladder. another gastroenteritis outbreak was observed in april, in the caserta province. the affected buffalo calves, - days of age, showed anorexia, apathy, yellow or bloody diarrhoea and dehydration, which led to the death of the affected animals within - h after the onset of clinical signs. the mortality rate was about %. the carcasses of two calves ( / -a, / -b) were submitted to necropsy, showing congestion and haemorrhages in the small and large intestines that were filled with abundant liquid content. in the same period, clinical signs of coronavirosis (loss of appetite, liquid diarrhoea) were observed in - -dayold calves form a buffalo herd of the foggia province (apulia region). in this case, the dams had been vaccinated against rotavirus, bcov and escherichia coli before calving in order to transfer the maternally derived immunity to their offspring. at necropsy on a dead buffalo calf ( / ), the typical gross lesions of coronaviral enteritis were observed and the intestinal content was collected for routine analyses. for bacteriological investigations, tissue samples and faeces or intestinal contents were plated out on % sheep blood agar and cultured aerobically at c for h for detection of aerobic pathogens. anaerobic pathogens were searched for by obtaining ten-fold dilutions of the samples in fluid thioglycolate medium and subsequent plating each sample dilution onto % sheep blood agar and egg yolk agar with d-cycloserine mg/ml. bacteria were allowed to grow overnight at c in anaerobic condition. bacteria identification was achieved by standard biochemical procedures and analytical profile index (api, biomé rieux italia s.p.a., rome, italy). to detect intestinal parasites in the faeces or intestinal contents, zinc sulphate flotation was used, whereas the ziehl nielsen staining was performed on the faecal samples or intestinal sections for identification of cryptosporidium spp. dna extraction from tissue samples and faeces or intestinal contents was carried out by means of the dneasy tissue kit (qiagen s.p.a., milan, italy), whereas rna was extracted from the fecal samples or intestinal contents and from tissues by using the qiaamp viral rna mini kit and qiaamp rneasy mini kit (qiagen s.p.a.), respectively. dna extracts were subjected to pcr assays for detection of bovine herpesvirus types (bohv- ) (vilcek, ) and (bohv- ) (boerner et al., ) using the kit la pcr kit ver. . (takara bio inc., shiga, japan). rna extracts were used for detection of bcov and bcov-like viruses (erles et al., ) , toroviruses (hoet et al., ) , rotaviruses (gouvea et al., ) , caliciviruses (jiang et al., ) , bovine viral diarrhea virus (bvdv) (sullivan and akkina, ) and bovine respiratory syncytial virus (valarcher et al., ) . rt-pcr assays were performed using superscript tm one- step rt-pcr for long templates (life technologies, invitrogen srl, milan, italy). the rna of bcov-like covs was quantified in the rt-pcr positive samples by means of a real-time rt-pcr assay recently established (decaro et al., b) . briefly, viral rnas were reverse-transcribed using geneamp rna pcr (applied biosystems, applera italia, monza, italy) and the c-dnas were real-time pcr-amplified in a ml-reaction bcov-like-positive samples representative of each outbreak ( / -c, / , / -b, / ) were subjected to virus isolation attempts on human rectal tumour (hrt- ) and madin darby bovine kidney (mdbk) cells, grown in dulbecco's minimal essential medium (d-mem) added with % foetal calf serum, as previously described (decaro et al., d) . viral growth was monitored by visual inspection of cytopathic effect and by an immunofluorescence assay using a bcov-positive bovine serum and a rabbit anti-bovine igg conjugated with fluorescein isothiocyanate (sigma aldrich srl, milan, italy). viral isolates were tested as previously described (hasoksuz et al., ; decaro et al., d) for their haemagglutination and receptor-destroying enzyme activities in comparison with the bucov prototype isolate / - and bcov enteric isolates / (decaro et al., c) and wbl (kindly provided by dr paolo cordioli, istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna, brescia, italy) and nasal isolate / - (decaro et al., a) . the end of the spike-protein (s) gene of the detected bucovs was amplified using primers bcv- f (tat-gatccgctaccaattattttgcttggca) and bcv- r (acaacaccagtgtctgtaaaatatgca) and super-script tm one-step rt-pcr for long templates (invitrogen srl, milan, italy), according to the manufacturer's instructions and a previous report (decaro et al., d) . the pcr-amplified products ( bp) were cloned into pcr . -topo vectors (topo ta cloning , invitrogen srl) and the recombinant clones individualized by blue/white screening. plasmid dnas were sequenced by genome express (meylan, france) and the obtained sequences were assembled and analysed using the bioedit software package (hall, ) and the ncbi's (http://www.ncbi.nlm.nih.gov) and embl's (http:// www.ebi.ac.uk) analysis tools. phylogenetic and molecular evolutionary analyses were conducted using mega (kumar et al., ) . phylogenetic trees based on nucleotides (nt) of the s-gene end were elaborated using both parsimony and neighbor-joining methods, supplying a statistical support with bootstrapping over replicates. the nt sequences of the partial s gene sequences of the four bubaline strains have been deposited in genbank under accession numbers gu -gu . all collected faecal samples and intestinal contents tested positive for bovine-like covs by a classical, gelbased rt-pcr targeting the s gene. a taqman assay confirmed the presence of the nucleic acid of bcov-like covs in all tested samples but intestinal content of calf / , with viral rna titres ranging from .  to .  viral rna copies ml À template (table ) . electrophoretic analysis of the real-time rt-pcr products showed the presence of the band of the expected size also for sample / , thus proving the presence of the viral rna in the tested sample as obtained by conventional rt-pcr. the band was excised form the gel, purified and sequenced, displaying a mismatch in the probe binding region, that could have prevented the annealing of the specific probe (data not shown). common parasites of ruminants were not recovered from any gastroenteritis outbreaks, whereas only the intestinal content of calf / was found to contain high amounts of clostridia, including clostridium perfringens type a and clostridium butyrricum. all bucovs inoculated on cell cultures but strain / were successfully isolated on hrt- cells (table ) , reaching at the rd passage viral titres higher than tcid ml À . in order to evaluate the growth characteristics of the bucov strains, prototype isolates from each outbreak ( rd passages on hrt- cells) were subjected to ten consecutive passages on mdbk cells and the cryolysates of the last passage were submitted to real-time rt-pcr. as shown in fig. , the bubaline strains were not able to replicate efficiently on the bovine cell line. in fact, by using real-time rt-pcr quantification, viral replication of strains / -c and / was no longer observed after the fifth and eighth passages, respectively, whereas the rna of strain / -b was detected at the th passage but at a low titre. the three bucov isolates as well as the bucov and bcov reference strains agglutinated mouse erythrocytes at high titres, whereas only the bucov isolate / was able to cause low-titre ha of chicken erythrocytes. in contrast, all bcov reference strains agglutinated chicken erythrocytes with high efficiency. as previously observed (decaro et al., d) , ha titres were higher at c than at c. using mouse erythrocytes, rde activity (loss of the ha pattern) was observed for the bovine and bubaline isolates (table ) . the end of the s gene of the four prototype bucov strains was pcr-amplified and sequenced as previously described (decaro et al., d) and the obtained sequences were compared to reference bucov strain / - and bcov strains mebus (old strain) and / (recently isolated in italy, decaro et al., c) . by sequence analysis of the end nt, three out of four bucov strains were found to be closely related to each other ( . - . % of nt identity) and to the prototype isolate / - ( . - . %), whereas strain / -c displayed the highest identity ( . %) to the recent italian bcov isolate / and only . - . % identity to prototype and new bucovs (table ) . phylogeny inferred with the neighbor-joining method showed that three bucov strains formed a unique cluster together with the prototype virus / - , which was separate from bcovs albeit distantly related to the recent italian bcov isolate / (fig. ) . in contrast, strain / -c was intermingled with classical bcov isolates, particularly with reference strains ok- - and lsu- lss- - that had been isolated in usa from calves with respiratory disease (chouljenko et al., ) . this pattern of segregation was confirmed by the tree constructed with the maximum parsimony method (data not shown). recently, a coronavirus strain was isolated from an outbreak of gastroenteritis in an italian water buffalo herd. the bubaline virus (bucov- / - ) was found to be strictly related to bcov at the genetic level, but displayed some unique biological properties such as poor growth on mdbk cells and lack of ha activity using chicken erythrocytes (decaro et al., d) . in this paper, we have reported the genetic characterisation of four additional bucov strains detected in buffalos with gastroenteritis. while three strains were strictly related to each other and to the prototype bucov isolate, strain / -c was more similar to bcovs by both sequence analysis and phylogeny of the end of the s gene. these findings seem to suggest a distinct origin of the two clusters of bucovs from bcov. however, as other genomic regions were not analysed in this study, a potential origin of strain / -c as recombinant virus between bucov and bcov cannot be ruled out definitively. the bcov origin or, alternatively, the presence of a common ancestor, has been hypothesised for several group a covs, including hcov-oc (vijgen et al., ) , phev (vijgen et al., ) , crcov (erles et al., ; lorusso et al., ) and some ruminant covs (hasoksuz et al., ; jin et al., ; alekseev et al., ; decaro et al., d) . we have already shown that more than one bcov strain or ancestor virus was likely involved in the origin of crcov, thus leading to the emergence of different canine strains (lorusso et al., ) . accordingly, a similar mechanism may have been involved in the emergence of genetically distinct bucov strains. three bucov strains were adapted to grow on hrt- cells but only one of those was able to replicate with low efficiency on mdbk cells and none displayed evident ha activity using chicken erythrocytes. these findings are in agreement with those already obtained with the prototype strain / - (decaro et al., d) , the giraffe isolate gicov-oh and recent bcovs (hasoksuz et al., ) . in the ha test with mouse erythrocytes, the results for the gicov-oh strain were similar to those for bcov strains at both c and c, whereas the ability to agglutinate chicken erythrocytes by gicov-oh was observed only at c. similar results were also obtained for one enteric bcov (bcov-ts) and two respiratory bcovs ( ns and ns) (hasoksuz et al., ) . the absent or poorly efficient adaptation to mdbk cells and the lack of ha activity with chicken red blood cells could be considered typical features of bucov. in fact, although other ruminant covs and even some bcov strains displayed similar characteristics, those were never combined (benfield and saif, ; hasoksuz et al., ) . two recent bcov isolates from southern italy (the enteric strain / and the respiratory strain / - ) exhibited ha and growth patterns very different from those of bucov isolates. however, more studies of contemporary bcov isolates in the same geographic area should be done to decide if these attributes apply only to bucov or if such differences are consistent among covs from different ruminant species. the bucov strains identified so far have displayed similar biological but not genetic features, thus requiring further studies aiming to isolate and characterise additional strains. the onward industrialisation of the bubaline farming has led to the emergence of diseases that until few years ago were considered typical of cattle herds but were rarely observed in water buffaloes. among the emergent pathologies, viral gastroenteritis, mainly associated with coronavirus (muniiappa et al., ; decaro et al., d) and rotavirus (sunil-chandra and mahalingam, ; martella et al., ) infections, is playing an increasing role in the economical losses related to the calf mortality, especially when female calves are affected. the changes in the hygienic conditions highlight the need for a more indepth investigation of these viral infections that are frequently associated to different clinical pictures in comparison with cattle. for instance, coronavirus infections can cause enteric as well as respiratory disease in cattle (lathrop et al., ; storz et al., ; decaro et al., a) , whereas respiratory distress has not been associated with bucov so far. in addition, the impact of these emerging infections in water buffalo herds should be evaluated as for the extensive use of vaccines. in conclusion, based on the present report, a potential role as enteric pathogen should be assigned to bucov and this virus should be included in the diagnostic panel for the infectious agents involved in the occurrence of acute gastroenteritis in buffaloes. to what extent bucov impacts on the buffalo productions should be assessed in the next future through extensive epidemiological and clinical survey in buffalo herds. bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences cell culture propagation of a coronavirus isolated from cows with winter dysentery a sensitive and specific pcr/southern blot assay for detection of bovine herpesvirus in calves infected experimentally nucleotide and predicted amino acid sequences of all genes encoded by the genomic portion ( . kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses revision of the family coronaviridae. taxonomic proposal of the coronavirus study group to the ictv executive committee an update on canine coronaviruses: viral evolution and pathobiology serological and molecular evidence that canine respiratory coronavirus is circulating in italy respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy detection of bovine coronavirus using a taqman-based real-time rt-pcr assay severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (bubalus bubalis) calves family coronaviridae detection of a group coronavirus in dogs with canine infectious respiratory disease isolation and sequence analysis of canine respiratory coronavirus identification of bovine and porcine rotavirus g types by pcr characterization of a coronavirus isolated from a diarrheic foal bioedit: a user-friendly biological sequence alignment and analysis program for windows / /nt isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe enteric and nasal shedding of bovine torovirus (breda virus) in feedlot cattle design and evaluation of a primer pair that detects both norwalk-and sapporo-like caliciviruses by rt-pcr analysis of the genome sequence of an alpaca coronavirus mega : integrated software for molecular evolutionary genetics analysis and sequence alignment association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots molecular characterization of a canine respiratory coronavirus strain detected in italy molecular analysis of the vp , vp , vp , nsp , and nsp / genes of a buffalo rotavirus strain: identification of the rare p[ ] rhesus rotavirus-like vp gene allele demonstration of coronavirus infection in buffaloes isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp. from cattle involved in two natural outbreaks of shipping fever a nested polymerase chain reaction assay to differentiate pestiviruses rotavirus-associated diarrhoea in buffalo calves in sri lanka evaluation of a nested reverse transcription-pcr assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc detection of the bovine herpesvirus- (bhv- ) genome by pcr characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child this work was supported by grants from the italian ministry of health, ricerca corrente izsme / ''studio della epidemiologia e delle caratteristiche genetiche del coronavirus del bufalo''. key: cord- - nxhvvm authors: lei, xi-mei; yang, yong-le; he, yong-qiang; peng, lei; zhao, pengwei; xu, shu-ya; cao, hongwei; fang, pengfei; qiu, wenying; qin, pan; wang, bin; huang, yao-wei title: specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: nxhvvm given the highly contagious and acute nature of porcine epidemic diarrhea (ped), especially in piglets, there is an urgent need for the development of rapid and sensitive diagnostic assays. the diagnostic potentials of specific porcine epidemic diarrhea virus (pedv) accessory and nonstructural proteins, if any, have not yet been investigated. in order to determine and compare which of the viral proteins may be useful as diagnostic antigens, whole virus (wv) particles and a panel of structural and nonstructural pedv proteins [spike subunit (s ), the c-terminal part of orf (orf c), envelope (e), nonstructural protein (nsp ), nsp , ac (acidic domain of nsp ), and adrp (adp-ribose- -monophosphatase domain of nsp ), expressed individually in bacterial and/or mammalian cells] were tested for reactivity with sera from pedv-infected pigs by elisa and/or western blot analysis. according to western blots, serum antibody interactions with the s protein were relatively more sensitive and specific than orf c, e and ac. furthermore, a total of serum samples from diarrheal pigs of different ages were analyzed by elisa, with most showing immune-reactivity towards the wv, s , orf c, and e proteins. the earliest igg antibody response was observed in the one-week-old piglets, with similar antibody ontogeny and patterns of seroconversion for s , orf c, e, and wv antigens. in addition, the pattern of neutralizing antibody was more similar to that of iga in weaning piglets after pedv infection. collectively, these data provide more reliable information on the host immune response to different viral proteins, which will be useful for development of novel serological assays and for design of vaccines that better stimulate protective immunity. porcine epidemic diarrhea (ped) is characterized by severe diarrhea, vomiting, and dehydration followed by high mortality in suckling piglets . the causative agent, porcine epidemic diarrhea virus (pedv), was initially identified in europe in , and its genome (˜ kb in size) consists of seven open reading frames (orfs) (kocherhans et al., ) . the ' two-thirds of pedv genome encodes orf (consisting of overlapping orf a and orf b), and the ' onethird harbors orfs encoding four structural proteins, the spike (s), envelope (e), membrane (m) and nucleocapsid (n), and an accessory orf between s and e kocherhans et al., ; tian et al., ) . rna synthesis in pedv is carried out by a replicase-transcriptase composed of nonstructural proteins (nsp - ) encoded by orf a and orf b. among them, nsp comprises multiple structural domains, including a highly acidic domain at the amino terminus (ac), and a highly conserved adp-ribose- -phosphatase (adrp) macrodomain. the ac domain of nsp is essential for virion assembly and plays a critical role in interaction with the viral nucleocapsid during early infection, whereas the adrp provides activities necessary for synthesis of genomic and subgenomic rnas (hurst-hess et al., ) . as pigs of all ages are susceptible to pedv (alvarez et al., ; annamalai et al., ) , there is an urgent need for the development of highly sensitive and specific diagnostic assays for use in the field (diel et al., ) . since the identification of pedv, several diagnostic tests based on pcr detection of viral rna have been described in the literature (diel et al., ) . another common diagnostic method is serological testing for the presence of specific antibodies against viral proteins, which is also fast and convenient for epidemiologic investigations. many tools have been developed for the detection of anti-pedv antibodies based upon the major structural proteins (such as s, m or n proteins) in serum, colostrum, milk, feces and oral fluid, including indirect immunofluorescence assays (ifa), virus neutralization assays (sn), enzyme-linked immunosorbent assays (elisa), and fluorescent microsphere immunoassays (fmia) (diel et al., ; gerber et al., ; gerber and opriessnig, ; gimenez-lirola et al., ; okda et al., ) . but comparative studies of the above assays using different pedv structural and nonstructural proteins as antigens are rarely conducted. meanwhile, the diagnostic potentials of specific pedv accessory and nonstructural proteins, if any, have not yet been investigated in details. in this study, a panel of recombinant pedv orfs encoding structural and nonstructural proteins were expressed in mammalian and/or bacterial cells and screened for reactivity with porcine sera from seven provinces of china by elisa and/or western blot analysis, in order to determine which antigen is most suitable as a diagnostic marker for pedv infection. several rabbit polyclonal antibodies against these recombinant proteins were also generated and validated for use as diagnostic tools upon pedv infection in vitro. vero cells (atcc® ccl- ™) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs). the pedv virulent strain zju/g / (genbank accession no. ku ) was used in the study qin et al., ) . a total of serum samples were collected in - from diarrheic pigs at commercial farms in the shandong, henan, jiangxi, hunan, jiangsu, heilongjiang and zhejiang provinces of eastern and northern china. there were samples from sows ( primiparous sows, and multiparous), from -week-old piglets, from week-olds, from week-olds, from week-olds, from week-olds, from week-olds, and from week-olds. another serum and fecal samples were obtained from weaning piglets experimentally challenged with pedv-zju/g / in three pedv challenged experiments. the procedure of the pedv challenge has been described previously . the briefly, the -dayold conventional piglets were inoculated with about ml infectious titer (tcid ) pedv in × pbs (ph . ). blood and fecal samples were collected prior to inoculation and at , , , , , , days postinoculation (dpi). samples from a total of survival piglets but not from dead pigs at each time point were collected. after centrifugation at ×g for min, serum was harvested, aliquoted and stored at − °c until use. fecal swab samples were individually mixed with ml of × pbs (ph . ) immediately after collection, placed in a ml cryogenic tube (bd falcon™), and stored at − °c until use. the animal experiments were approved by the experimental animal ethics committee of zhejiang university (approval no. zju ). the pedv-zju/g / strain was propagated in vero cells in dmem supplemented with μg/ml trypsin according to the standard method . briefly, a confluent cell monolayer was washed with minimum essential media (mem) twice before infecting with the virus [moi (multiplicity of infection) = ] for h at °c, after which additional culture medium was added without removing the inoculum. observed cytopathic effects (cpe) reached approximately % in - days, and the virus culture supernatant was collected after three freezethaw cycles, then clarified by high speed centrifugation ( ×g for min) and further purified by ultracentrifugation through a % (wt/ vol) sucrose cushion ( , ×g for h). the purified virus was further exposed by a negative staining technique, examined using electron microscopy, then stored at − °c until use. concentration of viral proteins was measured by bca protein assay kit (beyotime, shanghai, china). full-length pedv cdna was used as a template for amplification and cloning of various pedv genes, including those encoding the c terminus of pedv orf (orf c) and the complete sequences of e, nsp , nsp , ac, and adrp. the constructs used for this study are listed in table . for transient expression in mammalian cells, the pcdna- . vector was used, and the expression plasmid pcdna-pedv-s -fc has been described in the previous study (gerber et al., ) . for expression of his-tag fusion proteins in bacteria, pedv target genes were cloned in-frame with n-terminal ×his tags in the pet- a (novagen; for ac, adrp, nsp and nsp ) or the pet- a-derived psmart-i vector with an n-terminal sumo (small ubiquitin-related modifier) tag (smart-lifesciences, changzhou, china; for orf c and e). the oligonucleotide primer sequences and approaches used are available upon request. the sequences of all constructs were confirmed by dna sequencing (huada gene technology co., ltd). the above recombinant plasmids were transformed into bl (de ) competent cells, respectively, and grown in l of luria-bertani (lb) medium (invitrogen) containing μg/ml kanamycin at °c with shaking at rpm. when an od of . was reached, m isopropyl β-d- -thiogalactopyranoside (iptg) was added to the final concentration of . mm in the l of lb medium, and bacteria were grown for an additional h at °c. cells were chilled at °c and harvested by centrifugation at ×g for min, resuspended in ml lysis buffer ( mm tris−hcl, ph . ), and disrupted by ultrasonication. crude extracts were centrifuged at , ×g for min at °c, and soluble expression of the his-tagged fusion peptides was confirmed by sds-page analysis prior to purification by the ni-nta his•bind® resin system (transgen tech, dp , beijing, china) according to the manufacturer's instructions. for the polypeptides that were expressed in inclusion bodies, they were first solubilized in a denaturing buffer ( mm tris−hcl, with m urea, ph . ), and purified by ni-chelating chromatography (ge healthcare). elutions were pooled, dialyzed at °c against mm pbs (ph . ) with mm nacl and m urea, and analyzed by sds-page and western blot. five purified, recombinant pedv peptides (orf c, ac, adrp, nsp , and nsp ) were selected and separately used to immunize two new zealand white rabbits, using a custom antibody production service at hangzhou belta-biotechnology co., ltd. (hangzhou, china). rabbits were not immunized with recombinant e protein. the purified recombinant pedv s , orf c, e, adrp, nsp , nsp , and ac peptides were resolved on a - % bis-tris polyacrylamide gel (invitrogen) by electrophoresis and subsequently transferred onto a polyvinylidene difluoride (pvdf) membrane. proteins were then detected using an anti- ×his mab ( : dilution; protein-tech, wuhan, china) or rabbit polyclonal antisera at °c, followed by incubation with hrp-conjugated goat anti-mouse or anti-rabbit igg ( : , dilution; thermo fisher scientific, usa), as appropriate, at room temperature. after four washing steps using tris-buffered saline with . % tween (tbst), membranes were analyzed using an enhanced chemiluminescence kit (beyotime ecl plus kit, china). for serum western blot analysis, purified s , orf c, e, and ac peptides were incubated after sds-page and membrane transfer with individual porcine sera diluted : , or with anti-his mab or polyclonal rabbit serum as positive controls. hrp-conjugated rabbit antiswine igg and goat anti-mouse igg ( : , dilution; abcam, united states) were used, as appropriate, as secondary antibodies. antigen concentration and dilutions of sera and hrp-conjugated antibodies were optimized by checkerboard titrations. the optimal amount of pedv wv, s , orf c or e antigen used for coating was . , . , . or . ng/well/ μl. microtiter plates were blocked with μl/well blocking buffer (thermo fisher scientific, usa) for . h at °c. after coating, μl of serum samples ( : dilution) were transferred in triplicate and incubated at °c for h. afterwards, μl diluted hrp-conjugated goat anti-swine igg or iga ( : , dilution; thermo fisher scientific, usa) was added to each well and incubated at °c for h. wells were washed between incubation steps three times with μl pbs ( mm, ph . ) with . % tween- (pbs-t washing buffer). finally, μl tmb color liquid (solarbio, beijing, china) was added to each well and incubated for min at room temperature, after which the reaction was stopped by addition of μl/well of m sulfuric acid, and the plates were read at nm using a spectrophotometer. initial pedv-negative sera were obtained from the united states (a gift from dr. tanja opriessnig) that was subsequently used for screening negative porcine sera in china as reported previously (gerber et al., ) . the elisa positive cutoff values were calculated as the mean od of negative controls (n = ) plus three standard deviations. the positive and negative sera from experimentally-infected piglets were also confirmed by western blot on purified pedv wv and s protein antigens as previously described (huang et al., ) . positive and negative controls were run in duplicate on each elisa plate. antibody response in all tested samples was represented as a corrected sample-to-positive (s/p) ratio, calculated as follows: s/p ratio = (sample odmean od, negative controls) / (mean od, positive controlsmean od, negative controls). vero cells were seeded in confluent monolayers in -well plates (co star™, corning) for h, then infected with μl/well of pedv ( tcid /ml) for h at °c, followed by removal of the inoculum. mem supplemented with . % (w/v) trypsin (mmt) was added to each well and incubated at °c for h, then cells were fixed with % paraformaldehyde. for specific detection of pedv proteins, different rabbit anti-pedv polyclonal antibodies were used as appropriate, with a mouse anti-pedv s mab (cat no: , jbt, korea) used as a positive control. secondary alexa fluor -conjugated goat anti-mouse igg or goat anti-rabbit igg (invitrogen) were used at a : dilution, incubated for h at room temperature. plates were washed three times between antibody incubations with μl/well of pbs-t. nuclei were stained with ', -diamidino- -phenylindole (dapi; kpl, inc.) at a : dilution, and visualized under a fluorescence microscope. sera from challenged piglets were tested for neutralizing antibodies (na), according to the protocol with slight modification (kusannagi et al., ) . briefly, serum samples were inactivated at °c for min and then -fold serial dilutions ( : ˜ : ) were prepared in well plates. after mixing μl of each dilution with μl pedv ( tcid /ml), samples were incubated for h at °c and used to infect monolayers of vero cells in -well plates. after adsorption for h at °c, the inoculum was discarded, plates were washed three times with mem, and maintenance medium (containing μg/ml trypsin) was added to each well. after incubation at °c for h, cells were observed on an inverted microscope for cpe such as cell fusion and nuclear atrophy. sn titers were calculated using the reed and muench method and expressed as the reciprocal of the highest serum dilution resulting in % inhibition of pedv infection, relative to controls. all data were processed using spss (version . ) software and the graphpad prism program as described previously (gimenez-lirola et al., ; huang et al., ) . the cutoff value and diagnostic performance of each pedv antigen was determined by receiver operating characteristic (roc) analysis (sas version . , sas institute, inc., cary, nc, usa) based upon the elisa results. pedv-zju/g / strain was propagated in vero cells and purified by ultracentrifugation on a sucrose-gradient. electron microscopy revealed that the purified virus was comprised of a great number of vesicles with morphological heterogeneity and envelope fragments carrying spikes (fig. a) . previously, the virions of several coronaviruses such as transmissible gastroenteritis virus (tgev), turkey and bovine enteric coronaviruses have been observed with a diameter ranging between - nm (dea and garzon, ) . to our knowledge, the pleomorphic property of pedv virions containing not only the small or defective particles, but also the giant spherical particles ranging up to nm in diameter, is reported here for the first time. although a few of the pedv particles had lost partial spikes, they were relatively intact. as spike glycoproteins are known to be the most immunogenic proteins of coronaviruses , purification by ultracentrifugation through sucrose cushions in this study proved to be reliable. also, the level of pedv protein was relatively high as detected by bca protein kits, thus confirming that the quality, integrity and quantity of the purified virions were sufficient for use as the antigen in subsequent elisa assays. initially, we failed to detect the expression of the complete orf using the pet- a, the psmart or the other bacterial expression vectors under different conditions by western blot analysis (data not shown). therefore, the -aa of the c terminal part of orf (orf c) showing the predicted hydrophilicity profile was chosen as the target antigen for orf . the ac, e and orf c recombinant peptides were expressed in the inclusion bodies, whereas the nsp , adrp and nsp proteins displayed soluble expression in the cultured supernatants. expression yields of ac, e, and orf c were very low, hardly visible, when examined by coomassie blue staining after sds-page (data not shown); but confirmation of these three peptides with predicted sizes (ac:˜ kda; e fused with a sumo tag:˜ kda; orf c fused with a sumo tag:˜ kda) could be done using an anti-his-tag antibody by western blot (fig. b) . on the other hand, sds-page and western blot analyses of purified nsp , adrp and nsp soluble proteins showed bands that were consistent with the predicted sizes of , and kda, respectively (fig. c) . the purified s protein expressed in mammalian cells was also identified as a single band by sds-page (fig. d ) and by western blot using an anti-s monoclonal antibody as described previously (gerber et al., ) . due to glycosylation of the s protein, the size of the band in the gel was larger than its predicted size (˜ kda). . . antibodies generated against recombinant pedv ac, orf c, and nsp proteins resulted in specific fluorescence in vitro purified recombinant pedv peptides (orf c, ac, adrp, nsp , and nsp ) were used to immunize rabbits, generating polyclonal sera that were used to detect viral proteins on pedv-infected vero cells by ifa. the e protein was not used to immunize rabbits in this study. staining of anti-ac polyclonal serum resulted in specific fluorescence at h post-infection ( fig. a, b ) similar to the signal observed using the anti-s mab as the positive control (fig. g, h) . specific fluorescence was also detected with the anti-orf c (fig. c, d) and the anti-nsp polyclonal antibodies (fig. e, f) . in contrast, no specific fluorescence was observed when using the anti-adrp and anti-nsp polyclonal antibodies. ifa with pre-immune rabbit sera displayed no fluorescent signal in vero cells infected with pedv (fig. i, j) . the viral antigens were all detected in the cytoplasm of the infected cells. the anti-s mab reacted more strongly than the three positive rabbit antisera based on a comparison of the positive cell numbers and fluorescence intensities. infection of vero cells or vero cells expressing the entry receptor porcine apn with the other swine enteric coronaviruses , such as swine enteric alphacoronavirus , porcine deltacoronavirus (pdcov) and tgev, had no detectable fluorescence after ifa with the anti-pedv polyclonal antibodies described above (data not shown). therefore, anti-pedv-ac, anti-pedv-orf c and anti-pedv-nsp are pedv-specific and do not cross-react with these known porcine coronaviruses. previously, we have developed and validated indirect elisa based on the s protein to monitor serum anti-pedv igg and serum and fecal anti-pedv iga antibodies in postweaning pigs (gerber et al., ; gerber and opriessnig, ) . in this study, in order to determine the pattern of antibody response of weaning piglets in a -day weaning period after pedv infection, serum or fecal samples from experimentally-infected -day-old piglets were examined by elisa based on the pedv wv or the s protein, and by serum neutralization test (fig. ) . the results indicated that igg and iga responses against both antigens were detected in serum at different time points after pedv infection ( fig. a and b ). despite challenge with pedv in these piglets, levels of serum igg and iga decreased from dpi, reaching a minimum after dpi as detected by both wv and s antigens (fig. a, b) , and the pattern or trend of neutralizing antibody (na) was more similar to that of iga (fig. c) . a good linear relationship between the s -based iga elisa titers and na titers was observed (spearman's rank correlation coefficient of . ; p < . ), demonstrating the correlation between them (fig. d ). there were some differences in the sensitivity of the antigens to detect antibodies, as levels of serum iga were slightly higher when the s protein was used as the detection antigen. levels of fecal iga were also highest before challenge ( dpi), and continuously declined after challenge (fig. e) . the specificity and sensitivity of detection of s and wv antigens were similar for serum iga. the high igg and iga antibodies and na detected at the early stages of the weaning piglets are presumably maternal antibodies received from sows that were not pedv negative. in addition, piglets during weaning have not developed their own immunity to the virus. these results also demonstrated that the s -based elisa is an alternative (to wv-based) and ideal serological assay for detection of anti-pedv antibodies. . . antibodies against specific pedv peptides were detectable in sera from naturally infected pigs recombinant pedv s , orf c, e, ac and nsp peptides were used as antigens in western blots to detect antibodies in porcine sera from commercial farms since they produced antibodies in rabbits reacted to pedv antigens in infected cells except for the e protein that was not tested (fig. ) . the criterion for determining the seropositivity to a particular antigen of a sample was whether the expected protein band a mouse anti-s monoclonal antibody (g, h) and pre-immunization rabbit serum (i, j) were used as positive and negative controls, respectively. alexafluor -conjugated goat anti-rabbit igg and goat anti-mouse igg (green) were used as secondary antibodies, as appropriate. antibody staining merged with dapi nuclear staining (blue) is shown; magnification = × (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). was presented on the membrane. according to this, serum western blot analysis of the anti-nsp antibody showed high-background results and thus further investigation of nsp was not performed. a main specific band appeared for s , ac, e or orf c protein antigen tested (arrows indicated in representative samples in fig. ) , though additional "fuzzy" bands were present at low or high molecular weights that were likely from nonspecific reactivity of serum components. compared with the other proteins, the s band appeared to be more specific, with a cleaner background (fig. a) . pedv-positive sera were screened and verified viathis method, which were used to compare with the results of the elisa (fig. ; see below) , whereas negative sera from s -based elisa were also analyzed as negative controls, and none of them showed reactivity (fig. , the second lanes in all panels) . however, in the case of ac, the specific -kda bands also appeared in some sera that were negative by elisa (data not shown). furthermore, the specific igg antibody responses in sera from a total of diarrheic pigs from farms in china were analyzed using elisa assays based on pedv wv, s , orf c or e proteins (fig. a-d) . the results indicated good correlations among these antigens. overall, the levels of antibodies against pedv wv and the three proteins antigens were generally higher in primiparous and multiparous sows than those in - weeks-old pigs (p < . ). the trend of serum igg response to each pedv antigen from the -week-old to the -week-old pigs was similar to that of the experimentally-infected weaning piglets (fig. a) . antibodies were highest in the week-old piglets (p < . ) except for wv antigen, and then declined to a minimum by weeks-of-age significantly (p < . ) before increasing again to a relatively stable level from to weeks-of-age (fig. b ,c; p < . ). this pattern was in agreement with a recent report about the prevalence of pedv antibodies in swine farms (bertasio et al., ) , which might reflect that newborn piglets receive maternal igg antibodies from sows, though the level of the antibodies decline quickly, and the weaned piglets must begin to develop their own immunity to pedv. however, there were some differences in reactivity to the antigens used in the elisas. igg detected by wv-based elisa had a similar pattern to that detected by the other antigens, as the antibodies were in a relatively dispersed distribution, although its cutoff value ( . ) was closest to the orf c elisa ( . ). antibody levels against the pedv e protein were higher with a relatively concentrated distribution, especially in pigs older than weeks. however, the e protein-based elisa had the highest cutoff value ( . ), and the distribution of antibodies from to weeks-of-age was obviously different from the s -based elisa, which had the lowest cutoff ( . ). collectively, the data indicated that the pedv s protein was more specific and accurate as a detection antigen in elisa tests. with the introduction of pedv into the north american herd in - tian et al., ) , the need for a suitable diagnostic marker for the accurate, rapid, and early diagnose of pedv infection has become much more urgent. pedv herd-infection status is very important for biosecurity and the control of ped. compared to rt-pcr and other nuclei acid detection assays, serological tests are advantageous to study immune responses related to vaccination, wild-type virus infection, and to determine whether sow immunity is adequate in individual litters after pedv exposure (diel et al., ) . pedv infection is not always obvious in finishing pigs, which increases the risk of widespread disease in pigs of all ages (bertasio et al., ) . thus, sensitive serological tests would allow detection of recent infections, to avoid the introduction of these animals into naïve herds. however, there are many different structural and non-structural proteins to choose from when selecting an antigen to use in novel diagnostic assays. previously, we have developed and validated indirect elisa based on the s protein to detect anti-pedv igg and iga antibodies in postweaning pigs (gerber et al., ) . the present study was set up to investigate diagnostic potentials of specific pedv accessory and nonstructural proteins, which have not yet been reported systematically. fig. . detection of antibodies against pedv peptides in porcine sera by western blot. representative serum samples from diarrheic pigs were used in western blots to detect pedv s (a), ac (b), orf c (c), and e proteins (d); μg of recombinant peptides were loaded in each lane. hrp-conjugated goat anti-porcine igg was used as the secondary antibody, and pedv-negative serum was used as negative control. fig. . distribution of cumulative igg elisa sample-to-positive (s/p) ratios in serum samples collected from diarrheic pigs at commercial pig farms. various indirect elisa assays based on pedv whole virus (wv) (a), s (b), orf c (c), and e proteins (d) were used to test serum samples from commercial sows and pigs with diarrhea at different ages with unknown pedv infectious status. sera from naïve piglets were used as negative controls; samples above the determined s/p cutoff (dashed line) were considered positive. one-way analysis of variance (anova) was used for multiple comparisons between different ages among the individual antigens with an alpha value of . . the "ns" denoted no statistical differences. the sera and feces from the experimental infected piglets at weaning were first used for detection of antibodies using the pedv wv and s protein-based indirect elisas. the pattern of change in anti-pedv igg/iga from serum samples and in anti-pedv iga fecal samples (immediate decline post-infection) indicates that the piglets had obtained maternal antibodies at birth, and did not produced antibodies even after pedv challenge until their immune system matured. additionally, levels of na were more closely correlated with iga than igg (fig. b-d) , as seen in other studies (paudel et al., ) . abundant anti-pedv na have been demonstrated in colostrum on the day of birth, decreasing rapidly in milk by day , and gradually declining further from days - post-farrowing, which may contribute to variable protective capacity . the current study showed a similar decline, further confirming the reliability of the s -based elisa assays applicable to weaning pigs in addition to postweaning pigs (gerber et al., ; gerber and opriessnig, ) , and also highlighting the importance of accurate diagnosis in a short window for proper immunization of sows and piglets. compared to the pedv wv, s -based assays showed good reactivity and high sensitivity/specificity (figs. , a and b) . it is worth noting that the recombinant s protein was expressed in a eukaryotic expression system and should display a natural conformation with high glycosylation, as shown in fig. d , which may be one reason for its higher detection sensitivity. on the other hand, wv was mainly purified by sucrose density gradient centrifugation, differential centrifugation or polyethylene glycol (peg) precipitation (hoffmann and robert, ) . these methods would damage the integrity of the virus, especially the surface spike glycoprotein. therefore, the eukaryotic-expressed s protein is likely more advantageous than the wv as the antigen for pedv serological assays. for another two major structural proteins m and n, due to common epitopes shared by pedv, tgev and pdcov, several studies have previously demonstrated that pedv m or n presented some cross-reactivity to tgev or pdcov (gimenez-lirola et al., ; lin et al., ; ma et al., ) . in contrast, recombinant pedv-s had no cross-reactivity with sera from these porcine coronaviruses, showing the best diagnostic sensitivity (gimenez-lirola et al., ) . therefore, we did not pursue the development of serological assays based on m or n proteins in this study. the accessory orf protein is thought to have high potassium channel activity and may be associated with the virulence of pedv (wang et al., ) . the small structural e protein has important roles in the assembly of coronavirus virions, virus egress and in the host stress response (ruch and machamer, ) . besides the structural proteins, pedv has several non-structural proteins (nsp , nsp , nsp , among others) that express in the early stage of virus infection and have important functions in the viral replication cycle. the coronavirus nsp is a conserved component of the viral protein processing machinery, and may be incorporated in the virion viaits intimate association with viral rna (neuman et al., ) . the nsp is known as the largest replicase subunit, consisting of numerous distinct structural domains separated by disordered linkers. some of these, such as the ac and adrp (macrodomain), are well conserved across all genera of coronaviruses, though there have been no reports about serological assays based on pedv ac and adrp domains. considering their potential use in the study of host immune response, these proteins were specifically included in the current study. recombinant orf c, e, ac, adrp, nsp and nsp peptides were expressed and purified (fig. ) ; however, only orf c, ac and nsp produced functional rabbit antibodies recognizing pedv antigens in infected cells (fig. ; the anti-e was not generated and tested). subsequently, they were used individually as detection antigens in western blot and/or indirect elisas to detect anti-pedv igg antibodies in sera from diarrheic pigs (fig. ) . the pedv wv and the recombinant s expressed from mammalian cells was used for comparison. the s , orf c, and e peptides each reacted strongly with the sera, reflecting expected distributions of pedv-specific antibodies. the reactivity of ac, adrp, nsp and nsp peptides was less pronounced (data not shown), thus they were discarded from consideration as novel diagnostic antigens. the generally high level of igg against s , orf c, and e proteins in old-age pigs was consistent with recent reports that anti-pedv igg in infected pigs persisted for over than weeks after the onset of diarrhea symptoms (lin et al., ) . all antibody levels declined to their lowest levels at weeks-of-age, which was consistent with the result from experimentally-infected piglets at weaning (fig. ) . western blot was implemented to confirm the elisas, with additional "fuzzy" bands appeared when the orf c, ac and e were used as detection antigens, indicating non-specific recognition (fig. ) . this may be related to differences in antigens used and/or the sensitivity of the assays for detecting anti-pedv antibodies. the orf c antigen has moderate immune reactivity as evidenced by staining of anti-orf c in pedv-infected cells displaying specific fluorescence in ifa ( fig. c and d) , by serum western blot (fig. d) , and by indirect elisa (fig. c) . in future studies, we plan to employ the pedv mutants with the orf deletion generated by reverse genetics zhao et al., ) in comparison with the wild type pedv for more detailed evaluation of the protein for use as a marker in diagnostic assays. the accessory protein e showed intermediate sensitivity in western blot and extremely high sensitivity in elisa assays compared with the other antigens ( fig. c and d) . the elisa sensitivity was too strong, as nearly all of the sera from sows and - weekold piglets were strongly positive, thus it could not properly reflect the trend of pedv-specific antibodies. to our knowledge, this is the first report about the use of an orf -or e-based elisa on such a large scale. of the non-structural proteins, ac displayed strong immune reactivity ( figs. a and b) , suggesting that it may be released into circulation or is picked up by antigen-presenting cells (hurst-hess et al., ) . therefore, the study of anti-ac antibodies may contribute to a better understanding of the detailed function of nsp . our results also complement previous mass spec identification of nsp within purified virions (neuman et al., ) . in summary, this study is the first to dissect the range of antibody responses against pedv during infection, using different assays (elisa, western blot, sn) to comprehensively analyze pedv antibodies in porcine sera from china. the results confirmed high pedv prevalence in china (sun et al., ) . the antibody profiles provided by the study offer more reliable information on the host immune response to different viral proteins, and will be useful for design of vaccines that better stimulate protective immunity. above all, our data identified that besides s , the recombinant orf c and e proteins can also be used as diagnostic markers; but s represents greater sensitivity for a wide range of pedv-specific antibodies. the authors declare no conflict of interest. impact of porcine epidemic diarrhea on performance of growing pigs age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains identification of coronaviruses by the use of indirect protein agold immunoelectron microscopy porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states expression of the putative orf capsid protein of torque teno sus virus (ttsuv ) and development of western blot and elisa serodiagnostic assays: correlation between ttsuv viral load and igg antibody level in pigs serological profile of torque teno sus virus species (ttsuv ) in pigs and antigenic relationships between two ttsuv genotypes ( a and b), between two species (ttsuv and - ), and between porcine and human anelloviruses dissection of amino-terminal functional domains of murine coronavirus nonstructural protein aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate.pdf antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus discovery of a novel swine enteric alphacoronavirus (seacov) in southern china comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus (mrv ) from a diarrheic piglet and seroepidemiological survey of mrv in diarrheic pigs from east china the coronavirus e protein: assembly and beyond characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review evidence of recombinant strains of porcine epidemic diarrhea virus porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry pedv orf encodes an ion channel protein and regulates virus production identification of a peptide derived from the heptad repeat region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies this work was supported by the national key research and development program of china ( yfd ), the key research and development program of zhejiang province ( c ), and the national natural science foundation of china ( ). we thank the professional editing service nb revisions for technical preparation of the text prior to submission. key: cord- -yr zfxz authors: le devendec, laetitia; jouy, eric; paboeuf, frederic; de boisséson, claire; lucas, pierrick; drider, djamel; kempf, isabelle title: development of a pig infection model with colistin-resistant escherichia coli date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: yr zfxz colistin-resistant escherichia coli are isolated from pigs suffering from post-weaning diarrhea (pwd). this study was designed to develop an experimental model of pwd using mcr- -carrying shiga toxin-producing e. coli (stec) or enterotoxigenic e. coli (etec), for the future evaluation of control measures. three groups of eight piglets, kept in high biosecurity units, were orally inoculated with mcr- -positive stec or etec, and one unchallenged group was used as a control. clinical signs were recorded. regularly-collected fecal samples and samples obtained from the digestive tract of animals sacrificed one month after inoculation were cultured in selective media and isolates were characterized. blood samples were used to genotype the polymorphisms of the pigs’ intestinal receptors for f and f e. coli adhesins. diarrhea was more frequent and more fecal samples contained the inoculated strain in the group inoculated with the o -f etec strain than with the o -f or o -f stec strains. however, fewer positive samples were obtained from the two pigs with the f resistant genotype. the three inoculated strains could be re-isolated up to the end of the experiment. excretion peaked on the first week after inoculation with the o -f etec strain, and later for the other two. an mcr- gene transfer to other commensal isolates was observed only for o -f stec, while the loss of mcr- from the inoculated strain occurred in all groups. the o -f etec challenge may be used to evaluate alternative solutions to combat pwd caused by colistin-resistant e. coli in pigs. post-weaning diarrhea (pwd) and edema disease are common pathological conditions affecting piglets (rhouma et al., a) . various escherichia coli strains cause these diseases. indeed, different serogroups (o , o , o , o , o …), adhesive factors (f (k ), f , aida, eae…), and other virulence factors (enterotoxins lt, sta, stb, east…) have been reported in these strains (fairbrother and gyles, ) . the control of these infections must be based primarily on optimizing breeding management of the weaning piglets so as to avoid, insofar as possible, stressful conditions as they are separated from the sow, transported, mixed, placed in a new environment and provided with new feed. prophylactic measures may also include vaccination of the sow (rhouma et al., a) . however, these measures may be ineffective in preventing digestive troubles, and in the past, orally administered antimicrobials were almost systematically used to control colibacillosis. as a result, e. coli strains have developed antimicrobial resistance, particularly to the most frequently administered molecules. of note, resistance to colistin (cst) had been detected at relatively low levels for years in commensal isolates, but prevalence was sometimes high for pathogenic strains (kempf et al., ) , and after a publication on the emerging mcr- gene in china (liu et al., ) , it soon appeared that this gene was also present in many other countries, particularly in pig e. coli strains (kempf et al., ) . as these strains are frequently also resistant to many other antimicrobials (delannoy et al., ) , it became necessary to develop and evaluate novel control products or measures. to our knowledge, experimental models of colibacillosis in piglets have been developed with inoculation of cst-susceptible e. coli (madec et al., ; rhouma et al., rhouma et al., , b but not with cstresistant strains. however we believe that such models are needed to evaluate the impact or the efficacy of antimicrobials, including colistin, and alternative strategies on infections caused by strains with resistance or reduced susceptibilities to a number of molecules. furthermore, it has been shown that acquisition and expression of the mcr- gene in a e. coli cell may result in gross morphological changes, including cell membrane impairment, and reduced fitness and attenuated virulence in a galleria mellonella model. moreover the mcr- mediated lps modification reduces the stimulation of macrophages, and a lower production of il- and tnf was induced with lps extracted from mcr- strains compared to control lps (yang et al., ) . thus because of these potential differences of susceptibility and virulence of mcr- strains, we decided to develop an experimental model of colibacillosis in weaned piglets using multi-drug-resistant e. coli strains isolated from clinical cases and carrying the mcr- gene. . . preparation of strains, media and inocula and whole genome sequencing three e. coli strains ( - , - and - ) from our previously characterized collection of mcr- -positive e. coli isolates from diseased pigs were selected on the basis of their resistances, serogroups and virulence genes as detected by high-throughput real-time pcr microarray technology (delannoy et al., ) . the intent was to choose strains representative of cst-resistant isolates obtained from piglets with pwd. all three strains were hemolytic. strains - , - , and - belonged to phylogenetic groups (pgg) a, a, and e, and had markers o -f , o -f , and o -f respectively. all three strains were resistant to colistin, sulfamethoxazole, trimethoprim, tetracycline, and ampicillin. strains - and - were also resistant to gentamicin and chloramphenicol. a rifampicin-resistant mutant ( - m, - m, and - m) was prepared from each strain by culturing a large amount of inoculum on rifampicin-supplemented media ( mg/l). dna from the three mutants was prepared using qiamp dna mini kits (qiagen, courtaboeuf, france) according to the manufacturer's instructions. whole genome sequencing was performed with the ion proton system (ion torrent™, thermofisher, villebon sur yvette, france). we cleaned reads with trimmomatic (bolger et al., ) , then used bwa-mem (li, ) to align them with the e. coli e strain (ncbi reference sequence nz_jend . ) to obtain an estimated coverage depth of . we next performed spades assemblies on cleaned reads (bankevich et al., ) and mira assemblies (chevreux et al., ) on related raw reads. contigs were identified using megablast on a local nt database (chen et al., ) , and those corresponding to "uncultured soil fungus" were deleted. sequences were analyzed using a web tool (https://cge.cbs.dtu.dk/ services) to detect antimicrobial resistance genes and verotoxin virulence genes, and to determine the st of each strain (thomsen et al., ) . other virulence genes, including paa, sfpc, cnf , aida-i, ecf - , stce, and eibg (tseng et al., ), sita-d, iuca-d, iuta, hlyf, ompt, etsa-c, iss, irob-e, iron, cvaa-c, cvi, tsh, and eita-d (touzain et al., ) , tere, ured, and esc (ae . ), hlya (fm . ), orfa and orfb (gu ), were sought using blast (https://blast.ncbi.nlm. nih.gov/blast.cgi). to prepare the inocula, each mutant was cultured in mueller hinton (mh) broth containing colistin ( mg/l) for h at °c, under gentle agitation. each bacterial pellet obtained after centrifugation ( g, min) was re-suspended in peptone buffered solution in order to obtain a titer of colony-forming units (cfu)/ml. the experiment was performed in accordance with french animal welfare regulations and the protocol was approved by the anses/ enva/upec ethical committee (cometh authorization - (apafis no. )). the experiment was conducted in the anses ploufragan animal facilities. strict biosecurity measures were implemented to avoid contamination of the pigs, including the use of an air filtration system and airlocks for each unit, unit-specific clothes, and compulsory showering after visiting the pigs. the trials were conducted with specific-pathogen-free (spf) large white piglets. the spf piglets are naturally born from hysterectomy-derived sows controlled for presence of classical and african swine fever, aujesky's disease, footand-mouth disease, h n /h n swine influenza, transmissible gastroenteritis/porcine respiratory coronavirus, porcine parvovirus, reproductive and respiratory syndrome virus, porcine circovirus type , border disease, mycoplasma hyopneumoniae, pasteurella multocida, bordetella bronchiseptica, actinobacillus pleuropneumoniae, haemophilus parasuis, streptococcus suis type , salmonella, lawsonia intracellularis, brachyspira hyodysenteriae, yersinia, campylobacter, listeria monocytogenes, balantidium coli and trichomonas. the piglets are left with the sow until weaning and bred in a pathogen-free environment. the six-week-old piglets were randomized before the experiment. the animals did not receive any antibiotic treatment prior to the trial and were given the same non-supplemented feed. each room contained two pens (table ) . on day , five piglets in animal room e (group , five pigs) were orally inoculated with ml of sterile broth. on the same day, eight piglets per room (four piglets per pen) in animal rooms e , e , and e , (respectively named groups g , g , and g , eight pigs each) were orally inoculated with ml of the inocula prepared with strains - m, - m, and - m respectively. the weight of each animal was recorded once a week. during the week, daily clinical examinations consisted in looking for general clinical signs and taking rectal temperatures. individual fecal samples were collected from pigs on , , , , , , , , , , , , and (groups g , g , and g ) , and on , , and (group g ) . the pigs were sacrificed on day (group g ), day (group g ) or day (group g ). three pigs from the g group were also sacrificed on day , the others being kept as controls for another assay and necropsied on day following euthanasia. the sacrificed animals were necropsied and samples of the duodenum, jejunum, ileum, cecum, colon, and rectum were collected. the samples collected post-mortem contained both fecal material and scraped mucosa. for culture analysis, all the samples were diluted / in peptone buffered solution containing % glycerol. the / diluted aliquots were stored at - °c. before freezing (fecal samples) or after thawing (organ samples), decimal dilutions were prepared. one hundred microliters of decimal dilutions were inoculated using easyspiral® dilute (interscience, saint-nom-la-bretèche, france) onto macconkey media supplemented with rifampicin ( mg/l) and incubated overnight at °c. moreover, μl of the / thawed dilutions was enriched overnight in . ml of rifampicin-supplemented lb broth ( mg/l) before plating on rifampicin-supplemented macconkey plates. the limit of detection was cfu without enrichment and cfu/g after enrichment. whenever possible, two colonies (from samples positive only after enrichment) or three colonies (from samples positive without enrichment) per sample were stored. pcr was used to confirm that they belonged to the e. coli species (bej et al., ) , to detect the presence table arrangement of pigs inoculated with the different studied strains. groups all pigs were susceptible to e. coli f . a number of pigs resistant to e. coli f . of the mcr- gene (liu et al., ) , and to determine their phylogenetic group (clermont et al., ) . for isolates which did not exhibit the expected results (presence of mcr- and phylogenetic group of the inoculated strain), eric-pcr (rivera et al., ) and pfge profiles (ribot et al., ) after digestion with xbai were studied. the susceptibility of some of these isolates was compared to the susceptibility of the inoculated strains by determining the minimum inhibitory concentrations (mic) using a broth microdilution method on euvsec plates (sensititre, thermofisher scientific, dardilly, france). in addition, fecal samples collected on days , and were inoculated, after thawing, onto the chromagar orientation ™ agar medium (i a, montpellier, france) supplemented with colistin ( mg/l) and vancomycin ( mg/l) (cao-cv) as previously described (mourand et al., ) to detect colistin-resistant rifampicin-susceptible e. coli resulting from transfer of the mcr- gene to commensal e. coli. when e. coli colonies were observed, two colonies per sample were stored and further analyzed. their identity and the presence of the mcr- gene were checked by pcr, and their resistance to rifampicin was tested by culture on rifampicin-supplemented mh media ( mg/l). moreover, μl of the / dilutions was enriched overnight in . ml of colistin-supplemented lb broth ( mg/l) before plating onto rifampicinsupplemented macconkey plates (limit of detection: cfu/g). the colonies were identified and analyzed for the presence of the mcr- gene as previously described. for molecular analysis, thawed fecal and organ samples were tested with a slightly modified version of the protocol described by dona et al. (dona et al., ) . briefly, μl of the / diluted thawed fecal suspension was used to inoculate . ml of luria bertani (lb) broth supplemented with rifampicin ( mg/l). after overnight culture at °c under agitation, the broths were centrifuged at g for ten minutes and a cellular lysate was prepared from the pellet. e. coli dna and the mcr- gene were then detected by pcr in the lysate. pcr was performed on lysates obtained from enrichment in the colistin-supplemented broths ( mg/l), like for samples collected on days , , and . blood was obtained from each pig just before euthanasia. these samples were used to genotype the pigs for the different polymorphisms linked to the intestinal receptors for f and f e. coli adhesins. the porcine mucin (muc ) gene has been mapped on the q region of the pig chromosome , significantly associated with in vitro e. coli f ac adhesion to enterocytes, and muc is the most likely responsible gene governing susceptibility towards enterotoxigenic e. coli (etec) f ac diarrhea (ren et al., ) . the polymorphism of muc was analyzed based on the test described by rasschaert et al. (rasschaert et al., ) to determine the f ac/ab receptor status. briefly, a fragment of the mucin gene was amplified using hemo klen taq polymerase (neb, evry, france) and digested with xbai. the bp amplified muc pcr fragment of the resistant allele is indigestible, unlike the susceptible allele. muc was genotyped by pcr to detect the presence of amplicons of bp (muc a allele) and pb (muc b allele) as described by ren et al (ren et al., ) . susceptible animals usually carry at least one muc b allele. the e. coli f receptor locus is closely linked to the fucosyltransferase gene, and we used the test developed by meijerink et al. (meijerink et al., ) in which amplification followed by restriction with cfoi enables discrimination between the different alleles, based on the number of amplified fragments: two fragments for homozygous a/ a, three for homozygous g/g and four for heterozygous. the resistance is associated with allele a (meijerink et al., ) . the kruskall-wallis non-parametric test was used to compare weight gain. the distributions of pigs or isolates were analyzed using the chi -test or the fisher exact test. this whole genome shotgun project has been deposited at ddbj/ ena/genbank under the accession qzvz , qzwa and qzvy (respectively - m, - m and - m). sequencing enabled us to detect the resistance genes present in the isolates. thus, beside the previously described mcr- gene and the mutations in genes possibly involved in resistance to polymyxins (delannoy et al., ) , the strains harbored the genes coding for their other observed resistances: sulfonamides (sul , sul or sul ), trimethoprim (dfra ), tetracycline (tet(a)), chloramphenicol (cata , cmla ), ampicillin (bla tem a or bla tem b ) or aminoglycosides (aac( )-iia, aac ( )-iva, aada , aada , aada , aph( ′)-ia, aph( ′)- c, aph( )-ia, stra or strb). the virulence genes are listed in table . the genes previously detected by microarray (delannoy et al., ) were confirmed to be present. the two f stx -positive strains belonged to st and , whereas the o f strain containing genes coding for the lt, sta and stb toxins belonged to st- . the inoculum titers were . × cfu/ml, . × cfu/ml, and . × cfu/ml for - m, - m and - m respectively. only pigs could be tested as no blood was obtained from the two control pigs sacrificed on day . as expected, muc and muc gene results were in agreement, and only five animals were found to be of the genotype resistant to e. coli f ac/ab: one in group g , two in g , and two in g (table ) . for the f receptor, ten pigs were heterozygous ag and the other were homozygous gg. thus all pigs were susceptible to e. coli f . all pigs of group g had normal rectal temperature during the assay. moderate pyrexia ( . °c- . °c) was observed during the three days after inoculation in , and pigs in groups g to g respectively. no clinical signs were observed in group g . in groups g to g , moist, cow-dung appearance feces were observed in all pigs during the third week after inoculation. moreover, in group g , two pigs had diarrhea during the four days following inoculation. the mean weight gains of groups g to g from day to day were respectively . ± . , . ± . , . ± . , and . ± . kg (p = . ). the day -day weight gain of group g , but not those of groups g and g , was significantly different from the weight gain of the control group (p = . ). the weight gains from day to day of groups g to g were respectively . ± . , . ± . , . ± . , and . ± . kg and did not differ significantly (p > . ). no lesions could be observed post-mortem. the results are presented in tables and . all cultures from noninoculated pigs or from samples collected before inoculation were negative on rifampicin-supplemented media. some of these samples yielded e. coli colonies after enrichment, but none of the isolates tested had the mcr- gene. for cultures on rifampicin-supplemented media, with or without enrichment, and after mcr- -pcr control of isolates, all inoculated pigs were observed to be positive at least once. in groups g , g , and g , out of sampling days after inoculation, the pigs were positive on to (mean . ), to (mean . ) and to (mean . ) different days respectively. the total positive samples were out of , out of , and out of tested samples in groups g to g respectively, the proportion in g being significantly higher than the other two groups (chi- test, p < . ). in all groups, at least one pig was positive on the last sampling day, four weeks after inoculation. the maximum mean titers were observed on day ( . ) in group g , on days and ( . on each day) in group g , and on day ( . ) for group g . most isolates obtained directly on rifampicin-supplemented media ( / , %) or after enrichment in rifampicin-supplemented media ( / , %) harbored the mcr- gene and showed the expected characteristics of the inoculated strains. considering only isolates obtained without enrichment, for group g , out of isolates shared the characteristics of inoculated strain - m, and one isolate obtained on day , without enrichment, belonged to pgg a, had the eric and pfge profiles and the antimicrobial susceptibility of - m, including resistance to colistin, although it was negative for the mcr- gene. in group g , out of isolates obtained without enrichment shared the characteristics of inoculated strain - m, but nine isolates lacked the mcr- gene. six were fully analyzed and were found to belong to pgg a, and have the eric and pfge profiles of the - m isolate. they were resistant to tetracycline and gentamicin but, contrary to - m, susceptible to sulfamethoxazole, trimethoprim, colistin and ampicillin. the mcr- -negative isolates had been obtained on day , , , and from three pigs bred in the two pens of the animal room. in group g , out of isolates obtained without enrichment had the expected characteristics (presence of the mcr- gene and phylogenetic group e). six had the mcr- gene but belonged to pgg a, and their eric and pfge profiles were identical to each other but different to that of - m. they were obtained on day and day from three pigs placed in the two pens of the animal room. one isolate obtained on day was mcr- -negative. its pfge profile was identical to the inoculated (delannoy et al., ) . a: sample containing rifampicin-resistant, mcr- -positive e. coli of the expected phylogenetic group, obtained directly. b: sample containing rifampicin-resistant, mcr- -positive e. coli of the expected phylogenetic group, obtained after enrichment. c: sample containing rifampicin-resistant, mcr- -positive e. coli of an unexpected phylogenetic group and pfge profile, obtained directly. d: sample containing rifampicin-resistant, mcr- -positive e. coli of an unexpected phylogenetic group and pfge profile, obtained after enrichment (possibly transconjugant). e: sample containing rifampicin-resistant, mcr- -negative e. coli of the expected phylogenetic group and pfge profile, obtained directly (possibly inoculated strain having lost the mcr- gene). f: sample containing rifampicin-resistant, mcr- -positive e. coli detected on cao-cv medium. ns: no sample. grey cells: clinical signs (diarrhea or temperature > . °c). * : pig resistant to e. coli f . ; and for g , three pigs were positive on day . positive pigs were detected in the two pens for groups g and g (table ). there was no difference between the proportion of positive samples in the inoculated groups (fisher's exact test, p > . ). all the samples collected before inoculation were negative. for group g , nine out of samples were positive; they were obtained on days , , , and from seven different pigs. for group g , samples out of obtained on days , , , , , and were positive; seven pigs were positive. for the e group, out of samples were positive; they were obtained on days , , , , and from six different pigs. there was no difference between the proportion of positive samples in the inoculated groups (chi test, p > . ). only samples collected on days , , and were analyzed. all the results were negative except three fecal samples collected on day from two pigs in group g and one in g . for the same days , and , culture on rifampicin media detected positive samples out of samples from inoculated groups and was thus significantly more sensitive (p < . ). the results obtained on rifampicin-supplemented macconkey plates after enrichment in rifampicin-supplemented broth, are given in table . all the samples collected from the non-inoculated pigs and all the duodenum samples were negative. the proportion of positive samples in group g ( / ) was significantly lower than the proportion in g ( / ) and g ( / ) (fisher exact test, p < . ). besides the isolates with the characteristics of the inoculated strains, a few rifampicin-resistant mcr- -negative isolates belonging to the phylogenetic group of the respective inoculated strains were obtained in the jejunum or cecum from three different pigs in group g ; in the ileum or cecum from three different pigs in group g ; and in the ileum, colon, rectum or cecum from three different pigs in group g . the three strains proposed as an experimental model are relevant because they have been reported in literature and seem to be frequently involved in pig diarrhea. indeed sequencing confirmed that they were shiga toxin-producing (stec) ( - m and - m) or etec ( - m). o :f - m is of type st- , which has already been isolated from many different sources, such as humans, animals (including swine in italy and china), food (including pork in the usa) and the environment (water in france) (http://enterobase.warwick.ac. uk/species/ecoli/search_strains). an st- mcr- and bla ndm- e. coli of human origin was also characterized by zhang et al. (zhang et al., ) and several mcr- st- e. coli of healthy swine origin have also been reported in china . the o :f - m e. coli belongs to st- , like several pig or bovine pathogenic isolates of serogroups o :h :f ac reported in different european, american or asian countries, or an o poultry apec isolated in germany. the o :f - m strain belongs to st- , like o :f isolates obtained from pig diarrhea in hungary in (http://enterobase. warwick.ac.uk/species/ecoli/search_strains?query=st_search). an analysis of the different contigs carrying mcr- did not reveal other resistance or virulence genes or notifiable genes. symptoms, including hyperthermia and diarrhea, were more severe in group g . this group also had the highest excretion titers in the first week, and more fecal samples were found positive. however, at necropsy-more than one month after inoculation-the o :f + inoculated strain was less frequently isolated than the other two inoculated strains, both of type f + fimbriae. this may be related to the fact that susceptibility to f + or f + e. coli may vary with the pig's age. verdonck et al. (verdonck et al., ) previously observed a similar delay, with f + etec peak excretion days post-infection (pi), but no excretion days pi, in contrast to the peak excretion of f + vtec infection observed between and days pi, and excretion persisting up to days pi. interestingly, in group g , the only two pigs found to be genetically resistant to e. coli f had no diarrhea and gave only a total of eight positive fecal samples out of , whereas out of samples were positive for the six pigs which were genetically susceptible to e. coli f (p = . ); their mean log titer was . compared to a mean of . (p = . ) for the other pigs in group g . although all the pigs were genetically susceptible to e. coli f , the symptoms remained relatively moderate in groups g and g . the experimental model could be improved by using higher inocula doses, intra-gastric inoculation, withdrawing feed before inoculation, cold stress, pretreatment with antibiotics, co-inoculation of viruses, or inoculating piglets when younger (madec et al., ) , or using pigs known to be genetically susceptible to the inoculated strains. it is acknowledged that post-weaning digestive disorders are generally encountered in inadequate breeding conditions, contrary to the highstandard environment offered to our weaning piglets. using rifampicin-supplemented media, we could evaluate the recovery of the inoculated strain, and search for the possible loss of the mcr- gene from the inoculated strains. the use of colistin-supplemented media was used to try to detect the transfer of the mcr- gene from the inoculated strains to commensal ones. detection of the mcr- gene in colistin-enrichment broths was also performed but the sensitivity of this method was poor as previously observed in experiments with pigs inoculated with a commensal mcr- -positive e. coli strain (mourand et al., ) , and needs further improvements. the three inoculated strains could be recovered from fecal and post-mortem samples up to four and five weeks respectively after inoculation. for group g only, a few isolates carrying the mcr- gene were also obtained, but their other characteristics (phylogenetic group, pfge or eric-pcr profiles, and antimicrobial susceptibility) were different from those of the inoculated strain. as no mcr- -containing e. coli was present in the non-inoculated animals, the uncharacteristic isolates are very probably commensal isolates having acquired the mcr- gene or plasmid from e. coli - m. other analyses are necessary to determine the precise mechanisms of this transfer, which can occur through either conjugation or transposon mobilization. interestingly, in vitro, the mcr- gene was shown to be on a conjugative plasmid in e. coli - , but not in e. coli - or e. coli - . the transconjugant obtained in vitro from e. coli - acquired resistance to colistin, tetracycline, sulfamethoxazole and trimethoprim. different studies have shown that the in vitro acquisition of an mcr- -containing plasmid does not lead to a considerable fitness cost in e. coli as measured by growth kinetics, cytotoxicity, and virulence in a galleria mellonella model (tietgen et al., ; zhang et al., ) , but a high level of mcr- expression compromises growth rate, fitness, and the membrane's structural integrity while increasing cellular death (yang et al., ) . in our experiment, most isolates from samples collected from the e. coli - m-inoculated pigs shared the characteristics of this strain, suggesting that the commensal strains which had acquired the mcr- gene were perhaps not as competitive as e. coli - m. in the three inoculated groups, few of the isolates obtained were resistant to rifampicin, shared the phylogenetic group and pfge profile of the inoculated strain but lacked the mcr- gene. wu et al. ( ) studied the stability of various mcr- -harboring plasmids and showed that the inchi , inci , incx and incy plasmids were maintained after days of passage in a colistin-free medium, but the incfii plasmid was lost from the e. coli dh alpha transformant after day . the in vivo loss of the mcr- gene has already been detected for other strains (mourand et al., ) . interestingly, the loss of the mcr- gene was not accompanied by a loss of resistance to colistin for - m, suggesting that the previously described deletion in the pmrb protein, or other unnoticed mutations, probably played a role in colistin resistance. for e. coli - m, the isolates lacking mcr- were no longer resistant to colistin, trimethoprim, sulfamethoxazole, and ampicillin, suggesting that these resistance genes may be present on the same genetic element, but further analysis is needed to confirm this hypothesis, as the genes were present on contigs different from the mcr- contig. the loss of the colistin resistance consecutive to the absence of the mcr- gene also suggests that the previously detected phoq and phop mutations (delannoy et al., ) have probably no impact on colistin resistance). finally in group g , the isolate lacking the mcr- gene had only lost colistin resistance. globally, comparison of the clinical signs and culture results obtained in the inoculated and control groups, shows that the o -f etec challenge, resulting in more clinical signs and excretion, particularly in pigs with a f susceptible genotype, may be used to evaluate alternative solutions to combat pwd caused by colistin-susceptible or colistin-resistant e. coli in pigs. spades: a new genome assembly algorithm and its applications to single-cell sequencing detection of escherichia coli and shigella spp. in water by using the polymerase chain reaction and gene probes for uid trimmomatic: a flexible trimmer for illumina sequence data high speed blastn: an accelerated megablast search tool genome sequence assembly using trace signals and additional sequence information the clermont escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups characterization of colistin-resistant escherichia coli isolated from diseased pigs in france a sybr(r) green-based real-time pcr method for improved detection of mcr- -mediated colistin resistance in human stool samples colibacillosis colistin use and colistin resistance in bacteria from animals aligning sequence reads emergence of plasmid-mediated colistin resistance mechanism mcr- in animals and human beings in china: a microbiological and molecular biological study experimental models of porcine post-weaning colibacillosis and their relationship to post-weaning diarrhoea and digestive disorders as encountered in the field two alpha( , ) fucosyltransferase genes on porcine chromosome q are closely linked to the blood group inhibitor (s) and escherichia coli f receptor (ecf r) loci dissemination of the mcr- colistin resistance gene among pigs: an experimental study screening of pigs resistant to f enterotoxigenic escherichia coli (etec) infection susceptibility towards enterotoxigenic escherichia coli f ac diarrhea is governed by the muc gene in pigs in vivo therapeutic efficacy and pharmacokinetics of colistin sulfate in an experimental model of enterotoxigenic escherichia coli infection in weaned pigs post weaning diarrhea in pigs: risk factors and non-colistin-based control strategies the fecal presence of enterotoxin and f genes as an indicator of efficacy of treatment with colistin sulfate in pigs standardization of pulsed-field gel electrophoresis protocols for the subtyping of escherichia coli o :h , salmonella, and shigella for pulsenet enterobacterial repetitive intergenic consensus sequences and the pcr to generate fingerprints of genomic dnas from vibrio cholerae o , o , and non-o strains a bacterial analysis platform: an integrated system for analysing bacterial whole genome sequencing data for clinical diagnostics and surveillance impact of the colistin resistance gene mcr- on bacterial fitness characterization of plasmids harboring blactx-m and blacmy genes in e. coli from french broilers diverse virulence gene content of shiga toxin-producing escherichia coli from finishing swine different kinetic of antibody responses following infection of newly weaned pigs with an f enterotoxigenic escherichia coli strain or an f verotoxigenic escherichia coli strain complex dissemination of the diversified mcr- -harbouring plasmids in escherichia coli of different sequence types balancing mcr- expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms decreased fitness and virulence in st escherichia coli harboring blandm- and mcr- against a st strain with blandm- . front the authors would like to sincerely thank yann bailly (anses ploufragan) for technical assistance. the authors thank labocea for providing the three e. coli strains through resapath network. the authors indicate that there are no conflicts of interest. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord- -wzcas zd authors: lu, ti; seo, hyesuk; moxley, rodney a.; zhang, weiping title: mapping the neutralizing epitopes of f fimbrial adhesin subunit fedf of enterotoxigenic escherichia coli (etec) date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: wzcas zd k and f fimbrial enterotoxigenic escherichia coli (etec) are the major causes of post-weaning diarrhea (pwd) in pigs. a vaccine that induces broad immunity to prevent k and f fimbrial etec bacterial attachment and colonization in pig small intestines and to neutralize enterotoxin enterotoxicity would be effective for pwd. structure-based multiepitope-fusion-antigen (mefa) technology using a backbone immunogen to present neutralizing epitopes of representing virulence factors capacitates development of broadly protective etec vaccines. neutralizing epitopes have been identified from k fimbrial adhesin (faeg) and enterotoxins but not f fimbrial adhesin. in this study, we in silico identified immunodominant epitopes from f ac fimbrial subunit fedf which plays a critical role in f fimbrial adherence, genetically fused each epitope to a carrier, examined immunogenicity of each epitope fusion, and determined epitope-derived antibodies neutralizing activities against f fimbrial adherence. data showed that seven immune-dominant epitopes were identified from fedf subunit. fused to heterologous human etec adhesin subunit cfab, epitope fusions induced anti-f antibodies in subcutaneously immunized mice. moreover, antibodies derived from each fusion significantly blocked adherence of a f -fimbrial e. coli bacteria to pig intestinal cell line ipec-j . while all seven epitopes exhibited neutralizing activity, results from this study identified fedf epitopes # (ipsssgtltcqagt) and # (qpdatgswyd) the most effective for antibodies against f fimbrial adherence, and suggested their future application in pwd vaccine development. post-weaning diarrhea (pwd) is one of the most important swine diseases (fairbrother et al., ; usda, ) . piglets commonly develop diarrhea - days after they are weaned, a clinical condition called pwd. pwd is mainly caused by pathogenic bacteria and viruses including diarrheagenic escherichia coli, coronaviruses [transmissible gastroenteritis (tge) and porcine epidemic diarrhea virus (pedv)] and rotaviruses; however, diarrheagenic e. coli have a central role in the etiology of pwd (hampson, ) . pwd causes weight loss, slow growth and acute death in recently weaned pigs, resulting in economic losses to swine producers in the us and other countries (haesebrouck et al., ; nagy and fekete, ; verdonck et al., ; vu-khac et al., ) . diarrhea is also a main reason for using antibiotics on swine farms. antibiotic exposure is linked to antimicrobial resistance (amr), casting a major concern for animal and human health (docic and bilkei, ; mishra et al., ; torjesen, ) . however, a ban on the use of food animal growth promoting antibiotics in scandinavia and europe spiked pwd outbreaks (casewell et al., ) , urgently calling for alternative effective prevention strategies against pwd. vaccination would be the most economical and likely effective approach to control pwd and an effective means to reduce the use of antibiotics. though there are products on the market, truly effective pwd vaccines are urgently needed (fairbrother et al., ; melkebeek et al., ; zhang, ) . of the diarrheagenic e. coli, enterotoxigenic e. coli (etec) is the most common cause of pwd, though the stress of weaning, absence of maternally-derived enteric antibodies, and dietary change are important but indirect factors of clinical pwd (fairbrother et al., ) . etec strains causing pwd produce fimbriae and enterotoxins. fimbriae promote initial attachment to host cell receptors, enabling colonization (smith and linggood, ) ; colonized etec bacteria deliver enterotoxins to host enterocytes, causing water and electrolyte hypersecretion and diarrhea (nataro and kaper, ) . thus, fimbriae and enterotoxins are the major virulence determinants of etec, and have been targeted in intervention strategies. t etec fimbriae and enterotoxins are immunologically heterogeneous (gaastra and de graaf, ) . fimbriae of etec causing pwd include k (f ) and f , and occasionally k (f ), p (f ) and f (f ) (awad-masalmeh et al., ; casey and moon, ; frydendahl, ; moseley et al., ; nagy et al., ; zhang et al., ) . enterotoxins produced by etec are heat-labile toxin (lt), heat-stable toxin type i (sta), heat-stable toxin type ii (stb), shiga toxin e (stx e) and enteroaggregative heat-stable toxin type (east ) (frydendahl, ; lee et al., ; moon et al., ; nakazawa et al., ; osek, b; zhang et al., ) . clinical observations and epidemiological studies indicate that a typical etec strain expresses one and occasionally two types of fimbriae and one, two or more enterotoxins (francis, ; frydendahl, ; zhang et al., ) . laboratory experimental studies demonstrated that an etec strain expressing one type of fimbriae and lt, stb, or sta enterotoxin causes diarrhea in young pigs (berberov et al., ; erume et al., ; zhang et al., zhang et al., , . the optimal prevention approach would be to block attachment of different etec fimbriae to host receptors and to eliminate enterotoxicity of major enterotoxins (lt, sts) to host cells (walker, ; zhang, ; zhang and sack, ) . blocking attachment of all etec fimbriae and neutralizing against enterotoxicity of lt and sts have proven very challenging. however, a recent breakthrough in antigen preparation by using neutralizing epitopes and multiepitope-fusion-antigen (mefa) technology makes completion of such a task feasible nandre et al., ruan et al., a . additionally, molecular epidemiological studies showed that the vast majority of etec strains causing pwd express k or f fimbriae in conjunction with - toxins (frydendahl, ; zhang et al., ) . in the us, over % of pwd cases are caused by k or f fimbrial etec strains (francis, ; zhang et al., ) . thus, blocking attachment of k and f fimbriae to enterocyte receptors would be an effective means to prevent etec colonization. neutralizing epitopes from k fimbrial adhesin faeg (lu and zhang, ) and toxins including lt (huang et al., ) , sta (rausch et al., ; ruan et al., b; zhang et al., ) , and stb (rausch et al., ) were identified. however, epitopes from f adhesive subunit fedf are not mapped and neutralizing epitopes have not been identified. with neutralizing epitopes identified from all etec virulence determinants, we should be able to apply the mefa technology to develop a broadly protective vaccine against pwd. in this study, we in silico identified immunodominant epitopes from f fedf subunit, fused individual epitopes to protein carrier cfab (a structural subunit of heterologous human etec fimbria cfa/i), immunized mice with each epitope fusion protein, measured mouse anti-f antibody response, and examined epitope-derived antibodies for neutralizing activities against f fimbria adherence to determine fedf neutralizing epitopes. bacterial strains and plasmids used in this study are listed in table . recombinant cfab strain (ruan et al., ) were used as dna templates for cfab gene pcr amplification. cfab-epitope fusion genes were cloned in vector pet α (novagen, madison, wi) and expressed in e. coli bl -codonplus (de ). f fimbrial e. coli field isolate was used in antibody adherence inhibition assay. immunodominant epitopes from f fedf subunit protein was in silico identified with b-cell epitope prediction programs (larsen et al., ; saha and raghava, (table ), as we previously described huang et al., ; lu and zhang, ) . pcr generated cfab-epitope fusion genes were digested with nhei and eagi restriction enzymes (new england biolabs, ipswich, ma), and were cloned into pet a vector. recombinant cfab-epitope (cfab-fedf-ep) fusion proteins expressed by e. coli bl (de ) were extracted with bacterial protein extraction reagent (b-per; thermo fisher scientific, rochester, ny) and refolded as we previously described (huang et al., ; nandre et al., ; rausch et al., ) . refolded fusion proteins were examined in sds-page with coomassie blue staining, and were characterized in western blot or direct elisas with anti-f mouse antiserum. additionally, each cfab-epitope fusion protein was investigated for blocking f fimbriae from reacting with anti-f antiserum in competitive elisas. four μg cfab-epitope fusion protein was incubated with mouse anti-f serum dilutions ( : , to : , ) for min at room temperature. each mixture was added to elisa plate wells coated with f fimbriae ( ng per well). incubated at ⁰c for h, wells were washed with pbs- . % tween- (pbst), and incubated with horseradish peroxidase (hrp)-conjugated goat-anti-mouse igg ( : ; sigma, st. louis, mo). od was measured after exposure to , ′, , ′-tetramethylbenidine (tmb; kpl, gaithersburg, md) for min at room temperature. furthermore, cfab-epitope fusions were examined as competitive agents to prevent anti-f antibodies from inhibiting adherence of f fimbrial bacteria to pig intestine cell line ipec- . since protective anti-f antibodies inhibit adherence of f -fimbiral e. coli to host receptors, conformational f epitopes react with anti-f antibodies (derived from f fimbriae) thus abolish antibodies from adherence inhibition. nearly identical to antibody adherence inhibition assay previously described (rausch et al., ; ruan and zhang, ruan et al., a , , except the addition of cfab-epitope fusions as the competitive agent, this assay measured interference of cfab-epitope fusion protein to anti-f antibodies in inhibiting f fimbriae adherence to target host cells. ten ug cfab-epitope fusion protein together with . × cfu f -fimbrial e. coli bacteria were mixed with μl mouse anti-f antiserum and incubated at room temperature for each cfab-epitope fusion protein was used to subcutaneously immunize mice. a group of five -week old female balb/c mice was each administered with μg cfab-epitope fusion protein (in μl), adjuvanted with μg dmlt (double mutant lt, lt r g/l a ; provided by path). immunized mice received two boosters of the primary dose at the interval of two weeks. a group of mice without injection was used as the negative control. mouse serum samples collected before the primary and two weeks after the final booster were used to prepare serum samples. mouse serum samples were stored at - ⁰c until use. mouse immunization complied with animal welfare act under national research council guidance and was approved by kansas state university institutional animal care and use committee (protocol # ). serum samples from each immunized or control mouse were titrated for anti-f igg antibodies as we previously described (rausch et al., ; ruan and zhang, ; ruan et al., ) . briefly, wells of hb plates (fisher scientific) coated with heat-extracted f fimbriae ( ng per well) were incubated with mouse serum binary dilutions ( : to : , ) as primary antibodies and then hrp-conjugated goat-anti-mouse igg ( : ) as secondary antibodies. od measured after exposure to tmb were converted to antibody titers, in a log scale. mouse serum samples from the groups immunized with each cfabepitope fusion were examined for antibody adherence inhibition using f -fimbrial e. coli bacteria and ipec-j cells. briefly, × (cfus) bacteria exposed to μl serum pooled from each immunization group or the control group were added to ipec-j cell ( . × per well) and cultured for h in a co incubator. after washes with pbs to remove non-adherent bacteria, cells were dislodged, lysed, then diluted, and plated on agar plates for bacteria counting (cfus) after overnight growth at ⁰c. neutralizing epitopes were identified if mouse serum samples derived from the fusion protein showed significant inhibition against adherence from f -fimbrial e. coli bacteria, compared to the control mouse serum samples. data were analyzed statistically using graphpad prism software version . (la jolla, ca). anova (two-way) was used to compare data of competitive elisa of screening each cfab-epitope fusion protein competing against f fimbriae for reactivity with anti-f antiserum, whereas anova (one-way) was performed to analyze cfab-epitope fusions in competitive bacteria adherence inhibition assay, mouse serum anti-f igg antibody titration and antibody adherence inhibition assay data. the mean ± standard deviation (sd) was used to express the results; a p value of < . indicated differences significant. a total of seven immunodominant epitopes were in silico identified, at the length ranged from to amino acids (table ) . all seven epitopes are discontinuous, surface-exposed, and located on ẞ sheets or α-helix extension (fig. ) . seven cfab-epitope fusions were constructed, designed as cfab-fedf-ep , cfab-fedf-ep , cfab-fedf-ep , cfab-fedf-ep , cfab-fedf- t. lu et al. veterinary microbiology ( ) - ep , cfab-fedf-ep , and cfab-fedf-ep (fig. s ). epitope fusion protein were extracted and refolded ( fig. a) , and recognized by anti-f mouse antiserum (fig. b) . additionally, when coated on elisa plates, each of these seven fusion proteins reacted with anti-f antiserum (fig. c) . additionally, competitive elisas using f fimbriae as the coating antigen showed that cfab-epitope fusion proteins competed with coated f fimbriae for binding to anti-f antiserum (fig. a) . od readings were significantly lower in wells with the addition of individual cfabepitope fusion proteins or f fimbria (p < . ; as the positive control), confirming fedf epitopes presented in the fusion proteins retained native antigenic conformation. more importantly, cfab-epitope fusion proteins bound to anti-f antiserum and reduced anti-f antibodies from inhibiting the adherence f -fimbrial e. coli bacteria to pig cell line ipec-j (fig. b ). the addition of cfab-fedf-ep or cfab-fedf-ep fusion significantly reduced anti-f antiserum neutralizing activity for blocking the adherence of bacteria to ipec-j cells (p < . ) when compared to other epitope fusions, shown more bacteria adhered to ipec-j cells (fig. b) . mice immunized with cfab-epitope fusion proteins had anti-f igg antibody titers detected in serum samples at . ± . (cfab-fedf-ep ), . ± . (cfab-fedf-ep ), . ± . (cfab-fedf-ep ), . ± . (cfab-fedf-ep ), . ± . (cfab-fedf-ep ), . ± . (cfab-fedf-ep ), and . ± . (cfab-fedf-ep ) (fig. a) . no anti-f antibodies detected in the serum of the control mice or from the serum collected prior to the primary immunization. anti-f igg titers in the group immunized with cfab-fedf-ep were significantly lower than the titers of the groups immunized with the other cfab-epitope fusions (p < . ). serum samples of the mice immunized with cfab-epitope fusion proteins exhibited adherence inhibition activities against f -fimbrial e. coli (fig. b) . treated with the serum of mice immunized with cfab-fedf-ep ( ± . , %) or cfab-fedf-ep ( . ± , %), had the fewest bacteria adherent to ipec-j cells. one-way anova test indicated that serum antibodies of the mice immunized with cfab-fedf-ep or cfab-fedf-ep were most effective in inhibiting adherence. vaccination with live and subunit vaccines has been explored in the past decades, and remains the most promising for pwd prevention (fairbrother et al., ; melkebeek et al., ) . since etec strains expressing k or f fimbriae cause nearly all pwd cases, f and k fimbriae have been the main targets in vaccine development. while oral administration of purified k fimbriae induced k -specific antibodies and protected pigs against a homologous challenge ( van den broeck et al., a, b) , administration of purified f fimbriae did not induce protective immunity against f etec challenge in pigs . similarly, live vaccine candidates derived from avirulent e. coli field isolates expressing k fimbriae induced k specific antibodies and protected against colonization by a k etec challenge strain (bianchi et al., ; francis and willgohs, ; fuentes et al., santiago-mateo et al., . however, live strains expressing f were not effective in the induction of an f -specific fig. . competitive elisa and bacteria adherence inhibition assay to show cfab-epitope fusion proteins blocking binding of anti-f antiserum with f fimbriae or f -fimbrial e. coli bacteria . a: competitive elisas with f fimbriae as the coating antigen, and each cfab-epitope fusion protein as the competing agent. anti-f antiserum dilutions from : , to : , were used. b: antibody adherence inhibition assay using anti-f antiserum as the antibodies to inhibit f fimbiral e. coli strain binding to f- receptor positive pig intestine cell line ipec-j , and each cfab-epitope fusion protein as the agent to compete for anti-f antiserum thus to prevent anti-f antiserum from blocking the binding between bacteria to opec-j cells. pbs, no competing agent and no anti-f antiserum; control, control mouse serum only; anti-f , no competing agent but with anti-f antiserum. fig. . mouse serum anti-f antibody titration and antibody adherence inhibition assay. a: anti-f igg titers (in log ) from serum samples of the mice immunized with each cfab-epitope fusion protein; no anti-f igg titer detected from the negative control mice. b: mouse serum antibody inhibition assays to show epitopederived antibodies against adherence (in %) of f- fimbrial e. coli bacteria to ipec-j cells. pbs, no mouse serum; control, negative control mouse serum; anti-f , serum samples of mice immunized with f fimbriae. t. lu et al. veterinary microbiology ( ) - immune response, nor protective against challenge with an f etec strain (bertschinger et al., ; coddens et al., ) . f fimbriae, previously known as f , p and , consist of two antigenic variants: f ac and f ab nagy et al., ) , and are associated with pwd and edema disease in young pigs respectively (amezcua et al., ; frydendahl, ; osek, a; post et al., ) . the major structural subunit of f is feda, but the adhesive minor subunit fedf plays a central role in binding to host receptors (imberechts et al., ; smeds et al., smeds et al., , , and is highly conserved among f strains. a lt-k -f tripartite antigen that includes fedf peptide ( th - th aa), as a recombinant protein administered intramuscularly (ruan et al., ) or a holotoxin-structured protein expressed by a live e. coli strain when given orally (ruan and zhang, ) , induced antibodies blocking adherence of k -and f -fimbrial etec bacteria and protecting pigs against k -fimbrial etec challenge. this tripartite pwd vaccine candidate was not examined for efficacy against f -fimbrial etec challenge due to difficulty in identifying f susceptible pigs, and also needs to carry toxin antigens to induce protective antitoxin antibodies for effective protection against pwd. incorporating antigenic elements of all virulence determinants into a pwd vaccine product for effective protection against heterogeneous etec strains is challenging. structural based mefa platform allows a backbone immunogen to present multiple neutralizing epitopes for broad immunity. with neutralizing epitopes from k fimbrial adhesin subunit faeg, toxins including lt, sta and stb are already identified, we need to identify neutralizing epitopes from f fimbrial adhesin subunit fedf in order to have a pwd vaccine candidate for immunity against all etec virulence determinants. results from the current study indicated that epitope # (ipssgtltcqagt) and epitope # (qpda-tgswyd) are the top candidates of f adhesin subunit fedf neutralizing epitopes. imbedding these two fedf epitopes into a mefa that uses a lt toxoid as backbone and presents neutralizing epitopes of f , lt, sta and stb, we are a step closer for a broadly protective pwd vaccine, though future challenge studies are needed to confirm whether antibodies derived from these fedf epitopes are protective against f fimbrial attachment and colonization. in conclusion, seven epitopes identified from f fimbrial adhesin subunit fedf retained native antigenicity after being fused to heterologous carrier cfab protein, indicated by each epitope fusion protein recognized by anti-f antiserum but also ability to compete with f fimbria for binding to anti-f antibodies or to reduce anti- antibodies from inhibiting adherence of e. coli bacteria expressing f fimbriae. moreover, each cfab-epitope fusion protein induced antibodies specific to f fimbriae in subcutaneously immunized mice. more importantly, derived antibodies showed neutralizing activities against f fimbria adherence to pig intestine cell line ipec-j . among seven fedf epitopes that induce neutralizing anti-f antibodies, epitope and epitope displayed better in inducing neutralizing anti-f antibodies, suggesting their potential application in vaccine development against pwd. authors declare no conflict of interests from this study. drs. zhang and moxley are developing a broadly protective vaccine against pwd. presentation of postweaning escherichia coli diarrhea in southern ontario, prevalence of hemolytic e. coli serogroups involved, and their antimicrobial resistance patterns pilus production, hemagglutination, and adhesion by porcine strains of enterotoxigenic escherichia coli lacking k , k , and p antigens exploring the extremes of sequence/structure space with ensemble fold recognition in the program phyre relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic escherichia coli that produces multiple enterotoxins active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic escherichia coli with fimbriae f parenteral vaccination of mice and piglets with f + escherichia coli suppresses the enteric anti-f response upon oral infection the european ban on growth-promoting antibiotics and emerging consequences for human and animal health genetic characterization and virulence of enterotoxigenic escherichia coli mutants which have lost virulence genes in vivo the age-dependent expression of the f + e. coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens differences in antibiotic resistance in escherichia coli, isolated from east-european swine herds with or without prophylactic use of antibiotics mefa (multiepitope fusion antigen)-novel technology for structural vaccinology, proof from computational and empirical immunogenicity characterization of an enterotoxigenic escherichia coli (etec) adhesin mefa genetic fusion protein xsta-ovalbumin is an effective coating antigen in elisa to titrate anti-sta antibodies comparison of the contributions of heat-labile enterotoxin and heatstable enterotoxin b to the virulence of enterotoxigenic escherichia coli in f ac receptor-positive young pigs escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies enterotoxigenic escherichia coli infection in pigs and its diagnosis evaluation of a live avirulent escherichia coli vaccine for k +, lt+ enterotoxigenic colibacillosis in weaned pigs prevalence of serogroups and virulence genes in escherichia coli associated with postweaning diarrhoea and edema disease in pigs and a comparison of diagnostic approaches inoculation of nonpathogneic escherichia coli to control disease and reduce antibiotic usage host-specific fimbrial adhesins of noninvasive enterotoxigenic escherichia coli strains efficacy of vaccines against bacterial diseases in swine: what can we expect? postweaning escherichia coli diarrhoea in pigs significance of enterotoxigenic escherichia coli (etec) heatlabile toxin (lt) enzymatic subunit epitopes in lt enterotoxicity and immunogenicity characterization of f fimbrial genes fede and fedf involved in adhesion and length of enterotoxemic escherichia coli strain / fimbrial colonisation factors f ab and f ac of escherichia coli isolated from pigs with postweaning diarrhea and edema disease protein structure prediction on the web: a case study using the phyre server improved method for predicting linear b-cell epitopes characterization of the gene encoding heat-stable toxin ii and preliminary molecular epidemiological studies of enterotoxigenic escherichia coli heat-stable toxin ii producers identifying immuno-dominant and neutralizing epitopes from k fimbriae of enterotoxigenic escherichia coli (etec) etec vaccination in pigs vaccines and antibiotic resistance prevalence of pilus antigens, enterotoxin types, and enteropathogenicity among k -negative enterotoxigenic escherichia coli from neonatal pigs cloning of chromosomal dna encoding the f adhesin of enterotoxigenic escherichia coli and genetic homology between adhesins f and k enterotoxigenic escherichia coli (etec) in farm animals colonization of porcine intestine by enterotoxigenic escherichia coli: selection of piliated forms in vivo, adhesion of piliated forms to epithelial cells in vitro, and incidence of a pilus antigen among porcine enteropathogenic e. coli biological relationship between f ab and f ac fimbriae of enterotoxigenic and verotoxigenic escherichia coli from weaned pigs with oedema disease or diarrhoea virulence factors in escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in japan antibodies derived from an enterotoxigenic escherichia coli (etec) adhesin tip mefa (multiepitope fusion antigen) against adherence of nine etec adhesins: cfa/i, cs , cs , cs , cs , cs , cs , cs and etpa diarrheagenic escherichia coli prevalence of shiga toxin genes among escherichia coli strains isolated from pigs prevalence of virulence factors of escherichia coli strains isolated from diarrheic and healthy piglets after weaning frenquency of virulence factors in escherichia coli isolated from pigs with postweaning diarrhea and edema disease in north carolina. swine health prod antibodies derived from a toxoid mefa (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (lt), heat-stable toxins (sta, stb), and shiga toxin e (stx e) of porcine enterotoxigenic escherichia coli (etec) a tripartite fusion, faeg-fedf-lt( )a :b, of enterotoxigenic escherichia coli (etec) elicits antibodies that neutralize cholera toxin, inhibit adherence of k (f ) and f fimbriae, and protect pigs against k ac/heat-labile toxin infection multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of escherichia coli strains expressing colonization factor antigen i (cfa/i) characterization of heat-stable (sta) toxoids of enterotoxigenic escherichia coli fused to a double mutant heat-labile toxin (dmlt) peptide in inducing neutralizing anti-sta antibodies genetic fusions of a cfa/i/ii/iv mefa (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (sta) and heat-labile toxin (lt) of enterotoxigenic escherichia coli (etec) retain broad anti-cfa and antitoxin antigenicity oral immunization of a live attenuated escherichia coli strain expressing a holotoxin-structured adhesin-toxoid fusion ( faeg-fedf-lta : ltb) protected young pigs against enterotoxigenic e. coli (etec) infection prediction methods for b-cell epitopes avirulent k (f ) + escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic escherichia coli strain that expresses the same adhesin and enterotoxins characterization of the adhesin of escherichia coli f fimbriae mapping the binding domain of the f fimbrial adhesin observations on the pathogenic properties of the k , hly and ent plasmids of escherichia coli with particular reference to porcine diarrhoea current and future vaccines could help reduce reliance on antibiotics, amr says health monitoring syst. part ii: reference for swine health and health management in the united states receptor-dependent immune responses in pigs after oral immunization with f fimbriae different kinetic of antibody responses following infection of newly weaned pigs with an f enterotoxigenic escherichia coli strain or an f verotoxigenic escherichia coli strain mucosal immunization of piglets with purified f fimbriae does not protect against f + escherichia coli infection serotypes, virulence genes, intimin types and pfge profiles of escherichia coli isolated from piglets with diarrhoea in slovakia considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries progress and challenges in vaccine development against enterotoxigenic escherichia coli (etec) -associated porcine post-weaning diarrhea (pwd) progress and hurdles in the development of vaccines against enterotoxigenic escherichia coli in humans significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies prevalence of virulence genes in escherichia coli strains recently isolated from young pigs with diarrhea in the us escherichia coli constructs expressing human or porcine enterotoxins induce identical diarrheal diseases in a piglet infection model genetic fusions of heat-labile (lt) and heat-stable (st) toxoids of porcine enterotoxigenic escherichia coli elicit neutralizing anti-lt and anti-sta antibodies we thank dr. b schultz (kansas state university) for providing pig cell line ipec-j . this work was funded by usda-nifa agriculture and food research initiative competitive grant no. - - . supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -lnap kp authors: jung, kwonil; eyerly, bryan; annamalai, thavamathi; lu, zhongyan; saif, linda j. title: structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: lnap kp integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. it is maintained mainly by tight junctions (tjs) and adherens junctions (ajs). we conducted immunofluorescence (if) staining for in situ identification of zonula occludin (zo)- proteins for tj and e-cadherin proteins for aj in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (pedv). twenty -day-old piglets [pedv-infected (n = ) and mock (n = )] from pedv seronegative sows, were orally inoculated [ . log( ) genomic equivalents/pig] with pedv pc a strain or mock. at post-inoculation days (pids) – , infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. by immunohistochemistry, pedv antigens were evident in enterocytes lining the villous epithelium. at pids – , pedv-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of zo- or e-cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. the structural destruction and disorganization of tj and aj were extensive in pedv-infected pigs at pids – , but then appeared to reversibly recover at pid , as evident by increased numbers of zo- -positive epithelial cells and markedly improved appearance of e-cadherin-positive villous epithelium. our results suggest a possible involvement of structurally impaired tj and aj in the pathogenesis of pedv, potentially leading to secondary bacterial infections. integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. it is maintained mainly by tight junctions (tjs) and adherens junctions (ajs). we conducted immunofluorescence (if) staining for in situ identification of zonula occludin (zo)- proteins for tj and e-cadherin proteins for aj in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (pedv). twenty -day-old piglets and mock (n = )] from pedv seronegative sows, were orally inoculated [ . log genomic equivalents/pig] with pedv pc a strain or mock. at post-inoculation days (pids) - , infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. by immunohistochemistry, pedv antigens were evident in enterocytes lining the villous epithelium. at pids - , pedv-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of zo- or e-cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. the structural destruction and disorganization of tj and aj were extensive in pedv-infected pigs at pids - , but then appeared to reversibly recover at pid , as evident by increased numbers of zo- -positive epithelial cells and markedly improved appearance of e-cadherin-positive villous epithelium. our results suggest a possible involvement of structurally impaired tj and aj in the pathogenesis of pedv, potentially leading to secondary bacterial infections. ß elsevier b.v. all rights reserved. the intestinal epithelium provides a robust barrier that regulates absorption of nutrients and water and also restricts uptake of luminal bacteria (su et al., ) . the integrity of the intestinal epithelium is maintained by tight junctions (tjs), adherens junctions (ajs), desmosomes, and gap junctions. the tj is the major paracellular barrier and functions to separate apical and basolateral compartments or membranes. the aj forms a continuous belt between cells and is crucial for the maintenance of intercellular adhesion (hartsock and nelson, ; su et al., ) . the tj and aj are regulated by transmembrane tj (occludin and claudin) and aj (e-cadherin and catenins) proteins on the opposing cells' plasma membranes and interactions mediated by actin binding proteins such as the zonula occludin (zo) family, which link the aj complex to actin microfilaments (hartsock and nelson, ; su et al., ) . porcine epidemic diarrhea virus (pedv), which belongs to the genus alphacoronavirus in the family coronaviridae, causes high mortality of suckling pigs and substantial economic losses (saif et al., ) . epidemic pedv strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines, but the jejunum and ileum are the primary sites of infection (jung and saif, ; jung et al., ) . during the early stages of pedv infection, necrosis and exfoliation of infected villous epithelial cells is pronounced, resulting in acute, severe villous atrophy. to what extent the integrity and function of the pedv-infected villous epithelium is restored by intestinal stem cells located at crypt cell layers is currently unclear. the aim of the present study was to determine whether pedv infection causes structurally altered tj and aj in the villous and crypt epithelium of the small and large intestine by immunofluorescence (if) staining for in situ identification of the related proteins, zo- and e-cadherin for tj and aj, respectively. the wild-type us pedv strain pc a was obtained from the intestinal contents of a diarrheic -day-old piglet on an ohio farm in june (jung et al., ) . the original sample was serially passaged times in gn pigs. the original sample and gn pig-passaged pc a strain were confirmed to contain only pedv, as reported in our previous study (jung et al., ) . the titer of gn pig second-passaged pc a strain was . log ge/ml and was used as virus inoculum after dilution in minimal essential medium (mem). two seronegative, large white  duroc crossbred, pregnant sows to acquire nursing piglets, were obtained from a pedv-free spf (confirmed by history and seronegative sows; lack of qrt-pcr positive-fecal samples) swine herd of the ohio state university. the spf herd was seronegative for antibodies to prrsv, prcv, tgev and porcine circovirus type . twenty -day-old nursing piglets were randomly assigned to one of two groups: pedv inoculated (n = ) and mock (n = ). each experimental group of pigs was housed in a separate room in a high-security isolation facility (biosafety level ). nursing pigs were inoculated orally [ . log ge (% . log plaque forming units)/pig] (jung et al., ) with ml of pc a or mock inoculated with mem. inoculated and negative control pigs (n = - /group at each time-point) were euthanized for pathological examination at an acute-stage (pid ), at a mid-stage (pid ), and at a later-stage (pid ) of infection. after pedv inoculation, the pigs were monitored for clinical signs - times daily until necropsy. the institutional animal care and use committee (iacuc) of the ohio state university approved all protocols related to the animal experiments and care in this study. small (duodenum, proximal, middle and distal jejunum, and ileum) and large (cecum/colon) intestinal tissues and other major organs (lung, liver, heart, kidney, spleen, and lymph node) were examined grossly and histologically at pids , , and . tissues were placed in % phosphate buffered formaldehyde (ph . ), dehydrated in graded alcohol, embedded in paraffin, and cut in -mm sections onto microscope slides, fixed and stained with hematoxylin and eosin (h&e) then analyzed for histopathological changes. villous height and crypt depth were estimated by measuring at least villi and crypts throughout the section. mean ratios of jejunal villous height to crypt depth (vh:cd) were calculated as previously described (jung et al., ) . the formalin-fixed, paraffin-embedded tissues were prepared and tested by immunohistochemistry (ihc) for the detection of pedv antigens, using monoclonal antibody c - against the spike protein of pedv strain dr (provided by dr. daesub song, korea research institute of bioscience and biotechnology, daejeon, korea). the antibody was diluted : in pbst [phosphate-buffered saline (pbs) containing tween , . %]. ihc was conducted, as described previously (jung et al., ). the frozen jejunal/ileal and cecal/colonic tissues were prepared in tissue-tek oct compound (sakura, torrance, ca, usa) and tested by if staining for the detection of zo- and e-cadherin using monoclonal antibodies against human recombinant zo- and human e-cadherin (invitrogen, ca, usa). the anti-zo- antibody was diluted : in pbst and incubated on the tissues at c overnight. the anti-e-cadherin antibody was diluted : in pbst and incubated on the tissues at c overnight, and an anti-mouse antibody conjugated with alexa fluor (invitrogen) was used as the detection antibody and incubated on the tissues at c for h. the stained tissues were examined by fluorescence microscopy. zo- or e-cadherin-positive scores were computed by estimating the number of if-positive cells in the intestinal section per microscopic area, at  magnification based on the following criteria: , no positive cells; , - % of zo -or e-cadherin-positive villous or crypt epithelium showed staining; , - % of zo -or e-cadherin-positive villous or crypt epithelium showed staining; and , - % of zo -or e-cadherin-positive villous or crypt epithelium showed staining. because the entire intestinal villous and crypt epithelium of uninfected and pedv-infected pigs (e-cadherin-positive scores, all ) were positive for e-cadherin, and e-cadherin-stained villous epithelium (but not crypt epithelium) showed evident disorganization, e-cadherinstained tissues were additionally scored by the following system: , no positive cells; , - % of e-cadherin-positive villous epithelium showed disorganization; , - % of e-cadherin-positive villous epithelium showed disorganization; and , - % of e-cadherin-positive villous epithelium showed disorganization. disorganization was characterized by irregular distribution and mildly to moderately reduced expression of e-cadherin in villous epithelial cells of the small intestine. all values are expressed as the means ae standard deviation of the means (sdm). zo- -or e-cadherin-positive scores and disorganization scores of e-cadherin-positive villous epithelium between pedv-infected and uninfected nursing pigs, or between different time-points in the same group, were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). a value of p < . was considered statistically significant. clinical signs were first detected at pid in pedvinoculated nursing piglets. all inoculated nursing piglets at pids - exhibited acute, severe watery diarrhea and/or vomiting, followed by lethargy and dehydration. no negative control nursing pigs showed diarrhea or other clinical signs throughout the experiment. by macroscopic examination, all inoculated nursing pigs tested at pids - exhibited extensively thin and transparent intestinal walls and accumulation of large amounts of yellowish fluid in the small and large intestinal lumen. the other internal organs appeared normal. in inoculated nursing pigs, histologic lesions were limited to the small intestine, mainly jejunum and ileum, and included acute diffuse, severe atrophic enteritis. no histologic lesions were evident in the large intestine or other organs of the inoculated nursing pigs and negative controls. mean (aesdm) jejunal vh:cd ratios of uninoculated nursing piglets were . (ae . ) at pid , . (ae . ) at pid , and . (ae . ) at pid . on the other hand, mean (aesdm) jejunal vh:cd ratios of inoculated nursing piglets were . (ae . ) at pid , . (ae . ) at pid , and . (ae . ) at pid . all inoculated pigs exhibited pedv antigen-positive cells in the small and large intestine tested at pids - . under our ihc conditions, mean (aesdm) pedv antigen-positive cells per jejunal villus of inoculated nursing pigs were . (ae . ) at pid , . (ae . ) at pid , and . (ae . ) at pid . more detailed clinical disease, pathology, fecal shedding, and viremia (viral rna in serum) results are in a manuscript in preparation by jung et al., (unpublished data) . no other internal organs of the inoculated pigs showed ihc-positive staining. no pedv antigen-positive cells were detected in the negative control pigs. in frozen jejunal (or ileal) and colonic (or cecal) tissues of the uninfected nursing pigs at pids - , the major amount of zo- protein were regularly and discontinuously expressed on the apical surface of villous and crypt epithelial cells by if staining (fig. a-c) . transversely sectioned cells frequently exhibited rectangular, pentagonal, or hexagonal if staining for zo- (fig. a-f ). in the jejunum and colon of all uninfected nursing pigs tested at pids - , the entire villous (zo- -positive scores, all ) or crypt (zo- -positive scores, all ) epithelium was mostly positive for zo- protein ( fig. a -c, g and h). no differences in staining results and patterns were evident between jejunal and ileal tissues, or between cecal and colonic tissues. on the other hand, pedv-infected pigs at pids - had moderately to extensively disorganized, irregular distribution and reduced expression of zo- in villous epithelial cells of the small intestine, but not crypt epithelial cells ( fig. d and e) , compared to the negative controls ( fig. a and b). regardless of the infection stage, most of the crypt epithelium had mildly reduced expression of zo- protein or numbers of zo- -positive cells (mean zo- positive scores: . at pid , . at pid , and . at pid ) (fig. d , e, and h), compared to the negative controls. pedv-inoculated nursing pigs exhibited small numbers of zo- -positive cells at pids and (mean zo- -positive scores, all . ) ( fig. d and g) and moderate numbers of zo- -positive cells at pid (mean zo- -positive score, . ) ( fig. e and g) in the villous epithelium of the small intestine. mean zo- -positive scores at pid were significantly higher than those at pid or pid . at pids - , mean zo- -positive scores in the jejunal villous epithelium of infected nursing pigs were significantly lower than those in the uninfected pigs (p < . at pids and ; and p < . at pid ). however, there were no significant differences in mean zo- -positive scores in the colonic villous and crypt epithelium between uninfected and pedv-infected nursing pigs at pids - ( fig. c and f) . apart from zo- that was detected mainly on the apical surface of intestinal epithelial cells under the conditions tested, the large amount of e-cadherin protein were strongly expressed on the apical and basolateral surface and also mildly in the cytoplasm of the negative control pigs ( fig. a and c) . similar to zo- staining, the transversely sectioned cells frequently exhibited rectangular, pentagonal, or hexagonal if staining for e-cadherin ( fig. a-f) . in the jejunum (or ileum) and colon (or cecum) of all uninfected and pedv-infected nursing pigs tested at pids - , the entire villous (e-cadherin-positive scores, all ) and crypt (e-cadherin-positive scores, all ) epithelium was positive for e-cadherin (fig. a-f) . as a result, there were no differences in mean e-cadherin-positive scores between uninfected and pedv-infected pigs. also, no differences in staining results and patterns were detected between jejunal and ileal tissues, or between cecal and colonic tissues. pedv-infected pigs at pids - showed disorganized, irregular distribution and mildly to moderately reduced expression of e-cadherin in only villous epithelial cells of the small intestine ( fig. d and e) , compared to the negative controls ( fig. a and b ). e-cadherin-stained tissues were additionally scored based on the extent of disorganization of e-cadherin-positive villous epithelium. mean disorganization scores (aesdm) of e-cadherin-positive villous epithelium of the infected pigs were . (ae . ) at pid and . (ae . ) at pid , which were significantly higher than those (mean disorganization scores, all ) in the uninfected pigs at the same time-point (p < . at each note that transversely sectioned cells show rectangular, pentagonal, or hexagonal if staining for zo- (arrowheads) (b, e, and f). original magnification, all  . mean zo- -positive scores in the jejunal villous (g) or crypt epithelium (h) of pedv-infected nursing piglets compared to uninfected pigs. zo- positive scores were computed by estimating the number of if-positive cells in the intestinal section per microscopic area, at  magnification based on the following criteria: , no positive cells; , - % of zo -positive villous or crypt epithelium showed staining; , - % of zo -positive villous or crypt epithelium showed staining; and , - % of zo -positive villous or crypt epithelium showed staining. each bar represents the mean ae sdm. **p < . ; *p < . (statistically significant differences between the pedv-infected and uninfected nursing pigs by student's t-test). time-point) (fig. g) . at pid , however, there were no significant differences in mean disorganization scores of e-cadherin-positive villous epithelium between uninfected and pedv-infected nursing pigs (fig. g) . based on our data, pedv infection resulted in structurally altered tj and aj in the villous (not crypt) epithelium of the primary sites of infection, jejunum and ileum, but not the large intestine (cecum and colon). similar to pedv infection of intestinal epithelium, tj and aj disruption in pulmonary epithelium, which might contribute to the desquamation of the alveolar wall, was observed in lung biopsies from the betacoronavirus sars coronavirus (sars-cov)-infected macaques and patients (nicholls et al., ) . a previous study suggested that alteration of tj and aj may create a breach in the epithelial barrier allowing sars-cov to reach the basal matrix and eventually the systemic circulation, i.e. viremia (teoh et al., ) . by similar mechanisms, after pedv infection, viremia might be caused during acute infection stages, as reported previously (jung et al., ) . in this study, by qrt-pcr, all inoculated nursing pigs tested at pids - also had low to moderate viral rna titers in serum, ranging from . to . log ge/ml. another study showed that impaired tj in intestinal epithelium of patients with human immunodeficiency virus likely contributes to intestinal barrier dysfunction, resulting in note that transversely sectioned cells show rectangular, pentagonal, or hexagonal if staining for e-cadherin (arrowheads) (a, b, e, and f). original magnification, all  . (g) mean disorganization scores of e-cadherin-positive jejunal villous epithelium of pedv-infected nursing piglets compared to uninfected pigs. e-cadherin-stained tissues were scored by the following system: , no positive cells; , - % of e-cadherin-positive villous epithelium showed disorganization; , - % of e-cadherin-positive villous epithelium showed disorganization; and , - % of e-cadherin-positive villous epithelium showed disorganization. each bar represents the mean ae sdm. **p < . (statistically significant differences between the pedv-infected and uninfected nursing pigs by student's t-test). increased permeability and microbial translocation that cause severe, chronic diarrhea (chung et al., ) . the impaired tj and aj caused by pedv might be associated with high co-infection rates of infected nursing piglets with other enteropathogens, such as escherichia coli (turgeon et al., ; wang et al., ). an in vitro study using the porcine in testinal epithelial cell line, ipec-j , reported a significantly decreased expression (by western blotting) of tj and aj proteins at - mins after inoculation with another alphacoronavirus, transmissible gastroenteritis virus, or along with pedv, but not pedv alone (zhao et al., ) . whether pedv can alter tj or aj in vivo in the intestinal epithelium of pedv-infected pigs was uncertain. our study clearly demonstrated that at pids - , pedv-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of zo- or e-cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. structural alterations of tj and aj were extensive in the small intestine of pedv-infected pigs at pids - , but then appeared to recover at pid , as evident by increased numbers of zo- -positive epithelial cells and markedly improved appearance of e-cadherin-positive villous epithelium of the infected pigs. structural destructions and disorganizations of tj and aj in the intestinal villous epithelium with pedv infection might be reversibly recovered during the late stage of infection. our present study revealed evidence of a possible involvement of structurally impaired tj and aj and reduced expression of the related proteins (zo- and e-cadherin) in the pathogenesis of pedv. the pedv-infected intestine may have an impaired gut integrity, possibly leading to uptake of luminal bacteria and loss of water into the intestinal lumen with high osmotic pressure caused by pedv infection; however, further studies are needed to delineate the functional mechanisms involved. neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. progressive proximal-to-distal reduction in expression of the tight junction complex in colonic epithelium of virally-suppressed hiv+ individuals adherens and tight junctions: structure, function and connections to the actin cytoskeleton the effects of transplacental porcine circovirus type infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets 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 co-infections porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs sars: clinical virology and pathogenesis coronaviruses duodenal helminth infection alters barrier function of the colonic epithelium via adaptive immune activation the sars coronavirus e protein interacts with pals and alters tight junction formation and epithelial morphogenesis coronavirus-like particles associated with diarrhea in baby pigs in quebec porcine epidemic diarrhea virus variants with high pathogenicity transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with animal care; and xiaohong wang and john blakenship for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported by a grant from the oardc seeds, grant # oaoh (jung k, pi). key: cord- -ma oz o authors: ma, tianxin; xu, liwen; ren, mengting; shen, jie; han, zongxi; sun, junfeng; zhao, yan; liu, shengwang title: novel genotype of infectious bronchitis virus isolated in china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ma oz o recombination events are known to contribute to the emergence of novel infectious bronchitis virus (ibv) genotypes. in this study, we carried out detailed phylogenetic analysis and sequence comparisons based on complete nucleotide sequences of the ibv s gene, including strain i / and representative sequences from each genotype and lineage. the results showed that strain i / represented a novel genotype, designated as lineage within genotype vii (gvii- ). further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage within genotype i (gi- )-like virus with an as-yet-unidentified sequence, likely derived from another ibv strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. to the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/ch/lgx/ . these results emphasize the importance of limiting exposure to novel ibvs that may serve as a source of genetic material for emerging viruses, as well as the importance of ibv surveillance in chicken flocks. avian infectious bronchitis, caused by infectious bronchitis virus (ibv), was first described in north dakota, usa in the s (schalk and hawn, ) . it is a highly contagious viral respiratory disease that is considered as one of the most important causes of heavy economic losses to the poultry industry worldwide (cavanagh and gelb, ) . all ibv strains replicate primarily in the respiratory tract and cause respiratory diseases in birds. however, some virus strains are also able to replicate in many other epithelial surfaces, including kidneys and oviducts, and infection with these strains may thus result in nephritis, decreased egg production and quality, and significant mortality. numerous different ibv strains have been reported to date, with pathologies ranging from mild respiratory symptoms to severe kidney and oviduct diseases (cavanagh, ) . ibv is an avian coronavirus with a single-stranded, positive-sense, rna genome of approximately kb. the ′ end of the genome encodes four structural proteins, including spike glycoprotein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, and four non-structural accessory proteins, a, b, a, and b. the ′ end of the genome encodes two polyproteins ( a and ab) that contain proteins necessary for rna replication (boursnell et al., ) . similar to other coronaviruses, genetic diversity in ibv is created by both recombination events (kottier et al., ) and by mutations, including substitutions, deletions, and insertions (shi et al., ) , that occur during replication of the viral genome. the high mutation rates are attributed to the limit of replication fidelity although it has been shown that coronaviruses possess proofreading enzyme ( ′-to- ′ exoribonuclease) which is essential for replication fidelity (denison et al., ) , while recombination events are thought to result from a unique templateswitching copy-choice mechanism during rna replication (simon-loriere and holmes, ) . the spike protein comprises about amino acids and undergoes post-translational cleavage to form s and s subunits (cavanagh, ) . the s subunit contains virus-neutralizing epitopes and carries serotype-specific determinants. furthermore, the s gene has demonstrated the highest variability in the whole viral genome (niesters et al., ) , with mutations and recombination events in the s gene considered as critically important for the emergence of new virus genotypes, serotypes, and variants. many different ibv types have been https://doi.org/ . /j.vetmic. . . received october ; received in revised form january ; accepted january found worldwide, and new variants continue to emerge in different parts of the world. a new classification based on analysis of the whole s gene has recently been proposed (valastro et al., ) , including grouping and naming lineages, comprising six genotypes (gi- -gi- , gii-gvi), and a number of inter-lineage recombinants. some of these lineages are distributed in several continents, countries, or regions, such as gi- (formerly massachusetts; mass), / , or cr ) , , gi- (ck/ch/ldl/ i (ldl/ i) or q ), gi- (italy ), and gi- (var ) (de wit et al., ; valastro et al., ) , while others are geographically confined to specific regions. among the widely distributed ibv lineages, gi- , gi- , gi- , and gi- are currently circulating in china (de wit et al., ; valastro et al., ) , while strains assigned to some of these lineages, such as gi- and gi- , were also found to originate in china. in addition, ibv variants of indigenous lineages were also found to be circulating in chicken flocks in china. new lineages, gi- , gi- , and gi- (chen et al., ; jiang et al., ) , were recently found in chickens suffering from respiratory and urinary health problems. these results indicated that novel ibv lineages have been emerging continuously in china in recent years. an epidemiological study conducted in china from january to december based on the s gene sequence isolated and characterized a new variant, designated i / , in guangxi province in (xu et al., ) . despite the fact that most heterogeneity of ibvs occurs in the s gene, sequence analysis of this gene alone was not sufficient to characterize this novel strain. we therefore carried out a detailed investigation of the s gene and the complete genomic sequence of the i / strain to clarify its genetic characteristics and gain further insights into the origin of the strain. we also investigated the antigenicity and pathogenicity of the ibv strain i / in the present study. fertile white leghorn specific pathogen-free (spf) eggs and chickens were purchased from harbin veterinary research institute, chinese academy of agricultural sciences. ethical approval to carry out this study was obtained from the ethical and animal welfare committee of heilongjiang province, china (license no. hsy-iacuc- - ). we used the virus γcov/ck/china/i / , referred to as i / , in this study. this virus was previously isolated in spf chicken eggs from a disease outbreak associated with respiratory disease in a layer flock. the virus underwent six passages in spf eggs and was tentatively classified as a variant (xu et al., ) . a further eight ibv strains (h (gi- ) (chen et al., ) , / (gi- ) , ldl/ (ldl/ ) (gi- ) (liu et al., ) , ldl/ i (gi- ) (liu et al., ) , ck/ch/lsc/ i (lsc/ i) (gi- ) (liu et al., ) , ck/ ch/lgx/ (lgx/ ) (gi- ) , i / (gi- ) , and i / (gvi- ) (xu et al., ) ), representative of different ibv lineages circulating in china in recent years, were used in cross virus-neutralization (vn) tests. viral stock was prepared by inoculating each of the viruses into -day-old spf embryonated chicken eggs by the allantoic cavity route, followed by incubation for - h and chilling at °c overnight. allantoic fluid was harvested from the inoculated eggs and clarified by low-speed centrifugation at × g for min. cleared supernatant was stored in aliquots of . ml at − °c until further processing. virus titration was performed using -day-old spf chicken eggs, as described previously (liu et al., ). the eggs were examined for ibv lesions including curling and dwarfing up to days post-inoculation. viral titers were calculated according to reed and muench ( ) and expressed as the % egg infective dose per milliliter (eid /ml). . . rna extraction, reverse transcription-polymerase chain reaction, sequencing, and sequence determination viral rna was extracted from the allantoic fluid using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's protocol. the genomic sequence was amplified by reverse transcriptionpolymerase chain reaction (rt-pcr) using a primescript™ one-step rt-pcr kit ver. (takara bio inc., shiga, japan) according to the manufacturer's instructions. the strategy and primers used for amplifying, cloning, and sequencing the complete genome of strain i / have been described previously (liu et al., ) . the primers were previously designed for ibv complete genome sequencing (liu et al., ) , and if the primers failed to work due to sequence differences, new primers were designed based on the newly determined sequences flanking those genome regions. the ′/ ′ ends of the genome were determined using a ′/ ′ race kit (takara bio inc.) according to the manufacturer's protocol. the pcr products were cloned into the pmd -t vector (takara bio inc.) according to the manufacturer's instructions. each genome fragment was sequenced three times. chromatograms were analyzed using the program chromas (http:// technelysium.com.au/wp/chromas/) and sequences were aligned using bioedit (http://www.mbio.ncsu.edu/bioedit.htm). open reading frame (orf) predictions were carried out using the orf-finder program (https://www.ncbi.nim.nih.gov/orffinder/), and orfs were compared with the beaudette strain (genbank accession number: nc_ ). the complete genomic sequence of i / has been deposited in the genbank database, with the accession number mh . the full s gene sequence generated in this study was subjected to blast searches using the national center for biotechnology information database, and then analyzed phylogenetically using a dataset consisting of sequences (supplemental table ), including representative sequences for each genotype and lineage, as recently recommended (valastro et al., ; chen et al., ; jiang et al., ) , and the gx-nn strain, which was closely related to i / by blast analysis. the s gene sequences of the ibv strain i / and the selected viruses were aligned using clustalw, and phylogenetic analysis was carried out using mega version . software (http://www. megasoftware.net/) using the maximum likelihood method with the tamura-nei substitution model and bootstrap replicates to assess the robustness of the branches. only one ibv representative was selected from each lineage based on the phylogenetic trees to calculate the percentage identities between strain i / and the representative strains at both the nucleotide and amino acid levels. recombination events and the probable parental genotypes of strain i / were analyzed by selecting and downloading the complete genomes of ibv strains (supplemental table ) from genbank (www.ncbi.nlm.nih.gov/genbank/), representing sequences of ibv strains from all continents and each lineage if possible, three turkey coronavirus (tcov) strains, including north american and european strains, and the only available strain of gfcov. the complete genomic sequences of the ibv strain i / and the other selected viruses were aligned using clustalw, and phylogenetic trees were constructed using the above-mentioned method to reduce the database size, by collapsing the sequences into clusters with different sequence identities. sequence representative(s), including the complete genome sequences of ibv strains ck/ch/gd/gz and gx-yl , which showed close sequence identity to the i / strain, were examined using simplot version . . (http://sray.med.som.jhmi.edu/scropftware/simplot/) t. ma, et al. veterinary microbiology ( ) - to identify likely recombination breakpoints (lole et al., ) .the ibv strain gx-yl was used as a query virus. the window width and step size were set to bp and bp, respectively. to confirm these recombination breakpoints, three phylogenetic trees were constructed on the basis of the results of similarity plot (simplot) analysis for the nucleotide fragments - , ( ′ untranslated region (utr) to ′ end of gene ), , - , ( ′ end of gene to ′ end of gene ), and , - , ( ′ end of gene to ′ utr), from nine ibv strains, including i / , five strains (lgx/ , ck/ch/gd/gz , γcov/ck/china/i / (i / ), gx-yl , and gx-yl ) showing close relationships with i / based on the complete genomic sequence analysis, and three mass strains (beaudette, h , and m ) as an outgroup. in addition, the nucleotide identities between i / and the eight ibv strains were calculated. finally, blastn (http://blast.ncbi.nlm.nih.gove/blast.cgi) analysis using the fragment ( , - , ) of the recombined region was conducted using the genbank database. antisera against the viruses h , / , ldl/ , ldl/ i, lsc/ i, lgx/ , and i / chen et al., ; jiang et al., ) were used in this study. antisera against strains i / and i / were produced in this study following a standard protocol . briefly, -month-old spf chickens were inoculated intratracheally with approximately eid per bird, followed by an intravenous injection of the same dose weeks later. blood samples were collected after a further weeks, and serum was harvested, pooled, and inactivated for min at °c before being used in vn tests. we investigated the antigenic relationships between i / and the other reference strains by reciprocal β vn tests, using a fixed concentration of virus and serial dilutions of serum (ducatez et al., ) . two-fold serial dilutions of each antiserum were mixed with an equal volume of virus dilution containing eid in . ml and incubated for h at °c. each serum-virus mixture was then inoculated into embryonated spf chicken eggs via the allantoic sac route. the eggs were evaluated for non-specific mortality h after inoculation and for the presence of specific lesions at week after inoculation. end points corresponded to the serum dilutions that neutralized % of the virus. end-point titers were calculated by the method of reed and muench ( ) . vn end-point titers were used to calculate the percentage of antigenic relatedness (r) according to the method of archetti and horsfall ( ) . isolates with r values between % and % were considered to be antigenically related (gelb et al., ) . forty-five -day-old spf chickens were separated in three groups of birds each and housed in negative pressure isolators with ad libitum food and water. birds in groups and were inoculated via the ocular and nasal routes with eid /per bird of strain i / and lgx/ in μl volume, respectively, while birds in group served as a negative control. oropharyngeal and cloacal swabs and blood were collected from all birds in all groups at , , , , , , and days post-inoculation. the oropharyngeal and cloacal swabs were used to recover virus using -day-old spf chicken eggs via the allantoic cavity route (liu et al., ) and the allantoic fluids were used for rt-pcr to detect the presence of challenge viruses , to measure the duration of excretion of the strains i / and lgx/ during the course of the experiment. the sera were used to detect specific antibodies against ibv using a commercial enzymelinked immunosorbent assay (idexx corporation, westbrook, me, usa) according to the manufacturer's instructions. morbidity and mortality were recorded daily. five birds from each group were selected randomly and killed humanely at days post-inoculation. trachea, lung, proventriculus, cecal tonsil, and kidney tissues were collected from these birds and used for virus titration in day-old spf chicken eggs, as described previously . trachea and kidney were considered as the two targeted organs for ibv strains. lung is the representative of respiratory tract of chickens. proventriculus and cecal tonsil were representatives of upper and lower digestive tracts which were often used for ibv isolation (alvarado et al., ; yu et al., ) . data are expressed as mean ± standard deviation. virus titers were analyzed by a student's t-test using graphpad prism for windows version (graphpad software, la jolla, ca, usa). differences were considered significant if the p value was < . (*p < . , **p < . , ***p < . ). based on blast analysis using the complete nucleotide sequence of the s gene, the i / strain was found to be closely genetically related to gx-nn , which was isolated in guangxi province in . a phylogenetic tree comparing reference strains representing the well-established genotypes and lineages, strain gx-nn and the current strain i / are illustrated in fig. . the ibv strain i / , which was isolated in guangxi province in , was closely related to the reference strain gx-nn . strains i / and gx-nn were clearly separated from other ibv reference strains, including the six well-established genotypes and lineages. homology analysis of the ibv strains from the different lineages revealed similarities between isolate i / and the established lineages of . %- . % and . %- . % at the nucleotide and deduced amino acid levels, respectively. the deduced amino acid substitutions were scattered across the s subunit of the spike proteins in i / , compared with the other lineages (data not shown). however, strains i / and gx-nn shared % and . % nucleotide and amino acid identities, respectively, with each other. alignment of the s deduced amino acid sequences identified substitutions between i / and gx-nn , of which only two were located in the hypervariable region (hvr) (supplemental fig. a) . a consensus sequence of strain i / was obtained and the fulllength genome consisted of , nucleotides excluding the ′ poly a tails. orf analysis predicted ten orfs, resulting in a typical ibv genome organization of ′utr- a- b-s- a- b- c(e)-m- a- b-n- ′utr. in addition, there was also an orf with the potential to code for a protein of amino acids between gene m and a of strain i / which was identified previously in some ibv strains (bentley et al., ) . phylogenetic analysis of complete genomes was conducted to investigate the relationships between the obtained strain i / and different avian coronaviruses downloaded from genbank. strain i / was grouped together with all the ibv strains used in this study, which had been isolated from different regions at various times but was grouped apart from coronaviruses isolated from other avian species (fig. b) . within ibv strains, i / showed close relationships with ibv strains lgx/ ( . %), ck/ch/gd/gz ( . %), i / ( . %), ck/ch/ /jt- ( . %), gx-yl ( . %), and gx-yl ( . %), most of which were isolated from south china, particularly from guangxi province. we investigated the impact of probable recombination events on the origin of strain i / by conducting an analysis using simplot software ( fig. a) . two major recombination events were observed, one in the ′ end of gene (at approximately nucleotide , ) and another in the ′ end of gene (at approximately nucleotide , ). the phylogenetic trees (fig. b ) clearly supported the simplot results, with strain i / showing identities of > . % with lgx/ , ck/ch/gd/gz , i / , ck/ch/ /jt- , gx-yl , and gx-yl for the genomic region - , , compared with an identity of < . % with the mass-type strains. similar to this genomic region, i / showed an identity of . % with lgx/ for the genomic region , - , , compared with < . % with the mass-type strains. these results suggested that the lgx/ (gi- lineage) virus, circulating in chicken flocks in china , was a potential parent of recombinant i / . in contrast, in the second phylogenetic tree, the branch (genomic region , - , ) that included the spike gene separated the strain i / from all reference strain sequences, and shared no more than % nucleotide identity with any of the reference strains, making difficult to assess the origin of the fragment , - , . blastn analysis using this fragment of i / showed that this strain was closely related to strain gx-nn ( % nucleotide identity), but shared no more than % nucleotide identity with any other ibv strain in the genbank database. overall, these data suggest that gvii- viruses emerged as a result of replacement of the spike gene in strain lgx/ -like viruses (gi- ) through recombination. the antigenic properties of i / were compared to those of reference strains by vn tests. the calculated antigenic relatedness values, r, of strain i / against homologous and heterologous strains are listed in fig. . the r values confirmed the absence of relationships between i / and h , / , ldl/ , ldl/ i, lsc/ i, lgx/ , i / , and i / (all < %). the value of r for lgx/ was slightly higher but did not reach the % threshold for antigenic relatedness between strains, suggesting that the i / strain represented a novel serotype. respiratory signs started on day in four birds post-challenge, followed by the development of symptoms in nearly all birds. the t. ma, et al. veterinary microbiology ( ) - respiratory signs varied from mild respiratory distress to severe rales. generally, the respiratory signs of chickens challenged with strain lgx/ were more severe than those of chickens challenged with i / . one bird died on day after challenge with strain i / . in contrast, three birds died on days , and after challenge with strain lgx/ . macroscopic lesions in the dead birds were mainly confined to the kidneys, which showed pale, swollen, and mottled kidney parenchyma, and slightly distended tubules and urethras with uric acid crystals. petechiae were also observed in the trachea, pharynx, and larynx in most challenged birds. the clinical signs continued up to day in one bird that showed mild respiratory distress. no clinical signs were observed in the control birds throughout the experiment. no birds seroconverted before day post-challenge, . % and % birds seroconverted on day after challenge with i / and lgx/ , respectively. nearly all birds seroconverted on day after challenge with the two virus strains. as expected, no birds in the negative control group showed an antibody response against ibv. virus recovery from oropharyngeal swabs using -day-old spf chicken eggs challenged with i / revealed that % of birds were positive on day , % were positive on day , and . % were positive on day post-challenge. in contrast, using cloacal swabs, % were positive on days and , and . % were positive on day post-challenge. no birds tested positive on day or thereafter postchallenge. comparing with those of chickens challenged with i / , prolonged virus shedding were observed from both the respiratory tract and cloaca of chickens challenged with the lgx/ strain (fig. a) . no birds in the negative control group tested positive for virus recovery at any timepoint. the titers of the challenge viruses in trachea, lung, kidney, proventriculus, and cecal tonsil tissues were determined in five challenged chickens from each of the three groups, at days post-inoculation using -day-old spf chicken embryos . infection with strain lgx/ resulted in significantly higher production of infectious viruses in trachea, lung, kidney, proventriculus, and cecal tonsil tissues than those of corresponding tissues of chickens infected with strain i / (fig. b) . comparatively, infection with strain i / resulted in lower production of infectious virus in the trachea than those in the kidneys and cecal tonsils. no infectious viruses were detected in the lungs in the five chickens challenged with strain i / . no viruses were detected in any tissues in the five control chickens. some studies have reported that genotyping based on sequencing the hvrs of the s gene or partial sequencing of the s gene is representative of the grouping based on the complete s gene (lee et al., ) ; however, others disagree with these findings (moreno et al., ; schikora et al., ) , and this discrepancy is considered to be due to the presence of recombination events involving the s hvr. in the present study, we used the complete s gene for ibv typing, as suggested previously (manswr et al., ; valastro et al., ) , and found no evidence of recombination events in the i / s gene sequence. we did identify a novel genotype consisted by ibv isolate i / and the reference strain gx-nn , which clustered together in the phylogeny and did not fall within established lineages. in agreement with this result, the two strains shared high nucleotide and amino acid identities ( % and . %), compared with low identities with representative ibv strains from different lineages. based on these results, these two ibv strains should be grouped into a novel genotype, designated as gvii- , although the designation of lineage/genotypes should be assigned to monophyletic groups of at least three viruses sampled from at least two different outbreaks (valastro et al., ) . generally, it is considered that the chance of having the same serotype is higher between strains with the same genotype (wang and huang, ) , though these data showed that this relationship was not very strong, given that a change in a small percentage of the amino acids in the s protein could result in a change of serotype (cavanagh et al., ) . the current virus cross-neutralization results showed that gvii- was not only a novel genotype, but also antigenically different from the currently tested genotype lineages. strain gx-nn was isolated in guangxi province, china, in , suggesting that the novel gvii- had been circulating in china over a -year period. however, the source of this new virus introduction into the chicken population in china remains unknown. full-length genome analysis showed that the emergence of the i / strain involved a double crossover event that replaced the spike gene by that from an as-yet-unidentified ibv strain. however, the remaining parts of the genome originated from commonly known gi- ibv strains, suggesting that the i / strain was a mosaic, and that one of its putative parents was descended from gi- lineage viruses. the antigenic differences among ibv strains have been investigated, and previous work showed that s sequences had a stronger correlation with protective relatedness than with antigenic relatedness between strains (ladman et al., ) . the as-yet-unidentified parental virus of strain i / , which shared the same spike gene with strain i / t. ma, et al. veterinary microbiology ( ) - , might thus be antigenically different from vaccine strains used in china, such as h and / ; therefore vaccination with these live vaccines might not offer complete protection against infection with the parental virus of strain i / . similarly, considering the high prevalence of the gi- lineage in south china where the novel gvii- viruses were isolated, it is reasonable to speculate that the gi- lineage may co-infect birds more frequently than other lineages, although the current commonly used vaccines cannot offer complete protection against gi- viruses . vaccination with these vaccines is therefore likely to induce non-sterilizing immunity, which may allow prolonged replication, shedding, and circulation of both the deduced parental strains of gvii- viruses. consequently, although vaccination with attenuated vaccines is carried out intensively in china, this procedure may produce an environment where co-infection with gi- and the as-yet-unidentified parental viruses can enhance the likelihood of recombination. theoretically, the short persistence and shedding, and limited numbers of i / -like viruses isolated so far in china suggest that the probability of recombination between the as-yet-unidentified parental virus and gi- viruses may not be high, similar to the hypothesis suggesting that the shorter persistence and shedding of mass-based vaccines (bijlenga et al., ) and the low percentage of m -like viruses (chen et al., ; moreno et al., ) lgx/ fig. . infection of chickens with strains lgx/ and i / . virus recovery from oropharyngeal (upper) and cloacal (lower) swabs from chickens challenged with ibv strains lgx/ and i / (a). virus recovery was performed by inoculating -day-old embryonated, specific pathogen-free eggs through the allantoic route with supernatant from the swabs. replication of strains lgx/ and i / in trachea, lung, kidney, cecal tonsil, and proventriculus in chickens (b). one-dayold spf layer chickens in groups and were inoculated via the ocular and nasal routes with × eid /per bird of strains lgx/ and i / in . ml, respectively, and trachea, lung, kidney, cecal tonsil, and proventriculus tissues were collected from five birds at days post-challenge for virus titration in eggs. data are expressed as mean ± standard deviation. virus titers were analyzed by a student's t-test using graphpad prism for windows version (graphpad software, la jolla, ca, usa). differences were considered significant if the p value was < . (*p < . , **p < . , ***p < . ). t. ma, et al. veterinary microbiology ( ) - the ibv genome undergoes mutations and recombination, and the exchange of a long region of the genome by recombination may allow viruses to rapidly explore ample areas of the sequence space, potentially leading to the emergence of novel strains with different features in terms of virulence and disease pathogenesis, antigenicity, and cell and tissue tropisms (cavanagh et al., ; kant et al., ; simon-loriere and holmes, ) . the genome of strain i / was closely related to gi- viruses, except in the spike gene. this phenomenon was similar to that of tcov, the genome of which was also closely related to ibv, except in the spike gene. replacement of the spike gene of tcov was responsible for the host shift from chickens to turkeys and a pathogenicity shift from upper-respiratory disease to enteric disease (jackwood et al., ) . even though the recombination events that replaced the spike gene did not result in a tissue tropism shift in the kidneys of chickens after challenge, the i / strain showed low affinity to the respiratory tract compared with the parental virus lgx/ . in contrast, the ability of i / to reach the cecal tonsils and subsequently to replicate in this tissue was slightly higher compared with that of respiratory tract. the replicase gene of avian coronavirus ibvs is known to be a determinant of pathogenicity (armesto et al., ) , and we were therefore unable to compare the pathogenicity of i / with one of its deduced parental virus until a source has been identified for the replicase gene because it is as-yetunidentified, though another deduced parental virus (lgx/ ) showed more severe pathogenicity than i / in spf chickens. however, we cannot conclude that this difference in pathogenicity was due to replacement of the spike gene until the source of the gvii- spike gene has been identified, and alternate explanations for the observed differences between i / and gi- spike should be considered. the frequency of successful inter-typic genetic changes may depend on the viability of the recombinant progeny able to establish in the host population, and on the increase in fitness of the recombinant offspring in the host population compared with their non-recombination parents (smits et al., ) . the recombinant i / was only detected sporadically and persisted for a short time, and thus probably represented strains that were not sufficiently competitive with respect to one of their progenitor strains, lgx/ -like viruses . however, compared with the unidentified parental virus, i / -like strains may be sufficiently competitive, and the parental virus may have been circulating at below cut-off levels for detection, due to its lower replicative capacity and lower percentage. we isolated the i / ibv strain and passaged it in spf chicken eggs . the number of passages of the virus in eggs was high (six passages), and the possibility that some genetic changes were introduced as a result of its propagation in the eggs cannot be ruled out. no complete genomic sequences of gvii- lineage viruses are currently available in the public database. the present results thus provide valuable information on the characteristics of the i / strain and contribute to a better understanding of the origin of gvii- viruses. further studies on these isolates will provide information on the emergence, evolution, and effectiveness of current vaccines against these isolates. detection of massachusetts and arkansas serotypes of infectious bronchitis virus in broilers persistent antigenic variation of influenza a viruses after incomplete neutralization in ovo with heterologous immune serum the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and other gammacoronaviruses development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus coronavirus ibv glycopolypeptides: size of their polypeptide moieties and nature of their oligosaccharides coronavirus avian infectious bronchitis virus infectious bronchitis location of the amino acid differences in the s spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus molecular and antigenic characteristics of massachusetts genotype infectious bronchitis coronavirus in china identification and molecular characterization of a novel serotype infectious bronchitis virus (gi- ) in china 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 coronaviruses: an rna proofreading machine regulates replication fidelity and diversity characterization of a new genotype and serotype of infectious bronchitis virus in western africa serotype, antigenicity, and pathogenicity of a naturally recombinant tw i genotype infectious bronchitis coronavirus in china antigenic and s- genomic characterization of the delaware variant serotype of infectious bronchitis virus genetic, antigenic, and pathogenic characteristics of avian infectious bronchitis viruses genotypically related to /b in china genetics, antigenicity and virulence properties of three infectious bronchitis viruses isolated from a single tracheal sample in a chicken with respiratory problems emergence of a group coronavirus through recombination genome characterization, antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south china location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide experimental evidence of recombination in coronavirus infectious bronchitis virus infectious bronchitis virus s gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene genetic diversity of avian infectious bronchitis coronavirus strains isolated s gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives altered pathogenicity, immunogenicity, tissue tropism and '- kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos characterization of a recombinant coronavirus infectious bronchitis virus with distinct s subunits of spike and nucleocapsid genes and a ' untranslated region full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination evaluation of full s gene sequencing of classical and variant infectious bronchitis viruses extracted from allantoic fluid and fta cards a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain the peplomer protein sequence of the m strain of coronavirus ibv and its comparison with beaudette strains a simple method of estimating fifty per cent endpoints an apparently new respiratory disease of baby chicks genetic diversity of avian infectious bronchitis virus california variants isolated between and based on the s subunit of the spike glycoprotein genetic relationships of infectious bronchitis virus isolates from mississippi broilers why do rna viruses recombine? phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks genetic diversity of avian infectious bronchitis virus in china in recent years characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens sequencing and serologic identification of s genes of infectious bronchitis viruses isolated during - in guangxi province, china the authors declare that they have no competing interests. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -wkisfz m authors: han, mingyuan; yoo, dongwan title: engineering the prrs virus genome: updates and perspectives date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: wkisfz m porcine reproductive and respiratory syndrome virus (prrsv) is endemic in most pig producing countries worldwide and causes enormous economic losses to the pork industry. infectious clones for prrsv have been constructed, and so far at least different infectious clones are available representing both genotypes i and ii. two strategies have been taken for progeny reconstitution: rna transfection and dna transfection. mutations, insertions, deletions, and replacements of the viral genome have been employed to study the structure function relationship, foreign gene expression, functional complementation, and virulence determinants. essential regions and non-essential regions for viral replication have been identified in both the coding regions and non-encoding regions. foreign sequences have successfully been inserted into the nsp and n regions and in the space between orf b and orf a. chimeras between member viruses in the family arteriviridae have also been constructed and utilized to study cell tropism and functional complementation. this review discusses the advances and utilization of prrsv reverse genetics and its potential for future research. porcine reproductive and respiratory syndrome (prrs) was first reported in the united states in and subsequently in europe in and quickly became endemic in most pig producing countries worldwide (benfield et al., ; chand et al., ; murakami et al., ; shimizu et al., ; wensvoort et al., ) . the clinical manifestation of prrs is complicated but is characterized by severe reproductive losses including abortions, mummified fetuses, weak born and stillborn young, post-weaning pneumonia, increased mortality, and growth retardation of young pigs. the etiological agent is prrs virus (prrsv) . prrsv belongs to the family arteriviridae together with lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv). by comparative genome sequence analysis, prrsv isolates are divided into two distinct genotypes: the european type (genotype i) and north american type (genotype ii), represented by lelystad virus (lv) and vr- as the prototype virus for each genotype, respectively (benfield et al., ; wensvoort et al., ) . the sequence similarities between two genotypes are approximately % (allende et al., ; nelsen et al., ; wootton et al., ) . amino acid (aa) sequence alignments indicate that the major differences between two genotypes exist in the open reading frame (orf) a region and the structural protein region (kapur et al., ; murtaugh et al., ; nelsen et al., ) . natural deletions and insertions are observed in some isolates, especially in the orf a region (fang et al., ; gao et al., ; shen et al., ; tian et al., ) . the reverse genetics system has been developed for many rna viruses, and infectious clones have been utilized for the study of biology and the vaccinology of viruses. the availability of such a powerful molecular tool has revolutionized the structure function studies for viral genome and proteins and has facilitated the studies for virulence, pathogenesis, immune responses, and vaccine development. the first full-length genomic cdna clone was constructed for poliovirus more than three decades ago and its infectivity was demonstrated (racaniello and baltimore, ) . infectious clones have since been constructed for picornaviruses, caliciviruses, flaviviruses, togaviruses, influenza viruses, paramyxoviruses, rhabdoviruses, and coronaviruses to name a few (almazan et al., ; boyer and haenni, ; pu et al., ; scobey et al., ; sosnovtsev and green, ; yount et al., ) . for arteriviruses, eav and prrsv are the first for which the reverse genetics system has been developed van dinten et al., ) . this review will summarize our current knowledge on the principles of prrsv infectious clones and the application to the study of arteriviruses. the prrsv genome is a single-strand positive-sense rna of kb in length with a cap and -polyadenylated tail (fig. a ) (meulenberg et al., ; murtaugh et al., ; nelsen et al., ; wootton et al., ) . the prrsv genome is polycistronic and harbors two large open reading frames (orfs), orf a and orf b, followed by orf a, orf b, and orfs through , plus orf a within orf (firth et al., ; johnson et al., ; meulenberg et al., ; murtaugh et al., ; nelsen et al., ; wootton et al., ) . a - ribosomal frame-shifting has recently been identified for expression of nsp tf in the nsp -coding region. the nsp tf coding sequence is conserved in prrsv, ldv, and shfv but absent in eav (fang et al., ) . the coding sequences in the viral genome are flanked by the and un-translated regions (utrs) involved in translation, replication, and transcription (see review in snijder et al., ) . orf a and orf b code for two large polyproteins, pp a and pp ab, with the expression of the latter mediated by the - frame-shifting in the orf a/orf b overlapping region (fig. b; den boon et al., ; snijder and meulenberg, ) . thus, the pp b portion is expressed always as a fusion with pp a. the pp a and pp ab proteins are further processed to generate non-structural proteins (nsps). the polyprotein processing scheme involves the rapid auto-proteolytic release of three nterminal nsps, nsp a, nsp b, and nsp , mediated by papain like proteinase (plp) residing in each of them. the subsequent processing for the remaining portion of polyproteins is mediated by the serine protease in nsp resulting in individual nsps (den boon et al., ; van aken et al., ; ziebuhr et al., ) . the proteolytic cleavages for individual nsps were initially predicted by sequence comparisons in combination with some experimental data from eav, the prototype virus of the family arteriviridae (fang and snijder, ; ziebuhr et al., ) . the exact cleavage sites for prrsv nsp a#nsp b and nsp b#nsp have recently been confirmed to be m #a and g #a mediated by prrsv-plp a and prrsv-plp b, respectively sun et al., ; xue et al., ) . a set of -coterminal nested subgenomic (sg) mrnas, from which structural proteins are translated, is produced during infection (fig. b) . each mrna contains a common -end leader sequence identical to the proximal part of the genome and this sequence is referred to as transcription-regulatory sequence (trs). the fusion of the common sequence (leader trs) to the different -body segments of sg mrnas is mediated by discontinuous transcription which is a common strategy of nidoviruses (sawicki et al., ; snijder et al., ; sola et al., ) . during the negative-strand sg rna synthesis, transcription is attenuated at different body trs regions of the genomic template. the nascent subgenome-length minus-strand rna, having an antibody trs at its end, will then move and base-pair with the leader trs and completes the extension of sg rna. minus-strand sg rnas subsequently serve as a template for plus-strand sg mrna which is subsequently translated for structural protein (music and gagnon, ; sawicki et al., ; snijder et al., ) . the genome of negative-strand rna viruses is noninfectious, and its replication in permissive cells requires the ribonucleoprotein (rnp) complex as the infectious unit. in contrast, the genome from positive-strand rna viruses is fully infectious, and thus the assemly of full-length cdna clones corresponding to the rna genome is the kernel to the construction of infectious clones . the viral genome is polycistronic, harboring orf a and orf b, and structural genes of orf a, orf b, and orfs through , plus orf a within orf . (b) viral gene expression. non-structural proteins (black) are produced from pp a and pp ab after proteolytic processing. the prrsv replicase-processing scheme involves the rapid auto-proteolytic release of nsp a, nsp b, and nsp (yellow boxes), mediated by papain-like proteinase (plp) domains residing in each of them. the remaining polyproteins are processed by nsp , resulting in a set of individual nsps. the cleavage sites by plps and nsp are annotated by curved arrows and blue triangles, respectively. structural proteins (color-coded) are expressed from the subset of sg mrna. the -co-terminal nested set of minus-strand rnas is produced as a template for plus-strand sg mrna synthesis. trs, transcription regulatory sequence. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (boyer and haenni, ; meyers et al., ; moormann et al., ; sosnovtsev and green, ; van dinten et al., ) . non-retroviral rna viruses do not undergo a dna intermediate step in their replication cycle. to obtain a template which can be manipulated by molecular techniques, a full-length cdna clone is first generated using the reverse transcriptase and dna polymerase. once generated, two strategies have been established to generate virus progeny from the full-length copy of viral genome: rna transfection and dna transfection. in the rna transfection strategy, viral rna is synthesized by in vitro transcription using t or sp rna polymerase coupled with the respective promoter located immediately upstream of the viral genome. the synthesized rna genome is then introduced into cells to initiate an infection cycle. in the dna launch strategy, a full-length genomic clone is placed under a eukaryotic promoter such as a cytomegalovirus (cmv) promoter and the entire plasmid is introduced to cells for transcription by exploiting the nuclear function of the cell. the transcribed viral genome in the nucleus is exported to the cytoplasm where viral genome translation and replication occur. this strategy omits the steps of in vitro synthesis of genomic rna and rna transfection, thus the risk of rna degradation during transfection is reduced and transfection efficiency becomes consistent ). an arterivirus infectious clone was first made for eav. the peav full-length clone containing the . kb cdna copy of the eav genome was infectious (van dinten et al., ) , and the first prrsv infectious clone pabv was developed for the genotype i prrsv lelystad virus . subsequently, infectious clones for vr- which is the genotype ii prrsv, and the european-like genotype i prrsv sd - circulating in the us was developed (fang et al., a (fang et al., , b nielsen et al., ) . numerous clones have additionally been developed including the highly-pathogenic prrsv that emerged in china in (guo et al., ; lv et al., ; zhou et al., ) . to date, at least different infectious clones are available for prrsv (table ) . prrsv infectious clones have mostly been developed based on the rna launch strategy. bhk- , ma- , and marc- cells are cells of choice for transfection and progeny production. although bhk- cells are nonpermissive for prrsv infection, they provide a high efficiency of transfection and a good production of progeny . to eliminate the need for in vitro transcription and consistency associated with rna transfection, the cmv promoter has been used for construction of the p infectious clone. the p virus is an isolate recovered from an outbreak of highly virulent atypical prrs in the mid-western usa in yoo et al., ) . the cmv promoter-based infectious clone is convenient and simple to use and provides a consistency of transfection and recovery of progeny virus ). an infectious clone should genetically be identical to the parental virus. however, non-viral nucleotides are occasionally added to the viral genome at the or end to meet the engineering needs without impeding the infectivity of the clones truong et al., ) . to differentiate the reconstituted progeny virus from the parental virus, genetic markers of either restricted enzyme recognition sequences or certain nucleotide mutations have been introduced to infectious clones, and such modifications should be non-lethal and stable. to assure the starting position of the rna polymerase ii-mediated transcription, nucleotides are placed between the tata box and the genome start when constructing pcmv-s-p . the prrsv genome is usually divided to several fragments flanked by restriction sites for subsequent assembly nielsen et al., ) . as a cloning vector, a lowcopy-number plasmid is generally preferred as suggested in some studies sumiyoshi et al., ; nielsen et al., ) but has appeared unnecessary. progeny virus generated from an infectious clone should ideally retain the biological properties of the parental virus, such as growth rate, virulence, and transmissibility meulenberg et al., ; nielsen et al., ; truong et al., ; yuan and wei, ) . like most rna viruses, prrsv genome has evolved to optimal fitness, and most of the genetic information seems to be essential (verheije et al., ) . notably, the proximal portion of the genome is compact and organized to contain eight genes, most of which overlap with neighboring genes (snijder et al., ) . the prrsv genome is complex and the engineering of such a compact viral genome is a challenge. in addition, minor alternations in conserved regions or functional domains in the genome almost inevitably lead to non-viable consequences kroese et al., ; . despite such difficulties, some genetic manipulations for prrsv have been successful. mutation, deletion, insertion, and substitution are major approaches to viral genome manipulation. due to the large genome of prrsv, shuttle plasmids have been used as an intermediate platform to contain the target viral genomic sequence with a pair of unique enzyme sites at each end. mutations are introduced to target sites or sequences in the shuttle plasmid. the biological functions of plp a and plp b in nsp , conserved cysteine residues at c and c in the e protein, n-linked glycosylation sites in gp at n and gp at n , n , and n , cysteines at c , c , and c for homo-dimerization of n protein, and the motif for nuclear localization signal (nls) of n have been mutated to produce prrsv mutants kroese et al., ; pei et al., ; vu et al., ) . alanine scanning and protein surface accessibility predictions were conducted for identification of residues for type i ifns or tnf-a antagonism of nsp , and specific residues have been mutated in the infectious clones li et al., ; subramaniam et al., ) . mutations have also been introduced to knockout genes by changing the translation n/a, not application. a the individual time of prrsv infectious clones construction referred to the date of each publication. b genotype i and ii prrsv represents european and north america strains, respectively. c sequences of full-genome cdna clone rather than complete genome sequences of parental virus are listed. d the genome area in which the restricted enzyme sites are introduced is indicated with brackets. the position where single mutations are introduced is given. e genome-length cdna clones of pabv and pabv encode identical viral protein sequences except for one amino acid at position in orf a, which are a pro in pabv and a leu in pabv . f the abbreviation, na i, in the genotype column represents genotype i prrsv isolated in north america. initiation codon, and this approach destroys the expression of nsp and e protein tijms et al., ) . deletion of genomic sequences has been applied to identifying non-essential regions for prrsv replication or to obtaining attenuated live vaccine candidates (verheije et al., ) . inter-genotypic sequence alignments between genotype and genotype reveal the regions of sequence heterogeneity suggesting the potential to tolerate the deletions, and non-essential regions in the n gene and -utr (table ; sun et al., b; tan et al., ) . the hypervariable regions have been observed in the nsp gene (fang et al., ; gao et al., ; ni et al., ; shen et al., ; tian et al., ) , suggesting the existence of a non-essential region in nsp (chen et al., b; han et al., ; ran et al., ; xu et al., b) . deletion of orf or orf results in the absence of infectivity, suggesting the requirement of gp and gp proteins for prrsv infectivity . insertion of additional nucleotides to the viral genome expands the scope of modifications. an attempt was made to separate overlapping regions of prrsv structural protein genes, and three restriction enzyme sites were inserted between orfs / and orfs / (yu et al., ) , which produced viable viruses. the possibility of expressing foreign genes using prrsv has been explored; the nsp gene was utilized as an insertion site for expressions of green fluorescent protein (gfp) and flag tag (fang et al., b; kim et al., ). an alternate approach was taken to insert foreign genes within a structural gene; for example, a small portion of the influenza virus hemagglutinin (ha) gene into the or end of orf of prrsv (bramel-verheije et al., ) . however, the insertion of ha to n gene resulted in a nonviable virus. a strategy utilizing the mechanism of transcription of prrsv for foreign gene expression is of particular interest. using an infectious clone, two unique enzyme sites have been introduced between orf b and orf , and a copy of the trs sequence was inserted to replace the trs designed to synthesize the mrna for foreign gene expression (fig. ) pei et al., ; yoo et al., ) . the foreign genes including gfp, capsid protein of porcine circovirus type (pcv ), discosoma sp. (sea anemone) red fluorescent protein (dsred), renilla luciferase (rluc), ifn-a , ifn-b, ifn-d , and ifn-v have all been expressed as an independent transcript using this approach (table ; pei et al., ; sang et al., ) . multiple genes, a single gene, or partial sequence of the viral genome have been substituted with corresponding sequences from other arteriviruses for chimeric arterivirus construction. the first chimeric arterivirus was generated using an eav infectious clone as a backbone, and ectodomains of two membrane proteins, gp and m, were substituted with the corresponding sequences from prrsv or ldv (dobbe et al., ) . these chimeric viruses were viable, and additional chimeric arteriviruses have been constructed (table ). the construction of intra-or intergenotypic prrsv chimeras is maneuverable, and the regions of -utr, non-structural genes, and structural genes have been replaced (gao et al., ; lu et al., ; tian et al., tian et al., , vu et al., ; zhou et al., ) . to facilitate the intra-genotypic substitution, a gene-swapping mutagenesis technique has been used to substitute the structural genes (kim and yoon, ) . using this technique, individual replacement of orf a and orf through orf of vr- was successfully carried out with corresponding orfs from other strains of prrsv including ja , sdsu , prrs , and m (kim and yoon, ) . . . . nsp prrsv nsp is a multifunctional protein that undergoes remarkable genetic variations. the nsp protein consists of five regions: hypervariable region i (hv-i), plp cysteine protease core, hypervariable region ii (hv-ii), transmembrane regions, and a c-terminal tail ( fig. a ; han et al., ) . the plp cysteine protease domain possesses cisacting and trans-acting cleavage activities and mediates its rapid release from pp a and pp ab (han et al., ; snijder et al., ) . two sites were initially predicted for nsp /nsp cleavage at g/ g and somewhere at g/ g/ g, and recent studies showed the actual cleavage occurs at g/ g for vr- (allende et al., ; han et al., ; nelsen et al., ) . the corresponding cleavage for europrrsv sd - likely occurs at gg/ a (fang and snijder, ) . plp is as a member of the ovarian tumor domain (otu) family of deubiquitinating enzymes, and has shown to deconjugate ubiquitin (ub) and ifn-stimulated gene (isg) from cellular targets. this is an important viral strategy inhibiting the ub-dependent and isg -dependent host innate immune responses (frias-staheli et al., ; sun et al., a sun et al., , b van kasteren et al., ) besides the proteinase and deubiquitinase functions, nsp contributes to the major genetic differences between genotypes i and ii, sharing only less than % similarity at the amino acid level (allende et al., ; nelsen et al., ) . the nsp gene also contains naturally inserted sequences and deletions ( fig. a) in the hypervariable region (fang et al., ; gao et al., ; ni et al., ; shen et al., ; tian et al., ) . the deletion of amino acids in nsp was first found in a chinese prrsv isolate, hb- (sh)/ , in comparison with other north american isolates (gao et al., ) . sequence analysis of prrsv mn reveals three discontinuous deletions of , , and amino acids at the corresponding positions - , , and - of vr- , respectively (han et al., ) . discontinuous deletions were also identified in the highly pathogenic prrsv (hp-prrsv) associated with the outbreaks of porcine high fever disease in china (tian et al., ) . the amino acids discontinuous deletion consists of aa deletion at position and aa deletions at - , and the deletion region contains bcell epitopes (de lima et al., ) and t-cell epitopes (chen et al., b) . strikingly, cell culture passages of prrsv may generate a deletion in nsp , and a study shows the generation of a large deletion of aa at - in nsp during passages (ni et al., ) . a deletion in nsp is also found in genotype i prrsv. the europrrs sd- - virus in the us shows a aa deletion at positions - of nsp when compared to lelystad virus (fang et al., ) . biological significance of the genetic deletion in nsp remains to be determined. besides deletions, a aa insertion was also observed in the sp strain of prrsv, which is a vaccine strain, located between g and t of the sp nsp (shen et al., ) . given the tolerance of deletions and insertions in the hypervirable region of nsp , this region is considered as a site for foreign gene insertion ( fig. b and c) . the gfp gene was inserted into nsp of the sd - strain and fully infectious virus was rescued (fang et al., b) . the gfp insertion did not affect the growth of the virus, and the infectivity was comparable to that of parental virus. the capacity of deletion in nsp was determined by introducing a series of in-frame deletions (han et al., ) . the plp domain, the plp downstream flanking region, and the transmembrane domain were crucial for virus replication but deletions of - aa from the n-terminal portion of the hypervariable region and - aa from the hypervariable region appeared to be tolerable for viability. in the hypervariable region, the largest deletion that can be achieved was about aa at positions - , although a deletion of up to aa is preferable for infectivity (fig. b) . the insertion of gfp or other genes such as new castle disease virus nucleoprotein (np) gene has been successful as long as insertions reside in the hypervirable regions ( fig. c ) (kim et al., ; xu et al., a) . the deletion of inmmunodominant linear b-cell epitopes (es -es ) were attempted; deletion of es , es , or es allowed the generation of an infectious virus (chen et al., b; oleksiewicz et al., ) . prrsv n is a mutilfunctional protein. the specific domains and residues critical for virus replication have been identified in n (fig. a) . the n protein is comprised of or aa for the north american and european genotypes, respectively (music and gagnon, ) . n consists of the n-terminal rna-binding domain (rbd) at positions - and the c-terminal dimerization domain comprising a four-stranded antiparallel b-sheet floor flanked by a-helices (doan and dokland, ; yoo et al., ) . as the sole component of viral capsid, n interacts with itself via covalent or noncovalent interactions (doan and dokland, ; wootton and yoo, ) . the cysteine at position is responsible for the formation of an intermolecular disulfide bond, and aa - are essential for mediating noncovalent homodimers . a crystallographic study on n shows the imprtance of the c-terminal dimerization domain for n (doan and dokland, ; spilman et al., ) . prrsv n is a serine phosphoprotein which is a common property for n of eav and coronaviruses (music and gagnon, ; wootton et al., ) . one of the phosphorylation sites of n is at position , but its biological significance is still unknown. n contains nls in a stretch of basic amino acids -pgkknkk- which is overlapping with the rna-binding domain and particially with a nucleolar localization signal (nols) at aa - (rowland et al., . the nuclear export signal (nes) is found at positions - and is responsible for the nucleolar-cytoplasmic shuttling of n . the functional structure of n is compact and thus n is sensitive to structural modification. the secondary structure in the c-terminal residues - is an important determinant for conformational epitopes, and the mutations in this region change the monoclonal antibody (mab) reactivity (wootton et al., ) . insertion of a foreign sequence into the n gene was attemped and the influenza virus ha epitope was added at the n-terminus or cterminus. despite the initial rescue of the infectious virus, the ha expression was unsuccessful (bramel-verheije et al., ) . the gfp tag was inserted between orf and orf to moniter the orf mrna synthesis, but no mrna was made, indicating the end of the orf gene is essential for mrna synthesis . the n protein is inter-genotypically conserved but shares only % of its identity between lv and vr- (dea et al., ) . the c-terminus of n is heterogenous, and truncation of up to aa is tolerable (verheije et al., ) . in another study, deletions were made at the inter-genotypic variable region or conserved region of n, and regions at - , - , - , and - , were found to be dispensible for viability ( fig. b ) (tan et al., ) . no foreign gene can be incorporated in these rgions. the prrsv genome is flanked by -and -utr, and the utr sequences play a vital role for genomic replication, mrna transcription, and protein translation (pasternak et al., ; snijder et al., ) . the non-coding regions of the genome have been investigated. by serial deletions, the first nucleotides in -utr appears to be dispensible for viability in type ii prrsv first nucleotides are unique for each genotype, and a stretch of nucleotides is present in vr- but is absent in lv (allende et al., ; verheije et al., ) . a deletion study shows that nucleotides at the end of the -utr is tolerable for genotype i prrsv (verheije et al., ) . the -utr of genotype ii has also been studied, and at least nucleotides immediately following orf is dispensable for virus viability . the genetic information on the structural region of arteriviruses is organized in an extremely efficient manner. the genes for gp , gp , and gp overlap each other, and similarly the genes for gp , m, and n overlap each other for prrsv. this structural complexicity hampers the genetic manipulation of infectious clones. the importance of the overlapping gene arrangement for the life-cycle of virus has been studied (verheije et al., ; yu et al., ) . a series of full-length clones were engineered to separate overlapping genes for eav orfs / or orfs / by inserting small additional sequences containing a termination codon for the upstream gene, a unique restriction site, and a translation initiation codon for the downstream gene. the insertions result in the functional separation of overlapping orfs, and do not impair infectivity (de vries et al., ) . the orfs / separation in genotype i prrsv is also possible and progeny virus is produced (verheije et al., ) . for the north american prrsv, restriction sites were inserted between orfs / , orfs / , orfs / , orfs / , and orfs / -ntr, and progeny viruses are generated from these modifications. this indicates that gene overlap is dispensable for infectivity and that separation of each gene does not interrupt mrna synthesis (yu et al., ) . the development of infectious clones allows the construction of chimeric arteriviruses. an attempt was made to swab the ectodomains of gp and m. in engineered chimeric viruses using the eav clone as a backbone, the ectodomains were replaced by corresponding sequences from other arteriviruses. chimeric viruses containing the gp ectodomain from ldv and prrsv were infectious. these chimeric viruses however retain their cell tropism for bhk- cells, which are susceptible for eav but non-susceptible for ldv and prrsv (dobbe et al., ) . replacement of the m ectodomain of eav with the corresponding sequence from other arteriviruses does not produce infectious virus, but replacement of the m ectodomain of prrsv with the corresponding sequence from ldv, eav, and genotype ii prrsv produced an infectious virus. using the lv infectious clone as a backbone, substitutions with the eav m ectodomain or vr- m ectodomain is impossible, but removal of the gene overlap between the m and gp genes is required before swapping, indicating that the vr- m ectodomain and eav m ectodomain are incompatible with the remaining part of lv m. it is also possible that unintended mutations may have been introduced to gp during the ectodomain swap (verheije et al., ) . substitution of structural genes between arteriviruses has been extremely useful to identify viral factors for viral tropism. the substitution of gp or/and m do not alter their cell tropism (dobbe et al., ; lu et al., ; verheije et al., ) . in contrast, the substitution of minor envelope proteins and e protein using the prrsv infectious clone as a backbone allows the chimeric prrsv to acquire a broad cell tropism but to lose the ability to infect pams. it indicates that the gp /gp /gp minor proteins are determinants for cell entry and tropism (tian et al., ) . intra-genotypic or inter-genotypic gene-swapping have been conducted between eav and prrsv to study the genetic compatibility and viral-specific phenotypes, including neutralization, virulence, and pathogenesis. for neutralization, chimeric eavs were generated in which each construct contained individual orf from different isolates (balasuriya et al., ) . also, the role of individual envelope proteins of gp through m for cross-neutralization was studied using the vr- infectious clone as a backbone (kim and yoon, ) . the prrsv- strain is highly susceptible to serum neutralization and induces atypically rapid and robust neutralizing antibodies in pigs. analysis of structural genes of prrsv- reveals the absence of two n-linked glycosylation sites each in gp and gp . the significance of missing glycans for neutralization has been determined by replacing gp and gp genes from prrsv- (vu et al., ) . the major virulence determinants have also been identified by gene swapping experiments to locate in nsp through nsp and gp . highly pathogenic prrsv contains the aa deletion in nsp sequence (tian et al., ) . by gene swapping studies using nsp from an avirulent prrsv, the deletion in nsp was shown to be irrelevant to virulence and pathogenicity (zhou et al., ) . a recent study identified nsp -and nsp -coding regions together were essential for increased pathogenicity and fatal virulence for hp-prrsv by swapping these regions between the highly and low pathogenic strains (li et al., ) . the intergenotypic -utr swap between genotypes i and ii was investigated and shows that the -utr of genotype ii may be substituted with the corresponding sequence from genotype i, while the substitution of -utr of genotype i with its corresponding sequence from genotype ii is lethal (gao et al., ) . using this approach, the envelope proteins representing gp through gp of genotype i are shown to be fully functional for genotype ii when using genotype ii as a backbone (tian et al., ) . a random sequence shuffling has been employed to generate immunologic variants of prrsv zhou et al., zhou et al., , . gp sequences representing immunologically diverse strains of prrsv are randomly shuffled, and the shuffled gene is incorporated in the infectious clone to generate a new virus that contains a new gp gene, which may improve the cross neutralization . the breeding of gp and m have also been tried, and the rescued virus induces a broad spectrum of cross-neutralizing antibodies . the gp sequence from genetically diverse strains of prrsv and the gp -m sequence from different strains were subjected to breeding, and the shuffled genes were cloned in infectious clones for the generation of new viruses. two representative chimeric viruses, ds by gp shuffling and ds m by gp -m shuffling, were found to be clinically attenuated . this approach allows rapid generation of an attenuated virus and may be useful for vaccine development for antigenetically variable viruses zhou et al., ) . another approach to the rapid generation of attenuated prrsv is referred to as save (synthetic attenuated virus engineering). codon-pair bias is a phenomenon that certain codon pairs appear in a higher frequency in comparison to other synonymous codon pairs for the same amino acid, and the codon-pair bias is host species-dependent related to the efficiency of protein synthesis (coleman et al., ; moura et al., ; mueller et al., ) . by deoptimizing the codon pair of a virulence gene, an expression level of this protein decreases. the computer-aided deoptimization of codon-pairs modifies only naturally optimized pairs of codons and does not change the amino acid sequence (mueller et al., ) . using this approach, the gp gene was codon-pair deoptimized, and a new virus was generated. the modified gp sequence did not affect the viability of prrsv and the engineered virus was clinically attenuated in pigs (ni et al., ) . to fulfill serological discrimination between naturally infected and vaccinated animals, removing an immunodominant epitope has been applied to developing a liveattenuated differentiating infected from vaccinated animals (diva) vaccine against prrsv (de lima et al., ; vu et al., ) . the serologic marker antigen selected for diva vaccine should be highly immunodominant without disrupting protective well-conserved epitopes among prrsv isolates and stability during passages. besides, the removal of a selected epitope should not adversely affect the growth property or virulence of the mutant virus (de lima et al., ; vu et al., ) . two epitopes residing in nsp and m have been identified fulfilling the requirements for prrsv diva vaccine (de lima et al., ; vu et al., ) . two mutants, fldnsp / with a deletion of residues - within nsp , and q r disrupting antigenicity of epitope m in m protein have been designed and constructed accordingly (de lima et al., ; vu et al., ) . the immunogenicity of those two epitopes has been eliminated during infection of prrsv mutants, and both epitopes may be used as an immunologic marker for diva vaccine development (de lima et al., ; vu et al., ) . prrsv may serve as a vaccine vector. prrsv infectious clones have been developed as a gene delivery vector for foreign gene expression. identification of gene insertion sites in the viral genome and viral infectivity is critical for gene delivery. gfp and b-cell epitopes of the newcastle disease virus (ndv) nucleoprotein have been inserted into non-essential regions of nsp of prrsv (fang et al., b (fang et al., , kim et al., ; xu et al., a) . in this approach however, the stability of the inserted gene was of a concern. when prrsv expressing gfp in nsp , prrsv sd - -gfp, was cell-culture passaged, a population of gfp-expression negative-virus appears by the th passage. sequencing shows a deletion of gfp at the n-terminal half ( to ), leading to the loss of gfp expression. insertion of amino acids at position of gfp was also observed in some viral clones (fang et al., b) . the stability of the gfp gene in this recombinant virus was improved by deleting the es epitope located downstream of the gfp gene, and the gfp expression in this virus was stable for passages. however, r c mutation was found in gfp, and this mutation caused the loss of florescence (fang et al., ) . the loss of fluorescence was also observed in two other gfp recombinant viruses during serial passages. in another study, the gfp-coding sequence remained intact but point mutations were identified and these mutations caused amino acid changes to r c and n y (kim et al., ) . the expression of aa b-cell epitope of the ndv nucleoprotein in prrsv nsp remained stable in cell culture up to passages (xu et al., a; zhang et al., ) . the instability of foreign gene insertion in nsp is not fully understood. the length of insertion may be important for stability. an attempt was made to produce an additional mrna for foreign gene expression. the gfp gene was inserted between orf b and orf a for prrsv along with a copy of trs pei et al., ; sang et al., ; yoo et al., ) . compared to insertion in nsp , this site is suitable for foreign gene insertion since the recombinant virus was stable for up to at least passages without the loss of gene or fluorescence (pei et al., ). the genetic stability of genes inserted at this site has been confirmed by expressing other genes including dsred, renilla luciferease, ifna , ifnb, ifnd , and ifnv (sang et al., ) . this approach has the particular advantage of eliminating the need to alter the coding sequence of a viral gene and also of minimizing the effects on expression and post-translational modification of viral proteins (pei et al., ) . infectious clones are important molecular tools to study structure function relationships of proteins and genomic sequences at the infectious virus level in vivo. specific sequence motifs may be mutated or deleted from the virus and their phenotypes may be examined to determine their functions. the removal of n-linked glycosylation at n and n of gp results in a mutant virus with its phenotype of enhanced sensitivity to serum neutralization and high level induction of neutralizing antibodies . elimination of n -linked glycan is not in concert with a high-level neutralizing antibody response to wild type prrsv . meanwhile, introduction of multiple mutations at these n-linked glycosylation sites could significantly reduce virus yields . the e gene knock-out mutation allows for genome replication and transcription but does not produce infectious progeny, indicating that the e protein is essential for virion assembly . prrsv nsp is a multifunctional protein regulating the accumulation of genomic rna and mrnas. it also has the ability to modulate the host innate immunity by suppressing the type i ifn production (nedialkova et al., ; sun et al., a; yoo et al., ) . the motifs for zinc fingers, plps, and nuclease have been identified in nsp (fang and snijder, ; snijder et al., ; xue et al., ) . by deleting from the genome, nsp is shown to be dispensable for genome replication but crucial for mrna transcription. mutation in the catalytic sites of plp impairs both viral genome and mrna synthesis as well as the cleavage between nsp and nsp . mutations in the zinc finger motif abolished the mrna transcription, whereas genome replication was not affected (tijms et al., (tijms et al., , . when the catalytic sites of plp a are mutated using a prrsv infectious clone, the proteinase activity disappears and mrna synthesis is completely blocked. in contrast, mutations at the plp b catalytic sites result in no mrna synthesis and no viral infectivity, indicating that the normal cleavage of nsp and nsp is critical for viral replication (kroese et al., ) . to design effective vaccine candidates that may be useful to overcoming antigenic heterogeneity of prrs, extensive studies have been conducted to eliminate the ifn antagonistic function from the virus (see reviews snijder et al., ; sun et al., a; yoo et al., ) . among viral proteins, nsp a and nsp b have been identified as potent ifn analogists (beura et al., ; chen et al., a; han et al., han et al., , kim et al., ; song et al., ) . subsequent studies have identified specific residues regulating the ifn antagonism, and a mutant virus with a stretch of alanine substitution at positions - of nsp b showed the loss of ifn suppression . in another study, k and r were mutated to release the surface accessibility of nsp b, and mutant prrsv impaired the ifn antagonism (li et al., ) . motifs in the n protein have broadly been studied using mutant viruses. the importance of n protein dimerization has been examined by mutating c s which is responsible for the covalent interaction between n proteins. mutant viruses of c s, c s, and c s were constructed, and with the exception of c s, both c s and c s completely lost their infectivity. in another study however, the replacement of cysteines within n protein, either singly or in combination, did not impair the growth prrsv according . genome replication and mrna transcription were normal for both mutants, suggesting the dimerization of n may be important for particle assembly or maturation . the nuclear localization signal (nls) of n was also mutated to examine the biological consequence of n in the nucleus in prrsv-infected cells. compared to wild-type prrsv, nls-null mutant prrsv was attenuated in pigs and produced a significantly shorter mean duration of viremia and higher titers of neutralizing antibodies pei et al., ) , demonstrating that the n protein nuclear localization is a virulence factor. as an emerged and re-emerging disease in swine, prrs has extensively been studied for molecular biology, immunology, and prevention. the unusual immune responses in pigs and antigenic heterogeneity of the virus are two main obstacles to developing a satisfactory prrs vaccine. the availability of prrsv infectious clones and recent advances in recombinant dna technology have made possible to manipulate the viral genome to introduce specific mutations to targeted sequences and to create genetically modified mutant viruses. extensive efforts have been applied to making mutant viruses with modified phenotypes of immune responses including the evasion of neutralizing antibodies and suppressed innate immune responses. for viral heterogeneity, the rna-dependent rna polymerase (rdrp) is believed to cause frequent mutations in the prrsv genome. genetic swapping or modifications of nsp , the rdrp of prrsv, may be studied to make mutant viruses with reduced mutation rates during replication. the reverse genetics of prrsv is a powerful genetic tool and has the potential to apply to the basic understanding of the biology of prrsv and to the development of genetically modified vaccines and gene delivery. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions engineering the largest rna virus genome as an infectious bacterial artificial chromosome influence of nlinked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cdna clone characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation identification of amino acid residues important for anti-ifn activity of porcine reproductive and respiratory syndrome virus nonstructural protein infectious transcripts and cdna clones of rna viruses expression of a foreign epitope by porcine reproductive and respiratory syndrome virus pathogenesis of porcine reproductive and respiratory syndrome virus identification of two auto-cleavage products of nonstructural protein (nsp ) in porcine reproductive and respiratory syndrome virus infected cells: nsp function as interferon antagonist immunodominant epitopes in nsp of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response virus attenuation by genome-scale changes in codon pair bias development of a porcine reproductive and respiratory syndrome virus differentiable (diva) strain through deletion of specific immunodominant epitopes serologic marker candidates identified among b-cell linear epitopes of nsp and structural proteins of a north american strain of porcine reproductive and respiratory syndrome virus genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture development of genetic markers in the non-structural protein region of a us type porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development construction of a full-length cdna infectious clone of a european-like type prrsv isolated in the us heterogeneity in nsp of european-like porcine reproductive and respiratory syndrome viruses isolated in the united states a full-length cdna infectious clone of north american type porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the nsp region the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins efficient- frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses cis-acting structural element in utr is essential for infectivity of porcine reproductive and respiratory syndrome virus replacement of the heterologous untranslated region allows preservation of the fully functional activities of type porcine reproductive and respiratory syndrome virus genomic characterization of two chinese isolates of porcine respiratory and reproductive syndrome virus experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus identification of nonessential regions of the nsp replicase protein of porcine reproductive and respiratory syndrome virus strain vr- for replication in cell culture the porcine reproductive and respiratory syndrome virus nsp cysteine protease domain possesses both trans-and cis-cleavage activities complete genome analysis of rflp isolates of porcine reproductive and respiratory syndrome virus degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp alpha subunit novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states expression and stability of foreign tags inserted into nsp of porcine reproductive and respiratory syndrome virus modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells molecular assessment of the role of envelopeassociated structural proteins in cross neutralization among different prrs viruses the nsp alpha and nsp beta papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus rna synthesis infectious clonederived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones a dna-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties nsp and nsp contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china targeted mutations in a highly conserved motif of the nsp beta protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus chimeric viruses containing the n-terminal ectodomains of gp and m proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome infectious transcripts from cloned genomelength cdna of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cdna constructs infectious rna transcribed from an engineered full-length cdna template of the genome of a pestivirus large scale comparative codon-pair context analysis unveils general rules that fine-tune evolution of mrna primary structure live attenuated influenza virus vaccines by computer-aided rational design isolation and serological characterization of porcine reproductive and respiratory syndrome (prrs) viruses from pigs with reproductive and respiratory disorders in japan comparison of the structural protein-coding sequences of the vr- and lelystad virusstrains of the prrs virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis arterivirus np modulates the accumulation of minus-strand templates to control the relative abundance of viral mrnas porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents establishment of a dna-launched infectious clone for a highly pneumovirulent strain of type porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp region attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of virus envelope genes from genetically divergent strains computer-aided codon-pairs deoptimization of the major envelope gp gene attenuates porcine reproductive and respiratory syndrome virus generation of an infectious clone of vr- , a highly virulent north american type isolate of porcine reproductive and respiratory syndrome virus epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp fragment of the replicase polyprotein contains a cluster of b-cell epitopes nidovirus transcription: how to make sense functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association porcine reproductive and respiratory syndrome virus as a vector: immunogenicity of green fluorescent protein and porcine circovirus type capsid expressed from dedicated subgenomic rnas successful propagation of flavivirus infectious cdnas by a novel method to reduce the cryptic bacterial promoter activity of virus genomes cloned ppliovirus complementary-dna is infectious in mammalian-cells recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the nsp -encoding region the localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences replication-competent recombinant porcine reproductive and respiratory syndrome (prrs) viruses expressing indicator proteins and antiviral cytokines a contemporary view of coronavirus transcription reverse genetics with a fulllength infectious cdna of the middle east respiratory syndrome coronavirus determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with a unique insertion isolation of porcine reproductive and respiratory syndrome (prrs) virus from heko-heko disease of pigs arterivirus molecular biology and pathogenesis the molecular biology of arteriviruses the arterivirus nsp protease, an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases rna-rna and rna-protein interactions in coronavirus replication and transcription nonstructural protein alpha subunitbased inhibition of nf-kappab activation and suppression of interferon-beta production by porcine reproductive and respiratory syndrome virus rna transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require vpg for infectivity cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid amino acid residues in the non-structural protein of porcine reproductive and respiratory syndrome virus involved in down-regulation of tnf-alpha expression in vitro and attenuation in vivo infectious japanese encephalitis-virus rna can be synthesized from in vitro-ligated cdna templates interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus crystal structure of porcine reproductive and respiratory syndrome virus leader protease nsp -alpha the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-simulated gene identification of dispensable nucleotide sequence in ' untranslated region of porcine reproductive and respiratory syndrome virus identification of non-essential regions in nucleocapsid protein of porcine reproductive and respiratory syndrome virus for replication in cell culture arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype envelope proteins in the backbone of genotype emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark arterivirus subgenomic mrna synthesis and virion biogenesis depend on the multifunctional nsp autoprotease a zinc finger-containing papain-like protease couples subgenomic mrna synthesis to genome translation in a positive-stranded rna virus a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp arterivirus and nirovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription viable porcine arteriviruses with deletions proximal to the end of the genome chimeric arteriviruses generated by swapping of the m protein ectodomain rule out a role of this domain in viral targeting characterization of a serologic marker candidate for development of a live-attenuated diva vaccine against porcine reproductive and respiratory syndrome virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein n-linked glycosylation of gp of porcine reproductive and respiratory syndrome virus is critically important for virus replication in vivo construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates mystery swine disease in the netherlands -the isolation of lelystad virus antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages stable expression of foreign gene in nonessential region of nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus: applications for marker vaccine design identification of nonessential regions of the nsp protein of an attenuated vaccine strain (hun -f ) of highly pathogenic porcine reproductive and respiratory syndrome virus for replication in marc- cell the crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein nsp -beta reveals a novel metal-dependent nuclease modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin infectious cdna clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus reverse genetic manipulation of the overlapping coding regions for structural proteins of the type ii porcine reproductive and respiratory syndrome virus construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins disulfide linkages mediating nucleocapsid protein dimerization are not required for porcine arterivirus infectivity generation of an infectious clone of hun -f , an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the gp or m genes dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence virus-encoded proteinases and proteolytic processing in the nidovirales key: cord- -qcpua ck authors: park, su-jin; oh, eun-hee; park, sang-ik; kim, ha-hyun; jeong, young-ju; lim, guem-ki; hyun, bang-hun; cho, kyoung-oh title: molecular epidemiology of bovine toroviruses circulating in south korea date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: qcpua ck the prevalence of the bovine torovirus (btov) and its genetic characterization have been reported in north america, europe and japan. therefore, this study examined the prevalence and genetic diversity of the btov in a total of diarrheic fecal samples from korean native beef calf herds using rt-pcr and nested pcr with the primer pairs specific to a part of the btov membrane (m) gene. overall, ( . %) out of diarrheic samples from herds ( . %) tested positive for btovs by either rt-pcr or nested pcr. a comparison of the nucleotide (nt) and amino acid (aa) sequences of a part of the btov m gene ( bp) among the btovs showed the korean btovs to have comparatively higher sequence homology to the japanese and dutch btovs than to the american and italian btovs. generally, the korean btov strains clustered with the japanese and dutch btov strains. however, the american and italian btov strains clustered on a separate major branch, suggesting that these are more distantly related to other known btov strains. these results suggest that the btov infections are sporadic in diarrheic calves in south korea, and the korean btov strains are more closely related to the japanese and dutch btovs than to the american and italian btovs. toroviruses within the family coronaviridae are spherical, oval, elongated, or kidney-shaped enveloped viruses that possess a positive-sense single-stranded, polyadenylated rna genome of approximately - kb in length (cornelissen et al., ; horzinek, ; snijder and horzinek, ) . toroviruses have been detected in normal horses as well as in humans, cattle, pigs, and recently in turkeys with diarrhea (ali and reynolds, ; beards et al., ; duckmanton et al., duckmanton et al., , a kroneman et al., ; woode, ) . the first report of toroviruses described the identification of an unclassified virus www.elsevier.com/locate/vetmic veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] from diarrheic calves (woode et al., ) , which is now known as the breda virus, or bovine torovirus (btov) . fecal shedding of the btovs have been reported in diarrheic calves in several countries including the united states (hoet et al., (hoet et al., , a woode et al., woode et al., , , canada (duckmanton et al., a) , costa rica (pérez et al., ) , netherlands (koopmans et al., ) , germany (liebler et al., ) , hungary (matiz et al., ) , austria (haschek et al., ) , japan (ito et al., ) , and south africa (vorster and gerdes, ) . however, there are no reports of btov infections in south korea. since the molecular analysis of the btov has only been carried out in north american, european and japanese btov strains (cornelissen et al., ; draker et al., ; duckmanton et al., b; gonzálex et al., ; haschek et al., ; hoet et al., hoet et al., , a ito et al., ; smits et al., ) , it is unclear if the btovs circulating in other countries have distinct genetic characteristics. this paper reports the detection of btov shedding in diarrheic calves using rt-pcr and nested pcr, along with the genetic diversity of the btov strains based on a partial btov membrane (m) gene. according to farmers' request for the treatment of calf diarrhea, local veterinary clinicians visited the cattle farms, and treated diarrheic calves with - days fasting and the administration of an electrolyte fluid and antibiotics. all the diarrheic calves were aged between and days at the time of sample collection, were fed colostrum from their dams, and housed in the stall. the farm sizes ranged from to animals (average ). the farms were geographically distributed in chonnam province, which is the south-western province in south korea. in order to diagnose the enteric pathogens, local veterinary clinicians sampled one or two diarrheic fecal specimens from each farm and immediately submitted the sample to the college of veterinary medicine, chonnam national university. a total of diarrheic fecal specimens were collected from korean native beef (hanwoo) calf herds (aged - days) between january and december during the spring ( samples/ herds), summer ( samples/ herds), autumn ( samples/ herds) and winter ( samples/ herds). upon arrival of the fecal samples, they were immediately examined for common enteric viral, bacterial and protozoan pathogens including the btov, bovine coronavirus (bcov), groups a, b and c bovine rotaviruses (brv a-c), new bovine enteric calicivirus genus known as becovirus, bovine norovirus (bnov), bovine viral diarrhea virus (bvdv), salmonella spp., clostridium spp., campylobacter spp., shiga-toxin-producing escherichia coli, coccidium spp. and cryptosporidium spp. (asakura et al., ; park et al., in press-a,b; soulsby, ; timoney et al., ) . the results of bovine norovirus and bovine coronavirus have been described previously (park et al., in press-a,b) . data for other enteric viruses will be reported elsewhere. the rna was extracted from a ml starting volume of centrifuged % fecal suspensions using the trizol-ls (gibco-brl, life tech, grand island, usa) procedure. the total rna recovered was suspended in ml of rnase free water and stored at À c. the primer sets and rt-pcr and nested pcr conditions used for the detection of the btov, bcov, brv a-c, becovirus, bnov, and bvdv are described elsewhere (cho et al., ; park et al., , in pressa,b) . as a negative control, the rna was extracted from the normal feces of colostrum-deprived calf that had been inoculated with ml sterile pbs, and used for all rt-pcr assays. in order to confirm the absence of cross-contamination from rt-pcr to nested pcr, rt-pcr was performed with rna extracted from a normal mock-infected calf fecal sample, and nested pcr was then carried out using ml of this rt-pcr product. the amplification products were analyzed using . or % agarose gel electrophoresis and visualized by irradiating the ethidium bromide stained samples with uv. the nested pcr products for a portion of the m gene ( bp) were selected, purified and sequenced directly to verify the reaction specificity as well as to obtain the genomic data for phylogenetic analysis. the nested pcr products were purified using a genclean ii kit (bio , inc., lajolla, usa) according to the manufacturer's instructions. dna sequencing was carried out using an automated dna sequencer (abi system , applied biosystem inc., foster city, usa). the nucleotide (nt) and deduced amino acid (aa) sequences of the partial btov m gene were compared with those of the other known toroviruses using the dna basic module (dnasis max, alameda, usa) ( table ) . phylogenetic and bootstrap ( replicates) analyses based on the nt and aa alignments were constructed using the neighbor-joining method and the unweighted-pair group method with the average linkages of molecular evolutionary genetics analysis (mega, version . ) with a pairwise distance (kumar et al., ) . a sequence similarity search was carried out for the bovine calicivirus the rdrp protein using the lalign query program of the genestream network server at the institut de génétque humaine, montpellier, france (http://www.eng.uiowa.edu/$tscheetz/ sequence-analysis/examples/lalign/lalign-guess. html). a one-step rt-pcr assay, which targets a bp fragment of the m gene in the btov, detected three positive fecal samples from three herds. the nested pcr assay, which targeted a bp fragment of the m gene, detected positive fecal samples ( . %) from herds ( . %). the calculated percentage of btov infections in the diarrheic calves in south korea was comparatively low compared with that reported in other countries, where the reported fecal prevalence of the btov in calf diarrhea was % in lower saxony, germany (liebler et al., ) , . % in japan (ito et al., ) , . % in the usa (hoet et al., a) , and . % in southern ontario, canada (duckmanton et al., a) . this suggests that btov infections are sporadic in diarrheic calves in south korea. of the btov-positive fecal specimens from the calf herds, fecal samples from herds tested positive to the btov alone, while the other btovpositive fecal samples from the herds also tested positive to other enteric pathogens (table ). this suggests that other bovine enteric pathogens play an important role in the clinical and pathological presentation of this disease. an increase in the severity of the disease after a natural co-infection with other bovine enteric pathogens has been reported (duckmanton et al., a; hoet et al., a) . seasonally, btov infections were less prevalent in the diarrheic fecal samples of calves obtained in the spring than in the other seasons (table ) ; ( . %) out of fecal samples tested positive in spring; ( . %) out of fecal samples tested positive in autumn; ( . %) out of fecal samples tested positive in winter; and ( . %) out of fecal samples tested positive in summer. this result is very similar to that reported by hoet et al. ( a) , who showed that two peaks of btov shedding were observed, one during the winter months and the other during the summer season. thirteen nested pcr products were selected, purified and sequenced directly, and their nt and aa sequences were compared with those of other known toroviruses (table ) . table shows the sequence relationships of the nt and aa of the partial m gene among the torovirus strains. interestingly, a comparison of the nt and aa sequences among the btovs revealed all the korean btovs to share % nt and aa table sequence relationships of the nucleotide and amino acid of the partial membrane gene among torovirus strains identity to each other, except for the k strain ( . % nt and . % aa identity). in order to eliminate cross contamination, the negative control for rt-pcr and nested pcr was used when the field diarrheic fecal samples were analyzed by rt-pcr and nested pcr. however, there was no positive reaction detected in this negative control. therefore, this identical nt and aa sequence homology of the m gene among the korean btovs might be due to the fact that the positive fecal samples used for dna sequencing originated from the same county, muan, even though they had been sampled from different farms at different times (tables and ). the korean btovs shared higher nt and aa sequence homology to the japanese btovs ( . - . % nt and . - % aa identity) and the dutch btovs ( . - . % nt and . - % aa identity) than the italian ( . % nt and . - % aa identity) and american ( . % nt and . - . % aa identity) btovs. in addition, the btovs including the american, european, japanese and korean btovs had a lower nt and aa identity to the equine torovirus strain, berne ( . - . % nt and . - . % aa identity), than to porcine toroviruses ( . - . % nt and . - . % aa identity). from these results, the korean btovs were more closely related to the japanese and dutch btovs than to the american and italian btovs. in addition, the overall nt and aa sequence homology of the btov m gene in this study is higher than of the spike (s) protein gene (ito et al., ; koopmans and horzinek, ) . the relative conservation of the m protein of all btovs suggests that the structural constraints on this protein are stern, resulting in more limited evolution of this protein than the s protein (koopmans and horzinek, fig. . a phylogenetic tree of the nucleotide sequence of the m gene of the bovine torovirus strains was made using the neighbor-joining method of molecular evolutionary genetics analysis (kumar et al., ) . the names of the viruses used are listed in table . ). therefore, future studies will be needed to generate more information on the genetic diversity by including other parts of the genome particularly the s gene as well as including the fecal specimens from other regions in south korea. generally, phylogenetic data of nt sequence of the partial btov m gene revealed that the korean btov strains were closely related to the japanese and dutch btov strains (fig. ) . of the korean btov strains, all korean btov strains clustered together with the exception of the k strain, which clustered on a separate branch with the japanese btov strains, k- and k- . other japanese and dutch btovs made separate branches. in addition, the american breda and the italian b strains clustered on a separate major branch, suggesting these were the most distantly related to the other known btov strains. phylogenetic analysis of the aa sequence did not show any significant classification of the btovs because the homology of the aa sequence among the btov strains was higher than that of the nt sequence (data not shown). this data supports the above mentioned hypothesis that the korean btovs were more closely related to the japanese and dutch btovs than to the american and italian btovs. south korea imports live cows mainly from countries in north american and oceania, not from japan and european countries. hence, the similarity in phylogenetic data on the korean btov with the japanese and dutch btovs is quite surprising. therefore, continuous monitoring and intensive studies will be needed for a more detailed characterization of the btov with other genes. in conclusion, btov infections are sporadic in diarrheic calves in south korea. based on the partial sequence of the btov m gene, the korean btovs were more closely related to the japanese and dutch btovs than to the american and italian btovs. to our knowledge, this is the first report of the detection of btov shedding and its genetic diversity in diarrheic calves in south korea. characterization of the stunting syndrome agent: relatedness to known viruses detection and genetical characterization of shiga toxin-producing escherichia coli from wild deer preliminary characterization of torovirus-like particles of humans: comparison with berne virus of horses and breda virus of calves cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection hemagglutinin-esterase, a novel structural protein of torovirus the complete sequence of the bovine torovirus genome detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of breda virus characterization of torovirus from human fecal specimens a comparative sequence analysis to revise the current taxonomy of the family coronaviridae detection of bovine torovirus in neonatal calf diarrhea in lower austria and styria (austria) detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves enteric and nasal shedding of bovine torovirus (breda virus) in feedlot cattle molecular evolution of corona-and toroviruses epidemiological analysis of bovine torovirus in japan toroviruses of animals and humans: a review association of diarrhea in cattle with torovirus infections on farms identification and characterization of a porcine torovirus mega : integrated software for molecular evolutionary genetics analysis and sequence alignment the significance of bredavirus as a diarrhea agent in calf herds in lower saxony torovirus detection in faecal specimens of calves and pigs in hungary: short communication molecular epidemiology of bovine noroviruses in south korea detection and molecular characterization of calf diarrhea bovine coronaviruses circulating in south korea during detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons infectious agents associated with diarrhea of calves in the canton of tilaran costa rica. pre phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily primary structure and post-translational processing of the berne virus peplomer protein helminths arthropods and protozoa of domesticated animals, seventh ed. lea and febiger hagan and bruner's microbiology and infectious diseases of domestic animals the toroviruses: bovine (breda virus) and equine (berne virus) and the torovirus-like agents of humans and animals comparative studies on three isolates of breda virus of calves studies with an unclassified virus isolated from diarrheic calves this study was supported by grant no. rti - - from the regional technology innovation program of the ministry of commerce, industry and energy (mocie), republic of korea. the authors acknowledge a graduate fellowship provided by the korean ministry of education and human resources development through the brain korea project. key: cord- - so gqc authors: zhao, shanshan; gao, qi; qin, tao; yin, yinyan; lin, jian; yu, qinghua; yang, qian title: effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: so gqc virulent transmissible gastroenteritis virus (tgev) results in an acute, severe pathology and high mortality in piglets, while attenuated tgev only causes moderate clinical reactions. dendritic cells (dcs), through uptake and presentation of antigens to t cells, initiate distinct immune responses to different infections. in this study, an attenuated tgev (stc ) and a virulent tgev (shxb) were used to determine whether porcine dcs play an important role in pathogenetic differences between these two tgevs. our results showed that immature and mature monocyte-derived dendritic cells (mo-dcs) were susceptible to infection with shxb and stc . however, only shxb inhibited mo-dcs to activate t-cell proliferation by down-regulating the expression of cell–surface markers and the secretion of cytokines in vitro. in addition, after h of shxb infection, there was the impairment in the ability of porcine intestinal dcs to sample the antigen, to migrate from the villi to the lamina propria and to activate t-cell proliferation in vivo. in contrast, these abilities of intestinal dcs were enhanced in stc -infected piglets. in conclusion, our results show that shxb significantly impaired the functions of mo-dcs and intestinal dcs in vitro and in vivo, while stc had the opposite effect. these differences may underlie the pathogenesis of virulent and attenuated tgev in piglets, and could help us to develop a better strategy to prevent virulent tgev infection. transmissible gastroenteritis virus (tgev) belongs to the genus coronavirus of the family coronaviridae (masters, ) . virulent tgev infection leads to severe vomiting, acute villous atrophy, malabsorptive diarrhoea and dehydration, which approach % mortality in seronegative suckling pigs. even older piglets that recovered from virulent tgev infections had impaired immune function (woods, ) . however, the piglets exposed to attenuated tgev only had mild clinical signs and low mortality (frederick et al., ) . we presume that the interaction of tgev with the immune cells has an important role in this pathogenesis. dendritic cells (dcs), the most powerful antigenpresenting cells (ardavin, ) , are widely distributed in the mucosal tissue, which forms an extensive network monitoring the invasion of pathogens (haverson et al., ) . studies have demonstrated that viruses were directly or indirectly sampled by intestinal dcs (rescigno et al., ) . thus, dcs are proposed to be among the first cells encountered after mucosal exposure to viruses. tgev mainly infects and replicates in the porcine intestinal , through uptake and presentation of antigens to t cells, initiate distinct immune responses to different infections. in this study, an attenuated tgev (stc ) and a virulent tgev (shxb) were used to determine whether porcine dcs play an important role in pathogenetic differences between these two tgevs. our results showed that immature and mature monocyte-derived dendritic cells (mo-dcs) were susceptible to infection with shxb and stc . however, only shxb inhibited mo-dcs to activate t-cell proliferation by down-regulating the expression of cell-surface markers and the secretion of cytokines in vitro. in addition, after h of shxb infection, there was the impairment in the ability of porcine intestinal dcs to sample the antigen, to migrate from the villi to the lamina propria and to activate t-cell proliferation in vivo. in contrast, these abilities of intestinal dcs were enhanced in stc -infected piglets. in conclusion, our results show that shxb significantly impaired the functions of mo-dcs and intestinal dcs in vitro and in vivo, while stc had the opposite effect. these differences may underlie the pathogenesis of virulent and attenuated tgev in piglets, and could help us to develop a better strategy to prevent virulent tgev infection. ß elsevier b.v. all rights reserved. epithelial cells and must interact with the intestinal dcs. recent studies have been suggested that the interaction between dcs and the different virulence of viruses plays an important role in the induction or inhibition of the immune response. highly pathogenic avian influenza (hpai) infection of chicken dcs could produce a lower level of cytokine in contrast to low pathogenic avian influenza (lpai) infection (vervelde et al., ) . lassa virus infection of human dcs could inhibit the upregulation of surface marker expression and the development of an adaptive immune response, while the related naturally non-virulent arenavirus mopeia virus (mv) infection of dcs could increase the transcription of type i ifn mrna, il- mrna and cxcl- mrna and induce stronger t-cell responses (baize et al., ; pannetier et al., ) . similarly, virulent varicella-zoster virus (vzv) subverted secretion of th cytokines in human dcs by blocking tlr mediated innate signals, while the attenuated vzv vaccine strain enhanced secretion of th cytokines (gutzeit et al., ) . thus, different responses of dcs to the virulent and attenuated virus may underlie the differences in pathogenicity between them. whether the varied morbidity and mortality in piglets caused by the differing virulence of tgev infections is associated with the intestinal dcs is still unclear. in this study, the virulent tgev (shxb) and attenuated tgev (stc ) strains were first used to infect porcine monocytederived dendritic cells (mo-dcs) in vitro and evaluate the abilities of mo-dcs to take up and present the antigens of the different virulent strains of tgev, and stimulate the tcell responses. second, the shxb and stc strains were used to infect the piglets in vivo in order to further determine whether they could damage the ability of intestinal dcs to sample the heat-inactivated escherichia coli, migrate, and stimulate cd + , cd + and cd + t-cell proliferation at h postinfection. understanding these events may provide a theoretical basis for the study of the differences in pathogenesis of virulent and attenuated tgev infection in pigs. a total of six-week-old, yorkshire, landrace, and large white cross-bred pigs were bred and maintained in a unique high sanitary state characterised by freedom from a wide range of porcine pathogens, e.g., tgev, pedv, pcv- , prrsv. all animal experiments were carried out in accordance with the regulations and guidelines of laboratory animals of nanjing agriculture university (nanjing, china). two tgev strains [shxb, wild-virulent strain, verified by the animal attack virus test (see the supplementary data (additional files - )) and the stc live-attenuated strain (he et al., ) ] were provided by the jiangsu academy of agricultural sciences (jaas). the strains were propagated in swine testicle (st) cells and were purified using a sucrose gradient as described previously (krempl and herrler, ) . the viral % cell tissue infectious dose (tcid ) of the purified shxb and stc strains were calculated using the reed and muench method (haggett and gunawardena, ) . e. coli k (enterotoxigenic e. coli k , invitrogen, usa) was grown overnight in luria broth (lb). when the e. coli were at a concentration of - cfu ml À , they were heat killed in a water bath at c for min. they were then diluted to the appropriate concentration in fcs-free cell culture medium for testing that there was no live e. coli. porcine mo-dcs were generated as previously reported (carrasco et al., ) . briefly, porcine peripheral blood mononuclear cells (pbmc) were separated from the blood of six piglets by density centrifugation using histopaque ( . g l À ) (sigma, usa). pbmcs were washed three times in rpmi medium (gibco, usa), and resuspended in complete rpmi medium with % fbs (multicell, canada). pbmc were then placed in six-well plates and incubated overnight at c in % co . the nonadherent cells were removed leaving the adherent monocytes. the monocytes were cultured in complete rpmi medium containing ng ml À of pil- (biosource, usa) and ng ml À of pgm-csf (invitrogen, usa) at c in % co . cells were incubated for five days to allow for their differentiation into immature mo-dcs and replaced with cytokine-containing medium on day . immature mo-dcs were harvested on day and resuspended in rpmi medium. for further induction of maturation, immature mo-dcs were stimulated by the addition of ug ml À of lipopolysaccharide (escherichia coli o :b , sigma, usa) for h. immature and mature mo-dcs were inoculated with tgev (shxb or stc ) at a concentration of tcid cell À . the viruses were adsorbed for h at c. in order to eliminate the non-absorbed virus, cells were washed five times with pbs, centrifuged at  g for min at c, and re-suspended in fresh rpmi medium containing pil- and pgm-csf. mock-infected cells were treated in parallel. all mo-dcs were seeded onto -well plates (  cells/well) and cultured for the required incubation period. shxb-and stc -infected immature and mature mo-dcs were seeded onto -well plates (  ) and cultured for , , , h. the virus was collected by freezing and thawing the plates three times, and determined by the tissue culture infectious dose (tcid ) in st cells. . . determination of shxb-and stc -infected mo-dcs by flow cytometric analysis shxband stc -infected immature and mature mo-dcs were seeded onto -well plates (  ) and cultured for , , h. the cell samples (  cells) were washed with pbs twice and permeabilised for min with . % triton x- . the cells were incubated with a % solution of bsa for min at room temperature and stained with fitc-conjugated tgev polyclonal antibody (vmrd, usa) and pe-conjugated mouse anti-porcine monoclonal antibody to swine workshop cluster a (swc a) (abcam, hongkong) for min at c. cells were then washed twice with . m pbs and then were resuspended in ml of pbs. samples of  cells were analysed by flow cytometry. to determine the modulation of the surface markers on mo-dcs treated with tgev after h postinfection (p.i.), the cell samples (  cells) were washed with pbs twice, and incubated for min at c with appropriate dilutions of the following monoclonal antibodies: fitcconjugated mouse anti-swine monoclonal antibody to swine histocompatibility leukocyte ag ii-dr (sla-ii-dr) (lifespan biosciences, usa), fitc-conjugated mouse antihuman monoclonal antibody to the co-stimulatory molecules cluster of differentiation / (cd / ) (abcam, hongkong), fitc-conjugated mouse anti-porcine monoclonal antibody to cluster of differentiation a (cd a)(abcam, hongkong), and pe-conjugated mouse anti-porcine monoclonal antibody to swine workshop cluster a (swc a)(abcam, hongkong). cells washed twice with . m pbs and were resuspended in ml of pbs. samples of  cells were analysed by flow cytometry. the apoptosis of immature and mature mo-dcs treated with the shxb and stc strains was quantified at h postinfection (p.i.). the dual parameter analysis of annexin v-fitc (invitrogen, usa) and propidium iodide (pi; sigma, usa) was performed. samples containing  mo-dcs were labelled first with annexinv-fitc ( mg ml À ) for min and then with pi ( mg ml À ). the samples were analysed by flow cytometry. elisa was used to determine the concentration of il- , ifn-g, and il- in immature and mature mo-dcs (  cells/well) infected with shxb and stc at h p.i. the levels of the three cytokines were quantified in the supernatants using commercial elisa kits (r&d systems, usa), according to the manufacturer's recommendations. mlr was evaluated by flow cytometry using the florescent dye carboxyfluorescein succinimidyl ester (cfse; sigma, mo, usa). to isolate t cells, the pbmcs were labelled with mouse anti-cd antibody (abcam, hongkong) followed by incubation with rat anti-mouse igg microbeads (macs; miltenyi biotec, germany). purified t lymphocytes (  ) were stained with . mm cfse in rpmi- medium for min at c and % co in the dark. tgev-infected mo-dcs were incubated simultaneously with mitomycin c ( ug ml À ) for h. cfse-labelled t-lymphocytes (  ) were cocultured with tgev-infected dcs at a ratio of : ; : ; : (dcs: lymphocytes). co-cultures were incubated at c in % co for five days. finally, the cells were harvested, and the fluorescence intensity of the cfse was determined by flow cytometric analysis. the first animal experiment: a total of conventional . -month-old piglets were assigned to four groups, which were maintained in isolation facilities to prevent virus circulation. all piglets were starved h before surgery to insure the least amount of chymus possible in the intestine. the treatments of these groups were showed in table . in group i (n = ), the piglets were anesthetised with pentobarbital sodium, and a midline incision was table the first animal experiment design. inculation and numbers of segments per pig intestinal ligation i (n = ) pbs ( ml per segment, n = ) pbs ( ml per segment, n = ) shxb ( ml of  tcid ml À per segment, n = ) shxb ( ml of  tcid ml À per segment, n = ) h (from the first injection to sacrifice) min (from the second injection to sacrifice) h (from the first injection to sacrifice) min (from the second injection to sacrifice) ii (n = ) pbs ( ml per segment, n = ) pbs ( ml per segment, n = ) stc ( ml of  tcid ml À per segment, n = ) stc ( ml of  tcid ml À per segment, n = ) h (from the first injection to sacrifice) min (from the second injection to sacrifice) h (from the first injection to sacrifice) min (from the second injection to sacrifice) oral administ-ration iii (n = ) shxb ( ml of  tcid ml À per pig) h (from administration to sacrifice) iv (n = ) stc ( ml of  tcid ml À per pig) h (from administration to sacrifice) made just anterior to the navel (nielsen and sautter, ) . twelve jejunum segments containing the peyer's patches (pps) per piglet were divided into four treatments. the first treatment was injected with shxb strain h before sacrifice (n = ), the second treatment was injected with sterile . . m pbs h before sacrifice (n = ), the third was injected with shxb strain min before sacrifice (n = ), the fourth was injected with sterile . . m pbs min before sacrifice (n = ). during the procedure, pigs were kept warm on a c warming pad. the pigs were sacrificed and the twelve intestinal segments were removed. in group ii (n = ), except for stc strain, the surgery and the sample grouping were the same as groupi. piglets in group iii (n = ) and group iv (n = ) did not carry out the surgery, were separately inoculated with the shxb strain and stc strain by oral administration. h after the inoculation, the piglets were sacrificed and the three intestinal segments (n = ) were collected. upon washing with ice-chilled pbs, tissues were immediately fixed in % polyoxymethylene for histological processing. half of the fixed tissues were embedded in oct (a tissue-freezing medium, china), and then stored at À c until further use. frozen tissue sections ( -mm thick) were cut on a leica cm s cryostat and transferred to poly-l-lysine coated microscope slides. the slides were air-dried and stored for up to weeks at À c before immunofluorescence staining. another half of the fixed tissues were embedded in paraffin wax using standard techniques. slices ( -mm thick) were cut and stored for immunohistochemical staining. the second animal experiment: in brief, a total of conventional . -month-old piglets were divided into four groups and the treatments of these groups were showed in table . piglets in group i (n = ), group ii (n = ), group iii (n = ) and group iv (n = ) were, respectively, inoculated with sterile pbs, sterile pbs, shxb and stc by oral administration. at h after infection, all piglets were subjected to the intestinal ligation as described above. in group i, ml of pbs was injected into the intestinal segments (n = ) h and min before sacrifice as negative controls. in groups ii-iv, the heat-inactivated e. coli were injected into the intestinal segments (n = ) h and min before sacrifice. piglets were then sacrificed and the segments were removed. the following treatments were the same as described above. three-colour fluorescent immunohistology was carried out on cryosections. fc receptors were blocked for min with phosphate-buffered saline (pbs) containing % pig serum. combinations of fitc-sla-dr + pe-swc a + or cd + cd b + (abcam, hongkong) primary mabs were added to the slides overnight at c. subsequently, alexafluor -conjugated donkey anti-goat igg b and alexafluor -conjugated donkey anti-mouse igg (invitrogen, usa) were added for h at room temperature. cell nuclei were stained by a -min incubation with , diamidino- -phenylindole (dapi) solution (invitrogen, usa). each incubation step was followed by washing steps (  min each) in fresh changes of tbs-tween. labelled sections were sealed with nail varnish and stored at c. the negative control slides were treated in an identical manner except the primary antibodies were omitted. in brief, paraffin sections were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol. slides were blocked with serum as described above for immunofluorescence staining. subsequently, porcine cd + , cd + , cd + t cells primary antibody (abcam, hong kong) were, respectively, applied to the sections overnight at c. subsequently, the slides were incubated for another h with an abc-based system (biotinylated goat antimouse antibody was used as the secondary antibody with dab as a chromogen). finally, the slides were counterstained with hematoxylin and mounted for examination. the negative control slides were treated in an identical manner except the primary antibodies were omitted. slides were examined on an axioplan microscope (carl zeiss, oberkochen, germany) or on a leica dmre fluorescence microscope (leica, bensheim, germany). digital images were recorded on a spot rt digital camera by using spot advanced software (diagnostic instruments, sterling heights, mi, usa). measurements and cell counts were performed by using imagepro . (media cybernetics, silver spring, md, usa). dc positive results were measured by the mean number of the positive cells per mm of tissue section, and t cell positive results were expressed as the integral optical density (iod). the statistical analysis was performed using the statistical product and services solutions (spss) package, version . . duncan's test was conducted to determine the differences among treated groups. the means of paired groups were analysed by anova. significance was indicated by a probability of p < . . in order to differentiate into mo-dcs, porcine monocytes were cultured for five days using rpmi media with rpgm-csf and rpil- . the immature mo-dcs became semi-suspended or suspended, with a round appearance and dendritic processes, and often formed clusters at five days (fig. a, left) . mo-dcs stimulated with lps for h assumed a typical dc-like morphology, including a significant size, irregular shape, presenting either as single cells or in clusters on the cell surface at six days (fig. a, right) . it is already known that tgev could infect porcine alveolar macrophages (pams) (laude et al., ) . our results showed that the viral titres of shxb and stc strains in the immature and mature mo-dcs both persistently increased over time, and the two viral titres in the immature mo-dcs were higher than in the mature mo-dcs (fig. b) , which demonstrated that immature mo-dcs were more susceptible to the two strains. however, there was no significance between shxb titre and stc titre in the immature and mature mo-dcs. next, the percentage of infected mo-dcs at different times was detected by flow cytometry. the results showed that the percentage of infected immature and mature mo-dcs reached a peak at h. p.i., then gradually decreased at h. p.i. (fig. c) . to determine whether shxb and stc infection could affect mo-dc maturation, we analysed surface marker expression of cd a, cd / and sla-ii-dr on the mo-dcs. when compared to the immature mo-dcs, mature mo-dcs stimulated by lps significantly upregulated cd a and sla-ii-dr expression ( fig. a and b) . however, the expression of cd a clearly decreased in the immature and mature mo-dcs infected with shxb compared with the others, and the expression of sla-ii-dr in the immature mo-dcs infected with shxb was also lower than others ( fig. a and b) . the level of cd a and sla-ii-dr in the immature and mature mo-dcs infected with stc was similar with the control immature mo-dcs ( fig. a and b) . the results also showed that cd / expression in the immature and mature mo-dcs infected with shxb and stc remained unchanged ( fig. a and b) . the downregulation of co-stimulatory molecules on immature and mature mo-dcs may be a mechanism for virus immune escape for impairing dcs maturation, which may render ag-specific t cells anergy or suppress the t-cell response. next, the shxb and stc infection did not induce the early and late apoptosis of the immature and mature mo-dcs (fig. c) , which indicated that the downregulation of surface makers on immature and mature mo-dcs infected with shxb was not related to the apoptosis induced by the shxb. dcs are able to orchestrate immune responses not only through the expression of surface molecules, but also by secreting cytokines. our results showed that the mature mo-dcs stimulated by lps resulted in significant secretion of the three cytokines as compared with immature mo-dcs (fig. a) . the levels of il- and ifn-g in virulent shxbinfected immature mo-dcs was higher than in controls, but notably lower than those in stc -and lps-stimulated immature mo-dcs. however, the secretion of il- and ifn-g in shxb-and stc -infected mature mo-dcs was not significant. the levels of il- in both shxb-and stc infected immature and mature mo-dcs did not differ significantly from control immature mo-dcs (fig. a) . the results showed that the virulent shxb strain made immature and mature mo-dcs produce the limit of il- , ifn-g for clearance itself, while the attenuated stc could induce the immature mo-dcs to secrete a considerable amount of il- and ifn-g for activation of an immune response. however, neither shxb nor stc could stimulate the secretion of il- . the mixed leukocyte reaction was used to determine whether the ability of shxb-and stc -infected mo-dcs to stimulate t-cell proliferation had been damaged in vitro. our results showed that the mature mo-dcs treated by lps were more stimulatory than immature mo-dcs at dc/t cell ratios of : , : and : (fig. b) . shxb-infected immature and mature mo-dcs had almost no stimulatory capacity on t cell proliferation at dc/t cell ratios of : , : and : , while the stc -infected immature and mature mo-dcs had a stronger ability to activate t cells at a dc/t cell ratio of : (fig. b) . the shxb and stc strains were administered to the piglets, and the intestinal dcs were visualised in serial cryosections at min, h and h p.i. from fig. a -c, the cd b + cd + dcs and swc a + sla-ii-dr + dcs rapidly increased in small intestine villi and lamina propria after min of injecting shxb, but gradually decreased from to h after shxb infection. there was almost no change in the number of these dcs in the small intestinal villi and lamina propria after min, but the number gradually increased from to h after stc infection. these results implied that cd b + cd + dcs and swc a + sla-ii-dr + dcs had a strong ability to uptake and present the shxb in early infection of piglets, then significantly decreased with time, while the capability of these dcs to take up and present stc gradually enhanced with time. from the immunohistochemical staining pictures in vivo (fig. a) , the cd + , cd + and cd + t cells were mainly distributed in the lamina propria, and a small number were distributed in the intestinal villi when the piglets were infected with virulent and attenuated tgev. at min and h after shxb and stc infection, the iod of the cd + , cd + t cells in the porcine intestinal villi and lamina propria was significantly higher than in the control group, and the cd + t cells in the intestinal villi of shxb infected piglets was also significantly higher than in the control group ( fig. b-d) . after h of piglet infection with stc , the cd + t cells in the lamina propria and the cd + and cd + t cells in the intestinal villi and lamina propria were still significantly higher than in the control group, whereas they significantly decreased after h of infection with shxb (fig. b-d) . these results indicated that the number of cd + , cd + and cd + t cells rapidly increased to cause a strong immune response when piglets were infected early with virulent and attenuated tgev. after h of infection, t-cell proliferation was inhibited because of the damaged function of intestinal dcs in the shxb-infected piglets. however, t-cell proliferation was significantly enhanced due to the improved function of intestinal dcs in the stc -infected piglets. the shxb strain could damage the functions of intestinal dcs, and the stc strain could enhance these fig. . effect on the ability of intestinal cd b + cd + dcs and swc a + sla-ii-dr + dcs to sample the heat-inactivated e. coli, migrate and stimulate t cell proliferation in shxb-and stc -infected piglets. bar graphs show the number of cd b + cd + dcs (a) and swc a + sla-ii-dr + dcs (b) in the intestinal villus and lamina propria that sampled the heatinactivated e. coli and migrated at h postinfection with shxb and stc . bar graphs show the proliferation of cd + (c), cd + (d), and cd + (e) t cells in the intestinal villus and lamina propria when the heat-inactivated e. coli were injected into the shxb-and stc -infected piglets h later. data express the mean ae sem (n = ). bars labelled with different letters are significantly different from each other (p < . ). functions to some extent. next, the model antigen (the heat-inactivated e. coli) was used to further determine whether shxb and stc could influence the functions of intestinal dcs. the results showed that after min of injecting heat-inactivated e. coli, the cd b + cd + dcs and swc a + sla-ii-dr + dcs rapidly increased in the small intestine villi of only the injected heat-inactivated e. coli piglets, followed by the stc -infected piglets, and were lowest in the shxb-infected piglets (fig. a and b) . then, these dcs in the intestinal villi of all groups gradually decreased and in the lamina propria gradually increased after h. the number of these dcs in the intestinal villi and lamina propria in stc -infected piglets was still highest among all groups h after injecting heat-inactivated e. coli ( fig. a and b) . at h after injecting the heat-inactivated e. coli, the number of these dcs in villi of piglets infected with shxb was equal to the control group, but the cd b + cd + dcs in lamina propria of the piglets infected with shxb were significant lower than others ( fig. a and b) . these results indicated that stc -infected piglets could enhance the ability of intestinal dcs to uptake the heat-inactivated e. coli and migrate to the lamina propria, whereas this ability was impaired in shxb-infected piglets. the cd + , cd + , cd + t cells were mainly distributed in the intestinal lamina propria of piglets. (see the supplementary data (additional files )). these t cells rapidly increased in the small intestine villi and lamina propria of only injected heat-inactivated e. coli piglets and stc -infected piglets from min to h after injecting the heat-inactivated e. coli, while these t cells had no changes in small intestine villi and lamina propria of the piglets infected with shxb at a similar timepoint (fig. c-e) . this showed that the stc infected piglets had an enhanced ability of intestinal dcs to stimulate t-cell subset proliferation, while the shxbinfected piglets had an impaired ability of intestinal dcs to stimulate t-cell subset proliferation. a recent report showed that the intestinal dc network consisted of dcs derived from monocytes and pre-dcs (bogunovic et al., ) . therefore, porcine mo-dcs were used for researching the relationship between porcine dcs and tgev in vitro. phenotypically, porcine mo-dcs are characterised as cd a + sla-dr-ii + (mhc-ii + )cd / + and swc a + (cd a + ) (summerfield and mccullough, ) . in vivo, co-expression of cd , sla-dr-ii, cd b (cd r ) and swc a has previously been described (haverson et al., ) as being characteristic for dcs in the porcine intestinal lamina propria. in our work, the cd a + sla-dr-ii + cd / + swc a + mo-dcs and sla-dr-ii + /cd + / cd b + /swc a + -dcs were used for in vitro and in vivo analyses. a similar infectivity of sars coronavirus (sars-cov) in human immature and mature dcs was previously observed (law et al., ) . similar to sars-cov, both shxb and stc also infected immature and mature mo-dcs, but the infectivity in immature mo-dcs was higher than in mature mo-dcs. this may be because the major receptor of tgev (aminopeptidase n) is mainly distributed in immature mo-dcs, and the endocytosis of immature mo-dcs was also significantly higher than mature mo-dcs (these data were not shown). a previous study showed that the virulent tgev robustly infected and rapidly destroyed the villous epithelial cells in the porcine small intestine to cause the rapid death of piglets, while the attenuated virus had a poor ability to infect and evoke the sickness of the piglets (kim and chae, ) . our work was consistent with this, and we speculated that virulent tgev increased their replication in dcs for their dissemination. maturation of dcs is a critical process that results in presenting viral antigens to activate t cells and initiating an antiviral immune response (zhou et al., ) . the phenomenon has been reported in the classical swine fever virus (carrasco et al., ) and the prrs virus (peng et al., ) . it is known that dcs present antigen with cd a or sla-ii-dr as the first signal and cd / as the second costimulatory signal to activate t cells (bertram et al., ) . shxb may act by inhibiting mo-dc maturation, because it significantly down-regulated the expression of cd a and sla-dr-ii on the immature and mature mo-dcs. why did shxb cause a reduction in cd a and sla-dr-ii on the mature mo-dcs? it is possible that virus infection resulted in a loss of tlr- , which is essential for lps responsiveness (poltorak et al., ) . the surface markers of stc infected immature and mature mo-dcs changed slightly (fig. c) , which implied that stc caused a weak injury on the mo-dcs. similarly, the expression of cd , cd , mhc i and mhc ii did not significantly change in chicken mo-dcs infected by low pathogenicity avian influenza (lpai), while the markers notably decreased on mo-dcs infected by high pathogenicity avian influenza (hpai) (vervelde et al., ) . in addition, there was a negligible change in cd / expression on immature and mature mo-dcs infected by shxb or stc . this suggested that virulent tgev inhibit mo-dcs maturation through decreasing the antigen-presenting molecules, rather than the co-stimulatory molecules. furthermore, the downregulation of surface markers on the mo-dcs infected with shxb was not caused by their apoptosis. cytokine response induced by virus-infected mo-dcs is another critical process that results in presenting viral antigens to activate t cells. il- , believed to be the principal th -type cytokine, is necessary for cell-mediated immunity (cmi) (barchet et al., ) . ifn-g plays an important role in inducing immunostimulatory and immunomodulatory effects (chen et al., ) . il- is a predominant th -type cytokine that can downregulate il- production and cmi (yang et al., ) . in this article, the promotion of il- and ifn-g secretion both in immature and mature mo-dcs infected by shxb and stc strains implied that mo-dcs developed th immune responses to tgev infection, which was similar to the report that rotavirus (rv)-infected dcs promote their capacity to induce the polarisation of allogenic naive t cells towards a th response (narvaez et al., ) . however, the virulent shxb only produced a slight of il- and ifng, as little as possible to make the mo-dcs clear themselves, while stc , as a vaccine strain, induced the secretion of considerable cytokines to improve the immature mo-dcs' immune response. a recent report showed that only when the amount of a virulent virus reached a threshold, called virulence threshold, its virulence reached the strongest (lancaster and pfeiffer, ) . in this study, it might because the number of shxb did not reach its virulence threshold in the mature mo-dcs, like the attenuated stc strain, thus no difference in the expression of il- and ifn-g in shxb-and stc -infected mature mo-dcs was found in our work. the stc and shxb strains failed to stimulate il- secretion on the immature and mature mo-dcs. because dcs are central to the development of the acquired t-cell response, pathogens have evolved mechanisms to reduce the capacity of infected dcs to prime the adaptive immune response. virulent shxb infection inhibits mo-dc maturation and secreting cytokines, so that infected mo-dcs are potentially less able to stimulate t-cell responses, like dengue virus (dv) (dejnirattisai et al., ) . however, attenuated stc was unlike shxb because it induced the mo-dcs to stimulate t-cell proliferation at a dc/t ratio of : . for the phenomenon that there is a minor but significant difference between the shxb and stc infected mature cells only at a ratio of : . it might that stc did not inhibit the expression of cd a in the mature mo-dcs, while shxb suppress this compared with the control immature mo-dcs. moreover, the level of secreting il- in stc infected mature mo-dcs ( . pg ml À ) was higher than shxb infected mature mo-dcs ( . pg ml À ), thus it was possible that the significant difference between the shxb and stc infected mature cells stimulated the t cells proliferation. from the results, we observed that the more it was in the number of mo-dcs, the stronger it had the ability to activate the t cells proliferation. thus there is a minor but significant difference between the shxb and stc infected mature cells only at a dc/t ratio of : but not at the other ratios. in order to further verify that shxb strain severely impaired the functions of dcs in vitro, we carried out in vivo analyses. when encountering a virus, the dcs rapidly uptake the invading virus and migrate to the lymph nodes in order to activate t cells. because of stronger virulence, shxb robustly induced the intestinal epithelial cells to secrete the pro-inflammatory chemokines ccl , ccl and ccl , leading to a rapid recruitment of dcs and t cells to the intestinal villi (the data was not shown). therefore, after min of injection of shxb, the number of cd b + cd + dcs, swc a + sla-dr-ii + dcs and cd + , cd + and cd + t cells in intestinal villi rapidly increased; such a response did not occur after stc injection. having acquired the antigens, these dcs could migrate from intestinal villi to the lymph node for activating t cells, therefore, after h of shxb infection, the dcs gradually reduced in number in the intestinal villi, and increased in the lamina propria. after h of virus infection, the number of dcs and cd + , cd + and cd + t cells in the intestinal villi and lamina propria of piglets infected with shxb was significantly reduced; in contrast, there were significantly more of these cell types in stc -infected piglets. these results suggest that shxb significantly impaired the ability of porcine dcs to process and present antigens to activate t cells, and was consistent with that shxb significantly damaged the ability of porcine mo-dcs to process and present antigens to activate t cells in vitro. the stc strain was similar to the vaccine strain in that it enhanced the porcine dcs' ability to process and present antigens to stimulate cd + , cd + and cd + t-cell proliferation at h postinfection. it is known that the cd protein complex is an important t cell marker for the maturation of t cells (chetty and gatter, ) . cd + t cells play a central role in helping b cells make antibodies and inducing macrophages to develop enhanced microbicidal activity (zhu and paul, ) . cd + t-cells can kill virally infected target cells by secreting perforin and granzymes (mosmann et al., ; stenger et al., ) . from our results, cd + t cells accounted for - % of the total cd + t cells, while cd + t cells were only - % of the total cd + t cells. therefore, there was a tendency to develop humoral immunity for the intestinal mucosal immune response, and it also indirectly explained the important antiviral role of siga in mucosal immunity. lastly, the model antigen (the heat-inactivated e. coli) was used to further prove that virulent and attenuated tgev could influence the functions of intestinal dcs. the cd b + cd + dcs and swc a + sla-ii-dr + dcs in the piglets infected with stc had a higher ability to take up and migrate and stimulate t-cell proliferation. nevertheless, these two intestinal dcs had a significantly damaged ability to take up, migrate and stimulate t-cell subset proliferation in the piglets infected with shxb. this study indicated that the shxb strain severely impaired the ability of dcs to uptake, migrate, and induce the activation of t lymphocytes, while the stc strain enhanced these functions of dcs in vitro and in vivo. this might be due to the differences in pathogenesis of virulent and attenuated tgev in piglets, and could help us to develop a better strategy to prevent tgev infection by improving the functions of dcs. furthermore, this study also provides a reference for the mucosally transmitted infectious diseases (pneumonia, tuberculosis, measles, hiv/aids, avian flu, sars, and multidrug-resistant bacteria). the authors declare that they have no competing interests. origin, precursors and differentiation of mouse dendritic cellsnature reviews lassa virus infection of human dendritic cells and macrophages is productive but fails to activate cells complement-induced regulatory t cells suppress t-cell responses but allow for dendritic-cell maturation role of t cell costimulation in anti-viral immunity origin of the lamina propria dendritic cell network porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties interaction of classical swine fever virus with dendritic cells regulation of dendritic cells and macrophages by an anti-apoptotic cell natural antibody that suppresses tlr responses and inhibits inflammatory arthritis cd : structure, function, and role of immunostaining in clinical practice a complex interplay among virus, dendritic cells, t cells, and cytokines in dengue virus infections pathogenicity of an attenuated strain of transmissible gastroenteritis virus for newborn pigs identification of an important immunological difference between virulent varicella-zoster virus and its avirulent vaccine: viral disruption of dendritic cell instruction determination of population thresholds for settlement functions by the reed-muench method professional and non-professional antigen-presenting cells in the porcine small intestine studies on cell cultivation and pathogenicity of attenuated transmissible gastroenteritis virus stc experimental infection of piglets with transmissible gastroenteritis virus: a comparison of three strains (korean, purdue and miller) sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins viral population dynamics and virulence thresholds replication of transmissible gastroenteritis coronavirus (tgev) in swine alveolar macrophages chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells the molecular biology of coronaviruses functions of cd t-cell subsets secreting different cytokine patterns interaction of rotavirus with human myeloid dendritic cells infection of ligated intestinal loops with hemolytic escherichia coli in the pig human dendritic cells infected with the nonpathogenic mopeia virus induce stronger t-cell responses than those infected with lassa virus modulations of phenotype and cytokine expression of porcine bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria differential effects of cytolytic t cell subsets on intracellular infection the porcine dendritic cell family chicken dendritic cells are susceptible to highly pathogenic avian influenza viruses which induce strong cytokine responses efficacy of a transmissible gastroenteritis coronavirus with an altered orf- gene antiinflammatory properties of il- rescue small-for-size liver grafts role of dendritic cell synthesis of complement in the allospecific t cell response cd t cells: fates, functions, and faults this work was supported by grant number from the national science grant of china and the priority academic program development of jiangsu higher education institutions (papd). supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . / j.vetmic. . . . key: cord- -ipeup mp authors: chen, yuqiu; jiang, lei; zhao, wenjun; liu, liangliang; zhao, yan; shao, yuhao; li, huixin; han, zongxi; liu, shengwang title: identification and molecular characterization of a novel serotype infectious bronchitis virus (gi- ) in china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ipeup mp avian infectious bronchitis coronavirus (ibv) is a major poultry pathogen. a characteristic feature of ibv is the occurrence of many different strains belonging to different serotypes, which renders complete control of the disease by vaccination a challenging task due to the poor cross-protection between different serotypes. in this study, based on the results of s sequence analysis and virus cross-neutralization tests, ibv strain ck/ch/lgx/ was found to be genetically and antigenically different from other known ibv types, representing not only a novel genotype, but also a novel serotype (designated as gi- ). viruses belonging to this novel serotype have been isolated from several regions in china in recent years, suggesting endemic circulation of the serotype in various geographic locations in china. further studies by complete genomic analysis showed that strain ck/ch/lgx/ may have originated from recombination events involving lx genotype ibvs and an as-yet-unidentified ibv donating a s gene, or from the result of accumulation of mutations and selections, especially in the s gene, from a lx genotype virus. ck/ch/lgx/ is a nephropathogenic strain, although it had broader tissue tropism (respiratory, digestive, urinary, and reproductive tracts) among chickens challenged at one day old. infection of the oviducts with ck/ch/lgx/ found in this study may have severe implications because the virus will likely induce the occurrence of false layers. coronaviruses, family coronaviridae in the order nidovirales, are generally responsible for mild enteric and respiratory infections in both humans and animals (masters, ) . they are now recognized as emerging viruses with a propensity to cross into new host species, as have been shown by previous and recent outbreaks of severe acute respiratory syndrome and middle east respiratory syndrome (coleman and frieman, ) . to date, coronaviruses were classified into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the genus gammacoronavirus is mostly composed of viruses isolated from birds. however, gammacoronaviruses have also been detected in the beluga whale (mihindukulasuriya et al., ) and bottlenose dolphin (woo et al., ) . the most economically important of the avian gammacoronaviruses are infectious bronchitis virus (ibv) and turkey coronaviruses. ibv, which was the first identified coronavirus and was isolated in the s, causes highly contagious infectious bronchitis in domestic fowl, a respiratory, renal, and genital disease, which causes serious economic consequences worldwide (cavanagh, ) . coronaviruses are enveloped, positive-sense, and the largest known rna viruses, having a genome length of approximately kb. about two-thirds of the genome consists of two large overlapping open reading frames (orfs), orf a and orf b, which are translated as the polyproteins pp a and pp ab, and then processed by virus-encoded proteinases into or nonstructural proteins (ziebuhr, ) . the remaining one-third of the genome encodes the virus structural spike (s), membrane (m), enveloped (e), and nucleocapsid (n) proteins, as well as four nonstructural accessory proteins ( a, b, a and b). the s glycoprotein can be cleaved into two subunits, s and s , and contains the regions of both b-and t-cell epitopes that are important for virus neutralization and the reorganization of virus-infected cells (reguera et al., ; satoh et al., ) . the s domain occupies the amino proximal half, which contains the receptor-binding domain, and the s domain occupies the other half, which contains elements involved in membrane fusion. in contrast to species belonging to the alpha-and betacoronaviruses, which occur as only one or two different serotypes, there are many different serotypes/genotypes of the chicken coronavirus ibv (wickramasinghe et al., ) . typing of ibv strains is based on the feature of the s protein, especially s domain (cavanagh, ) . the high mutation and recombination rate of ibv, especially in the s subunit of the s protein gene, has led to the emergence of new variants, particularly in europe and north america, and more recently in intensive poultry farms in china. among the ibvs, two very important genotypes, lx (qxlike) and ck/ch/ldl/ i, are believed to have originated in china in mid- and have since spread to other regions of the world (ababneh et al., ; marandino et al., ) . more recently, another novel genotype, guandong/xindadi (xdn), which is also believed to have originated in china from independent recombination events between the lx and /b ibv genotypes, have emerged in italy and spain (moreno et al., ) . the continuous emergence of new ibv genotypes in china that later spread to other regions of the world has not only prompted the development of appropriate control programs aimed to mitigate the occurrence of the disease caused by those new ibv genotypes, but also emphasize the importance of continuous surveillance of ibv in chicken flocks in china. meanwhile, extensive investigations, involving molecular characterization (especially the complete genome), antigenicity, and pathogenicity, are necessary to clarify the origin of the viruses and are significant for the control of diseases caused by new variants. in this study, a novel serotype (designated as gi- ) of ibv was found to be mainly circulated in south china in recent years and the complete genome, antigenicity, and pathogenicity were investigated to elucidate the origin and evolution of the novel serotype. the ibv strain ck/ch/lgx/ was isolated from the proventriculus of a diseased -day-old yellow broiler that had been vaccinated against ibv with live attenuated h vaccine at days old and boosted at days old with live attenuated / vaccine. the bird showed obvious respiratory signs at days old. in this flock, morbidity was about % and mortality was %. gross lesions were mainly mild with tracheitis, proventriculitis, and nephritis in some chickens. the proventriculi of the dead chickens were enlarged and had pale or mottled pale serosa. kidneys of the birds were enlarged and pale. virus isolation was performed as described by . briefly, the proventriculi were homogenized, clarified by centrifugation, filtered, and then inoculated into specific pathogen-free (spf) eggs (harbin veterinary research institute, harbin, china) on embryonation day via the allantoic cavity. the isolate were purified and propagated by inoculating and passaging three times in the allantoic cavity of -day-old spf embryonated chicken eggs until characteristic ibv lesions, such as dwarfing, stunting, or curling of embryos, were observed hewson et al., ). the infectious allantoic fluid was clarified by centrifugation at g for min at c. total viral rna was extracted from the supernatant using trizol reagent (invitrogen corporation, carlsbad, ca, usa), following the manufacturer's protocol, and then stored at À c for further use. ibv-specific primers (liu et al., ) spanning the entire viral genome were used to reverse transcribe and amplify the viral rna contained in ml of extracted total rna using the primescript tm one-step rt-pcr kit ver. (takara bio inc., shiga, japan) as described by the manufacturer. briefly, a -ml reaction was set up on ice containing ml of rna, ml of mm forward, ml of mm reverse primer, . ml of  step buffer (as supplied by the manufacturer), ml of primescript step enzyme mix, and water to a final volume of ml. the reaction was incubated at c for min to allow cdna synthesis, and then increased to c for min, followed by cycles of c for s, c- c (depending on the primer set) for s, and c for min. the reaction underwent a final extension phase at c for min and was then held at c. the and ends of the viral genomes were confirmed by rapid amplification of cdna ends using a commercial kit (takara bio, inc.) as described by liu et al. ( ) . for each amplicon, ml of pcr products were separated on a % agarose/ tris/borate/ethylenediaminetetraacetic acid gel to confirm the pcr product size and to estimate the amount of dna by comparison with standards. the pcr products were sequenced directly and/or cloned into a pmd -t vector (takara bio inc.) following the manufacturer's instructions. for each amplicon, three to five clones were sequenced. the s gene of strain ck/ch/lgx/ was used to search the genbank database (https://blast.ncbi.nlm.nih.gov/blast.cgi) using the blastn program for ibv sequences. six sequences sharing more than % nucleotide identity with ck/ch/lgx/ were selected for construction of a phylogenetic tree for sequence comparison. in addition, the s gene sequences of ibv reference strains with different genotypes were also selected for comparison in this study ( fig. ) (valastro et al., ) . the phylogenetic tree was constructed using a neighbor-joining algorithm with bootstraps based on an alignment generated using molecular evolutionary genetics analysis (mega) software version . . (tamura et al., ) . the s nucleotide and amino acid sequences of selected viruses (based on the phylogenetic tree results) were also aligned and compared with those of strain ck/ch/lgx/ using lasergene software (dnastar, inc., madison, wi, usa). percent similarities were calculated to determine distances between nucleic acid and amino acid pairs. to determine a consensus sequence for the complete genome of strain ck/ch/lgx/ , the sequence contigs were combined, manually edited, and assembled using lasergene software. the genomic sequences between ck/ch/lgx/ and other reference ibvs were analyzed using lasergene software to map each of the genes in the genome of strain ck/ch/lgx/ . the sequence of the complete genome of strain ck/ch/lgx/ was submitted to the genbank database under the accession number kx . to identify the recombination events, the blastn program was used to search the genbank database for ibv sequences that were homologous to strain ck/ch/lgx/ . simplot analyses were performed using a -bp window with a -bp step. ibv strain h was used as the query strain. six ibv strains, including ck/ch/lgx/ in this study and five other strains representing five serotypes ( / for /b serotype, h for massachusetts serotype, ck/ch/ldl/ for tw i serotype, ck/ch/ldl/ i for ck/ch/ldl/ i serotype, and ck/ ch/ldl/ for lx serotype) were subjected to one additional fig. . phylogenetic trees constructed with the nucleotide sequence alignments of the s glycoprotein genes using the nearest neighbor-joining method with bootstrap calculations (valastro et al., ) . the genbank accession numbers of the ibv strains are shown in parentheses. the strain ck/ch/lgx/ is indicated by a $. all the ibv strains were clustered into genotypes (gi-gvi) and a number of inter-lineage recombinants. ninety-three ibv reference strains in gi were further grouped into distinct viral lineages (gi- -gi- ) (valastro et al., ) . our ck/ch/lgx/ strain was clustered into a novel lineage in gi with other ibv reference strains and designated as gi- . passage in embryonated chicken eggs for virus stock preparation and cross-neutralization tests. the virus titers were determined in -day-old spf chicken embryos by the allantoic route of inoculation as described by chen et al. ( ) . in addition to embryo microscopic changes, ibv rna in allantoic fluid was detected by rt-pcr as described by chen et al. ( ) to determine virus replication and subsequently calculate the virus titer. the median embryo infectious dose (eid ) was calculated using the method of reed and muench ( ) . the antisera used for this test included anti- / , anti-h , anti-ck/ch/ldl/ , anti-ck/ ch/ldl/ i, anti-ck/ch/ldl/ , and anti-ck/ ch/lgx/ , which were prepared as described by gao et al. ( ) . for virus neutralization, the b vn method with constant virus and diluted serum was employed in spf chicken embryos for serotyping. briefly, sera were serially diluted two-fold and mixed with eid of the ibv strains. after incubation for h at c, each of the virus-serum mixtures was inoculated into the allantoic cavity of five spf chicken embryos, which were observed for days. similarly, ibv rna in allantoic fluid was detected by rt-pcr and used to determine virus replication and subsequently calculate the end-point titer of serum. the end-point titer of each serum sample was calculated using the method of reed and muench ( ) . thirty -day-old spf white leghorn layer chickens were separated into two groups of chickens per group, which were housed in separate isolation units. birds in group were challenged with the ck/ch/lgx/ strain at -day-old via the intranasal and ocular routes with eid in . ml of diluent per bird. birds in group were not challenged and served as negative controls. the chicks were examined daily for clinical signs of infection . morbidity and mortality were recorded daily. five chickens in each group were humanly killed using carbon dioxide/oxygen. the dead chickens were carefully examined, especially for lesions in the trachea, kidneys, and proventriculi. then, tissues of the tracheal, lungs, kidneys, and cecal tonsils were collected, fixed in % neutral buffered formalin, embedded in paraffin, sectioned at a thickness of mm, and mounted on glass slides, which were stained with hematoxylin and eosin. immunohistochemical (ihc) analysis was performed to detect the ibv antigen using monoclonal antibody d against the n protein as described by han et al. ( ) and gao et al. ( ) . blood samples were collected on day from birds and on days , , , , and post-challenge from all birds to confirm the presence of antibodies against ibv using a commercial enzymelinked immunosorbent assay kit (idexx laboratories, inc., westbrook, me, usa) in accordance with the manufacturer's instructions. the percentage of seropositive birds for each group in this study was calculated. all surviving birds were killed humanely using carbon dioxide/oxygen at months of age, which was followed by exsanguination. post-mortem examinations were performed with special attention to abnormalities in the oviducts and kidneys. the kidney and oviduct samples were collected to detect the presence of ibv by ihc analysis. genotyping based on the phylogenetic analysis of the s gene from our ck/ch/lgx/ and reference ibv strains assigned the viruses into different clusters (fig. ) . the ibv reference strains were clustered into genotypes that together comprise distinct viral lineages (gi- -gi- ; gii-gvi) and a number of inter-lineage recombinants (valastro et al., ) . the isolate ck/ ch/lgx/ in this study was clustered into a novel lineage in genotype i (gi) with ibv strains isolated in china and clearly set apart in the phylogenetic tree from the reference strains; hence, we designated the novel genotype as gi- . viruses of the gi- genotype possessed more than . % and . % nucleotide and amino acid identities, respectively, between each other in this study, supporting the classification of these viruses as a novel genotype. one ibv strain lie together with the qxibv strain, and have been described previously as lx genotype or qx-like viruses . comparatively, the lx genotype (qx-like) viruses are closely related to the gi- genotype, sharing approximately . % and . % nucleotide and amino acid identities, respectively, with the s protein of the gi- genotype viruses selected in this fig. . simplot analysis of the complete genomic sequences of strains ck/ch/lgx/ and ck/ch/ldl/ . analysis was performed using simplot software version . . to identify potential recombination breakpoints (chen et al., ) . a -bp window with a -bp step was used. strain h was used as the query strain. study. the massachusetts genotype h had less than % nucleotide and amino acid identities with the s protein of gi- genotype viruses. the tw i genotype, which has been described previously (xu et al., ) . the s protein of genotypes gi- and tw i shared approximately % nucleotide and amino acid identities. the strains ck/ch/ldl/ i and q formed a unique cluster that shared the lowest identities with the gi- genotype (nearly %). the /b genotype shared about % and % nucleotide and amino acid identities, respectively, with the s protein of the gi- genotype. the genome of ibv strain ck/ch/lgx/ is , nucleotides in length, excluding the poly(a) tail, and shows typical ibv organization. the end of the viral genome contains a -nt untranslated region (utr) followed by a -nt replicase gene encoding for two large polyproteins, pp a and pp ab, which occupied about two-thirds of the viral genome. four structural and accessory proteins were downstream of the replicase gene followed by a -nt utr at the end of the genome. results of a blastn search showed that not only the s gene, but the complete genome of the ck/ch/lgx/ strain, showed the closest genetic relatedness with lx genotype strains. hence, the lx genotype strain ck/ch/ldl/ was used for simplot analysis with strain ck/ch/lgx/ . as illustrated in fig. , a fragment from approximately nt position , to , in the genome of strain ck/ch/lgx/ was obviously different from that of strain ck/ch/ldl/ . this approximate -nt fragment occupied most of the s gene and small parts of sequence at the end of the s gene. the remaining regions in the genome were very similar, although some regions showed slight diversities between the two strains, implicating the involvement of lx genotype ibvs in the origin of ck/ch/lgx/ -like strains. the results of the cross-neutralization tests using the ibv strain ck/ch/lgx/ and antisera against the five ibv strains, which represented the massachusetts, /b, lx , ck/ch/ldl/ i, and tw i serotypes, showed that the strains belonged to different serotypes (table ). in line with the genotyping results, strain ck/ ch/lgx/ was antigenically distinct from the massachusetts, /b, ck/ch/ldl/ i, and tw i serotypes. comparatively, the cross-neutralization results showed that strain ck/ch/lgx/ was antigenically close to the lx genotype strain ck/ch/ldl/ (table ) with an r value of . . these results demonstrated that strain ck/ch/lgx/ represents a new serotype (also designated as gi- ) that is antigenically distinct from other ibv serotypes. all of the chickens exhibited mild respiratory signs at - days post-challenge (dpc) with the ibv strain ck/ch/lgx/ , and three of them died at , , and dpc, respectively. the most remarkable lesions were confined in the kidneys of the five chickens killed at days pdc and the three dead chickens. the affected kidneys were swollen and pale with tubules and ureters distended with urates (fig. a) . the most interesting observation was the dilatation and serous fluid accumulation in the oviducts of two of the three layer hens that survived dpc (fig. b) . no clinical signs were observed and no birds died in the control group during the experimental period. no lesions were found in the kidneys or oviducts of the birds in the control group at the end of the experiment. viral antigens were detected by ihc analysis in the kidneys, secondary bronchi ( fig. a and b) , and enterocytes of the cecal table results of reciprocal b virus neutralization tests using ck/ch/ldl/ and other ibv strains (serum dilution using a constant amount of virus). tonsils of the five chickens infected with strain ck/ch/lgx/ at dpc ( fig. c and d) . obvious lesions (nephritis) were found in the kidneys of the five chickens killed at days pdc and the three dead chickens, and ihc-positive cells were observed in the kidneys of the five chickens killed at days pdc with ibv strain ck/ch/lgx/ as nephropathogenic strains ( fig. e and f) . most of the birds seroconverted by dpc and all seroconverted by dpc (table ). there was no seroconversion in any chicken in the negative control group. ibv has progressively emerged as the cause of moderate to severe disease in chickens, with different variants/genotypes/ serotypes being detected in recent years in china. the ibv isolate ck/ch/lgx/ , investigated in the present study, displayed distinct molecular features with respect to other genotypes and was clearly set apart from those that are not only used as vaccines in china like massachusetts and /b, but also from chinese variants of ibv, such as genotypes lx , ck/ch/ldl/ i, and tw i. dead survival ck/ch/lgx/ / / / / / / / / / / / negative control / / - / - / / / / / / a the post-mortem examinations were only conducted for chickens that died from challenge with ck/ch/lgx/ strain, as well as for the chickens that survived days post-challenge. all of the dead chickens, but none of the surviving chickens, had nephritis. b antibody responses against ibv were examined from to days post-challenge. c only three of the surviving chickens were hens, which were examined for oviduct abnormalities. the closest relative genotype was ibv lx , the most predominant variant in china , which shared approximately % nucleotide and amino acid identities with isolate ck/ch/lgx/ . there are several publications of the ibv genotype that included ck/ch/lgx/ , but unfortunately these reports do not agree on genotype designations, such as a by ji et al. ( ) and ch iii by luo et al. ( ) and feng et al. ( ) . also, the virus genotype designations were inconsistent even in reports by the same research group (ji et al., ; feng et al., ) . the ch iii and a genotypes have previously been used to classify other ibv genotypes in china (liu et al., ; xu et al., ) . herein, by comparison with the results from valastro et al. ( ) , the ibv reported in this study was designated as the gi- genotype. comparison of the s genes of the gi- genotype with those of other genotypes selected in this study revealed not more than . % nucleotide and amino acid identities, which could explain the distinct antigenic relationship of ck/ch/lgx/ , as compared with other serotypes (cavanagh, ) . analysis of complete genomic sequence of the ck/ch/lgx/ revealed that the virus exhibited high identity in the entire genome with lx genotype ibvs with the exception of the s protein gene, which is markedly diverse. we proposed that the emergence of gi- genotype viruses in chicken flocks in china resulted from recombination events involving lx genotype ibvs and an asyet-unidentified ibv donating a s gene that encoded a protein of low amino acid identity to those of other ibvs (approximately . %). this was very similar to that of turkey coronavirus, which was thought to have arose through recombination of the s gene (jackwood et al., ) . alternatively, the gi- serotype might have originated from the accumulation of mutations and selection, especially in the s gene, because s domain showed the high sequence diversity in ibv (wickramasinghe et al., ) . if this is the case, strain ck/ch/lgx/ might have undergone extensive divergent evolution and acquired the abilities to infect and spread among chicken flocks. the higher sequence similarity in other regions in the genome outside of the s gene suggest a common origin of these sequences and could explain the closer antigenicity between ck/ch/lgx/ and ck/ch/ldl/ , as compared with those of other serotypes, because in addition to the five conformation-dependent neutralizing antigenic sites mapped on s , another immunodominant region was mapped in the nterminal region of s (wickramasinghe et al., ) and some antigenic epitopes that have roles in the protection have also been identified on n proteins of ibv (ignjatovic and sapats, ) . in this study, mild respiratory signs developed in the chicks at - dpc and severe nephritis present in the dead chickens was caused by infection with the isolate ck/ch/lgx/ and no obvious lesions were observed in the proventriculus. this is in contrast to the observation in the field cases, where obvious respiratory signs and proventriculitis were observed. there are at least two possible reasons for this discrepancy: first, ibv strain ck/ ch/lgx/ may not be the only causative pathogen responsible for the occurrence of the outbreak, and secondary infections caused by other microorganisms enhance the severity and contribute to the disease development. second, it is also possible that different genetics of the chicks in the field cases and of the chicks experimentally infected are responsible for the difference in tropism of the virus; it is believed that genetics of chickens play an important role in differences in ibv susceptibility and tropism (ignjatovic et al., ) . it is believed that ibv mainly infects the respiratory and urogenital tracts, but it has long been known that ibv can be isolated from digestive organs, such as the proventriculus, intestines, and cloaca, and persists in the cecal tonsils (ambali and jones, ; ambali, ; montgomery et al., ) . similarly, the results of this study showed that obvious positive immunohistochemical reactions developed not only in the cytoplasm of the tubular epithelial cells and in the mucous membrane of the ureters and collecting ducts in the kidney, and in the epithelial cells of the tracheal mucosa of the secondary bronchi at dpc, but the viral antigens were also detected in the cecal tonsils. this result suggests a broad tissue tropism of strain ck/ch/lgx/ , similar as that of its deduced parental lx genotype viruses (benyeda et al., ; de wit et al., ; mork et al., ) . the clinical manifestations of ibv in the oviduct are of high economic importance in the poultry industry because an infection of the oviduct may have severe implications comprising a drop in egg production, poor egg quality, and the occurrence of so-called "false layers." it has been reported that ibv genotypes lx (benyeda et al., ; de wit et al., ) , massachusetts serotype h (duff et al., ) , is/ / -like virus isolated in the middleeastern region (awad et al., ) , and nrtw i serotype viruses isolated in china related to cystic oviducts in layer flocks and induce false layers. an interesting observation in this study was that the lesions of cystic oviducts were observed in the surviving chickens that were challenged at day old with strain ck/ ch/lgx/ . this finding is in agreement with the field cases, in which false layers were observed in the layer flock from which strain ck/ch/lgx/ was isolated. the gi- type first emerged in in guangdong province and has mainly circulated in south china in recent years (fig. ) . meanwhile, some strains of this type were also isolated in other regions of central china, implicating the wide distribution and spread of this ibv type and emphasizing the importance of continuous surveillance of ibv in chicken flocks in china. in addition, it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control gi- type ibv strains in future because the gi- viruses related to cystic oviducts in layer flocks and might induce false layers. ). presence of infectious bronchitis virus strain ck/ch/ldl/ i in the middle east early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus recent studies on the enterotropic strain of avian infectious bronchitis virus experimental infection of is/ / -like infectious bronchitis virus in specific pathogen free and commercial broiler chicks comparison of the pathogenicity of qx-like, m and /b infectious bronchitis strains from different pathological conditions coronavirus avian infectious bronchitis virus molecular and antigenic characteristics of massachusetts genotype infectious bronchitis coronavirus in china coronaviruses: important emerging human pathogens 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 infection of day old chicks with infectious bronchitis (ib) virus and subsequent anatomical abnormalities analysis of s gene of avian infectious bronchitis virus isolated in southern china during 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 rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus susceptibility of three genetic lines of chicks to infection with a nephropathogenic t strain of avian infectious bronchitis virus emergence of a group coronavirus through recombination phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in china comparative analysis of four massachusetts type infectious bronchitis coronavirus genomes reveals a novel massachusetts type strain and evidence of natural recombination in the genome genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between phylogenetic analysis of the s glycoprotein gene of infectious bronchitis viruses isolated in china during phylodynamic analysis of avian infectious bronchitis virus in south america the molecular biology of coronaviruses identification of a novel coronavirus from a beluga whale by using a panviral microarray attempts to reproduce a runting/stunting-type syndrome using infectious agents isolated from affected mississippi broilers a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus ibv strains qx and b are not related to the sialic acid binding properties of their spike proteins a simple method of estimating fifty percent endpoints structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies screening and identification of t helper and linear immunodominant antibody-binding epitopes in the spike domain and the nucleocapsid protein of feline infectious peritonitis virus mega : molecular evolutionary genetics analysis (mega) software version . s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification the avian coronavirus spike protein discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus isolation and identification of four infectious bronchitis virus strains in china and analyses of their s glycoprotein gene the coronavirus replicase the authors declare that they have no competing interests. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j. vetmic. . . . key: cord- - ydh jev authors: meng, x.j title: heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ydh jev porcine reproductive and respiratory syndrome virus (prrsv) continues to be a major problem to the pork industry worldwide. increasing data indicate that prrsv strains differ in virulence in infected pigs and are biologically, antigenically, and genetically heterogeneous. it is evident that the current vaccines, based on a single prrsv strain, are not effective in protecting against infections with the genetically diverse field strains of prrsv. the recent outbreaks of atypical or acute prrs in vaccinated pigs have raised a serious concern about the efficacy of the current vaccines and provided the impetus for developing more effective vaccines. special attention in this review is given to published work on antigenic, pathogenic and genetic variations of prrsv and its potential implications for vaccine efficacy and development. although there are ample data documenting the heterogeneous nature of prrsv strains, information regarding how the heterogeneity is generated and what clinical impact it may have is very scarce. the observed heterogeneity will likely pose a major obstacle for effective prevention and control of prrs. there remains an urgent need for fundamental research on this virus to understand the basic biology and the mechanism of heterogeneity and pathogenesis of prrsv. porcine reproductive and respiratory syndrome (prrs), characterized by severe reproductive failure in sows and respiratory diseases in young pigs, was ®rst recognized in the united states (u.s.) in (keffaber, ; hill, ) . since its appearance, prrs has devastated the swine industry with tremendous economic losses (polson et al., ) . the causative agent of prrs, porcine reproductive and respiratory syndrome virus (prrsv), was ®rst isolated by wensvoort et al. ( ) in the netherlands using porcine alveolar macrophages (pam) and was designated as lelystad virus (lv). in the u.s., prrsv was ®rst isolated and characterized in a continuous cell line atcc cl collins et al., ) . prrsv is a small, enveloped, single positive-stranded rna virus (wensvoort et al., ; ben®eld et al., ; meulenberg et al., a, b) . prrs has now been recognized worldwide (plana et al., ; saito et al., ; valicek et al., ; madsen et al., ; chueh et al., ) and is considered to be an economically important global disease. although prrsv strains identi®ed from around the world cause similar diseases in pigs, increasing data indicate that prrsv strains differ in virulence in infected animals and are antigenically and genetically heterogeneous. more recently, swine herds in the u.s. have experienced outbreaks of a severe form of prrs characterized by abortion and high mortality in pregnant sows (botner et al., a; bell, ; mengeling et al., ; lager et al., ; osorio et al., ) . this form of prrs has been referred to as sow abortion and mortality syndrome, atypical prrs, severe prrs and acute prrs. surprisingly, many of the affected herds were vaccinated, suggesting that the current prrs vaccines do not confer % protection and that a new generation of vaccines is needed. this review will summarize published data describing the heterogeneity of prrsv and discuss the potential implications for current vaccine ef®cacy and future vaccine development. recent reviews on other aspects of prrsv (rossow, ; botner, b; molitor et al., ; meulenberg et al., a; zimmerman et al., ; van reeth, ) also discuss some of the work cited here. meulenberg et al. ( a) ®rst cloned and sequenced the genome of lv, a european strain of prrsv. subsequently, partial sequences of another european strain (conzelmann et al., ) and two north american strains of prrsv (mardassi et al., ; meng et al., ) were reported. more recently, the complete genomes of two north american strains of prrsv have been determined (allende et al., ; nelsen et al., ) . the genome of prrsv is an kb positive strand rna molecule that encodes eight overlapping open reading frames (orfs) organized similarly to the orfs of coronaviruses (lai and cavanagh, ; meulenberg et al., a; allende et al., ; nelsen et al., ) . the overlaps between the orfs of lv range from bp (between the orfs and ) to bp (between the orfs and ). the u.s. strains of prrsv have a bp noncoding region separating orfs and morozov et al., ) . orfs a and b comprise about % of the viral genome and are predicted to encode the viral rna polymerase (meulenberg et al., a; allende et al., ; nelsen et al., ) . the c-terminus of orf a overlaps the n-terminus of orf b by nucleotides. a heptanucleotide slippery sequence, uuuaaac, located just upstream of the uag stop codon of orf a, and a pseudo-knot structure downstream of the slippery sequence are believed to be essential for the expression of orf b of prrsv via a mechanism of ribosomal frame-shifting (meulenberg et al., a; allende et al., ; nelsen et al., ) . orfs , and of prrsv encode virion-associated proteins designated as gp , gp and gp , respectively (meulenberg et al., , b meulenberg and petersen-den besten, ; van nieuwstadt et al., ) . however, the gp protein of a canadian prrsv isolate encodes a nonvirion-associated, soluble protein (mardassi et al., ) , which is similar to the orf protein encoded by lactate dehydrogenase-elevating virus (ldv) (faaberg and plagemann, ) . the reason for the discrepancy in whether gp protein of prrsv is a structural protein is not clear, but genetic variation of the orf gene might be responsible. therefore, the gp protein of other diverse strains of prrsv should also be evaluated. orfs , and of prrsv encode envelope (gp ), membrane (m) and nucleocapsid (n) proteins, respectively (mardassi et al., ; meulenberg et al., meulenberg et al., , a . monoclonal antibodies directed against gp and gp proteins are neutralizing (meulenberg et al., b; pirzadeh and dea, ) . the m protein is an unglycosylated protein of kda which has the same hydrophobicity pro®le as the m proteins of equine arteritis virus (eav) (de vries et al., ) and ldv (godeny et al., ) . the n protein is not n-glycosylated, although it contains or potential n-glycosylation sites . the order of prrsv genes, -viral polymerase (orfs a/ b)-virion-associated proteins gp (orf )-gp (orf )-gp (orf )-gp (orf )-m (orf )-n (orf )- , is the same as in eav (den boon et al., ) , ldv (plagemann and moennig, ) and simian hemorrhagic fever virus (shfv) . therefore, prrsv, along with eav, ldv and shfv, is now classi®ed within a single genus arterivirus in the family arteriviridae in the order nidovirales (cavanagh, ) . the expression and replication of prrsv requires the production of at least six subgenomic mrnas (sg mrnas) (conzelmann et al., ; meulenberg et al., a meulenberg et al., , b, a meng et al., meng et al., , b snijder and meulenberg, ) . these sg mrnas, together with the genomic virion rna, form a -coterminal nested set. each of these sg mrnas contains a common leader sequence of about bp in size (meulenberg et al., a, b; morozov et al., ; nelsen et al., ; allende et al., ; oleksiewicz et al., ) . the leader-mrna junction sequence of prrsv, in which the leader joins to the body of the sg mrnas, is a conserved sequence motif of six nucleotides (ucaacc) or a highly similar sequence (meulenberg et al., a, b; meng et al., b meng et al., , b nelsen et al., ) . the sg mrnas of prrsv are not packaged into the virions , suggesting that the encapsidation signal of prrsvis likely localized within the orf region that is unique to the viral genome, but not present in the sg mrnas. northern blot analysis with orf-speci®c probes indicates that sg mrnas are polycistronic . it is generally believed that only the orf at the end of each sg mrna is translationally active and, thus, each of the sg mrnas is functionally monocistronic. the precise mechanism of transcription and translation of prrsv is not understood, although it is believed to be similar to that of coronaviruses (lai and cavanagh, ) . the recently constructed infectious cdna clone of prrsv should facilitate investigation of the mechanism of prrsv replication. biological variations among prrsv isolates have been reported. the european prrsv isolates were preferentially propagated in pam cultures (wensvoort et al., ; wensvoort, ) , whereas the north american isolates were grown in pam cultures as well as in three continuous cell lines, cl , marc- and crl collins et al., ; kim et al., ; meng et al., meng et al., , a . swine testis (st) cells were also reported to support prrsv replication (plana et al., ) . however, the st cells cannot propagate a high virulence prrsv isolate vr . variations in the susceptibilities of cl cells and pam to prrsv infection were reported. not all prrsv isolates growing in cl cells replicated in pam, and vice versa (bautista et al., a) . failure to propagate some strains of prrsv in certain cell cultures indicates the existence of prrsv variants and, thus, both pam and other cell lines should be used when attempting virus isolation from clinical samples. it has been reported that ®eld strains of prrsv vary in their susceptibility to antibodydependent enhancement (ade) of infection . therefore, the altered ability to infect may be due to the selection of variants that can facilitate infection of macrophages through ade. antigenic variations among prrsv isolates have been well documented. wensvoort et al. ( ) and wensvoort ( ) reported that, antigenically, four european isolates resembled each other closely, but differed from the u.s. isolates, and that three u.s. isolates differed from each other. serologic survey of the ®eld samples by immunouorescence assay indicated that about % of the samples were positive for european lv, but negative for u.s. isolate vr , and that about % of the samples were positive for vr , but negative for lv (bautista et al., b) . in another study, north american isolates were found to be more closely related serologically to each other than to the european isolates (frey et al., ) . of canadian swine sera tested, samples were positive for vr antibody, but only samples were positive for lv. when swine sera from the netherlands were tested, samples were positive for lv antibody, but only samples were positive for vr . western blot analysis indicated that the orf protein of mn- isolate reacted with only % of prrsv-infected pig sera tested (kwang et al., ) . differential reactivity of monoclonal antibodies (mabs) with different prrsv isolates was also reported. two mabs to n protein recognized a conserved epitope in u.s. and european prrsv isolates, but four other mabs to n protein reacted with u.s. isolates only (nelson et al., ) . six mabs raised against a british isolate of prrsv did not react with u.s. isolates tested (drew et al., ) . five mabs against gp protein of a canadian isolate did not react with lv (pirzadeh and dea, ; pirzadeh et al., ). an mab to m protein reacted with all north american prrsv isolates tested, but failed to react with any of the european isolates . the reactivity of mabs against gp , gp and n proteins with european and american prrsv isolates also revealed antigenic differences not only between the u.s. and european isolates, but among different european or u.s. isolates as well (katz et al., ; wieczorek-krohmer et al., ) . in addition, antigenic variation was demonstrated between an isolate and its progeny recovered after in vivo passages (le gall et al., ) , suggesting that a relatively high rate of mutations occurs during prrsv replication in its natural host. given the degree of antigenic diversity observed among prrsv strains, it is unlikely that a vaccine based on one strain of prrsv will effectively protect against antigenically different enzootic ®eld strains of prrsv. in fact, lager et al. ( ) recently showed that gilts inoculated with one strain of prrsv did not completely protect against heterologous challenge with an antigenically distinct prrsv strain. therefore, the effectiveness of a vaccine against heterologous enzootic ®eld strains of prrsv will largely depend on the antigenic relatedness of the virus strain to which the vaccinated animals were exposed. the design of future vaccines will have to take into consideration the antigenic diversity. the author believes that a multivalent vaccine consisting of multiple antigenically distinct strains of prrsv is the most promising candidate for the next generation of vaccines. the mechanism of prrsv pathogenesis is poorly understood. it is generally believed that prrsv initiates an infection in pigs via entry through nasal epithelial, tonsillar, and pulmonary macrophages. prrsv replicates in these cells, causes viremia and, subsequently, results in pneumonia, myocarditis, encephalitis, rhinitis, vasculitis, lymphadenopathy, etc. in target organs (rossow et al., (rossow et al., , . it has been well documented that prrsv causes persistent infections in pigs (albina et al., ; christopher-hennings et al., a, b; wills et al., a, b) . in experimentally infected boars, prrsv can be detected by pcr in semen samples at days postinoculation (dpi) (christopher-hennings et al., a, b) . pigs persistently infected with prrsv can transmit the virus to naive pigs by direct or indirect contact, and the transmission by direct contact occurs up to weeks after infection (albina et al., ; wills et al., a, b) . bilodeau et al. ( ) showed that when speci®c-pathogen-free (spf) pigs were introduced into a barn that had housed prrsv-infected pigs as much as months after clinical signs of infection had disappeared, the newly introduced spf pigs became infected. this study indicates that subclinical prrsv infection can persist in the animals. wills et al. ( a, b) demonstrated that prrsv can be isolated from oropharyngeal samples for up to dpi. pigs persistently infected with prrsv may appear clinically normal, but can still transmit virus to pigs in naive swine herds. therefore, persistent infection of prrsv plays an important role in prrsv survival and transmission, and will likely pose a major obstacle in prrs control programs. despite the ample data documenting prrsv persistence, little has been done to understand the mechanism of persistent infection or what clinical impact it may have. clearly, more studies are needed in the future to determine the host and virus factors that lead to the persistent state and to fully elucidate the mechanism of prrsv pathogenesis. marked differences in virulence among prrsv strains have been observed in experimentally-infected pigs . signi®cant differences in severity of clinical respiratory disease, rectal temperatures, gross lung lesions and microscopic lung lesions were observed among nine different u.s. isolates of prrsv. the european lv and the low-virulence u.s. prrsv isolate vr (isu ) induced mild transient pyrexia, dyspnea and tachypnea, but several high virulence u.s. isolates induced labored respiration, pyrexia, lethargy, anorexia and patchy dermal cyanosis. at dpi, mean lung lesion scores estimating the percentage of lungs affected by pneumonia ranged from . % for lv, . % for vr (isu ), . % for vr , to . % for isu- . despite the observed difference in virulence among prrsv isolates, tissue tropism and distribution of prrsv antigen or nucleic acid within tissues and organs were very similar in pigs inoculated with different strains of prrsv haynes et al., ) . strains of prrsv also vary in virulence for their ability to cause reproductive failure (mengeling et al., ) . mengeling et al. ( ) reported that the effects of prrsv on reproductive performance are strain-dependent. in addition, apathogenic ®eld isolates of prrsv have been reported (ohlinger et al., ; van alstine, ) , indicating that ®eld isolates of prrsv differ in virulence. the recent outbreaks of severe atypical or acute prrs further indicate that the recent atypical prrsv strains circulating in the u.s. swine herds are more virulent than those strains isolated earlier (botner et al., a; mengeling et al., ; bell, ; lager et al., ; osorio et al., ) . mengeling et al. ( ) demonstrated that a ®eld strain of atypical prrsv produced especially severe clinical signs of disease and reproductive failure in experimentally infected gilts. rossow et al. ( ) reported that marked neurovirulence in neonatal pigs was found to be associated with infection by some ®eld isolates of prrsv. prrsv was identi®ed in macrophages or microglia of brain lesions by immunohistochemical staining of brain sections. the replication of the virus in the brain was veri®ed by in situ hybridization. the mechanism for the observed prrsv neurovirulence in neonatal pigs is not known, but genetic changes in prrsv genome may alter the tissue tropism of prrsv. the mechanism for pathogenic variation observed among prrsv strains remains unknown, but the genetic make-up of a particular virus strain will likely determine the virulence of the virus in animals. therefore, it is important to genetically characterize ®eld strains of prrsv with differing virulence. one recent breakthrough in prrsv research is the construction of an infectious cdna clone of prrsv . using this infectious clone, one should be able to construct viruses that are chimeras of low and high virulence strains of prrsv or to mutant genes of interest to study the structural and functional relationship of prrsv genes. the availability of this infectious cdna clone will eventually aid prrsv researchers in identifying the genetic virulence determinant(s) of prrsv. prrsv is genetically heterogeneous. extensive sequence variation was found between the european and the u.s. isolates (mardassi et al., ; meng et al., meng et al., , a meng et al., , b, b morozov et al., ; murtaugh et al., ; nelsen et al., ; allende et al., ) . the nucleotide sequence identity between lv and the u.s. isolates is ± % in orf , ± % in orf , ± % in orf , and ± % in orf (meng et al., a, b) . orfs and genes are relatively conserved among the u.s. isolates or among the european isolates, but extensive genetic variation was observed in the orfs and genes between european and u.s. isolates. it has been shown that the nucleotide sequence identity was ± % in orf , ± % in orf , ± % in orf , and ± % in orf among six u.s. isolates . interestingly, the least virulent u.s. isolate, isu (atcc vr ), has the most divergent sequence compared to the other ®ve u.s. isolates. the nucleotide sequence identity between isu and the other u.s. isolates was ± % in orf , ± % in orf , and ± % in orf . the orf of isu has a three-nucleotide deletion and shares ± % nucleotide sequence identity compared to that of the other u.s. isolates. the sequence variation between the least virulent strain isu and other u.s. isolates appears to be randomly distributed throughout the genome , thus it is dif®cult to speculate regarding any correlation between prrsv virulence and a particular gene or sequence. kapur et al. ( ) analyzed the nucleotide sequence of orfs ± of u.s. prrsv isolates, and found that the genetic distance ranges from . ± . % among these u.s. isolates and is about % between lv and the u.s. isolates. simple accumulation of random neutral mutations cannot explain the substantial nucleotide differences among prrsv isolates, and the mechanism for generating the genetic heterogeneity remains unknown. the leader sequence of prrsv strains also varies signi®cantly. the bp leader sequence of vr strain is bp shorter than that of lv, and has a sequence identity of % with that of lv . the leader sequence of another north american strain, b, is bp in length and also differs considerably in nucleotide sequence with that of lv (allende et al., ) . like the leader sequence, the orf gene sequence also differs extensively between the u.s. and the european strains (allende et al., ; nelsen et al., ) . the orf a of vr strain shares only about % nucleotide sequence identity with that of the lv strain. orf b is more conserved than orf a and shares about % nucleotide sequence identity with that of lv. however, a stretch of amino acids at the carboxyl terminus of orf b of vr has only % similarity with that of lv . allende et al. ( ) also showed that north american strain b shares only about % amino acid identity in the orf a polyprotein region with that of lv. the greatest divergence is found in the nonstructural protein (nsp ), which shares only about % amino acid identity with the corresponding region of lv. surprisingly, the nsp of strain b is amino acids longer than that of lv (allende et al., ) . like strain vr , strain b also exhibits greater divergence in the carboxyl terminal region of orf b (cp protein), with only % identity with the corresponding region of lv. considering the striking differences in the leader sequence and in all orfs between european and north american strains, it is surprising that both european and north american strains cause a similar disease. although the origin of prrsv remains unknown, these data strongly suggest that the european strains and the north american strains of prrsv have undergone divergent evolution on separate continents from a common ancestor. based on the sequence and evolution analyses of u.s. isolates, kapur et al. ( ) estimated that the time for generating the amount of nucleotide variation in the midwestern u.s. isolates takes about ± years of virus evolution, suggesting that prrsv probably emerged as a swine pathogen approximately a decade ago. it has been speculated that european and north american prrsv strains may have evolved from an ldv-like ancestor (plagemann, ; nelsen et al., ) . however, the almost simultaneous emergence of prrs in swine on two continents makes the theory of divergent evolution dif®cult to believe. more likely, the simultaneous emergence of prrs on two continents might relate to global changes in commercial swine management and husbandry . the sg mrnas of prrsv are heterogeneous (meulenberg et al., b; meng et al., b; snijder and meulenberg, ; faaberg et al., ; nelsen et al., ) . meng et al. ( b) reported that, in addition to the genomic rna, a nested set of six or seven sg mrnas is present in cells infected with different isolates of u.s. prrsv that differ in virulence. prrsv isolates isu , isu and isu produce seven sg mrnas, whereas isolates isu and isu produce only six sg mrnas. the additional species of sg mrna (designated as sg mrna - ) is located between sg mrnas and , and is generated from the sequence upstream of orf . however, there is no apparent correlation between virus virulence and the additional sg mrna - . interestingly, a small orf with a coding capacity of amino acid residues at the -end of the sg mrna - was identi®ed. thus, the sg mrna - in some isolates is potentially bicistronic. however, whether this small orf - is actually translated or has any biological functions remains to be studied. morozov et al. ( ) reported that there are two leader-body junction sites for sg mrnas and . nelsen et al. ( ) also demonstrated that vr strain of prrsv utilizes two leader-body junction sites for sg mrna transcription: one site at bp upstream of the orf start codon for most mrna transcripts, and the other site at bp upstream of the orf start codon for a minority of transcripts. in contrast, lv only utilizes the site at bp upstream of the orf start codon. faaberg et al. ( ) has also shown that the sg mrna is transcribed with different leader-body junction sites. the differences in sg mrnas and leader-body junction sites among prrsv isolates further re¯ect the heterogeneous nature of the virus. future studies are needed to elucidate the biological signi®cance of the heterogeneity. at least two distinct genotypes of prrsv have been reported: the european and the north american . recently, an impressive amount of sequence data on prrsv isolates has been generated. to gain a better understanding of the genetic relationship and evolution of prrsv, phylogenetic analyses were performed based on the sequences of orf (fig. a) and orf (fig. b) genes of strains of prrsv worldwide. these sequences were either published (andreyev et al., ; casal et al., ; chueh et al., ; conzelmann et al., ; drew et al., ; gagnon and dea, ; kapur et al., ; le gall et al., ; madsen et al., ; mardassi et al., ; meulenberg et al., a; murtaugh et al., ; nelsen et al., ; pirzadeh et al., ; plana et al., ; rodriguez et al., ; saito et al., ; suarez et al., ; sur et al., ; valicek et al., ; wesley et al., ; wootton et al., ; yang et al., ) or are available in genbank (af , af , af , af , af , af , x , af , u , u ). phylogenetic analysis was also performed on the basis of the complete sequences of the structural genes (orfs ± ), which are available for isolates of prrsv (fig. c) . all three phylogenetic trees, based on different regions of the genome, indicate that the north american and european isolates of prrsv represent two distinct genotypes as reported previously. however, within each of the two major genotypes, several minor genotypes (or variants) of prrsv were also identi®ed ( fig. a and c) . the n gene of prrsv is relatively conserved within each of the two major genotypes (fig. b) . in contrast, the major envelope protein gene (gp ) of prrsv exhibits greater genetic diversity within each major genotype (fig. a) . similarly, the phylogenetic tree based on the complete sequence of the structural genes ( fig. c ) displays greater genetic diversity than the n gene within each major genotype. interestingly, the strains from japan, china, taiwan, guatemala and three danish strains of prrsv are all clustered within the north american genotype. the three danish strains, the chinese strain (s ), a canadian strain (pa ), and a strain from nebraska (ne b) are all found to be closely related to the modi®ed-live vaccine (mlv) virus and the vaccine strain vr . the three danish strains of prrsv are believed to originate from the mlv vaccine virus (vr ) that was used in danish swine herds (madsen et al., ) . phylogenetic trees (fig. a and c) con®rm that these three danish strains are most closely related to the mlv respprrs vaccine virus, but are less related to other north american strains, further indicating that the introduction of north american type of prrsv in denmark was due to the spread of vaccine virus vr (madsen et al., ) . the origin of other strains that are closely related to the mlv and strain vr is not known. it is possible that these strains isolated from different geographic regions also evolved from the mlv vaccine virus vr as a result of largescale vaccination programs in swine herds around the world. quasispecies is de®ned as a population of closely related, yet heterogeneous sequences that are variants of one dominant sequence (bukh et al., ) . viral quasispecies are closely related mutant and recombinant viral genomes subjected to continuous genetic variation, competition and selection (domingo et al., ) . within a single infected animal, many rna viruses have been found to exist as quasispecies. this heterogeneity can cause persistent infection resulting from selection of mutants that escape neutralizing antibody or cytotoxic t lymphocytes (ctl) or as a result of the presence of defective particles (duarte et al., ; ahmed et al., ; domingo et al., ) . extensive genetic variations have been observed among different strains of prrsv; however, little is known about the mechanism of generating genetic diversity. recently, rowland et al. ( ) reported evidence for quasispecies evolution and emergence of a virus subpopulation during in utero infection of pigs with a prrsv isolate. a single nucleotide change in the ectodomain of gp protein was identi®ed during infection of pigs with prrsv strain vr . this ®nding suggests that the genetic variability in the ectodomain of the orf may be due to positive or negative selection forces, such as selection by antibody or other host defenses. other factors such as rna secondary structure, especially in orf , the diverse leader-body junction sites, and the size and sequence difference of the leader sequence among prrsv strains should also be considered as potential driving forces for heterogeneity and quasispecies evolution of prrsv. quasispecies has been well documented in other viruses that cause persistent infections including ldv, an arterivirus closely related to prrsv (duarte et al., ; plagemann et al., ; plagemann, ; bukh et al., ; ahmed et al., ; domingo et al., ) . the extent and nature of prrsv quasispecies evolution and whether quasispecies evolution are related to prrsv persistency remain to be determined. in ldv, quasispecies have been identi®ed and biologically characterized (chen et al., (chen et al., , a (chen et al., , b, . the ldv quasispecies were found to differ in neuropathogenicity, and the neuropathogenic ldvs are incapable of establishing persistent infection in mice. the existence of a quasispecies population during virus infection will affect vaccine ef®cacy and may lead to vaccine failure (domingo and holland, ; duarte et al., ; domingo et al., ) . the ®rst report of prrsv quasispecies evolution by rowland et al. ( ) should stimulate further study of the nature of prrsv quasispecies evolution and its clinical implications. rna recombination can provide a powerful and effective mechanism for evolution of an rna virus. the ability to exchange genetic information may allow rna viruses to adapt a changing environment and to escape a selection pressure (such as neutralizing antibodies), thus providing the recombinant virus with an evolutionary advantage (lai and cavanagh, ) . recently, yuan et al. ( ) provided evidence for homologous rna recombination between prrsv isolates propagated in cell culture. recombinant viral particles containing chimeric orf and orf proteins were identi®ed in ma- cells co-infected with two prrsv isolates. nucleotide sequence analyses con®rmed independent recombination events. the frequency of recombination was estimated from < % up to % within the -bp fragment analyzed. sequence analyses of ®eld isolates of prrsv suggest that rna recombination of prrsv may also occur in nature (yuan et al., ) . by analyzing ®eld isolates of u.s. prrsv, kapur et al. ( ) also provided evidence for intragenic recombination in orfs ± and in orf among prrsv isolates. whether rna recombination plays any roles in generating genetic heterogeneity of prrsv is not known. direct experimental evidence for in vivo rna recombination of prrsv is still lacking. most rna virus recombination studies have been performed in cell cultures, although rna recombination has also been demonstrated in the natural host of picornaviruses, coronavirus and ldv (lai and cavanagh, ; minor et al., ; li et al., ) . for example, a case of poliovirus vaccine-associated poliomyelitis in a human was caused by recombination between two poliovirus vaccine strains (minor et al., ) . high frequency homologous genetic recombination was reported in mice dually infected with two strains of ldv . therefore, future studies are warranted to determine whether rna recombination of prrsv is a phenomenon unique to cell culture or whether this actually occurs in vivo. the frequency of recombination, crossover sites and the clinical implications of prrsv rna recombination also need to be studied. the sg mrna species may also be involved in rna recombination events during infection. thus, future studies are needed to examine rna recombination not only at genomic rna level, but at the sg mrna level as well. several prrs vaccines are currently available; however, there are mixed results regarding the ef®cacy of these vaccines against the genetically diverse ®eld strains of prrsv (lager and mengeling, ; plana-duran et al., ; christopher-hennings et al., ; van woensel et al., ; osorio et al., ; madsen et al., ; mengeling et al., a, b, c; wesley et al., ) . respprrs/repro (boehringer ingelheim), an mlv, is recommended for use in ± week-old pigs and in nonpregnant females (lager and mengeling, ; dee and joo, ) . the prime pac prrs vaccine (schering plough animal health corporation) (hesse et al., ) is also an mlv which has been shown to reduce the severity and duration of disease following challenge. however, it did not prevent infection of vaccinated pigs by a virulent heterologous strain. a live vaccine based on a european isolate of prrsv (porcilis prrs) was found to protect fattening pigs against the respiratory manifestations of prrs (mavromatis et al., ) . osorio et al. ( ) compared three commercial vaccines in their ability to induce protection against prrsv strains of high virulence, and found that these vaccines confer protection against clinical disease, but not against infection. the use of mlvs in boars resulted in vaccine virus shedding in semen and reduced semen quality, but there was reduced or no shedding of wild-type prrsv after challenge (christopher-hennings et al., ) . in another study, vaccination with a mlv in boars resulted in marked reduction in viremia and shedding of virus in semen, but vaccination with inactivated vaccine did not change the onset, duration or level of viremia or shedding of virus in semen (nielsen et al., ) . by using a restriction-site marker that is present in the vaccine virus (vr ), mengeling et al. ( b) demonstrated that the marker was not detected in any of the ®eld strains of prrsv isolated before use of the vaccine. however, the restriction-site marker was detected in of ®eld strains isolated after the introduction of the vaccine, and these ®eld strains were believed to be direct-line descendants of the vaccine virus (mengeling et al., b) . more importantly, these putative vaccine-related strains produced more pronounced pathological changes than did the vaccine virus alone (mengeling et al., b) . wesley et al. ( ) also showed that the restriction fragment length polymorphism (rflp) patterns change as the vaccine virus spreads among a swine population. a glycine marker in the orf gene of the vaccine virus is rapidly lost and replaced with arginine. the use of mlvs in herds may lessen the clinical signs of prrs following infection. however, the potential risk for reversion of mlvs to virulent phenotypes cannot be overlooked. in the absence of a new generation of vaccines, more studies are needed to fully evaluate the safety and ef®cacy of the current mlvs. the emergence and re-emergence of viral infectious diseases is often in¯uenced by the genetics of the viruses (domingo and holland, ; duarte et al., ; domingo et al., ) . genetic heterogeneity of prrsv, due to quasispecies evolution and rna recombination, could lead to the selection of virulent viruses and to the emergence or reemergence of new forms of prrs. quasispecies evolution of prrsv in response to positive or negative selection pressures may signi®cantly change the genomic sequence of mlvs over time as the vaccine virus spreads among swine herds, and ultimately, these genetic changes may revert mlvs to virulent phenotypes. it is also possible that virulent strains of prrsv could be generated through rna recombination between mlvs used in the vaccination programs and enzootic ®eld strains of prrsv. the recent outbreaks of the atypical or acute prrs re¯ect the need to further study this virus to better understand its biology and develop more effective vaccines. most of the herds affected by the atypical prrs had been vaccinated with the current vaccines (bell, ; lager et al., ) . it is possible that a mutant strain(s) of prrsv may be responsible for the recent outbreaks of acute prrs. the heterogeneous nature of prrsv suggests that complete elimination of the virus from the environment is unlikely. the observed genetic diversity among ®eld isolates of prrsv will continue to be the major obstacle for prrs control. therefore, the design for the next generation of vaccines will have to take into consideration the genetic heterogeneity of prrsv, or prrs will remain dif®cult to control. intensive research is required to answer the many questions that remain. the recently constructed infectious cdna clone of prrsv should enable us to study the basic biology of prrsv. using the infectious cdna clone, one can monitor the nature of quasispecies evolution of prrsv in pigs infected by a homogeneous virus derived from the infectious clone. one can also genetically engineer the virus to produce a modi®ed avirulent strain that could be used as an mlv. the immunogenic gene(s) of the genetically-engineered avirulent strain of prrsv can be replaced with that of other antigenically and genetically distinct prrsv strains to produce avirulent virus strains that can be used as a multivalent mlv. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions persistence of viruses genetic variation and phylogenetic relationships of porcine reproductive and respiratory syndrome virus (prrsv) ®eld strains based on sequence analysis of open reading frame comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody serologic survey for lelystad and vr- strains of porcine respiratory and reproductive syndrome (prrs) virus in u.s. swine herds characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) persistence of porcine reproductive and respiratory syndrome virus infection in a swine operation appearance of acute prrs-like symptoms in sow herds after vaccination with a modi®ed live prrs vaccine diagnosis of prrs genetic heterogeneity of hepatitis c virus: quasispecies and genotypes identi®cation of a common antigenic site in the nucleocapsid protein of european and north american isolates of porcine reproductive and respiratory syndrome virus nidovirales: a new order comprising coronaviridae and arteriviridae coexistence in lactate dehydrogenase-elevating virus pools of variants that differ in neuropathogenicity and ability to establish a persistent infection neuropathogenicity and susceptibility to immune response are interdependent properties of lactate dehydrogenase-elevating virus (ldv) and correlate with the number of n-linked polylactosaminoglycan chains on the ectodomain of the primary envelope glycoprotein lactate dehydrogenase elevating virus variants: cosegregation of neuropathogenicity and impaired capability for high viremic persistent infection selective antibody neutralization prevents neuropathogenic ldv from causing paralytic disease in immunocompetent mice sequence analysis of the nucleocapsid protein gene of the porcine reproductive and respiratory syndrome virus taiwan md- strain persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars detection of porcine reproductive and respiratory syndrome virus in boar semen by pcr effects of a modi®ed-live virus vaccine against porcine reproductive and respiratory syndrome in boars isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group strategies to control prrs: a summary of ®eld and research experiences structural proteins of equine arteritis virus equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily complications of rna heterogeneity for the engineering of virus vaccines and antiviral agents quasispecies structure and persistence of rna virus production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus rna virus quasispecies: signi®cance for viral disease and epidemiology orf of lactate dehydrogenase-elevating virus encodes a soluble, nonstructural, highly glycosylated, and antigenic protein subgenomic rna is transcribed with different leader-body junction sites in prrsv (strain vr ) infection of cl cells diagnostic testing for sirs virus at the national veterinary service laboratory (nvsl) differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their orfs and genes complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) development of a streptavidin-biotin immunoperoxidase procedure for the detection of porcine reproductive and respiratory syndrome virus antigen in porcine lung immunohistochemical identi®cation of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two u.s. porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparison of the antigen distribution of two u.s. porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine u.s. porcine reproductive and respiratory syndrome virus (prrsv) isolates in a ®ve-week-old cesarean-derived, colostrum-deprived pig model temporal and morphologic characterization of the distribution of porcine reproductive and respiratory syndrome virus (prrsv) by in situ hybridization in pigs infected with isolates of prrsv that differ in virulence ef®cacy of prime pac prrs in controlling prrs respiratory disease: homologous and heterologous challenge overview and history of mystery swine disease (swine infertility and respiratory syndrome) genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states antigenic differences between european and american isolates of porcine reproductive and respiratory syndrome virus (prrsv) are encoded by the carboxyterminal portion of viral open reading frame reproductive failure of unknown etiology enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line cloning, expression and sequence analysis of the orf gene of porcine reproductive and respiratory syndrome virus mn- b current status of vaccines and vaccination for porcine reproductive and respiratory syndrome acute prrs evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challengeexposed with an antigenically distinct prrsv isolate the molecular biology of coronaviruses antigenic variability of porcine reproductive and respiratory syndrome (prrs) virus isolates: in¯uence of virus passage in pig molecular variation in the nucleoprotein gene (orf ) of the porcine reproductive and respiratory syndrome virus (prrsv) high-frequency homologous genetic recombination of an arterivirus, lactate dehydrogenase-elevating virus, in mice and evolution of neuropathogenic variants sequence analysis of porcine reproductive and respiratory syndrome virus of the american type collected from danish swine herds differential reactivity of a monoclonal antibody directed to the membrane protein of porcine reproductive and respiratory syndrome virus identi®cation of major differences in the nucleocapsid protein genes of a quebec strain and european strains of porcine reproductive and respiratory syndrome virus intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus a subset of porcine reproductive and respiratory syndrome virus gp glycoprotein is released into the culture medium of cells as a non-virionassociated and membrane-free (soluble) form field evaluation of a live vaccine against porcine reproductive and respiratory syndrome in fattening pigs molecular cloning and nucleotide sequencing of the terminal genomic rna of porcine reproductive and respiratory syndrome virus phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the usa and europe sequence comparison of open reading frames to of low and high virulence united states isolates of porcine reproductive and respiratory syndrome virus characterization of a high-virulence u.s. isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from ®eld cases of atypical prrs safety and ef®cacy of vaccination of pregnant gilts against porcine reproductive and respiratory syndrome identi®cation and clinical assessment of suspected vaccine-related ®eld strains of porcine reproductive and respiratory syndrome virus diagnostic implications of concurrent inoculation with attenuated and virulent strains of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav subgenomic rnas of lelystad virus contain a conserved leader±body junction sequence characterization of proteins encoded by orfs to of lelystad virus identi®cation and characterization of a sixth structural protein of lelystad virus: the glycoprotein gp encoded by orf is incorporated in virus particles molecular characterization of lelystad virus posttranslational processing and identi®cation of a neutralization domain of the gp protein encoded by orf of lelystad virus infectious transcripts from cloned genome-length cdna of porcine reproductive and respiratory syndrome virus antigenic and molecular evolution of the vaccine strain of type poliovirus during the period of excretion by a primary vaccine immunity to prrsv: double-edged sword sequence analysis of open reading frames (orfs) to of a u.s. isolate of porcine reproductive and respiratory syndrome virus characterization of leader-body junction sites in subgenomic mrnas of a u.s. prrsv isolate comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies examination of virus shedding in semen from vaccinated and from previously infected boars after experimental challenge with porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents some aspects of the virus causing prrs in germany determination of -leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs prrsv: comparison of commercial vaccines in their ability to induce protection against current prrsv strains of high virulence monoclonal antibodies to the orf product of porcine reproductive and respiratory syndrome virus de®ne linear neutralizing determinants genetic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope gp glycoprotein lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus, a new group of positive strand rna virus lactate dehydrogenase-elevating virus: an ideal persistent virus? lactate dehydrogenase-elevating virus and related viruses porcine epidemic abortion and respiratory syndrome (mystery swine disease). isolation in spain of the causative agent and experimental reproduction of the disease ef®cacy of an inactivated vaccine for prevention of reproductive failure induced by porcine reproductive and respiratory syndrome virus financial evaluation and decision making in the swine breeding herd epitope mapping of the nucleocapsid protein of european and north american isolates of porcine reproductive and respiratory syndrome virus pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterized by marked neurovirulence the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- characteristics of major structural protein coding gene and leader-body sequence in subgenomic mrna of porcine reproductive and respiratory syndrome virus isolated in japan phylogenetic relationships of european strains of porcine reproductive and respiratory syndrome virus (prrsv) inferred from dna sequences of putative orf- and orf- genes porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis isolation and identi®ca-tion of porcine reproductive and respiratory syndrome virus in cell cultures isolation of sirs virus from nursery pigs of two herds without current reproductive failure proteins encoded by open reading frames and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion pathogenesis and clinical aspects of a respiratory porcine reproductive and respiratory syndrome virus infection european serotype prrsv vaccine protects against european serotype challenge whereas an american serotype vaccine does not organization of the simian hemorrhagic fever virus genome and identi®cation of the sgrna junction sequences mystery swine disease in the netherlands: the isolation of lelystad virus antigenic comparison of lelystad virus and swine infertility and respiratory syndrome virus lelystad virus and the porcine epidemic abortion and respiratory syndrome differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from north american ®eld strains by restriction fragment length polymorphism analysis of orf evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): monoclonal antibodies detect common epitopes on two viral proteins of porcine reproductive and respiratory syndrome virus: a persistent infection antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus comparative sequence analysis of open reading frames to of the modi®ed live vaccine virus and other north american isolates of the porcine reproductive and respiratory syndrome virus antibody-dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs field isolates of porcine reproductive and respiratory syndrome virus (prrsv) vary in their susceptibility to antibody-dependent enhancement (ade) of infection recombination between north american strains of porcine reproductive and respiratory syndrome virus general overview of prrsv: a perspective from the united states acknowledgements i wish to thank drs. roger avery and thomas toth of virginia±maryland regional college of veterinary medicine, and dr. jill sible of department of biology at virginia tech for their critic reviews of the manuscript; drs. prem paul and patrick halbur of iowa state university's college of veterinary medicine for their continuous support and collaboration, and mr. denis guenette for editorial assistance. i apologize to the authors of important papers not cited here because of the narrow scope of this review and of space limitations. key: cord- -p bl authors: gao, mengying; wang, qiuling; zhao, wenjun; chen, yuqiu; zhang, tingting; han, zongxi; xu, qianqian; kong, xiangang; liu, shengwang title: serotype, antigenicity, and pathogenicity of a naturally recombinant tw i genotype infectious bronchitis coronavirus in china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: p bl since , strains of the naturally recombinant tw i genotype of infectious bronchitis virus (ibv) have caused considerable damage to the chinese poultry industry. to better understand the antigenicity and pathogenesis of this genotype, the characteristics of the ck/ch/ldl/ strain were compared to those of four commercial ib vaccine strains that are used commonly in china, as well as four attenuated viruses that represent two types of ibv strains, which are believed to have originated in china and are the predominant ibv types circulating in chicken flocks in china and many other parts of the world. the results showed that all eight strains were genetically and serotypically different from the strain ck/ch/ldl/ . furthermore, neither the vaccine strains nor the attenuated viruses could provide complete respiratory protection of chickens against a challenge with the ck/ch/ldl/ strain, indicating that it is necessary to develop new live vaccines or to evaluate the use of established vaccines in combination to control naturally recombinant tw i-type ibv strains in the future. our results showed that strain ck/ch/ldl/ is very pathogenic, and that it is able to cause cystic oviducts in a high percentage of birds, as well as mortality due to nephritis and respiratory distress with complete tracheal ciliostasis, especially in chickens infected at day of age. being a coronavirus and, therefore, a single-stranded rna virus, infectious bronchitis virus (ibv) has an enormous capacity to change by spontaneous mutation and genetic recombination. it is believed that spontaneous mutations and genetic recombination can occur randomly in the ibv genome; however, if these events occur in the spike (s) gene, especially in its hypervariable regions, these events are most likely to result in the emergence of many different antigenic or genotypic types, which are commonly referred to as variants. therefore, ibv is ubiquitous in most parts of the world where poultry are reared, and it is able to spread very rapidly in non-protected chicken flocks, leading to heavy economic losses in poultry industries (cavanagh and gelb, ) . in general, while many new variants are unable to replicate or survive for a long time, a few variants that are of economic importance have emerged worldwide or in restricted geographic areas. therefore, effective surveillance, which is primarily based on the isolation and identification of the virus type causing disease, is of great importance. classification or typing of ibv strains is very important for implementing control measures, research purposes, and understanding the epidemiology and evolution of ibvs. thus far, two major groups of classification systems, including functional tests that examine the biological functions of a virus (immunotypes, protectotypes, and serotypes) and non-functional tests that assess the viral genome (genotypes), are commonly used for ibv typing (de wit, ) . for a specific ibv strain, although evidence from some studies suggests that there is a high correlation between the genotype and serotype, other studies have presented conflicting data (de wit, ; zhang et al., ; chen et al., ) , which may lead to contradictory results. the disadvantages of each system are that they only analyze one or several characteristics of a virus strain. hence, data from only one system has to be interpreted with caution, while a more objective and accurate conclusion can be drawn by completely analyzing the results from different systems, although it is suggested that the preferred typing system usually depends on the goal (e.g., selection of vaccination programs or epidemiological studies), available techniques, experience, and costs (de wit, ) . a large number of ibv genotypes and variants have been isolated in china in recent years (han et al., ) , among which two genotypes, the lx and ck/ch/ldl/ i types (also known as qx-and q -like, respectively), were first isolated in china and subsequently have become widespread worldwide (valastro et al., ) . among these ibv genotypes, the tw type was first isolated in in taiwan, and it was considered to have a different genotype than all of the other ibvs (wang and tsai, ) . the majority of tw ibvs isolated from taiwan are nephropathogenic, and they have mortality rates ranging from to % in -day-old specific-pathogen-free (spf) chickens (wang and tsai, ) . however, the naturally recombinant tw i (nrtw i) genotype was first isolated in in china, and it was thought to have originated from a natural recombination between lx and tw viruses (xu et al., ) . despite a previous study that characterized the genetic characteristics of an nrtw i type virus and its nephropathogenicity in -day-old spf chickens (xu et al., ) , there is no further information on this important ibv variant. to better understand the nrtw i type, a series of experiments was performed to investigate its antigenicity and pathogenicity in the oviducts of spf layers, and to evaluate the protection provided by commercial vaccines and attenuated viruses. nine ibv strains, including four vaccine strains (h , ldt -a, / , and connecticut (conn)), four attenuated strains (ck/ch/ldl/ [ldl/ ], ck/ch/lsd/ [lsd/ ], ck/ch/ lgx/ [lgx/ ], and ck/ch/ldl/ i [ldl/ i]) and the ck/ch/ldl/ (nrtw i) strain (xu et al., ) , were used in this study. the ibv strains ldl/ , lsd/ , and lgx/ (chen et al., ) were isolated in china and belong to the lx type. these three strains were attenuated by serially passaging them (p ), (p ), and (p ) times, respectively, in -day-old spf chicken eggs. the ldl/ p , lsd/ p , and lgx/ p strains were shown to be fully attenuated in spf chickens (liu et al., a) . furthermore, vaccination with attenuated viruses provided complete protection against their virulent parental viruses (data not shown). the ldl/ i strain was also passaged times (p ) in spf chicken eggs (liu et al., b) . each of the virus stocks was prepared in -dayold embryonated spf chicken eggs by the allantoic route of inoculation, and the infectious allantoic fluid was collected h post-inoculation as previously described (liu et al., a) . the titers of the viruses were determined as previously described (chen et al., ) , and the median embryo infectious dose (eid ) was calculated using the method of reed and muench ( ) . white leghorn spf layer chickens and fertile spf chicken eggs were obtained from the harbin veterinary research institute. the birds were maintained in isolators with negative pressure, and food and water were provided ad libitum. all experimental procedures were approved by the ethical and animal welfare committee of heilongjiang province, china. the s genes of strains lsd/ p and lgx/ p were amplified and sequenced as previously described . the sequences have been submitted to genbank, and they have been assigned accession numbers kt and kt , respectively. the s gene sequences of the remaining eight viruses, including the h , ldt -a, / , conn vaccine, ldl/ p , and ldl/ i p , nrtw i and the tw i-type tw / strains, were selected from the genbank database and used for s gene comparison. both the sequences of amino acid and nucleotide were assembled and the similarities were calculated using the clustal w method available in the bioedit software package (version . . . ., available at: http://www.mbio.ncsu.edu/bioedit/bioedit). in addition, other reference strains in which the s subunit sequences were available in genbank (www.ncbi.nlm.nih.gov/genbank/) were also selected for a phylogenetic analysis. the characteristics of the reference viruses are listed in table . a phylogenetic tree based on the s gene was constructed from aligned amino acid sequences by the neighbor-joining method with bootstraps using the mega program (tamura et al., ) . four ibv vaccine strains (ldt -a, / , h and conn), the attenuated ldl/ i p and nrtw i were used for virus crossneutralization test in this study. because strains ldl/ p , lsd/ p and lgx/ p belong to the same genotype (lx -type or qx-like), hence, only the strains ldl/ p and lsd/ p were used. in addition, the ibv strain ck/ch/lsc/ i, which represents another important genotype in china (han et al., ) , was also used for virus cross-neutralization test in this study. sera against these ibv strains were prepared as previously described (guo et al., ) . briefly, -day-old spf chickens were inoculated by a combined intraocular and intranasal route using a total dose of eid of each virus per bird, respectively. after weeks, a booster dose of each virus with eid was administered by intravenous inoculation to the bird. birds were exsanguinated week after the last inoculation and the separated sera from chickens inoculated with the same virus were pooled. all sera were inactivated at c for min and stored in . ml aliquots at À c until required. for virus neutralization, sera were serially diluted two-fold with sterile phosphate-buffered saline (pbs) and mixed with eid of the ibv strains. after incubation for h at c, virus-serum mixtures were inoculated into the allantoic cavity of spf chicken embryos, which were observed for d. the end-point titer of each serum sample was calculated using the method of reed and muench ( ) . fifty-five -day-old spf layer chickens were separated into four groups, and they were provided with food and water ad libitum. groups - included birds, and they were challenged with the nrtw i strain when they were , , and days old, respectively. the challenge strain ( eid in . ml of diluent per bird) was applied by the intranasal and ocular routes. group , which served as a negative control, included birds that were not challenged. the chicks were examined daily for clinical signs of infection, such as tracheal rales, nasal discharge, watery eyes, and wheezing. the clinical signs from all of the birds in each group were counted by three people over a -min period. morbidity and mortality were recorded daily. gross lesions were also carefully examined from the dead chickens, and the kidneys of the dead chickens were subjected to immunohistochemistry (ihc) using monoclonal antibody d , which is directed against the nucleoprotein, as previously described (de wit et al., ; xu et al., ) . blood samples were collected on , , , , , and d post-challenge (dpc) from all birds, and they were examined for the presence of antibodies against ibv using a commercial enzyme-linked immunosorbent assay (elisa) kit (idexx, portland, me, usa) according to the manufacturer's instructions. at months of age, the birds were killed humanely using carbon dioxide/oxygen, followed by exsanguination. a post-mortem examination was performed, and special attention was paid to abnormalities in the oviducts and kidneys. meanwhile, samples from the kidney were collected to detect the presence of ibv by ihc. ami no acid substit uti ons (x . phylogenetic trees constructed from the nucleotide sequences of the s subunit gene of the four vaccine strains, four attenuated viruses, and reference ibv strains. the trees were computed using the neighbor-joining method. the significance of the tree topology was assessed by bootstrapping calculations. genbank accession numbers are indicated in table . one hundred -day-old spf chickens were separated into experimental groups, each containing birds. birds in groups - were vaccinated with eid of the h , ldt -a, / , and conn vaccines strains, and the ldl/ p , lsd/ p , lgx/ p , and ldl/ i p attenuated strains, respectively, in a total of . ml of pbs per bird, by the intranasal and ocular routes. birds in groups and received ml of sterile pbs via the same routes. blood samples were collected at , , , , and d post-vaccination (dpv) . at dpv, birds in groups - were challenged with eid of the virulent nrtw i strain, in a total of ml of pbs per bird, by the intranasal and ocular routes. birds in group were not exposed to virus and served as a negative control. nasopharyngeal and blood samples were collected from all of the birds in each group at , , , , and dpc. nasopharyngeal swabs were used to recover viruses from day-old spf chicken eggs, and viruses were subsequently detected by reverse transcription polymerase chain reaction (rt-pcr) as previously described (liu et al., a) . the serum was stored at À c for detecting antibodies against ibv using the aforementioned elisa kit as previously described (liu et al., a) . as illustrated in fig. , the four commercial vaccine strains (h , ldt -a, / , and conn), which represent different serotypes (massachusetts (mass), tl/ch/ldt / , /b, and connecticut, respectively) of ibv, were genetically different from the nrtw i strain, as indicated by an analysis of their s genes. of the four attenuated strains, the ldl/ p , lsd/ p , and lgx/ p strains belonged to the lx genotype, while the ldl/ i p strain belonged to the ck/ch/ldl/ genotype. these two genotypes were also genetically different from those of the nrtw i strain and the four vaccine strains. in accordance with these results, strain nrtw i did not share more than % nucleotide and amino acid similarities with the s genes and s proteins of the vaccine and attenuated virus strains in this study (table ). in addition, similar to previous results (xu et al., ) , strain nrtw i shared the highest nucleotide ( . %) and amino acid ( . %) similarities with the tw i prototype strain tw / . the results of the two-way cross-neutralization tests using the ibv strain nrtw i and the antisera against the four vaccine strains, which represented the mass, tl/ch/ldt / , /b ( / ), and conn serotypes, showed that the strains belonged to different serotypes ( table ). the results using the nrtw i strain and the antisera against the deduced parental virus types, the ldl/ p , lsd/ p , lgx/ p , and ldl/ i p strains, also showed non-detectable levels of cross-neutralization. none of the chickens that were vaccinated with the ldt -a vaccine strain or the attenuated ldl/ and ldl/ i viruses showed clinical signs or mortality when challenged with the ibv strain nrtw i, thereby demonstrating that vaccination with these three viruses provided a high degree of clinical protection. in contrast, respiratory signs (sneezing, nasal discharge, and tracheal rales) developed at - dpc and lasted until dpc in some of the chickens in the groups that were vaccinated with the h , / , and conn vaccines, and the attenuated lsd/ and lgx/ viruses when challenged with the nrtw i ibv strain at days of age. in addition, some of the chickens died in these groups, indicating that vaccination with these viruses could not provide complete protection against the nrtw i strain. no birds died during the experiment and no clinical signs were observed in the negative control group. as listed in table , the nrtw i challenge virus was re-isolated from the tracheas of nearly all of the birds in the eight vaccinated groups at dpc, as well as from non-vaccinated birds that were challenged. as expected, none of the chickens in the negative control group tested positive for virus isolation. the serological responses induced by the ibv vaccines and the challenge virus are presented in table . all of the birds in each of the vaccinated groups seroconverted at dpv, as well as at dpc with the nrtw i strain. none of the birds in the negative control group seroconverted during the experiment. table nucleotide and amino acid similarities of the s gene and protein among strain nrtw i and the eight vaccine/attenuated strains. a amino acid identity (%) -, not tested. a reciprocal titer. all of the chickens in the -and -day-old groups that were challenged with the nrtw i strain exhibited the aforementioned respiratory signs at - dpc, and nine and two chickens, respectively, died at - dpc. similar to the negative control group, no birds died and no clinical signs were observed in the day-old group (table ) . obvious hyperemia of the tracheal mucosa with catarrhal exudation was observed in all of the dead chickens. the most remarkable lesions were detected in the kidneys of the dead chickens. the affected kidneys were enlarged and pale, and urate deposition was observed in the tubules and ureters ( fig. a) . similar to our previous results (xu et al., ) , viral antigen was detected by ihc in the kidneys of all of the dead birds. antigen was found in the cytoplasm of the tubular epithelial cells and in the mucous membrane of the ureters and collecting ducts (fig. b) . interestingly, neither gross lesions nor ihc-positive cells were observed in the kidneys of the surviving chickens at the end of the experiment. in addition, characteristic dilatation of the oviduct developed in most ( / ) of the birds in the -day-old group. in contrast, one and two of the birds in the -and -day-old groups, respectively, exhibited cystic oviducts ( fig. ; table ). surprisingly, the dilatation and serous fluid accumulation in the oviducts were observed not only in the chickens in the -and -day-old groups, but also in chickens in the -day-old group, although only / of the birds in the -day-old group showed these lesions. as expected, no such lesions were observed in the birds of the negative control group. all of the birds in the -and -day-old groups seroconverted at and dpc, respectively, with strain nrtw i, which was earlier than that in the -day-old group, in which all of the birds seroconverted at days of age (table ) . we did not observe seroconversion in any chickens of the negative control group. in this study, most ib vaccines, including two types of government-approved vaccines (h and ldt -a) and two types of non-government-approved vaccines ( / and conn) that are used commonly in china, as well as the nrtw i type strains, were selected for genotyping, an s gene comparison, serotyping, and vaccination-challenge tests. in addition, we also selected four attenuated strains representing two types (the lx type strains ldl/ , lsd/ , and lgx/ , and the ck/ch/ldl/ i type strain ldl/ i) of ibv strains that are believed to have originated in china and are the predominant ibv types circulating in chicken flocks in china and many other parts of the world (valastro et al., ) . a phylogenetic analysis and an s gene comparison showed that the aforementioned six types of viruses are genetically different from each other and from strain nrtw i. this is in accordance with the results of the cross-neutralization tests that showed that the serotype of strain nrtw i differs from those of the selected six serotypes in this study and likely represents a novel serotype. the cross-neutralization tests did not include the tw i prototype strain tw / from taiwan, china, which was unavailable for the comparison. table results of the vaccination-challenge tests (groups of chickens were vaccinated with ibv vaccine or attenuated strains, and challenged with the nrtw i strain). / / / / / / / / / / / / / / / / / ldt -a / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / conn / / / / / / / / / / / / / / / / -ldl/ p / / / / / / / / / / / / / / / / -lsd/ p / / / / / / / / / / / / / / / / -lgx/ p / / / / / / / / / / / / / / / / -ldl/ i p / / / / / / / / / / / / / / / / / nrtw i d / / / / / / / / / / / / / / / / -negative control / / / / / / / / / / / / / / / / / a number of chickens that seroconverted/number inoculated. b two procedures, including virus isolation and subsequent rt-pcr, were used for virus recovery after challenge as described previously (liu et al., ) . the results from the two procedures were identical. number of chickens that were positive for virus recovery/number challenged. c days after challenge. d the chickens in this group were not vaccinated with the vaccine or attenuated viruses, but were challenged at days of age with the ibv nrtw i strain. the nrtw i strain was isolated from chickens that had been vaccinated against ibv with the commercial live attenuated h vaccine at day of age and then boosted at days of age (xu et al., ) , possibly indicating that the vaccine does not provide sufficient protection against a challenge with strain nrtw i. thus, further examination of the protection conferred by commercially available ibv vaccines and attenuated viruses against challenge with the novel serotype nrtw i strain was important for evaluating the extent of protection against distinct antigenic types. comparatively, the vaccine strain ldt -a and two attenuated viruses, ldl/ p and ldl/ i p , provided good clinical protection against challenge with the nrtw i strain, although all the strains analyzed in this study have actually percentages of similarity with nrtw i that are of almost the same value (they ranged from . to % of similarity at a nucleotide level, and from . to . % of similarity at an amino acid level). some antigenic epitopes that have roles in the protection have also been identified on the s and n proteins of ibv (ignjatovic and sapats, ) and may attribute to the difference of the cross-protection. further studies are needed to better understand the relationship between antigenic epitopes on s (both s and s ) and n proteins and the level of protection provided in challenge studies. as illustrated in table , the currently available vaccines and attenuated viruses did not provide sufficient respiratory protection against nrtw i challenge, which is in agreement with the genotyping and serotyping results. the results showed that the challenge virus was shed by some chickens in all of the vaccinated groups for at least dpc. additionally, the high rates of challenge virus isolation (less than % at dpc) from chickens that were vaccinated with the heterologous vaccines indicated that none of these vaccines and attenuated viruses provided sufficient protection against the ibv nrtw i strain, especially if a reduction of ibv transmission is considered to be important. these results revealed that it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control nrtw i-type ibv strains in the future. over the past years, nephropathogenic ibv strains have emerged as the most predominant ibv strains in the poultry industry, and they are responsible for many outbreaks of kidney disease on chicken farms (de wit et al., ) . generally, the nephropathogenic ibv strains have a tropism for the epithelial cells of the respiratory tract, which leads to clinical signs such as excessive water consumption and wet droppings, followed by a severe renal infection that leads to increased mortality, as shown in the case for the nrtw i strain in our previous study (xu et al., ) , which was corroborated in the present study. our results also confirmed that the morbidity and mortality caused by nrtw i infection was age-dependent (cavanagh and gelb, ) . it was shown that many of the infections in young chickens caused by ibv strains result in permanent damage to the oviduct (broadfoot et al., (broadfoot et al., , , which leads to a significant reduction in egg production and quality upon sexual maturity. the effect of ibv on the oviduct of chickens is extremely variable. there are at least four major factors that influence the severity of oviduct lesions following ibv infection, as well as the outcome regarding the induction of false layers: the ibv strain that is involved, the age at infection, the presence of strain-specific, virus-neutralizing, maternally-derived antibodies at the time of infection, and the early protection induced by vaccinating young chickens (crinion and hofstad, ; de wit et al., ) . infections with different strains of lx type (qx-like) viruses can induce cystic oviducts with water-like fluid accumulation in some chickens that survive the infection, and cystic oviducts were observed at different ages after challenge (benyeda et al., ; de wit et al., ) , whereas no lesions could be observed in the oviducts after infection with the pathogenic / strain (benyeda et al., ; de wit et al., ) . for the mass serotype viruses, it appears that the induction of cystic lesions/false layers differs among different strains (crinion, ; jones and jordan, ; benyeda et al., ). these differences are not related to virulence because it was shown that a live attenuated strain, h , was capable of producing similar pathological changes (duff et al., ) . a recent study showed that cystic oviducts were also found in some spf female chicks that were infected with an is/ / -like virus (awad et al., ) , which was first detected in israel (meir et al., ) and later found in other parts of the world (awad et al., ) . in this study, we found that the nrtw i strain could induce cystic oviducts in spf chickens of different ages within month post-challenge. the nrtw i strain emerged recently in china, and it originated from recombination events between tw i-and lx -like (qx) viruses (xu et al., ) . one of the parental viruses, the tw ibv strain, was first isolated in in taiwan, and it is a nephropathogenic ibv strain; however, it was unknown whether it could induce cystic oviducts (wang and tsai, ) . in contrast, it was clearly shown that another parental virus (the lx type or qx-like) could induce both nephritis and cystic oviducts in chickens that survived infection (benyeda et al., ; de wit et al., ) . the nrtw i strain acquired the sequences of the nsp and s genes from a tw i-like virus, and the rest of its genome from an lx -like virus, although the tissue tropism and pathogenicity determinants of ibv remain unknown (xu et al., ) . in addition, our results also confirmed that as they age, chickens become more resistant to oviduct damage caused by nrtw i infections (cavanagh and gelb, ) . similar to other reports (benyeda et al., ) , our results showed that viral antigens in the kidneys and oviducts of the surviving chickens became undetectable immunohistochemically by the end of the experiment; this is in agreement with our field experience, which showed that ibvs could not be isolated (in most cases) from chickens exhibiting cystic oviducts. in conclusion, these experiments confirmed previous observations that showed that the nrtw i strain has a novel genotype, and that it is a pathogenic ibv strain that can result in high mortality rates by causing nephritis in susceptible birds. our results also showed that nrtw i infection induced age-dependent cystic oviducts. in addition, a cross-neutralization test showed that the nrtw i strain of the nrtw i genotype has a novel serotype that differs from those of the other strains used in this study. a vaccination-challenge test using different heterologous live vaccines and attenuated viruses in -day-old chickens did not induce high levels of protection against challenge at days of age, which suggests that it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control nrtw i type ibv strains in future. experimental infection of is/ / -like infectious bronchitis virus in specific pathogen free and commercial broiler chicks comparison of the pathogenicity of qx-like, m and /b infectious bronchitis strains from different pathological conditions effects of infectious bronchitis on egg production effects of infectious bronchitis in baby chicks infectious bronchitis molecular and antigenic characteristics of massachusetts genotype infectious bronchitis coronavirus in china pathogenicity of four serotypes of avian infectious bronchitis virus for the oviduct of young chickens of varying ages egg quality and production following infectious bronchitis virus exposure at one day old 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 infection of day old chicks with infectious bronchitis (ib) virus and subsequent anatomical abnormalities a comparative study of pigeons and chickens experimentally infected with ppmv- to determine antigenic relationships between ppmv- and ndv strains a -year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china fine level epitope mapping and conservation analysis of two novel linear b-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus persistence of virus in the tissues and development of the oviduct in the fowl following infection at day old with infectious bronchitis virus altered pathogenicity, immunogenicity, tissue tropism and - kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/ i strain of infectious bronchitis coronavirus characterization of a recombinant coronavirus infectious bronchitis virus with distinct s subunits of spike and nucleocapsid genes and a untranslated region identification of a novel nephropathogenic infectious bronchitis virus in israel a simple method of estimating fifty percent endpoints mega : molecular evolutionary genetics analysis (mega) software version . s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification genetic grouping for the isolates of avian infectious bronchitis virus in taiwan emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks serotype shift of a /b genotype infectious bronchitis coronavirus by natural recombination the authors declare that they have no competing interests. key: cord- - dikelw authors: pujols, joan; segalés, joaquim title: survivability of porcine epidemic diarrhea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: dikelw bovine plasma was inoculated with porcine epidemic diarrhea virus (pedv) at an average final titer of . log tcid( )/ml to determine the effect of spray drying on viral inactivation. using a laboratory scale drier, inoculated plasma was spray dried at °c inlet temperature and either or °c throughout substance. both liquid and dried samples were subjected to three passages on vero cell monolayers to determine pedv infectivity. results indicated liquid samples contained infective virus, but none of the spray dried samples were infectious. also, survivability of pedv inoculated on spray dried bovine plasma (sdbp) and stored at , or °c was determined for , and days. commercial sdbp powder was inoculated with pedv to an average final titer of . log tcid( )/g. five samples per time and temperature conditions were subjected to three passages on vero cell monolayers to determine pedv infectivity. the virus was non-infectious for all samples stored at °c at , and days. pedv was infective in out of samples stored at °c at days, but none of the samples stored for and days were infectious in cell culture. for samples stored at °c, out of samples were infectious at days, out of samples were infectious at days, but none were infectious at days. in summary, pedv was not infectious on cell culture within days when stored at room temperature and within days when stored at refrigerated temperature. porcine epidemic diarrhea (ped) virus (pedv) is an rna enveloped virus ranging in diameter from to nm, which is taxonomically classified into the coronaviridae family (song and park, ) . pedv is antigenically unrelated to other porcine coronaviruses like gastroenteritis transmissible virus and hemaglutinating encephalomyelitis virus (kusanagi et al., ) . pedv is highly infectious and transmission is primarily through pig-to-pig contact or indirect contact with fecal material from infected pigs. ped was linked to diarrhea in nursery and fattening pigs in europe in , and pedv was initially recognized in in belgium (pensaert and debouck, ) . ped has also been documented in south-east asia since the s (song and park, ) . however, variant strains of pedv causing severe outbreaks of disease in suckling pigs with high morbidity and mortality appeared in in china (li et al., ) . furthermore, and since april , very similar pedv strains to the chinese variants have been veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] bovine plasma was inoculated with porcine epidemic diarrhea virus (pedv) at an average final titer of . log tcid /ml to determine the effect of spray drying on viral inactivation. using a laboratory scale drier, inoculated plasma was spray dried at c inlet temperature and either or c throughout substance. both liquid and dried samples were subjected to three passages on vero cell monolayers to determine pedv infectivity. results indicated liquid samples contained infective virus, but none of the spray dried samples were infectious. also, survivability of pedv inoculated on spray dried bovine plasma (sdbp) and stored at , or c was determined for , and days. commercial sdbp powder was inoculated with pedv to an average final titer of . log tcid /g. five samples per time and temperature conditions were subjected to three passages on vero cell monolayers to determine pedv infectivity. the virus was noninfectious for all samples stored at c at , and days. pedv was infective in out of samples stored at c at days, but none of the samples stored for and days were infectious in cell culture. for samples stored at c, out of samples were infectious at days, out of samples were infectious at days, but none were infectious at days. in summary, pedv was not infectious on cell culture within days when stored at room temperature and within days when stored at refrigerated temperature. ß elsevier b.v. all rights reserved. detected in north america resulting in significant pig mortality and economic loss (huang et al., ) . phylogenetic analyses suggested that the pedv in the usa likely originated from china (huang et al., ) . taking into account that the exact source of origin of the us strains has not been identified, the potential continued spread of the virus worldwide is a critical concern. under this scenario, feed or feed ingredients, including spray-dried porcine plasma (sdpp), have recently been suggested as potential sources for transmission of pedv (pasick et al., ) . spray-dried plasma (sdp) proteins are used extensively in nursery pig feed to enhance feed intake, growth, and feed efficiency during the post-weaning period (coffey and cromwell, ; van dijk et al., ) and represents an excellent alternative to antibiotics (torrallardona, ) . the spray drying process used to manufacture sdp involves sudden changes in temperature and pressure that causes rapid evaporation of water which is detrimental to a variety of pathogens including bacteria and viruses (polo et al., (polo et al., , . a series of studies have demonstrated that manufacturing conditions to produce commercial spray-dried porcine plasma (sdpp) inactivate several viruses of importance to the swine industry such as porcine reproductive and respiratory syndrome virus (prrsv), pseudorabies virus (prv) (polo et al., ) or swine vesicular disease virus (svd) (pujols et al., ) . in addition, several studies have reported that orally consumed commercial sdpp does not transmit either porcine parvovirus (ppv) or porcine circovirus type (pcv ) (polo et al., ; pujols et al., pujols et al., , shen et al., ) , which are two of the most heat and chemically resistant swine viruses. the first objective of this study was to determine the survival of pedv inoculated in liquid bovine plasma that was then submitted to two different spray drying conditions ( and c throughout its substance). a second objective was to determine survival time of pedv inoculated on spray-dried bovine plasma when stored under different temperatures ( , and c) for different periods of time ( , , and days). the cv strain (pensaert and debouck, ; debouck and pensaert, ) , prototype pedv strain from belgium, belongs to group g - and is similar to isolates br- / (britain), js- (china), p- v (japan) and kped- , sm - and dr- (south korea) (park et al., ) . this strain of pedv is well known to propagate on vero cells and was selected as a model swine coronavirus to study inactivation of pedv by spray drying. pedv was propagated in vero cell line, grown in eagle's minimum essential medium (mem), supplemented with . % tryptose phosphate broth (sigma), % fetal bovine serum and an antibiotic combination (penicillin iu/ml, streptomycin mg/ml). virus stock was produced in vero cells seeded in cm flask to confluent monolayers, cells were rinsed times with phosphate buffered saline and inoculated with ml of pedv . log tcid /ml (moi of . ) to each bottle. the virus-cells mixture was incubated h at c, and then the inoculum was replaced with post-infection media (pm), which consisted of mem supplemented with . % tryptose phosphate broth (sigma), . mg/ml trypsin (sigma) and the above mentioned antibiotic combination (hofmann and wyler, ) . cultures were checked at and h after infection to assess syncytia formation and the evolution of cytopathogenic effect (cpe) until reaching nearly % cpe. liquid supernatants and scraped cells were collected. the pooled suspensions were centrifuged at g for min at c and supernatants were stored at c. the resulting pellet was suspended in pm and subjected to freeze-thaw, centrifuged and supernatants were harvested and added to bulk supernatants. stock virus was stored at À c or used immediately after its production (hofmann and wyler, ) . a pedv virus stock was produced to a virus titer ranging between . log and . log tcid /ml (hofmann and wyler, ; song et al., ; oh et al., ) . total pedv rna genome copies of virus production was estimated by quantitative reverse-transcriptase pcr (qrt-pcr) with a swine enteric panel assay for pedv and other enteric viruses (thermo fisher scientific inc., waltham, ma). correlation between pedv loads by tcid /ml on cell culture and ct values by qrt-pcr was calculated. for this, -fold dilutions of . ae . log tcid of strain pedv cv were extracted two times and amplified times each one. the reason for using bovine plasma was mainly to avoid the presence of potential antibodies against pedv european strains or cross-reaction with antibodies against other porcine coronavirus that may be present in swine plasma. bovine plasma was collected from an eu inspected slaughter facility. animals were inspected and approved for slaughter for human consumption. blood was collected into stainless steel pans containing sodium phosphate as anticoagulant. blood was transported refrigerated to the laboratory and plasma was separated by centrifugation and three . -l bottles were prepared and frozen at À c. a -ml sample of each of the . -l batches was stored at À c and used as controls to determine absence of virus neutralization antibodies and viral contamination. each batch of plasma was inoculated with stock pedv at : dilution. pedv was added to each . l of bovine plasma to get an approximate virus titer of . log - . log tcid /ml. each of the three . -l batches was sampled in three ml tubes just after the virus inoculation and immediately frozen at À c. the three . -l batches were dried using a bü chi mini spray dryer (bü chi labortechnik ag, flawil, switzerland). all the bottles were dried with an inlet temperature of ae c and outlet temperature of ae c. before spray-drying plasma and virus mixture, the spray-drier was drying control bovine plasma at the target temperature for min to ensure that the system was processing at the target parameters when the virus inoculated plasma was dried. the spray drying parameters used were similar to those previously established (polo et al., ) . airflow (at c) through the column was set at m h À and the suspension flow to the nozzle was set at . m h À . the airflow through the feeding nozzle was set at . m h À (at c). estimated dwell time was . s. the dwell time of industrial size driers used in manufacturing plants to produce commercial sdp ranges between and s, depending on size of the spray drier and configuration (fluid bed, etc.), whereas the dwell time of the lab drier was < s. in an attempt to more closely simulate industrial size driers and conditions, immediately after the spray-drying process, sub-samples ( . g each) of sdp were placed in . cm (inner diameter) glass tubes and held at - c in a water bath for a dwell time of s or s to achieve an interior plasma powder temperature of . c and . c, respectively. the temperature was determined with thermocouple probe (crison instruments sa, alella, spain) inserted in the glass tube containing the sdbp. dried samples were immediately frozen in dry ice and stored at À c prior to analysis. liquid and dried frozen samples were analyzed for infectivity in vero cell cultures, using the microtiter assay procedure. dried samples were reconstituted by adding . g of plasma to . ml of distilled water to each sample. to determine that no minute quantities of virus had survived, ml of each of the reconstituted spray-dried samples were inoculated on a cm flask of vero cell monolayers. after h of incubation at c the inoculum was discarded and cultures were rinsed times with pbs and then pm media was added. after four days of incubation at c and % co , cell cultures were frozen-thawed, harvested, gently vortex to full homogenization and ml supernatants seeded onto new cm bottles with vero cell monolayers. three serial passages were made. if cpe was observed, then the initial sample was titrated on vero monolayers grown in -well tissue culture plates. a virus neutralization (vn) test was performed on the three liquid samples to verify that the bovine plasma used had no neutralizing antibodies against pedv. briefly, log dilutions of bovine plasma were incubated h at c with tcid /ml of pedv. then . ml of this mixture was added to a vero cells monolayer grown in -well plates that previously had been rinsed times with pbs, and were incubated for h at c. after adsorption, inoculum was removed and plates were rinsed times with pbs. pm was added and plates were incubated for days at c. virus neutralization was expressed as the reciprocal of highest serum dilution that abrogates the cpe (oh et al., ) . . . experiment : survival of pedv inoculated on spray dried bovine plasma stored over time at different temperatures spray dried bovine plasma (sdbp) was selected from commercial production for use in the experiment. a -g sample of the sdbp was stored at À c and used as a control to determine absence of pedv neutralization antibodies and pedv contamination. sixty grams of sdbp contained in a large petri dish were inoculated with . ml of pedv cell culture by spraying the liquid virus culture onto the sdbp using a spray bottle. the petri dish was sealed with plastic paraffin film and shaken vigorously for min to ensure a good homogenization of the virus with the sdbp. one gram of inoculated sdbp was added to different glass tubes ( . cm inner diameter). one set of tubes was stored at ae c in the lab refrigerator, another set of tubes was stored at ae c in a calibrated water bath and the last set of tubes was stored at room lab temperature of ae c. for each temperature storage condition, tubes were selected at , and days and stored at À c until their analysis. at the time of analysis, the À c stored samples (the initial samples and the samples stored at different time by temperature conditions) were analyzed to detect the survival of pedv in cell culture. each dried sample was reconstituted by adding g of sdbp to ml of distilled water. to determine that no minute quantities of virus survived, ml of each of the reconstituted sdbp samples were inoculated on a cm flask of vero cell monolayers and, after h incubation at c, the inoculum was discarded and cultures were rinsed times with pbs and then pm media was added. after four days, cell cultures were thawed, harvested, clarified by centrifugation and passaged to new vero cell cultures. three serial passages were made. if cpe was observed, then virus was titrated on vero cell monolayers grown in -well tissue culture plates. a tissue culture vn test was performed on the control sdbp used to ensure lack of neutralizing antibodies against pedv in the original sample. briefly, log dilutions of sdbp re-suspended in distilled water ( . g sdbp to ml distilled water) were incubated h at c with tcid /ml of pedv. then, the same procedure explained before for liquid bovine plasma was followed. statistical analysis were performed on qrt-pcr results by glm procedure and turkey-kramer multiple comparison test (ncss , statistical systems, ncss llc, kaysville, ut). correlation between pedv titers expressed as tcid /ml and pedv loads expressed in qrt-pcr ct values are displayed in fig. . under the range of detection on cell culture, the qrt-pcr results displayed an increased range of standard deviation, which was attributed to non-encapsidated virus material, still present at higher dilutions. the regression results showed a close correlation (r = . ; y = À . x + . ). the non-inoculated liquid bovine plasma stored at À c was determined negative for presence of pedv and negative for neutralizing antibodies against the virus. total concentration of pedv in the pellet used to inoculate the liquid bovine plasma was determined to be . log tcid /ml (ct = . ae . by means of qrt-pcr). results of the liquid and the sdbp samples are presented in table . analyses of the initial batches of inoculated liquid bovine plasma stored at À c gave an average titer of . ae . log tcid /ml (calculated for dry product: . ae . log tcid /g). the samples processed at both temperatures ( and c) throughout its substance were negative for pedv infectivity on cell culture. the calculated inactivation rate at these temperatures was at least greater than the initial titer of . log tcid tcid /g. the qrt-pcr ct results for the virus inoculated plasma were . ae . , and for the spray dried plasma at and c throughout its substance were . ae . and . ae . , respectively. the non-inoculated sdbp stored at À c was determined negative for pedv in vero cell culture and qrt-pcr. total concentration of pedv in the pellet used for sdbp inoculation was titrated to be . log tcid /ml (ct = . ae . ). the expected titer after virus inoculation was . log tcid /g. results of the sdbp pedv inoculated samples are presented in table . analyses of the initial contaminated sdbp samples that were immediately stored at À c averaged . log tcid /g (average ct = . ae . ). for samples stored at ae c, out of the samples contained infectious pedv at day of storage, but at day only out of the samples contained infectious virus and none of the samples contained infectious pedv by day of storage. the ct value for samples stored at ae c averaged . ae . . for samples stored at ae c, only out of the samples stored for days contained infectious virus and by days and none of the samples retained infectivity. the ct value for samples stored at ae c averaged . ae . . for samples stored at room temperature ( ae c), none of them remained infectious at , and days of storage. the ct value for samples stored at c averaged . ae . . the primary route of infection of pedv is direct contact with infected pigs or from the manure of infected pigs (oie, ) . other routes of infection responsible for spreading the virus have not been clearly identified, but transport vehicles for swine and feed or feed ingredients have been shown to contain pedv genome by rt-pcr (dee et al., ; oie, ) . the role of feed or feed ingredients in the spread of pedv was suggested after the first report of ped in ontario (canada). the subsequent investigation conducted by the canadian food inspection agency (cfia) concluded that nursery feed containing sdpp might have been a source of pedv transmission. although the cfia reported that infective virus was detected in samples of sdpp, infective virus could not be detected in the feed containing the corresponding sdpp (pasick et al., ) . in spite of this conflicting data, the opinion that sdpp may contain infective pedv as a reason to explain the spread of pedv across north america has been adopted by some swine industry professionals. the results obtained in the present study indicated that pedv inoculated in bovine plasma was apparently inactivated when spray-dried at both simulated commercial processing conditions of and c throughout its substance. these results are in agreement with previous publications indicating that pedv is a low thermal stable virus (song and park, ) . in addition, these results agree with previous reports with different enveloped the spray dryer concentrated liquid plasma times; the expected virus titer in the dry product was . ae . log tcid /g. c the theoretical limit of detection of the method used was estimated to be able to detect as low as . viral particles/g. viruses like prrsv and prv (polo et al., ) and a nonenveloped virus like svd (pujols et al., ) . these studies demonstrated that the commercial spray drying conditions used for the manufacturing process of commercial sdp should be sufficient to eliminate low-medium heat resistant viruses like pedv. the normal processing temperature condition used by commercial sdp manufacturing plants is > c throughout its substance. the present data indicated that drying at c throughout its substance was already adequate to inactivate at least . log tcid /g of this virus. therefore, the heat treatment used during the commercial spray-drying process is theoretically more than adequate to inactivate pedv if present in the raw material. recent data would support such conclusion opriessnig et al., ) . in the first study, researchers used raw plasma from a non-infected pig spiked with wild type us strain of pedv and either fed directly as raw liquid spiked plasma or spray-dried it (in a laboratory drier at c outlet temperature) and fed to naïve pigs. their results indicated that the raw liquid spiked plasma was infective but the spray dried spiked plasma was not infective, indicating that spray drying at outlet temperature around c should be enough to inactivate the pedv . in the second study, piglets received a diet with % sdpp containing . ae . log pedv rna copies/g and showed no evidence of infectivity by the existing pedv rna present in the sdpp lot utilized . pedv viremia has been reported only once in gnotobiotic pigs experimentally infected with pedv genogroup (jung et al., ) . it is usually accepted that pedv rna detected in serum is possible at the peak of disease, but viremia is reported to be short and of low magnitude (pensaert, ) . therefore, it appears unlikely that pedv viremia and utilization of blood from viremic pigs is a main source of pedv contamination of sdpp. probably, the presence of this virus in raw blood is more likely attributed to carcass contamination at time of slaughter opriessnig et al., ) . the present data studying the survival of pedv inoculated on sdbp appeared to be dependent upon storage temperature and time, with virus survivability associated with lower temperature storage conditions. however, virus concentration diminished considerably for the samples stored at c or c for days compared to samples immediately stored frozen at À c. importantly, samples stored at room temperature did not contain infective pedv by days of storage, which is consistent with previous data reported by other researchers. they demonstrated that pedv-positive feces spread evenly on the bottom surface aluminum tray and treated at c for days were non infective when feed-back to naïve pigs by oral-gastric tube (thomas et al., ) . also, it is important to highlight that, even at c, the virus was not infectious in cell culture after days of storage. the fact that spray dried blood products are powders with low moisture (below %) and water activity (< . ) are important, since pedv does not survive very long under dry conditions (oie, ) . the ct values for samples stored at , and c for days were . ae . , . ae . and . ae . , respectively. these results indicated that qrt-pcr ct values were not different between samples (p > . ), independently of the storage conditions. however, differences between days (p < . ) at each temperature condition should be indicative of progressive genome degradation. moreover, the qrt-pcr positive results compared to those generated by cell culture of the virus indicated that the former technique is not a good indicator to establish pedv infectivity. however, it is also true that virus isolation might not be highly sensitive; therefore, a swine bioassay would be the most definitive way to demonstrate the putative infectivity of those qrt-pcr positive samples which were negative after inoculation of vero cells. in summary, results of the present work demonstrated that pedv (at least the european prototype strain, cv ) was apparently inactivated by spray drying conditions mimicking those used by the spray-dried blood products industry. moreover, if sdp would become contaminated use of spray-dried animal plasma in diets for weanling pigs experimental infection of pigs with a new porcine enteric coronavirus cv an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naive pigs following consumption via natural feeding behavior: proof of concept the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate new variants of porcine epidemic diarrhea virus, china comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection infection with porcine epidemic diarrhoea virus porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naive pigs sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada porcine epidemic diarrhea a new coronavirus-like particle associated with diarrhea in swine efficacy of spray-drying to reduce infectivity of pseudo rabies and porcine reproductive and respiratory syndrome (prrs) viruses and seroconversion in pigs fed diets containing spray-dried animal plasma neutralizing antibodies against porcine circovirus type in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma inactivation of swine vesicular disease virus in porcine plasma by spray-drying lack of transmission of porcine circovirus type to weanling pigs by feeding them spray dried porcine plasma commercial spray-dried porcine plasma does not transmit porcine circovirus type in weaned pigs challenged with porcine reproductive and respiratory syndrome virus commercially produced spray dried porcine plasma contains high levels of porcine circovirus type (pcv ) dna but did not transmit pcv when fed to naïve pigs porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf evaluation of time and temperature sufficient to inactivate porcine epidemic diarrhea virus in swine feces on metal surfaces spray dried animal plasma as an alternative to antibiotics in weanling pigs -a review growth performance of weanling pigs fed spray-dried animal plasma: a review funding for this study was provided by apc europe, s.a., granollers, barcelona, spain. the authors thank dr. hans nauwynck, laboratory of virology, faculty of veterinary medicine (ghent university) for supplying the pedv cv strain and the vero cell line. we also thank nuria navarro, nuria pujol, marta valle and lorena có rdoba from cresa and carmen rodríguez and neus saborido from apc europe, s.a. for their assistance providing the raw material and with the laboratory work. the authors declare no conflict of interest. key: cord- - mhjtitv authors: kim, hyun-jeong; park, sang-ik; ha, thi phuong mai; jeong, young-ju; kim, ha-hyun; kwon, hyoung-jun; kang, mun-il; cho, kyoung-oh; park, su-jin title: detection and genotyping of korean porcine rotaviruses date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: mhjtitv porcine group a rotavirus (garv) is considered to be an important animal pathogen due to their economic impact in the swine industry and its potential to cause heterologous infections in humans. this study examined fecal samples from farms located in provinces across south korea. rt-pcr and nested pcr utilizing primer pairs specific for the garv vp gene detected garv-positive reactions in ( . %) diarrheic fecal samples. a total of porcine garv strains isolated from the garv-positive feces were analyzed for g and p genotyping. based on the sequence and phylogenetic analyses, the most predominant combination of g and p genotypes was g p[ ], found in garv strains ( . %). the other combinations of g and p genotypes were g p[ ] ( strains [ . %]), g p[ ] ( strains [ . %]), g p[ ] ( strains [ . %]), and g p[ ] ( strain [ . %]). the counterparts of g or p genotypes were not determined in three g , five p[ ], and one p[ ] strains. interestingly, phylogenetic analysis indicated that all korean g strains were more closely related to lineage vi porcine and human viruses than to other lineages (i–v) of garvs and to korean human g strains (lineage iii). these results show that porcine garv infections are common in diarrheic piglets in south korea. the infecting strains are genetically diverse, and include homologous (g p[ ]), heterologous (g p[ ]), and reassortant (g p[ ]), as well as emerging g garv strains. group a rotavirus (garv), a member of the reoviridae family, is one of the major pathogens that cause severe, acute dehydrating diarrhea in young children and in a wide variety of domestic animals (estes and kapikian, ; gentsch et al., ; glass et al., ) . the rotavirus genome consists of segments of double-stranded (ds) rna enclosed in a trilaminar capsid and encodes six structural (vp -vp , vp , and vp ) and six nonstructural proteins (nsp -nsp ) (estes and kapikian, ; gentsch et al., ; parashar et al., ) . due to the segmented nature of the genome, garvs can undergo genetic reassortment if two different garvs of the same group co-infect one cell (estes and kapikian, ; gentsch et al., ; parashar et al., ) . recently, a new rotavirus classification system was proposed, in which nucleotide percentage identity cut-off values define different genotypes for all the genomic rna segments (matthijnssens et al., ) . the vp and vp outer capsid proteins independently elicit neutralizing antibody responses and are used to classify garvs into g (for glycoprotein) and p (for protease-sensitive) types (ciarlet and estes, ; estes and kapikian, ; glass et al., ) . currently, g and p genotypes have been described for garvs of humans and animals (abe et al., ; ursu et al., ) . as many more reassortant or new genotypes are predicted to appear, continuous monitoring of circulating rotaviruses is important for improving regional epidemiological information and updating the vaccine strains. porcine garvs can cause enormous economic losses in the swine industry and are a potential source of heterologous garv infections in humans (jain et al., ; leite et al., ; martella et al., ; timenetsky et al., ; unicomb et al., ) . thus, molecular epidemiology on porcine garvs in south korea is needed to determine the prevalence, as well as the extent of diversity in the circulating strains to improve vaccination programs by updating the vaccine strains. this paper reports the prevalence of porcine garvs in diarrheic piglets, along with the genetic diversity of the porcine garvs based on the characterization of the g and p genotypes. from to , fecal specimens from diarrheic pigs were obtained from farms across provinces in south korea during the spring ( samples/ farms), summer ( samples/ farms), autumn ( samples/ farms), and winter ( samples/ farms). the ages of the pigs tested from these provinces ranged from to days old. the fecal samples were examined for common bacterial enteric pathogens including escherichia coli (e. coli) and salmonella spp. using specific agar media. brachyspira hyodysenteriae was detected by pcr with the specific primers b.hyo nest ( -ctgctgccttcttca-taaat- ) and b.hyo nest ( -aagaatgggtattg-ttgctg- ) (la et al., ) . for the extraction of viral rna, fecal suspensions of each sample were prepared immediately by diluting the feces : in . m phosphate-buffered saline (pbs), ph . . the suspensions were then vortexed for s, centrifuged (  g for min), and then the supernatants were collected and stored at À c until needed. the rna was extracted from a ml starting volume of centrifuged % fecal suspensions and from the lysates of garv-infected fetal rhesus monkey kidney (tf- ) cells using the sv total rna isolation system reagent (promega corporation, madison, wi) according to the manufacturer's instructions. the total rna recovered was suspended in ml of rnase free water and stored at À c until used. rt-pcr assays with different primer sets (table ) for the detection of porcine groups a-c rotaviruses (garvs-gcrvs), porcine sapovirus (psav), porcine norovirus (pnov), porcine torovirus (ptov), transmissible gastroenteritis coronavirus (tgev), and porcine epidemic diarrhea coronavirus (pedv) were performed using a standard one-step rt-pcr as previously described (jeong et al., ) . in order to increase the sensitivity and specificity of rt-pcr, nested pcr assays with the primer pairs specific to porcine garv, gcrv and psav (table ) were performed as previously described (jeong et al., ) . the amplification products were analyzed by . or % agarose gel electrophoresis and visualized by uv after ethidium bromide staining. monolayers of tf- cells (a cloned derivative of ma- monkey kidney cells) grown for or days in -well plates were used to isolate garvs, as previously described (bohl et al., ; park et al., ) . the isolated garvs were confirmed by direct immunofluorescence (if) tests and rt-pcr (bohl et al., ; park et al., ) . to obtain genomic data on the g and p genotypes of korean porcine garvs, porcine garvs isolated from the diarrheic fecal samples were subjected to rt-pcr with primer pairs specific to each vp and vp gene of garvs (table ) . rt-pcr products amplified by each primer pair were selected based on the intensity of the bands shown by agarose gel electrophoresis and ethidium bromide visualization. before sequencing, the rt-pcr products from each gene fragment were purified using a qiaex ii gel extraction kit (qiagen, valencia, ca) according to the manufacturer's instructions. dna sequencing was carried out using an abi system automated dna sequencer (applied biosystems, foster city, ca). using the dna basic module (dnasis max, alameda, ca), the nucleotide and deduced amino acid sequences of the partial vp gene ( bp, devoid of primer pair sequences) and vp gene ( bp, devoid of primer pair sequences) were compared with those selected from other known garvs (table ) . phylogenetic analysis based on the nucleotide alignments was constructed using the neighbor-joining method and the upgma method of molecular evolutionary genetics analysis (mega version . ) with a pair-wise distance comparison (tamura et al., ) . a sequence similarity search was performed for the garv vp and vp genes using the lalign query program of the genestream network server at institut de gé né tique humaine, montpellier, france (http://www. eng.uiowa.edu/$tscheetz/sequence-analysis/examples/ lalign/lalign-guess.html). in order to determine the prevalence of porcine garvs in diarrheic korean piglets, a total of fecal samples from diarrheic pigs in farms across south korea were screened by rt-pcr and nested pcr using two sets of primer pairs (table ) . by rt-pcr, out of diarrheic fecal samples tested positive for porcine garvs. in nested pcr, an additional samples were found to be positive for porcine garvs. overall, ( . %) out of diarrheic fecal samples were positive for porcine garvs (table ) . of the porcine garv-positive diarrheic fecal specimens, fecal samples ( . %) tested positive only for the porcine garvs, while the other fecal samples ( . %) were also positive for other enteric pathogens, including gbrv, gcrv, psav, ptov, e. coli, salmonella and b. hyodysenteriae (table ). in addition, fecal specimens ( . %) that tested negative for porcine garvs were positive for other enteric pathogens (table ) . no enteric pathogens were detected in the remaining fecal samples ( . %). porcine garv infections were more prevalent in fecal samples of pigs in summer than in the other seasons: ( . %) out of fecal samples were positive in spring; ( . %) out of fecal samples were positive in summer; ( . %) out of fecal samples were positive in autumn; and ( . %) out of fecal samples were positive in winter. of the porcine garv-positive fecal samples by rt-pcr or nested pcr, porcine garvs were isolated from fecal samples. after the second or third passage, cytopathic effect (cpe), characterized by rounded and detached cells in clusters, was observed in the cultures inoculated with each fecal sample from diarrheic piglets at post inoculation days - . no differences in cpes were observed among the isolates. cpe was not observed in the mock-infected tf- cells. the direct if test detected garv-specific cytoplasmic fluorescence in the tf- cells inoculated with each of these samples at the second or third passage. a specific band was detected after amplification of all isolates using a rt-pcr assay targeting a bp fragment of the vp gene of garvs. using rt-pcr to amplify full length sequence ( nucleotides in length) of the vp gene, amplicons could be achieved for out of strains and could be sequenced. a comparison of the nucleotide and deduced amino acid sequences of the vp gene between all korean porcine garv strains and the garv strains representing all g genotypes was performed with a bp fragment (excluding the primer sequences) (tables and ). sixty-six korean strains showed high nucleotide ( . - . %) and deduced amino acid ( . - %) identities with the g strains, which include the porcine osu and jl strains, and the bovine kj strains. on the other hand, these strains had comparatively lower nucleotide ( . - . %) and deduced amino acid ( . - . %) identities with the other g genotypes (data not shown). phylogenetic analysis also confirmed that the vp gene of korean porcine garv strains was closely related to the g strains and clustered with the porcine g strains (fig. a) . seventeen of the korean porcine garv strains had . - . % nucleotide and . - . % deduced amino acid identities to the g garvs including the bovine brv , sun , kag , and ngrbg strains (table ) , whereas they showed relatively lower nucleotide ( . - . %) and deduced amino acid ( . - . %) identities with other g genotypes (data not shown). phylogenetically, these strains are grouped with g strains, including the bovine brv , sun , kag , and ngrbg strains. the remaining nine korean porcine garv strains showed high nucleotide ( . - . %) and deduced amino acid ( . - . %) identities with the g strains (table ). in contrast, these strains shared lower nucleotide ( . - . %) and deduced amino acid ( . - . %) identities with the other g genotypes (data not shown). phylogenetic analysis showed that all g korean porcine strains clustered with those of lineage vi. in addition, all korean human g strains were found to be grouped with those of lineage iii (fig. b) . a part of the vp gene ( nucleotides in length) was able to be amplified in out of isolated strains. the nucleotide and deduced amino acid sequences encoding amino acids representing vp * and the amino terminus of vp * of the strains were compared with garv strains representing all the p genotypes. of the korean strains, had high nucleotide ( . - . %) and deduced amino acid ( . - . %) identities with the p[ ] garvs including the porcine osu, jl , and sw / strains, and the bovine pp- strain (table ), but less than . % nucleotide and . % deduced amino acid identities with the other p genotypes (data not shown). phylogenetic analysis of the vp gene provided a molecular basis for their similarity to the p[ ] genotype strains (fig. ) . the sequences of the korean strains, - and - , were most closely related to the bovine brv , ncdv, c , and rf (table ). in contrast, these strains showed lower nucleotide ( . - . %) and deduced amino acid ( . - . %) identities to the representatives of other p genotypes (data not shown). phylogenetically, these two strains clustered with those of the p[ ] genotype (fig. ) . the remaining two strains, - - and - , shared high nucleotide ( . - . %) and deduced amino acid ( . - . %) identities to the p[ ] strains (a , jp - , and hokkaido- strains) ( table ), but less than . % nucleotide and . % deduced amino acid identities compared to representatives of the other p genotypes (data not shown). phylogenetic analysis of the vp gene showed that these strains were grouped with those of the p[ ] genotype (fig. ) . based on the sequence and phylogenetic analyses of korean porcine garvs, g and p genotype combinations were determined in the korean porcine garvs (table ) . the most common combination of g and p genotypes was g p[ ], which was detected in garvs. sixteen garvs had the g p[ ] combination, while g p[ ] garvs were detected in strains. two garvs showed the g p[ ] combination, and one strain had the g p[ ] combination. in addition, the counterparts of g and p genotypes were not determined in three g , five p[ ], and one p[ ] garv strains (table ) . epidemiological information related to the prevalence and genotype specificities of porcine garvs are beneficial for the development of effective vaccines (rosen et al., ) . therefore, we investigated the prevalence of porcine garv infections as well as their genotype diversities in south korea. the fecal prevalence of porcine garv infections in diarrheic piglets has been reported to be . % in argentina (parra et al., ) , % in southern germany (wieler et al., ) , . % in canada (morin et al., (khamrin et al., ) and . % in brazil (rá cz et al., ) . in this study, porcine garv infections in south korea were found widespread and highly prevalent at . %, similar to brazil at . % (rá cz et al., ) . this suggests that porcine garv infections are epidemic in diarrheic piglets in south korea. this is the first large-scale, epidemiological study on the prevalence of porcine garv infections in diarrheic piglets in south korea. epidemiological studies have demonstrated that five g genotypes (g , g , g , g , and g ) and p[ ] ) are the most frequent vp and vp types associated with porcine garv infections (kobayashi et al., ) . in this study, two-thirds of the vp and vp genotypes were comprised of g and p[ ] genotypes, respectively. the other g and p genotypes including g , g , p[ ], and p[ ] were a minority of the vp and vp genotypes. however, g , g , g , p[ ], p[ ], p[ ], and p[ ] genotypes, which were known to be common, were not detected in this study. it is unclear whether the data in this study exactly reflected the true prevalence of g and p genotypes in the field farms due to the difficulty in cultivating some rotaviruses in cell culture (zaberezhny et al., ) . for example, p[ ] porcine garvs are quite fig. . (a) phylogenetic tree of the complete vp genes of the sixty-six g , seventeen g , and nine g strains of korean porcine garvs indicating their genetic relationships with other g genotypes. black triangles contain rotavirus g , g , and g strains. (b) a detailed phylogenetic tree of the complete vp genes of the nine korean porcine g strains with other known g strains indicating their genetic relationships with other known vi lineages of g genotype. reference sequences used in the analysis (a and b) were obtained from the genbank database (table ) . reference sequences used in the analysis were obtained from the genbank database (table ) . common in nature, but are not usually cultivatable (martella et al., ; zaberezhny et al., ) . since we analyzed only cell culture cultivated porcine garv strains, future studies should use the fecal samples for g and p genotyping of porcine garvs to generate a more accurate picture of garv genotypes in south korea (zaberezhny et al., ) . in this study, we isolated g garvs ( . %) in combination with p[ ] and p[ ] across south korea, which were ranked the second most frequently detected g and p types, indicating that these strains may be prevalent throughout south korea. the discovery of these g garvs is important to the swine industry, veterinary practitioners, and garv vaccine producers in south korea. it should be noted that the serotype g is one of the major bovine serotypes in combination with p[ ] and p[ ] genotypes (alfieri et al., ; chang et al., ; fukai et al., ; gentsch et al., ) . in addition, g gavr serotype has been detected in rare cases in humans (adah et al., ; cunliffe et al., ; fischer et al., ; matthijnssens et al., ; palombo et al., ; steele et al., ) , and pigs . among the g garvs, one strain contained bovine-like p[ ]-vp gene, indicating bovine-like g p[ ] strains can infect heterologous species in nature, such as pigs. the remaining g strains contained the porcine-like p[ ]-vp gene. this result implies that reassortant events between porcine and bovine garvs occur in nature. in previous reports (ha et al., ; park et al., unpublished data) , we demonstrated that reassortant garvs between bovine and porcine, and heterologous garvs whose genome segments are of pig origin infect calves and induce diarrhea. therefore, interspecies transmission of garvs between bovine and porcine, either as whole virions or by gene segment reassortment, appear to occur in nature at a relatively high frequency in south korea. since g garv was first detected in a child with gastroenteritis in the united states in (clark et al., ) and subsequently in other countries (das et al., ; nakagomi et al., ; urasawa et al., ; zizdić et al., ) , g garvs have not been reported in humans for a decade. from mid- s, g garvs reemerged and efficiently spread throughout the world as the fifth globally important serotype (ramachandran et al., ; santos and hoshino, ) . recently, g garvs have been classified into i-vi lineages, with i-ii consisting of strains isolated in the s, and iii-vi composing of strains isolated from the mid- s (phan et al., ) . of these, lineages iii and vi were found in both humans and pigs (phan et al., ) . in the present study, g garvs were isolated and identified as the third most important genotype in the diarrheic pigs. all korean strains were clustered in lineage vi of known porcine and human g garvs. thus, continuous genotypic characterization of the garvs and cautions against the increase of the g is necessary in south korea. moreover, human g garv infections belonging to lineage iii have been emerging in south korea since (kang et al., ) , meaning that korean porcine g garvs are different from korean human g garvs. human garvs showed a striking seasonal pattern of infection in developed countries, with epidemic peaks occurring in the cooler months of each year (estes and kapikian, ) . this may be related to the influence of low relative humidity as a factor facilitating the survival of garvs on surfaces (brandt et al., ) . studies describing the seasonal pattern of porcine garvs in diarrheic piglets have rarely been published, and those published data varied widely svensmark et al., ) . in one of the few comparable studies, the seasonal curves of porcine garv infections were highest in winter and the slightly higher in late summer and early autumn in iowa, usa . in contrast, danish porcine garv infections showed a slight increase during the autumn (svensmark et al., ) . in this study, however, porcine garvs occurred throughout the year with the highest prevalence during the summer months. the reason for the seasonal pattern variations around the world is not yet known. therefore, more intensified epidemiological studies throughout the world will be needed to fully understand the seasonal pattern of porcine garv infections and to establish porcine garv surveillance programs to prevent infections. in summary, this study demonstrates that porcine garv infections are epidemic and widespread in diarrheic piglets in south korea. the infecting strains are genetically diverse, and include homologous (g p[ ]), heterologous (g p[ ]), and reassortant (g p[ ]), as well as emerging g garv strains. molecular epidemiology of rotaviruses among healthy calves in japan: isolation of a novel rotavirus bearing new p and g genotypes molecular epidemiology of rotaviruses in nigeria: detection of unusual strains with g p[ ] and g p[ ] specificities g and p genotypes of group a rotavirus strains circulating in calves in brazil isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses rotavirus gastroenteritis and weather the characterization of vp (g type) and vp (p type) genes of bovine group a rotaviruses from field samples using rt-pcr and rflp analysis rotaviruses: basic biology, epidemiology and methodologies rotavirus isolate wi representing a presumptive new human serotype rotavirus g and p types in children with acute diarrhea in characterization of the g serotype and genogroups of new delhi newborn rotavirus strain e rotaviruses characterization of incompletely typed rotavirus strains from guinea-bissau: identification of g and g types and a high frequency of mixed infections molecular characterization of novel p[ ],g bovine group a rotavirus, sun , isolated in japan serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs identification of group a rotavirus gene types by polymerase chain reaction rotavirus vaccines at the threshold vp typing of bovine and porcine group a rotaviruses by pcr sequence analysis of unusual p[ ]g bovine rotavirus strains reveals evidence of interspecies transmission great diversity of group a rotavirus strains and high prevalence of mixed rotavirus infections in india genetic diversity of porcine sapoviruses molecular epidemiological profile of rotavirus in south korea novel porcine rotavirus of genotype p[ ] shares new phylogenetic lineage with g porcine rotavirus strain diversity of g-type and p-type of human and animal rotaviruses and its genetic background development of a duplex pcr assay for detection of brachyspira hyodysenteriae and brachyspira pilosicoli in pig feces rotavirus g and p types circulating in brazil: characterization by rtpcr, probe hybridization, and sequence analysis identification of a novel vp genotype carried by a serotype g porcine rotavirus strain recommendations for the classification of group a rotaviruses using all genomic rna segments g rotavirus strains isolated in the democratic republic of congo belong to the ds- -like genogroup neonatal diarrhea of pigs in quebec: infectious causes of significant outbreaks isolation and molecular characterization of a serotype human rotavirus strain characterization of a ''european-like'' serotype g human rotavirus isolated in australia rotavirus and severe childhood diarrhea molecular characterization of novel g bovine rotavirus strains phylogenetic analysis of porcine rotavirus in argentina: increasing diversity of g strains and evidence of interspecies transmission genetic heterogeneity, evolution and recombination in emerging g rotaviruses molecular characterization of porcine rotaviruses from the southern region of brazil: characterization of an atypical genotype g[ ] strain molecular characterization of serotype g rotavirus strains from a global collection serotypic differentiation of rotaviruses in field samples from diarrheic pigs by using nucleic acid probes specific for porcine vp and human and porcine vp genes global distribution of rotavirus serotypes/ genotypes and its implication for the development and implementation of an effective rotavirus vaccine comparative studies of human rotavirus serotype g strains recovered in south africa and the united kingdom epidemiological studies of piglet diarrhoea in intensively managed danish sow herds. i. pre-weaning diarrhoea mega : molecular evolutionary genetics analysis (mega) software version . survey of rotavirus g and p types associated with human gastroenteritis in sao paulo, brazil, from to evidence of high-frequency genomic reassortment of group a rotavirus strains in bangladesh: emergence of type g in antigenic and genetic analysis of human rotaviruses in chiang mai, thailand: evidence for a close relationship between human and animal rotaviruses molecular analysis of the vp gene of pheasant rotaviruses identifies a new genotype, designated g prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern germany evaluation of rotavirus infection and diarrhea in iowa commercial pigs based on an epidemiologic study of a population represented by diagnostic laboratory cases prevalence of p types among porcine rotaviruses using subgenomic vp gene probes newly discovered rotavirus serotypes in years of collecting samples from children with diarrheal syncromes this study was supported by the national veterinary research and quarantine services (nvrqs), ministry of agriculture and forestry, the korea science and engineering foundation (kosef) grant ( - ), and the regional technology innovation program of the ministry of commerce, industry and energy (mocie), republic of korea. the authors would like to acknowledge a graduate fellowship from the korean ministry of education and human resources development through the brain korea project. key: cord- -a uhy ex authors: huang, fen; zhou, junfang; yang, zhibiao; cui, li; zhang, wen; yuan, congli; yang, shixing; zhu, jianguo; hua, xiuguo title: rna interference inhibits hepatitis e virus mrna accumulation and protein synthesis in vitro date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: a uhy ex hepatitis e virus (hev) is a zoonotic pathogen to which several species, including human beings, pigs and rodents, are reported to be susceptible. to date, vaccines developed against hev still need to be improved and a structural gene (orf ), which encodes a capsid protein with high sequence conservation found across hev genotypes, is a potential candidate. to exploit the possibility of using rna interference (rnai) as a strategy against hev infection, four small interference rna (sirna) duplex targeting orf gene were constructed. a challenge against hev infection by rnai was performed in a cells. real-time quantitative polymerase chain reaction (real-time qpcr) and western blot assay demonstrated that four hev specific sirnas (si-orf - , si-orf - , si-orf - and si-orf - ) were capable of protecting cells against hev infection with very high specificity and efficiency. the results suggest that rnai is a potent anti-hev infection prophylaxis strategy. hepatitis e virus (hev) is a zoonotic pathogen to which several species, including human beings, pigs and rodents, are reported to be susceptible. to date, vaccines developed against hev still need to be improved and a structural gene (orf ), which encodes a capsid protein with high sequence conservation found across hev genotypes, is a potential candidate. to exploit the possibility of using rna interference (rnai) as a strategy against hev infection, four small interference rna (sirna) duplex targeting orf gene were constructed. a challenge against hev infection by rnai was performed in a cells. real-time quantitative polymerase chain reaction (real-time qpcr) and western blot assay demonstrated that four hev specific sirnas (si-orf - , si-orf - , si-orf - and si-orf - ) were capable of protecting cells against hev infection with very high specificity and efficiency. the results suggest that rnai is a potent anti-hev infection prophylaxis strategy. ß elsevier b.v. all rights reserved. ). moreover, immune escapes can also occur as new virulent hev variants evolve, as the case with severe acute respiratory syndrome coronavirus (sars-cov) evolved from human coronavirus (lin et al., ; saif, ) . in this respect, the mutational events in cell cultures that have been reported during the primary propagation and consecutive passages (p , p , and p ) reported in (lorenzo et al., ; tanaka et al., ) are particularly worrisome. in this paper, rna interference (rnai) was used as a way of circumventing some of these issues regarding vaccines, such as high specificity and efficiency. rnai is a phenomenon in which small double-stranded rna molecules induce sequence-specific degradation of homologous singlestranded rna (hannon, ) . rnai is a powerful tool to investigate gene function through specific suppression of a particular mrna, and has been employed in therapeutic studies of human diseases, including cancer, neurogenerative diseases and viral infectious diseases. similar strategies were capable to reduce significantly sars-cov replication (akerstrom et al., ) and hepatitis c virus mrna accumulation (liu et al., ) , which suggests that rnai may be one of the most useful antiviral therapy strategies currently being investigated. as the field of rnai has expanded, much research has been reported for many genes of interest, including oncogenes and viral genes, indicating their successful silencing in both cells and animal models (akerstrom et al., ; kirchhoff, ; kleinman et al., ; liu et al., ) . there have also been a number of clinical trials using rnai strategies, including one on agerelated macular degeneration (amd) (kleinman et al., ) , and more applications are expected. the hev used in this study was characterized as genotype hev (genbank accession no. ef ) and was isolated from swine feces from the shanghai, china. this was used as template for a reverse transcription nested polymerase chain reaction pcr (rt-npcr) to amplify the hev orf gene. the orf gene encodes the major structural or capsid protein of hev (riddell et al., ) . the external forward primer and reverse primer were -cgattttgcgctt-gagcttga- (p ) and -tggagaccgagcgcacggcac- (p ), respectively. the internal forward primer and reverse primer were -ctcggcgggctcccgacag- (p ) and -aggtgcgaggacaccaacggcag- (p ), respectively. rt-npcr analysis was conducted using an amv reverse transcriptase xl kit for rt-pcr (takara, tokyo, japan) according to the manufacturer's directions. the rt-pcr protocol performed included a reverse transcription phase at c for min and c for s. two microliters of the cdna synthesized were then amplified by nested pcr at c for min, followed by c for s, c for s and c for min, and repeated for cycles. the pcr products were detected by electrophoresis on agarose gel containing . mg/ml ethidium bromide. the amplified dna fragment was inserted into the multicloning site of a eukaryotic expression vector pegfp-n (clontech, a gift from dr. wei liu) which contains the reporter gene of enhanced green fluorescence protein (egfp), using standard cloning procedures with the restriction sites of ecori and bamhi. the egfp gene was located downstream of the target genes. the recombinant plasmid was named pegfp-orf . four nt sirnas with dna ends corresponding to the target genes ( fig. ) were designed according to qiagen's guideline (http://www.qiagen.com). the sirna duplexes have been designed using the hiperformance design algorithm licensed from novartis ag, integrated with a stringent in-house homology analysis tool. the highestranking sirna duplexes generated by the algorithm were chosen as representing the best combination of activity and specificity. scrambled sirna, constructed from a random sequence heterology with the hev sequence, served as a negative control for identifying the specificity of hev sirna ( table ). the synthesized non-modified sirnas were diluted with rnase-free buffer to obtain a mm solution. the solution was denatured by heating at c for min, incubated at c for min, then either used immediately or stored at À c in an rnase-free environment. a (human lung carcinoma, atcc, va, usa) cell line was used in this study and maintained as described previously (huang et al., ) . a cells were trypsinized and transferred to ml antibiotic-free growth medium in -well plates at . - .  /well. cells were cultured h before transfection. one microgram of pegfp-orf scramble sirna sense -r(uuc ucc gaa cgu guc acg u)dtdt- antisense -r(acgugacacguucggagaa)dtdt- recombinant plasmid was diluted in ml of serum-free dmem medium, and mixed gently with . ml each of the five nm sirnas and . ml lipofectamine (invitrogen, ca, usa) according to the manufacturer's instructions in ml of serum-free dmem medium for min, and then co-transfected to the cells. meanwhile, each sirnas were either co-transfected with pegfp vector or transfected alone into a cells to test the interferon response. the optimal combination of four parameters for each cell line, including the highest transfection efficiency, the lowest non-specific effects, the conditions for the most efficient delivery of sirna, and the concentration of lipofectamine , was first determined in preliminary experimentation. four to six hours post-transfection, the culture medium was replaced with fresh complete medium and the cells were incubated at c for h for mrna detection, and h for protein detection. to determine transfection efficiency, the egfp fluorescence intensity of transfected cells was monitored with an inverted fluorescence microscope (nikon te , tokyo, japan) before real-time qpcr analysis. the efficiency of suppression of the hev orf gene by various hev specific sirnas was evaluated by fluorescence microscopy, real-time qpcr and western blot. specificity of the inhibition was confirmed by co-transfection of the pegfp-orf recombined plasmid with scrambled sirna. hev has been successfully cultured in a cells as previously described (huang et al., ) . for hev infection challenge, a cells were transfected with each of sirnas (si-orf - , si-orf - , si-orf - , si-orf - , and scrambled sirna) as described previously h before the virus challenge. the viral challenge was performed as the previously described (huang et al., ; tanaka et al., ) . the virus at a viral count of -  /ml as calculated by viral genomic titer determined by real-time quantitative pcr (li et al., ; kasorndorkbua et al., ) was inoculated into a cells, and cpes were observed daily. the cells were harvested h postinoculation, when the cpe was observed, for real-time qpcr analysis (downstream of the target in orf ), and h for western blot assay. a cells ( . -  ) per well seeded in -well plates were incubated as described above except that the amounts of sirnas and virus/pegfp in each well were onefourth of those in -well plates. forty-eight hours post-inoculation with viruses, the culture medium was removed, and the cells were lysed by freeze-thaw three times. the total rna was extracted by trizol (invitrogen, ca, usa) according to the manufacturer's directions and reverse transcribed into cdna using an amv reverse transcriptase xl kit. the synthesized first strand cdna ( ml) was added as a template for real-time qpcr using sybr premix ex taq tm (perfect real-time, takara, tokyo, japan) according to the manufacturer's directions. the forward primer for orf was -cgcacct-cactctgcgctg- ( - nt), and reverse primer was -attggaaagcgcagccctg- ( - nt). the mixtures were reacted at c for s, followed by c for s and c for s repeated for cycles. the product was expected to be bp. the housekeeping gene gapdh served as a loading control. the forward primer of gapdh was -tgggctacactgagcaccag- , and reverse primer was -aagtggtcgttgagggcaat- . the pcr protocol was same as the cells except repeated for cycles. the real-time qpcr analysis was performed in the abi prism real-time pcr system (abi, ct, usa). all procedures were performed in triplicate and data are expressed as means ae s.d. the infected a cells were harvested h postinoculation and lysed with buffer ( mm tris-hcl, ph . , mm nacl, . % sodium azide, % triton x- , mg/ ml aprotinin, and mg/ml pmsf). equivalent amounts of total protein were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto a nitrocellulose membrane. after blocking non-specific-binding sites with % skim milk, the membrane was incubated with primary antibodies at c overnight. the primary antibodies used in this experimental were rabbit polyclonal anti-hev (abr, ca, usa, : dilution), and a rabbit polyclonal anti-gapdh for loading control (proteintech group, co, usa, : dilution). after washing with tbs buffer, the blots were incubated with hrp conjugated goat anti-rabbit igg (promega, wi, usa, : dilution) at room temperature for h. the bands were then exposed to x-ray films with supersignal west pico trial kit (pierce, rockford, usa). the bp orf fragment of hev ( - nt) was amplified by rt-npcr and introduced into the pegfp-n vector with the restriction sites of ecori and bamhi to yield pegfp-orf . the recombinant plasmid was identified by digestion with restriction enzymes (ecori and bamhi) and sequence. a cells transfected with pegfp-orf recombinant plasmid with each sirnas were observed by fluorescence microscope at - h post-transfection (fig. pegfp-orf ). the efficiency of transfection was confirmed by the highly expressed reporter gene egfp protein. fluorescence assays showed an obvious reduction effect induced by all hev specific sirnas except the scrambled one (fig. ) , which suggested that a high efficiency and specificity of inhibition effects by sirna had been achieved. to further study the effect of sirnas on protecting a cells against hev destruction, a mts assay was performed. the od values (mean ae s.d.) of solutions in wells treated with each sirnas (si-orf - , si-orf - , si-orf - , and si-orf - ) were shown in fig. . for the absorbance at nm is directly proportional to the number of living cells in culture, the number of living cells in a well treated with each of hev specific sirnas was more than that treated with scrambled sirna or untransfected controls cells. interferon response was not observed in a cells transfected with each sirnas alone or co-transfected with pegfp vector. the reduction of mrna level of hev was evaluated by real-time qpcr. the percentage of hev mrna in cells previously transfected with sirna over that in cells inoculated with hev alone was calculated according to pfaffl method (pfaffl, ) . the suppression of hev mrna h post-inoculation was decreased about . -fold, . fold, . -fold and . -fold in cells transfected with nm/well sirnas (si-orf - , si-orf - , si-orf - and si-orf - , respectively), while no significant changes were noted in cells transfected with scrambled sirna (fig. a) . the gapdh transcription level was rarely changed in either untransfected or transfected cells (data not shown). to optimize the inhibitory effect of sirna on hev transcription, a viral mrna yield reduction assay was conducted by transfection a cells with each sirnas at indicated doses. as shown in fig. b , transfection of nm/ well of each sirnas just induced an approximately % reduction in viral mrna. however, with the dosage of sirnas increasing, the hev mrna decreased. when the fig. . each of sirnas co-transfected with pegfp-orf /pegfp in a cells. the expression of hev orf protein was observed h post-transfection. hev specific sirnas induced an obvious reduction, whereas the expression level of egfp protein co-transfected with scrambled sirna showed no significant change. each of sirnas (si-orf - or si-orf - ) was co-transfected with pegfp vector to test the interferon response. pegfp-orf : pegfp-orf recombinant plasmid transfection alone; pegfp+ si-orf - : pegfp vector co-transfected with si-orf - ; pegfp+ si-orf - : pegfp vector co-transfected with si-orf - ; scrambled sirna: pegfp-orf recombinant plasmid co-transfection with scrambled sirna; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection. pictures were taken at h post-transfection with a nikon te fluorescence microscope. fig. . protection of a cells from hev infection by sirnas. rnai inhibited a cells growth as determined by mts assay. cells transfected with hev specific sirnas, scrambled sirna, and untransfected (infected) cells were served as controls. absorbance was read at nm and the results were obtained in triplicate wells. od value shown is the mean ae s.d. amount of sirna reached nm/well, the highest inhibition of hev transcription was obtained. the data indicated that the suppression of hev transcription by sirna was dose-dependent. to analyze further the efficiency of suppression of the hev protein synthesis by sirna, a cells were collected for western blot analysis. only the cells infected with hev expressed the expected $ kda protein band , while uninfected a cells showed no protein band reacting with anti-hev antibody. western blot showed that hev putative structural protein expression was reduced significantly in cells transfected with hev specific sirnas (fig. -si-orf - to si-orf - ) compared with cells infected with hev alone or transfected with scrambled sirna (fig. -hev and -scrambled) . gapdh was used as a loading control in this experiment. in conclusion, the results demonstrated that highly efficient inhibition of hev transcription by hev specific sirnas in a cells was obtained. hev is a major cause of enterically transmitted acute hepatitis of adults and is highly lethal in pregnant women. the hev orf gene is highly conserved in four genotypes and is the most important structural region of the virus. evidence from epidemiological, animal transmission and vaccine studies had indicated that the orf gene is a suitable region for hev treatment studies. therefore, the hev orf gene was chosen as the target for sirnas gene silencing in the current study. rna interference has showed great promise as an antiviral therapy since its discovery in mammalian cells (elbashir et al., ) . its application in vitro and in vivo has shown it to be a new and efficient approach for inhibition of viral infection. however, as also shown in this study, different sirna sequences have different interference efficacies, which depending on the characteristics of the target rna, including local rna folding and the accessibility of the sirna-binding site on the target rna (kurreck, ; shao et al., ) . the highest potential efficiency of interference is considered a high knockdown that avoids the degradation of untargeted genes (off-target effects) with the fewer sirnas (ladunga, ) . rna interference in viral infection has been the focus of numerous studies (liu et al., ; mungall et al., ) , and an effective and specific interference in hev transcription was obtained in a cells in the current study. rnai, its exquisite specificity in seeking targets, which determines the exclusive protection against hepatitis e virus. hev specific sirnas were capable of preventing hev infection in a cells. antiviral activity was demonstrated both in mrna transcription level by real-time qpcr and in protein expression level by western blot. these findings indicate that this protocol may be an effective antiviral strategy for protecting host cells against viral invasion. the sirna is very effective in vitro; however, there are many obstacles to application of rnai in vivo as a therapy. the main obstacles are prevention of degradation of sirna and avoidance of side effects to the host induced by sirna itself. couzin ( ) and grimm and kay ( ) mentioned the toxicity of both sirna or shrna expression vectors in vitro and in vivo, and cell apoptosis was observed when vectors were introduced at a high concentration. although the rnai pathway is a promising treatment for cancer, virus and hepatitis therapy, there are serious safety issues that first need to be addressed (mcbride et al., ) . there needs to be a focus on decreasing the toxicity that accompanies sirna treatment. in conclusion, the present study demonstrated that hev specific sirnas could specifically and effectively suppress hev transcription and translation in a cells. these results indicate that rnai can be a potential antiviral therapy for suppression hev infection. further study is required to determine the effectiveness and the safety of rna interference as a means of protection against hev infection in vivo. viral hepatitis in travelers hepatitis e: an overview and recent advances in clinical and laboratory research inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, a/ b, a/ b and s prevalence of antihepatitis e virus antibodies in different indian animal species hepatitis e virus rna in commercial porcine livers in the netherlands rnai safety comes under security duplexes of -nucleotide rnas mediate rna interference in cultured mammalian cells detection and characterization of infectious hepatitis e virus from commercial pig liver sold in local grocery stores in the usa cross-species infection of specific-pathogen-free pigs by a genotype strain of human hepatitis e virus macaque models of human infectious disease therapeutic application of rnai: is mrna targeting finally ready for prime time? comparative pathogenesis of infection of pigs with hepatitis e virus recovered from a pig and a human rna interference expression and diagnostic utility of hepatitis e virus putative structural protein expressed in insect cells identity of a novel swine hepatitis e virus in taiwan forming a monophyletic group with taiwan isolates of human hepatitis e virus cell culture of sporadic hepatitis e virus in china routes of transmission of swine hepatitis e virus in pigs silencing hiv in vivo sequence-and target-independent angiogenesis suppression by sirna via tlr hepatitis e vaccine-ready for prime time? sirna efficiency: structure or sequence-that is the question more complete gene silencing by fewer sirna: transparent optimized design and biophysical signature safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine quantitative detection of hepatitis e virus rna and dynamics of viral replication in experimental infection rna interference effectively inhibits mrna accumulation and protein expression of hepatitis c virus core and e genes in human cells mutational events during the primary propagation and consecutive passages of hepatitis e virus strain je - f in cell culture artificial mirnas mitigate shrna-mediated toxicity in the brain: implications for the therapeutic development of rnai prevalence of antibodies to hepatitis e virus in veterinarians working with swine and in normal blood donors in the united states and other countries inhibition of henipavirus infection by rna interference hepatitis e virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention presence of antibodies to hepatitis e virus in japanese pet cats a new mathematical model for relative quantification in real-time rt-pcr identification of immunodominant and conformational epitopes in the capsid protein of hepatitis e virus by using monoclonal antibodies animal coronavirus vaccines: lessons for sars effect of target secondary structure on rnai efficiency safety and efficacy of a recombinant hepatitis e vaccine efficient recombinant hepatitis e virus vaccine: mission accomplished? development and evaluation of an efficient cell-culture system for hepatitis e virus mutational events during the primary propagation and consecutive passages of hepatitis e virus strain je - f in cell culture zoonotic transmission of hepatitis e virus from deer to human beings elisa for antibody to hepatitis e virus (hev) based on complete open-reading frame- protein expressed in insect cells: identification of hev infection in primates serological evidence of hepatitis e virus infection in different animal species from the southeast of brazil key: cord- -wkrj n d authors: zhang, pengfei; yu, linyang; dong, jianguo; liu, yanling; zhang, leyi; liang, pengshuai; wang, lei; chen, bin; huang, li; song, changxu title: cellular poly(c) binding protein interacts with porcine epidemic diarrhea virus papain-like protease and supports viral replication date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: wkrj n d porcine epidemic diarrhea virus (pedv) belongs to the alphacoronavirus genus in the coronaviridae family. similar to other coronaviruses, pedv encodes two papain-like proteases. papain-like protease (plp) has been proposed to play a key role in antagonizing host innate immunity. however, the function of plp remains unclear. in this study, we found that overexpression of plp significantly promoted pedv replication and inhibited production of interferon-β. immunoprecipitation and mass spectrometry were used to identify cellular interaction partners of plp . host cell poly(c) binding protein (pcbp ) was determined to bind and interact with plp . both endogenous and overexpressed pcbp co-localized with plp in the cytoplasm. overexpression of plp upregulated expression of pcbp . furthermore, overexpression of pcbp promoted pedv replication. silencing of endogenous pcbp using small interfering rnas attenuated pedv replication. taken together, these data demonstrated that plp negatively regulated the production of type interferon by interacting with pcbp and promoted pedv replication. porcine epidemic diarrhea virus (pedv) was identified as the causative agent of porcine epidemic diarrhea (ped) in (pensaert and de bouck, ) and has had catastrophic impacts on the global pig industry. the clinical signs and symptoms of ped include severe enteritis, vomiting, watery diarrhea, and high mortality. in china experienced and outbreak of a mutant pedv, leading to huge economic losses (sun et al., a) . pedv has a positive-sense single stranded rna genome that is approximately kb in size and contains six open reading frames (orfs). orf ab encodes polyprotein (pp) a and pp ab, which are further cleaved into non-structural protein (nsp) - . the structural spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are encoded by orf , orf , orf and orf , respectively. orf encodes the accessory protein orf (kocherhans et al., ) . nsp is the largest nsp, and comprises papain-like protease (plp) and plp domains (lei et al., ) . the innate immune response is the first line of host defense against viral infection (o'neill and bowie, ) . type interferons (ifns) play a key role in host resistance to viral infections. rna viruses induce the production of ifns through toll-like receptor (tlr ) and retinoic acid-inducible gene (rig)-i-like receptor-dependent pathways (kawai and akira, ) . the virus can evade host innate immune response in two major ways: by modifying or hiding pathogen-associated molecular patterns (pamps), and by encoding specific proteins to block immune responses. coronaviruses have evolved specific mechanisms to evade or inhibit host antiviral innate immune j o u r n a l p r e -p r o o f responses (g devaraj et al., ; zhou and perlman, ) . many pedv proteins are involved in escaping the innate immune response. nsp , nsp , nsp , nsp , nsp , and nsp , as well as the structural e, m and n proteins, demonstrated ifn antagonism . the mechanisms underlying the ifn antagonism of nsp , plp , nsp , and n protein have been elucidated (wang et al., ; xing et al., ; zhang et al., ) . pedv nsp inhibits production of type i ifns by degrading cyclic adenosine monophosphate responsive element-binding protein-binding protein and inhibiting immune stress granule expression (dragan et al., ; zhang et al., ) . pedv nsp is a c-like protease that proteolytically cleaves the nuclear transcription factor kappa b (nf-κb) essential modulator (nemo) at glutamine , impairing the ability of nemo to activate ifn production (wang et al., ) . the pedv n protein interacts with the tank-binding kinase, blocking its association with interferon regulatory factor (irf ) and thus inhibiting irf activation and type i ifn production (ding et al., ; hu et al., ) . pedv can also resist immune responses by co-opting host cell proteins. poly(c) binding protein (pcbp ) belongs to a class of proteins that bind poly(c) sequences in both rna and dna and are involved in maintaining mrna stability, regulating translation and cellular antiviral responses (makeyev and liebhaber, ) . pcbp expression is induced following viral infection and acts as a negative regulator of mitochondrial antiviral signaling protein (mavs), triggering its degradation (you et al., ) . it was previously reported that pcbp interacted with porcine reproductive and respiratory syndrome virus (prrsv) nsp β and supported viral replication (beura et j o u r n a l p r e -p r o o f wang et al., ) . pcbp also antagonized vesicular stomatitis virus growth by affecting viral gene expression . it is unclear whether pcbp is also involved in pedv replication. although significant progress has been made in understanding pedv evasion of innate immune responses, the mechanism of interaction between viral and host cell proteins remains unclear. in the present study, we demonstrated that plp interacted with pcbp to inhibit ifn-β production and promote pedv replication. human embryonic kidney (hek) t cells, porcine intestinal epithelial ipec-j cells and african green monkey vero e kidney cells were maintained in dulbecco's modified eagle's medium supplemented with % heat-inactivated fetal bovine serum (fbs), units/ml penicillin and μg/ml streptomycin. pedv strain gdgh (genbank accession number: mg ) was isolated and stored in our laboratory. a dna sequence encoding pedv plp protein was cloned into pcmv-ha as previously described to yield the pcmv-plp -ha expression vector (yu et al., ) . pcbp and pcbp were amplified by pcr and cloned into the pecmv- ×flag-n vector using the primers listed in table . all plasmids were verified by dna sequencing. hek t cells were seeded on coverslips in -well plates and cotransfected with j o u r n a l p r e -p r o o f ng of ifn-luc, ng of tk-luc and ng of pcmv-plp -ha or empty pcmv-ha vector. twenty-four hours post-transfection (hpt), cells were stimulated with ng/ml tumor necrosis factor (tnf)-α for h. cell lysates were prepared for analysis of luciferase activity using a luciferase enzyme assay system in accordance with the manufacturer's instructions (promega, beijing, china). total cellular rna was extracted using the minibest universal rna extraction kit (takara, japan) in accordance with the manufacturer's protocol. total rna was reverse transcribed to cdna using prime script™ rt master mix (takara, japan) and relative gene expression levels were quantified by quantitative rt-pcr using sybr® green real-time pcr master mix (toyobo, japan) using the cycle threshold method. βactin was used as the internal control. all primers were designed using primer premier . software and are listed in table . hek t cells were grown to % confluence in a mm dish and transfected with empty pcmv-ha vector or pcmv-plp -ha using lipofectamine ltx reagent (thermo scientific, china) and following the manufacturer's instructions. at hpt, cells were washed with phosphate-buffered saline (pbs) and lysed with lysis buffer ( mm tris, ph . , containing mm nacl, % triton x- , sodium pyrophosphate, β-glycerophosphate, ethylenediaminetetraacetic acid, na vo , and leupeptin) supplemented with mm phenylmethylsulfonyl fluoride. lysates were centrifuged at j o u r n a l p r e -p r o o f , g for min at °c. the supernatant was collected and incubated with µl of anti-ha agarose (pierce® ha tag ip/co-ip kit, thermo scientific) at °c with shaking overnight. immunoprecipitates were washed with tbst buffer ( mm tris-hcl, ph . , containing . m nacl and . % tween ) and eluted in non-reducing sample buffer. the samples were separated by sds-page and either transferred to a nitrocellulose membrane for western blotting or silver stained for mass spectrometry (pierce tm, thermo scientific). the silver stained gel was analyzed, and one differentially expressed protein spot was selected for liquid chromatography-mass spectrometry (lc-ms/ms) analysis. briefly, the excised band was cut into small cubes approximately . - mm in size, decolorized in a °c water bath, further decolorized in decolorizing solution ( mm potassium ferricyanide and mm sodium thiosulfate) in a °c water bath, reduced with mm dithiothreitol at °c for h, then alkylated with mm iodoacetamide for min at room temperature in the dark. the samples were digested with trypsin overnight. tryptic peptides were analyzed using easy-nl c and q-exactive (thermo scientific, waltham, ma). trapped peptides were separated on an analytical c column ( μm × cm, thermo scientific). the mobile phases consisted of % acetonitrile (acn) a and % acn b, both containing . % formic acid. a gradient of %- % solvent b was used to elute the peptides at a constant flow rate of nl/min for min. data were acquired using a ms scan range (m/z) of - , j o u r n a l p r e -p r o o f acquisition mode dda and a resolution of , . thermo xcalibur . software (thermo) was used for data acquisition. protein identifications were assigned using the ncbi nr databank and swissprot/uniprot databank. cells were harvested and lysed with lysis buffer. protein samples were mixed with sds sample loading buffer, electrophoresed on % or % polyacrylamide gels and transferred to a nitrocellulose membrane. the membrane was incubated with primary antibodies for h at °c. plp was detected using a primary anti-ha-tag rabbit monoclonal antibody (cell signaling technology, danvers, ma). an anti-flag mouse antibody (sigma-aldrich, st. louis, mo) was used to detect flag-tagged proteins and an anti-tubulin antibody (cell signaling technology) was used to detect tubulin. rabbit polyclonal pcbp antibody (proteintech, chicago, il) was used to detect endogenous pcbp protein. horseradish peroxidase-conjugated anti-mouse igg and goat anti-rabbit igg antibodies were used as secondary antibodies. chemiluminescence was detected using the gel imaging system tanon- multi (tanon, shanghai, china). vero e cells were grown in glass slides and cotransfected with pcmv-plp -ha, pecmv- ×flag-pcbp or empty vector (pecmv- ×flag). at hpt, the cells were washed three times with pbs, fixed with . % paraformaldehyde for h at room temperature, and blocked with % bovine serum albumin for h. subsequently, the cells were permeabilized with . % triton x- for min, and then incubated at °c primers for pcbp quantitative rt-pcr are shown in table . anti-tubulin antibody and rabbit polyclonal pcbp antibody (proteintech) were used to assess expression levels of tubulin and pcbp proteins. all results were presented as the means ± standard errors of the means of three independent experiments. data were analyzed using graphpad prism . (graphpad software, inc., la jolla, ca). differences between and among groups were assessed j o u r n a l p r e -p r o o f using the student's t-test and two-way analysis of variance, respectively. values of p < . were considered statistically significant and were indicated as follows: *p< . , **p< . , and ***p< . . ifn-α/β is a key component of the host innate immune response to viral infection. pedv plp has been proposed to play a key role in antagonizing innate immunity (xing et al., ) . to investigate the effect of plp on pedv replication, vero e to investigate the effect of plp on type i ifn promoter activation, hek t cells were cotransfected with ifn-luc and pcmv-plp -ha or empty pcmv-ha vector. tk-luc was cotransfected as an internal control. at hpt, the cells were stimulated with ng/ml tnf-α for h and prepared for luciferase analysis. as shown in fig. b , tnf-α significantly activated the ifn promoter and plp overexpression significantly inhibited tnf-α induced ifn-luc activity. to investigate the effects of plp on type i ifn production, hek t cells were stimulated with tnf-α ( ng/ml), then cells were cotransfected with pcmv-plp -ha or empty pcmv-ha vector. at , and hpt, cells were harvested for total j o u r n a l p r e -p r o o f rna extraction and ifn-β mrna abundance was assessed by quantitative rt-pcr. as shown in fig. c , tnf-α stimulated ifn-β mrna transcription, whereas overexpression of plp significantly inhibited tnf-α-induced transcription of ifn-β mrna. these results indicated that plp acted as a negative regulator of ifn-β, thus augmenting pedv infection. to further investigate the mechanism through which pedv plp inhibited expression of ifn-β, we identified cellular interaction partners of plp using immunoprecipitation and mass spectrometry. hek t cells were transfected with pcmv-plp -ha or pcmv-ha empty vector. at hpt, the cells were lysed and proteins were immunoprecipitated using the pierce® ha tag ip/co-ip kit (thermo scientific, china). the eluent was collected and analyzed by sds-page. silver staining showed multiple and distinct protein bands immunoprecipitated in plp overexpressing cells but not in cells transfected with empty vector. we chose the band with the largest expression difference for mass spectrometry analysis (fig. b ). gene ontology (go) and kyoto encyclopedia of genes and genomes (kegg) analysis was performed to analyze the functions of target proteins (fig. ) . the identified host proteins were classified based on molecular function (mf), cellular component (cc) and biological process (bc) (fig. a) . the proteins were involved in cellular processes, metabolic processes and biological regulation. the majority of proteins pulled down with plp are listed in table . analysis of the top pathways showed that the majority of proteins played a role in organismal systems, human diseases and viral j o u r n a l p r e -p r o o f infection (fig. b) . identification of cellular interaction partners showed that pcbps were involved in the immune response against pedv infection. on the basis of the mass spectrometry results, we initially identified two proteins (pcbp and pcbp ) related to immune responses for further study. the genes encoding these two proteins were amplified and cloned into pecmv- ×flag, and hek t cells were cotransfected with pcmv-plp -ha and pecmv- ×flag-pcbp or pecmv- ×flag-pcbp . at hpt, cells were lysed and immunoprecipitated with mouse anti-flag antibody. the results showed that pcbp and pcbp were both pulled down, and that pcbp interacted most strongly with plp ( fig. ) . therefore, pcbp was selected for further studies. to examine the co-localization of pcbp with plp , vero e cells were cotransfected with pcmv-plp -ha and pecmv- ×flag-pcbp . at hpt, the cells were stained with anti-ha and anti-flag antibodies and analyzed by confocal microscopy. overexpressed plp and pcbp were mainly co-located in the cytoplasm (fig. a) . we further analyzed the co-localization of plp and endogenous pcbp in vero e cells. the cells were transfected with pcmv-plp -ha or pcmv-ha, then analyzed by confocal microscopy following staining with anti-ha and anti-pcbp antibodies. as shown in fig. b , plp was mainly co-localized with endogenous pcbp in the cytoplasm. to study whether plp promoted pcbp upregulation, vero e cells were transfected with different concentrations of pcmv-plp -ha or empty pcmv-ha vector and western blotting was performed using anti-ha, anti-pcbp , and anti-tubulin antibodies. plp overexpression enhanced endogenous accumulation of pcbp protein in a dose-dependent manner (fig. ) . to investigate the effect of endogenous pcbp on viral replication, expression of pcbp was silenced using sirnas. three sirnas ( to ) were designed. following transfection of ipec-j cells, the effect of knockdown was assessed by western blotting. levels of pcbp protein were significantly decreased in all sirna-treated groups compared with the control group, and sirna showed the most efficient pcbp silencing (fig. a) . next, ipec-j cells were treated with sirna or a negative control (nc) sirna for h and then infected with pedv at a moi of . . levels of pcbp mrna and pedv rna were determined at and hpi by quantitative rt-pcr. as shown in fig. b and c, pcbp mrna levels in sirna-treated ipec-j cells were significantly reduced compared with nc-sirna-treated cells, and pedv replication was significantly suppressed (fig. b and c ). to further investigate whether overexpression of pcbp would affect pedv replication, ipec-j cells were transfected with pecmv- ×flag-pcbp or empty vector. at hpt, the cells were infected with pedv at a moi of . and pedv viral loads were determined at , and hpi by quantitative rt-pcr. we found that pcbp overexpression enhanced pedv replication (fig. d) . these results indicated j o u r n a l p r e -p r o o f that plp acted as a negative regulator of ifn-β, and plp functions to augment pedv infection by interacting with pcbp . this work was supported by the national key technologies r&d program nsp is the largest protein encoded in the coronavirus genome and comprises two subdomains (plp and plp ), which are mainly responsible for cleavage of nsp /nsp and nsp /nsp , respectively (barretto et al., ; harcourt et al., ) . not all coronaviruses have a plp domain, while the plp domain is conserved in all coronaviruses. plp proteins encoded by various viruses have been shown to inhibit innate immunity (zheng et al., ) . severe acute respiratory syndrome coronavirus plp inhibits irf activation by blocking ubiquitination of rig-i, tnf-receptor associated factor , and stimulator of interferon genes (a lindner et al., ) . transmissible gastroenteritis virus plp and prrsv plp rely on deubiquitination activity to antagonize ifn production hu et al., ; j o u r n a l p r e -p r o o f b). similar to other coronaviruses, pedv can also inhibit the production of type ifns (zheng et al., ) . pedv plp has been shown to have deubiquitination activity and is an ifn antagonist, but plp has no deubiquitination activity (xing et al., ) . our results showed that plp is also an ifn antagonist. overexpression of plp enhanced pedv infection (fig. a) . using a dual fluorescent reporter, we found that plp could inhibit tnf-α-induced production of ifn-β. in hek t cells transfected with plp expression vectors, induction of ifn-β by tnf-α was inhibited (fig. c) . these results suggested that plp could enhance pedv infection by inhibiting ifn-β production. studies have shown that the plp of pedv cv strain has no ifn antagonism (zheng et al., ) . there are amino acid substitutions distinguishing the plp s of pedv gdgh and cv strains (data not shown). we suspect that these substitutions may lead to differences in plp function. future studies should investigate the effects of amino acid mutations at these sites on plp function. the innate immune response is an important line of defense, and type ifns are essential for host defense against viral infection. the double-stranded rna (dsrna) molecules produced by viral rna replication viral are pamps and can be recognized by pattern recognition receptors (prrs) (matzinger, ) . tlrs and rig-i-like receptors are two crucial prrs that recognize pathogens and stimulate downstream signaling to activate immune responses (huang et al., ; kawai and akira, ) . mavs is a downstream adaptor protein of rig- . rig- mediates the activation of nf-κb and irf by interacting with the caspase-recruiting-like domain of mavs to inhibit the production of type i ifns (biacchesi et al., ; kawai et al., ; meylan et al., j o u r n a l p r e -p r o o f seth et al., ) . pcbp is a negative regulator of mavs whose expression is induced following viral infection and results in mavs degradation (you et al., ) . we found that overexpression of pcbp promoted pedv replication (fig. d) . pedv replication could be inhibited by silencing pcbp expression (fig. c ). pcbp has been reported to play a role in a variety of viral infections luo et al., ; wang et al., ) . we found that plp interacted with pcbp ( fig. and fig. ), and that overexpression of plp induces the expression of pcbp (fig. ) . these results indicated that plp plays an important role in the antagonism of ifn responses. therefore, the study of plp proteins is important for understanding viral escape from host immune responses. compared with plp , the function of plp has received less attention. the plp s of many coronaviruses have deubiquitination activity, and can interfere with host antiviral responses (barretto et al., ) . we found that pedv plp also has ifn antagonism and contributes to viral replication. our results indicate that plp interacted with pcbp , thereby helping pedv escape from host immune responses. our findings reveal a new mechanism evolved by pedv to circumvent the host antiviral response, and contribute to our understanding of the role of coronavirus selectivity in isg and ubiquitin recognition by the sars coronavirus papain-like protease the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity cellular poly(c) binding proteins and interact with porcine reproductive and respiratory syndrome virus nonstructural protein beta and support viral replication mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against rna and dna viruses proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk antagonistic effects of cellular poly(c) binding proteins on vesicular stomatitis virus gene expression mechanisms of activation of interferon regulator factor : the role of c-terminal domain phosphorylation in irf- dimerization and dna binding regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity transmissible gastroenteritis virus papain-like protease antagonizes production of interferon-beta through its deubiquitinase activity tank-binding kinase (tbk ) isoforms negatively regulate type i interferon induction by inhibiting tbk -irf interaction and irf phosphorylation type i interferons and interferon regulatory factors regulate tnf-related apoptosis-inducing ligand (trail) in hiv- -infected macrophages antiviral signaling through pattern recognition receptors ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction completion of the porcine epidemic diarrhoea coronavirus (pedv nsp of coronaviruses: structures and functions of a large multi-domain protein polyc-binding protein interacts with '-untranslated region of enterovirus rna in membrane-associated complex to facilitate viral replication the poly(c)-binding proteins: a multiplicity of functions and a search for mechanisms the danger model: a renewed sense of self cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus sensing and signaling in antiviral innate immunity a new coronavirus-like particle associated with diarrhea in swine identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf outbreak of porcine epidemic diarrhea in suckling piglets nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo interaction of cellular poly(c)-binding protein with nonstructural protein beta is j o u r n a l p r e -p r o o f beneficial to chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip porcine epidemic diarrhea virus nsp induces pro-inflammatory cytokine and chemokine expression inhibiting viral replication in vitro suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by key: cord- -up q k q authors: dortmans, j.c.f.m.; li, w.; van der wolf, p.j.; buter, g.j.; franssen, p.j.m.; van schaik, g.; houben, m.; bosch, b.j. title: porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: up q k q porcine epidemic diarrhea virus (pedv) is the highly contagious, causative agent of an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality and growth retardation. in the last few decades, only classical pedv was reported sporadically in europe, but in outbreaks of pedv were described in germany. phylogenetic analysis showed a very high nucleotide similarity with a variant of pedv that was isolated in the us in january . the epidemiological situation of pedv infections in the netherlands in was unknown and a seroprevalence study in swine was performed. in total, blood samples from sows from farms and samples from wild boars were collected from may till november and tested for antibodies against pedv by elisa. the apparent herd prevalence of . % suggests that pedv was not circulating on a large scale in the netherlands at this time. however, in november a clinical outbreak of pedv was diagnosed in a fattener farm by pcr testing. this was the first confirmed pedv outbreak since the early nineties. sequence analyses showed that the viruses isolated in and in the netherlands cluster with recently found european g b strains. this suggests a one event introduction of pedv g b strains in europe in , which made the netherlands and other european countries endemic for this type of strains since then. porcine epidemic diarrhea (ped) is an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality -particularly in neonatal pigletsand growth retardation. the causative agent is porcine epidemic diarrhea virus (pedv), which is an enveloped, positive singlestranded rna virus, belonging to the family of coronaviridae (pensaert and de bouck, ) . the genome of pedv is approximately kb long and about two-third encodes for non-structural proteins and one-third of structural proteins (kocherhans et al., ) . among these proteins, the main research interest is focused on the spike (s) gene and its glycoprotein product s, mediating receptor binding and membrane fusion (li et al., ) . although only one serotype has been described, phylogenetic studies of the s gene showed that pedv can be genetically separated into two groups: genogroup (g ) and genogroup (g ). each genogroup can be further divided into subgroups a and b, and a and b, respectively (lee, ) . classical ped, now grouped g a, was first recognized as a severe swine enteric disease separate from transmissible gastro enteritis (tge) in the united kingdom in and first described in belgium in (pensaert and de bouck, ) . in the eighties and nineties, the virus was detected in many countries in europe including the netherlands and from europe pedv spread to asia, where it caused large outbreaks with considerable losses in the pig industry (song and park, ) . until , north america was considered to be free of pedv infections (cima, ) , but in that same year highly virulent strains of pedv emerged in the united states of america (us), causing diarrhea, vomiting and loss of appetite in pigs of all age groups and up to % of mortality in suckling piglets (chen et al., ; huang et al., ; stevenson et al., ) . this strain, typed as g b, rapidly spread across the us, canada, mexico and several countries in south america . in the last few decades, only classical pedv (or g a) was reported sporadically in europe (alborali et al., ; martelli et al., ) . in , outbreaks of pedv were described in germany and phylogenetic t analysis showed a very high nucleotide similarity with a variant of pedv (oh ) containing nucleotide insertions and deletions in the s gene (s-indel) that was isolated in the us in january stadler et al., ) . this variant, typed as g b, caused mild clinical signs and lower mortality rates in suckling piglets compared to other circulating pedv g b strains in the us . since the reports of outbreaks in germany, more reports about outbreaks of this particular s-indel virus in several european countries have been published, among which france, belgium, spain, portugal and austria (efsa, ; grasland et al., ; mesquita et al., ; steinrigl et al., ) . this suggests that this mild pedv variant is circulating in europe since the beginning of . the aim of this study was to determine the status of pedv in the netherlands with a serological survey and to investigate the first pedv outbreak in the netherlands since the early nineties. the number of required blood samples from animals and farms to estimate the seroprevalence of pedv in dutch sow herds was calculated based on the following assumptions: pedv is highly contagious and no vaccination against this virus was carried out in the netherlands. as a result, it was expected that, if pedv was present in a sow herd, the within herd prevalence would be very high (bertasio et al., ; goede et al., ) . the required number of blood samples per herd was calculated using winepiscope . (thrusfield et al., ) . to detect infection in a herd with % probability, an estimated within herd prevalence of %, and an average herd size of sows per farm (wur, ), three blood samples per farm were required. in , there were approximately sow farms in the netherlands (wur, ) . in order to show with a high probability ( %), that less than % of the dutch farms (n = ) were infected, farms would need to be tested (thrusfield et al., ) . herds were randomly selected stratified by pig density per province to represent the total dutch sow herd population ( fig. a and b). statistical analyses were performed using stata/se version . software (stata corporation, ). for the serological survey, blood samples were selected from samples collected for the obligatory monitoring of aujeszky's disease (pseudorabies), swine vesicular disease (svd) and classical swine fever (csfv). additionally, blood samples were collected from sows at slaughter (vion, groenlo, the netherlands). herds were identified based on their unique herd number (ubn). samples were randomly selected given the availability of enough serum to perform all assays. samples were collected from may till up to and including november . commissioned by the dutch government, gd animal health was also monitoring wild boars for aujeszky's disease, svd, foot and mouth disease, csfv, trichinellosis and african swine fever. because it is suggested that wild boars may play an important role as a pedv reservoir (lee et al., ) , blood samples of wild boar collected as part of this monitor were also tested. these blood samples were collected from regions close to the borders with germany and belgium, from april till august . blood samples collected were stored at − °c, before dispatched to the virology division of the faculty of veterinary medicine, at utrecht university (uu). for the detection of pedv antibodies in serum samples an in-house indirect elisa based on the viral spike (s) protein s -part of the g b strain gdu (non-s-indel, genbank ku . ) was used, similar as the elisa previously described (gerber et al., ) . the s antigen used in this study is produced in hek t cells, a mammalian expression system. to facilitate the purification from the cell culture supernatant, the protein is associated with the fc part of murine igg. per well ng of protein was used to coat the plates. the in-house elisa was optimized by uu with sera from pedvinfected (g b) pigs from the us and with hyperimmune sera of animals vaccinated with the pedv s protein. sera were tested in duplicate at a : dilution, the absorbance was measured with an elisa plate reader at nm. the mean od value of pedv-negative sera was . . sera with od values of > . ( × od value pedv negative sera) were considered positive. the aforementioned pedv-positive sera from infected animals were high positive (> . od) in this elisa ( supplementary fig. ). the calculated sensitivity and specificity of the elisa was % (ci %: - %; n = ) and % (ci %: - %; n = ), respectively. for the detection of virus-specific antibodies in serum samples the virus neutralization test (vnt) was used as an alternative sero-diagnostic assay. a previously described vnt was used to test all samples positive by elisa. the validation of this test is shown in fecal swabs or eswabs (copan diagnostics incorporated) of individual animals were taken by the local practitioner and sent in the same day for pcr testing. also three pigs of the index farm with severe diarrhea were euthanized and submitted for post mortem examination. rna was isolated from fecal samples and the detection pcr was performed as previously described (kim et al., ; lowe et al., ) . in order to type the viruses the pedv s gene was sequenced with primers previously described (chen et al., ; oka et al., ) and designed in this study (table ) , and compared with sequences available in genbank. a phylogenetic tree was constructed using mega software by neighbor-joining method maximum likelihood method. branch lengths represent the predicted number of substitutions and are proportional to the differences between the isolates (tamura et al., ) . after confirmation of the first g b outbreak in the netherlands it was unquestionable to try to prevent the further spread of the outbreak. therefore, it was decided to monitoring and follow up of specific management advices to the farmers. five fattening herds, five sow herds and one nursery herd were selected after pedv infection was confirmed by pcr on feces. for control and eradication of pedv, a tailor made advice per farm, mainly based on biosecurity measures, was given. advise was mainly focused on the separation between pedvinfected and non-infected healthy animals. this separation was applied in stables, animal categories and compartments. it was made using the following measures: dress code (change between compartments), additional disinfection cleaning and disinfection of compartments and corridors and improving pest control. furthermore, all professional herd visitors were informed and required to take measures, in particular those aimed at cleaning and disinfection, which would prevent the transfer of the disease to another pig farm. regular testing of pooled fecal samples was done to monitor the effect of interventions. in sow herds, nursery piglets and replacement gilts were sampled; in fattening herds a random sampling in all age groups was performed. after introduction of pedv, the virus could be detected in a compartment for - weeks. at farm level the virus was detectable much longer due to transmission to new susceptible animals. farms were presumed negative for pedv if three sampling rounds with at least -weeks interval, of thirty randomly taken individual fecal samples each, proved to be pcr negative. in total, sera from sows originating from farms, and sera from wild boar were collected. the blood samples came from all provinces in the netherlands and are fairly representative for the distribution of farms across provinces (fig. b ). all sera were tested in the indirect pedv s -based elisa (group , table ). nine samples, originating from nine different farms located in four different provinces, tested seropositive. the od values were low in all seropositive samples (od: . - . ) relative to positive control sera (convalescent or hyperimmune sera) with values od: . - . . two of the nine elisa-positive sera from the serological study also tested positive in the vnt. with two confirmed positive samples of the samples the animal prevalence was . % (ci %: - . %). the herd prevalence with out of farms was . % (ci %: - . %). for all herds, three samples were tested. the additional samples of seven herds with a seropositive sample of which one vnt positive, were examined. these samples, in total, were all negative in the pedv elisa. the proportion of seropositive samples ( / ) falls within the expected proportion of false positives given the specificity of the elisa which is estimated at > %. so we either detected pedv outbreaks that had not spread yet or the seven original samples were false positives. for two herds no additional samples from the monitoring programs were available (including one sow farm with a vnt positive sample). all tested sera obtained from wild boar were negative for pedv antibodies in the used indirect elisa (group , table ). in the first week of november , gd animal health received a report of pigs showing lethargy and anorexia for up to h after which profuse diarrhea occurred in almost all pigs in nine compartments within the fattening barn. diarrhea was watery, light greyish, sometimes yellow or green colored. body temperature in the clinical phase reached . °c. after the first days of disease lethargy subsided and lack of appetite diminished, a profuse diarrhea became more prominent. in later stages of the infection the consistency of the diarrhea changed to slightly more solid. pigs did not seem to suffer much and no animals died, although some pigs did not grow for a week and within the group body weights started to differ. after an extra week of feeding, pigs did recover and had a normal weight at slaughter. the fattening barn, located in a pig dense area of the netherlands, consisted of compartments with pigs each, divided over pens. first symptoms occurred on october th in one compartment followed by symptoms in consecutive compartments in the following days. initial diagnostic tests for the presence of e. coli, salmonella, lawsonia intracellularis and brachyspira pilosicoli were, except for low numbers of pathogenic e. coli, negative. based on the low numbers of pathogenic e. coli and the age of the pigs involved, e. coli was ruled out as causative agent in this case. subsequently, it was decided to test for porcine deltacoronavirus (pdcov) and pedv. six fecal samples were found to be negative for pdcov, but positive for pedv rna on november th . on the same day, three pigs with severe diarrhea were euthanized and were submitted for post mortem examination. pathological examination showed severe villus atrophy in the small intestine, and pcr tested positive for pedv. during the outbreak, in total % of the animals ( compartments) were affected by pedv as confirmed by pcr on fecal samples. based on the results of the serological survey, over % of the dutch farms were pedv negative in ; it was decided to try to prevent the further spread of the outbreak. to control and eradicate pedv the biosecurity measures taken, as described in the materials and methods section, seemed to be of great importance. also pet control was applied since some farms suffered from mice and rats. furthermore, it became clear that in compartments in which the infection was present, after thorough cleaning and disinfection, pedv free piglets could be introduced and that those stayed free from pedv infection. three fattening and three sow herds were presumed free of pedv within months after the diagnosis of ped was confirmed. the nursery herd was depopulated, and, after double cleaning and disinfection of all compartments, repopulated. despite the immediate action of all parties in the dutch pig production industry to optimize their hygiene measures to ensure that infection by pedv would be avoided as much as possible, it could not be prevented that pedv spread to other farms. most infected farms were located on the east side of the netherlands at the border of germany (fig. c) . at the end of , in total farms were confirmed pedv positive by gd animal health. in fig. the number of new pedv pcr positive farms per month since the beginning of the outbreak is shown. in order to characterize the virus originating from outbreaks in and , the s gene of three isolates of different farms was sequenced and the sequences ned/gd / , ned/gd / and ned/ gd / were deposited in the genbank database and received accession numbers kr (index farm), kr and mf , respectively. together with sequences available in genbank, the isolates were compared in a phylogenetic tree (fig. ) . sequence analyses showed that the isolates had a . % homology with the usa/oh strain and a . % homology with the german g b strains. furthermore, the european strains available in genbank ( - ) cluster together with this oh strain, or the so called s-indel strain (fig. ) . this study showed that most likely pedv and particularly the genogroup b (s-indel) strains did not circulate in the netherlands on a large scale till the end of . only a very small part of the dutch sow farms tested positive on antibodies against pedv ( . % (ci %: - . %)) and no pedv antibodies were detected in wild boars. the used elisa based on the s protein of a g b strain showed a high sensitivity and specificity against antibodies raised against g a, g b en g b strains. however, the elisa was validated with a limited amount of samples ( supplementary fig. ) and field samples may react differently, just like in a similar elisa recently described (gerber et al., ) . the viral spike (s) protein is prominent on the virus surface and is very immunogenic. all animals that have been infected with pedv have antibodies against the s protein and particularly against the s part. the s -part of the spike protein is the most variable part between related coronaviruses and there is no cross reactivity with other coronaviruses such as transmissible gastroenteritis coronavirus (tgev), porcine respiratory coronavirus (prcov) and porcine delta coronavirus (pdcov) as previously described (gimenez-lirola et al., ; lin et al., b) . because results of immunoassays based on the s protein correlate well with viral neutralization (paudel et al., ) , as shown in supplementary table , we decided to use the vnt as an alternative sero-diagnostic assay. based on case reports from germany stadler et al., ) , austria (steinrigl et al., ) and the netherlands (gd animal health) a pedv infection with the european circulating strain (g b) will spread very rapidly within a herd and a single pedv positive animal on a farm is not likely to occur. the number of elisa positive animals ( %, table , group ) on the index farm after the clinical signs started, seemed to justify the assumption of the high within herd prevalence of % for sample size calculation for the serological survey. eventually, there were fewer herds sampled than planned ( instead of ) since it was decided to stop the serological survey, because the first case of pedv was diagnosed in the netherlands on november th . sequence analyses of the viruses isolated in - showed a % homology with oh , a less virulent pedv strain found in the us and in germany in . although the g b virus strain present in europe is less virulent (efsa, ; grasland et al., ; hanke et al., ; mesquita et al., ; steinrigl et al., ) compared to the strain that caused the us outbreaks in , this european strain showed to be very contagious and still could cause severe economic damage in pedv naive herds. after infection on sow farms loss off piglets could range up to % in the sucking piglets ( (lin et al., a) and individual case reports, gd animal health). in the foreseeable future, vaccination will not be possible. therefore, the authorities in the netherlands advised all parties in the pig production industry to optimize their hygiene measures to ensure that infection by this virus would be avoided as much as possible. the strict preventive biosecurity measures taken in these herds demonstrated that most herds that were monitored could prevent the transfer to pedv naive compartments within the herd and some were able to obtain a presumed pedv free status for the whole farm. additionally, a higher biosecurity level helps preventing the introduction of other pathogens and controls the spread of infection within that herd (fao, ) . however, an increasing number of herds became infected (fig. ) and pedv spread across the country after the initial outbreak (fig. c) . the transport trucks with positive animals between herds seemed to have the highest transmission risk (data not shown). the number of new pedv pcr positive farms per month since the beginning of the outbreak is most likely an underestimation (fig. ) . since ped is not a notifiable disease, nor have all veterinarians performed diagnostic testing in all cases, as the clinical signs of an outbreak are very typical. it seems that pedv g b became endemic in the netherlands since its initial outbreak in november , just like in most countries in europe. that g b viruses isolated in europe in - phylogenetically cluster together with a high similarity (fig. ) suggests a onetime introduction event. however, pedv is endemic and many other coronaviruses are circulating that may result in coronavirus variants through mutation or recombination (fehr and perlman, ) . therefore, it is of upmost importance that the presence of circulating pedv is being monitored and genetically analyzed, in order to update diagnostic tools where necessary. we declare no conflict of interest. surveillance and control of ped coronavirus in pigs in italy porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states viral disease affects u.s. pigs: porcine epidemic diarrhea found in at least states updated epidemiological data on ped good practices for biosecurity in the pig sector -issues and options in developing and transition countries coronaviruses: an overview of their replication and pathogenesis detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france comparison of porcine epidemic diarrhea viruses from germany and the united states origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states multiplex realtime rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus wild boars harboring porcine epidemic diarrhea virus (pedv) may play an important role as a pedv reservoir manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination cellular entry of the porcine epidemic diarrhea virus experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains role of transportation in spread of porcine epidemic diarrhea virus infection epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy outbreak of porcine epidemic diarrhea virus in portugal cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines emergence of porcine epidemic diarrhea virus in southern germany first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega : molecular evolutionary genetics analysis version . win episcope . : improved epidemiological software for veterinary medicine distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus pig farming the authors would like to thank d. oorburg en c. sima of vion, groenlo, the netherlands, for providing blood samples from slaughter sows, g. spierts and laboratory staff for collecting and archiving blood samples and n. schuurman and j. de jong for performing the elisa. furthermore, r. dijkman for implementing the pcr at the gd animal health lab, a. van lenthe for advice during the survey, m. meijerink for assisting in sample collection during herd visits and a. veldhuis and h. brouwer-middelesch for making the maps of the netherlands (fig. ) . the authors thank the owner of the index farm and the practitioner involved in this farm for their information and their cooperation in collecting the samples.the authors would like to thank the dutch ministry of economic affairs and the product board for livestock and meat for funding the monitoring system for pig health in the netherlands. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -i pqiwx authors: abro, shahid hussain; renström, lena h.m.; ullman, karin; isaksson, mats; zohari, siamak; jansson, désirée s.; belák, sándor; baule, claudia title: emergence of novel strains of avian infectious bronchitis virus in sweden date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: i pqiwx infectious bronchitis virus (ibv) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. recent ibv infections in sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. the complete spike gene of selected isolates from ibv cases was amplified and sequenced using conventional rt-pcr. nucleotide and amino acid sequence comparisons have shown that the recent isolates bear . % genetic similarity with strains of the qx-like genotype. the phylogenetic analysis revealed that strains predominant in the nineties, which were of the massachusetts type, have been replaced by d /qx-like strains, however the evolutionary link could not be established. the homology between the two genotypes was and %. remarkably, a strong positive selection pressure was determined, mostly involving the s subunit of the s gene. this strong selective pressure resulted in recombination events, insertions and deletions in the s gene. two new isolates generated from recombination were found with nucleotide sequence diverging . – . % from the d /qx-like branch, indicating the emergence of a new lineage. the study demonstrates a constant evolution of ibv that might be in relation to increased poultry farming, trade and vaccine pressure. the findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario. infectious bronchitis virus (ibv) causes avian infectious bronchitis (ib), a highly contagious disease that affects poultry worldwide and produces severe economic losses. the disease is clinically manifested by respiratory distress, drop in egg production, poor egg quality in layers and some strains causes nephritis (cavanagh, ; cavanagh, ; mahmood et al., ) . the disease may lead to loss of weight and predisposes to secondary bacterial infections that may be fatal (cavanagh, ) . initially ibv infects the respiratory tract, however, infections of kidneys and oviduct may follow (cavanagh and naqi, ) . also a shift of tissue tropism in the virus has been reported (liu et al., ; zhou et al., ) , resulting in infection of a wide range of avian host species especially those reared close to domestic fowl. for example, ibv cases were found in chinese peafowl (pavo), guinea fowl (numida meleagris), partridge (alectoris) and teal (anas) (cavanagh, ) . ibv belongs to the order of nidovirales, family coronaviridae and to genera of gamma-coronavirus group (gonzalez et al., ) . the genome is positive-sense veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] infectious bronchitis virus (ibv) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. recent ibv infections in sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. the complete spike gene of selected isolates from ibv cases was amplified and sequenced using conventional rt-pcr. nucleotide and amino acid sequence comparisons have shown that the recent isolates bear . % genetic similarity with strains of the qx-like genotype. the phylogenetic analysis revealed that strains predominant in the nineties, which were of the massachusetts type, have been replaced by d /qx-like strains, however the evolutionary link could not be established. the homology between the two genotypes was and %. remarkably, a strong positive selection pressure was determined, mostly involving the s subunit of the s gene. this strong selective pressure resulted in recombination events, insertions and deletions in the s gene. two new isolates generated from recombination were found with nucleotide sequence diverging . - . % from the d /qx-like branch, indicating the emergence of a new lineage. the study demonstrates a constant evolution of ibv that might be in relation to increased poultry farming, trade and vaccine pressure. the findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario. ß elsevier b.v. all rights reserved. single stranded rna of about . kb and contains and untranslated regions (utrs). the proximal two-thirds of the genome encode two overlapping open reading frames (orfs) a and b that are translated into large polyproteins. these are then cleaved to generate nonstructural proteins (nsp - ), comprising a main protease, an rna-dependent rna-polymerase and other nonstructural proteins that carry different functions in transcription and replication. the remaining third of the genome encodes four main structural proteins: the envelope protein (e), the spike glycoprotein (s), the nucleocapsid protein (n) and the membrane protein (m). ibv has two accessory genes and that express accessory proteins a, b and a, b, respectively. (cavanagh, ; pasternak et al., ) . generally, the spike glycoprotein of the virus is translated as a pre-cursor protein (s o ) and later cleaved into the s and s subunits. however, this cleavage of the spike glycoprotein is not observed in all coronaviruses (holmes, ) . the spike glycoprotein of ibv consists of amino acids and its functions include attachment to the host cell, neutralization and induction of protective immunity (ignjatovic and galli, ; johnson et al., ) . it is the most variable of the ibv proteins, with most of the variability mapping to the s part (adzhar et al., ; farsang et al., ; keeler et al., ; kingham et al., ) due to mutations or recombination in this segment. the sequence variability in the s coding gene determines serotype specificity of ibv. more than serotypes have been recognized to date, differing by - % and sometimes % at amino acid level (adzhar et al., ; gelb et al., ) . as a result, cross protection between serotypes is poor (kuo et al., ) and changes as small as % in the amino-terminal half (s ) of the s protein have been shown to alter the protection ability of a vaccine (cavanagh, ) . the several different ibv variants are present around the globe some of them exist in particular region, while others are generally distributed (de wit et al., ) . the increasing number of new serotypes and genotypes of ibv is a major challenge for the prevention and control of the disease. ib had never been reported in sweden until the first outbreak of ib in commercial poultry farms in s. from a series of outbreaks have been reported in layer flocks and later in parent flocks for broilers in the south of the country. due to this situation a decision was made in to vaccinate with a live attenuated vaccine of massachusetts (ma) type ib ma . vaccination reduced the frequency and seriousness of the outbreaks. subsequent to introduction of vaccination, our study has found indications of outbreaks initiated by the vaccine virus (farsang et al., ) . investigations as to the origins of ib cases during have shown presence of antibodies reacting with the / variant. in and , the d variant of ibv was identified in chickens. despite the immunization of a number of swedish poultry flocks with live attenuated vaccines, cases of infections were detected again from to in broilers. reports of similar situations abound also in other countries (beato et al., ; handberg et al., ; worthington et al., ; indicating a sub-optimal protection provided by the vaccines. the appearance of two new economically important field strains of ibv qx-like and italy- in commercial poultry flocks in western europe (beato et al., ; worthington et al., ) has further raised the need for a better understanding of the epidemiology of ib all over the world. clinically, the d /qx-like and ita- genotypes are characterized by nephritis, production of severe disease symptoms and mortality (ammayappan and vakharia, ). such symptoms have not been observed in sweden, where signs of infection are limited to poor growth in broilers. the objective of the present study was to perform molecular characterization of ibv isolates in sweden in order to better understand the epidemiology and the factors behind the occurrence of new infections. samples from ib cases (trachea, bronchi, ceaca), collected in sweden from the different outbreaks between and , were obtained from the national veterinary institute (sva) sample bank. available information of the isolates is summarized in table . all the ibv strains have been propagated in specific pathogen-free (spf) embryonated chicken eggs (lohmann tierzucht, cuxhaven, germany) to maintain the stock. virus isolation was performed by inoculation of - day old embryonated hen's eggs with ml of % tissue homogenates. the eggs were incubated at c for h. at the end of the incubation the eggs were chilled at c for h and the allantoic fluid was harvested and centrifuged at rpm for min. the rna was extracted using trizol reagent as recommended by the manufacturer (invitrogen, carlsbad, table list of selected swedish ibv strains isolated during - used in this study. year country ncbi accession number sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn sweden jn a sweden jn usa). synthesis of cdna was performed with a primer complementary to the ibv spike gene and using super-script ii reverse transcriptase (invitrogen tm life technologies, carlsbad, usa) according to the manufacturer's instructions, in a total reaction volume of ml. two sets of primers were used to amplify the spike gene by rt pcr using the phusion high-fidelity pcr enzyme (finnzymes, espoo, finland). the pcr mix contained ml of distilled water, ml of  phusion gc buffer, ml of dntp mix, ml of each primer, ml of phusion tm high-fidelity dna polymerase and ml of cdna. the thermoprofile was initial denaturation at c/ s; and cycles of c/ s, c/ s, and c/ min. a final extension at c/ min was included. pcr products of were run on . % agarose gel containing ethidium bromide. the specific bands were excised from gels and purified using wizard sv gel and pcr clean-up system (promega, madison, wi, usa). sequencing reactions were carried out using big dye terminator sequencing kit (applied biosystems, foster city, ca) according to the manufacturer's instructions. a set of internal primers was used to generate overlapping sequences in both directions. due to sequence diversity, primers have been designed as new sequence data were generated. the final sequence data enables the selection of consensus primers for sequencing of similar strains. the sequences of the primers are available on request. the thermal profile was setup at c/ min; and cycles at c/ s, c/ s and c/ min. the reactions were precipitated in sodium acetate and % ethanol. thereafter, they were centrifuged. the pellets were washed with % ethanol and dried at room temperature. sequencing was carried out on a dna analyzer (applied biosystems, foster city, ca). the sequences of s and s obtained were edited and assembled to the bp of the s gene. for sequence analysis, the sequences data set was pair wise aligned and edited using software lasergene dnastar (madison, usa). selective pressure of the spike glycoprotein was determined by using snap (korber, ) services available at http://hcv.lanl.gov/content/sequence/snap/snap.html. the difference between synonymous (ds) and non-synonymous (dns) substitutions in the codons was calculated to evaluate the substitution rate in the spike gene of the virus. the sequences were analysed for recombination events, as confirmed by analysis with a recombination detection program, rdp v. . . the window size was adjusted to with the highest p value . . the detection of recombination events were applied between sequences sharing and % identity. phylogenetic analysis was performed with sequences generated in the present study and sequences of the partial s and complete spike gene downloaded from the genbank (accession numbers are presented in tables and ). all the gaps in the sequences created by insertions were deleted by using software mega version (tamura et al., ) . phylogenic trees were constructed by the neighbour-joining method with the nucleotide substitution model of kimura- parameter model. the tree reliability was evaluated by bootstrap replicates. the data set was kept similar for the analysis. the tree topology was prepared in the fig-tree_v . . . between and , the sva received specimens from different outbreaks of avian infectious bronchitis that had been submitted by commercial poultry farms for diagnosis. initially outbreaks were confirmed as ibv positive by real-time pcr. twenty swedish ibv isolates from different outbreaks were selected for comprehensive study of the complete spike gene of the virus. the isolates were assigned into two categories: one comprising isolates from nineties to and the other containing isolates from to . the complete spike gene sequences were subject to blast searches in genbank and the results confirmed the isolates as ibv. comparative analysis of the nucleotide sequences of the complete spike gene was carried out to determine the relatedness among swedish isolates ( the sequences were also analysed for the presence of nucleotide insertions or deletions in the spike gene. the deduced amino acid sequences of the spike glycoprotein of the swedish ibv isolates from to exhibited . - % homology. the isolates from to contained three amino acid insertions, aspartic acid, glycine and serine at positions , and , respectively. the new isolates and had a deletion of arginine at position , whereas isolates , and have lost threonine, asparagine, glutamine and arginine at positions , , and , respectively, consequence to the nucleotide deletions. the swedish ibv isolates from to had shared homology of . - . %. hypervariable regions were found encompassing positions - , - and - within the s in all the analysed swedish isolates. positions and were highly subjected to amino acid substitutions. overall, high rates of amino acids substitution were observed in the sequences of spike glycoprotein of the virus. a bioinformatics and pair wise comparison approach was applied to estimate the synonymous and nonsynonymous substitution rates and selective evolutionary pressure in the spike glycoprotein. the analyses revealed that most of the s subunit was under strong positive selective pressure, showing positive codon specific differences and a high level of genetic diversity across the s gene (fig. ) . in particular, codon regions - and - contained a high number of non-synonymous substitutions. overall, the s gene showed negative selective pressure, however, the codon regions encompassing positions - and - were found to singapore dq taiwan eu taiwan gq taiwan dq taiwan dq taiwan eu taiwan eu taiwan eu h taiwan eu taiwan gu thailand gq thailand gq thailand gq kku thialand gq contain non-synonymous substitutions, thus contributing to overall variation of the s gene. there was evidence of recombination events in the s subunit of the swedish isolates from to . recombination events were mapped to begin with break point position at amino acid in isolates and . the ending break point strong signals were observed at amino acid position . phylogenetic analyses of the swedish ibv isolates were carried out based on the partial s and complete spike codon positions in the sequence fig. . the selective pressure analysis in the spike glycoprotein of ibv. the graph illustrates the s part of the spike glycoprotein was mostly subject to positive selective pressure. in s , the selective pressure was mostly negative. gene. published ibv sequences of massachusetts type, m , h , baudette, italy , qx-like and / available in the genbank were used to determine likely origin, genetic characteristics and molecular epidemiology of the swedish isolates. the phylogenetic analysis based on the partial s gene revealed the sequences distinguished into four main groups (fig. ) . one group was composed of massachusetts-type, m , h and beaudette strains, where the swedish isolates from to clustered, together with strains from spain, korea thailand, china and other vaccine strains. this group was classified as massachusetts genotype. the second group was divided into two sub-groups: one subgroup comprising sequences from sweden, france, the netherlands, spain, italy, uk, israel, korea and china known to belong qx-like genotype. this sub-group was further composed of three clusters from sweden, spain and mixed strains from france and the netherlands. three isolates from israel, italy and uk occupied a separate position near to these subgroups. the recent swedish isolates from to were found distributed in this sub-group as a separate cluster and closely related to the dutch strain nl . two new swedish isolates and branched out separately from the other swedish isolates. the remaining two sub-groups in this group consisted mostly of korean and chinese qx strains. the third group consisted of two isolates from uk and italy related to ita- and belonging to the ita- genotype. the fourth group has shown relation to the / genotype and consisted of reference strain / and two isolates from iraq. there were no complete spike gene sequences of the european d /qx-like genotype available in the gen-bank. thus the comparison was limited to available sequences in the databases. the phylogenetic relationship based on the complete spike gene of ibv revealed that these sequences were separated into two main groups (fig. ) . the first group contained sequences of the strains from usa, sweden, china and singapore related to baudette, h , ark and massachusetts genotype. this group was further divided into subgroups. swedish isolates from to were found in this group. the swedish isolates , and a have fallen in the subgroup which is closely related to mass isolates from usa. the swedish isolates , , , , , and clustered together and showed identity as a separate subgroup of massachusetts type. one swedish isolate occupied a distinct place in this group. the second group consisted of two subgroups. one of the subgroup mostly contained isolates from taiwan. the second sub-group consisted of two main clusters. the swedish isolates from to were found forming a separate cluster in this sub-group of the sequences. the closest relationship of these swedish isolates shared with the ck/ita/ / strain. ck/ita/ / has occupied a distinct place within this subgroup. the other cluster consisted of different chinese isolates. ibv has been detected in the poultry population in sweden in the nineties and then in more recent years cases were reported. despite current immunization with live attenuated vaccines, occasional cases of ib have occurred in poultry flocks in - . therefore, this study was designed to characterize strains of ibv associated with outbreaks in the nineties and in recent years in order to understand the molecular epidemiology of this disease in sweden. analysis of the nucleotide sequences of spike glycoprotein of swedish isolates from to showed that they comprise distinct sets of ibv variants; differing among themselves along that timeline (table ). it is obvious that the outbreaks in the nineties and those in s were caused by different ib viruses. the isolates and , standing alone had the genetic signature of a new variant, as they have shown unique substitutions compared to the remaining sequences in this cluster. the nucleotide sequence data showed different regions of hypervariability in the s subunit of the spike glycoprotein. this is consistent with the notion that the s is prone to mutations, as demonstrated in different studies (adzhar et al., ; farsang et al., ; keeler et al., ; kingham et al., ) . previously it has been reported that ark-like strains have a deletion of three nucleotides in the s gene at nucleotides positions - (ammayappan and vakharia, ) but in this context, we have observed insertion rather than deletion in s unit of the sequences of d / qx-like strains. these insertions and deletions possibly have some significance in the properties related to s functions. further studies are needed to determine the role of these insertions and deletions in the spike glycoprotein for example in neutralization phenotype or virus tropism, which could explain the reported change in the clinical manifestation of infectious bronchitis. the s gene of the isolates showed much higher variation as compared to s gene that was mostly conserved, as previously reported (adzhar et al., ; keeler et al., ; kingham et al., ) . however, certain regions in the s were determined to be also variable, and, to an extent, contributing to sequence discrimination based on analysis of the complete s gene. therefore, it is plausible that the s subunit may have some role in development of new field variants. it has been reported that the evolutionary rates in the s of vaccinated birds were . - . % per year. nucleotide point mutations, deletions, insertions, and rna recombination in the sequences of spike gene lead to generation of different ibv serotypes (wang and huang, ) . the deduced amino acid sequence data revealed different patterns of substitutions resulting from point mutations and insertions in the swedish ibv isolates especially in s subunit. the insertions of aspartic acid, glycine and serine in the sequences of the isolates may affect antigenicity of the virus, and this will be a question for further studies on serotyping of the isolates. our results are consistent to those were obtained by previous studies (ammayappan and vakharia, ; liu et al., ) . the strong positive selective pressure resulting from mutations in s gene is likely responsible for the genetic diversity and in the appearance of new phenotypic and antigenic variants. recombination events were found in the spike gene of recent swedish ibv isolates. in the particular case of strains and the recombination event was mapped to amino acid and is believed to have determined a new lineage within swedish d /qx-like cluster. in order to obtain more information concerning to molecular epidemiology of ib, we have carried out a comprehensive phylogenetic study based on partial s and complete spike gene of the selected swedish isolates. the phylogenetic studies, based on the partial s gene regions, showed that swedish ibv isolates from to were related to the massachusetts genotype. the findings confirmed our previous observation (farsang et al., ) . it is hypothesized that these genotypic variants have been reduced by vaccine and did not appear through diagnosis after . the swedish isolates from to were found to be closely related to other western european isolates from comparison of sequences available in the genbank especially to one isolate from the netherlands (fig. ) . the qx strains were first isolated in china in and were found associated mostly with proventriculitis, diarrhea and loss of body weight in - day old chickens (wang et al., ) . it was interesting that the chinese qxlike genotype of ibv was isolated from a backyard flock in a vladimir oblast (russia) where the prevalence of chinese qx-like genotype was common and this region is close to europe (bochkov et al., ; gough et al., ) . it is unclear how the qx viruses spread to europe. there were more cases of qx-like virus infection with presentation of nephritis rather than proventriculitis, and false layers in mature hens reported from belgium, china, france, germany, the netherlands, russia, taiwan and uk (ammayappan and vakharia, ; beato et al., ; bochkov et al., ; worthington et al., ; zhou et al., ) . similar type of ibv isolates has been reported in israel and korea (meir et al., ; park et al., ) . a qxlike strain in chicken associated with nephritis, rare proventriculitis and mortality was reported in poland in (domanska-blicharz et al., . these results demonstrated that qx-like strains comprise new emerging ibvs and presented evidence of its involvement in epidemics in many countries. it is noteworthy that despite a number of outbreaks in europe caused by qx-like strains, only a few sequences are available in the genbank. the lack of sufficient information in this aspect is limiting the comprehensible tracing of routes of infection in the continent. for example in denmark qx-like ibv variants have been reported (handberg et al., ) but the sequences of these viruses are not available in genbank. there is a possibility that trade and import of day old chicks and poultry products from other european countries or indirect routes may have contributed to the spread of the qx-like strains in sweden. the role of wild or migratory birds in dissemination of the ibv new variants still remained unclear. however, previous studies on other coronaviruses and ibv in wild birds have indicated a potential role of wild birds in dissemination of various coronaviruses, including ibv in europe (gough et al., ; muradrasoli et al., ) . taken together, the complete spike sequence data revealed different isolates of ibv of the massachusetts type and european d /qx-like strains circulating over a time-line in sweden. so far, there was no evidence of presence of the ita- and / genotypes in sweden. the data suggest that the massachusetts type strains have been replaced recently with european d /qx-like strains in sweden. sequence diversity and different molecular characteristics were observed among the swedish isolates from to . the investigations revealed that presumably, d /qx-like viruses were introduced from other european countries through imports. considering the number of investigated viruses, the present study provides the most comprehensive sequence data on ibv variants related to the d /qxlike genotype, detected in europe to date. thus, the sequence data of this article will provide valuable reference for future studies on the evolution of genotypes, molecular epidemiology, improved diagnosis and on the virus-evolution in relation to vaccine development. ultimately, the information provided in these studies, will contribute to a more effective control of ib, this viral disease in poultry of global importance. molecular analysis of the /b serotype of infectious bronchitis virus in great britain complete nucleotide analysis of the structural genome of the infectious bronchitis virus strain md reveals its mosaic nature evidence of circulation of a chinese strain of infectious bronchitis virus (qxibv) in italy molecular epizootiology of avian infectious bronchitis in russia severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus infectious bronchitis coronaviruses in poultry and other birds coronavirus avian infectious bronchitis virus infectious bronchitis virus variants: a review of the history, current situation and control measures new variant of ibv in poland molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel a comparative sequence analysis to revise the current taxonomy of the family coronaviridae chinese qx strain of infectious bronchitis virus isolated in the uk vi international symposium on avian corona-and pneumoviruses and complicating pathogens coronaviridae and their replication the s glycoprotein but not the n-protein or m-protein of avian infectious-bronchitis virus induces protection in vaccinated chickens a recombinant fowl adenovirus expressing the s gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s ) gene identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s gene gerald and learn, hiv signature and sequence variation analysis. computational analysis of hiv molecular sequences evolution of infectious bronchitis virus in taiwan: characterisation of rna recombination in the nucleocapsid gene genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between complete genome sequence analysis of a predominant infectious bronchitis virus (ibv) strain in china isolation and molecular characterization of sul/ / avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east identification of a novel nephropathogenic infectious bronchitis virus in israel prevalence and phylogeny of coronaviruses in wild birds from the bering strait area (beringia) variations in the nucleocapsid protein gene of infectious bronchitis viruses isolated in korea nidovirus transcription: how to make sense molecular evolutionary genetic ananlysis (mega) software version . relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus isolation and identification of glandular stomach type ibv (qx ibv) in chickens a reverse transcriptasepolymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from new genotype of infectious bronchitis virus in chickens in scotland characterization of an avian infectious bronchitis virus isolated in china from chickens with nephritis key: cord- -xbszmiql authors: clark, k.j; grant, p.g; sarr, a.b; belakere, j.r; swaggerty, c.l; phillips, t.d; woode, g.n title: an in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: xbszmiql crude theaflavin was extracted from black tea and then fractionated by hplc into five components (initial peaks (ip), tf( ), tf( a), tf( b), and tf( )). the crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. the mean effective concentration (ec( )) was calculated and compared. activity varied from the most active being the uncharacterized theaflavin-like initial peaks (ip) with an ec( ) of . μg/ml to the least active being theaflavin- monogallate (tf( a)) with an ec( ) of . μg/ml. the combination of tf( )+tf( a)+tf( b)+tf( ) was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. only the crude extract was assayed for activity against coronavirus; the ec( ) was . μg/ml. gastroenteritis due to rotavirus and coronavirus infections has a significant economical impact on agriculture. yet, despite numerous trials and decades of research on these two veterinary microbiology ( ) ± viruses, effective vaccines are still not available. although research efforts have appeared promising, vaccine field trials continue to produce inadequate results or fail entirely (de leeuw et al., ; myers and snodgrass, ; snodgrass et al., ; waltner-toews et al., ) . following infection the only treatment available is supportive therapy such as fluid replacement. thus, viral gastroenteritis is still largely uncontrolled and alternative methods to control infection appear necessary. one of the goals of our laboratory has been to investigate a broad spectrum antiviral agents that is not discriminatory among various serotypes and strains of viruses. provided that these agents could be used as food additives, they would have a distinct advantage over vaccines in prevention of infection and/or disease due to their broad spectrum of activity and because most agents that cause gastroenteritis are contracted orally, usually via contaminated food and water. the broad spectrum antiviral agents previously investigated in this laboratory included phyllosilicate clays and charcoal as viral adsorbents. extensive research on these adsorbent materials indicates that they have high affinity adsorbing properties, although the adsorbed virus retains some infectivity in vitro. therefore, although the use of phyllosilicate clays and charcoal are an ideal method of concentrating virus particles, they do not appear to be an effective method of eliminating the infectivity of rotavirus or coronavirus as judged from in vitro studies (clark et al., ) . to pursue further the idea of using broad spectrum methods to inhibit infection, we looked for a natural substance that could neutralize the infectivity of both bovine rotavirus and bovine coronavirus. the intent was to find a substance that is active either alone or in conjunction with a phyllosilicate clay to reduce or eliminate viral infectivity. among the chemicals considered for testing were phenol, formalin, theaflavin and theaflavin gallate derivatives. theaflavins are extracted from black tea and were selected because they are derived from a natural source. the oral administration of tea is reported to be used as a therapy for enteric diseases; a japanese folk legend discusses the medicinal value of tea for curing gastroenteritis in children (mukoyama et al., ) . theaflavin is thought to be responsible for the medicinal value of black tea and can be fractionated into four parts: theaflavin (tf ), theaflavin- -monogallate (tf a ), theaflavin- h -monogallate (tf b ), and theaflavin- , h digallate (tf ) (hara et al., ) . tf has been found to have antiviral activity against influenza a and b (green, ; nakayama et al., nakayama et al., , , poliovirus (strain sabin ) coxsackie virus b (strain nancy), and human rotavirus group a strains of serotypes ± , and (mukoyama et al., ) . in our studies, the crude extract of theaflavin and each of the purified derivatives were studied (individually and in combination) for their antiviral effect against rotavirus and coronavirus. theaflavin and theaflavin gallate derivatives were extracted from three sources of black tea, one sample each from the united states, india, and china. all solvents and reagent materials used were of the highest purity commercially available. theaflavin, theaflavin monogallates, and theaflavin digallate were extracted from tea following a modified version of the method described by roberts and myers ( ) . tea ( g) was added to . l of water, boiled for min and filtered. the filtrate was cooled below c and then ml of concentrated sulfuric acid was added. a precipitate formed and the mixture was left in a cool, dark place overnight. the tea was then centrifuged at Âg for min to separate the precipitate from the aqueous portion. the wet precipitate was resuspended in approximately ml water and extracted four times with ml ethyl acetate (ea), and centrifuged at Âg for min. ea extracts were combined and washed with ml of . % nahco and then with . m sulfuric acid. the extracts were centrifuged each time at Âg to remove the aqueous layer. the ea extracts were evaporated to dryness under reduced pressure. the dry extract was reconstituted in ml acetone and then precipitated with ml chloroform. the precipitate was collected and centrifuged at Âg for min. light petroleum ether was added and the suspensions were centrifuged at Âg for min. the precipitate was dried at c and the yield was calculated. further purification was performed by high performance liquid chromatography (hplc). the mobile phase for the hplc consisted of acetonitrile, ethylacetate, and . % phosphoric acid at the ratio: : : . all hplc procedures were conducted using waters equipment which includes: pumps (model ), automatic sampler (wisp model ), column: m bondapak c ( .  mm), variable uv wavelength adsorbance detector (model , wavelength at nm), surface interface module, professional , and la printer. tea extracts were reconstituted in the mobile phase at . mg/ml. the flow rate was set at ml/min. the samples were analyzed for min with expected retention times at approximately ± , ± , ± , and ± min for tf , tf a , tf b , and tf , respectively (hara et al., ) . fractions were collected according to their retention times and then evaporated to dryness. the precipitate was then dissolved in ml of % ethanol to determine the concentrations. each individual fraction was evaluated by hplc again to verify that each sample was pure. the absorbance (a) of each ml sample of individual peaks was obtained by a du- spectrophotometer. the concentration (c) of each peak was calculated using the lambert±beer law. the molecular weight (mw) and ae are known values (table ) for (hara et al., ) . the c (in mg/ml) was calculated by substituting the known values plus a into the equation below. lambertÀbeer law x c mw  a  ae . . molecular modeling of tf , tf a , tf b , and tf the compounds were modeled by drawing the structures in isis draw . (mdl information systems, san leandro, ca) and then importing it into hyperchem . . hyperchem was used on a pentium processor-based computer with mbytes of memory. the structure was energy minimized using the molecular mechanical mm method (anonymous, ) . the structural information was then imported to chemplus conformational search module for the determination of probable conformers (anonymous, ). the conformational search was a random seed process that was allowed to run overnight and resulted in more than iterations for theaflavin, greater than iterations for each of the theaflavin monogallates, and greater than iterations for the digallic ester of theaflavin. the viruses selected for study were bovine rotavirus, ncdv-lincoln strain, (fernelius et al., ) and bovine coronavirus, bcv atcc p (mebus et al., ; sharpee et al., ) . culturing and assaying for infectivity was in the bsc- cell line for bovine rotavirus and the hrt- cell line for bovine coronavirus. these methods have been previously described (woode et al., ; storz et al., ) . these assays use ± -day-old bsc- cells or ± -day-old hrt cells cultured in well tissue culture microtiter plates, monoclonal antibody b e (mab e ) which is made into an epitope on vp of b rotavirus (zheng et al., ) , gnotobiotic calf # (gc ) antisera to bovine coronavirus (woode, pers. comm.) , and fluorescein-labeled affinity-purified antibodies, goat anti-mouse igg (gam) and goat anti-bovine igg (gab) (kirkegaard and perry). crude theaflavin was assayed for its ability to inactivate both bovine rotavirus and coronavirus. in addition to the crude extract, the component parts of the extract including the initial peaks and peaks tf , tf a , tf b , and tf (individual and combined) were assayed and compared for their ability to neutralize the infectivity of bovine rotavirus. all the original work used theaflavins extracted from the u.s. source of tea. crude theaflavin extractions were later made from indian and chinese black tea and compared to the u.s. source for inactivation activity against bovine rotavirus. neutralization of infectivity assays has been previously described for rotavirus (woode et al., ) and for coronavirus . briefly, theaflavin was made into two-fold dilutions in microtiter plates and then an equal volume of virus was added. for bovine rotavirus ± immunofluorescent focus-forming units (iffu) of virus were used, and for bovine coronavirus, tcid . the theaflavin plus virus was incubated for . h before being transfered to the microtiter plates of cultured bsc- or hrt cells ( ml/well). at least three wells per dilution were used for rotavirus samples and at least four wells per dilution for coronavirus samples. bsc- cells were used at ± days of age and were washed with sf/mem prior to use. hrt- cells were used at ± days of age and were washed with sf/rpmi prior to use. microtiter plate(s) of bsc- rotavirus-infected cells were incubated at c in the presence of % co for h and hrt- coronavirus-infected cells were incubated under the same conditions but for days. post-incubation, plates were fixed with % acetone and dried for approximately h. the plates were rehydrated for at least min in pbs prior to staining. bsc- plates were first stained with mab e and then with gam ( h each). hrt- plates were first stained with gc and then gab ( h each). each stain was washed off using pbs. plates were read by counting iffu in the case of rotavirus and by counting fluorescent wells in the case of coronavirus for each dilution. the neutralizing activity of theaflavin was expressed as the mean effective concentrations in mg/ml that inhibited % of viral infection (ec ). the ec was calculated by applying a linear equation using power of regression (microsoft excel) for bovine rotavirus and the method of reed and muench for bovine coronavirus (reed and muench, ) . the crude theaflavin extract was measured to quantitate the yield of each extraction with the indian source of tea yielding the highest quantity of crude extract. each of the extracts was then examined by hplc. the chromatograms of the crude extracts showed the same four peaks: tf , tf a , tf b , and tf at the expected retention times of ± , ± , ± , and ± min and in addition, multiple peaks prior to peaks tf through tf which we refer to as initial peaks (fig. ) . the initial peaks are suspected to be theaflavin-like substances but as of now they remain uncharacterized. the u.s. source of tea was selected for additional studies. after fractionation of each of the peaks tf through tf of this source, each individual fraction was evaluated by hplc again to verify that each sample was pure. proposed chemical structures of tf , tf a , tf b , and tf are shown in fig. (takino et al., ) . the structures were modeled and the possible conformations were examined. it was found in the conformational search of tf that the hydroxylated bynzopyranyl (hbp) functional groups were commonly found parallel to one another and perpendicular to the benzocyloheptenone (bch) ring structure. an additional gallic acid group attached through an ester bond on one of the hbp functional groups formed the tf a and tf b fig. . chromatograms of crude extract of black tea. compounds (fig. ) . in reviewing the results of the conformational search for tf a , it was noted that the two hbp groups would form a conformer with their orientation parallel to one another and the gallic ester group would lie parallel to the bch group (fig. ) . this was similar to the three-dimensional structure of tf , except for the placement of the gallic ester group. however, when the conformers of tf b were reviewed the parallel conformation of the hbp functional groups was found but the placement of gallic ester was not along the bch ring structure. the lowest energy conformer of the tf had both gallic esters parallel over the bch ring system. the orientation of the gallic esters is interdependent on the orientation of the hbp functional groups which were also found parallel to one another and perpendicular to the main ring system (fig. ) . all the theaflavin and gallate derivatives were shown to have inactivation activity (in vitro) against bovine rotavirus. by separating the theaflavins by hplc and testing their activity we found that the most active components were the uncharacterized initial peaks with an ec of . mg/ml. all individual fractions have at least some antiviral activity with tf being most active. our results indicate that there is synergism between the component fractions tf , tf a , tf b , and tf which have an activity level that is greater when combined and when any one fraction is tested individually. the order of activity, from highest to lowest, is as follows: uncharacterized initial peaks > crude theaflavin extract >tf tf a tf b tf >tf >tf >tf b >tf a (table ) . limited studies were carried out to assess the activity of the crude theaflavin extract against bovine coronavirus; results of the assays conclude the mean ec for coronavirus is . mg/ml (standard error mg/ml). theaflavins are polyphenolic compounds extracted from black tea; their precursors are found in the unfermented green tea leaf and their concentration varies according to genetics, season, and growing conditions. as expected, we found that the source of tea varies widely in yield of overall crude extract and thus quantity of individual fractions which should be considered when purchasing tea. according to our data, the activity of the crude extract and the theaflavin and theaflavin gallate derivatives is constant in individual samples but there is a large variation among samples as a result of the source of tea used. the results of this study support that theaflavin and theaflavin gallate derivatives have inactivation activity (in vitro) against both rotavirus and coronavirus. neutralization activity varies among the crude extract, the uncharacterized initial peaks and each of the individual fractions. an explanation of the differences in effectiveness of the theaflavin compounds could be due to conformational differences. the simplest structure was tf which also had the greatest effect on infectivity of the individual fractions ( table ). the theaflavin monogallate compounds are very similar in their structure (the same functional groups, same molecular weights) but have significant differences in their effect on infectivity. it is suggested since the compounds differ only by the placement of the gallic ester group that a conformation could be responsible for the difference in the effects on infectivity. steric interactions may restrict access to the bch group in tf a and thus reduce its effect on neutralizing infectivity. the conformations of tf , tf b , and tf had conformers that left the functional groups of the bch ring structure sterically accessible while the gallic ester on tf a would wrap around the bch structure which could reduce accessibility to this functional group. since the initial peaks appear to have such great activity, future work should include defining their chemical composition. black teas are consumed in large amounts by people worldwide for pleasure and according to both japanese folk legends and anecdotal data from egypt and india, black teas are used for the cure of gastroenteritis. scientific data indicate that theaflavins have both bactericidal (vibrio cholerae, salmonella typhi, staphylococcus aureus) and virucidal (influenza virus, vaccinia virus, herpes simplex virus, coxsackie virus, poliovirus- , and human rotavirus) activity. is drinking a cup of tea in fact therapeutic for rotavirus infection? we of course cannot extrapolate from our in vitro studies to answer that. however, according to our extractions, one ounce cup ( ml) of brewed tea (u.s. source, . gm of tea leaves) contains approximately . mg of crude theaflavin extract, which is equal to . mg/ml. there is . mg/ml of crude theaflavin required for one ec in vitro and thus approximately times that amount in each cup of tea. this would be expected to vary according to the source of tea; different sources of tea vary in their amount of theaflavin concentration (unpublished data). chemplus: extension for hyperchem. hypercube, ont., canada, pp. ± . anonymous, . hyperchem: computational chemistry. hypercube, ont., canada in vitro studies on the use of clay, clay minerals, and charcoal to adsorb bovine rotavirus and bovine coronavirus cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in nebraska inhibition of multiplication of influenza virus by extracts of tea angiotensin i converting enzyme inhibiting activity of tea component comparison of bovine coronavirus (bcv) antigens: monoclonal antibodies to the spike glycoprotein distinguish between vaccine and wild-type strains neonatal calf diarrhea: propagation, attentuation, and characteristics of a coronavirus-like agent inhibition of rotavirus and enterovirus infections by tea extracts colostral and milk antibody titers in cows vaccinated with a modified live rotavirus±coronavirus vaccine inhibition of influenza virus infection by tea inhibition of the infectivity of influenza virus by tea polyphenols the phenolic substances of manufactured tea. vi. the preparation of theaflavin and of theaflavin gallate characterization of a calf diarrheal coronavirus passive immunity in calf diarrhea: vaccination with k antigen of enterotoxigenic escherichia coli and rotavirus monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus the structure of theaflavin, a polyphenol of black tea crouch c.f. a field trial to evaluate the efficacy of a combined rotavirus±coronavirus/escherichia coli vaccine in dairy cattle protection between different serotypes of bovine rotavirus in gnotobiotic calves: specificity of serum antibody and coproantibody responses comparative studies of the antigenic polypeptide species vp , vp , and vp of three strains of bovine rotavirus this research was funded by a grant from the texas advanced technology program, grant number - . key: cord- - crnh a authors: zhu, zhaozhong; xiao, chao-ting; fan, yunshi; cai, zena; lu, congyu; zhang, gaihua; jiang, taijiao; tan, yongjun; peng, yousong title: homologous recombination shapes the genetic diversity of african swine fever viruses date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: crnh a the african swine fever virus (asfv) has severely influenced the swine industry of the world. currently, there is no effective vaccine or drugs against the asfv. how to effectively control the virus is challenging. in this study, we have analyzed all the publicly available asfv genomes and demonstrated that there was a large genetic diversity of asfv genomes. interestingly, the genetic diversity was mainly caused by extensive genomic insertions and/or deletions (indels) instead of the point mutations. further analyses showed that the indels may be attributed much to the homologous recombination, as supported by significant associations between the occurrence of extensive recombination events and the indels in the asfv genomes. besides, the homologous recombination also led to changes of gene content of asfvs. finally, repeated elements of dozens of nucleotides in length were observed to widely distribute and cluster in the adjacent positions of asfv genomes, which may facilitate the occurrence of homologous recombination. this work highlighted the importance of homologous recombination in shaping the genetic diversity of the asfvs, and could help understand the evolution of the virus. african swine fever virus (asfv), the causative agent of african swine fever (asf), is a complex, large, icosahedral multi-enveloped dna virus. it is classified as the only member in the family asfarviridae (galindo and alonso, ; arias et al., ) . the genome of the virus belongs to double-stranded dna, with the size ranging from kb to kb (dixon et al., ) . asfv mainly infect suids and soft ticks. the suids include domestic pigs and wild boars, and were reported as the natural hosts of the virus (sanchez-cordon et al., ; costard et al., ) . asfv was firstly discovered in kenya in (arzt et al., ) . it remained restricted in africa till , when it was reported in spain and portugal. up to now, the virus has caused asf outbreaks in more than fifty countries in africa, europe, asia, and south america (costard et al., ) . the latest reports showed that the virus has caused outbreaks in nearly all provinces of china (ge et al., ; world animal health information and analysis department, ; zhou et al., ; food and agriculture organization of the united nations, ). because of the high lethality of asfv in domestic pigs, the most commonly used strategies to control the virus were the massive culling campaigns and the restriction of pig movement (sanchez-cordon et al., ) . both strategies have resulted in a huge economic loss for pig industry and affected people's livelihoods. how to effectively control the virus is still a great challenge for the globe. the large genetic diversity of asfvs, which was supposed to hinder the development of effective vaccines or drugs against the virus (sanchez-cordon et al., ; escribano et al., ; arabyan et al., ) , has been investigated in many studies. the asfv genome encodes over proteins, including viral enzymes, viral transcription and replication-related proteins, structural proteins, other proteins involved in the virus assembly, the evading of host defence systems and the modulation of host cell function, etc (dixon et al., ; alejo et al., ; kessler et al., ) . for example, the transcription of the virus is independent on the host rna polymerase because the virus contains relevant enzymes and factors (dixon et al., ) . the viral genome contains a conservative central region of about kb and two variable ends, which results in the variable size of the genome (dixon et al., ; chapman et al., ; de villiers et al., ) . there are significant variations among the asfv genomes due to the genomic insertion or deletion, such as the deletion of the multigene family (mgf) members (dixon et al., ) . although much progress have been made on genetic diversity of the virus, the extent and mechanisms are still not clear. besides, most of these studies either only investigated the genetic diversity of some common genes, such as p and p (fraczyk et al., ; michaud et al., ) , or only used one to twelve isolate genomes (dixon et al., ; chapman et al., ; de villiers et al., ) . the number of discovered viral genomes has increased rapidly as the development of dna sequencing technology. therefore, a comprehensive study on the genetic diversity of asfvs is necessary. homologous recombination, which has been reported to occur in several groups of viruses (roossinck, ; nagy and bujarski, ; wang et al., ) , such as herpesvirus, retroviruses, and coronaviruses, has played an important role in viral evolution (nagy and bujarski, ) . a few studies on several asfv genes have suggested the occurrence of homologous recombination in the evolution of asfvs (dixon et al., ; fraczyk et al., ) . however, a comprehensive study on the homologous recombination in asfv at the genomic scale is lacking, and the role of the recombination on the genetic diversity and the evolution of the virus is still unknown. in this study, we have systematically investigated the genomic diversity and the homologous recombination of asfvs based on the analysis on all the publicly available asfv genomes. the results demonstrated that the homologous recombination contributed much to the genetic diversity of asfvs. this work would help to understand the evolution of the asfv and thus facilitate the prevention and control of the virus. all the asfv genomic sequences with over , bp were obtained from ncbi genbank database on february , (agarwala et al., ) . after removing the genomic sequences derived from a patent, a total of asfv genomes were kept in the analysis (table s ). the genomic sequences were aligned by mafft (version . b) with the default parameters (katoh and standley, ) . unless otherwise specified, all the analyses in this study were conducted based on the alignment by mafft. genes encoded in asfv genomes were predicted with the help of genemarks (version . ) (besemer et al., ) with the default parameters, which is available at http://opal.biology.gatech.edu/ genemark/. the protein sequences for these genes were also provided by genemarks (table s ). all the inferred proteins of asfvs were grouped based on sequence homology using orthofinder (version . . ) (emms and kelly, ) with the default parameters. manual check was conducted to ensure that each protein group contains one type of protein. a total of protein groups were obtained, including groups with more than proteins and groups with only one protein (table s ) . to name each protein group, the proteins included in the group were blast against the asfv proteins downloaded from ncbi protein database on february , . the names of the blast best hits were used to infer the name of the protein group (table s ). the functions of the protein groups were adapted from dixon's (dixon et al., ) and alejo's work (alejo et al., ) (table s ) . to determine insertion and deletion events of genes between two asfv genomes, an asfv gene set was defined as all the genes encoded by the asfv genome. the order of the gene, no matter which strand it was encoded, was determined by the coding region of the gene in the direction of ′ end to the ′ end on the plus strand. each gene set was firstly sorted by the gene order. then, each gene was named as the group name of the protein which the gene encoded. finally, the global alignment of pairwise gene sets was conducted using the needleman-wunsch algorithm. to reduce the uncertainty of grouping mgf genes, the genes of the same mgf family were considered to be the same in the alignment. rdp (version ) (martin and rybicki, ) was used to detect the recombination events in the aligned asfv genomes. a total of nine methods, i.e., rdp, geneconv, bootscan, maxchi, chimaera, sisscan, phylpro, lard, seq, were used to infer the recombination events with the default parameters. only the recombination events with significant p-values (< . ) were recorded for each method. for each recombination event, rdp outputted the recombination region, the recombinant virus, the potential major and minor parents, and the support by each method. for robustness, only the recombination events which were detected by at least two methods were used for further analysis (table s ). all retrotransposons in the databases of repbase (version . ) (genetic information research institute, ) and trep (wicker et al., ) were downloaded on november , . all asfv genomes were searched against these retrotransposons using blastn (altschul et al., ) . no hits were obtained under the e-value cutoff of . . maximum-likelihood phylogenetic trees were inferred using mega (version . ) (tamura et al., ) with the default values of parameters. bootstrap analysis was conducted with replicates. the phylogenetic tree was visualized using dendroscope (version . ) (huson et al., ) . to illustrate the recombination event, several maximum-likelihood phylogenetic trees were built based on genomic sequences with and without the recombination regions. to determine the genotype of asfvs analyzed, the c-terminal sequences ( bp) of b l gene of the asfvs were used to build the maximum-likelihood phylogenetic tree. the genotype of each asfv was assigned based on previous studies (quembo et al., ; bishop et al., ; boshoff et al., ) . all the statistical analyses were conducted in r (version . . ) (r core team, ). the t-test was used to test whether the ratios of the gaps in the recombination regions were similar to those in other regions, and whether the number of indels in the recombination regions was similar to that in other regions. the paired t-test was used to test whether the genomic differences caused by the insertions and deletions (indels) were similar to those caused by the point mutations, and whether the number of repeated elements in the windows ( - , bp in length) including recombination was similar to those without recombination. the t-test and paired t-test was conducted by the function of t.test() in r. z. zhu, et al. veterinary microbiology ( ) . results a total of genome sequences of asfvs were obtained from the ncbi genbank database, which were listed in table s . they were mostly isolated from africa and europe during the years from to . besides, two isolates from china in , i.e., /anhuixcgq and sy , were also included. the size of the asfv genomes ranged from , bp to , bp, averaged at , bp. the viral isolate kenya_ had the largest size, while the isolate ba v had the smallest size. no increasing or decreasing trend in the genome size was observed from to (fig. s ), suggesting the dynamic changes of the viral genomes. pairwise comparisons between asfv genomes were conducted after the multiple sequence alignment of genomes. the pairwise genomic differences between asfv genomes ranged from to , bp ( fig. s ) , with an average of , bp, which accounts for more than % of the genome alignment. interestingly, the genomic differences caused by the insertions and deletions (indels) were much larger than those caused by the point mutations (p-value < . e- in the paired t-test) (fig. s a) . specifically, the genomic differences caused by indels ranged from to , bp, with an average of , bp; while that caused by point mutations ranged from to , bp, with an average of , bp. among these point mutations, a median ratio of . % happened in the coding regions, and a median ratio of . % belonged to non-silent substitutions (fig. s ). for robustness of the results, we also conducted the analysis based on the genome alignment by clustalw (larkin et al., ) , and found that the indels caused larger genomic differences than the point mutations did (p-value = . e- in the paired t-test) (fig. s b) . the size and the number of indels in asfv genomes were also analyzed. % of indels were no longer than bp, and about % of indels were more than bp (fig. s ). the number of indels in each genome ranged from to , with a median of . the occurrence of indels was much more frequent in both ends of the genome, especially in the ′ end (indicated by the red line in fig. ). the distribution of the indel size was similar along the genome, but the large indels with over bp (marked by a blue line in fig. ) were mostly observed in both ends. the impact of the indels on gene function for each genome was further analyzed. the number of indels which occurred in coding regions was counted for each genome. about % of indels were located in the coding regions (fig. s ) . among them, about % of indels had length of or multiple of (colored in blue in fig. s ), which were likely to cause amino acid indels; the remaining indels were likely to cause changes of reading frames (colored in red in fig. s ). as numerous indels have been revealed in the asfv genomes, then, we investigated the mechanism of generating indels. according to the results in previous studies, three factors may contribute to the extensive indels in asfvs: replication slippage, retrotransposition and recombination. replication slippage mainly produced duplications of short genetic sequences (viguera et al., ) and may cause short indels, but it is unlikely to generate large indels observed in asfvs. retrotransposition can result in duplication of large genetic sequences or genes (wicker et al., ) , but no retrotransposons were observed in the analyzed asfv genomes (as described in materials and methods). finally, we investigated the role of recombination in the generation of indels in the asfv genomes. the analyses on the recombination showed that there were a total of unique recombination events, and each asfv genome had - recombination events ( fig. & table s ). the virus isolate mkuzi_ experienced the largest number of recombination events. on average, each virus experienced a median of recombination events. the sizes of recombination region ranged from to , bp. the ratio of recombination region in each genome, i.e., the proportion of genomic regions involved in the recombination events, ranged from % to %. in total, the regions in the asfv genomes involved in all recombination events covered a total of , nucleotide sites, accounting for % of the aligned genome. most recombination events were genotype-specific. there were genotypes among the asfvs analyzed ( figs. a and s ). the genotype ii constituted nearly half of asfvs, including the isolates from east europe and china in recent years. more than ten recombination events were genotype ii-specific (fig. b) . the genotype ix, which included six viruses from uganda and kenya, had the least recombination events. fig. illustrates the recombination event in genotype i and vii (colored in red), including two viral isolates from africa (mkuzi_ and benin_ / ) and eight viral isolates from europe. these viral isolates formed a separate lineage in the phylogenetic tree. the recombination region ranged from , to , bp of the genome alignment, located in the central conservative region of the genome (shown by the black arrow in fig. b ). in the phylogenetic tree built with genomic sequences without the recombination regions, the recombinants are the neighbors of a clade containing viruses from eastern europe countries (fig. a) ; while in the tree built with genomic sequences of the recombination regions, the recombinants are the descendants of viruses from africa (fig. b) . most recombination events happened at both ends, especially at the ′ end (fig. b) . interestingly, the recombination event in the aligned genomes was observed to be consistent with the ratio of the gap in the genome (the bottom of fig. ) . almost all the recombination events happened in or close to the gap-rich regions where the indels were observed. the ratios of the gaps in the recombination regions were found to be significantly higher than those in other regions (pvalue < . in the t-test) (fig. s ) . further comparison of the number of indels in the recombination regions and other regions showed that for indels of varying length, such as those greater than , , or bp, the number of indels in the recombination regions was much larger than those in other regions (p-values < . in the t-test) (fig. s ) . then, we investigated the functional consequences of the recombination events in terms of gene content. in of recombination events, there was at least one gene included in the recombination region. the genes included in the recombination region of the recombinants were compared to those of the inferred major parent virus, which was supposed to provide the larger fraction of the recombinant sequence except the recombination region. in of recombination events, there were at least one gene difference in the recombination region between the recombinant and the major parent virus, either by gene insertion, deletion or replacement (table ) . for example, in the ninth recombination event, a gene l l was inserted in the recombination region of the recombinant virus ken /tk , compared to that of the major parent virus kenya_ . in another recombination event, the major parent virus kenya_ encoded the genes of dp r and dp l in the recombination region, the latter of which was lost in the recombinant virus pretorisuskop/ / . interesting to note, the genes of the mgf families were involved in the gene content variation of the recombination region in of these recombination events. we further compared the gene content between the asfv genomes. the asfv genome encoded - genes, with an average of genes (table s ). after pairwise alignment of the gene set encoded by genomes, the number of different genes between genomes was calculated. on average, there were different genes between the gene set of genomes, which was about % of the gene set. the number of gene deletions and insertions between genomes was further calculated and shown in fig. . the matrix referred to the number of gene deletions when comparing the gene set of asfv genomes in the row to those in the column. it showed that in most cases there were both gene deletions and insertions when comparing pairwise gene sets of asfvs. even for the virus ken /tk , which encoded the largest number of genes among all asfvs analyzed, there were still gene insertions when comparing to other genomes (marked by the black star). among the genes which were involved in gene insertions or deletions, the member of mgf families, especially the mgf- and mgf- , accounted for nearly % of gene insertions or deletions (fig. s ) . unfortunately, most of them had unknown functions. besides, the genes of dp l, which was reported to have the function of neurovirulence, and p , which was reported to be one of the antigen protein, were also widely involved in gene insertions or deletions. repeated elements could facilitate the homologous recombination. in this study, lots of repeated elements ranging from to bp were z. zhu, et al. veterinary microbiology ( ) identified, and then the distribution of the repeated elements in the asfv genomes was analyzed. as shown in fig. s , the number of repeated elements in asfv genomes decreased monotonously as the size of elements increased. then, the distances between adjacent elements for a given repeated element was investigated (fig. a) . as the size of the elements increased from to , the average distance between the adjacent elements also increased because the number of repeated elements in the genome decreased and the repeated elements became more disperse in the genome. interestingly, the average distance decreased as the size of the elements increased from to ; it reached to the minimum ( bp) when the size was ; then the distance kept unchanged as the size increased from to ; finally, it increased as the size of repeated element increased from to . it should be noted that the average distance was still less than bp even for the repeated elements of bp. these phenomena suggested that the repeated elements of bp or larger tended to cluster in the genome, especially for those of - bp. for example, when the size of elements was bp, each genome had a median of types of elements which repeated at least two times in the genome. some elements appeared for over ten times in the genome, such as the element "aggcgttaaacattaaaattattactactg" in the viral strain ba v. the region covered by repeated elements accounted for %- % of the genome in asfvs. the median distance between repeated elements was bp, suggesting they tended to cluster in adjacent regions. fig. b shows the distribution of repeated elements in the aligned genome. most repeated elements were located at both finally, the contribution of repeated elements to the recombination was investigated. for elements of or more nucleotides, the number of repeated elements in the windows ( - , bp in length) table gene content variation in the recombination region between the major parent and the recombinant virus in two recombination events. genes highlighted in black bold referred to those different between the recombinant virus and the major parent virus in the recombination region. including recombination was significantly larger than those without recombination (p-values < . in the paired t-test) (table s ) . fig. c showed the comparison of the number of repeated elements ( bp in length) in the windows of , bp with and without the recombination in viral genomes. the windows including the recombination had a mean of repeated elements, which was four times of that in the windows without recombination. this work systematically analyzed the genetic diversity of asfvs. large diversity was observed among the genomes and the genes of asfvs, which may lead to diverse phenotype, such as the diversity in antigen and virulence. indels were found to have a larger contribution to the genetic diversity of asfvs than the point mutations. this was similar to that observed in a previous study by lin, during which the author found that insertion/deletion of simple sequence repeat (ssr) could cause large genetic variations in phages (lin, ) . compared to point mutations, indels could introduce a larger variation to the genome, and cause a more severe damage to the genome structures, which may lead to the death of viruses (wang et al., ; singh et al., ; sharma et al., ) . therefore, only few indels were observed in viruses with small genomes, such as influenza viruses (taubenberger and kash, ) and hepatitis c viruses (hcv) (torres-puente et al., ) . however, it was more robust for the indels to occur inside the viruses with large genomes, such as asfvs (dixon et al., ) , poxviruses (elde et al., ) and phages (lin, ) , because the viruses with large genomes had lots of repeated elements (such as ssrs) and duplicated genes (such as mgfs). moreover, indels may provide a more efficient way of survival than the point mutations under the natural zhu, et al. veterinary microbiology ( ) selection pressure (dixon et al., ; lin, ; elde et al., ) , since the virus with indels could rapidly change its phenotype, such as antigen, virulence, or ability of replication and transcription. for example, the deletion of some mgf genes in asfv could reduce the viral replication or virulence, which may help with the viral infection of soft ticks (dixon et al., ; burrage et al., ) . as dixon et al. pointed out, gene families are commonly involved in the evolution of dna viruses with large genomes (dixon et al., ) . for example, the poxviruses can rapidly acquired fitness via recurrent amplification of a key anti-host defence gene (elde et al., ) . the asfv genomes have five mgf families, each of which has - mgf genes (dixon et al., ) . previous studies have shown that most genome variation in asfvs was as the result of gain or loss of mgf genes (dixon et al., ) . this study found that the member of mgf families were frequently involved in recombinations. they accounted for more than / of gene insertions or deletions when comparing the proteome of asfvs. although most of them had unknown functions until now, they are supposed to play important roles in rapidly changing the phenotypes of the virus. several factors could contribute to the indels, including replication slippage, retrotransposition and recombination (zhang, ) . the replication slippage may introduce short indels which were widely observed in asfv genomes, but it is unlikely to cause large indels. this study suggested that the ectopic homologous recombination, during which the segments with unequal length were exchanged (freitas-junior et al., ) , may contribute much to the extensive indels observed in asfv genomes (fig. a) . as a proof, significant associations were observed between the occurrence of recombination events and the indels. the clustered repeated elements observed in asfv genomes may facilitate the homologous recombination (fig. ) . taken together, the homologous recombination should be the effective strategy of asfvs to generate the genetic diversity, which further leads to the diverse phenotypes, including antigen, virulence, replication and transcription ability, and the "weapons" of escaping from the host immunity (fig. b) . the widespread distribution of repeated elements in the asfv genomes may have important implications for the viral evolution. the short repeated elements may facilitate the replication slippage, leading to short indels. for example, dixon et al. have identified short tandem repeats within the asfv genes (dixon et al., ) , such as e l and b l, which cause large variations of these genes. besides the short repeated elements, there are also an abundance of long repeated elements, such as those longer than bp. the clustered long repeated elements can facilitate the ectopic homologous recombination, which lead to large indels including the gene insertions/deletions. this work provides some insights into the prevention and control of the asfvs. since the virus can rapidly change its phenotypes, such as the antigen, traditional methods of developing vaccines or drugs may be ineffective, as was demonstrated in previous studies (escribano et al., ; arabyan et al., ; sanchez et al., ) . identification of the conservative antigenic epitopes or drug targets may help for development of effective vaccines and drugs. besides, development of drugs targeting host proteins instead of viral proteins may be an alternative strategy (han et al., ) . moreover, since the recombination play a large role in shaping the genetic diversity of the virus, coupling the drugs which inhibit the recombination process with the traditional drugs or vaccines, may help prevention and control of the viral infection. there were some limitations to this study. firstly, the number of asfv genomes was limited, which hindered a comprehensive analysis on the evolution of asfv genomes. previous studies have identified over twenty genotypes of asfvs (boshoff et al., ; bastos et al., ) , among which only seven genotypes were included in this study. fortunately, the isolates included in this study covered a long time period from to , and also covered a large area including africa, europe and china, which were the major areas of the asfv circulation. besides, the genotype ii, the most widely spread genotype in recent years (ge et al., ; zhou et al., ; quembo et al., ; garigliany et al., ) , were also included and constituted nearly half of all asfvs. thus the results based on these isolates could reflect the genetic diversity of the asfvs to a large extent. secondly, the location and size of the indels observed in asfv genomes may be affected by the sequence alignment algorithm. two common methods, i.e., mafft and clustalw, for the alignment of asfv genomes were used in this study. in both methods, the indels were observed to contribute much more to the genetic diversity than the point mutations did (fig. s ) , suggesting the robustness of the results. lastly, the extensively repeated elements in asfv genomes could facilitate the frequent occurrence of recombination events. however, some of recombination events cannot be detected by the recombination detection method because of the exchange between the genomic segments with small indels. such kinds of recombination events are difficult to detect. increasing the sensitivity of the recombination detection method can help detect them, but may also bring false positives. therefore, the sensitivity and specificity should be balanced in the recombination detection methods.overall, this work provided a systematic view of the genetic diversity of asfvs. extensive homologous recombination detected in this study may contribute much to the widespread indels observed in asfv genomes, which further lead to the large genetic diversity of asfvs. the results on the causes of the diversity of asfvs would help with the understanding of the evolution of the virus and thus facilitate the prevention and control of asfvs. the authors have declared that no competing interests exist. ( -i m- - ). database resources of the national center for biotechnology information a proteomic atlas of the african swine fever virus particle gapped blast and psi-blast: a new generation of protein database search programs genistein inhibits african swine fever virus replication in vitro by disrupting viral dna synthesis gaps in african swine fever: analysis and priorities agricultural diseases on the move early in the third millennium genotyping field strains of african swine fever virus by partial p gene characterisation genemarks: a self-training method for prediction of gene starts in microbial genomes. implications for finding sequence motifs in regulatory regions comparative analysis of the complete genome sequences of kenyan african swine fever virus isolates within p genotypes ix and x genetic characterisation of african swine fever viruses from outbreaks in southern africa ( - ) african swine fever virus multigene family genes affect virus replication and generalization of infection in ornithodoros porcinus ticks comparison of the genome sequences of non-pathogenic and pathogenic african swine fever virus isolates epidemiology of african swine fever virus phylogenomic analysis of complete african swine fever virus genome sequences african swine fever virus replication and genomics poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses orthofinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy antibody-mediated neutralization of african swine fever virus: myths and facts food and agriculture organization of the united nations evolution of african swine fever virus genes related to evasion of host immune response frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of p-falciparum african swine fever virus: a review phylogeographic analysis of african swine fever virus molecular characterization of african swine fever virus, china genetic information research institute human enterovirus protein interaction network prompts antiviral drug repositioning dendroscope: an interactive viewer for large phylogenetic trees mafft multiple sequence alignment software version : improvements in performance and usability the intracellular proteome of african swine fever virus clustal w and clustal x version . simple sequence repeat variations expedite phage divergence: mechanisms of indels and gene mutations rdp: detection of recombination amongst aligned sequences comprehensive phylogenetic reconstructions of african swine fever virus: proposal for a new classification and molecular dating of the virus homologous rna recombination in brome mosaic virus: au-rich sequences decrease the accuracy of crossovers genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype r: a language and environment for statistical computing. r foundation for statistical computing mechanisms of plant virus evolution development of vaccines against african swine fever virus african swine fever: a reemerging viral disease threatening the global pig industry comparative genetic variability in hiv- subtype c p gene in early age groups of infants a proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology mega : molecular evolutionary genetics analysis version . influenza virus evolution, host adaptation, and pandemic formation contribution of insertions and deletions to the variability of hepatitis c virus populations replication slippage involves dna polymerase pausing and dissociation origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis crispr/cas -derived mutations both inhibit hiv- replication and accelerate viral escape a unified classification system for eukaryotic transposable elements world animal health information and analysis department evolution by gene duplication: an update emergence of african swine fever in china supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -pzixgqcy authors: benetka, viviane; kübber-heiss, anna; kolodziejek, jolanta; nowotny, norbert; hofmann-parisot, margarete; möstl, karin title: prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: pzixgqcy feline coronaviruses (fcov) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (fip). independent of virulence variations they are separated into two different types, type i, the original fcov, and type ii, which is closely related to canine coronavirus (ccv). the prevalence of fcov types in austrian cat populations without fip has been surveyed recently indicating that type i infections predominate. the distribution of fcov types in cats, which had succumbed to fip, however, was fairly unknown. pcr assays have been developed amplifying parts of the spike protein gene. type-specific primer pairs were designed, generating pcr products of different sizes. a total of organ pools of cats with histopathologically verified fip was tested. a clear differentiation was achieved in cats, % of them were type i positive, % type ii positive, and % were positive for both types. these findings demonstrate that in fip cases fcov type i predominates, too, nonetheless, in % of the cases fcov type ii was detected, suggesting its causative involvement in cases of fip. feline infectious peritonitis (fip) is a fatal, immune-mediated disease of domestic and wild fe-et al., ; wege et al., ) . barlough et al. ( ) showed that an infection with ccv caused seroconversion but no clinical signs in the cats examined, neither was the course of the subsequently experimentally induced fip disease more severe. in contrast to these findings, mcardle et al. ( ) demonstrated that after infection with ccv the course of fip disease was more severe, and that ccv induced in some cats similar symptoms as in the dog. furthermore, one ccv strain caused in a cat clinical symptoms which were not discernible from fip. after all, the importance of ccv for the cat remains uncertain (sparkes et al., ) . two biological types of fcovs are known, feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv) (pedersen, b (pedersen, , (pedersen, , pedersen et al., ) . the genome of some fecv strains proved to be . kb shorter, suggesting a deletion of bp at the -end (vennema et al., ) . molecular studies showed that fipv arises by mutation from fecv (pedersen et al., ; evermann et al., ; hök, ; poland et al., ; herrewegh et al., ; vennema et al., vennema et al., , . both fipv and fecv may, depending on their virulence, cause viremia (herrewegh et al., ; fehr et al., ; gunn-moore et al., ; horzinek, ) . fcovs are separated into two different types based upon their growth ability in vitro, their antigenic relationship to ccv, their neutralisation reactivity with sprotein-specific mabs (fiscus and teramoto, a,b; hohdatsu et al., hohdatsu et al., , and upon sequence analysis of the s-protein gene (motokawa et al., ) . while type i shows no or little replication in cell culture (fipv ucd , ucd , ucd , ucd , tn- , nw , yayoi, ku- , dahlberg, fecv ucd), type ii induces a lytic cytopathic effect (fipv - , nor (df ) , cornell- , fecv - ) . the ability of an fcov strain to propagate in cell culture does not correlate with its virulence in vivo (mochizuki et al., ) . among the fcov types i and ii, both fipv and fecv strains are represented. the s-protein gene of type ii is closely related to those of tgev and ccv, showing a similarity of the nucleotide sequence of and %, respectively, but of only % with the s-protein gene of type i (motokawa et al., ) . herrewegh et al. ( ) demonstrated that fcov type ii resulted from recombination of fcov type i with ccv. recent studies indicate that type ii uses the feline aminopeptidase n (fapn), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, as receptor, and that fapn may also bind ccv, tgev and hcv. it is not clear whether or not this receptor specificity of type ii plays a role in the pathogenesis or pathological alterations of fip (williams et al., ; de groot and horzinek, ; hohdatsu et al., ; tresnan and holmes, ) . the prevalence of types i and ii has been surveyed in two studies from austria and japan, respectively, both suggesting that the majority of fcov infections is due to type i (hohdatsu et al., ; posch et al., posch et al., , . fcovs are ubiquitous in the cat population, highly infectious by the oronasal route and therefore endemic in multi-cat households, catteries and shelters. investigations showed that a high percentage of cats without fip symptoms from exposed environments were positive for fcov infection: - % were seropositive, - % viremic and - % excreted virus in their faeces (addie and jarrett, b; sparkes et al., ; herrewegh et al., ; foley et al., a,b; gunn-moore et al., ) . posch et al. ( posch et al. ( , found % seropositive cats in austrian cat populations without signs of fip, % of these cats tested positive for fcov nucleic acid in blood. there is strong evidence for the existence of persisting and chronic infections, with virus persisting in the intestine and other organs of healthy cats. asymptomatic carriers may excrete virus over a period of months or even years (foley et al., a; herrewegh et al., herrewegh et al., , . asymptomatic carriers and shedders represent coronavirus reservoirs and therefore the main problem in the prevention of fip in multi-cat environments (addie and jarrett, a,b; addie et al., addie et al., , foley et al., a,b; herrewegh et al., ) . approximately - % of seropositive cats develop fip, with the highest incidence in cats between months and years of age, and the majority of cases occurring in cats ≤ year of age (scott, ; addie and jarrett, a) . the higher incidence of fip among purebred cats (scott, ) , cheetahs (evermann et al., ) and cats from fip-susceptible bloodlines may be an indication for a genetic predisposition. in addition, the sex of the host may influence the outbreak of the disease. while pedersen ( a) found no generic disposition, potkay et al. ( ) and binder and hartmann ( ) observed a higher incidence of fip among males than among females. although serological testing by immunofluorescence assay (ifa) (moestl, ) or elisa (mochizuki and furukawa, ) is a helpful tool for fip diagnosis, results can only be interpreted in correlation with clinical symptoms (sparkes et al., ) . at present, the only conclusive fip diagnosis can be established by histopathological examination of a biopsy or post mortem material. the recently developed reverse transcriptase polymerase chain reaction (rt-pcr) assays, using primers targeted to highly conserved regions of the viral genome ( -utr (untranslated region) (herrewegh et al., ; fehr et al., ) , or s-protein gene (li and scott, ; gamble et al., ) ), which are common to all fcov strains, became a valuable tool for the detection of fcov nucleic acid in blood, body cavity effusions, faeces and tissue samples of infected cats. in particular the n-terminal domain of the s-protein gene allows a differentiation between the two types i and ii. posch et al. ( posch et al. ( , developed an rt-pcr using primers targeted to the s-protein gene to study the prevalence of the two fcov types in cats without fip symptoms, and showed that % of the pcr-positive cats proved positive for type i, % for type ii and % for both types. with the retrospective study presented here we investigated the prevalence of the two types of fcovs in cats with histopathologically verified fip using nested and seminested rt-pcr assays, with primers targeted as well to the s-protein gene. the aim of this study was to investigate the distribution of the two fcov types in fip diseased cats. furthermore-since fcov types i and ii may use different receptors-we wanted to investigate whether the two types are associated with differences in the clinical course of the disease and/or distinct histopathological changes. finally we intended to get more information about the importance of ccv for the cat. ccv itself may infect the cat, or it may be involved indirectly, regarding the possibility that recombinations between fcov type i and ccv may happen in the field at any time (horzinek, ) . between and a total of cats were examined at the institute of pathology and forensic veterinary medicine of the university of veterinary medicine, vienna, and of these cats were diagnosed with fip. the analysis of breed, gender and age of the cats with fip compared to the cats without fip symptoms is shown in table . the statistical evaluation of the parameters breed, gender and age in the two groups "cats with fip" and "cats without fip" was carried out by χ -test using the program spss for ms windows, version . . from of the cats with histopathologically confirmed fip organ samples (lung, liver, spleen, kidney, gut) were available, either formalin-fixed paraffinembedded tissues (pet) (n = , - ) or fresh organ samples (n = , - ) . pet samples had been fixed in buffered formaldehyde for h and were then embedded in paraffin. fresh organ samples were taken during section and either processed immediately or stored at − • c until used. the preparation of pet samples was carried out essentially as described by sorg and metzler ( ): four to six m thick sections of paraffin-embedded organs from each cat were pooled and deparaffinised by incubating for min in xylene and washing twice for min in ethanol at room temperature. after centrifugation and air drying for min at • c, - l proteinase k and - l (depending on the sample size) buffer atl (qiagen, valencia, ca, usa) were added and the samples were then incubated at • c for days. when necessary, another equivalent of proteinase k and buffer atl was added at the second or third day of incubation. after inactivation of proteinase k at • c for min and centrifugation, rna was extracted from the upper aqueous phase using a commercially available kit (qiaamp viral rna mini kit, qiagen, valencia, ca, usa). the extracts were then stored at − • c. one to three grams of each organ sample were pooled and homogenised with sterile sand, and resuspended in - ml diethyl pyrocarbonate (depc)treated water. after centrifugation the rna was the general screening for fcov was carried out by rt-and nested (n) pcr as described by herrewegh et al. ( ) using the primers p and p for rt-pcr and p and p for npcr, respectively. samples positive in these assays were submitted to further analysis employing oligonucleotide primers, which had been designed in regions of the s-protein gene allowing a differentiation between fcov types i and ii. to improve the sensitivity of the pcr assays, a second round of amplifications (npcr with primers b, seminested with primers a) was carried out following rt-pcr. the primers were selected with the help of the primer designer program (scientific and educational software, version . ) and are shown in table . rt-pcr was carried out as a single-tube assay with a reaction volume of l ( . l pcr mixture and . l template) using a commercially available kit (access rt-pcr system, promega, madison, wi, usa). the mgso concentration was optimised at and mm using the primers fecv and fecv , respectively. negative samples were re-tested by employing the one step rt-pcr kit from qiagen (valencia, ca, usa). cycler schemes were carried out following the instructions of the manufacturers. an amount of . l of the rt-pcr product was added to . l of the master mix for npcr and seminested pcr, respectively, containing mm tris-hcl (ph at • c), mm kcl, . % triton x- , m each -deoxynucleoside -triphosphate, . mm mgcl , . u taq polymerase and pm of each primer. forty-five cycles of denaturation at • c, primer annealing at • c and primer extension at • c, s each, were employed. as the possibility of false positive results due to carryover of amplification products in particular during npcr cannot be ruled out, a number of precautions were taken to minimise the risk of contamination. these included the physical separation of all pcr procedures, the use of at least four negative controls of rnase free water for each assay and of three or more primer pairs for each sample. the rt-pcr amplification product was added to the master mix for the npcr in a laboratory specifically installed for this purpose. finally sequence analysis of amplification products served as an additional control. twenty microlitres of each pcr product were analysed by electrophoresis in a % agarose gel for h min at v, and visualised by ethidium bromide staining. the bp ladder (amersham pharmacia biotech inc., piscataway, nj, usa) served as molecular weight marker. bands were visualised with uv illumination and photographed using the eagle eye tm ii uv gel imaging system (stratagene, la jolla, ca, usa). sequence analysis was performed after gel extraction of the amplified product (qia quick gel extraction kit, qiagen, valencia, ca, usa) and sequencing pcr (abi prism big dye tm terminator cycle sequencing ready reaction kit, perkin elmer, alameda, ca, usa) using the sequence analyser abi prism genetic analyser (pe applied biosystems). partial nucleotide sequences (a stretch of bp within the s-protein gene region) of selected type i and type ii positive samples, as well as one of the five samples which had tested positive for both types, were determined; their alignment is shown in fig. . extracts of cell culture supernatants from five different fcov-strains (type i: fipv ku , fipv nw ; type ii: fipv - , fecv - , fipv df ) were submitted to rt-pcr, nested and seminested pcr employing the primers fecv a, fecv b, fecv a and fecv b. gel extracts of the strains fipv ku and fipv - , containing . and . pmol/l dna, respectively, obtained after rt-pcr with the primers fecv b and fecv b, were diluted in rnase free water with a concentration of % trna, and served as template for npcr. as far as antibody titres had been recorded in the case histories, they were compared to the pcr results. the results of the pathological examination were analysed according to the following criteria: during section the presence and amount of fip typical effusion (low, medium, high amount) and of fip suspicious granulomas and pyogranulomas (granulomas yes, no and localisation) were recorded. the subsequent histopathological examination confirmed the diagnosis fip only in the presence of the typical vasculitis with central necrosis and perivascular infiltration with plasma cells, macrophages, lymphocytes and neutrophils. general data of fip-diseased cats were analysed and compared to those of cats without fip symptoms examined during the same period of time ( ) ( ) ( ) ( ) at the institute of pathology and forensic veterinary medicine of the university of veterinary medicine in vienna (table ). the statistical examination showed that the incidence of fip was significantly higher among males versus females (p = . ), among purebred versus domestic short hair cats (p = . ) and among young animals up to year (p = . ). a significantly greater number of males among fip-diseased cats was found in the age category - year (p = . ), but in cats older than year this trend could not be observed (p > . ). while of the pet samples tested negative, nucleic acid could be detected in all fresh organ samples with the primers described by herrewegh et al. ( ) . the differentiation of the two types was accomplished in of the pet samples which tested positive for fcov (the pcr result of one additional sample was questionable) and in out of fresh organ samples (one additional questionable result). in total, a differentiation was possible in samples. among these, ( %) tested positive for type i, ( %) for type ii and ( %) for both types i and ii (table ) . of the samples positive for type i, tested positive with the fecv b primers, with both the fecv a and fecv b primer pairs, and with the fecv a primers only. of the samples with a questionable pcr result, tested questionably positive with the fecv a primer pair and negative with the fecv b primers, the second one vice versa. of the samples positive for type ii, tested positive employing the fecv a primers and using the fecv b primers. of these results ( %) were already achieved after rt-pcr. extracts of cell culture supernatants of five different fcov strains, the type i strains fipv ku and fipv nw , and the type ii strains fipv - , fecv - and fipv df , were subjected to rt-pcr and nested or seminested pcr with the primer pairs fecv a, fecv b, fecv a and fecv b. employing the primers fecv a and fecv b, the type i strains ku and nw showed amplification products of the estimated size, whereas the type ii strains - , - and df tested negative (fig. , primers fecv b ). with the primers fecv a and fecv b the strains - , - and df tested positive whereas the type i strains remained negative (fig. , primers fecv b). gel extracts of the strains fipv ku and fipv - , obtained after rt-pcr with the primer pairs fecv b and fecv b, tested positive in the nested pcr assays up to a dilution of − and − , respectively. antibody titres to fcov were known from the case history for cats, of them had titres of ≥ : , of : , of : and was indicated as serologically negative. in the group of cats with fcov antibody titres of ≥ : twelve tested positive for fcov nucleic acid of type i and were negative for type ii, and one was positive for type ii but negative for type i; for seven cats a differentiation between the two types could not be achieved. all three cats with titres of : tested positive for type i and negative for type ii. the single cat with a titre of : and the sero-negative cat tested both negative for fcov nucleic acid. partial nucleotide sequences of the s-protein gene of type i and type ii positive samples as well as of one sample positive for both types were deter-mined. the sequences were compiled (resulting in a readable stretch of bp) and aligned using the sequence of the type i strain fipv ku as a reference. in the alignment, also the corresponding sequences of the fcov type ii reference strain - and of the ccv reference strain insavc- were included. the analysis of the samples revealed a nucleotide identity of - % for the type i specimens, and of - % for the type ii samples, respectively, in reference to the type i strain fipv ku (fig. ) and of and % for the type ii specimens in reference to the ccv strain insavc- . histopathologic examination exhibited no differences related to the type of fcov detected. both types were found in effusive and non-effusive fip as well as in cases with signs of both forms; a statistical evaluation was not possible due to the small number of type ii positive samples. the comparison of the two groups, cats with fip (n = ) and cats without fip symptoms (n = ) examined in the years - at the institute of pathology and forensic veterinary medicine of the veterinary university in vienna showed significant differences. the statistical evaluation using the χ -test of the parameters breed, gender and age in the two groups showed that the incidence of fip was significantly higher among purebred cats, males, and among cats year of age or younger. the percentage of purebred cats in fip-diseased cats was more than twice as high as in the comparative group ( . % versus . %, p ≤ . ). these findings are sustained by earlier studies (pedersen, ) . foley and pedersen ( ) observed a higher susceptibility for fip in purebred cats when a first degree relative succumbed to fip. due to inbreeding a genetic predisposition may have evolved in certain breeds of cats, which may allow fcov to propagate more efficiently in these cats than in cats with a wider genetic history. in the group of the cats with fip, the majority was male ( . %), only . % were female. although in the comparative group the percentage of males was slightly higher as well ( . % males versus . % females), the difference between the groups was significant (p ≤ . ). these findings are in contrast to pedersen ( a) , who did not find a sexual predisposition, but in accordance with potkay et al. ( ) and binder and hartmann ( ) , who also reported a higher incidence of fip among males. the majority of the cats with fip was -year-old or younger ( . %), in the comparative group only . % were in this age class. addie and jarrett ( a) found as well as scott ( ) a higher incidence of fip in cats of up to year. when comparing the incidence of males and females in the age groups - year and older than year between the cats with and without fip, we found that among younger cats the incidence of males was significantly higher in the cats with fip than in the comparative group. the role of sex-specific differences in the immune system, in particular the cell mediated immunity and the importance of these factors in neutered animals (hormonal influence) are still not clear. in a total of . % of the samples, fcov nucleic acid could be detected. only six pet samples tested negative, whereas all fresh organ samples tested positive. in respect to the expected lower rna concentration in the pets due to the formalin fixation procedure on one hand and due to the long storage time ( - years) on the other hand, we chose primers which amplified, compared to those employed by posch et al. ( posch et al. ( , , a smaller segment of the viral genome. with these primers we achieved a differentiation of the two types in of pet specimens and in of fresh organ samples, in addition two samples exhibited a questionable pcr result. as expected, the percentage of positive pcr results was lower in the pet samples than in the fresh organ samples. specificity was tested on five different fcov strains with four different primer pairs (figs. and , primers fecvb). we found no false positive results and all amplification products showed bands of the expected size. despite the use of different primer pairs, in some samples the pcrs remained negative, probably due to the variability in the s-protein gene of fcov. the oligonucleotide primers employed in the pcr assays exhibited high sensitivity, the fecv b primers proved to amplify specific nucleic acid up to a dilution of − , and the fecv b primers showed amplification even up to a dilution of − . since the original rna concentration was similar in both samples, these findings indicate a higher sensitivity for the detection of type ii viruses. thus, in those samples, in which the differentiating pcr was unsuccessful, fcov type i may predominate as well. on the other hand, due to the lower sensitivity of the type i pcr, we cannot exclude a causative involvement of type i in the type ii positive cats. of the samples in which a differentiation was achieved, ( %) tested positive for type i, ( %) for type ii and ( %) for both types i and ii, thus identifying type i as the causative agent in the majority of the fip cases we examined. whereas posch et al. ( posch et al. ( , identified type ii in % (including those samples with both types) of fcov-positive cats without fip symptoms, we found type ii only in % of cats with fip involved, among them % showing a double infection with both types i and ii. these findings are in contrast to the results of hohdatsu et al. ( ) in japanese cats, in which none of the healthy cats tested positive for type ii, whereas among the chronically diseased cats without fip symptoms over % and among the fip-diseased cats even more than % were infected with fcov type ii. due to their close antigenic relationship in the s-protein gene and the need to choose primers from this region for a possible differentiation between the two fcov types, an infection with ccv would also have been detected (data not shown). therefore, a causative involvement of ccv in the type ii positive fip cases cannot be ruled out in this study. the importance of ccv for the cat still remains unclear (barlough et al., ; mcardle et al., ) , but the possible recombination between fcov type i and ccv horzinek, ) requires further investigation, in particular the role of ccv in double infections with both fcov types observed especially in multi-cat households. the temperature-sensitive fipv strain used as fip vaccine is a type ii strain (fipv df ). regarding the fact that the majority of the fip cases we examined was due to type i, the question arises whether this fact contributes to some of the observed vaccine failures, and whether the inclusion of also a type i strain in the vaccine should be considered. the sequencing results show even in the very short region, which had been sequenced, a clear discrimination between fcov type i, fcov type ii and ccv strains (fig. ) . the partial sequences of the austrian fcov type i samples show significant differences, compared to the reference strain ku , which resulted in identity rates of (only) - % to the reference strain; also within the austrian fcov type i samples several nucleotide changes can be noticed, indicative of a quite high mutation rate. the fcov type ii samples form an own group with an identity to the fcov type i reference strain of only - %, respectively. ccv exhibits an identity to fcov- ku- of %; its much closer relationship to fcov type ii than to fcov type i can nicely be observed by the similarity of several nucleotide changes of fcov type ii and ccv. these findings support the observation that fcov type ii may arise from recombinations with ccv . nonetheless, ccv also exhibits several unique nucleotide changes. the sequencing data of one of the five samples showing double infections (fig. ) demonstrate clearly the plausability of such co-infections. among the cats with known antibody titres, all cats positive for fcov nucleic acid showed antibody titres of : or higher. neither during section nor in the histopathologic examination any differences related to the fcov type detected could be identified. in the group of the type i positive cats % showed signs of both forms (effusive and non-effusive) of fip, followed by % with non-effusive and % with effusive fip. in the type ii positive samples all forms of fip were represented as well. these findings do not point towards a pathogenetic importance of the receptor-specificity of type ii (hohdatsu et al., ) , but emphasises the role of the immune response and the genetic predisposition of the individual in the outbreak of the disease. our findings suggest an involvement of each of both fcov types in fip which is in accordance with earlier reports that both types of fcov are able to cause fip; they also correlate with the results obtained in healthy fcov-infected cats, supporting the predominance of fcov type i infections in both fcov-infected healthy and fip-diseased cats. fcov type ii, the probable recombination between type i and canine coronavirus, was involved in % of the fip cases investigated. it has to be assumed that these recombinations occur in the field and therefore contact with dogs excreting canine coronavirus may play a role in the emergence of new type ii fcov. however, the samples positive for fcov type ii need further investigations with respect to their relationship and even differentiation to ccv. special interest should also be paid to cats with double infections concerning the role of field infections with ccv and their role in the development of fip. finally, no differences in the histopathological changes were found related to the fcov type detected. a study on naturally occurring feline coronavirus infections in kittens feline coronavirus antibodies in cats risk of feline infectious peritonitis in cats naturally infected with feline coronavirus feline coronavirus in the intestinal contents of cats with feline infectious peritonitis experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus klinik der felinen infektiösen peritonitis. feline infectious peritonitis biological and pathological consequences of feline infectious peritonitis virus infection in the cheetah perspectives on the epizootiology of feline enteric coronavirus and the pathogenesis of feline infectious peritonitis nachweis feliner coronaviren mittels rt-pcr: grundlage zum studium der pathogenese der felinen infektiösen peritonitis (fip) functional differences in the peplomer glycoproteins of feline coronavirus isolates antigenic comparison of feline coronavirus isolates: evidence for markedly different peplomer glycoproteins the inheritance of susceptibility to feline infectious peritonitis in purebred catteries patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments risk factors for feline infectious peritonitis among cats in multiplecat environments development of a nested pcr assay for detection of feline infectious peritonitis virus in clinical specimens detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis detection of feline coronavirus rna in feces, tissues and body fluids of naturally infected cats by reverse transcriptase pcr persistence and evolution of feline coronavirus in a closed cat-breeding colony feline coronavirus type ii strains - and - originate from a double recombination between feline coronavirus type i and canine coronavirus antigenic analysis of feline coronaviruses with monoclonal antibodies (mabs): preparation of mabs which discriminate between fipv strain - and fecv strain - the prevalence of types i and ii feline coronavirus infections in cats differences in virus receptor for type i and type ii feline infectious peritonitis virus morbidity, mortality and coronavirus antigen in previously coronavirus free kittens placed in two catteries with feline infectious peritonitis das fip-paradoxon: eine nichtkontagiöse infektionskrankheit antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses detection of feline coronaviruses in cell cultures and in fresh and fixed feline tissues using polymerase chain reaction induction and enhancement of feline infectious peritonitis by canine coronavirus an enzyme-linked immunosorbent assay using canine coronavirus-infected crfk cells as antigen for detection of anti-coronavirus antibody in cat antigenic and plaque variations of serotype ii feline infectious peritonitis coronaviruses nachweis von antikörpern gegen das virus der felinen infektiösen peritonitis in katzenseren und peritonealexsudaten molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type i serologic studies of naturally occurring feline infectious peritonitis feline infectious peritonitis: something old, something new feline infectious peritonitis and feline enteric coronavirus infections. part ii. feline infectious peritonitis virologic and immunologic aspects of feline infectious peritonitis virus infection an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus differentiation of feline coronaviruses type i and ii strains by rt-pcr feline coronaviren: differenzierung der typen i und ii mittels rt-pcr und deren vorkommen in österreichischen katzenpopulationen feline infectious peritonitis in a closed breeding colony feline infectious peritonitis: transmission and epidemiology detection of borna disease virus rna in formalin-fixed, paraffin-embedded brain tissues by nested pcr feline infectious peritonitis: a review of clinicopathological changes in cases, and a critical assessment of their diagnostic value coronavirus serology in healthy pedigree cats feline aminopeptidase n is a receptor for all group i coronaviruses genomic organization and expression of the end of the canine and feline enteric coronaviruses a comparison of the genomes of fecvs and fips: what they tell us about the relationship between feline coronaviruses and their evolution genetic shift and genetic drift during feline coronavirus evolution the biology and pathogenesis of coronaviruses receptor for mhv is a member of carcinoembryotic antigen family of glycoproteins we cordially thank helga lussy, claudia pallan and dr. barbara bauder for their excellent technical assistance. key: cord- - ceq av authors: blum, shlomo; elad, daniel; zukin, nonna; lysnyansky, inna; weisblith, limor; perl, shmuel; netanel, orly; david, dan title: outbreak of streptococcus equi subsp. zooepidemicus infections in cats date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ceq av streptococcus equi subsp. zooepidemicus (s. zooepidemicus) is a commensal of the mucous membranes and skin of animals, notably equine, and is associated with various infections in animals and humans. here, we describe an outbreak of respiratory disease in a cattery, which, to the best of our knowledge, is the first report of s. zooepidemicus infection in cats. clinical disease was characterized firstly by abundant purulent nasal discharges and cough, progressing to sinusitis, dyspnea, symptoms of pneumonia and death. pathological examination revealed different degrees of inflammation of the lower respiratory tract. s. zooepidemicus was the main bacteria isolated. sequencing of the v fragment of the s gene revealed that the isolates were distributed in two previously described genogroups. streptococcus equi subspecies zooepidemicus (s. zooepidemicus), a lancefield c beta-hemolytic streptococcus, is a commensal of the mucous membranes and skin of various animals species, notably equine, and has been associated with different infections in animals and humans (quinn et al., ; ruoff et al., ) . feline respiratory disease is mostly multifactorial. among the pathogens associated with respiratory infections in cats are feline herpes virus- (fhv- ), feline calicivirus (fcv), chlamydophila felis, and different bacteria, such as mycoplasma felis and mycoplasma gateae, pasteurella multocida, and streptococcus canis. the role of bacteria in the disease process is usually considered to be secondary (greene, ) . here we describe an outbreak of respiratory disease in a cattery, which, to the best of our knowledge, is the first report of s. zooepidemicus infection outbreak in cats. approximately cats were raised freely in a cattery (cattery a) that comprised two m , covered enclosures, which were previously used to raise poultry. practically no compartmentalization was present in any of the enclosures except for a small separated area for kittens in one enclosure and one separated area for treatment of sick cats in the second enclosure. the original cats' population was formed by two main groups of animals: one formed of cats rescued during the evacuation of israeli settlements in the gaza strip in late and the other formed of cats raised in a shelter (cattery b) situated about km to the north of cattery a and that was closed due to poor conditions. small groups of cats were accepted to cattery a after its establishment. no history of vaccination was available for any of these populations. prior to entry to the cattery, cats were castrated/neutered, but not vaccinated. the animals were feed with commercial cat food and received water ad libitum. public access to the cattery was limited. a veterinarian visited the cattery approximately three times veterinary microbiology ( ) - streptococcus equi subsp. zooepidemicus (s. zooepidemicus) is a commensal of the mucous membranes and skin of animals, notably equine, and is associated with various infections in animals and humans. here, we describe an outbreak of respiratory disease in a cattery, which, to the best of our knowledge, is the first report of s. zooepidemicus infection in cats. clinical disease was characterized firstly by abundant purulent nasal discharges and cough, progressing to sinusitis, dyspnea, symptoms of pneumonia and death. pathological examination revealed different degrees of inflammation of the lower respiratory tract. s. zooepidemicus was the main bacteria isolated. sequencing of the v fragment of the s gene revealed that the isolates were distributed in two previously described genogroups. ß elsevier b.v. all rights reserved. a week. most of the sick cats were treated in the cattery, but a few were treated off-site by volunteers. in , a few months after the establishment of cattery a, an outbreak of respiratory disease was reported. nasal and pharyngeal swabs and bronchoalveolar lavage (bal) from sick cats were sent for bacteriological culture in the kimron veterinary institute (kvi). following the first cases, dead cats started to be sent for post-mortem examination in the kvi and internal organs were sampled for histopathological and bacteriological exams. bacteriological culture was performed from bal, nasal or pharyngeal swabs of sick cats and internal organs of dead cats (mostly lungs, also spleen, liver, kidney and intestine). samples were inoculated onto nutrient agar, macconkey agar and % sheep blood agar plates, incubated at c and examined after and h. an additional blood agar plate was incubated for h in anaerobic conditions (pack anaero, mitsubishi, usa). bacteria that grew under aerobic conditions were identified by morphology and standard biochemical methods (quinn et al., ) . s. zooepidemicus final identification was confirmed with api rapid id strep (biomerieux, france). anaerobic bacteria were identified with api id a (biomerieux, france). bal, swab and lung specimens were inoculated in mycoplasma broth (rosengarten et al., ) under c, % co for h, and plated onto mycoplasma agar (rosengarten et al., ) afterwards. mycoplasma agar plates were incubated in a moist chamber under the same conditions and observed for mycoplasma colonies growth every days for up to days. mycoplasma final identification was performed by immunofluorescence with antibodies for m. felis, m. gateae, mycoplasma arginini and mycoplasma felifaucium (purdue university school of veterinary medicine, west lafayette, in). slides for chlamydophila spp. immunofluorescence stain (chlamydia cel lps, cellabs pty. ltd., brookvale, nsw, australia) were prepared from bal, swab and lung samples. total dna was extracted directly from bacteria grown on blood agar using qiagen dneasy tm according to the manufacturer instructions. a bp segment of the v fragment of the s rrna gene was amplified as described before (abdulmawjood and lammler, ) . the reaction was carried out using ng of each primer v -f ( -gagagtttgatcctggctcagca- ) and v -r ( -ttaccgcggctgctggcacgt- ) in ml containing ml template dna, mm tris-hcl ph . , mm (nh )so , . mm mgcl , % dmso, mm dxtp, and units taq dna polymerase (amplitap, perkin-elmer). pcr included a denaturation step at c for min followed by cycles of c for s, c for s and c for s. pcr products were purified (wizard pcr prep dna purification system, promega, madison, wi, usa) and sequenced using an applied biosystems automatic sequencer with the two previous pcr primers. sequences were assembled and aligned using clustal x and the neighbor-joining method implemented in mega . . the robustness of branching patterns was tested by bootstrap pseudoreplications. from june to january , a total of dead cats (approximately % of the population in cattery a) were received for post-mortem examination in the kvi. fortynine cats showed signs of respiratory disease prior to death. twenty-eight clinical specimens, including bal, nasal and pharyngeal swabs from cats with clinical symptoms of respiratory disease were also received for bacteriological examination. clinical disease was characterized firstly by abundant purulent nasal discharge and cough, progressing then to sinusitis, dyspnea, symptoms of pneumonia and death. complete pathological examination revealed different degrees of inflammation of the lower respiratory tract in cats. the major gross pathology findings were severe, acute and diffuse bronchopneumonia or bronchioalveolar pneumonia, either suppurative or necrosuppurative. interstitial multifocal pyogranulomatous pneumonia was present in a few cases. pleuritis was found in four cases with pyothorax present in one of these cases. pyogranulomatous meningoencephalitis was found in four cases, two of them showing necrotizing lesions. in one case, necrosuppurative peritonitis was observed. the most common histopathological changes observed were a diffuse mixed infiltrate of neutrophils and histiocytes, with lymphocytes to a lesser extent, thickening of the inter-alveolar septa and multifocal bacterial colonies with cocoid forms. pathological results were strongly consistent with bacterial infections. in ten cats with clinically suspected respiratory disease, no gross pathology changes could be found. bacteriology results are summarized in table . s. zooepidemicus was the main bacterium isolated from clinical specimens and from dead cats with signs of respiratory disease. from dead animals, s. zooepidemicus was isolated from the lungs in all the cases, and also from the sinuses in a few (data not shown). s. zooepidemicus was isolated from the pleura in two out of four cases of pleuritis, from the brain in three out of the four cases of meningoencephalitis and from the peritoneum in one case of peritonitis. s. zooepidemicus was mostly isolated alone and was mostly dominant in mixed cultures. interestingly, one strain of s. zooepidemicus (ct / ), isolated from the brain in a case of meningoencephalitis, grew only in an anaerobic atmosphere at first, requiring a % co atmosphere for growth thereafter. s. zooepidemicus was not isolated from any of the dead cats that were received for post-mortem examination without clinical and pathological signs of respiratory disease, and only from two of ten cases in which respiratory disease was suspected prior to death, but no gross pathological signs were found in the post-mortem examination. feline respiratory disease is multifactorial and the role of bacteria is usually regarded to be secondary to viral infection (greene, ) or stressful conditions. because the vaccination status in cattery a was unknown, the possibility of s. zooepidemicus infection being secondary to viral infections could not be ruled out. lesions suggesting feline infectious peritonitis were found in a few cases during the pathological examination, but the presence of feline coronavirus was discarded by immunohistochemical stains (data not shown). lesions resembling feline distemper were also observed in a few cats, but most of these were not included in this report due either to lack of respiratory symptoms or s. zooepidemicus isolation. the prevalence of fhv- and fcv was not assessed, but we assumed that these viruses were most probably present in the cattery, as suggested by typical clinical signs of infection with these viruses in some cats. infections by fhv- and fcv are limited mostly to the upper respiratory tract and although they may predispose to secondary infections they do not directly cause pneumonia (greene, ) . stress caused by the transfer and merging of different populations of cats in a single enclosure may have greatly predisposed to the respiratory infections reported here. adequate hygienic conditions and ventilation were present in cattery a and the enclosures were apparently not over crowded. clinically ill cats were treated with antibiotics, but not always fully separated from the other animals. in addition, s. zooepidemicus was isolated also from cats showing minor signs of respiratory disease (data not shown), which probably shed the organism long before being detected and treated. thus, s. zooepidemicus may have become persistent in the cattery in spite of sufficient hygienic practices and treatment. twenty-two isolates of s. zooepidemicus were subjected to molecular typing. analysis of the v fragment of the s gene sequences and comparison to reference strains and published sequences revealed two genogroups described before (baverud et al., ) (fig. ) . israeli cat isolates belonging to the same genogroup showed % homology. cat isolates in genogroup ii showed a two nucleotide difference compared to s. zooepidemicus strain no. , which was isolated from a case of metritis in a mare. s. zooepidemicus isolates belonging to genogroup i were closely related to streptococcus equi subsp. equi reference strains ef , similarly to previous work (baverud et al., ) . no correlation was found between genogroup and time of isolation during the outbreak. the earliest typed strains isolated ct / and ct / were clustered in genogroup ii, but ct / , which was isolated within months from the above strains, was clustered in genogroup i. since some strains isolated early in the outbreak were not typed, it is unknown which genogroup appeared first in the outbreak. in addition, no correlation between genogroup and virulence (as determined by source, dead cat or clinical specimen) could be found. one strain of s. zooepidemicus (ct / ) included in the molecular analysis was isolated from a fibrin purulent infiltrate in the brainstem region of the skull of a dead cat that was raised close to the place where cattery b was located. the owner of this cat volunteered in cattery b, but it is unknown if this cat was previously raised in that cattery. cattery b was closed due to poor conditions and all the animals were transferred to cattery a at the time of its establishment. this population constituted most of the cats in cattery a at that time. close to the place where that cat was raised there was a petting farm, with goats, sheep and other farm animals. it is possible that s. zooepidemicus has been introduced to cattery a by the population of cats brought from cattery b and that those cats were somehow infected either by direct contact with animals in the petting farm or through stray cats in the same region. strain ct / was included in genogroup i, whereas early strains in the outbreak were included in both genogroups. by the time this communication has been written, only one other case of s. zooepidemicus outside the aforementioned catteries a and b was found. in this case, s. zooepidemicus was isolated from a nasal swab from a house cat (ct / ) and it was apparently not linked to the outbreak described here. outbreaks of s. zooepidemicus infections have been reported in different species, such as clinical mastitis in sheep (las heras et al., ) , hemorrhagic pneumonia in dogs (garnett et al., ; kim et al., ; pesavento et al., ) and human dairy-born septicemia (edwards et al., ; kuusi et al., ) . the direct transmission of s. zooepidemicus from horses to a human has been suggested (rose et al., ) and the zoonotic potential of this pathogen should not be over-looked. based on the bacteriological and consistent pathological results described above, we concluded that s. zooepidemicus was the main etiological agent in this outbreak of feline respiratory disease. to the best of our knowledge, this is the first report of an outbreak of s. zooepidemicus infections in cats. determination of intraspecies variations of the v region of the s rrna gene of streptococcus equi subsp real-time pcr for detection and differentiation of streptococcus equi subsp. equi and streptococcus equi subsp. zooepidemicus a milk-borne outbreak of serious infection due to streptococcus zooepidemicus (lancefield group c) hemorrhagic streptococcal pneumonia in newly procured research dogs infectious diseases of the dog and cat outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs an outbreak of streptococcus equi subspecies zooepidemicus associated with consumption of fresh goat cheese a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus streptococcus zooepidemicus (group c) pneumonia in a human antigen heterogeneity among isolates of mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins streptococcus key: cord- -unwehrr authors: zhou, haisheng; zhang, meihong; tian, xue; shao, hongxia; qian, kun; ye, jianqiang; qin, aijian title: identification of a novel recombinant virulent avian infectious bronchitis virus date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: unwehrr the emergence of new infectious bronchitis virus (ibv) variants is often disastrous in the poultry industry. in this study, an ibv, ck/ch/ /jt- , was isolated from an h - and / -ibv-vaccinated flock in china. antisera against vaccine strains h and / could not provide effective protection against ck/ch/ /jt- in virus neutralization assays. ck/ch/ /jt- could cause . % mortality with respiratory and severe renal lesions in inoculated chickens. phylogenetic analysis of the s gene showed that ck/ch/ /jt- and other isolates could be grouped as a new genotypic cluster. recombination analysis revealed that three recombination events could be found in the genome of ck/ch/ /jt- at positions - , - and - . whole-genome sequence analysis showed that ck/ch/ /jt- originated from multiple template switches among qx-like, ck/ch/lsc/ i-, tl/ch/ldt / - and / -type ibvs. all of these data demonstrated that ck/ch/ /jt- is a new recombinant genotype ibv with high virulence. our findings suggest that the surveillance of new genotype strains of ibv is very important for developing more effective anti-ibv strategies. infectious bronchitis (ib) is a highly contagious disease caused by infectious bronchitis virus (ibv), which belongs to the family coronaviridae and the genus coronavirus. ibv infection can cause clinical pathological signs mainly in the respiratory tract, kidney and reproductive tract of chickens, resulting in huge economic losses in affected flocks (cook et al., ) . little or no crossprotection occurs between different serotypes of ibv, and the increasing number of new serotypes of ibv is a major challenge for the prevention and control of ib (cavanagh, ) . although the ibv vaccine plays a vital role in controlling ib, it is still an epidemic across the world. ibv variants have been continuously emerging in china xue et al., ; zhao et al., ) . the nature of the large, single-rna genome of ibv makes the virus variable through recombination or mutation. recombination can cause the emergence and evolution of different ibv genotypes and different species of coronaviruses (jackwood et al., ) . more and more recombination events have been reported in ibv, and they are distributed throughout the entire genome (brooks et al., ; kuo et al., ; thor et al., ) . recently, a new recombinant cluster of nephropathogenic ibvs able to cause death, respiratory signs and nephritis in infected chickens emerged in korea (lim et al., (lim et al., , . therefore, studies on the function of recombination in the antigenicity and pathogenicity of ibv are very important, potentially allowing ibv evolution to be predicted and better strategies for ibv control to be developed. in this study, an ibv, ck/ch/ /jt- , was isolated from a vaccinated flock in china, and its complete genomic sequence was determined. genomic sequence analysis and pathogenicity studies revealed that ck/ch/ /jt- is a virulent recombinant strain that belongs to a novel ib genotype. in this study, we identified and isolated an ibv (ck/ch/ /jt- ) from a broiler flock vaccinated with ibv h and / , in which the birds showed severe respiratory symptoms and some of them died. gross examination showed lesions in the different organs, especially in trachea and kidney. the morbidity and mortality in this flock were as high as approximate % and % respectively. trachea, lung and kidney specimens of five -day-old chickens were collected from the broiler flock. sample suspensions ( % w/ v) of tissues prepared in sterile phosphate-buffered saline (pbs) were used for virus isolation . ten passes were performed, and characteristic embryo changes, such as dwarfing, stunting, curling or death of the embryos, were observed between and days post-inoculation (dpi). cross virus neutralization tests as described in the oie manual (http://www.oie.int/international-standard-setting/terrestrialmanual/access-online/) were performed using anti-sera against mass serotype vaccine strain h and / serotype vaccine strain / to determine their antigenic relationship. in brief, the viral titres of the mass-type m and / -type ck/ch/ /tm strains were determined by inoculation of -fold dilutions into groups of five -day-old embryonated specific-pathogen-free (spf) chicken eggs. the b method of cross-virus neutralization testing was performed using constant ( % embryo infectious doses [eid s]) viral titres and -fold diluted serum against h and / in spf chickens embryos. the eid and the end-point of each serum sample were calculated using the methods of reed and muench. viral rna was extracted from ml of allantoic fluid from the inoculated eggs using trizol reagent (introvigen, grand island, usa). first-strand cdna was synthesized using random hexamers (introvigen, grand island, usa). the complete genomic sequence of strain ck/ch/ /jt- was amplified with pairs of primers, including the -and -terminal segments. primer sequences and locations were designed corresponding to the genomic sequence of strains ck/ch/ibtz/ and ck/ch/ljl/ (genbank accession numbers kf and kc , respectively). the polymerase chain reaction (pcr) conditions for amplification included c for min; cycles of c for min, c for min for p -p or c for min for p -p , c for min; followed by c for min. the -and -ends of the viral genome were amplified using -and -random amplification of cdna ends (race) kits (takara, dalian, china) according to the operating instructions. the amplified products were analysed by . % agarose gel electrophoresis, then purified with a gel extraction kit (qiagen, hilden, germany) and ligated into the pgem-t easy vector (promega, madison, usa). each fragment of the viral genome was sequenced at least three times, and the consensus sequence was determined. the complete genome and gene sequences of isolate ck/ch/ /jt- and of ibv reference strains obtained from genbank were aligned and analysed using the clustalw multiple alignment method in the megalign program of dnastar software (version . ; dnastar, madison, usa). a phylogenetic tree was constructed from the nucleotide sequences using mega . software (www. megasoftware.net) with upgma or neighbour-joining statistical methods. the phylogeny test method chosen was bootstrap with bootstrap replicates. evolutionary distances were computed by the pairwise distance method using the maximum composite likelihood model. blastn analysis was performed by services available on http://blast.ncbi.nlm.nih.gov/blast.cgi the aligned nucleotide sequences of the complete genomes were analysed with the recombination detection program (rdp , version . ) to detect potential within-gene recombination events. seven detection methods in rdp v. . , including rdp, geneconv, bootscan, maxchi, chimaera, siscan and seq, were used to confirm the recombination events. only transferred gene fragments where at least detection methods p-value  À were accepted. recombination events and recombination breakpoints were further confirmed by simplot and bootscan analysis using the simplot program (version . . .). nucleotide identity was performed by the kimura ( -parameter) method with a transitiontransversion ratio of , and the window width and step size were and bp, respectively. the genomic sequence of ibv strain ck/ch/ /jt- was submitted to the genbank database and assigned the accession number ku . the genbank accession numbers of strains m and ck/ch/ /tm are dq and ku , respectively. twenty-one -day-old spf white leghorn chickens were randomly divided into two groups. sixteen birds in group were inoculated intranasally with eid s of ibv strain ck/ch/ / jt- in . ml pbs. five birds in group were inoculated with . ml pbs as non-infected controls. the birds were housed in isolators. all birds were observed daily for signs of disease (e.g., dishevelled feathers, depression, respiratory signs or diarrhoea) and death for dpi. gross pathologic changes in tissues such as trachea, lung and kidney were observed. serum samples collected from chickens that survived were checked for ibv antibodies using a commercial enzyme-linked immunosorbent assay (elisa) kit (idexx laboratories, westbrook, me, usa). the endpoint titres were calculated according to the manufacturer's instructions, and titres of more than were considered positive for ibv antibody. all experiments complied with the institutional animal care guidelines and were approved by the university of yangzhou animal care committee. a histopathological assay and haematoxylin-eosin (he) staining of the tissues were performed as previously described (zhang et al., ) . briefly, the tracheae, lungs, and kidneys of the sick chickens were fixed in % neutral formalin, dehydrated in alcohol, and embedded in paraffin. they were then stained with he and screened via light microscopy. . . sera against mass-and / -type ibv could not completely neutralize isolate ck/ch/ /jt- the last dilution of each serum against h and / , which protected % of the embryos against eid s of strains m and ck/ch/ /tm , was tested at : . and : . , respectively. however, serum against h could not neutralize eid s of ck/ch/ /jt- , while the end-point of serum against / that could neutralize eid s of ck/ch/ /jt- was only : . . these results indicate that the ck/ch/ /jt- isolate was antigenically distinct from the ibv mass and / serotypes. by the third passage, typical symptoms, such as curling, dwarfing and stunting, were observed in the eggs. inoculated egg death appeared at the th passage, and the inoculated eggs had all died by the th- th passages. the virus was identified as ibv by rt-pcr and named ck/ch/ /jt- . the complete genome sequence of ck/ch/ /jt- was obtained by assembling overlapping sequences, with a -nucleotide genome. the complete genome encodes several different genes, and components of the related genes are ordered utr - a- b-s- a,b,c(e)-m- b- a,b-n-utr . sequence analysis showed that ck/ch/ / jt- shares the highest complete genome sequence identity with gx-yl ( . %) and gx-yl ( . %) but only . %- . % s nucleic acid identity with reference strains (fig. ) . blastn analysis showed that the s gene of ck/ch/ /jt- shares %- % similarity with isolates from china in recent years. phylogenetic analysis of the s gene of ibvs revealed that ck/ch/ /jt- and other isolates described in blastn analysis are grouped as a new cluster (fig. ) , which is separated from the previously identified serotypes and genotypes. to examine the sequence characteristics of isolate ck/ch/ / jt- , potential recombination events were analysed. three recombination events were identified in the genome of ck/ch/ /jt- . the assessment using rdp software revealed four recombination events in the ck/ch/ /jt- genome ( table ) . the parent strains cq - , saibk and gx-yl were grouped into genotype ck/ch/lsc/ i, while lx and ck/ch/ldl/ were qx-like genotypes, and / and partridge/gd/s / were genotypes / and tl/ch/ldt / (fig. ) . three recombinant events were further confirmed by simplot ( fig. a) and bootscan analysis (fig. b) . recombinant gene fragments were considered recombinants if any crossover event appeared between two putative parental strains. obvious recombination signals were found in simplot and bootscan analyses of recombinant events , and . the similarities and breakpoints also agreed with the rdp software. however, the recombinant gene fragments shared low homology with the parental sequences, similarity analysis could not further confirm recombination in event . furthermore, whole non-structural protein (nsp) ; genes a, b and n; the utr and parts of the utr; nsp and nsp ; and genes s and m were involved in the recombination events. to further track the origins of the other parts of isolate ck/ch/ /jt- s genome, we cut the complete genome sequence into different fragments according to recombination analysis and conducted a pairwise comparison of the fragment sequences of isolate ck/ch/ /jt- with four qx-like, three ck/ch/lsc/ itype, two tl/ch/ldt / -type and two / -type strains. genomic positions at - of ck/ch/ /jt- appeared very similar to ck/ch/lsc/ i-type strains gx-yl and gx-yl , while genomic positions at - and - fell into the same group with tl/ch/ldt / -type ibvs. however, the ck/ch/ /jt- isolate fell into the group of qx-like strains from location - in the phylogenetic tree (fig. a) . these results strongly suggested that ck/ch/ /jt- originated from homologous rna recombination events from multiple template switches among qx-like, ck/ch/lsc/ i-, tl/ch/ldt / -and / -type ibvs (fig. b) , and the genome approximate positions - and - , - and - , - and - , and - possibly arose from ck/ch/lsc/ i-type, qx-like, tl/ch/ldt / -type and / -type ibvs, respectively. animal experiments for ck/ch/ /jt- in chicken were performed to evaluate the pathogenicity of isolate ck/ch/ / fig. . phylogenetic tree of the s gene from infectious bronchitis viruses (ibvs). a phylogenetic tree was constructed with the upgma method using mega version . . bootstrap values were determined from replicates of the original data. the ck/ch/ /jt- strain is marked with a black solid triangle, and strains described in blastn analysis are marked with black hollow triangles. rdp, geneconv, maxchi, chimaera, seq ( .  À , .  À , .  À , .  À , and .  À ) a only transferred gene fragments where at least detection methods p-value  À are included in the table. b "genes" indicates the coding sequences contained within the fragment introduced by recombination. c the "minor parent" is the sequence closely related to that from which sequences in the proposed recombinant region may have been derived. d the "major parent" is the sequence closely related to that from which the greater part of the recombinant's sequence may have been derived. jt- . at dpi, birds in group inoculated with ck/ch/ /jt- appeared depressed with ruffled feathers and huddled together. at dpi, the birds in group developed respiratory symptoms, most of the birds breathing with head shaking and a few breathing with open mouth and lifted up neck. notably, infected birds began to die at dpi, which continued until dpi, with the death rate reaching . % (fig. a) . no deaths or clinical signs were observed in group . among the seven dead birds in group , slight haemorrhage with serous catarrhal exudates could be seen in the trachea (fig. b) . typical kidney lesions were found in all dead chickens, the affected kidneys from the dead chickens showing predominant gross lesions that were pale, mottled, and swollen, with renal tubules and ureters that were distended with excess urate (fig. d) . clinical signs in the surviving birds tended to disappear gradually, and the rest of the pathological changes were present by dpi. no gross lesions were observed in any bird in group . antibody responses in birds that survived at dpi were measured. all of the chickens showed positive reactions (fig. b) , and the mean titre induced by the ck/ch/ /jt- strain was . at dpi. these data clearly demonstrated that strain ck/ch/ /jt- isolated here was a virulent ibv. microscopic examination of the tracheal tissues revealed extensive degeneration and necrosis of the ciliated epithelial cells, sometimes with pseudoacinar structures resulting from dropout of dead cells (fig. b) . lung lesions included haemorrhage, congestion, and lymphocytic infiltration in the alveolar lumen (fig. d) . severe renal lesions were characterized by degeneration and necrosis of renal tubular epithelial cells, lymphocytic infiltration in the interstitium, and exfoliated renal tubular epithelial cells, with erythrocytes being frequently observed (fig. f) . recently, various live-attenuated and inactivated vaccines have been widely and extensively used to prevent ib disease in chicken farms in china. however, the ib vaccine program has not been very effective, as new ibv serotypes, genotypes and variants are sometimes isolated from vaccinated chickens (feng et al., ; han et al., ) . this study identified an ibv, ck/ch/ /jt- , and other isolates grouped into a novel genotypic cluster different from previously identified genotypes. although of the isolates were reported in other five papers, we found that among the isolates, isolates were clustered into a genotype with reference strains a and qxibv (luo et al., ) , isolates were grouped with reference strains / and uk / (feng et al., ; ji et al., ; li et al., ) , and one isolate showed far evolutionary distances with reference tl/ch/ldt / strains, but grouped into tl/ch/ldt / -type . in our study, the ck/ch/ /jt- and strains showed a low s nucleic acid identity with reference strains. phylogenetic analysis of the s gene showed these strains could be grouped as a new genotypic cluster which was different from the previously identified genotypes ibv in china. some vaccines, such as mass-type h and / -type / vaccine strains, could not provide effective protection against ck/ch/ /jt- infection. in addition, the ck/ ch/ /jt- -like isolates form a new genotypic cluster, which is similar to a new ibv cluster in korea (lim et al., ) . coronaviruses are identified in a wide variety of animals and divided into three distinct groups based on genotypic and serological characterization (brian and baric, ; lai and cavanagh, ) . coronaviruses have a high frequency of recombination due to the unique mechanism of viral replication. ibv is a member of the genus gamma coronavirus. novel ibv variants could emerge mainly via two approaches: recombination and mutation toro et al., ) . in this paper, we found the ck/ch/ /jt- -like strains underwent three recombination events. the recombination fragments were located in approximate positions - , - and - . the genome approximate positions - and - , - and - , - and - , and - of the ck/ch/ /jt- -like ibv possibly arose from ck/ch/lsc/ i-type, qx-like, tl/ch/ldt / -type and / -type ibvs respectively (fig. b ). however the chronological order of the recombination events needs further investigation. recently, nine genotypes of ibv have been circulating in china. the predominant genotype is qx-like ibv, which has spread widely in asia, europe and africa since it was first reported in in china (cook et al., ; knoetze et al., ; toffan et al., ) . the second dominant genotype detected in china is the ck/ch/lsc/ i-type . the co-circulation of qx-like, ck/ch/ lsc/ i, vaccine, tl/ch/ldt / and / genotype strains in the same chicken farm provided the possibility of new recombinants like ck/ch/ /jt- . previous studies found that the recombination hot spots tended to be located in the a (nsp and nsp ), b (nsp ), s , envelope, membrane genes and the utr . most of the recombination breakpoints identified in isolate ck/ch/ /jt- are located in these genes. to our knowledge, this recombinant virus is different from those in other reports wu et al., ; xu et al., ; zhao et al., ) . all these data indicate that the new isolate is recombined with homologous rna recombination events from multiple template switches among qx-like, ck/ch/lsc/ i-, tl/ch/ldt / -and / -type ibv strains. a new genotype virus could acquire new biological characteristics, including virulence from gene recombination. the ibv spike and replicase genes are known to be determinants of pathogenicity (armesto et al., ; wickramasinghe et al., ) . the spike gene also plays a key role in ibv tissue tropism (wickramasinghe et al., ) . the mass-type m and / -type /b could cause the respiratory signs without death in infected chickens (benyeda et al., ) . recently, the chinese qx-like ibv virulent strains were found to cause %- % mortality in infected chickens (feng et al., ; liu et al., ; shi et al., ; sun et al., ) , while the virulent ck/ch/lsc/ i-type strains yn and ck/ch/ldl/ i and the tl/ch/ldt / -type strain tl/ch/ldt / caused %, %, and % mortality with severe kidney lesions in infected chickens, respectively (feng et al., (feng et al., , liu et al., ) . the isolate ck/ch/ /jt- could cause respiratory and severe renal disease and resulted in deaths of . % experimental infections of -day- old spf chickens. these results indicate the ck/ch/ /jt- isolate is highly virulent. the replicase gene of isolate ck/ch/ / jt- was derived from qx-like, ck/ch/lsc/ i-and tl/ch/ldt / type strains, and the spike gene possibly originated from / -and tl/ch/ldt / -type strains. all of these features yield . % mortality with respiratory and severe renal disease in ck/ch/ / jt- infected chickens. the altered / fragment in the spike gene may not affect the tissue tropism of isolate ck/ch/ /jt- ; the tissue tropism of the virus may have more than one pathogenicity factor, an issue to be studied in the future. in this study, an ibv, named ck/ch/ /jt- , was identified from an ibv-vaccinated flock in china. ck/ch/ /jt- -like viruses are grouped as a new genotypic cluster different from previously identified genotypes. genome sequence analysis suggested that isolate ck/ch/ /jt- originated from qx-like, ck/ch/lsc/ i, tl/ch/ldt / and / genotypes. ck/ch/ / jt- of ibv is very virulent in chickens. such novel recombinants should receive more attention during viral surveillance for better development of anti-ibv strategies. comparison of the pathogenicity of qx-like, m and /b infectious bronchitis strains from different pathological conditions coronavirus genome structure and replication comparisons of envelope through b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes coronavirus avian infectious bronchitisvirus the long view: years of infectious bronchitis research virulent avian infectious bronchitis virus, people's republic of china analysis of s gene of avian infectious bronchitis virus isolated in southern china during development and efficacy of a novel live-attenuated qx-like nephropathogenic infectious bronchitis virus vaccine in china a -year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china emergence of a group coronavirus through recombination molecular evolution and emergence of avian gammacoronaviruses phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in china during two genotypes of infectious bronchitis virus are responsible for serological variation in kwazulu-natal poultry flocks prior to evolution of infectious bronchitis virus in taiwan: positively selected sites in the nucleocapsid protein and their effects on rna-binding activity the molecular biology of coronaviruses isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in south china during an emerging recombinant cluster of nephropathogenic strains of avian infectious bronchitis virus in korea successful cross-protective efficacy induced by heatadapted live attenuated nephropathogenic infectious bronchitis virus derived from a natural recombinant strain isolation of avian infectious bronchitis coronavirus from domestic peafowl (pavo cristatus) and teal (anas) histopathologic analysis (haematoxylin-eosin stain) of chickens infected with ibv strain ck/ch/ /jt- a, normal trachea tissue control b, trachea tissue infected with ck/ch/ /jt- displays extensive dropout, degeneration, and necrosis of ciliated epithelial cells and lymphocytic infiltration (black arrow). scale bar = mm d, lung tissue infected with ck/ch/ /jt- displays haemorrhage and congestion (black arrow) jt- displays severe renal lesions, including degeneration, necrosis of renal tubular epithelial cells, and exfoliated renal tubular epithelial cells (black arrow) molecular characterization and pathogenicity of infectious bronchitis coronaviruses: complicated evolution and epidemiology in china caused by cocirculation of multiple types of infectious bronchitis coronaviruses origin and characteristics of the recombinant novel avian infectious bronchitis coronavirus isolate ck/ch/ljl/ phylogenetic analysis of the s glycoprotein gene of infectious bronchitis viruses isolated in china during evaluation of recombinant fowlpox virus expressing infectious bronchitis virus s gene and chicken interferon-gamma gene for immune protection against heterologous strains phylogenetic analysis of infectious bronchitis coronaviruses newly isolated in china, and pathogenicity and evaluation of protection induced by massachusetts serotype h vaccine against qx-like strains recombination in avian gamma-coronavirus infectious bronchitis virus qx-like infectious bronchitis virus in africa genetic diversity and selection regulates evolution of infectious bronchitis virus binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity genome sequence and origin analyses of the recombinant novel ibv virulent isolate saibk characterization and analysis of an infectious bronchitis virus strain isolated from southern china in complete genome sequence of a recombinant nephropathogenic infectious bronchitis virus strain in china comparison of hematoxylin-eosin staining and methyl violet staining for displaying ghost cells molecular characterization of an infectious bronchitis virus strain isolated from northern china in this study was made possible by funding from the priority academic program development of jiangsu province higher education institutions (papd). the authors declared that they have no conflict of interest. key: cord- - lp mmu authors: kuo, shu-ming; wang, ching-ho; hou, ming-hon; huang, yuan-pin; kao, hsiao-wei; su, hong-lin title: evolution of infectious bronchitis virus in taiwan: characterisation of rna recombination in the nucleocapsid gene date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: lp mmu avian infectious bronchitis virus (ibv) belongs to the coronaviridae family and causes significant economic loss in taiwan (tw), even in flocks that have been extensively immunised with massachusetts (mass)-serotype vaccines. phylogenetic analysis of all non-structural and most structural genes shows that tw ibv is genetically distinct from the us strain and more similar to chinese (ch) ibv. in contrast, the nucleocapsid (n) gene of tw ibv presents phylogenetic incongruence. rna recombination at the ′ end of the n gene between tw and us ibv is shown to be responsible for this discordance. surprisingly, the recombinant n gene is found in all of tested tw ibv isolates, suggesting that a recombination event gave origin to a founder lineage. our data indicate that rna recombination in the recombinant ′ end of the n gene may have caused the emergence of the current ibv population in taiwan. infectious bronchitis virus (ibv) is highly contagious and pathogenic in chickens. although the current massachusetts (mass)-and connecticut-serotype vaccines usually offer sufficient protection, they are less effective in certain areas, such as china (ch) and taiwan (tw) wang and khan, ; yu et al., ) . frequent point mutations and evolutionary fitness in the hypervariable regions (hvrs) of the spike (s ) gene contribute to most of the antigenic determinants of ibv. therefore, the s gene is often referred to as the index of genetic diversity and of viral evolution, due to its high variability and close genotype/serotype correlation (dolz et al., ; liu et al., ; wang and huang, ; wang and tsai, ) . in addition to point mutation, rna recombination is also involved in the generation of new antigenic variants. rna recombination in a coronavirus was first demonstrated experimentally in mouse hepatitis virus (mhv) (lai et al., ; makino et al., ) , and a high frequency of recombination was found both in vitro and in vivo after mhv infection (keck et al., ; makino et al., ) . recombination sites were detected throughout nearly the entire genome, and many recombinants with multiple crossover sites were found (liao and lai, ) . the high frequency of rna recombination in coronaviruses is likely caused by the unique mechanism of rna synthesis, which involves discontinuous transcription and polymerase jumping (cavanagh, ; lai et al., ; liao and lai, ; makino et al., ) . putative ibv genetic recombination in the s gene has been documented in different field isolates, including a japanese strain (kb ), european avian infectious bronchitis virus (ibv) belongs to the coronaviridae family and causes significant economic loss in taiwan (tw), even in flocks that have been extensively immunised with massachusetts (mass)-serotype vaccines. phylogenetic analysis of all non-structural and most structural genes shows that tw ibv is genetically distinct from the us strain and more similar to chinese (ch) ibv. in contrast, the nucleocapsid (n) gene of tw ibv presents phylogenetic incongruence. rna recombination at the end of the n gene between tw and us ibv is shown to be responsible for this discordance. surprisingly, the recombinant n gene is found in all of tested tw ibv isolates, suggesting that a recombination event gave origin to a founder lineage. our data indicate that rna recombination in the recombinant end of the n gene may have caused the emergence of the current ibv population in taiwan. ß elsevier b.v. all rights reserved. isolates (dutch d and british / ), texas local strains (pp and se ) and a new york isolate (cu-t ) (jia et al., ; kusters et al., ; wang et al., ) , based on sequence comparison and phylogenetic incongruence. additionally, half of the nucleocapsid (n) gene of cu-t was further shown to be replaced by that of the holland (h ) vaccine strain, indicating that genomic rna recombination in ibv may occur in multiple genes (jia et al., ) . in taiwan, phylogenetic analysis revealed that few tw ibv strains, such as / and / , may undergo genetic recombination with ch ibv (chen et al., ; huang et al., ) . this experimental evidence and the sporadic detection of recombinant variants in nature support the notion that recombination occurs in coronaviruses (kottier et al., ) . however, available evidence remains insufficient to allow the conclusion that coronavirus evolution can be driven by interspecies recombination. tw ibv, first isolated in (lu et al., ) , can be classified into two individual serological groups, tw i and tw ii, based on the sequences of the hvrs of the s proteins (wang and huang, ) . the mass-serotype vaccine has shown a modest protective effect on the ibv outbreak in taiwan (huang and wang, ; lin et al., ) , possibly due to the considerable variation in s sequences between tw and us strains. in this study, we analyse tw ibvs and show that they are genetically closer to ch isolates than to us strains. the n gene of tw ibv is the only exception and shows phylogenetic incongruence. we further demonstrate that rna recombination has contributed to this genetic discordance. the recombinant n gene is detected in all the analysed tw ibvs, suggesting that rna recombination drives the evolution of this coronavirus at the population level. tw ibv strains were isolated and propagated through the allantoic sac route by inoculating - -day-old specific pathogens free (spf) embryonated eggs from the animal health research institute, council of agriculture in taiwan (gelb and jackwood, ) . tw ibvs had been purified by three consecutive limiting dilutions (wang and huang, ; wang and tsai, ) . sequences of both us and ch ibvs were obtained from genbank maintained by ncbi. we listed the tested ibvs and their genbank numbers in supplementary table , including tw strains, us strains and ch strains. inquired from the genbank up to , we found tw and us ibvs are decoded in both s and n genes and we included all these up-to-date ibvs in this study. among the tw strains, ibvs in the same clade of me tree (supplementary fig. s ) were chosen for the analysis of n gene in figs. and . both tw / and tw / were excluded because of their phylogenetic discordance in s (fig. s ). both / and / was excluded due to the discordance in n (fig. s ) . the length of the tested genes is all intact and no -utr deletion was found in these ibv strains. total rna was extracted from tw / -infected allantoic fluid using trizol tm c&t (protech, taiwan), according to the manufacturer's protocol. rna ( . mg) was reverse transcribed into cdna using superscript tm iii reverse transcriptase (rt) (invitrogen, usa). pcr was performed with a proofreading dna polymerase (kod-plus, toyobo, japan). generally, the pcr conditions included cycles of c for s, c for s, and c for min, on a pcr machine (astec , japan). consecutive viral genomic fragments were cloned into the pcrii-topo vector (invitrogen, usa) by ta cloning and were subjected to sequencing. the -untranslated regions (utr) of ibv / was analysed using a smart tm race cdna amplification kit (clontech, usa). first, cdna was prepared from mg of total rna from allantoic fluid, using the powerscript tm rt and a unique reverse primer ( -tac ttg caa gac aag ttc c- , nt - of the viral genome). after the rt reached the end of the mrna template, it added - dc residues to the newly synthesised cdna. the smart ii a oligonucleotide ( -aagcagtggtat-caacgcagagtacgcggg- ) annealed to the dc tag and served as an additional extending primer for rt. consequently, the single-stranded cdna was used as the pcr template with the nested primer ( -aag-cagtggtatcaacgcagagt- ) and a new reverse primer, -ccaacaagcctgcgcattgccatag- (nt - of the viral genome). amplified dna products were cloned into the pgem-t easy vector (promega, usa) and sequenced. phylogenetic trees were constructed by bayesian inference (bi), maximum likelihood (ml) and minimum evolution (me) methods. the best-fit models and parameters for initial settings of the phylogenetic programs were selected by modeltest . for bi and ml algorithms on the basis of the akaike information criterion (posada and crandall, ) . estimates of evolutionary divergence over all sequence pairs were computed by averaging the number of base substitutions per site in mega . . all positions containing gaps and missing data were eliminated from the dataset. standard error estimates were obtained by a bootstrap procedure from replicates. for the s data set in fig. a , the best-fit model of substitution was a gtr model (rodriguez et al., ) with a gamma substitution parameter ( mrbayes . . was used for bi analysis. random starting trees were used. a total of two million generations of markov chains were run. trees were saved every generations, resulting in , trees in the initial samples. the stationary phase of log-likelihood was reached within , generations. thus the burn-in (number of initial trees that were discarded) was set to . a majority-rule consensus tree was generated from the remaining samples ( , trees), and the percentage of samples recovering any particular clade represented the clade's posterior probability. we conducted nonparametric bootstrap replicates for ml analysis using garli v . . we used the heuristic search option with substitution models set to the best-fit model chosen by modeltest . . me analysis was performed using mega . with replicates. twelve ibv strains, including tw / , tw / , tw / , tp/ , mass , cal , beaudette, cu-t , ch-bj, ch-s , ch-lx and ch-ldt , were subjected to the recombination detection program (rdp) . (martin and rybicki, ) . genes s and n were aligned by clustalw and analysed by seven algorithms in rdp . , including rdp, bootscan, geneconv, maxchi, chimaera, siscan, and seq. based on the results automatically calculated by rdp, tw / , tw / , cu-t and ch-lx were chosen as representative strains for which data is shown below. data were further subjected to the genetic algorithms for recombination detection (gard) program to confirm the putative single breakpoint position (kosakovsky pond et al., ) . to elucidate the phylogenetic relationships of tw ibv, ibvs were analysed in this study, including tw strains, ch strains and us strains. minimum evolution (me) test showed that tw ibvs form a unique branch independent of the us and ch groups (fig. s ). some exceptional strains showing the phylogenetic incongruence were noticed, such as the tw / (chen et al., ; huang et al., ) , tw / (huang et al., ) , tw / (chen et al., ) , china ck/ch/ ldl i and china ck/ch/ldl i (chen et al., ) . in their respective clades, tw strains, ch strains, us strains were chosen for the analyses of nucleotide-sequence identity, maximum likelihood (ml) and bayesian inference (bi). the nucleotide-sequence identity between tw s genes and those of us or ch isolates was similar ( - %). the phylogenetic tree shown in fig. a , which were constructed by bi, shows that tw ibv is a branch independent of the us and ch groups. analysis of ml reproduces similar tree topology and the bootstrap values are illustrated together with the posterior probability (fig. a) . interestingly, the evolutionary divergence and me methods indicated that tw ibvs are more closely related to the ch cluster than to the us group (table , fig. b and fig. s ), supported by less difference between tw-ch strains (table ) and the high bootstrap value ( %) for the branch separating the tw-ch groups from us group (fig. b) , respectively. a virulent taiwanese field isolate, tw / , was fully sequenced ( , bps; dq ). the g/c content of the viral genome was . %. orfs a, b, e and m of tw / were compared to those of other ibv genomes (fig. ) . phylogenetic trees of the a and b genes of tw ibvs consistently show a closer relationship to the ch strains than to the us strains ( fig. a and b) . analysis of the -utr and the orf -encoded proteins (including the papain-like proteinase, helicase, rna-dependent rna polymerase, p and hd protein) using the neighbourjoining algorithm reproduces the same topology (data not shown). similar results are also seen in the trees based on the full-length e and m genes of ibv, as shown in fig. c and d, further illustrating that tw ibv is genetically closer to the ch virus. in contrast to the above results, the n gene of tw ibv showed surprising phylogenetic incongruence and was more closely related to that of us isolates according to the me algorithm (fig. s , fig. a and b) and the evolutionary divergence analysis (table ) . a fragment containing nt - and its encoded proteins ( fig. c and d) also reflected the same phylogenetic relationship as the full-length n region. interestingly, results based on the -terminus of the tw ibv n gene and its encoded protein ( fig. e and f) were inconsistent with those from the full-length n gene, and were similar to those for the genes described above and shown in figs. and . to further examine this phenomenon, unrooted phylogenetic trees produced by bi clearly illustrate that the tw n gene is phylogenetically closer to us ibv than to ch ibv (fig. a, c and e) . crucially, fig. c illustrates that the termini of the n genes of tw and us ibv belong to the same monophyletic group, rather than being separate isolated branches. the k a /k s ratio of tw / n versus all ibvs analysed in this study is . - . , indicating the purifying selection of n protein during evolution. to further clarify whether the phylogenetic incongruence is caused by different evolutionary rates in different parts of the n gene, a ml test that are not biased by evolutionary rate variation was applied to recheck the phylogenetic relationships (holmes and rambaut, ) . no major difference was observed between the results from the bi and those from the ml test (fig. b , d and f), confirming that rna recombination probably occurred between tw and us ibvs in the terminal region of the n gene and that the phylogenetic incongruence was not caused by point mutation or variation in local evolutionary rates. to further test our hypothesis, genes and n of ibvs were subjected to the recombination detection program (rdp) (martin and rybicki, ) and the genetic algorithms for recombination detection (gard) program (kosakovsky pond et al., ) to examine the interspecies recombination. rdp analysis and three gard algorithms all consistently showed high significance (p value = .  À to .  À ) to support the hypothesis of interspecies recombination with the breakpoint at nt of gene . rdp-bundled algorithms also revealed one candidate crossover region between the tw and us genes, located between nt and of the queried sequences, covering the gene and the -terminus of the n gene (fig. a ). for instance, the p values of the rdp and bootscan algorithms were .  À and .  À , respectively. alignment of the first base pairs (bp) of n clearly indicated high identity between the tw and us strains (fig. b) . moreover, the deduced protein sequences also showed that the ch ibvs were significantly different from the tw and us strains ( fig. c and fig. ). in addition, all the analysed tw ibvs shared the recombinant n gene, including one of the earliest isolates found in taipei in , tp/ (figs. and ) . this finding suggests that the recombination in the n gene produced a novel ibv founder distinct from ibv lineages present before . the recombination event may have provided replication benefits, enabling the offspring of this founder to become dominant. the variation in the s region often represents viral genetic diversity and strain evolution (dolz et al., ; liu et al., ; wang and huang, ; wang and tsai, ) . the ratio of k a /k s can be an indicator of selective pressure acting on an open reading frame. the k a /k s of nucleotides in the s region was . ae . % among evaluated tw ibvs. although previous studies of ibv s genes have argued that tw ibv is an independent branch of the us group (huang et al., ) , the relationship between tw ibv and ch isolates remains unclear. in this study, we have shown that taiwanese ibv features a recombinant ntd in the n locus and has evolved into a unique population. the interspecies recombination is evidenced by the incongruent phylogenetic relationship inferred for this gene region, high consensus in the n-terminus between tw and us strains, rare synonymous mutations in the conserved n protein, and wellsupported results from rdp analyses. a fast rate of mutation caused by an error-prone rnadependent rna polymerase is a major factor driving the evolution of rna viruses. nevertheless, as the restraint of error threshold of lethal mutations, the coronavirus is speculated to have a relatively low mutation rate due to its large genome size (eigen, ; maynard smith and szathmá ry, ) . to increase viral adaptability and fitness, coronavirus has evolved a unique discontinuous transcription system and 'copy-choice' genomic synthesis to achieve high efficiency of rna recombination (cavanagh, ; lai et al., ; liao and lai, ; makino et al., ) . however, only sporadic recombinant variants have been detected in nature. we here show that the recombinant n gene was detected in all the isolated tw ibvs, supporting the participation of recombination in viral evolution and showing that a recombinant rna virion can emerge as a local dominant strain. since the emergence of severe acute respiratory syndrome coronavirus (sars-cov) in humans, coronavirus has become a new focus of pathogenic research. in the present, little is known about the original strain and the natural host of the sars-cov. plausibly, the sars-cov may have originated, like tw ibv, through rna recombination in an unidentified host and subsequently emerged as a new virus in human beings by crossing the host boundary (holmes and rambaut, ; hon et al., ; magiorkinis et al., ; stavrinides and guttman, ; zhang et al., ) . however, present evidence is far from supporting this conclusion, and extensive sampling of the coronavirus genome from candidate hosts is required to solve this important issue. although studies of mhv have demonstrated that rna recombination can occur throughout the genome, genetic crossovers in coronaviruses are usually detected after tas sequences, referred to as putative hotspot sites (keck et al., ; kusters et al., ; liao and lai, ) . in this study, both the rdp/bootscan and gard analyses suggested the possibility of a putative recombination crossover nick at nt of gene in the tw ibv genome. alternatively, given the remarkable discrepancy between the first nt of ch n and those of tw and us n (fig. ) , the start codon of n after the tas sequence may also serve as a breakpoint candidate for genetic recombination within tw ibv genome. it is noteworthy that one of the earliest tw ibvs, tp/ , shares high sequence identity in the first nt of n with other tw ibvs and us ibvs (fig. ). during the s in taiwan, flocks were extensively immunised with the mass-serotype h vaccine (lu et al., ) . in this study, our finding suggests that the recombination in the n gene produced a novel ibv founder distinct from ibv lineages present before . however, we did not exclude the possibility that the vaccination may also contribute to the antigenic shift of tw ibvs. the recombination event may have provided replication benefits, enabling the offspring of this founder to become dominant. to increase fitness under the pressure of h vaccination, the tw ibv with n recombinant might have undergone positive selection to escape from immune surveillance or to gain a replication advantage within quasi species. coronavirus avian infectious bronchitis virus identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan molecular epidemiology and evolution of avian infectious bronchitis virus in spain over a fourteen-year period new concepts for dealing with the evolution of nucleic acids a laboratory manual for the isolation and identification of avian pathogens viral evolution and the emergence of sars coronavirus evidence of the recombinant origin of a bat severe acute respiratory syndrome (sars)-like coronavirus and its implications on the direct ancestor of sars coronavirus s and n gene analysis of avian infectious bronchitis viruses in taiwan development of attenuated vaccines from taiwanese infectious bronchitis virus strains a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains rna recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus a and fusion-negative mouse hepatitis virus multiple recombination sites at the -end of murine coronavirus rna gard: a genetic algorithm for recombination detection experimental evidence of recombination in coronavirus infectious bronchitis virus sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus recombination between nonsegmented rna genomes of murine coronaviruses rna recombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments protective effect of vaccination in chicks with local infectious bronchitis viruses against field virus challenge genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between the incidence and virus isolation of infectious bronchitis in chickens in taiwan phylogenetic analysis of the full-length sars-cov sequences: evidence for phylogenetic discordance in three genomic regions high-frequency rna recombination of murine coronaviruses rdp: detection of recombination amongst aligned sequences the major transitions of evolution modeltest: testing the model of dna substitution the general stochastic model of nucleotide substitution mosaic evolution of the severe acute respiratory syndrome coronavirus isolation, pathogenicity, and h protection efficacy of infectious bronchitis viruses isolated in taiwan relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus genetic grouping for the isolates of avian infectious bronchitis virus in taiwan evidence of natural recombination within the s gene of infectious bronchitis virus molecular characterization of an infectious bronchitis virus strain isolated from an outbreak in vaccinated layers characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens testing the hypothesis of a recombinant origin of the sars-associated coronavirus this work was supported by the bureau of animal and plant health inspection and quarantine, council of agriculture in taiwan (grant number as- . . -bq-be). it is highly appreciated. we also thank dr. yi-ling lin at the institute of biomedical sciences in academia sinica for critical reading of this manuscript and valuable comments. supplementary data associated with this article can be found, in the online version, at doi: . /j.vetmic. . . . key: cord- -vmprhd g authors: zhang, li; luo, yuzi; wang, wei; sun, yuan; zhang, jiaoer; fatima, madiha; jia, xiangdong; qiu, hua-ji title: efficient inactivation of african swine fever virus by ozonized water date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: vmprhd g african swine fever virus (asfv) is the causative agent of african swine fever (asf), which is a devastating disease of domestic pigs and wild boar, causing significant economic losses to the pig industry worldwide. to evaluate the ability of ozonized water as a disinfectant to inactivate asfv, ozonized water of different concentrations was tested, and the viral reduction was determined by infectivity assay on porcine primary alveolar macrophages. the results showed that log ( %) reduction in viral titer was observed when ( . ) tcid( )/ml wild-type or reporter asfv was inactivated with ozonized water as lower as mg/l within min at room temperature; while a viral reduction of approximately log ( %) wasobserved when ( . ) tcid( )/ml wild-type or reporter asfv was inactivated with mg/l ozonized water within min, and log ( . %) virus was inactivated by or mg/l ozonized water within or min, respectively; furthermore, mg/l ozonized water inactivated log ( %) reporter asfv as higher as ( . ) tcid( )/ml in min, and a viral reduction of approximately log ( . %) in reporter asfv or log ( %) in wild-type virus was observed when inactivated with mg/l ozonized water in min; meanwhile, a viral reduction of log ( . %) was observed when mg/l ozonized water was applied to the wild-type asfv of ( . ) tcid( )/ml in min. overall, ozonized water can rapidly and efficiently inactivate asfv, representing an effective disinfectant for asf control. african swine fever (asf) is an oie (world organization for animal health)-listed devastating disease of domestic pigs and wild boar, causing significant economic losses to the pig industry worldwide. the disease is caused by african swine fever virus (asfv), a large, enveloped, double-stranded dna virus belonging to the genus asfivirus of the family asfarviridae. the virus can infect pigs of all ages. acute clinical forms of asf are accompanied by hyperthermia, skin cyanosis and organ bleeding with a high mortality up to % (costard et al., ; dixon et al., ) . asf mainly remained in africa and rarely introduced to other continents until , when it was reported in georgia (rowlands et al., ) . then it rapidly spread across the caucasus and into the russian federation and reached the european union (eu) in (gogin et al., ; oie, ) . in august , asf emerged in china for the first time (zhou et al., ) and then spread rapidly across all provinces and municipalities. and subsequently it spread to a number of asia countries (oie, ) . the continuous spread of the disease through africa, europe, russian federation and asia countries, especially china, the biggest pig producer in the world, make the situation worsen. the worldwide spread of asf has led to high socio-economic impacts on the global pig industry and trade (dixon et al., ) . asfv can be transmitted through different routes such as direct or indirect contact with infected pigs and their secretions, excretions, blood, tissues, pork and j o u r n a l p r e -p r o o f pork products, as well as contaminated transport vehicles, feed, water, personnel, etc. (dixon et al., ; luo et al., ) . the survivability of asfv in the environment and different biological matrices means that contaminated materials play an important role in transmission (dixon et al., ; petrini et al., ) . as there are no effective vaccines and treatment available for asf, quarantine and strict biosecurity measures are important for prevention and control of the disease. considering the severe situation of asf spreading in china and other countries, there is a pressing need to improve the control strategies for asf. effective disinfectants for inactivating asfv are extremely essential for implementation of strict biosecurity measures. the ideal disinfectant should have the advantages of fast and high efficiency, low toxicity, broad antimicrobial spectrum and stable nature (bicknell et al., ) . ozone (o ), also known as superoxide, is an allotrope of oxygen (o ). ozone is a special odor gas at room temperature and soluble in water. ozonized water has strong oxidation ability, broad spectrum of sterilization and disinfection, and fast sterilization and disinfection functions, which is stable and non-toxic and harmless. furthermore, oxygen is generated after decomposition, without any residue and side effects, so it is widely used in food industry and medical areas (wang, ; ye, ) . precious studies have shown that ozonized water can inactivate rna viruses, such as vesicular stomatitis virus (zhang et al., ) , norwalk virus (shin, et al., ) , poliovirus (shin, et al., ) , severe acute respiratory syndrome coronavirus (zhang et al., ) and porcine epidemic diarrhea virus (guo et al., ) , indicating that ozonized water has a good inactivation ability for these viruses. so far, there is no report regarding the inactivation effect of ozonized water on dna viruses. in view of the urgent need for asf biosecurity prevention and control in china, in this study we evaluated the inactivation effects of ozonized water produced by j o u r n a l p r e -p r o o f electrolytic ozonize generator on asfv. primary porcine alveolar macrophages (pams) were prepared from -week-old specific-pathogen-free (spf) pigs and maintained in rpmi medium (gibco, usa) containing % fetal bovine serum (fbs) (gibco, usa), mg/ml streptomycin and iu/ml penicillin. pams ( cells/ml) were seeded into -well plates with ml per well or -well plates with µl per well and incubated at °c in a humidified incubator with % co . following tests were conducted after cell adherence ( to h or overnight). all experiments with asfv in this study were performed in the biosafety level (bsl- ) facilities in harbin veterinary research institute (hvri) of the chinese academy of agricultural science (caas). the wild-type asfv sy strain (wt-asfv) or the reporter asfv (asfv-Δmgf-egfp, with deletion of the mgf gene and introduction of the egfp reporter based on the sy strain) (zhou et al., ) was inoculated into pams ( -well plates) and incubated at °c for to h. after two freeze-thaw cycles, the cell cultures were centrifuged at , g for min to remove cells and other debris and the supernatant were collected and stored at - °c. the viral titer was determined by the method of reed and muench (reed and muench, ) . ozonized water was prepared by using an electrolytic ozonize generator produced by hangzhou qingwei science and technology co. ltd. firstly, tap water was put into a non-conductive container (a glass beaker), and the electrical conductivity was measured using a conductivity meter and adjusted to be to j o u r n a l p r e -p r o o f s/cm. subsequently, electrode was inserted into the water and energized. after stirring for to min, the ozonize concentration in water was determined by the iodometric method (shi et al., ) . the stability of ozonized water ( to h) was determined. to evaluate the cytotoxicity of ozonized water, neutralizer and neutralizing products on cells, reporter asfv ( . tcid /ml) were incubated at room temperature with mg/l of ozonized water ( : , v/v) for min, following by addition of neutralizer (pbs containing g/l sodium thiosulfate and % fbs) (group in table ) or rpmi medium (group ) ( : , v/v). after -min incubation at room temperature, pams (in -well plates) were inoculated with the μl mixtures and incubated at °c for days with % co . the same procedures for group to as shown in table . also, different concentrations of ozonized water ( , and mg/l), neutralizer or tap water were separately tested without asfv. the effect of these treatments on the cells was determined using cell counting kit- (cck- ) (dojindo) according to the manufacturer's instructions and the technical specification for disinfection (the ministry of health of the people's republic of china, ). at the same time, the ph values of ozonized water and neutralizer were determined, which were . ± . and . ± . , respectively. meanwhile, the inactivation effect of ozonized water on the reporter asfv was evaluated by infectivity assays on pams and the viral titer was determined as described previously (reed and muench, ; li and yang, ) . average values and standard deviations of three independent experiments were calculated. wt-asfv or reporter asfv ( . , . and . tcid /ml) were incubated at the stability of ozonized water was assessed by determination of the concentration over time ( - h). the results showed that the half-life of the ozone water was approximately h and which was completely degraded within to h (table ) . the cytotoxicity assay showed that over % to near % cell livability could be achieved following addition of different concentrations of ozonized water ( , and mg/l), neutralizer or tap water from to h, and also the viral titers in treatment groups to were near to . log ( / of starting titers), indicating that these treatments did not significantly affect cell viability (tables and ) . different concentrations of ozonized water were applied to . tcid /ml wt-asfv or reporter asfv and incubated for , , and min, respectively, and then stopped by the neutralizer and the viral reductions were determined by infectivity assays on pams. as shown in figs. and , and table , log reduction ( %) of the either wt-asfv or reporter asfv was observed when inactivated with ozonized water as lower as mg/l within min, indicating that ozonized water can inactivate asfv rapidly and efficiently. further, different concentrations of ozonized water ( , and mg/l) were applied to wt-asfv or reporter asfv of . tcid /ml for , , and min, respectively. the results showed that a viral reduction of approximately log ( %) was observed when inactivated with mg/l ozonized water within min, and log ( . %) virus was inactivated with or mg/l ozonized water within min or min, respectively (figs. and , table ). different concentrations of ozonized water ( , or mg/l) were applied to . tcid /ml wt-asfv or reporter asfv for , , and min, respectively. as shown in figs. and , and table , mg/l ozonized water inactivated log ( %) reporter asfv in min, and a viral reduction of approximately log ( . %) in reporter asfv or log ( %) in wild-type virus was observed when inactivated with mg/l ozonized water in min; meanwhile, a viral reduction of log ( . %) was j o u r n a l p r e -p r o o f observed when mg/l ozonized water inactivated wild-type asfv of . tcid /ml in min, indicating that ozonized water ( to mg/l) can also effectively inactivate the high dose of asfv within a short time. the continuous spread of asf worldwide has revealed big challenges for control. thus, biosecurity measures are urgently needed to reduce the risk of the pathogen expansion and spread (jancovich et al., ) . effective disinfectants are necessary for this issue. as a strong oxide, ozone can destroy the structure of microorganisms such as bacteria and viruses in a short period of time, making it incapable of viability. there are many disinfectants that can kill microorganisms by oxidizing properties to achieve disinfection effects, including chlorine, bleaching powder, potassium permanganate, and so on. however, these disinfectants have shown slow antimicrobial ability and harmful to humans and animals. ozone is different from other disinfectants, and excess ozone can be quickly decomposed into oxygen, which is non-toxic and harmless. high efficiency and rapid sterilization of microorganisms can be achieved by ozone disinfection in an instant. high concentration of ozonized water (> mg/l) can be produced simply with an electrolysis method in several minutes. ozonized water disinfection is not limited by space, region and temperature. it has been widely used in food production and processing fields such as dairy products, beverages, water, melons, pork and pork products. previous studies have shown that ozonized water directly destroys the nucleic acid (rna or dna) of the virus through oxidation, leading to the inactivation of the virus (roy et al., ; shin et al., ) . this study evaluated the inactivation of j o u r n a l p r e -p r o o f within min, and as high as . tcid /ml asfv could be inactivated by to mg/l ozonized water within min, indicating that ozonized water can be used as an effective disinfectant for asf control. asfv can be transmitted by direct or indirect contact between infected animals and/or other contaminated materials, such as drinking water and feed. fomites such as clothing, transport trucks or feed supplies may act as a source of infection (dixon et al., ) . ozonized water can be used for all-round disinfection of pig farms, as well as cleaning and disinfection of the environment and potential pollutants in the whole pig industry chain, such as slaughterhouses and meat processing plants. ozonized water can be used by spraying, rinsing, soaking, wiping, etc., according to the environmental conditions. it can be used for air disinfection in offices and other areas by spraying or wiping; disinfection of drinking water; disinfection of food, clothes, boots, kitchen waste and other materials by soaking; disinfection of environments, transport vehicles, pig houses and others by rinsing. it is suggested that the epidemic areas can be disinfected with ozonized water of mg/l or above, and that the non-epidemic area should be disinfected with ozonized water of mg/l. ozone disinfection of drinking water -technology transfer and policy issues epidemiology of african swine fever virus african swine fever epidemiology and control african swine fever in the north caucasus region and the russian federation in years efficacy of sodium trioxide (o ) water against porcine epidemic diarrhea virus immunization of pigs by dna prime and recombinant vaccinia virus boost to identify and rank african swine fever virus immunogenic and protective proteins statistical evaluations of viral clearance studies for biological products african swine fever: a major threat to the chinese swine industry survival of african swine fever virus (asfv) in various traditional italian dry-cured meat products a simple method of estimating fifty percent endpoints african swine fever virus isolate mechanism of enteroviral inactivation by ozone the methods of measuring ozone concentration in water reduction of norwalk virus, poliovirus , and bacteriophage ms by ozone disinfection of water the ministry of health of the people's republic of china advances in the application of ozone disinfection world animal health information database (wahid) world organization for animal health (oie). . world animal health information database (wahid) comprehensive application of ozone in controlling microbial hazard in meat processing enterprises key: cord- -j yh v g authors: zhao, dongmin; zhang, lijiao; han, kaikai; liu, qingtao; yang, jing; huang, xinmei; liu, yuzhuo; li, yin; zhao, peng title: peptide inhibitors of tembusu virus infection derived from the envelope protein date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: j yh v g the outbreak and spread of tembusu virus (tmuv) has caused very large losses in the waterfowl-breeding industry since . the viral envelope (e) protein, the principal surface protein of viral particles, plays a vital role in viral entry and fusion. in this study, two peptides derived from domain ii (dii) and the stem of the tmuv envelope protein, tp and tp , respectively, were tested for their antiviral activity. tp and tp inhibited tmuv infection in bhk- cells, and their % inhibitory concentrations (ic( )) were . mg/l and . mg/l, respectively. viral inhibition assays in different cell lines of avian origin showed that the inhibitory effects of tp and tp are not cell type dependent. moreover, tp also exhibited inhibitory activity against japanese encephalitis virus (jev) infection. the two peptides inhibited antibody-mediated tmuv infection of duck peripheral blood lymphocytes. co-immunoprecipitation assays and indirect enzyme-linked immunosorbent assays (elisas) indicated that both peptides interact with the surface of the tmuv virion. rnase digestion assays confirmed the release of viral rna following incubation with tp , while incubation with tp or tp interfered with the binding between tmuv and cells. taken together, these results show that tp and tp may be developed into antiviral treatments against tmuv infection. tembusu virus (tmuv), a member of the flaviviridae, is a newly emerging pathogen of avian origin that causes paralysis and ovarian haemorrhage in mainly ducks and geese. tmuv-infected ducks and geese exhibit a rapid drop in egg production and daily food intake. the sudden outbreak and fast spread of tmuv have caused heavy economic losses in the major waterfowl-breeding provinces of china since . concerningly, the range of tmuv hosts is expanding. in addition to ducks and geese, chickens, pigeons and house sparrows can be infected by tmuv. therefore, tmuv has a severe impact on development of the domestic poultry industry (chen et al., ; chen et al., b; zhang et al. ). the whole tmuv genome encodes three structural proteins and seven nonstructural proteins. among these viral proteins, the envelope (e) protein is located on the surface of mature virions and forms head-to-tail homodimers (kuhn et al., ) . the e protein consists of an ectodomain (containing three domains: di, dii and diii) and a stem-transmembrane domain that anchors the protein to the membrane. the first step in the process by which tmuv enters host cells is binding of the e protein to cellular receptors on the cell surface (rodenhuis-zybert et al., ) . to date, several cellular receptors have been identified; these include glucose-regulated protein (grp ) on bhk- cells, heat shock protein a on df- cells and heparin sulfate on both bhk- and duck embryo fibroblasts (defs) (zhao et al., b; liu et al., ; wu et al., ) . these receptors play an important role in the first step of tmuv entry and viral attachment. subsequently, attached tmuv enters cells by the clathrin-mediated endocytosis pathway, after which endocytic vesicles carrying the virus are delivered to endosomes (smit et al., ) . under the low-ph conditions of the endosome, the e homodimer dissociates into monomeric proteins, exposing the fusion loop in dii. subsequently, the fusion loop inserts into the target membrane, and three copies of the e protein interact to form the e protein trimer. diii then folds back against the trimer, directing j o u r n a l p r e -p r o o f the e stem anchor towards the fusion loop. the energy released by these conformational changes induces fusion between the viral membrane and host cell membrane. finally, a fusion pore is formed, and delivery of the nucleocapsid into the cytosol initiates replication (pierson and kielian, ) . previous studies have shown that artificially synthesized small peptides based on a protein domain can bind intact proteins and inhibit their biological activity through interfering with proper protein folding. envelope protein-derived peptides were reported to bind the envelope protein and inhibit viral infection (hall and frieden, ) . jiang et al. ( ) reported that a synthetic peptide corresponding to the envelope protein of human immunodeficiency virus type i (hiv-i) could block virion infectivity. subsequently, other synthetic inhibitory peptides of the fusion proteins of paramyxoviruses, herpesviruses, orthomyxoviruses, retroviruses and coronaviruses have also been identified (porotto et al., ; okazaki and kida, ; markosyan et al., ; yao and compans, ; bosch et al., ) . to date, several peptides corresponding to different domains of the flavivirus e protein have been shown to inhibit flavivirus infection. dn , an inhibitory peptide corresponding to the stem region of dengue virus (denv) e protein, almost completely inhibited denv infection in llc-mk cells and exhibited cross-inhibitory activity against west nile virus (wnv) infection. the wn inhibitor, a direct mimic of dii of the wnv e protein, reproducibly inhibited wnv infectivity at a low-μm concentration (hrobowski et al., ) . the inhibitory peptide z , which was synthesized based on the stem region of the zika virus (zikv) e protein, could penetrate the placental barrier of pregnant icr mice and inhibit vertical transmission of zikv in pregnant c bl/ mice (yu et al., ) . the peptide p , derived from the stem region of the japanese encephalitis virus (jev) e protein, could block jev and zikv infection in vitro. p also protected mice from jev-induced lethality and j o u r n a l p r e -p r o o f reduced zikv-induced histopathological damage by decreasing viral load (chen et al., a) . moreover, dn and oan could inhibit antibody-dependent enhancement (ade)-mediated denv infection in vitro, suggesting peptide inhibitors as a strategy to inhibit flavivirus ade (nicholson et al., ) . although multiple peptide inhibitors corresponding to the e proteins of other flaviviruses have been studied, inhibitory peptides against tmuv have not been reported. in this study, the peptides tp and tp derived from dii and the stem region of the tmuv e protein, respectively, were synthesized, and their ability to prevent tmuv infection was investigated. the data demonstrated that tp inhibited tmuv infection through destroying the integrity of the viral particles, while both tp and tp exerted inhibitory effects by interfering with the binding of tmuv to cells. additional results suggested that tp cross-inhibited jev infection. these findings provide new insight into the development of drugs against tmuv. tmuv strain js (genbank accession no. jf ) was isolated by our laboratory . jev strain sa - - (genbank accession no. af ) was obtained from dr. xiuli feng at nanjing agricultural university. tmuv strain js and jev strain sa - - were propagated in baby hamster kidney cells (bhk- cells) grown in rpmi- medium (hyclone, south logan, usa) containing % foetal calf serum (fcs, hyclone, south logan, usa). viral titres in bhk- cells were determined by plaque assay as previously described (zhao et al., ) . specific-pathogen-free (spf) ducks were purchased from the harbin veterinary research institute in china. this study was approved by the animal care and use committee of jiangsu province. monoclonal antibody against the e protein of tmuv strain js was produced by our laboratory . anti-β-actin monoclonal antibody was obtained from cowin biotechnology company (cowin, beijing, china). hrp-labelled goat anti-mouse igg antibody was purchased from zsbio biotechnology company (beijing, china). the peptides tp (swlvnrdwfhdlnlpwtgssagtwq) and tp (mvalgdtawdfgsvggvltsigkgihqvfgsafksl), the negative peptide stp (mtpltrwhssgeehhlvtkrmevatwlvgaapgqgewirsi, panya et al., ) , tp -biotin, tp -biotin and stp-biotin at a % purity ( fig a) were synthesized by the nanjing genscript biotechnology company. the peptides were solubilized in dimethyl sulfoxide (dmso) and diluted in serum-free nanjing, china). the primers used for qrt-pcr were ns af primer (forward, '-gcagcccaggagattttgag- ') and ns ar primer (reverse, '-ctaacgcaacgccaagca- '). the viral titres in the supernatants of cultured cells were determined by a plaque assay as described previously (zhao et al., ) . briefly, ten-fold serial dilutions of the supernatants were used to inoculate a monolayer of bhk- cells. after h of incubation at °c, the cells were washed with pbs and covered with cell culture medium containing % agarose. after h of incubation at °c, the cells were stained with . % neutral red to visualize the plaques. the amounts of tmuv e protein in the cell lysates were detected by western blotting as described previously . briefly, tmuv-infected cells were lysed using cell lysis buffer containing mm phenylmethylsulfonyl fluoride (beyotime, shanghai, china). samples containing equal amounts of protein were separated by sds-page, and the proteins were then electrophoretically transferred to a pvdf membrane which was blocked at °c for h with % ecl prime blocking agent (ge, buckinghamshire, uk) in pbs containing . % tween- . the membrane was then incubated with anti-tmuv or anti-β-actin monoclonal antibody at °c overnight. after washing three times with pbst, the membrane j o u r n a l p r e -p r o o f was probed with hrp-labelled goat anti-mouse igg antibody at °c for h. the membrane washing procedure was repeated three times, and the proteins were detected using the beyoecl plus kit (beyotime, shanghai, china). cells were treated with mg/l tp or tp for h at °c. to determine whether the two peptides are toxic to cells, cellular viability was detected with a cck- kit (dojindo, kumamoto, japan) according to the manufacturer's instructions. the ic values of the two peptides were measured as previously described (panya et al., ) . briefly, bhk- monolayer cells were grown in a -well plate. two-fold serial dilutions of the peptides were mixed with tmuv at a concentration of tcid and then incubated at °c for h before their infection of bhk- cells. after h of incubation at °c, supernatants were removed, and the cells were covered with a mixture of % agarose and rpmi- medium containing % fcs. the cells were further incubated for h and then stained with . % neutral red to visualize plaques. the tmuv plaques were counted and analysed by comparison with control cells infected with tmuv treated with negative peptide (set as % inhibition). the nonlinear regression function of graphpad prism software (graphpad software, la jolla, usa) was used to calculate ic values of the peptides against tmuv. duck anti-tmuv serum was prepared and stored in our laboratory. the neutralizing antibody titre of the antiserum was assessed by the plaque reduction neutralization test as j o u r n a l p r e -p r o o f previously described (zhao et al., a) . duck peripheral blood lymphocytes were isolated using a duck peripheral blood lymphocyte isolation kit (haoyang, tianjin, china) according to the manufacturer's protocol. briefly, ml of sterile peripheral blood with anticoagulant was obtained from ducks and then diluted with ml of rpmi- medium. ten millilitres of separation medium was added to the centrifuge tube. the diluted whole blood was carefully layered onto the separation medium. the tube was centrifuged with a horizontal rotor at ×g for min at °c. cells at the interface were carefully transferred into a new centrifuge tube containing ml of washing buffer and mixed gently. the tube was centrifuged with a horizontal rotor at ×g for min at °c. the cell pellet was then washed twice at °c with washing buffer. finally, the cells were resuspended in rpmi- medium containing % fcs and inoculated into a -well plate. ade inhibition assays were carried out as previously described (nicholson et al., ) . briefly, inactivated duck anti-tmuv serum with a neutralizing antibody titre of : was diluted : with rpmi- medium. then, . ml of diluted inactivated duck anti-tmuv serum was incubated with an equal volume of virus at a concentration of tcid for h at °c. then, the serum-virus mixtures were incubated with peptides at different concentrations for an additional h. the mixtures were added to duck peripheral blood lymphocytes and incubated for h at °c. the cultures were harvested, and viral titres were determined by plaque assay and qrt-pcr. a co-immunoprecipitation assay was conducted as previously described (zhao et al., b) . briefly, tmuv was incubated with biotin-peptide on a rocker at °c overnight, j o u r n a l p r e -p r o o f followed by incubation with anti-biotin antibody (sigma, st. louis, usa) for h. subsequently, protein a/g agarose beads (beyotime, shanghai, china) were added to the mixture and incubated for h. the beads were washed with pbs five times and collected for qrt-pcr detection. ninety-six-well elisa plates were pre-coated with purified tmuv at °c overnight. after washing with pbst three times, the plates were blocked with pbst containing % bsa at °c for h. after washing three times, . ml of diluted biotin-peptides was added to each well and incubated at °c for h. the plates were washed with pbst three times, followed by the addition of . ml of hrp-labelled streptavidin diluted : . after incubation at °c for h and three further washes, tetramethyl benzidine (tmb) substrate was added and incubated for min at room temperature. the reaction was stopped with the addition of m h so , and the plates were read by a biotek microplate reader. an rnase digestion assay was carried out as previously described (lok et al., ; yu et al., ) . briefly, tmuv at a concentration of approximately × tcid was incubated with tp or tp at °c for h, after which the released genomic rna was digested with micrococcal nuclease (neb, ipswich, usa) for h at °c. genomic rna was extracted and detected by qrt-pcr as described above. a viral binding inhibition assay was conducted as described previously (costin et al., ) . confluent bhk- cell monolayers were washed once with chilled pbs. tmuv and j o u r n a l p r e -p r o o f peptides were co-incubated at °c for h before their addition to the monolayers and incubation for h at °c. the monolayers were washed with chilled pbs three times and then harvested for rna extraction and detection. statistical analyses were performed using statistical package for social science (spss) statistical software. differences with a p value < . were considered statistically significant. tmuv was incubated with tp or tp before its infection of bhk- cells. at h post-inoculation, tp -or tp -mediated inhibition of tmuv infection was assessed by qrt-pcr (fig b) , plaque assay (fig c) , and western blotting (fig d) . the qrt-pcr results showed that both peptides inhibited tmuv infection in a dose-dependent manner. incubation with tp or tp at a concentration of mg/l led to slightly decreased virus production, but these differences were not statistically significant. however, incubation with mg/l and mg/l tp or tp caused a significant decrease in virus production. compared with negative peptide treatment, the virus production following mg/l and mg/l tp treatment was decreased by . % and . %, respectively. similarly, mg/l and mg/l tp treatment reduced virus production by . % and . %, respectively. the results of the plaque assay and western blot analysis were similar to those obtained by qrt-pcr. in addition, the ic values suggested that tp derived from the stem region exhibited a stronger inhibitory effect than tp derived from dii ( fig e) . as expected, the negative peptide did not show an inhibitory effect. a toxicity assay was conducted to exclude j o u r n a l p r e -p r o o f the possibility that the antiviral effects of the two peptides were due to underlying cytotoxicity. the results showed no significant differences between bhk- cells with or without the peptides, suggesting that the two peptides are non-toxic at the indicated concentrations ( fig f) . these results suggest that tp and tp can effectively inhibit tmuv infection. to determine whether the inhibitory effects of tp and tp are cell type dependent, different cell types of avian origin (chicken embryo fibroblasts (cefs), df- cells (immortalised cefs) and duck embryo fibroblasts (defs)) were used to test the effects of tp and tp on tmuv infection. at h post-infection, qrt-pcr, plaque assays, and western blotting were conducted to detect virus production with peptide treatment. as shown in fig , in the presence of mg/l and mg/l tp or tp , virus production in the three cell types was significantly decreased (fig a) . the results of the plaque assay were similar to those obtained by qrt-pcr. compared with negative peptide treatment, treatment with mg/l and mg/l tp reduced virus production by . % and . % in cefs, . % and . % in df- cells and . % and . % in defs, respectively. however, mg/l and mg/l tp decreased virus production by . % and . % in cefs, . % and . % in df- cells and . % and . % in defs, respectively ( fig b) . the antiviral effects of tp and tp were also confirmed by western blotting (fig c) . moreover, tp and tp showed no cytotoxic effect on cefs, df- cells or defs (fig d) . these results indicated that the peptides exhibited similar inhibitory effects in different cell types. because the e proteins among flaviviruses share sequence homology, whether the j o u r n a l p r e -p r o o f peptides would inhibit jev infection was investigated. the inhibitory effects of the peptides were determined by the plaque assay at h post-infection. tp efficiently protected against jev infection, demonstrating that tp could cross-inhibit jev infectivity. however, tp did not exhibit inhibitory activity against jev (fig ) . previous studies have shown that antibodies can mediate flavivirus infection to facilitate viral entry, leading to enhanced infection and severe disease outcomes, in a process known as ade (halstead et al., ; hawkes et al., ) . liu et al. ( ) reported tmuv ade when anti-tmuv antibody levels were low. earlier studies in our laboratory indicated that duck anti-tmuv serum could enhance infection in duck peripheral blood lymphocytes, with this effect peaking with anti-tmuv serum (with a neutralizing antibody titre of : ) diluted : (data not shown). as shown in fig , anti-tmuv serum diluted : facilitated infection in duck peripheral blood lymphocytes. however, incubation of anti-tmuv serum mixed with tp or tp inhibited ade in a concentration-dependent manner. although the inhibitory peptides did not block ade completely, treatment with tp or tp led to a significant reduction in virus production compared with that following treatment with the negative peptide. although tp and tp inhibited tmuv ade, the two peptides showed no cytotoxic effect on duck peripheral blood lymphocytes (fig c) . several studies have shown that the interaction between inhibitory peptides and the virion causes a reduction in infection (zhang et al., ) . in this study, the binding of peptides to tmuv was examined by the addition of biotin-peptides at different concentrations j o u r n a l p r e -p r o o f to an elisa plate pre-coated with purified tmuv that were then detected using hrp-labelled streptavidin. biotin-tp or biotin-tp at increasing concentrations increased the signals indicating binding to the tmuv that had been coated on the plates in a concentration-dependent manner, but signals from negative peptide and non-enveloped tmuv were very low (fig a) . the binding between tmuv and the peptides was further confirmed by co-immunoprecipitation assays (fig b) . these results verified the direct interaction between biotin-peptides and tmuv. lok et al. ( ) reported that the inhibitory peptide dn could inhibit denv by interacting with virions and inducing the release of viral genomic rna. here, the release of viral genomic rna induced by the interaction between tmuv and the peptides was investigated by exposing peptide-treated virions to rnase digestion. the rnase digestion assay showed that genomic rna released from tmuv virions co-incubated with tp at increasing doses was sensitive to micrococcal nuclease. approximately . % of the genomic rna released by tmuv virions treated with mg/l tp was degraded by rnase. however, the genomic rna released by virions treated with negative peptide or tp was protected from rnase digestion (fig a) . to explore the mechanism of the inhibitory effects of tp , a virus binding inhibition assay was performed to determine whether the peptides interfered with the binding between tmuv and cells. tmuv was incubated with tp or tp before its addition to bhk- cells at °c for h to allow viral attachment. unbound virus was repeatedly washed with chilled j o u r n a l p r e -p r o o f pbs, and the amount of tmuv remaining on the cells was then detected by qrt-pcr and western blotting. compared to the rna of virus treated with negative peptide, relative tmuv rna was significantly decreased after tp or tp treatment. the results of western blot analysis were consistent with those of qrt-pcr and suggested that the binding of tmuv to the cells was reduced after tp or tp bound tmuv (fig b and c) . at present, several commercial inactivated and attenuated live vaccines have been approved by the chinese ministry of agriculture for tmuv prevention. however, ducks and geese in some farms can still be infected by tmuv because of the lack of immunization or failure to induce adequate antibodies after immunization. however, there are no effective and specific treatments for tmuv infection. the need to develop treatments to control the spread of tmuv is urgent. in this study, the peptide tp , derived from dii of the tmuv e protein, and peptide tp , corresponding to a sequence in the stem region of the tmuv e protein, were highly effective in inhibiting tmuv infection, indicating that tp and tp are potential candidates for tmuv treatment. the stem region of the e protein is highly conserved among flaviviruses. previous studies reported that peptide inhibitors derived from the stem region exhibited cross-antiviral activity. dn , derived from the stem region of the denv e protein, had inhibitory effects against denv as well as wnv (hrobowski et al., ) . p , corresponding to the stem region of the jev e protein, effectively blocked zikv infection with an ic at the micromolar level (chen et al., a) . in this study, the tp peptide exhibited antiviral activity against tmuv as well as jev. since the corresponding stem regions of tmuv, jev and other flaviviruses are highly conserved (fig ) , other flaviviruses may be inhibited by tp . therefore, tp , tp derivatives or analogous peptides probably act as broad-spectrum flavivirus inhibitors. in this study, co-immunoprecipitation assays and indirect elisa indicated that tp or tp could bind tmuv in a concentration-dependent manner. the direct binding of tp or tp to tmuv, but not to target cells, might explain why both peptides exhibited similar inhibitory effects in the different cell types bhk- cells, cefs, df- cells and defs. the mechanism by which flavivirus infection is inhibited by peptide inhibitors is not clearly understood. previous studies reported that once dn bound the virion, it likely inserted itself between the e protein ectodomain and the membrane during the dynamic "breathing" process of the viral particle (johnson, ; lok et al., ) . this insertion resulted in the formation of holes in the viral membrane or disrupted the membrane bilayer structure. subsequently, negatively charged genomic rna escaped from the viral particles. similarly, the binding of z to the e protein inactivated zikv by disrupting the integrity of the zikv membrane (yu et al., ). an rnase digestion assay showed that tmuv treated with tp , but not tp , was sensitive to rnase digestion, indicating disruption of the viral membrane and the escape of genomic rna from viral particles. however, the underlying mechanism by which tp disrupts the viral membrane is still unclear. as described in previous studies, possible explanations are hydrophobic and electrostatic effects or viral dynamic "breathing", but determining this mechanism lies beyond the scope of this study. the rnase digestion assay showed that tp treatment did not destroy the integrity of tmuv. therefore, a virus binding inhibition assay was conducted to investigate the mechanism by which tp inhibited tmuv infection. the results demonstrated that treatment j o u r n a l p r e -p r o o f with either tp or tp prevented tmuv from binding cells. costin et al. ( ) reported that the surface of denv particles changed from smooth to rough and that denv particles lost their icosahedral symmetry after incubation with the peptides dn opt and oan . this structural deformation probably interfered with cell binding. alhoot et al. ( ) demonstrated that the inhibitory peptides det and det caused structural abnormalities and altered the arrangement of the viral e protein, which interfered with viral binding and entry. tp and tp are likely to interfere with viral attachment by a mechanism similar to that described above. nevertheless, the idea that the decrease in tmuv attachment after incubation with tp was due to the release of the genome cannot be excluded. thus, further research to elucidate the precise mechanism of the inhibitory effects of tp and tp is needed. the antibody induced by an inactivated vaccine may enhance tmuv infection through ade. this phenomenon increases the challenge in developing an effective and safe vaccine to provide protection against tmuv. tp and tp , which show inhibitory activity against ade, are probably favourable for the inoculation of infected ducks and geese without the concern of ade. in addition, unlike most small-molecule antiviral drugs, the nontoxicity of tp and tp also ensures the safety of these peptide inhibitors for antiviral therapy. however, why tp and tp inhibit tmuv ade is still unclear. yu et al. ( ) could be further developed as novel treatments against tmuv. the authors declare no conflict of interest. degradation of genomic rna released from tmuv due to tp or tp in an rnase digestion assay. b and c, pre-incubation of tmuv with either tp or tp reduced the binding of tmuv to bhk- cells compared to that of tmuv pre-incubated with negative peptide. b, viral genomic rna was detected by qrt-pcr. the data were analysed using the comparative ct (ΔΔct) method. gapdh was chosen as a reference gene for internal control. negative peptide treatment was used as a reference for each comparison. data are the means±sds of triplicate experiments. the asterisk indicates a statistically significant difference (p< . ). c, cells lysates were subjected to western blotting to study the levels of tmuv e proteins. β-actin was used as a loading control. j o u r n a l p r e -p r o o f inhibition of dengue virus entry into target cells using synthetic antiviral peptides the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex antiviral activity of peptide inhibitors derived from the protein e stem against japanese encephalitis and zika viruses avian tembusu virus infection effectively triggers host innate immune response through mda and tlr -dependent signaling pathways avian interferon-inducible transmembrane protein family effectively restricts avian tembusu virus infection structural optimization and de novo design of dengue virus entry inhibitory peptides protein fragments as probes in the study of protein folding mechanisms: differential effects of dihydrofolate reductase fragments on the refolding of the intact protein neutralization and antibody-dependent enhancement of dengue viruses enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls peptide inhibitors of dengue virus and west nile virus infectivity identification and molecular characterization of a novel flavivirus isolated from geese in china inhibition of hiv- infection by a fusion domain binding peptide from the hiv- envelope glycoprotein gp virus particle dynamics structure of dengue virus: implications for flavivirus organization, maturation, and fusion identification of heat shock protein a as a tembusu virus binding protein on df- cells. virus res an adapted duck tembusu virus induces systemic infection and mediates antibody-dependent disease severity in mice release of dengue virus genome induced by a peptide inhibitor binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins a study of low ph-induced refolding of env of avian sarcoma and leukosis virus into a six-helix bundle viral entry inhibitors block dengue antibody-dependent enhancement in vitro development of double antibody sandwich elisa for detection of duck or goose flavivirus a synthetic peptide from a heptad repeat region of herpesvirus glycoprotein b inhibits virus replication a peptide inhibitor derived from the conserved ectodomain region of denv membrane (m) protein with activity against dengue virus infection flaviviruses: braking the entering molecular determinants of antiviral potency of paramyxovirus entry inhibitors dengue virus life cycle: viral and host factors modulating infectivity flavivirus cell entry and membrane fusion heparin sulfate is the attachment factor of duck tembus virus on both bhk and def cells peptides corresponding to the heptad repeat sequence of human parainfluenza virus fusion protein are potent inhibitors of virus infection a peptide-based viral inactivator inhibits zika virus infection in pregnant mice and fetuses the stem region of premembrane protein plays an important role in the virus surface protein rearrangement during dengue maturation an updated review of avian-origin tembusu virus: a newly emerging avian flavivirus screening and identification of b-cell epitopes within envelope protein of tembusu virus domain i and ii from newly emerging goose tembusu virus envelope protein functions as a dominant-negative inhibitor of virus infectivity the study was supported by national natural science foundation of china (no. the authors also acknowledge the financial support from china scholarship council program (file no. ). key: cord- -ncvvmkca authors: labarque, g; van reeth, k; van gucht, s; nauwynck, h; pensaert, m title: porcine reproductive–respiratory syndrome virus infection predisposes pigs for respiratory signs upon exposure to bacterial lipopolysaccharide date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ncvvmkca this study examined whether an infection with porcine reproductive and respiratory syndrome virus (prrsv) potentiates respiratory signs upon exposure to bacterial lipopolysaccharides (lps). five-week-old conventional pigs were inoculated intratracheally with the lelystad strain of prrsv and received days later one or two intratracheal lps administrations. the necessary controls were included. after lps administration, pigs were intensively monitored for clinical signs. additionally, some pigs were euthanatized after a second lps administration for broncho-alveolar cell analysis and virological examinations of the lungs. broncho-alveolar lavage (bal) cells were counted and differentiated. lung suspensions and bal fluids were titrated for prrsv. exposure of pigs to prrsv only resulted in a fever for time periods ranging from to days and slight respiratory signs. exposure of pigs to lps only resulted in general signs, characterized by fever and depression, but respiratory signs were slight or absent. prrsv–lps exposed pigs, on the other hand, developed severe respiratory signs upon lps exposure, characterized by tachypnoea, abdominal breathing and dyspnoea. besides respiratory signs, these pigs also showed enhanced general signs, such as fever and depression. lung neutrophil infiltration was similar in non-infected and prrsv-infected pigs upon lps exposure. prrsv quantities were similar in lungs and bal fluids of pigs infected with prrsv only and prrsv–lps exposed pigs. these data show a clear synergism between prrsv and lps in the induction of respiratory signs in conventional pigs. the synergism was observed in % of the pigs. so, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures. porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus, causes infections in pigs worldwide. the virus replicates highly in the respiratory tract and shows a distinct tropism for broncho-alveolar macrophages (duan et al., ) . however, a single prrsv infection, particularly under experimental circumstances and with european isolates, fails to induce overt respiratory disease (done and paton, ) . also under field circumstances, most pigs become infected with prrsv at growing age without respiratory disease. still, the frequency and severity of respiratory disease have increased since the enzootic occurrence of prrsv (done and paton, ) . this has stimulated research into the combined effects of prrsv and other infectious agents. consequently, experimental dual infections have been performed with prrsv followed by various bacteria such as haemophilus parasuis, pasteurella multocida, streptococcus suis, bordetella bronchiseptica, and salmonella choleraesuis (cooper et al., ; galina et al., ; brockmeier et al., ; wills et al., ) . we ourselves have performed dual infections with prrsv followed by enzootic viruses, notably porcine respiratory coronavirus (prcv) or swine influenza virus (siv) (van reeth et al., ) . the clinical effects of these combinations were extremely severe in some cases, but almost completely subclinical in other. most important, none of the dual infections mentioned provides a reliable model to study pathogenetic features or to test control measures. we hypothesized therefore that the clinical outcomes of dual inoculations with two infectious agents are influenced by factors that are too difficult to control, such as the stage of replication and the viral or bacterial load. bacterial lipopolysaccharides (lps) or endotoxins, a major constituent of the cell wall of gram-negative bacteria, are released in high concentrations in the lungs upon infection with gram-negative bacteria (lamp et al., ; kadurugamuwa and beveridge, ) and these endotoxins are present in varying concentrations in dust in swine buildings (rylander, ; zejda et al., ) . the release of lps by gram-negative bacteria, such as h. parasuis, p. multocida, b. bronchiseptica, and s. choleraesuis may explain the more severe disease in the experimental dual infections with prrsv and these bacteria (cooper et al., ; brockmeier et al., ; wills et al., ) . van reeth et al. ( ) recently demonstrated that dual inoculations with prcv followed by bacterial lps seriously aggravate respiratory signs in gnotobiotic pigs, while the respective single inoculations were subclinical. therefore, we wanted to examine if exposure of prrsvinfected pigs to lps similarly enhances respiratory signs. prrsv may lend itself excellently as a predisposing agent for synergism with lps, because all pigs become infected at ages varying from weeks to fattening age (albina et al., ; houben et al., ) . also, prrsv persists in the lungs for (labarque et al., ) to (mengeling et al., ) days. we have examined the clinical course of inoculations with prrsv followed by lps, and the effect of the timing and frequency of lps administrations. additionally, some preliminary investigations of cellular and virological aspects in the lungs were performed. a fifth passage on pulmonary alveolar macrophages (pams) of the lelystad strain of prrsv (wensvoort et al., ) was used in this study. the inoculation dose was . tcid /pig. escherichia coli lps (o :b ) was obtained from difco laboratories and used at a dose of mg/kg body weight. this dose was based on data from previous experiments in gnotobiotic pigs, and selected to cause no respiratory signs . forty-six conventional pigs, originating from prrsv-negative sows, were used. pigs were weaned at weeks of age and placed in isolation. they were allowed to acclimatize during days before initiation of the experiments. prrsv inoculations and lps administrations occurred intratracheally as described by van reeth et al. ( ) . briefly, the pigs were held in vertical position with their neck extended. a needle was inserted through the skin cranial to the sternum and the inoculum was injected. the intratracheal administration was chosen to ensure that all the pigs received exactly the same dose in the lungs. three experiments were performed. in the first experiment, pigs were inoculated with prrsv and received one lps administration days later. seven pigs were inoculated with prrsv only. eight pigs, not previously inoculated with prrsv, received one lps administration. clinical monitoring was performed daily during consecutive days after prrsv inoculation and every h during the first h after lps administration. in the second experiment, eight pigs were inoculated with prrsv and, days later they received two lps administrations with a h interval. four pigs, not previously inoculated with prrsv, received two lps administrations with a h interval. clinical monitoring was performed daily during consecutive days after prrsv inoculation and at , , , and h after the second lps administration. in the third experiment, out of the prrsv-lps exposed pigs, described in the first experiment, received a second lps administration, h after the first one. these pigs were divided in two subgroups. one subgroup of six pigs was again monitored for clinical signs every h until h after the second lps administration. one subgroup of five pigs was euthanatized between and h after the second lps administration for study of the broncho-alveolar lavage (bal) cell population and for virological and bacteriological examinations of the lungs. from the seven prrsv control pigs, described in the first experiment, four pigs were again monitored for clinical signs every h for h at day after prrsv inoculation. the remaining three pigs were euthanatized at time points corresponding to those of the prrsv-lps exposed pigs and served as controls for the broncho-alveolar cell and virological examinations. all eight lps exposed pigs, described in the first experiment, received a second lps administration, h after the first one. four pigs were again monitored for clinical signs every h until h after the second lps administration and four pigs were euthanatized between and h after the second lps administration for broncho-alveolar cell and virological examinations. four non-inoculated pigs were euthanatized for the same purpose. pigs were monitored for general signs, notably fever and depression, and for respiratory signs, notably tachypnoea, abdominal breathing and dyspnoea. scores were given for these five clinical parameters. body temperatures . c were scored as , temperatures between ! . and . c were scored as and temperatures ! . c were scored as . respiration rates were scored as , rates between ! and were scored as and rates ! were scored as . depression, abdominal breathing and dyspnoea were scored as (absent) or (present). scores were added up and a mean of the cumulative general and respiratory scores per group was calculated. at necropsy, the lungs were removed. the right lung was used for broncho-alveolar cell examination after bal using the method described by van reeth et al. ( ) . the bal fluid was centrifuged (  g, min, c) to separate the cells and the cell-free lavage fluid. aliquots of the cell-free lavage fluid were stored at À c until virus titration on pams. bal cells were counted in a türk chamber and cytocentrifuge preparations were stained with diffquik (baxter, düdingen, switzerland) to determine the percentage of mononuclear cells and neutrophils. the left lung was used for virological and bacteriological examinations. twenty percent suspensions of lung lobes were made in a phosphate-buffered saline, clarified by centrifugation and the supernatant was used for prrsv titration. virus titration of lung suspensions or bal fluids was performed on pams, as described by labarque et al. ( ) . for bacteriology, samples of lung tissue were plated on bovine blood agar and cultured aerobically. a nurse colony of coagulase-positive staphylococcus species was streaked diagonally on each plate. plates were inspected for bacterial growth after and h. colonies were then identified by standard techniques. non-parametric tests were used, because of lack of normality in the data. standard twosample mann-whitney tests were used to compare general and respiratory clinical scores between groups. p < : was taken as the level of statistical significance. statistical analyses were performed using spss . . twenty-six of the total of prrsv-infected pigs developed fever for time periods ranging from to days. respiratory signs were absent, except for two pigs, which showed tachypnoea and abdominal breathing. in experiment , six of the seven prrsv control pigs showed fever until the end of the monitoring period. respiratory signs were slight in one pig and absent in the other pigs. the mean respiratory score was . (table ) . in experiment , all four prrsv control pigs showed fever until the end of the monitoring period. respiratory signs, characterized by increased respiration rates, were observed in one of the four pigs. the mean respiratory score was . (table ) . in non-infected pigs, a single lps administration induced transient general signs (fig. ) . respiratory signs were slight or absent and the mean respiratory score was only . ( table ). in prrsv-infected pigs, however, lps induced severe clinical signs with fever table mean general and respiratory scores after the last lps administration in prrsv-lps exposed pigs and their controls in all the pigs and respiratory signs in % of the pigs (fig. ) . respiratory signs were characterized by tachypnoea (peak breaths/min), abdominal breathing and dyspnoea and lasted until the end of the monitoring period. two out of the pigs did not show respiratory signs after lps administration. mean general and respiratory scores were significantly higher in prrsv-lps exposed pigs than in singly lps exposed pigs (table ) . in non-infected pigs, which received two lps administrations with a h interval, both general and respiratory signs were observed (fig. ) . clinical signs were significantly higher in prrsv-infected pigs not only with regard to the number of affected pigs but also with regard to the clinical scores. all pigs reacted severely. the mean clinical scores after the second lps administration are presented in table . non-infected pigs had recovered at the time of the second lps administration, h after the first one. this second lps administration again induced general signs within h, but no respiratory signs (fig. ) . prrsv-infected pigs had not yet recovered h after the first lps administration (fig. ) . the second lps administration however increased the number of pigs with general and respiratory signs and mean clinical scores (fig. ) . here again, mean general and respiratory scores were significantly higher in prrsv-lps exposed pigs than in singly lps exposed pigs. total bal cell numbers and differentials are shown in table . bal cell numbers and differentials in prrsv-lps exposed pigs were essentially similar to those of pigs, exposed to prrsv or lps only. however, there was great individual variation within all three groups. mean prrsv titres in lungs and bal fluids are shown in table . virus titres were similar in lungs and bal fluids of singly prrsv-inoculated pigs and prrsv-lps exposed pigs. the lungs and bal fluids of lps controls and non-inoculated controls were negative for prrsv. bacterial culture of lung tissue yielded negative results for all pigs. it has become generally accepted that prrsv plays an important role in respiratory disease problems in the field, particularly in multi-factorial respiratory disease. however, it has been most difficult to reproduce respiratory signs in experimental infection studies with prrsv and a second infectious agent. the present prrsv-lps combination induces clear respiratory signs in % of the pigs. unlike in our previous studies with prrsv-siv table bal cell study of prrsv-lps exposed pigs and their controls at - h after a second lps administration table virological study of lungs and bal fluids of prrsv-lps exposed pigs and their controls at days after prrsv inoculation exposure number of pigs mean prrsv titres (range) lungs (log tcid /g) bal fluids (log tcid /ml) prrsv-lps . ( . - . ) . ( . - . ) prrsv only . ( . - . ) . ( . - . ) lps only negative negative none negative negative and prrsv-prcv combinations (van reeth et al., ) , mean clinical scores were higher than those of control pigs in every experiment. only two out of the prrsvinfected pigs did not develop respiratory signs upon lps exposure. it is important to mention, however, that individual variation in disease severity is unavoidable with respiratory pathogens. such an individual variation has even been reported in experimental infection studies with primary respiratory pathogens such as actinobacillus pleuropneumoniae (baarsch et al., ) or siv (van reeth et al., ) . we used two lps administrations with the purpose to extend the duration of clinical signs. the clinical effect of a second lps administration was dependent on the time interval between the two lps administrations. in non-infected pigs, a second lps administration at a h interval caused milder clinical signs than the first one. on the other hand, a second lps administration within a h interval seriously aggravated and prolonged general and respiratory signs. these observations suggest that two lps administrations within a short time interval lead to an accumulation of lps in the lungs. indeed, it has been demonstrated that the clinicopathological manifestations of lps are strictly dose-dependent. for example, if sufficient amounts of lps are given to animals and man, cytokine induction, lung inflammation and decreased lung function are observed. slightly smaller lps doses, on the other hand, will cause only a mild lung inflammation (michel et al., ) . in prrsv-infected pigs, the clinical effect of a second lps administration was difficult to assess since pigs had not yet recovered at the moment of the second lps administration, or h after the first one. there is little information on the effect of repeated lps administrations to farm animals in the literature. it appears logical, however, that the mediators or mechanisms responsible for the clinical effects of lps may become exhausted if high lps doses are administered frequently. respiratory signs following prrsv-lps exposure could not be explained by the extent of virus replication or inflammatory changes in the lungs. indeed, virus titres were similar in prrsv-lps or singly prrsv-inoculated pigs. total bal cell numbers and neutrophil infiltration were similar in prrsv-lps or singly lps exposed pigs. also, the two prrsv-lps exposed pigs, which remained healthy, had similar bal cell profiles as their clinically affected group mates. this suggests that inflammatory changes in the lungs have little or no effect on the synergism between prrsvand lps. we hypothesize therefore that functional lung changes, such as bronchial hyper-responsiveness, are more important in the pathogenesis of prrsv-lps induced disease than structural changes. similar findings were made in a previous experimental infection study with prcv followed by lps . in this study, disease development was tightly correlated with lung production of proinflammatory cytokines, among which tumor necrosis factor-alpha (tnfa). interestingly, tnf-a has been shown to cause bronchial hyper-responsiveness in laboratory animal models (kips et al., ; thomas et al., ) . under field circumstances, most pigs become infected with prrsv at growing age and they are continuously exposed to airborne endotoxins. also, during gram-negative infections of the lungs, excessive amounts of endotoxins are released locally. the present prrsv-lps infection model therefore is relevant for the study of prrsv-induced respiratory problems in the field. the synergism was observed in % of the pigs. so, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units pathophysiologic correlates of acute porcine pleuropneumonia effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, bordetella bronchiseptica, or a combination of both organisms in pigs porcine reproductive and respiratory syndrome: neb- prrsv infection did not potentiate bacterial pathogens porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to prrsv interaction between streptococcus suis serotype and porcine reproductive and respiratory syndrome virus in specific pathogenfree piglets pattern of infection with the porcine reproductive and respiratory syndrome virus on swine farms in belgium natural release of virulence factors in membrane vesicles by pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release tumor necrosis factor causes bronchial hyperresponsiveness in rats effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs influence of antibiotic and e monoclonal immunoglobulin m interactions on endotoxin release from escherichia coli and pseudomonas aeruginosa diagnosis of porcine reproductive and respiratory syndrome dose-response relationship to inhaled endotoxin in normal subjects endotoxins tumor necrosis factor-a increases airway responsiveness and sputum neutrophilia in normal human subjects dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study broncho-alveolar interferon-a, tumor necrosis factor-a, interleukin- and inflammation during acute influenza in pigs: a possible model for humans? a potential role for tumour necrosis factor-a in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs mystery swine disease in the netherlands: the isolation of lelystad virus synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine respiratory health status in swine producers relates to endotoxin exposure in the presence of low dust levels this work was supported by grant a from the belgian ministry of agriculture. the authors would like to thank fernand de backer, krista de winne, lieve sys, chantal vanmaercke and carla de winter for excellent technical assistance. geoffrey labarque was supported by grant o d from the research council of the ghent university. key: cord- -fxgx c v authors: di martino, barbara; di felice, elisabetta; ceci, chiara; di profio, federica; marsilio, fulvio title: canine kobuviruses in diarrhoeic dogs in italy date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: fxgx c v canine kobuviruses (cakvs) are newly recognized picornaviruses recently detected in dogs in the us. by molecular analysis of the whole genome, cakv that appeared genetically closest to the murine kobuvirus (mukv) and to the human aichi virus (aiv), may be classified in the kobuvirus genus as new genotype (cakv type ) within the species aichivirus a. to date, there are no information on the epidemiology of these novel viruses in other continents. in this study, by screening a collection of dog fecal samples either from diarrhoeic or asymptomatic animals, cakv was identified in six specimens with an overall prevalence of . % ( / ). all the positive dogs presented diarrhea and were found to be infected by cakv alone or in mixed infections with canine coronavirus (ccov) and/or canine parvovirus type (cpv- ). by molecular analysis of the partial d gene, all the strains detected displayed a close relatedness with the cakvs recently identified in the us. this study provides evidence that cakvs circulate in diarrhoeic dogs in italy and are not geographically restricted to the north american continent, where they were first signaled. picornaviridae are small non-enveloped viruses of approximately - nm in diameter with single stranded, positive-polarity rna genomes of . (knowles et al., ) , aichivirus a (formerly human aichi virus) (yamashita et al., ) , aichivirus b (formerly bovine kobuvirus) (yamashita et al., ) and aichivirus c (porcine kobuvirus) (reuter et al., ) . novel kobu-like viruses have been identified in sheep, goats, wild-boars, rodents and dogs (reuter et al., ; barry et al., ; kapoor et al., ; li et al., ; phan et al., ; lee et al., ; carmona-vicente et al., ; reuter et al., ) . human aichi virus (aiv) was first recognized in as the cause of oyster-associated nonbacterial gastroenteritis in humans in aichi prefecture, japan (yamashita et al., ) . several investigations worldwide have revealed that aivs are involved in . - . % of sporadic cases of pediatric gastroenteritis (ambert-balay et al., ; reuter et al., b; kaikkonen et al., ; jonsson et al., ) . canine kobuviruses (cakvs) have been recently discovered in dogs in the us (kapoor et al., ; li et al., ) , demonstrating for the first time that pets can be infected with members of this genus. based on the sequence analysis of the complete genome, cakv was found to be genetically closest to the recently identified mouse veterinary microbiology ( ) - a b s t r a c t canine kobuviruses (cakvs) are newly recognized picornaviruses recently detected in dogs in the us. by molecular analysis of the whole genome, cakv that appeared genetically closest to the murine kobuvirus (mukv) and to the human aichi virus (aiv), may be classified in the kobuvirus genus as new genotype (cakv type ) within the species aichivirus a. to date, there are no information on the epidemiology of these novel viruses in other continents. in this study, by screening a collection of dog fecal samples either from diarrhoeic or asymptomatic animals, cakv was identified in six specimens with an overall prevalence of . % ( / ). all the positive dogs presented diarrhea and were found to be infected by cakv alone or in mixed infections with canine coronavirus (ccov) and/or canine parvovirus type (cpv- ). by molecular analysis of the partial d gene, all the strains detected displayed a close relatedness with the cakvs recently identified in the us. this study provides evidence that cakvs circulate in diarrhoeic dogs in italy and are not geographically restricted to the north american continent, where they were first signaled. ß elsevier b.v. all rights reserved. kobuvirus m- /usa/ ( . % amino acid [aa] identity) (phan et al., ) and to human aivs ( . % aa identity). according to the criteria established by the international committee on taxonomy of viruses (ictv) (http://www.picornastudygroup.com/definitions genus_ definition.htm), cakv may be considered a distinct genotype (cakv type ) from both murine kobuvirus (mukv type ) and human aiv (aiv type ), within the species aichivirus a (knowles et al., ) . the detection of cakvs in dogs raises interesting questions regarding the distribution, the pathogenic role and, given the interactions between pets and humans, the zoonotic potential. to date, the newly described cakvs were detected from healthy and diarrhoeic dogs in the us (kapoor et al., ; li et al., ) . however, there is no additional information on the epidemiology of these novel kobuviruses in other countries of the world. to further investigate the presence of cakvs in dogs, a surveillance study was initiated by testing fecal specimens from animals with and without diarrhea. a total of fecal samples from dogs aged - months were collected from december to november in municipal shelters and veterinary clinics located in different areas of central italy. the fecal panel consisted of samples of dogs with signs of mild to severe gastroenteritis and from asymptomatic dogs. fecal specimens were placed in isothermal boxes using ice bags and transferred in the lab. samples were kept frozen at À c until tested. the rna was extracted from ml of % (w/v) fecal suspension by using the trizol ls (invitrogen, ltd., paisley, uk) procedure. the final rna pellet was resuspended in ml of rnase free water and used directly in rt-pcr assays or stored at À c. viral dna was extracted from the supernatants of fecal homogenates by boiling for min and chilling on ice. to reduce residual inhibitors of dna polymerase activity to ineffective concentrations, the dna extracts were diluted : in distilled water as previously described (decaro et al., ) . kobuvirus rna was assessed by rt-pcr using a broadly reactive primer pair, univ-kobu-f/univ-kobu-r which amplifies a -bp fragment of the d gene of all kobuvirus species, following the reaction conditions previously described (reuter et al., a) . cakv specific primers (cakv/f: -ccctggaacac-ccaaggccgct- , corresponding to nucleotides [nts] - of the strain cakv/us-pc and cakv/r: -tctggttgccatagatgtggtg- , corresponding to nts - of cakv/us-pc ), targeting a -bp fragment at the d region, were designed by visual inspection of an alignment containing the sequences obtained in this study and the corresponding conservative regions of the cakv and aiv-like sequences available on ncbi website. rt and pcr were performed in one-step procedure, using the superscript iii one-step system (invitrogen, ltd., paisley, uk). the rt-pcr conditions were c for min, c for min, and cycles of c for s, c for min and c for s, with a final extension of c for min. all the amplicons were purified by using a qiaquick gel extraction kit (qiagen gmbh, hilden, germany) and subjected to direct sequencing using bigdye terminator cycle chemistry and dna analyzer (applied biosystems, foster, ca). basic local alignment search tool (blast; http://www.ncbi.nlm.nih.gov) with default values was used to find homologous hits. phylogenetic analysis with bootstrap statistical support ( replicates) was conducted using the mega software package, version . (kumar et al., ) . the cakv sequences detected in this study were made available in genbank under the accession numbers kc -kc . samples were screened for cpv- by pcr amplification of a bp segment at the carboxy terminus of cpv- open reading frame using primers and reaction conditions described by buonavoglia et al. ( ) . detection of ccov was carried out by hemi-nested rt-pcr using external primers ccv /ccv and the internal primer ccv targeting a bp fragment of the m gene, as previously described (pratelli et al., a) . out of samples, ( . %) were found to be positive for kobuvirus rna using the primer set univ-kobu-f/ univ-kobu-r. by fasta and blast analysis, the -bp d fragments displayed the closest identity ( . - . % nt) to the cakv-like sequences available on the databases. a total of six samples ( . %), included those amplified with the generic primers, were positive to cakvs, when rescreening the specimens using the specific primer set designed in this study. all cakv-positive samples were identified from diarrhoeic dogs ( / ), with a prevalence rate of . %. fecal samples were also tested for the presence of cpv- and ccov. cpv- was detected in a total of samples, all collected from animals with gastroenteritis signs ( . %), while ccov was found in specimens either from healthy ( . %) or from symptomatic dogs ( . %) ( table ) . mixed infections with two pathogens were identified in a total of samples (cakv + ccov, n = ; cpv- + ccov, n = ) and were positive for all three viruses. cakv was the single identified enteric virus in two specimens (table ) . upon sequence analysis of the partial d region with primers cakvf/cakvr, the six strains detected in this study (cakv/ c/ (kapoor et al., ; li et al., ) . furthermore, a high sequence match ( . % nt and . % aa identities) was found to a novel kobuvirus (kov-sewktm), recently detected in untreated sewage in nepal (ng et al., ) . nucleotide identity to the mukv m /usa/ (phan et al., ) was . % ( . % aa), while identity to human aiv-like sequences ranged from . % to . % ( . - . % aa). neighbor-joining phylogenetic analysis based on the -nt d sequences was carried out with a selection of strains representative of the kobuvirus genus, including kov-sewktm (ng et al., ) , mukv-m (phan et al., ) , aivs and the prototype strains porcine kobuvirus s- -hun (reuter et al., ) and bovine kobuvirus u (yamashita et al., ) . based on the inspection of the tree (fig. ) , all the italian sequences formed a tight cluster with the cakvs recently identified in the us (kapoor et al., ; li et al., ) sharing, in accordance with the pairwise identity detected in the partial d gene, a common root with the kobuvirus strain detected in sewage, the murine kobuvirus and aivs. to our knowledge, this is the first study in which evidence was collected for the occurrence of cakvs in italy. using a combination of generic and specific primers, cakv strains were identified in a samples collection obtained from young dogs. the novel kobuviruses were detected exclusively in diarrhoeic animals with a prevalence rate similar to that previously found in the us (li et al., ) . in our analysis, the majority of cakv positive dogs were also infected by other canine pathogens such as cpv- or ccov, whilst single infections by cakv were detected only in two of the symptomatic animals. these findings are not unexpected, as mixed infections are not infrequent, especially in animal communities such as kennels or shelters. although these results demonstrated that cakvs are common viruses of dogs, it is not clear whether they also play a role as enteric pathogens. furthermore, in the prevalence study performed on a mostly adult canine population of the us, cakvs was detected by real time rt-pcr not only in symptomatic dogs, but even in healthy animals (li et al., ) . accordingly, experimental infections are necessary to elucidate the pathogenic role of cakv and to understand whether it can act by itself as a primary causative agent of gastrointestinal disease or whether it can be involved in mechanisms of co-infection with other pathogens, as observed between ccov and cpv- (appel, ; pratelli et al., b) . sequence and phylogenetic analyses of the partial d gene, allowed us to obtain information on the genetic relationship among the various members of the kobuvirus genus. the six sequences detected in this study revealed little genetic variation in the region analyzed suggesting limited strain heterogeneity of cakvs. however, further characterization of these strains was not possible, since no other sequence data were obtained, but efforts are presently being made to attempt additional genome information. the close genetic relationship observed among aivs, mukv and cakvs reinforces the notion that the evolution of aivs is intermingled with that of animal kobuviruses (phan et al., ) and highlights the need to further explore the genetic diversity of these viruses in animals and humans. in conclusion, the findings of this investigation demonstrate that cakvs are not geographically restricted to the north american continent, where they were first signaled (kapoor et al., ; li et al., ) . extensive epidemiologic surveillance and comprehensive characterization of these viruses from other geographic areas might help clarify the global distribution, heterogeneity and possible association of cakv with enteric diseases in dogs. . phylogenetic tree based on the -nt d sequence. phylogenetic tree was generated using the neighbor joining method and kimura- parameters distance correction, supplying a statistical support with bootstrapping of replicates. labels denote cakv-like sequences detected in the present study. furthermore, large age-stratified serological investigations are necessary in order to assess precisely the patterns of canine seroprevalence. finally, given the close genetically relatedness of these novel kobuviruses with aivs, investigations in humans will be useful to address their zoonotic potential. prevalence and genetic diversity of aichi virus strains in stool samples from community and hospitalized patients does canine coronavirus augment the effects of subsequent parvovirus infection? first detection of kobuvirus in farm animals in brazil and the netherlands antigenic analysis of canine parvovirus strains isolated in italy phylogeny and prevalence of kobuviruses in dogs and cats in the uk a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs aichi virus infection in elderly people in sweden aichi virus infection in children with acute gastroenteritis in finland characterization of a canine homolog of human aichi virus virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses mega : integrated software for molecular evolutionary genetics analysis and sequence alignment kobuvirus in south korean black goats viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses high variety of known and new rna and dna viruses of diverse origins in untreated sewage the fecal viral flora of wild rodents development of a nested pcr assay for the detection of canine coronavirus fatal coronavirus infection in puppies following canine parvovirus b infection candidate new species of kobuvirus in porcine hosts complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus kobuvirus, family picornaviridae detection of aichi virus shedding in a child with enteric and extraintestinal symptoms in hungary kobuvirus in domestic sheep porcine kobuvirus in wild boars (sus scrofa) isolation and characterization of a new species of kobuvirus associated with cattle isolation of cytopathic small round viruses with bs-c- cells from patients with gastroenteritis we thank the dr. roberto speranza and dr. roberta campanella for their technical support during the fecal samples collection. the work was financed by grants from the university of teramo, italy, and from the italian ministry of university and research. all authors declare that there are no financial or other relationships that might lead to a conflict of interest. all authors have seen and approved the manuscript and have contributed significantly to the work. key: cord- -rv efgg authors: decaro, nicola; martella, vito; elia, gabriella; desario, costantina; campolo, marco; lorusso, eleonora; colaianni, maria loredana; lorusso, alessio; buonavoglia, canio title: tissue distribution of the antigenic variants of canine parvovirus type in dogs date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: rv efgg twelve dogs dead as consequence of natural infection caused by canine parvovirus (cpv) type a (n = ), type b (n = ) or type c (n = ) were investigated for determining the viral dna loads in different tissue samples. by means of a real-time pcr assay, cpv dna was detected in all tissues examined, with the highest titres observed in the lymphoid tissue and the lowest loads in the urinary tract. surprisingly, the nervous tissue was found to contain considerable amounts of cpv nucleic acid. similar patterns of tissue distribution were observed in all the examined dogs irrespective of the antigenic variant causing the disease. parvovirus was first described as a clinical entity causing haemorrhagic gastroenteritis in dogs in (appel et al., ) and the aetiological agent was named canine parvovirus type (cpv- ) to distinguish it from the antigenically unrelated virus, the canine parvovirus type (cpv- ), also known as minute virus of canines. virus infection spread rapidly worldwide causing high rate of mortality in pups (carmichael and binn, ) . the dogs are infected through the oronasal route and after - days they develop an acute gastroenteritis characterised by loss of appetite, vomiting, fever, diarrhoea (from mucoid to haemorrhagic) and leukopenia. cpv- contains a single-stranded dna genome of about nucleotides, enclosed in an icosahedral capsid made up of two proteins, vp and vp . amino acids substitutions in vp sequence are known to cause changes in genetic and antigenic properties www.elsevier.com/locate/vetmic veterinary microbiology ( ) - (parrish, ; truyen et al., truyen et al., , . cpv- belongs to the feline parvovirus (fpv) subgroup of the genus parvovirus, together with feline panleukopenia virus (fplv), mink enteritis virus (mev), raccoon parvovirus (rpv), raccoon dog parvovirus (rdpv) and blue fox parvovirus (bfpv) (berns et al., ) . its origin is unknown, but it was suggested that it arose as a mutant from fplv in dog or cat populations or from related viruses in wild carnivores truyen, truyen, , . after the first outbreak of cpv- infection, there were consecutive emergences of new antigenic variants of this virus, designated type a and b (parrish et al., . starting from and in a relatively short period, these variants replaced the original type worldwide in dogs (mochizuki et al., ; de ybanez et al., ; greenwood et al., ; truyen et al., truyen et al., , sagazio et al., ; steinel et al., ; buonavoglia et al., ; pereira et al., ) . cpv- a and cpv- b differ from the original type cpv- by amino acid changes affecting the vp protein and by their extended host range which includes canine and feline cells in vitro and dogs and cats in vivo. subsequently, further mutations in the cpv capsid protein have been described in several countries, but there is no evidence for a further spread of these mutants (ikeda et al., ) . analysis of italian cpv isolates revealed the onset in of an unusual cpv- mutant with a change (asp ! glu) occurring in the strategic residue of the capsid (buonavoglia et al., ) . the glu- mutant, also detected in other countries (nakamura et al., ; decaro et al., d) , is widely distributed in italy and is currently replacing cpv- b in the italian dog population desario et al., ; decaro et al., d decaro et al., ,e, b . in contrast with other mutations previously reported, such as asp- and pro- , the mutation at residue has probably provided the mutant with an evolutionary benefit. we are currently investigating whether this change really represents an advantage for viral spread and whether it has biological consequence (decaro et al., c) . since the mutation asp glu affects the major antigenic region, that has been taken into account for classification of the variants a and b (parrish et al., , the glu- mutant has been referred to as new antigenic variant c (decaro et al., c (decaro et al., , b ). molecular methods have been developed to quantify the viral loads in the faeces of dogs infected with cpv- (decaro et al., e) , characterise the cpv variants (decaro et al., b) and discriminate between vaccine and field strains (decaro et al., a,c) . in this study, by using real-time pcr, we investigated the pattern of distribution of the cpv variants in different tissues of dogs infected naturally. the carcasses of twelve - -month-old dogs, that had died as a consequence of single cpv infection within - days after the onset of the clinical signs, were selected from a previous study (decaro et al., b) . all dogs were mixed-bred and had been housed in different animal shelters of italy until the occurrence of disease or death. by using real-time pcr assays based on minor groove binder (mgb) probe technology (decaro et al., b) , the dogs were found to be infected by cpv- a (n = ), cpv- b (n = ) or cpv- c (n = ). traditional and molecular methods ruled out co-infections by other common pathogens of dogs, such as bordetella bronchiseptica, pasteurella multocida, leptospira interrogans (gravekamp et al., ) , reoviruses (leary et al., ; decaro et al., b) , rotaviruses (gouvea et al., ) , caliciviruses (jiang et al., ; marsilio et al., ) , canine adenoviruses (hu et al., ) , canine distemper virus (elia et al., ) , canid herpesvirus (schulze and baumgartner, ) , canine coronavirus (ccov) (decaro et al., ) . faecal samples from the rectal ampulla and tissue samples were collected from the dead dogs, including brain, cerebellum, cerebral bulb, tonsils, retropharyngeal and mesenteric lymph nodes, thymus, myocardium, lungs, liver, spleen, kidneys, bladder, bone marrow, jejunum, colon, rectum. each tissue sample was withdrawn using disposable sterile scalpels. faecal samples were homogenised ( %, w/v) in phosphate buffered saline (pbs, ph . ) and subsequently clarified by centrifuging at  g for min. dna was extracted from faecal homogenates by boiling for min and chilling on ice (schunck et al., ; uwatoko et al., ) , whereas nucleic acid purification from tissue samples was achieved by using the dneasy tissue kit (qiagen s.p.a., milan, italy), followed by final elution of dna with ml of ae buffer and storage at À c until use. to reduce residual inhibitors of dna polymerase activity to ineffective concentrations, the dna extracts from both faecal and tissue samples were diluted : in distilled water (decaro et al., e) . the dna extracts were tested by a taqman assay able to recognise all the cpv- strains (decaro et al., e) . the assay is internally controlled by using as exogenous dna the nucleic acid extracted from ovine herpesvirus type (decaro et al., ) . the reaction of real-time pcr ( ml) contained . ml of master mix (bio-rad laboratories srl, milan, italy), nm of primers ( -aaacaggaattaactatactaa-tatattta- ) and cpv-rev ( -aaatttgac-catttggataaact- ), nm of probe cpv-pb ( -tggtcctttaactgcattaaataatgtacc- ) and ml of standard or template dna. all standard dilutions and unknown samples were tested in duplicate. the following thermal protocol was used: activation of itaq dna polymerase at c for min and cycles consisting of denaturation at c for s, primer annealing at c for s and extension at c for min. the viral dna titres found in the different tissues of the dogs infected naturally by types a, b and c are reported in fig. . cpv- dna was demonstrated in all tissues analysed, showing a very wide distribution of the virus in the organism. the highest viral loads were detected in the lymphoid tissues, with maximal titres observed in the tonsils of dogs infected with cpv- c (median titre = .  dna copies/ ml of template) and in the spleen of dogs infected with cpv- b (median titre = .  dna copies/ ml of template). very high dna titres were observed in the bone marrow (median titre of .  in dogs infected with cpv- a). the urinary tract was found to contain the lowest cpv- dna amounts, with median titres of about dna copies/ ml of template in the bladder of all examined dogs. surprisingly, viral dna was detected also in the nervous tissues, including brain, cerebellum and cerebral bulb, with median titres above dna copies/ ml of template in all dogs. faecal samples contained lower viral quantities than internal organs, with titres ranging between .  and .  dna copies/ ml of template for types c and a, respectively. cpv infects dogs through the oronasal route and reaches the intestinal mucosa after an initial spread to lymphoid tissues (appel and parrish, ) . viraemia may reach very high titres of viral dna and persist for several weeks, even after the virus has disappeared from the intestinal content (decaro et al., unpublished data) . in this study, all tissues analysed were shown to contain cpv dna, probably as a consequence of viral spread in the organism through the blood. the number of dogs was limited, with only four dogs analysed per each of the three cpv variants. in fact, in order to limit the variability of viral titres due to different ages, breeds and clinical courses of the disease, only pups were chosen that were mixed-bred, had approximately the same age and had died after a similar duration of disease ( - days) as a consequence of single cpv infection. moreover, it was difficult to recruit dogs dead due to infection with cpv- b, since this variant is disappearing progressively from the italian dog population decaro et al., c decaro et al., ,e, a , and only one type b strain has been detected in italy in (decaro et al., unpublished data) . tissue distribution of cpv was found to have similar patterns in dogs infected by types a, b and c, revealing that the variants have the same biological behaviour. parvovirus replication in dogs and cats takes place mainly in highly mitotically active tissues, such as bone marrow, lymphoid organs and intestinal crypts (appel and parrish, ) . involvement of the nervous tissues has been described in cats (csiza et al., ; wilcox et al., ; url et al., ) , whereas in dogs cpv antigen has been never detected in neurons, despite the presence of neurodegeneration (agungpriyono et al., ; url and schmidt, ) . in contrast with previous studies, we have demonstrated the presence at high titres of cpv nucleic acid in all tissues examined including brain, cerebellum and bulb. whether the presence of viral dna is associated to effective replication and expression of viral proteins in neurons should be assessed by identification of cpv antigens using supplementary techniques, such as immunohistochemistry. cpv dna titres detected in the faeces were generally lower than those observed in lymphoid organs. it has been shown previously that shedding of cpv dna in the faeces reaches maximal loads in the first days after infection (with a peak at - days postinfection), with a rapid decrease already at - days post-infection (decaro et al., a; elia et al., ) . moreover, in the late stage of infection the high antibody levels in the gut lumen may sequestrate most of the virions, so that traditional tests, such as haemagglutination and virus isolation, give frequently false negative results (decaro et al., e; desario et al., ) . the results of the present work indicate that in laboratories where molecular methods are not employed routinely, traditional diagnostic methods for detection of cpv in dead dogs should be carried out on the internal organs rather than on the faeces or intestinal contents. subacute massive necrotizing myocarditis by canine parvovirus type infection with diffuse leukoencephalomalacia in a puppy isolation and immunization studies of canine parvo-like virus from dogs with haemorrhagic enteritis canine parvovirus type family parvoviridae evidence for evolution of canine parvovirus type- in italy antigenic analysis of canine parvovirus strains isolated in italy new enteric viruses in the dog respiratory signs and central nervous system lesions in cats infected with panleukopenia virus. a case report identification of types of canine parvovirus circulating in spain first two confirmed cases of malignant catarrhal fever in italy quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr maternally-derived antibodies in pups and protection from canine parvovirus infection virological and molecular characterization of a mammalian orthoreovirus type strain isolated from a dog in italy clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant new approaches for the molecular characterization of canine parvovirus type strains a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs a minor groove binder probe real-time pcr assay for discrimination between type -based vaccines and field strains of canine parvovirus characterisation of the canine parvovirus type variants using minor groove binder probe technology diagnostic tools based on minor groove binder technology for rapid identification of vaccine and field strains of canine parvovirus type b first detection of canine parvovirus type c in pups with haemorrhagic enteritis in spain canine parvovirus infection: which diagnostic test for virus? antibody levels and protection to canine parvovirus type detection of canine distemper virus in dogs by real-time rt-pcr identification of bovine and porcine rotavirus g types by pcr detection of seven species of pathogenic leptospires by pcr using two sets of primers comparison of isolates of canine parvovirus by monoclonal antibody and restriction-enzyme analysis detection and differentiation of cav- and cav- by polymerase chain reaction predominance of canine parvovirus (cpv) in unvaccinated cat populations and emergence of new antigenic types of cpvs in cats design and evaluation of a primer pair that detects both norwalk-and sapporo-like caliciviruses by rt-pcr detection of mammalian reovirus rna by using reverse transcription-pcr: sequence diversity within the l -encoding l gene nested pcr for the diagnosis of calicivirus infections in the cat a canine parvovirus mutant is spreading in italy surveillance activity for canine parvovirus in italy antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in japan a novel antigenic variant of canine parvovirus from a vietnamese dog mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones natural variation of canine parvovirus rapid antigenic-type replacement and dna sequence evolution of canine parvovirus molecular characterisation of canine parvovirus in brazil by polymerase chain reaction assay antigenic characterization of canine parvovirus strains isolated in italy nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies a simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces antigenic and genetic analysis of canine parvoviruses in southern africa emergence and recent evolution of canine parvovirus evolution of canine parvovirus-a need for new vaccines? characterization of the feline host range and a specific epitope of feline panleukopenia virus evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo antigenic type distribution among canine parvoviruses in dogs and cats in germany survey on viral pathogens in wild red foxes (vulpes vulpes) in germany with emphasis on parvoviruses and analysis of a dna sequence from a red fox parvovirus distribution of antigenic types of canine parvovirus in switzerland, austria and germany evidence of parvovirus replication in cerebral neurons of cats do canine parvoviruses affect canine neurons? an immunohistochemical study rapid method utilizing the polymerase chain reaction for detection of canine parvovirus in feces of diarrhoeic dogs recovery of viral agents from the central nervous system of cats key: cord- -s t qxj authors: soliman, h.; el-matbouli, m. title: reverse transcription loop-mediated isothermal amplification (rt-lamp) for rapid detection of viral hemorrhagic septicaemia virus (vhs) date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: s t qxj a one step reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for detection of viral hemorrhagic septicaemia virus (vhs). a set of six primers were designed, based on the g-protein sequence of the vhs virus serotypes (he, f , . , klapmolle and rindsholm). the assay was optimised to amplify vhs rna by incubation at °c for only h, and required only a simple water bath or heating block to provide a constant temperature of °c. rt-lamp amplification products were detected by visual inspection using sybr green i stain and had a ladder-like appearance when electrophoresed on an agarose gel. the detection limit of the rt-lamp assay was found to be similar to the commonly used rt-pcr method: both methods detected vhs rna at a dilution of ( ). the assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for vhs virus. reverse transcription loop-mediated isothermal amplification (rt-lamp) for rapid detection of viral hemorrhagic septicaemia virus (vhs) animal rhabdoviruses, belonging to the family rhabdoviridae, infect a broad range of hosts throughout the animal kingdom, including insects, mammals and fish. aquatic rhabdoviruses cause significant diseases in fish reared as part of the worldwide salmonid farming industry (rocha et al., ) . among fish rhabdoviruses, viral hemorrhagic septicaemia virus (vhs), of the genus novirhabdovirus, often causes pronounced cumulative mortality of salmonids, especially rainbow trout oncorhynchus mykiss (miller et al., ) . the virus is enzootic in much of continental europe (wolf, ) and has been found in north america (king et al., ; snow and smail, ) . vhs is a notifiable disease, included in list ii of the european union directive / ( ), and is known by several names including egtved disease and infectious kidney-swelling and liver degeneration. www.elsevier.com/locate/vetmic veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the virus' aetiology was established by jensen ( ) , and it is known to infect primarily rainbow trout, oncorhynchus mykiss, brown trout, salmo trutta, and to a lesser extent northern pike, esox lucius (jorgensen, ; meier and jorgensen, ) , grayling, thymallus thymallus, and whitefish, coregonus sp. (wizigmann et al., ; ahne and thomsen, ; meier et al., ) . the virus has also been isolated from free-living marine fish in the northern pacific and atlantic oceans, the north sea and the baltic sea (meyers and winton, ; mortensen et al., ) . virus isolates from wild marine fish are serologically indistinguishable from normal fresh water isolates (jorgensen and olesen, ) . vhs virus is transmitted by direct contact with either infected fish or contaminated water (vestergard jorgensen, ) . survivors from infection become carriers and shed the virus with urine and sexual fluids (neukirch, ) . although the virus can be present on the surface of eggs, it is readily dissipated and there is no true vertical transmission (vestergard jorgensen, ) . vhs infection in susceptible fish species is often lethal, due to impairment of osmotic balance; clinical signs include oedema and red haemorrhagic spots around the eyes and on the gills, and blood clots in body fat, viscera and muscle (wolf, ) . rapid diagnosis of vhs is necessary to prevent further spread of the disease (miller et al., ) . serological tests have been used to diagnose vhs virus, and include the virus neutralisation test (jorgensen, (jorgensen, , , a fluorescent antibody technique (jorgensen, ; enzmann, ) and enzyme linked immunosorbent assay (elisa) (olesen and jorgensen, ). yet serological tests along with other traditional means of diagnosing viral infection, including epizootiological data, direct light or electron microscopic observation and isolation of the virus in cell culture, are often time-consuming and expensive (amos, ; sanz and coll, ) . recently, molecular biological techniques have been developed to improve diagnosis of fish pathogens by rapid and sensitive detection of pathogen rna and dna. a reverse transcriptase-polymerase chain reaction (rt-pcr) has been developed to detect vhs (bruchhof et al., ; einer-jensen et al., ; miller et al., ; strommen and stone, ; guillou et al., ; williams et al., ) . however, diagnosis of vhs using this pcr test requires expensive equipment and reagents, rendering it unfavourable for use on a large-scale basis, or onsite. a novel, rapid and sensitive technique called loop-mediated isothermal amplification (lamp) has been developed by notomi et al. ( ) and is capable of amplifying dna or rna under isothermal conditions with high specificity and efficiency. lamp is based on the principle of autocycling strand displacement dna synthesis. the reaction is performed by a dna polymerase with high strand displacement activity and a set of two specific inner primers and two outer primers (notomi et al., ) . a reverse transcriptase coupled lamp assay has been used to detect west nile virus and for rapid detection of sars (severe acute respiratory syndrome) coronavirus (thai et al., ) . the aim of the present study was to develop a onestep, single-tube, accelerated rt-lamp reaction for rapid detection of different serotypes of vhs virus. the specificity and sensitivity of the method were assessed, and its applicability as a diagnostic test was evaluated. vhs virus serotypes (he, klapmolle, rindsholm, and . ) were kindly provided by dr. fichtner, federal research institute for animal health, insel riems, germany. the vhs virus serotype (f ) and infectious hematopoietic necrosis virus (ihnv) were provided by dr. widemann, department of virology, central institute of animal health, bavaria, germany. fish tissues suspected of being infected with vhs (spleen, kidney and brain) were preserved in rna later. these were then processed by grinding thoroughly in liquid nitrogen with a mortar and pestle. twenty milligram of tissue powder was then placed into a rnase-free, liquid nitrogen-cooled ml microcentrifuge tube with ml lysis buffer. the lysate was transferred directly into a qiashredder spin column (qiagen gmbh, hilden, germany) and centrifuged at maximum speed ( ,  g) for min. one volume ( ml) of % ethanol was added to the supernatant and extraction completed as per the manufacture's instructions. rna was eluted in rnase-free water ( c) and stored at À c. genomic viral rna was extracted from ml of infected culture supernatant using the qiaamp viral rna kit (qiagen gmbh, hilden, germany) according to the manufacture's instructions. after elution of the rna in elution buffer, it was stored at À c until required. six lamp primers were designed, based on the gprotein sequence of the vhs virus serotypes: f , klapmolle, rindsholm, he and . (genbank accession number af , af , af , u and u , respectively). the forward inner primer, fip, consisted of complementary sequence of f ( nt), a tttt linker and the sense sequence of the f ( nt): -gatccaccga-tactgtttttggggttttcccgttcttc cctg aaccc- . the backward inner primer, bip, contained a sense sequence of b ( nt), a tttt linker and the complementary sequence of the b ( nt): -argggg tytgcacarcctcgc tttt cgack-ygggrcaakgggc- . the outer primers consisted of the sense sequence of f ( ) -ggsaagcaaggaycacgag- ; and complementary sequence of b ( nt) -caggtgtc-cytctagtgtttc- . to accelerate amplification, two loop primers were used: a loop forward primer (loop-f, nt): -gttatgtccttatggacattg- ; and a loop back-ward primer (loop-b; nt): -gtcaaact-cattggcaggg- . the location of the primers within the gene fragment is shown in (fig. ) . the reverse transcriptase-polymerase chain reaction used specific primers vg and vgr, which amplified a bp segment, and seminested primers vd and vd , which amplified a bp segment of vhs cdna (bruchhof et al., ) : vg : -atggaatggaacacttttttc- , vgr: -tcagaccgtctgacttctgga- ; vd : -tcccgctatcagtcaccag- ; vd : -tgtgatcatgggtcctggtg- . rt-pcr was performed using a titanium tm onestep rt-pcr kit (bd clontech, heidelberg, germany). for a ml reaction volume, mg of rna was mixed with  one-step buffer, dntps mix, recombinant rnase inhibitor, thermostabilising reagent, gc-melt, oligo (dt) primer, mm of each vg and vgr vhs specific primers, and rt-titanium tm taq enzyme mix. the reaction conditions comprised: incubation at c for h, then denaturation at c for min; followed by cycles of c for s; c for s and c for s. there was a terminal extension step of c for min. following rt-pcr, ml of the product was added to ml of semi-nested pcr reaction mixture: comprising .  reddymix pcr master mix (abgene, hamburg, germany: mm tris-hcl (ph . ), mm (nh ) so , . mm mgcl , . % tween , . mm each of datp, dctp, dgtp, dttp; . u taq dna polymerase and red dye for electrophoresis), and pmol of each vhs specific primers vd and vd . the reaction entailed initial denaturation at c for min, followed by cycles of c for s, c for s and c s. there was a terminal extension step of c for min. the lamp reaction was performed in a ml reaction volume containing: mm tris-hcl (ph . ), mm kcl, . mm mgso , mm (nh ) À so , . % triton x- , . m betaine, deoxynucleotide triphosphates . mm each, . mm each fip and bip, . mm each loop-f and loop-b, . mm each f and b primers, u bst dna polymerase (new england biolabs, gmbh, frankfurt, germany), . u avian myeloblastosis virus (amv) reverse transcriptase (invitrogen, groningen, the netherlands) and ml rna template. rna template was omitted from one reaction as a negative control. the mixture was incubated at c for min and then heated at c for min to terminate the reaction. rt-pcr and rt-lamp reaction products were analysed by gel electrophoresis with . % agarose in tris acetate-edta buffer, tae ( . m tris acetate, mm edta), stained with ethidium bromide and visualised on a uv transilluminator. a bp dna molecular weight standard ladder (cambrex bio science, inc., rockland, me, usa) was used. success of the rt-lamp reaction was also gauged by visual inspection: ml of : diluted sybr green i nucleic acid gel stain, ,  concentration in demso (cambrex bio science, rockland, inc., me, usa) was added to the reaction tube and any colour change noted. the ability of the assay to amplify only vhs rna was appraised by testing it with rna from infectious hematopoietic necrosis virus a related rhabdoviruses. rna from non-infected fish was used as a negative control to determine any non-specific amplification. ten-fold serial dilutions of the used vhs serotypes rna were tested by both rt-lamp and rt-pcr to determine the detection limits of each assay. the suitability of the assay to diagnose vhs was evaluated by comparing detection results of rt-lamp assay on experimentally infected rainbow trout with vhs serotype f (mattes, ) against rt-pcr results on the same samples. different primer concentrations, primer combinations, amplification temperatures, and amounts of reverse transcriptase were tested to determine the optimal rt-lamp conditions. optimal amplification of vhs rna was achieved by incubation of fip, bip, f , b primers, u of bst dna polymerase and u of amv reverse transcriptase enzyme with the target rna at c for h. the amplification product appeared as a ladder-like pattern (many bands of different molecular weights) on the gel (fig. ) . there was no amplification of the negative control. the assay was able to amplify all five serotypes of vhs rna (fig. ) . two additional primers, loop-f and loop-b, were subsequently added to enhance and accelerate the reaction down to min (fig. ) . there was no difference in amplification pattern using the additional primers. colour changes were noted on visual inspection of rt-lamp reaction tubes after addition of diluted sybr green i: positive samples turned green, while negative samples and no template control reactions remained orange (fig. ) . these observations agreed with gel electrophoresis results. the specificity of the vhs-lamp primers and conditions to vhs virus was confirmed by its aptitude to amplify only rna from vhs virus serotypes, with no amplification of ihnvor non-infected fish (fig. ) . amplification of -fold serial dilutions of vhs rna by rt-pcr and rt-lamp revealed that both tests could detect viral rna to a dilution of in ( figs. and ) . the same result was obtained with the all tested vhs serotypes. rt-lamp detected vhs rna from infected samples, which also tested positive by the rt-pcr test. this result indicates that the developed rt-lamp assay can feasibly be used as a diagnostic test of vhs infection. viral infections pose a serious threat to the aquaculture industry and are responsible for significant financial losses (caipang et al., ) . it is clearly important to rapidly identify and differentiate the causative agents of fish diseases to prevent further disease transmission or outbreaks (bruchhof et al., ) . vhs is one of several rhabdoviruses that fig. . agarose gel highlighting the specificity of the rt-lamp primers to vhs virus rna. the reaction was carried out at c for h using the primer set. lanes: f , he, . , klapm; and rinds, amplification products of vhs virus rna serotypes (f , he, . , klapmolle and rindsholm); ihnv, no amplification product was detected for ihn virus rna; nf, no amplification product was detected using rna from noninfected fish; mar = bp molecular weight marker. fig. . agarose gel showing rt-lamp reactions using -fold serial dilutions of template rna. the assay detected vhs rna at a dilution one in . lanes: À = template concentration À ; À = À ; À = À ; À = À ; À = À ; À = À ; À = À ; mar = bp dna molecular weight standard. affects the aquaculture industry, and causes disease with high mortality among salmonids (miller et al., ) . difficulties associated with isolation of viruses from clinical specimens has lead to development of rapid and reliable virus detection assays including many rt-pcr assays developed for diagnosis of vhs (bruchhof et al., ; einer-jensen et al., ; miller et al., ; strommen and stone, ; guillou et al., ; williams et al., ) . despite the simplicity of the rt-pcr reaction, its requirements for a high precision thermacycler and elaborate methods for detecting the amplified products have restricted its wide use as a routine diagnostic tool (thai et al., ) . to overcome these issues, notomi et al. ( ) developed a novel nucleic acid amplification assay: loop-mediated isothermal amplification. the lamp reaction has been used successfully to detect fish viral infections (caipang et al., ; gunimaladevi et al., gunimaladevi et al., , , and has been applied to the detection of vhs in the present study. the rt-lamp technique is rapid, straightforward and includes a simple visual method for detecting positive results. the reaction is executed in a single tube and only requires a simple water bath or heating block to provide a constant temperature of c for h. the lamp reaction relies on autocycling strand displacement dna synthesis and utilises a set of four specially designated inner and outer primers (notomi et al., ) which are used during the initial steps of the reaction. during the subsequent cycling reactions, only the inner primers are used for strand displacement dna synthesis . each inner primer contains two distinct sequences corresponding to the sense and antisense sequences of the target dna. the primers form stem-loop structures which initiate self-priming dna synthesis, and serve as the starting material for the subsequent lamp cycling reaction (kuboki et al., ) . to further reduce the reaction time, two additional loop primers were used to hybridise to the stem-loops formed by the outer primers. the loops hybridised by the inner primers subsequently prime strand displacement dna synthesis (nagamine et al., ) . due to the variation in the glycoprotein gene sequence of the vhs serotypes, degenerative primers were designed from an alignment of the g-protein sequences of vhs serotypes he, f , . , klapmolle and rindsholm. these primers were then aligned against g-protein sequences of ihnv serotypes, wrac, srcv and rb, to check that they would not bind; which was confirmed by testing in a reaction with ihnv rna (fig. ) . the lamp primers are suitable for amplification of at least five vhs serotypes (fig. ) . the specificity of the reaction was extremely high because it uses six primers that recognise eight distinct regions on the target dna (notomi et al., ; nagamine et al., ) . the amplified products appeared as a ladder-like pattern on the gel due to the formation of a mixture of stem-loop dna products with varying stem lengths, and cauliflower-like structures of multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand (thai et al., ) . the assay used sybr green i dna staining as a rapid, fig. . agarose gel showing vhs rt-pcr using -fold serial dilutions of template rna. the rt-pcr produced a bp amplification product and detected vhs rna at a dilution of in . lanes: À = template concentration À ; À = À ; À = À ; À = À ; À = À ; À = À ; À = À ; mar = bp dna molecular weight standard. specific method for detection of amplification products. visual detection is possible due to the high specificity and amplification efficiency of lamp (iwamoto et al., ) , and to the high binding affinity of sybr green i to dna (karlsen et al., ) . visual inspection is a superior detection method as there is no need for gel electrophoresis and staining with ethidium bromide; only ml of diluted sybr green i was required to visualise the reaction products: a distinct green colour indicated a positive result while orange indicated negative. all samples which tested positive by visual inspection were positive also when analysed by electrophoresis; there were no samples which were negative by visual examination but which tested positive by electrophoresis. the rt-lamp assay detected vhs rna at a dilution of in , which was equivalent to the limit of rt-pcr. the amplification efficiency of the lamp method was extremely high under isothermal conditions at the optimal temperature for the polymerase; any inhibition reactions at the latter stages of amplification (kalinina et al., ) were less likely to occur compared with pcr (mori et al., ) . the suitability of the rt-lamp assay for detection of vhs rna in infected samples was successfully validated. in conclusion, the developed rt-lamp assay is extremely rapid, cost effective, sensitive and specific for detection of vhs rna. the test requires only a water bath and is completed in h compared with - h for rt-pcr. considerably less time is required to obtain a result using sybr green i stain, compared with traditional gel electrophoresis. the assay is suitable to be used under field conditions for the rapid diagnosis of vhs, which would allow expedited control and hygiene measures to be implemented to prevent spread of infection. occurrence of viral hemorrhagic septicaemia virus in wild whitefish coregonus sp procedures for the detection and identification of certain fish pathogens differential diagnosis of fish pathogenic rhabdoviruses by reverse transcriptase-dependent polymerase chain reaction rapid detection of a fish iridovirus using loop-mediated isothermal amplification (lamp) use of polymerase chain reaction (pcr) to differentiate serologically similar vhs virus isolates from europe and america rapid identification of vhvs from trout by immunofluorescence detection of viral hemorrhagic septicaemia virus (vhs) in rainbow trout (oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. diagnostic validation detection of koi herpesvirus in common carp, cyprinus carpio l., by loop-mediated isothermal amplification a loop mediated isothermal amplification (lamp) method for detection of infectious hematopoietic necrosis virus (ihnv) in rainbow trout (oncorhynchus mykiss) loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples research on the virus of egtved disease serological identification of egtved virus egtved virus: antigenic variation in virus isolates examined in neutralization tests and by means of the fluorescent antibody technique egtved virus: the susceptibility of brown trout and rainbow trout to eight virus isolates and the significance of the findings for the vhs control cod ulcus syndrome rabdovirus is indistinguishable from the egtved (vhs) virus nanoliter scale pcr with taqman detection sybr green i dann staining increases the detection sensitivity of viruses by polymerase chain reaction distribution of viral hemorrhagic septicaemia virus in wild fish species of the north sea, north east atlantic ocean and irish sea loop-mediated isothermal amplification for detection of african trypanosomes untersuchungen zur empfänglichkeit zweier regenbogenforellen-stämme gegenüber tetracapsuloides bryosalmonae, yersinia ruckeri und dem viralen hämorrhagischen septikämie-virus isolation of vhs virus from pike fry (esox lucius) with hemorrhagic symptoms fish viruses: viral hemorrhagic septicaemia in whitefish (coregonus sp vhs-virus in north america rapid and sensitive reverse transcriptase polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation isolation of vhsv from wild marine fish species in the baltic sea, kattegat, shagerrak and the north sea accelerated reaction by loop-mediated isothermal amplification using loop primers uptake, multiplication and excretion of vhsv in rainbow trout loop-mediated isothermal amplification of dna rapid detection of viral hemorrhagic septicaemia virus in fish by elisa real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus conformation-and fusion-defective mutations in the hypothetical phospholipidsbinding and fusion peptides of viral hemorrhagic septicaemia salmonid rhabdovirus protein g techniques for diagnosing viral diseases of salmonid fish experimental susceptibility of turbot scophthalmus maximus to viral hemorrhagic septicaemia virus isolated from cultivated turbot detection of vhs virus in fish tissues by semi-nested polymerase chain reaction (pcr) development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of sever acute respiratory syndrome coronavirus the survival of vhs virus associated with trout eggs artificial transmission of vhs of rainbow trout multiplex reverse transcriptase pcr assay for simultaneous detection of three fish viruses isolation of viral hemorrhagic septicemia virus from fry of rainbow trout, pike, and grayling fish viruses and fish viral diseases we would like to express our grateful thanks to dr. d. fichtner and dr. bergmann, federal research institute for animal health, insel riems, germany and dr. widemann, department of virology, central institute of animal health, bavaria, germany for providing us with the vhs and ihn viruses. we also thank dr. marianne mattes for providing samples of the experimentally infected rainbow trout. key: cord- - ybfiz f authors: decaro, nicola; lorusso, alessio title: novel human coronavirus (sars-cov- ): a lesson from animal coronaviruses date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: ybfiz f the recent pandemic caused by the novel human coronavirus, referrred to as severe acute respiratory syndrome coronavirus (sars-cov- ), not only is having a great impact on the health care systems and economies in all continents but it is also causing radical changes of common habits and life styles. the novel coronavirus (cov) recognises, with high probability, a zoonotic origin but the role of animals in the sars-cov- epidemiology is still largely unknown. however, covs have been known in animals since several decades, so that veterinary coronavirologists have a great expertise on how to face cov infections in animals, which could represent a model for sars-cov- infection in humans. in the present paper, we provide an up-to-date review of the literature currently available on animal covs, focusing on the molecular mechanisms that are responsible for the emergence of novel cov strains with different antigenic, biologic and/or pathogenetic features. a full comprehension of the mechanisms driving the evolution of animal covs will help better understand the emergence, spreading, and evolution of sars-cov- . eighteen years after the emergence of severe acute respiratory syndrome (sars) in china and years after the emergence of middle east respiratory syndrome (mers) in saudi arabia, a novel coronavirus (cov) epidemic, recently classified as pandemic by the who, is threatening the human population worldwide (zhou et al., ) . the disease, now referred to as coronavirus disease , is caused by a novel human cov, which was initially denominated novel coronavirus ( -ncov) and later renamed as sars coronavirus (sars-cov- ) the by coronavirus study group of the international committee on taxonomy of viruses (gorbalenya et al., ) . covid- emerged in december in wuhan city, hubei province, china, in humans exposed to wildlife at the huanan seafood wholesale market, which is the largest seafood market in central china, and where different species of farm and wild animals are commonly sold (lorusso et al., ) . the epidemic has then expanded not only to neighbouring asian countries, but also to other continents (https://www.who.int/ docs/default-source/coronaviruse/situation-reports/ -sitrep- -covid- .pdf?sfvrsn=c ea _ ). . a list of human covs is showed in table . historically, only two human covs (hcovs) had been known before the sars emergence, namely hcov- e, an alphacoronavirus originated in bats and transmitted to humans through alpacas, and hcov-oc , a betacoronavirus which had passed from rodents to humans through cattle (corman et al., (corman et al., , . after - sars epidemic, the renovated interest in hcovs allowed the discovery of two additional viruses, the alphacoronavirus hcov-nl and the betacoronavirus hcov-hku , derived from bats and rodents, respectively (tao et al., ) . all these four viruses are usually responsible for mild respiratory symptoms in immunocompetent patients. sars-cov and mers-cov are two unrelated betacoronaviruses originated in bats and transmitted to humans by wild carnivores and dromedary camels, respectively. in contrast to other hcovs, these two viruses displayed an increased virulence, causing severe pneumonia and even the death of affected people, with mortality rates of about % and %, respectively (guarner, ) . the occurrence of three highly pathogenic covs with a zoonotic origin in less than two decades, highlights the role of animals in generating covs with increased virulence that can adapt to humans, causing epidemics (and eventually pandemics) with high impact on human health. indeed, cov infections of veterinary interest have been known since almost a century (cavanagh, ; pedersen, ; decaro et al., ) , so that animal covs are paradigmatic of how this large family of viruses evolves, generating strains with different biological properties. in addition, the efforts done in veterinary medicine https://doi.org/ . /j.vetmic. . received march ; received in revised form april ; accepted april evolve rapidly, changing their antigenic profile, tissue tropism or host range by means of two distinct mechanisms. the viral replicase (an rna dependent-rna polymerase) does not possess a good proof reading activity, therefore the incorporation of wrong nucleotides at each replication cycle and the consequent accumulation of mutations in the viral genome lead to a progressive differentiation of the viral progeny from the parental strain. this mechanism, which is well known for influenza viruses being responsible for the so called antigenic drift, may cause the progressive adaptation of the viral surface proteins to the cell receptors of new animal species, increasing the viral fitness. in addition, the particular replicating machinery of covs facilitates recombination events due to the presence of consensus sequences upstream each gene. therefore, in the case of coinfection by more than one cov strain, the rna polymerase can jump from the rna of a strain to that of the other one, synthetizing a hybrid rna containing sequences from both viruses. recombination can occur not only with genomic sequences of other covs (homologous recombination), but also with rnas of different viruses and other organisms (heterologous recombination) (luytjes et al., ; banner and lai, ; lai, ; zeng et al., ; huang et al., ) . recombination is an alternative mechanism that let covs acquire novel biological properties in terms of virulence, host range and tissue tropism, so that cov strains, which are non-pathogenic or lowpathogenic in the original host, may increase their pathogenicity in the same species or adapt to different species spreading in the new host with exceptional rapidity (banner and lai, ) . the occurrence of three human cov epidemics in less than years, along with the emergence of less pathogenic human covs, arises some questions on how these viruses that have their reservoirs in bats and rodents may overcome the species barriers jumping to humans. the animal-to-human transmission of viruses has been already occurred in the past, but it seems that its frequency has been increased in the last decades, involving in a short time span not only covs, but also a plethora of genetically and biologically different viruses with zoonotic potential, such as ebola virus, influenza viruses, flaviviruses, hendra and nipah viruses (mcmahon et al., ) . climate changes that are intensifying in this first quarter of the st century are favouring the spread of vector-borne diseases through increasing the proliferation of vectors and predisposing to their occupation of new ecological niches. the emergence in temperate climate areas such as europe of vectorborne diseases caused by viruses considered exotic until few years ago (west nile virus, usutu virus, chikungunya virus) accounts for a progressive geographic expansion of tropical diseases thanks to the ongoing phenomenon of tropicalisation (mcmahon et al., ) . deforestation and urbanization are other major factors that facilitate the spill-over of zoonotic agents to humans by reducing the habitat of wildlife and increasing the chances of contacts between wild animals (like bats, rodents and birds) and human beings (beena and saikumar, ; lorusso et al., ) . this could be the case of ebola virus, hendra and nipah viruses, hantavirus and coronavirus infections. in addition, the close contact between human beings and different animal species sold at the wet markets of east asia represents the optimal situation for the host species jump and adaptation to humans of potentially zoonotic agents like covs. it is not a coincidence that two of the most severe zoonoses of the last two decades (highly pathogenic h n avian influenza and sars) have emerged in the same chinese province of guangdong where the contact between humans and animals is closer (lorusso et al., ) . table reports the most important avian cov species recognised so far and their associated diseases. the number of avian species in which covs have been detected in the last years is humongous. since the emergence of sars-cov in , there has been increased interest in table main coronaviruses in domestic and domesticated avian species. schalk and hawn ( ); beach and schalm ( ) ; beaudette and hudson ( ) turkey ( respiratory and kidney disease spackman et al. ( ) n. decaro and a. lorusso veterinary microbiology ( ) covs in other species, including birds. prior to that time, our knowledge of covs in avian species was limited largely to three birds of the order galliformes, i.e., domestic fowl (gallus gallus), turkeys (genus meleagris) and pheasants (phasianidae), with their infectious bronchitis virus (ibv), turkey coronavirus (tcov), and pheasant coronavirus (phcov), respectively. these three viruses were considered for a long-time different species for several reasons such as the diverse pathotype (enterotropic or respirotropic), host range and genetic relatedness of the s protein (cavanagh, ) . this scenario radically changed after the discovery of several novel covs with high genetic diversity from different avian species and the novel rules for species designation of the coronavirus study group (csg https://talk.ictvonline.org/ictv-reports/ ictv_ th_report/positive-sense-rna-viruses- /w/posrna_viruses/ /coronaviridae). all these viruses as well as analogous ibv-like covs detected in other birds including penguins, pigeons, peafowl, parrots, waterfowl, teal, quail, duck and whooper swan (cavanagh et al., ; circella et al., ; domanska-blicharz et al., ; torres et al., ; hughes et al., ; liu et al., ; wille et al., ; jordan et al., ; bande et al., ; suryaman et al., ) have been assigned to the same viral species known as avian coronavirus (acov) within the subgenus igacovirus of genus gammacoronavirus. ibv and ibv-like strains are commonly detected in both gallinaceous and non-gallinaceous birds, also asymptomatically (cavanagh, ) . this might suggest that these species would act as wild reservoirs, spreading ibv strains over the world (de wit et al., ) . as for the huge economic impact of the disease it causes, ibv is one of the most studied covs over the last decades. ibv causes the infectious bronchitis (ib), a term adopted in for describing the main clinical characteristics of a transmissible respiratory disease of poultry detected for the first time in north dakota (usa). ib has been now diagnosed worldwide and is one of the most important viral diseases of poultry characterised by respiratory signs, but it can also affect the kidneys and reproductive tract following viremia with a severity that differs depending on the involved viral strain (cavanagh and gelb, ) . the disease also affects wild and ornamental birds chen et al., ) . ib control has been hampered by the intricate ibv evolution, which has been entailed, over the years, by the emergence of many different antigenic or genotypic types, commonly referred to as variants, with divergent molecular, biological, and antigenic properties. being a cov, ibv has, indeed, a considerable ability to change both by mutation and by homologous recombination events, which may cause, along with replicase stuttering or slippage, also insertions and deletions in the genome (cavanagh and gelb, ) . if these mechanisms involve the hypervariable region s , they frequently result in the emergence of new ibv variants. although many new variants are not successful, a few may emerge, spread, causing devastating disease either worldwide or in limited geographic areas. currently lineages have been recognized, categorized into six genotypes (gi to gvi) (valastro et al., ) . through their s protein, ibv and ibv-like viruses recognise as cellular host receptor the α , -linked sialic acid glycan, widely distributed in the respiratory tract and in several other host tissues, factor which may explain the tropism also for several organs of the infected host (winter et al., (winter et al., , shahwan et al., ; ambepitiya wickramasinghe et al., ) . extensive use of vaccines has greatly contributed to the high variability of ibv strains thorough recombination between vaccine and field viruses and viral selection pressure resulting from vaccination and presence of partially immune birds (gandon and day, ; gandon et al., ; bande et al., ) . important ibv-like strains are tcov, responsible for enteritis in turkey (also known as bluecomb disease), guinea fowl coronavirus (gfcov) and quail coronavirus (qcov) responsible for fulminating enteric disease in guinea fowl and quail, respectively cavanagh, ; liais et al., ) . tcov, gfcov and qcov are evolutionarily distant from acov based on the s protein. while ibv is a primarily respiratory pathogen, tcov causes gastrointestinal disease (liais et al., ; guy, ) . enterotropism has also been observed for some ibv serotypes; however, all ibv strains infect primarily the respiratory tract, resulting in mild to severe inflammation of the nasal and tracheal epithelia (cavanagh, (cavanagh, , . the s domain of the s protein is highly variable, with the amino acid sequences of ibv and tcov from the usa sharing < % sequence identity. phylogenetic analysis of the s gene shows, indeed, grouping of ibv and ibv-like viruses on the one hand and tcov-us, gfcov and qcov on the other hand (ambepitiya wickramasinghe et al., a) . accordingly, the emergence of covs in turkeys in the usa was proposed to have resulted from recombination events involving ibvs and an as-yet-unidentified cov donating a novel s gene. this switch contributed largely to determine the in vivo tissue tropism of tcov and related viruses. intriguingly, the s protein of these covs requires nonsialylated type poly-lacnac structures on n-glycan cores for binding. this is in marked contrast to the α , -linked sialic acid glycan binding of ibv and ibv-like viruses (ambepitiya wickramasinghe et al., b) . the s subdomain of a tcov isolate from france in (tcov-fr) had only % sequence identity to that of the tcov-us strain (maurel et al., ) . this diversity was biologically evident by the prominent tropism for the epithelium of the bursa of fabricius and only mild tropism for the small intestine of turkey. tcov-fr s protein did not show, indeed, affinity for nonsialylated type poly-lacnac (ambepitiya wickramasinghe et al., a) . this genetic diversity between tcovs is in accordance with several recombination events involving ibvs on different continents with several unknown covs. on the one hand, the s genes of gfcov/fr/ (isolated in france in ) and tcov-us share significant genetic relationships, and thus these viruses must have acquired their s gene from a common ancestor. on the other hand, gfcov/fr/ and fr tcov have a very similar genetic background in other genes. two recombination events may be responsible for the genesis of tcov-us and fr tcov. a first event occurred between an ibv eu recipient strain and an unknown acov donor, resulting in a virus with a new s gene, whose evolution would have resulted in fr tcov and gfcov/fr/ . a second recombination event involving a us ibv recipient and gfcov/fr/ would have generated us tcov viruses, which share a stronger s gene similarity with gfcov/fr/ than with fr tcov . additional covs distinct from acovs and mainly circulating in ducks (duck coronavirus, dcov), pigeons (pigeon coronavirus, pcov), or geese (goose coronavirus, gcov) have been identified (cheng et al., ; jonassen et al., ; muradrasoli et al., ; kim and oem, ; zhuang et al., ; papineau et al., ) . although their genome seems to fulfill the official ictv criteria required to distinguish a new species within the gammacoronavirus genus, ictv approval is still pending. historically, covs of birds were all included in the gammacoronavirus genus and, in turn, all covs belonging to this genus were identified only in birds. however, this suggestion was rebutted by the evidence of a cov belonging to the gammacoronavirus genus in a beluga whale first discovered in (viral species beluga whale coronavirus sw species, subgenus cegacovirus, genus gammacoronavirus) (mihindukulasuriya et al., ) , and of three novel covs, bucov hku , thcov hku , and mucov hku in birds of the order passeriformes, namely bulbuls (pycnonotus jocosus), thrushes (turdidae) and munias (lonchura punctulate), respectively, which did not cluster phylogenetically with extant covs identified in birds. these latter three viruses were distinct from known covs forming a unique cluster in the phylogenetic tree, which was the basis for generation of the deltacoronavirus genus (woo et al., ) . importantly, additional novel viruses belonging to this novel genus were detected in wild birds chu et al., ; durães-carvalho et al., ; torres et al., ) . these viruses cluster with previously unclassified covs detected in various asian carnivores, i.e., the asian leopard cat (prionailurus bengalensis) and chines ferret badger (nyctereutes procyonoides) (dong n. decaro and a. lorusso veterinary microbiology ( ) et al., ) . covs belonging to the betacoronavirus genus, which are strictly related to mouse hepatitis virus (mhv), were also described in wild birds, including parrots, in brazil (durães-carvalho et al., ) . interestingly, this was not the first detection of viruses belonging to the betacoronavirus genus in birds. often overlooked is the discovery over years ago of a cov from the manx shearwater (puffinus puffinus), a bird that visits the shores of britain in summer (nuttall and harrap, ; cavanagh et al., ) . this virus was also related to mhv. however, at that time, considering the unusual finding and that the virus was isolated by passage of shearwater material in the brains of mice, it was speculated that the detected virus was an mhv strain already present in the mice before inoculation (cavanagh, ) . bats are an ancient and heterogeneous group of ecologically important mammals, representing nearly a quarter of all mammalian diversity on earth. they belong to the order chiroptera and further classified in two suborders yinpterochiroptera and yangochiroptera. the first includes the non-echolocating pteropodidae family (megabats) and five echolocating rhinolophoidea microbat superfamilies. yangochiroptera contain thirteen echolocating microbat families (tsagkogeorga et al., ) . bats are thought to host a large plethora of viruses. these include, amongst the others, lyssaviruses, filoviruses, henipaviruses, and reoviruses (calisher et al., ) . before sars-cov epidemic, bats were not known to host covs. indeed, the first evidence of a bat cov was published in . after the sars epidemic, there was a boost in interest regarding searching for novel covs in various animals, including bats. to date, over novel covs have been identified in bats and approximately % of the bat virome sequenced to date is composed of covs (chen et al., ) . this data has been made available following the massive surveillance, coupled with the advent of next-generation sequencing (ngs) technology, which has been performed in wild animals banerjee et al., ) . just a small portion of these covs have been officially recognised by the ictv; many others are still pending for official designation. cov species detected in bats and officially recognised by the ictv are listed in table and the following chapter reasonably discusses only officially recognized bat cov species. bats can carry and transmit covs into local bat populations via migration even though little is known about the migratory patterns of these animals. closely related covs can be detected in the same bat species living at locations separated by thousands of miles (drexler et al., ) and different cov species or genera can be found in different bat species living at the same roosting sites. however, some covs have been shown to be species-specific. accordingly, regional patterns of bat cov outbreaks at species level can be deduced from the population distribution of their respective bat hosts. although bats seem to develop clinical diseases induced by several viruses and bacteria (mühldorfer et al., ) , generally covs do not cause apparently overt disease in these mammals, also experimentally. this phenomenon seems to be related with peculiar characteristics of their immune system (ahn et al., ; brook et al., ) . based upon genomic data available so far, it is widely accepted that while birds represent the reservoir for covs belonging to genera gammacoronavirus and deltacoronavirus, bats are the natural reservoir for alpha-and betacoronaviruses. however, only betacoronaviruses of subgenera sarbecovirus, merbecovirus, nobecovirus and hibecovirus have been detected in bats so far. given that several betacoronaviruses from the subgenus embecovirus have been discovered in rodents, it was speculated that rodent covs may be the ancestors of currently circulating viruses belonging to this subgenus . covs have been detected at high frequency in bats in all continents, with alphacoronaviruses being more widespread than betacoronaviruses . subgenus colacovirus (genus alphacoronavirus) officially comprises the viral species bat coronavirus cdphe , so far composed by two bat covs strains named cdphe /usa/ and myotis lucifugus cov (myl-cov), which share a . % nucleotide identity across the whole genome. both strains have been detected in myotis lucifugus bats (vespertilionidae) also known as the northern american little brown bats. the former was detected in in colorado (genbank acc. no. kf ), while the latter was reported in in canada. this virus was identified in the intestines and lungs and associated with minimal pathology or inflammation (subudhi et al., ) . subgenus decacovirus (genus alphacoronavirus) comprises the species rhinolophus ferrumequinum alphacoronavirus hub- composed so far by btms-al-phacov/gs and btrf-alphacov/hub strains discovered in china in myotis spp. and rhinolophus ferrumequinum bats, respectively. these two viruses share very high sequence identities (higher than %), which dramatically decrease in the s genes (only % nucleotide identity) (wu et al., ) . woo et al., ) shares an % sequence identity with severe acute diarrhoea syndrome-coronavirus (sads-cov) of pigs . these two viruses are now included in the same viral species rhinolophus bat coronavirus hku (subgenus rhinacovirus, genus alphacoronavirus). viral strains btkynl - a, btkynl - b, btkynl - and btkynl - a, identified in in triaenops afer bats from kenya, form the viral species nl -related bat coronavirus strain btkynl - b that is part of the subgenus setracovirus (genus alphacoronavirus) along with human coronavirus nl (tao et al., ) . in this regard, a bat origin has been strongly suggested for two of the less-pathogenic hcovs causing mild respiratory symptoms in immunocompetent people, namely hcov- e and hcov-nl , both belonging to the alphacoronavirus genus. whereas hcov- e (subgenus duvinacovirus) recognises as direct ancestor an alphacoronavirus from alpacas, which in turn derives from e-related covs identified in hipposiderid bats (corman et al., ) , hcov-nl is likely a recombinant virus originating from the distantly related e-related covs associated with hipposiderid bats and covs associated with triaenops afer bats (tao et al., ) (table ). the s protein of hcov-nl is more closely related to that of e-related covs, whereas the rest of the genome with covs included in the nl -related bat coronavirus strain btkynl - b species (tao et al., ) . different from the bovine coronavirus (bcov)-like viruses that cause enteric disease, in a novel alpaca cov was associated to respiratory disease in california, usa. full-length genome analysis showed that this respiratory alpaca cov was closely related to the alphacoronavirus hcov- e (subgenus duvinacovirus) (crossley et al., ) . more recently, close relatives of hcov- e were detected in african hipposiderid bats. interestingly, both bat and alpaca viruses displayed an intact accessory gene orf located at the genomic ' end, while hcov- e retained only a conserved trs preceding remnants of this orf, suggesting its loss after acquisition of a e-related cov by humans. therefore, hcov- is likely a descendant of the alpaca alphacoronavirus (corman et al., ) . strains forming the viral species bat hp-betacoronavirus zhejiang (subgenus hibecovirus, genus betacoronavirus) were discovered in hipposideros pratti bats from china in (wu et al., ) . strain ro-batcov gccdc was identified from stools of rousettus leschenaultii, a species of fruit bats (pteropodidae) of southern asia, which were collected in yunnan province, china, in (huang et al., ) . ro-batcov gccdc shows a small intact orf of nucleotides embedded between the n and ns a genes. this orf has no homology to any known coronavirus, and the encoded protein exhibited . % amino acid identity with the p protein encoded by the first orf of segment s of bat fusogenic orthoreoviruses (genus orthoreovirus, species nelson bay orthoreovirus, also known as pteropine orthoreovirus). these viruses are double-stranded segmented rna viruses, belonging to the family reoviridae, and are able to cause severe pneumonia in humans (chua et al., ; lorusso et al., ) . ro-batcov gccdc is included in the viral species rousettus bat coronavirus gccdc within the subgenus nobecovirus, genus betacoronavirus. rousettus bat coronavirus hku , belonging to subgenus nobecovirus, was also identified in rousettus leschenaultii and in other bat species (mendenhall et al., ) . this virus was first detected in in guangdong province in china (woo et al., ) . subsequent studies suggested that the virus was widely distributed and is circulating in different bat species (ge et al., ) . covs from the bthku -like cluster were also detected in hipposidereos commersoni and rousettus aegyptiacus bats in kenya (tong et al., ) . being a fruit bat, rousettus leschenaultii has a wider flying range than most of the insectivorous bats in china, thus it may carry viruses over long distances. a comparison of the reported hku -cov sequences showed a high genetic diversity within this viral species (luo et al., a, b; lau et al., ; ge et al., ) . when mers-cov was first isolated in the middle east in and its genome sequenced, it was found that it was most closely related to ty-batcov hku discovered in tylonycteris pachypus and pi-batcov hku discovered in pipistrellus abramus, which were the only known members of subgenus merbecovirus at that time. these two viruses are now the prototype strains of tylonycteris bat coronavirus hku and pipistrellus bat coronavirus hku viral species, respectively, within subgenus merbecovirus, genus betacoronavirus. although mers related covs (mers-rcovs) were lately discovered, mers-cov was much closer in the s region to hku -cov than to mers-rcov or hku -cov. indeed, dipeptidyl peptidase (dpp ), the receptor for mers-cov, is also the receptor for hku , but neither for hku nor for early discovered mers-rcovs. however, hku prefers bat dpp over human dpp , whereas mers-cov shows the opposite trend . so far, hku -covs are only carried by tylonycteris spp. bats (t. pachypus and t. robustula) and are relatively conserved; hku -covs are found in different pipistrellus spp. bats, including p. abramus, p. pipistrellus and p. minus . due to the current sars-cov- pandemic, attention should be given to the viral species severe acute respiratory syndrome-related coronavirus (sars-rcov,subgenus sarbecovirus, genus betacoronavirus) and middle east respiratory syndrome-related coronavirus (mers-rcov, subgenus merbecovirus, genus betacoronavirus), which enclose sars-cov and mers-cov, the first two highly pathogenic covs that were discovered in humans. in , at the beginning of the sars epidemic, almost all early human index patients had animal exposure in a market place, in guangdong province, before developing disease. after sars-cov was identified, its rna and/or specific antibodies were found in masked palm civets (paguma larvata) and animal handlers in a market place. however, later investigations of farmed and wild-caught civets revealed that sars-cov strains found in market civets were transmitted to them by other wild animals (tu et al., ; kan et al., ) . subsequently, novel covs related to human sars-cov (sars-rcovs) were discovered in horseshoe bats (genus rhinolophus) in china and hong kong lau et al., ) . these sars-rcovs showed genome sequence identity of - % among themselves and - % identity to human or civet sars-cov isolates. sars-rcovs were detected in rhinolophus spp. bats of other regions of china (tang et al., ; woo et al., ; yuan et al., ; ge et al., ) . sars-rcovs with higher genetic diversity with respect to chinese strains were also detected in rhinolophid bats from slovenia, bulgaria and italy in europe (drexler et al., ; rihtaric et al., ; balboni et al., ) . covs related to sars-rcov were also detected in hipposideros spp. and chaerophon spp. bats from ghana, kenya and nigeria (hu et al., ) . these evidences suggested that bats may be the natural hosts for sars-cov and that wild carnivores were only intermediate hosts. although these sars-rcovs showed high sequence identity to sars-cov, they were demonstrated to be unable to bind to the human cell angiotensin converting enzyme ii (ace ) receptor, the receptor of sars-cov, as a consequence of deletions in their s protein (ren et al., ) . besides, the theory of bat origin of sars-cov lacked a powerful support due to the failure of direct isolation of this virus from bats. thus, considering that no direct progenitor of sars-cov was found in bats and that rna recombination is the fuel for cov evolution, it has been proposed that sars-cov emerged through recombination of bat sars-rcovs. this hypothesis was made after the evidence of a single bat cave in yunnan, china, with very high covs diversity and considering that, within the identified covs, all genetic elements needed to form sars-cov have been identified in that single cave (ge et al., ) . recombination analysis also strongly supported the hypothesis that the civet sars-cov strain sz originated following a recombination event of two existing bat strains, wiv and rf (hu et al., ) . moreover, wiv , the closest relative to sars-cov that has been found in bats so far (more than % nucleotide identity, higher than that of any other bat sars-rcovs ( - %)), likely arose through recombination of two other prevalent bat sars-rcov strains. the most frequent recombination breakpoints were within the s gene and upstream of orf , which encodes an accessory protein. these genes were also involved in the crucial adaptation pathways of sars-cov from bats to wild carnivores, from wild carnivores to humans, and from human to human (cui et al., ) . wiv has been shown to have the capacity to bind to the human, civet and bat cell ace receptor (ge et al., ) . the isolation in cellculture of a highly related sars-cov strain, coupled with the evidence of a functional s protein capable of using the same ace receptor, provided robust and conclusive evidence for the bat origin of sars-cov. an additional sars-rcov strain has been shown, by reverse genetics studies, to have the capacity to bind to the human ace receptor (menachery et al., ) . quite the opposite, a direct bat cov highly related to mers-cov of humans was never detected. indeed, the genome sequences of mers-cov in human and dromedaries possess only around - % nucleotide identities to those of the other members of subgenus merbecovirus from different bats. human mers-covs were instead almost identical to mers-covs identified in dromedary camels (camelus dromedaries). lately, genomic sequence analyses indicated that covs now belonging to the mers-rcov species were found in several bat species from two bat families, vespertilionidae and nycteridae (lelli et al., ; de benedictis et al., ; corman et al., a, b; anthony et al., ; moreno et al., ; wong et al., ) . however, none of these mers-rcovs is a direct progenitor of mers-cov, as their s proteins differ substantially from that of the human virus. the closest relative to mers-cov of humans and dromedary camels is mers-rcov strain neoromicia/ isolated from neoromicia capensis bats in south africa (geldenhuys et al., , table ). a short sequence (around nucleotides) of viral rna identical to that of mers-cov was also detected in a taphozous perforates bat in saudi arabia (memish et al., ) . overall, although it is widely accepted that mers-cov ancestor is in bats, further studies are warranted in order to discover the precise mechanisms of its emergence in dromedary camels and humans. it was suggested that mers-cov ancestors had been circulating in bats for very long time. mers-cov has evolved to adapt to use human receptor and the dpp -recognising bat coronaviruses like hku may follow up, thereby posing a serious risk to human health. recent mers-rcovs were shown to have the capacity to bind to the dpp as entry cell receptor as they acquired the s through recombination with hku -like viruses (luo et al., a, b) . as for the recent and threatening covid- outbreak in humans, we certainly know that sars-cov- belongs to the species sars-rcov together with sars-cov from humans and sars-rcovs from wild carnivores and horseshoe bats (genus rhinolophus) (gorbalenya et al., ; zhou et al., ; wu et al., ) . epidemiological investigations revealed that many initial patients were exposed to wildlife at the huanan seafood wholesale market (south china seafood market), which is the largest seafood market in central china (lorusso et al., ) . sars-cov- has been assigned to an existing species of hundreds of known viruses largely isolated from bats. these viruses have names derived from sars-cov, but only the viral isolates originating from the - outbreak have been confirmed to cause sars in humans (gorbalenya et al., ) . importantly, it has also been confirmed that sars-cov- uses the ace receptor through the receptor binding domain (rbd) of the s protein (hoffmann et al., ; zhou et al., ) . likely, also sars-cov- has a bat origin. according to genome sequences available so far, the most closely related virus ( . % of nucleotide sequence identity) to sars-cov- is strain batcovratg identified from a bat, rhinolophus affinis, from yunnan province, china, followed by sars-rcovs identified from pangolins (tang et al., ) . the receptor-binding spike protein of sars-cov- is highly divergent from other covs with less than % nucleotide sequence identity to all previously described sars-rcovs, except for a . % nucleotide identity to batcovratg (zhou et al., ) . although sars-cov- uses the ace receptor, five out six critical amino acid residues in rbd were different between sars-cov- and sars-cov; the same residues were instead identical to those of pangolin sars-rcovs and, in turn, only one of these residues was identical to those of batcovratg (tang et al., ) , although this latter shows the highest nucleotide sequence identity with sars-cov- along the whole genome. thus, it was tempting to speculate that sars-cov- rbd region might have originated from recent recombination event in pangolins or that sars-cov- and sars-rcovs of pangolins represent the result of coincidental evolution (lam et al., ; tang et al., ) . overall, it remains to be solved whether also sars-cov- needed an intermediate (and amplification) host before being able to infect humans as it was the case for sars-cov and other hcovs. since a mammal reservoir has not yet been identified, a prudent use of specific antigens is strongly recommended for serological diagnosis of sars-cov- in animals as cross-reactions with viruses of the alphacoronavirus genus, widespread in animals, might occur (sun and meng, ) . analogously to bats, but with a lesser extent, also rodents have been recently demonstrated to play a significant role in the evolution of cov, in particular of those belonging to subgenus embecovirus of genus betacoronavirus. rodentia (rodents) is the largest order of mammals with more than species worldwide, representing a major source of zoonotic infectious diseases (han et al., ) . for decades, only one species of coronavirus, murine coronavirus (subgenus embecovirus, genus betacoronavirus), has been associated with rodents. the prototype virus, which was named mouse hepatitis virus (mhv), was first isolated in mice in (cheever et al., ) . a mhv variant was lately identified in rats in (parker et al., ) . rat coronavirus (rcov) causes epidemics of respiratory disease in laboratory rat colonies. the two prototype strains of rcov are sialodacryoadenitis virus (sdav) and parker's rcov (rcov-p) (bhatt et al., ; parker et al., ) . both strains infect the respiratory tract, and sdav can also infect the eye, salivary and lacrimal glands. young rats are especially susceptible to rcov with the infection occurring in the lower respiratory tract and developing into interstitial pneumonia (parker et al., ) . together with feline infectious peritonitis virus (fipv) and ibv, mhv has been one of the most strictly animal cov studied ever. mhv is a natural pathogen of mice, normally infecting the liver, gastrointestinal tract, and central nervous system, causing a wide range of disease, including hepatitis, gastroenteritis, and acute and chronic encephalomyelitis. importantly, it served as model for cov replication and pathogenesis, with emphasis for neuro-invasion and neurovirulence (weiss and navas-martin, ) . as for the additional structural protein he, some strains (such as jhm) of mhv contain the he protein, while others (such as a ) do not yokomori et al., ) . the role of rodents in the evolution of covs belonging to embecoviruses has been recently highlighted by means of the discovery of a novel betacoronavirus in norway rats (rattus norvegicus) in china. this virus forms a separate species named china rattus coronavirus hku (chrcov hku ) within the embecovirus subgenus. although designated as a novel species, this virus possessed genome characteristics that resemble to those of both betacoronavirus- and murine coronavirus, suggesting that chrcov hku represents the murine origin of betacoronavirus- , with interspecies transmission from rodents to other mammals having occurred centuries ago (lau et al., ) . genus betacoronavirus consists of five subgenera, with bat covs being including in all but one of subgenus embecovirus, where rodent, human and bovine covs are included (https://talk.ictvonline.org/ taxonomy/). this supports the hypothesis that rodent covs were the ancestors of embecoviruses of other animals, while bats are the natural reservoirs for all other betacoronaviruses. importantly, rodent covs are not restricted to genus betacoronavirus. a deep virological screening was performed in rodents sampled in zhejiang province, china, during - , with nearly % of rodents testing positive for cov . in particular, covs were detected in striped field mice (apodemus agrarius), norway rats, lesser ricefield rats (rattus losea), asian house rat (rattus tanezumi) and chinese white-bellied rat (niviventer confucianus). amplicons of the replicase gene sequences were recovered from ( %) of the cov rna positive rodent samples described above and whole genome or nearly whole genome sequences (> %) were recovered from and cov positive samples, respectively. by means of whole genome sequence analysis, authors were able to identify a divergent alphacoronavirus, which was lately officially designated as species lucheng rn rat coronavirus (lrnv) within the subgenus luchacovirus, and two novel betacoronaviruses termed longquan aa mouse coronavirus (lamv) and longquan rl rat coronavirus (lrlv) and assigned to the two established species betacoronavirus- and murine coronavirus, respectively . moreover, lrnv seems to be a recombinant virus as its n protein gene is more closely related to those of the genus betacoronavirus. overall, the discovery of rodent-associated covs belonging to subgenera that are distinct from those including bat covs warrants further investigations upon the role played by rodents in the evolution and emergence of these viruses. sars-cov replication has been studied in mice, syrian golden and chinese hamsters. the most severe symptoms of sars were observed in aged animals. indeed, aged mouse model of sars-cov has been generated (gretebeck and subbarao, ) . transgenic mice expressing human ace were also developed to closely mimic sars-cov infection in humans. some animal models have been tested and analysed on the genomic and proteomic level to study the pathogenesis of sars-cov. therefore, we have reason to believe that such models would work also for sars-cov- . quite the opposite, studies have demonstrated that mice, guinea pigs and hamsters are not susceptible to experimental mers-cov infection, mainly because their homologous dpp molecules table coronaviruses in domestic swine and associated diseases. do not function as receptors for mers-cov entry (cockrell et al., ) . the first mouse model of mers infection reported in involved transducing animals with recombinant adenovirus encoding human dpp (hdpp ) molecules intranasally, and this resulted in replication of mers-cov in the lungs. this mouse model also showed clinical symptoms of interstitial pneumonia, including inflammatory cell infiltration, and thickened alveolar and mild oedema (song et al., ) . currently, six covs are circulating in swine (table ). these include four alphacoronaviruses, transmissible gastroenteritis virus of swine (tgev) and its derivative porcine respiratory coronavirus (prcov) (subgenus tegacovirus), porcine epidemic diarrhoea virus (pedv) (subgenus pedacovirus) and sads-cov (subgenus rhinacovirus), one betacoronavirus, porcine haemagglutinating encephalomyelitis virus (phev) (subgenus embecovirus), and one deltacoronavirus, porcine deltacoronavirus (pdcov) (subgenus buldecovirus). tgev, pedv, sads-cov and pdcov are responsible for acute gastroenteritis in swine, with fatal infections in piglets born to seronegative sows, prcov causes a mild respiratory disease and phev is the causative agent of neurological and/or digestive disease in pigs (mora-díaz et al., ; wang et al., ). tgev was first described in uk in s, representing the oldest known swine cov. tgev and prcov are closely related to canine coronavirus (ccov) and feline coronavirus (fcov) forming with these carnivore covs a unique species, referred to as alphacoronavirus- . based on the analysis of the accessory protein gene orf , it has been postulated that tgev has originated from ccov type ii (ccov-ii), since while ccov type i (ccov-i) exhibits an intact gene, both ccov-ii and tgev, which are strictly related in the s gene, have only remnants of orf (lorusso et al., ) . prcov, in turn, has derived from tgev through the deletion of ≈ nucleotides at the ' end of the s gene (corresponding to ≈ amino acids at the n-terminus of the spike protein) and consequent change of the major tissue tropism from the enteric to the respiratory epithelium. this large deletion caused the loss of sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins, so that tgev but not prcov is able overcome the intestinal mucus barrier, having access to the gut mucosa . prcov shares some epitopes for neutralising antibodies with tgev, so that its extensive circulation in swine herds has resulted in a drastic reduction of tge outbreaks worldwide. pedv was introduced in the pig population in the s, likely as a consequence of a spillover event from bats. the virus was first described in europe and had been primarily maintained as an endemic pathogen in european and asian swine populations until its introduction into north america in . pedv is more strictly related to a scotophilus bat coronavirus than to other known alphacoronaviruses, including tgev and human alphacoronaviruses hcov- e and hcov-nl . therefore, pedv and btcov/ / likely have a common evolutionary precursor and a cov cross-species transmission may have occurred between bats and pigs (banerjee et al., ) . accordingly, pedv contains signature motifs at the ′-untranslated region that are shared by bat covs, thus providing further support of the evolutionary origin of pedv from bats and potential cross-species transmission (huang et al., ) . currently, different pedv genotypes are described based on the s gene: i) g a pedv, including classical european and asian strains with moderate virulence; ii) g pedv, also called "original us pedv", comprising highly virulent strains that originated in asia and are now widespread in the usa; iii) g b pedv, which is represented by the so-called s-indel strains, i.e., strains presenting insertions and deletions in the s gene that are associated with mild clinical outbreaks. these strains are natural recombinant pedvs with a g -like genomic backbone carrying an s region of g a strains; iv) s n-terminal domain-deletion (ntd-del) strains that are g -like strains containing a to -aa deletion within the n-terminal domain of the s subunit, also associated to mild clinical forms (hou and wang, ) . recombinant strains between pedv and tgev have been also reported in europe (akimkin et al., ; belsham et al., ; boniotti et al., ) . sadv-cov, now referred to as swine enteric alphacoronavirus (seacov), is another virulent swine enteric alphacoronavirus that originated from bats, sharing an % sequence identity with a bat alphacoronavirus hku -cov. since viruses displaying a - % sequence identity to sads-cov were detected in rhinolophus spp. bats, sads-cov and hku -cov likely descend from a common ancestor . accordingly, both viruses now belong to the unique species rhinolophus bat coronavirus hku . in contrast, phev, which was first described in in nursery pigs with encephalomyelitis in ontario, canada, has not derived from bat covs, but its evolutionary history is tightly intermingled with other two closely related betacoronavirus, hcov-oc and the oldest known bcov, with which phev may have common ancestors (vijgen et al., ) and is included in the same viral species, betacoronavirus- (corman et al., ) . most probably, hcov-oc and phev descend from a rodent betacoronavirus through preliminary adaptation to bcov, from which they may have emerged in the context of a pandemic recorded historically at the end of the th century (corman et al., ) . pdcov was recently detected in in hong kong during cov molecular surveillance in avian and mammalian species. this swine deltacoronavirus seems to recognise another different ancestor, likely emerging from a host-switching event between avian and mammal covs. the most closely related pdcov relative has been identified in quail deltacoronavrus uae-hku and the virus has been proposed to be a recombinant between other two avian deltacoronaviruses, sparrow cov hku and bulbul cov hku . all these deltacoronavirus are now members of the same species coronavirus hku (lau et al., ) . pigs were found to be susceptible to experimental infection with the betacoronavirus mers-cov (vergara-alert et al., ), while sars-cov rna was detected in pigs and wild boars wang et al., ) . in contrast, a recent experimental infection demonstrated that pigs are not susceptible to sars-cov- . few studies have been carried out to assess the circulation of covs in farmed or free-ranging wild boars (sus scrofa). antibodies against tgev/prcov were detected in some animals in slovenia (vengust et al., ) and croatia (roic et al., ) and pedv rna was demonstrated in south korea (lee et al., ) . a wild boar sold at a live animal market of guangzhou, china, was positive for sars-cov rna . the main covs infecting ruminants are reported in table . the oldest known ruminant cov is bcov, which is also the prototype of the species betacoronavirus- (subgenus embecovirus, genus betacoronavirus). this virus is able to cause a variety of clinical forms, including enteric disease with high mortality rates in neonate calves, winter disease (a severe enteric form) in lactating cows (decaro et al., b) , and a respiratory disease, also known as shipping fever, in cattle of all ages, with a higher prevalence in - month-old calves (decaro et al., a) . it was postulated that the presence of genetic signatures differentiates enteric and respiratory bcovs (hasoksuz et al., ) , but it was ultimately evident that the same virus strain could be responsible for simultaneous appearance of enteric and respiratory disease in the same animals (chouljenko et al., ) . it has been postulated that bcov originated from a rodent cov (corman et al., ) . very recently, a novel cov, representing a new viral species, referred to as china rattus coronavirus hku (chrcov-hku ), was detected in norway rats in china. this virus was phylogenetically distinct from mhv and hcov-hku and displayed genome features that were intermediate between bcov and mhv. therefore, chrcov hku may represent the murine origin of bcov and rodents are likely an important reservoir for ancestors of subgenus embecovirus (lau et al., ) . bcov is paradigmatic of how covs are able to cross the interspecies barriers, establishing its derivatives as separate viral lineages affecting the respiratory and/or enteric tract of humans (hcov-oc ), swine (phev), horses (equine coronavirus, ecov), and dogs (canine respiratory coronavirus, crcov). a number of bcov-related viruses, all currently included in the unique species betacoronavirus- , have been detected in the enteric and/or respiratory tract of domestic and wild ruminants. these bcov-like covs include viruses of domestic and domesticated ruminants that were reported in sheep and goats (reinhardt et al., ; yang et al., ) , water buffalo (bubalus bubalis) (decaro et al., c) , llamas (lama lama) and alpacas (vicugna pacos) (cebra et al., ; jin et al., ) . in the wild, bcov-like covs were demonstrated in six species of the cervidae family, which are caribou/ reindeer (rangifer tarandus caribou), elk/wapiti (cervus elephus), samber deer (cervus unicolor), white-tailed deer (odocoileus virginianus), sika deer (cervus nippon yesoensis) and water deer (hydropotes inermis) (amer, ) . similar viruses were also found to circulate in the giraffe (giraffa camelopardalis) (hasoksuz et al., ) , several species of antelopes (alekseev et al., ; chung et al., ) , wisent (bison bonasus), himalayan tahr (hemitragus jemlahicus) (chung et al., ) , and dromedary camels (camelus dromedarius) (woo et al., ) . the last strain, detected in the united arab emirates and consequently named dromedary camel coronavirus uae-hku- (dccov uae-hku ), was slightly divergent from other bcov-like viruses (woo et al., ) . dromedary camels are susceptible to mers-cov infection, developing asymptomatic infections or mild upper respiratory disease, so that they are considered the natural host of mers-cov, with adult animals in many countries in the middle east as well as in north and east africa showing > % seroprevalence to the virus (hemida et al., b) . although human-to-human transmission has occurred outside middle east due to travel-associated patients with mers and has caused large clusters of human cases within healthcare facilities in saudi arabia, jordan and united arab emirates, it remains inefficient and sustained community transmission has not being documented so far, thus suggesting multiple virus introduction into the human population by infected dromedaries (hemida et al., b) . more recently, a phylogenetic study of mers-cov full-genome sequences revealed recombination signatures that defined five major phylogenetically stable lineages, all of which contained human and camel mers-cov sequences (sabir et al., ) . in the same study, an alphacoronavirus strictly related to hcov- e was found in the respiratory tract of dromedary camels of saudi arabia (sabir et al., ) . although some studies ruled out the susceptibility of other domestic ruminants to mers-cov (reusken et al., ; adney et al., ) , a recent study detected specific antibodies and rna in sera and nasal secretions, respectively, of domestic ruminants raised in africa, including sheep, goats and cattle (kandeil et al., ) . llamas were found to be susceptible to experimental infections with mers-cov (vergara-alert et al., ). the only cov that has been so far known in horses is ecov, which is a bcov-descendant betacoronavirus (subgenus embecovirus). ecov was first isolated from the faeces of a diarrhoeic foal in (ecov-nc ) in north carolina, usa (guy et al., ) , and was initially believed to only affect foals. since , the virus has been recognised in japan, europe and the usa as a new, clinically important, enteric virus of adult horses (pusterla et al., ) . despite mers-cov was successfully adapted to the in-vitro growth in equine cell lines (meyer et al., ) , serological and molecular table coronaviruses in domestic and domesticated ruminants and associated diseases. sabir et al. ( ) n. decaro and a. lorusso veterinary microbiology ( ) investigations have demonstrated that horses are not naturally infected by mers-cov (meyer et al., ; hemida et al., a) , nor they are susceptible to experimental infection (adney et al., ; vergara-alert et al., ) . however, surprisingly, mers-cov rna was detected in respiratory specimens of three donkeys of from egypt (kandeil et al., ) , a finding that requires further confirmation. a molecular survey aimed to assess cov circulation in horses in saudi arabia and oman has detected two dccov uae-hku strains in enteric samples of horses (hemida et al., a) . scarce data are available about cov circulation in donkeys. these equids are susceptible to ecov infection since positive rt-pcr results were obtained from a donkey in ireland (nemoto et al., ) . in addition, three donkeys ( . %) of from egypt tested positive for mers-cov rna in their nasal secretions (kandeil et al., ) . covs of carnivores are listed in table . three covs are known in dogs, i.e., two alphacoronaviruses of the subgenus tegacovirus, namely ccov-i and ccov-ii, and one betacoronavirus of the subgenus embecovirus, namely crcov. ccovs (species alphacoronav-irus- ) are commonly responsible for mild, self-limiting enteritis in pups . although they are neglected viruses and vaccination is not recommended due to the absence of an effective challenge model, two independent studies have demonstrated their significant involvement in the onset of acute canine enteritis (duijvestijn et al., ; dowgier et al., ) . the evolutionary history of ccovs is tightly intermingled with that of tgev and fcovs. ccov-i possesses a divergent spike protein and the intact form of an additional gene, orf , whose remnants are present in ccov-ii and, at a lesser extent, in tgev. therefore, ccov-ii has likely emerged as a consequence of recombination between the original ccov-i and an unknown cov in the s gene and of progressive loss of orf (lorusso et al., ) . a further recombination occurred in the very ' end of the s gene between ccov-ii and tgev, giving rise to back recombinant ccov-ii strains, also known as tgevlike ccovs, having a spike protein n-terminus of tgev in a ccov-ii backbone (decaro et al., , . consequently, the ccov taxonomy was revised, with classical and tgev-like strains being referred to as ccov-iia and ccov-iib, respectively. while ccovs are usually involved in mild forms of diarrhoea, there are some hypervirulent strains that are associated to severe, haemorrhagic, sometimes fatal gastroenteritis. in addition, ccov-iia strains, designated pantropic ccov, that are able to spread systemically and cause severe disease and the death of infected dogs have been reported in italy (buonavoglia et al., ; alfano et al., ) , other european countries (decaro et al., ) and south america (pinto et al., ) . genomic sequences from pantropic ccovs were analysed, but no obvious genetic signatures that may have caused the switch in pathogenicity were found (decaro and buonavoglia, ; decaro et al., ) . different from ccov-i and ccov-ii, the betacoronavirus crcov is associated with mild respiratory signs and has been proposed as an etiological agent of canine infectious respiratory disease (cird) together with other viral and bacterial agents . the virus was first detected firstly in uk in (erles et al., ) and subsequently in other european and extra-european countries (decaro et al., (decaro et al., , mitchell et al., ; maboni et al., ; piewbang et al., ; more et al., ) . being a bcov derivative, crcov possesses the same genomic organisation, with some differences in accessory orfs located between the s and e protein genes. in particular, while some crcovs possess a unique . kda protein gene directly downstream of the s protein gene, other canine bcov-like covs display the canonical set of bcov accessory genes but with truncated forms of the . kda protein gene . in cats, two alphacornavirus- genotypes are known, namely fcov type i (fcov-i) and fcov type ii (fcov-ii), the latter being generated as table coronaviruses in domestic and domesticated carnivores and associated diseases. jakob, jacob ( , pedersen et al. ( ) cat ( erles et al. ( ) n. decaro and a. lorusso veterinary microbiology ( ) a consequence of recombination events between ccov-ii and fcov-i that generated viruses with a ccov-ii genomic region, encompassing orf b, orf (s gene), orf abc, orf (e gene), and partial orf (m gene), in the context of an fcov-i backbone (pedersen, ) . both genotypes are involved in the development of feline infectious peritonitis (fip), a perivascular pyogranulomatosis of cats that may occur in two clinical forms, effusive and non-effusive fip, which are characterised by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip occurs as a consequence of a change in tissue tropism of an enteric fcov strain (feline enteric coronavirus, fecv), infecting enterocytes of the intestinal villi, that acquires the ability to infect monocytes/macrophages switching to the more virulent fipv, which is responsible for systemic infections and dysregulation of the proinflammatory cytokines (addie et al., ) . the changes responsible for the pathogenetic shift have been investigated for many decades, being suggested to be variably represented by point mutations located in the s gene (rottier et al., ) , deletion/insertion in the group-specific genes c (vennema et al., ; chang et al., ) , b (vennema et al., ) or a (kennedy et al., a) . however, none of these differences appeared to consistently correlate with disease phenotype. more recent studies have identified specific genetic signatures in the s gene of fcov-i that are implicated in monocyte/macrophage tropism. two amino acid substitutions, m l and/or s a, corresponding to nucleotide mutations a t/c and t g, respectively, in the viral genome, together distinguished fcovs found in the tissues of fip cats from those found in the faeces of healthy cats without fip in > % of cases (chang et al., ) . however, subsequent studies concluded that these mutations are likely to be markers of systemic fcov infection rather than fip per se (porter et al., ; barker et al., ) . two alphacoronaviruses, both belonging to subgenus minacovirus, are currently known in mustelids, namely mink coronavirus (mcov- ) and ferret coronavirus (frcov). mcov- has been recently identified as the etiological agent of mink epizootic catarrhal gastroenteritis (ecg), an infectious disease of farmed american (neovison vison) and european (mustela lutreola) mink first described in (larsen and gorham, ) and later affecting several million mink in different countries (vlasova et al., ) . the disease is observed at greater frequency in mink of ≥ months and is characterised by seasonality, high morbidity (approaching %) and low mortality (< %). recent full-genome analysis demonstrated that mcov- is phylogenetically distant from ccovs and fcovs, being closely related to frcov (vlasova et al., ) . presently, the two viruses are considered separate species within subgenus minacovirus (https://talk.ictvonline.org/taxonomy/). frcov has been recognised as the causative agent of epizootic catarrhal enteritis (ece), first described in in domestic ferrets (mustela putorius furo) in the eastern part of the usa (williams et al., ) and subsequently reported in domestic and laboratory ferrets throughout the world (murray et al., ) . analogous to fcov, frcov exists in two different pathotypes: i) ferret enteric coronavirus (frecv) is associated to ece, a highly contagious diarrhoeal disease also known as green slime disease, which affects mainly young ferrets with morbidity and mortality rates similar to those of ecg; ii) ferret systemic coronavirus (frscv) is responsible for a systemic diseases of ferrets, which is characterised by pyogranulomatous perivasculitis and peritonitis resembling to those of fip (murray et al., ) . similar to fip, wise et al. ( ) have shown that frecv and frscv differ significantly in spike protein and that deletions in frcov c may also correlate with the severe pathotype of frscv. recombination in the s, c and e genes between different frcov has been also reported (lamers et al., ) . different covs were found to circulate in wild carnivores. ccovs were detected in wolves (canis lupus), red foxes (vulpes vulpes), eurasian otters (lutra lutra), common genets (genetta genetta) (alfano et al., ; rosa et al., ) . ccov-like viruses were also found in african wild carnivores, including spotted hyenas (crocuta crocuta) and silver-backed jackals (canis mesomelas) (goller et al., ) . fcovs have a wide circulation in non-domestic felids (kennedy et al., (kennedy et al., , , with fip cases being reported in servals (felis serval) (juan-salles et al., ) , cheetah (acinonyx jubatus) (kennedy et al., b) , mountain lion (puma concolor) (stephenson et al., ) , and european wildcat (felis silvestris) (watt et al., ) . divergent alphacoronavirus- viruses were detected in chinese ferret badger (nyctereutes procyonoides) and raccoon dog (melogale moschata) (dong et al., ) . the same study reported the identification in asian leopard cat (prionailurus bengalensis) and chinese ferret badger of an unclassified cov, which was closely related to gammacoronaviruses in most parts of the genome, whereas the s gene displayed the highest sequence identity to alphacoronaviruses (dong et al., ) . with the discovery of deltacoronaviruses, these viruses were later included in this novel genus along with avian and porcine strains (woo et al., ; wang et al., ) . some domestic and wild carnivores are also susceptible to sars-cov infection. while the potential natural reservoirs are horseshoe bats, sars-like cov strains were found to be widespread in masked palm civets (paguma larvata) and raccoon dogs, which were suspected to be intermediate hosts (guan et al., ) . full-genomic comparative analysis has shown that sars-like covs isolated from palm civets are under strong selective pressure and are genetically most closely related to sars-cov strains infecting humans early in the outbreaks (song et al., ) . sequence analysis of the sars-cov-like virus in masked palm civets indicated that they were highly homologous to human sars-cov with nucleotide identity over . %, indicating the virus has not been circulating in the population of masked palm civets for a very long time (shi and hu, ) . a chinese ferret-badger (melogale moschata) was found to have neutralising antibodies against sars-cov (guan et al., ) , whereas sars-cov rna was detected in naturally infected cats and red foxes (vulpes vulpes), but not in domestic dogs . there was, however, a single dog testing positive for sars-cov (https://apps.who.int/iris/bitstream/handle/ / /who_cds_csr_gar_ . _eng.pdf). among carnivores, sars-cov- is able to infect cats, ferrets and, at a lesser extent, dogs . in , a highly divergent cov, tentatively named sw , was discovered a deceased beluga wale (delphinapterus leucas) with pneumonia and hepatic necrosis (mihindukulasuriya et al., ) . the virus was only distantly related to ibv, so that it now represents the prototype of the single mammalian cov species belonging to the genus gammacoronavirus, namely beluga wale coronavirus sw (bwcov-sw ) (subgenus cegacovirus). few years later, related gammacoronaviruses were retrieved from faecal samples of three indo-pacific bottlenose dolphins (tursiops aduncus), which were named bottlenose dolphin cov (bdcov) hku . comparative genome analysis showed that bdcov-hku and bwcov-sw have similar genome characteristics and structures, displaying a % nucleotide sequence identity each to other (woo et al., ) . a novel betacoronavirus distantly related to mers-cov was detected in the faeces of european hedgehogs (erinaceus europaeus), an insectivorous mammal belonging to a related order of chiroptera, from germany. the virus was tentatively referred to as erinaceus cov (ericov) (corman et al., b) and covs found in hedgehogs in france, england and italy had an identity from % to % with the ericov (monchatre- leroy et al., ; saldanha et al., ; delogu et al., ) . these hedgehog covs are are now included in a unique species, hedgehog coronavirus (subgenus merbecovirus). the virus was not associated to any form of disease, so that western european hedgehog is a reservoir host of ericov in the absence of apparent disease, suggesting that hedgehogs in addition to bats may contribute to the evolution of merbecovirus (saldanha et al., ) . a slightly divergent merbecovirus was later found in amur hedgehogs (erinaceus amurensis) in china and was poposed as a prototype of a separate species, namely erinaceus amurensis hedgehog coronavirus hku (ea-hedcov hku ) . a novel coronavirus, named wénchéng shrew coronavirus (wesv) was detected in shrews (suncus murinus) in china . wesv is highly divergent from other alphacoronaviruses, exhibiting less than . % amino acid similarity to any known members of the genus alphacoronavirus in the coronavirus-wide conserved domains of the replicase polyprotein pp ab and less than . % amino acid similarity to the other three coronavirus genera. however, taking into account the current ictv criteria, wesv is sufficiently divergent to be considered a distinct member of the genus alphacoronavirus, but not a new genus of the subfamily orthocornavirinae . covs have been known in veterinary medicine since many decades; some of these viruses, such as ibv, swine enteric covs, bcov and mustelid covs, can cause diseases that have a great impact on the farm industry. other covs, namely fipv, frscv and mhv, cause severe disease in companion (cats, ferrets) or laboratory (mice) animals. animal covs are paradigmatic on how covs evolve through accumulation of point mutations and homologous (and heterologous) recombination, generating different genotypes and pathotypes. these virus variants may have different antigenic properties, escaping the host immunity induced by vaccines, as is the case of ibv. alternatively, they may have a different tissue tropism in the same host that can increase or decrease the virus pathogenicity, as observed for the virus pairs fecv/fipv or frecv/frscv and tgev/prcov, respectively. in other circumstances, the cov evolution may result in the switch of the host range from one animal species to another one or from animals to humans. the former event is well documented in veterinary medicine, with a plethora of viruses being originated from ibv and bcov that adapted to different animal species. however, the most interesting scenario is the jumping and further adaptation of an animal cov to humans. there is increasing evidence that all hcovs currently known recognise an animal origin, with bat or rodent covs being the most probable ancestors. in most instances, it was suggested that other mammals served as intermediate hosts prior to final adaptation to humans, i.e., alpacas and cattle for the low-pathogenic hcov- e and hcov-oc , respectively, and wild carnivores and dromedary camels for the high-pathogenic sars-cov and mers-cov, respectively. other two hcovs, namely hcov-nl and hcov-hku , were likely derived from bats and rodents, respectively, but whether this transmission required an intermediate mammalian host is presently unknown. the origin of sars-cov- should be zoonotic, since highly related sequences were detected in bats, but a definitive intermediate host has been not identified so far. what should we expect from the current pandemic? when hcov-oc crossed the species barrier to infect humans from domestic livestock around , an epidemic of respiratory infection was recorded. even though, several years later, influenza was suspected to be the cause of it, in that pandemic involvement of central nervous system was more pronounced than in other influenza outbreaks. this evidence is further supported by molecular studies claiming that the most recent common ancestor of bcov and hcov-oc emerged around (vijgen et al., ) and by the fact that hcov-oc can be neuroinvasive (arbour et al., ) . likely, hcov-oc crossed species to infect dogs becoming established in this species as crcov . a similar scenario could be observed with sars-cov- with dogs and, at a greater extent, cats. apparently, cats represent, within the domestic animals which have been experimentally infected, the host, together with ferrets, which is able to sustain more efficiently sars-cov- replication . furthermore, based on structural studies and biochemical experiments, sars-cov- seems to have an rbd that binds with high affinity to ace also from ferrets and cats (andersen et al., ) . reasonably, a full comprehension of the animal cov molecular evolution, host range and pathobiology is beneficial to better understand the mechanism driving the emergence and adaptation to humans of zoonotic covs. the present review has highlighted that in the last years, also thanks to the availability of novel sequencing technologies, we have witnessed a large number of novel covs being discovered in a large number of animals. truth to be told, it was difficult for us to summarise, in this single review, all covs detected in animals and the tight interaction existing between them and human covs. among animals, it is evident that bats are the group of mammals that harbor the largest number of covs and that many other animal covs recognise their ancestors in bat covs. in an excellent review (cui et al., ) written by the group coordinated by dr. zheng-li shi of the wuhan institute of virology, hubei, (china), city infamously known for being the epicenter and origin of the covid- outbreak, authors stated that "...given the prevalence and great genetic diversity of bat sars-rcovs, their close coexistence and the frequent recombination of covs, it is expected that novel variants will emerge in the future". this forecasting statement was not surprising to coronavirologists and it was not, importantly, surprising to those scientists that daily deal with the plethora of viruses existing at the human/animal health interface. although scientists were well aware of this hazard, no substantial actions were taken forward the limitations of strict and repeated contacts between humans and wildlife. indeed, whereas biological mechanisms underlying viral evolution are not under human control, social and cultural habits can be modified accordingly through a deep and pounding informative campaigns. if to the human habits we sum the impact of modern agricultural practices and urbanization and the decrease of vital space for wildlife, it is quite easy to understand that, if countermeasures are not taken, we will face novel serious health emergencies of animal origin in the following years with tremendous social and economic impact on our lives. as clearly demonstrated by the sars-cov- emergence, covs are the main characters of this intricate puzzle characterised by the interactions of viral biological mechanisms and human habits. our review was reasonably prepared also to highlight (once more!) how covs originate, evolve, jump, mutate and infect their host. could have the current covid- outbreak been avoided? answering this question is not relevant now, but actions to avoid the next viral spillover from animals to humans is certainly a priority. this task needs to be coupled with massive genomic surveillance in wild animals not limited to covs. massive sequencing of sars-cov- strains detected in humans and covs of wildlife will help further assess the origin of this novel human pandemic and plan future measures able to reduce the risk of emergence of new cov spillover events. however, additional tasks should be provisionally addressed in order to reduce the risk of future cov pandemic like the current one. these include: i) prevention of animal-to-human infections through a ban of the wet markets and a more friendly management of the environment; ii) studies on cov-host interactions to be performed both in vitro (cell cultures, ex-vivo explants of the respiratory tract) and in vivo (animals susceptible to sars-cov- infection); iii) development of new anticoronaviral drugs and evaluation of their efficacy in cell cultures and animal models. feline infectious peritonitis. abcd guidelines on prevention and management inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding dampened nlrp -mediated inflammation in bats and implications for a special viral reservoir host new chimeric porcine coronavirus in swine feces bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences dentification of pantropic canine coronavirus in a wolf (canis lupus italicus) in italy circulation of pantropic canine coronavirus in autochthonous and imported dogs binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity novel receptor specificity of avian gammacoronaviruses that cause enteritis host tissue and glycan binding specificities of avian viral attachment proteins using novel avian tissue microarrays bovine-like coronaviruses in domestic and wild ruminants the proximal origin of sars-cov- further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus neuroinvasion by human respiratory coronaviruses detection of a virus related to betacoronaviruses in italian greater horseshoe bats isolation and metagenomic identification of avian leukosis virus associated with mortality in broiler chicken global distributions and strain diversity of avian infectious bronchitis virus: a review random nature of coronavirus rna recombination in the absence of selection pressure bats and coronaviruses limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis cultivation of the virus of infectious bronchitis emerging horizon for bat borne viral zoonoses. virus dis characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group recovery and characterization of a coronavirus from military dogs with diarrhea porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus coronavirus genome structure and replication accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence first complete genome sequence of european turkey coronavirus suggests complex recombination history related with us turkey and guinea fowl coronaviruses canine coronavirus highly pathogenic for dogs bats: important reservoir hosts of emerging viruses coronaviruses in poultry and other birds coronavirus avian infectious bronchitis virus infectious bronchitis coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys potential pathogens in feces from unweaned llamas and alpacas with diarrhea feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral c gene spike protein fusion peptide and feline coronavirus virulence a murine virus jhm causing disseminated encephalomyelitis with extensive destruction of myelin. isolation and biological properties of the virus sars-associated coronavirus transmitted from human to pig identification and survey of a novel avian coronavirus in ducks dbatvir: the database of bat-associated viruses detection of specific antibodies to the nucleocapsid protein fragments of severe acute respiratory syndrome-coronavirus and scotophilus bat coronavirus- in three insectivorous bat species comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia coronaviruses in bent-winged bats (miniopterus spp genomic characterizations of bat coronaviruses ( a, b and hku ) and evidence for co-infections in miniopterus bats avian coronavirus in wild aquatic birds a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans detection and characterization of bovine-like coronaviruses from four species of zoo ruminants coronavirus associated with an enteric syndrome on a quail farm mouse dipeptidyl peptidase is not a functional receptor for middle east respiratory syndrome coronavirus infection rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs evidence for an ancestral association of human coronavirus e with bats hosts and sources of endemic human coronaviruses identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus e origin and evolution of pathogenic coronaviruses infectious bronchitis virus variants: a review of the history, current situation and control measures alpha and lineage c betacov infections in italian bats canine coronavirus: not only an enteric pathogen serological and molecular evidence that canine respiratory coronavirus is circulating in italy respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (bubalus bubalis) calves recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs an update on canine coronaviruses: viral evolution and pathobiology recombinant canine coronaviruses in dogs european surveillance for pantropic canine coronavirus molecular surveillance of traditional and emerging pathogens associated with canine infectious respiratory disease eco-virological preliminary study of potentially emerging pathogens in hedgehogs (erinaceus europaeus) recovered at a wildlife treatment and a transmissible gastroenteritis in pigs covid- from veterinary medicine and one health perspectives: what animal coronaviruses have taught us detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern a molecular survey for selected viral enteropathogens revealed a limited role of canine circovirus in the development of canine acute gastroenteritis genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences enteropathogen infections in canine puppies: (co-)occurrence, clinical relevance and risk factors coronaviruses detected in brazilian wild birds reveal close evolutionary relationships with beta-and deltacoronaviruses isolated from mammals detection of a group coronavirus in dogs with canine infectious respiratory disease bat coronaviruses in china a previously undescribed coronavirus associated with respiratory disease in humans evidences of parasite evolution after vaccination imperfect vaccines and the evolution of pathogen virulence a hemagglutinating virus producing encephalomyelitis in baby pigs metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in china isolation and characterization of a bat sars-like coronavirus that uses the ace receptor a metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in neoromicia bats within south africa coronavirus genotype diversity and prevalence of infection in wild carnivores in the serengeti national park the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- . coronaviridae study group of the international committee on taxonomy of viruses animal models for sars and mers coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china three emerging coronaviruses in two decades turkey coronavirus enteritis characterization of a coronavirus isolated from a diarrheic foal a new virus isolated from the human respiratory tract middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation rodent reservoirs of future zoonotic diseases isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe coronavirus infections in horses in saudi arabia and oman dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor emerging highly virulent porcine epidemic diarrhea virus: molecular mechanisms of attenuation and rational design of live attenuated vaccines bat origin of human coronaviruses discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states a bat-derived putative cross-family recombinant coronavirus with a reovirus gene genetically diverse coronaviruses in wild bird populations of northern england studies on broiler's ibv and ib-like virus from guinea fowl therapeutische, kasuistische und statistische mitteilungen aus der klinik für kleine haustiere an der reichstierarzneischule in utrecht (holland) analysis of the genome sequence of an alpaca coronavirus molecular identification and characterization of novel coronaviruses infecting graylag geese anser anser feral pigeons columbia livia and mallards anas platyrhynchos identification of avian coronavirus in wild aquatic birds of the central and eastern usa an outbreak of feline infectious peritonitis in captive servals (felis serval): clinical, pathological, and immunohistochemical findings calf diarrhea coronavirus. the lancet molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis detection of feline coronavirus infection in captive cheetahs (acinonyx jubatus) by polymerase chain reaction detection of feline coronavirus in captive felidae in the usa detection of feline coronavirus infection in southern african nondomestic felids surveillance of avian coronaviruses in wild bird populations of korea a novel coronavirus associated with severe acute respiratory syndrome identifying sars-cov- related coronaviruses in malayan pangolins naturally occurring recombination in ferret coronaviruses revealed by complete genome characterization a new mink enteritis: an initial report severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku belonging to a novel betacoronavirus subgroup recent transmission of a novel alphacoronavirus, bat coronavirus hku , from leschenault's rousettes to pomona leaf-nosed bats: first evidence of interspecies transmission of coronavirus between bats of different suborders discovery of a novel coronavirus, china rattus coronavirus hku , from norway rats supports the murine origin of betacoronavirus and has implications for the ancestor of betacoronavirus lineage a discovery and sequence analysis of four deltacoronaviruses from birds in the middle east reveal interspecies jumping with recombination as a potential mechanism for avian-to-avian and avian-to-mammalian transmission identification of a novel betacoronavirus (merbecovirus) in amur hedgehogs from china. viruses wild boars harbouring porcine epidemic diarrhea virus (pedv) may play an important role as a pedv reservoir detection of coronaviruses in bats of various species in italy novel avian coronavirus and fulminating disease in guinea fowl isolation of avian infectious bronchitis coronavirus from domestic peafowl pavo cristatus and teal anas gain, preservation, and loss of a group a coronavirus accessory glycoprotein molecular characterization of a canine respiratory coronavirus strain detected in italy a new member of the pteropine orthoreovirus species isolated from fruit bats imported to italy novel coronavirus (sars-cov- ) epidemic: a veterinary perspective longitudinal surveillance of betacoronaviruses in fruit bats in yunnan province discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus sequence of mouse hepatitis virus a mrna : indications for rna recombination between coronaviruses and influenza c virus canine infectious respiratory disease: new insights into the etiology and epidemiology of associated pathogens first full-length sequences of the s gene of european isolates reveal further diversity among turkey coronaviruses recovery in tracheal organ cultures of novel viruses from patients with respiratory disease ecosystem change and zoonoses in the anthropocene middle east respiratory syndrome coronavirus in bats, saudi arabia a sars-like cluster of circulating bat coronaviruses shows potential for human emergence identification of a lineage d betacoronavirus in cave nectar bats (eonycteris spelaea) in singapore and an overview of lineage d reservoir ecology in se asian bats serologic assessment of possibility for mers-cov infection in equids identification of a novel coronavirus from a beluga whale by using a panviral microarray european surveillance of emerging pathogens associated with canine infectious respiratory disease identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs porcine hemagglutinating encephalomyelitis virus: a review a serological survey of canine respiratory coronavirus in new zealand detection and full genome characterization of two beta cov viruses related to middle east respiratory syndrome from bats in italy diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain prevalence and phylogeny of coronaviruses in wild birds from the bering strait area (beringia) ferret coronavirus-associated diseases the first detection of equine coronavirus in adult horses and foals in ireland isolation of a coronavirus during studies on puffinosis, a disease of the manx shearwater (puffinus puffinus) isolation and characterization of viruses associated with transmissible enteritis (bluecomb) of turkeys genome organization of canada goose coronavirus, a novel species identified in a mass die-off of canada geese rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats isolamento di un coron-avirus simile da quaglie giapponesi (coturnixcoturnix japonica) con sindrome respiratoria:prima descrizione della malattia ed isolamentodel virus an update on feline infectious peritonitis: virology and immunopathogenesis cross-sectional investigation and risk factor analysis of community-acquired and hospital-associated canine viral infectious respiratory disease complex characterization of pantropic canine coronavirus from brazil pathogenic differences between various feline coronavirus isolates isolation of a porcine respiratory, nonenteric coronavirus related to transmissible gastroenteritis identification of a novel coronavirus in bats 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 enteric coronavirus infection in adult horses two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea the behaviour of recent isolates of human respiratory coronavirus in vitro and in volunteers: evidence of heterogeneity among e-related strains diagnosis of coronavirus in sheep in valdivia province difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan identification of sars-like coronaviruses in horseshoe bats (rhinolophus hipposideros) in slovenia electron microscopy of coronavirus-like particles characteristic of turkey bluecomb disease prevalence of antibodies to selected viral pathogens in wild boars (sus scrofa) in croatia in - and - unveiling patterns of viral pathogen infection in free-ranging carnivores of northern portugal using a complementary methodological approach acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia extension of the known distribution of a novel clade c betacoronavirus in a wildlife host an apparently new respiratory disease of baby chicks transmissible gastroenteritis virus infection: a vanishing specter sialic acid binding properties of soluble coronavirus spike (s ) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus a review of studies on animal reservoirs of the sars coronavirus susceptibility of ferrets, cats, dogs, and different domestic animals to sars-coronavirus- identification of a new transcriptional initiation site and the corresponding functional gene b in the murine coronavirus rna genome cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human from sars to mers, thrusting coronaviruses into the spotlight isolation of infectiousbronchitis virus from pheasants feline infectious peritonitis in a mountain lion a persistently infecting coronavirus in hibernating myotis lucifugus, the north american little brown bat antigenic cross-reactivity between the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus and polyclonal antisera of antigenic group i animal coronaviruses: implication for sars diagnosis isolation and characterization of avian coronavirus from healthy eclectus parrots (eclectus roratus) from indonesia prevalence and genetic diversity of coronaviruses in bats from on the origin and continuing evolution of sars-cov- surveillance of bat coronaviruses in kenya identifies relatives of human coronaviruses nl and e and their recombination history detection of novel sars-like and other coronaviruses in bats from kenya an avian coronavirus in quail with respiratory and reproductive signs gammacoronavirus and deltacoronavirus in quail phylogenomic analyses elucidate the evolutionary relationships of bats enteric coronavirus-like coronavirus particles in sheep s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification a serological survey of selected pathogens in wild boar in slovenia feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses livestock susceptibility to infection with middle east respiratory syndrome coronavirus complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc molecular characterization of a new species in the genus alphacoronavirus associated with mink epizootic catarrhal gastroenteritis surveillance on severe acute respiratory syndrome associated coronavirus in animals at a live animal market of guangzhou in detection and genetic characterization of deltacoronavirus in pigs discovery, diversity and evolution of novel coronaviruses sampled from rodents in china discovery of a highly divergent coronavirus in the asian house shrew from china illuminates the origin of the alphacoronaviruses emerging and re-emerging coronaviruses in pigs an extended outbreak of infectious peritonitis in a closed colony of european wildcats (felis silvestris) coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol high prevalence and putative lineage maintenance of avian coronaviruses in scandinavian waterfowl coronavirus-associated epizootic catarrhal enteritis in ferrets sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent comparative sequence analysis of the distal one-third of the genomes of a systemic and an enteric ferret coronavirus an apparently new syndrome of porcine epidemic diarrhoea global epidemiology of bat coronaviruses characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia molecular diversity of coronaviruses in bats comparative analysis of twelve genomes of three novel group c and group d coronaviruses reveals unique group and subgroup features comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c coronavirus coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases a new coronavirus associated with human respiratory disease in china serosurveillance of viral diseases in korean native goats (capra hircus) receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans structure of coronavirus hemagglutinin-esterase offers insight in corona and influenza virus evolution isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic analysis and surveillance of the coronavirus dominant in ducks in china funding were provided by the italian ministry of health, izs am / rc , ricerca corrente "ngs e diagnostica molecolare in sanità animale: fast d ", recipient alessio lorusso. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . key: cord- - bvwxci authors: clark, k.j; sarr, a.b; grant, p.g; phillips, t.d; woode, g.n title: in vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: bvwxci rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. as an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. this report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. percent adsorptions ranged from . % to . % for rotavirus and . % for coronavirus; while sand (negative control) was < . %. a high affinity binding was present as determined by a low percent desorption ( . – . %). however, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity. gastroenteritis is a clinical term referring to an acute diarrheal disease; in practice however, enteritis is a more correct term because infections are usually limited to the veterinary microbiology ( ) ± small and large intestines. gastroenteritis primarily effects neonatal but also adult animals, birds and humans and is a major cause of disease and often death worldwide. viruses, bacteria, protozoa, toxins and heavy metals are among the unrelated causes of the disease but environmental, immunological, and nutritional factors play an important role as well. to date, viruses that are implicated in this disease complex include adenoviruses, astroviruses, calicivirus-like agents, coronaviruses, parvoviruses, rotaviruses, and toroviruses. it has been shown that viruses are among the most common agents associated with diarrhea of children, calves, (house, ; woode and crouch, ; tzipori, tzipori, , bern and glass, ) and foals (dwyer et al., ) . currently, rotaviruses are the leading cause and coronaviruses are major contributors of acute gastroenteritis in the young of various mammalian and avian species. the livestock industry carries a great financial burden due to the animal deaths and costs associated with prevention and treatment of disease as a result of infections by these two viruses. in epizootic infections of rotavirus, morbidity rates can be as high as ± % and although mortality rates are usually in the range of ± % they can be as high as %. mortality rates are probably dependent upon the virulence of the virus strain, other pathogens present, and the host's phenotype and genotype. implementation of fluid replacement therapy can greatly reduce or eliminate death rates (bywater and woode, ; black et al., ; bywater, ) . gastroenteritis due primarily to rotavirus and coronavirus infections but also to other viruses has a significant economic impact on agriculture. yet, despite the numerous trials and decades of research, effective vaccines are still not available for these or other viral agents that cause gastroenteritis. although, research efforts have appeared promising, vaccine field trials continue to produce inadequate results or fail entirely (de leeuw et al., ; myers and snodgrass, ; snodgrass et al., ; waltner-toews et al., ; yuan et al., ) . the inadequacy of rotavirus vaccines may be explained, in part, by the complex antigenic diversity of the group a rotaviruses (woode et al., , zheng et al., ; hardy et al., ; parwani et al., ; , and by doubts concerning which of the two main antigens, vp or vp , is the most important antigen to induce immune protection (ward et al., ; . in addition, immunizing neonates with modified live rotavirus or coronavirus vaccine is difficult because of interference by colostral antibodies which contain neutralizing activity and effectively block the host's immune response (waltner-toews et al., ) . post infection, the only treatment available is supportive therapy such as fluid replacement. thus, viral gastroenteritis is still largely uncontrolled at present. because of the above problems and the number of enteric pathogens, alternative methods to control infection should be investigated. one of our goals has been to investigate broad spectrum methods which are not discriminatory among various serotypes and strains of viruses. anecdotal data showed that clays have been incorporated into diets for centuries by many different societies to prevent gastroenteric diseases and clays have been used in the treatment of a variety of illnesses including abscesses, acne, allergies, arthritis, headaches, and shingles (abehsera, ) . more recently, a new approach has been directed towards the use of clays and clay minerals to alter the bio-availability of toxins. a hydrated sodium calcium aluminosilicate clay (hscas) has been found to bind aflatoxins and when added to the food greatly reduces the adverse effects of aflatoxin in leghorn and broiler chicks (phillips et al., ) . various clays have also been shown to adsorb and inactivate the thermo-labile (lt) enterotoxins of escherichia coli and the cholera toxin (ct) enterotoxins of vibrio cholerae which has been reviewed recently (ramu et al., ) . there is also evidence of virus adsorption to clays; the viruses most studied include poliovirus, encephalomyocarditis virus and reovirus (kelly et al., ; schaub and sagik, ; taylor et al., ; lipson and stotzky, ; preston and farrah, ) . this paper is the first of the two investigating the use of non-specific methods to inhibit the infectivity of rotavirus and coronavirus. the viruses used in these experiments include bovine rotavirus, ncdv-lincoln strain (fernelius et al., ) and bovine coronavirus, bcv atcc p (mebus et al., ; sharpee et al., ) . culturing and assaying for infectivity were performed in the bsc- cell line for rotavirus and the hrt- cell line for coronavirus. these methods have been described previously (woode et al., ; storz et al., ) . the various adsorbent materials that were tested included: alumina-boehmite (alumina oxyhydroxide), attapulgite (palygorskite), charcoal, kaolinite, manu, muscuvite (mica), various smectites: acid activated clay (x- -h), acid activated polkville, hydrated sodium calcium aluminosilicate (hscas i, hscas ii, and hscas iii), and sodiumbentonite and two zeolites: clinoptilolite and mordenite. sand has low ion exchange ratios and a low surface area which results in low or no adsorption of viruses and thus, was used as a negative control for all experiments. sand was washed prior to use and sand as well as all of the adsorbent materials were autoclaved prior to use. experiments were designed to test the ability of various adsorbent materials to tightly bind bovine rotavirus and coronavirus. bovine rotavirus was chosen to be the model for the experimental assays and as such was tested multiple times with all the adsorbent materials. bovine coronavirus was tested with selected materials; various sorbents were screened while hscas i and ii were repeatedly tested for their ability to bind bcv. hscas i and ii were tested more extensively because of their chemical selectivity which has been well documented (phillips et al., ) . for each adsorption assay, . g of clay was weighed in a centrifuge tube, ml of media was added and then equilibrated for min. the virus (at a titer of ± immunofluorescent focus forming units/ml) was added to each clay sample to result in a final % w/v concentration of clay. two controls were used in each experiment: ( ) media plus virus and ( ) washed sand plus virus. the samples were incubated for min at room temperature on a glas-col rotator at rpm. afterwards, all samples were centrifuged at relative centrifugal force (rcf) of Âg for min. supernatants of each sample were then transferred into snap cap tubes (one sample per tube). the clay sample was reserved for desorption and infectivity studies. all supernatants were titrated from À to À in serum free (sf) media. the original supernatants and each of their dilutions were assayed for infectivity. the pellet of clay that was reserved for the desorption experiment was resuspended in sf-media at a final % w/v concentration of clay. the sample was mixed well and incubated at room temperature for h on the rotator at rpm. (if an infectivity study was to be run on the clay sample, an aliquot of clay suspended in media was removed post incubation.) the sample was centrifuged at rcf Âg for min. the supernatant was then transferred to a snap cap tube and titrated from À to À in sf/media and assayed for infectivity. the suspended samples of clay bound virus or clay/virus complex retained from the adsorption and desorption experiments were used for infectivity testing. an aliquot of the clay sample was removed and placed in a snap cap tube and then titrated from À to À in sf/media. these clay samples were assayed for infectivity. virus dilutions were inoculated ( ml/well, wells/dilution) into microtiter plates of ± days old bsc- cells. microtiter plate(s) were incubated at c for h and then fixed with % acetone in water (À c) for min; the acetone was removed and the plates were dried for approximately h. the plates were rehydrated in pbs prior to a two part staining procedure. plates were stained for h with approximately . ml/well of monoclonal antibody b e (mab e ) which was made to an epitope on vp of b rotavirus. (mab e was diluted : in pbs, ph . .) plates were washed with pbs and then stained with approximately . ml/well of fluorescein-labeled affinity purified antibody-goat anti-mouse igg (gam) for h at room temperature and then washed with pbs. (gam was diluted : in pbs.) the fluorescein-tagged cells were viewed by a leitz inverted uv microscope. titers were calculated by counting fluorescent cells/well or immunofluorescent focus forming units. the methodology described by reed and muench was used to determine the % endpoint (reed and muench, ). virus dilutions were inoculated ( ml/well, wells/dilution) into six well plates of -day-old bsc- cells. after a min absorption time at c, the virus dilutions were removed and ml of nutrient agar (equal volumes of % agar and double strength mem) was added to each well. the plates were then incubated at c for ± days. post incubation, plates were fixed with % formalin for at least h. the formalin and agar was removed and the cells were immediately stained with crystal violet. titers were calculated by counting the number of plaques at each dilution. percent of adsorbed virus, desorbed virus, and infectivity of clay bound virus were calculated. the following symbols were used in the formulas listed below: t v titer of virus t a titer of supernatant from adsorption study t d titer of supernatant from desorption study (dilution factor) t i titer of clay/virus complex from infectivity study a variety of adsorptive materials were tested extensively using bovine rotavirus. charcoal, sodium bentonite, attapulgite, kaolinite, and hscas iii were found to adsorb greater than . % of bovine rotavirus while the other materials assayed ranged in their adsorptive capabilities from less than % to greater than % (table ) . charcoal, sodium bentonite, attapulgite, kaolinite, mordenite, mica, hscas iii were screened for their adsorptive capability of bovine coronavirus; their mean percent adsorption was . %. hscas i and hscas ii tested more extensively due to their chemiselectivity and their mean percent adsorption was found to be . ae . % and . ae . %, respectively. sand was used as a negative control in all adsorption assays. because sand did not adsorb either virus, it was not assayed for desorption or infectivity. all adsorbents tested had a high affinity binding to both viruses as determined by a low percent desorption. mean percent desorptions ranged from . % to . % for bovine rotavirus with charcoal and attapulgite having the lowest percent desorptions (table ) . desorption assays of bovine coronavirus were performed on the chemiselective substances, hscas i and hscas ii; no desorption ( . %) was detected by our assay. table results of ncdv adsorption, desorption, and infectivity assays sample as both bovine rotavirus and coronavirus bound to various adsorbent materials, including hscas i and ii, with high affinity, both were tested for infectivity when in the bound state. however, as indicated by the infectivity assays, neither virus was inactivated by the adsorbent materials. according to the microtiter assay method at least some infectivity remained in all samples of both viruses. mean percent infectivity of clay/ rotavirus complex ranged from . % to . % with charcoal and hscas iii having the lowest percent infectivity (table ) . as with the other assays using coronavirus, only the chemiselective adsorbents (hscas i and hscas ii) were tested. complexes of hscas i/coronavirus and hscas ii/coronavirus both retained % infectivity. the plaque assay method was used to compare the infectivity results of rotavirus and hscas ii to the results in the microtiter assay. the percent infectivity of hscas ii in the plaque assay was . % compared to a mean of . % in the microtiter assay. the plaque assay results confirmed the accuracy of the data from the microtiter assays. we have looked at a variety of inorganic adsorbent materials and have found that all adsorbent agents tested had good ( ± % adsorption) to excellent (> % adsorption) capability of adsorbing bovine rotavirus and excellent capability of adsorbing bovine coronavirus, both with a high affinity. we have characterized a high affinity binding as an adsorbent material that has a high percent adsorption of virus and a low percent desorption. the structure of the adsorbent materials is very complex and the relationship of binding is not completely understood. however, the diameter of rotavirus and coronavirus particles are ± and ± nm, respectively which are significantly larger than the pores of the adsorbent material particles. therefore, the virions must be attached to the outer surface of each adsorbent particle. furthermore, we believe this interaction to be a non-specific binding of protein. this is supported by the binding of viruses including rotavirus, coronavirus, poliovirus, reovirus, and enterovirus, ct and lt enterotoxins, and casein which was found to block the adsorption of ct and lt enterotoxins which has been reviewed previously (ramu et al., ) . adsorbent materials appear promising as a method of adsorbing virus from solution and may be useful as such in filtering aqueous systems. they may have a distinct advantage in prevention of infection and/or disease because most agents that cause gastroenteritis, including rotavirus and coronavirus, are generally contracted orally, usually via contaminated food and water. according to our studies, adsorbent materials will allow between % and % of virions to remain in solution. as a result they may not eliminate infection but may eliminate water borne disease by effectively reducing the infectious dose by greater than %. it was our hypothesis that the prophylactic use of adsorbent agents (feeding adsorbent agents daily in grain) could decrease enteric viral infections by binding with the viral pathogens in the gut and thus reducing the infectious dose. however, after completing the infectivity studies we lack support of this hypothesis. the infectivity data of the adsorbent material/virus complex (over virus alone) indicates that the complexes are not only infectious but many complexes are more infectious than the virus was originally. these results correspond with prior investigations which found that the persistence of reoviruses and enteroviruses is maintained and often enhanced when associated with clay and clay minerals (schaub and sagik, ; lipson and stotzky, ) . there is a potential error of titrating a solid in a liquid; however, the infectivity data is consistent and reproducible. the plaque assay is accepted as a more sensitive assay method ( . ± log dilution) than the microtiter assay. the microtiter assay was used primarily in these studies because it is efficient and conserves time and cost over the plaque assay method. results from the microtiter assay were confirmed by plaque assay using bovine rotavirus. it is our opinion that the adsorbent materials have enhanced the infectivity of the virus. one possibility to explain this observation is an improved presentation of virus to cells. a previous study (bridger and woode, ) indicates that there are two particle types of calf rotavirus: infectious and non-(or low) infectious particles. the complete rotavirus virion is composed of both inner and outer protein shells and is highly infectious as opposed to an incomplete virion which is missing its outer protein shell and is non-infectious. the outer protein shell is composed of viral proteins and (vp and vp ) which are responsible for the binding of virion to cell which is necessary for infection and thus replication. bridger and woode found that the ratios of particle number to infectivity were  : for the complete virion vs.  : for the incomplete virion. our hypothesis for a more efficient presentation of virus is that an increased fraction of these incomplete particles are entering cells as a result of adsorbent materials being engulfed/phagocytosed and thus carrying virus into the cell with them. if this is the case, it may prove to be a method of culturing viruses that are difficult to propagate because they are comprised of a large proportion of incomplete virions. it is difficult of course to speculate what effect this will have in vivo but it is possible that diet may enhance infection. future work includes separating complete and incomplete virions and repeating the infectivity studies, exploring both the reasons for infectivity of the complexed virus/adsorbent materials and the methods to significantly reduce the infectivity levels, and investigating the mechanisms of viral concentration within aqueous systems. characterization of two particle types of calf rotavirus oral fluid replacement by a glucose glycine electrolyte formulation in e. coli and rotavirus diarrhoea in pigs rotavirus infections in calves: efficacy of oral vaccination in endemically infected herds a study of the etiology and control of infectious diarrhea among foals in central kentucky cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in nebraska comparative amino acid sequence analysis of vp for vp serotype bovine rotavirus strains ncdv, b , and uk economic impact of rotavirus and other neonatal disease agents of animals removal of enteroviruses from sewage by activated sludge adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area effect of proteins on reovirus adsorption to clay minerals neonatal calf diarrhea: propagation, attentuation, and characteristics of a coronavirus-like agent colostral and milk antibody titers in cows vaccinated with a modified live± rotavirus±coronavirus vaccine characterization of field strains of group a bovine rotaviruses by using polymerase chain reaction-generated g and p type-specific cdna probes hydrated sodium calcium aluminosilicate: a high affinity sorbent for aflatoxin selective chemisorption and detoxification of aflotoxins by phyllosilicate clay activation thermodynamics of virus adsorption to solids association of enteroviruses with natural and artificially introduced colloidal solids in water and infectivity of solids-associated virions characterization of a calf diarrheal coronavirus passive immunity in calf diarrhea: vaccination with k antigen of enterotoxigenic escherichia coli and rotavirus monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus influence of ph and electrolyte composition on adsorption of poliovirus by soils and minerals the aetiology and diagnosis of calf diarrhoea the relative importance of enteric pathogens affecting neonates of domestic animals a field trial to evaluate the efficacy of a combined rotavirus±coronavirus/escherichia coli vaccine in dairy cattle immunodominance of the vp neutralization protein of rotavirus in protective natural infections of young children antigenic relationships among some bovine rotaviruses: serum neutralization and cross-protection in gnotobiotic calves protection between different serotypes of bovine rotavirus in gnotobiotic calves: specificity of serum antibody and coproantibody responses naturally occurring and experimentally induced rotaviral infections of domestic and laboratory animals immunodominant neutralizing antigens depend on the virus strain during a primary immune response in calves to bovine rotaviruses studies on the role of vp of g serotype rotavirus (b ) in the induction of the heterologous immune response in calves antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus comparative studies of the antigenic polypeptide species vp , vp , and vp of three strains of bovine rotavirus this research was funded by a grant from the texas advanced technology program, grant number - .