key: cord- -u cnsdl authors: lin, z.; kato, a.; kudou, y.; umeda, k.; ueda, s. title: typing of recent infectious bronchitis virus isolates causing nephritis in chicken date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: u cnsdl four isolates of infectious bronchitis viruses (ibv) from chickens with nephritis, were characterized by polymerase chain reaction (pcr) and restriction enzyme fragment length polymorphism (rflp), and were found to be genetically different from the other twelve strains which we previously studied. . amplification of cdna from four isolates. the strains listed at the top of the lanes were amplified by pcr in cycles. ibv m was also amplified as a standard strain. amplified cdnas were analyzed by electrophoresis on . % agarose gel in tris-borate buffer containing . ~tg/ml of ethidium bromide. hinfi digested puc dna was used as the molecular size markers ( //), and their sizes are indicated in base pairs (bp) on the left in this study, essentially the same procedure was used in an attempt to characterize four ibv isolates f- , y- , m-l, and ni- from chickens with nephritis in different endemic areas of japan between and . three of them, f- , m-l, and ni- , were obtained from affected kidneys, while y- was from a gizzard, cdna fragments of the four isolates were amplified by pcr. the amplified dnas comigrated exactly in a . % agarose gel, giving an identical size approximately bp corresponding to the length between the two primers ( fig. ). this indicates that the amplified region was well conserved without apparent deletion or insertion. to see the rflp of these amplified dnas, they were cleaved by each of restriction enzymes (hpaii, maeiii, xhoii, hinfl, scai, ddei, hincii, haeiii, and psti) under the conditions recommended by the enzyme suppliers, and the digestants were run on % polyacrylamide gels. figure shows the representative cleavage patterns of the amplified dna of m- and y- isolates. the dna of m- was cleaved at one site by all of the restriction enzymes, except hpaii and psti (fig. a) . the dna of y- dna was cleaved at two sites by scai and at one site by maelii, xhoii, ddei, and hincii (fig. b) . the differences in cleavage sites between the two isolates are shown in hinfi, scai, and haeiii digestion. the dna from the other two isolates, f- and ni- , gave an identical cleavage pattern with that of the m- isolate, indicating that these three isolates are closely related to each other, and may be derived from a common origin (data not shown). to facilitate the classification of strains, a pairwise comparison of cleavage sites was performed. there are cleavage sites by restriction enzymes as shown in fig. . for each pair of strains, the difference in restriction sites was counted ( - ). the results with four new isolates and previously defined strains were schematically illustrated in fig. . the four new isolates are obviously closer with each other than with the other strains which previously classified (group i-v) [ ] , and therefore classified into a new distinct group (vi). serological data based on the cross-neutralization test measured by plaque reduction in chicken kidney (ck) cell culture showed that the m- strain was not neutralized by the antiserum against any other previous strains (y. kudou, unpubl, data). this suggests that our pcr and rflp data are consistent with serological relationships. the results of the present study, indicate that the four recently obtained ibv isolates causing nephritis are, by our genetic criteria, similar to each other but different from previous isolates, hence are classified together into a new subtype (group vi). because there is no comparison between our japanese isolates and the australian t strain, it is not clear, despite their similar ne- z. lin et al. the grouping from i to vi is essentially according to lin et al. [ ] phrotropic features, if there is any serological or genetic relationship between them. to control ibv infection, it is necessary to survey whether the prevalent ibv strains are similar to or different from current vaccine strains. several investigators have characterized the isolates obtained from ibv outbreaks, and showed that they had a strong relationship with the vaccine strain, suggesting that the prevalent strains may have originated by recombination with live vaccine strains [ , ] . our present observation that the four new isolates used were different from the reference strains including current vaccine strains suggests that they were not derived from the live attenuated vaccine. however, it is totally u n k n o w n and remains to be clarieed how these new strains evolved. oligonucleotide fingerprinting of ribonucleic acids of infectious bronchitis virus strain infectious avian nephrosis (ureamia) in australia occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the netherlands serological comparisons of strains of infectious bronchitis virus using plaque-purified isolants molecular epidemiology of infectious bronchitis virus in the netherlands a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism etiology of an infectious nephritis-nephrosis syndrome of chickens key: cord- -c qcjve authors: faaberg, k. s.; plagemann, p. g. w. title: membrane association of the c-terminal half of the open reading frame a protein of lactate dehydrogenase-elevating virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: c qcjve orf a of lactate dehydrogenase-elevating virus, strain p (ldv-p), encodes a protein of amino acids. eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the c-terminal half of the molecule (amino acids – ) that flank the serine protease domain. cdnas encoding orf a protein segments encompassing transmembrane segments to and its amphipathic c-terminal end as well as the n-terminal amino acids of the downstream orf b protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. the synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. when synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, ph . , and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase k. no n-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. the results indicate that the c-terminal half of the orf a protein represents a non-glycosylated integral membrane protein. potential modes of synthesis and function of the protein are discussed. in addition, the results showed that the synthesis of the orf a protein was generally terminated at its termination codon, but that read-through into the orf b gene occurred with low frequency. summary. orf la of lactate dehydrogenase-elevating virus, strain p (ldv-p), encodes a protein of amino acids. eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the c-terminal half of the molecule (amino acids - ) that flank the serine protease domain, cdnas encoding orf la protein segments encompassing transmembrane segments to and its amphipathic c-terminal end as well as the n-terminal amino acids of the downstream orf lb protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. the synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. when synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, ph . , and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase k. no n-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. the results indicate that the c-terminal half of the orf la protein represents a non-glycosylated integral membrane protein. potential modes of synthesis and function of the protein are discussed. in addition, the results showed that the synthesis of the orf la protein was generally terminated at its termination codon, but that read-through into the orf lb gene occurred with low frequency. lactate dehydrogenase-elevating virus (ldv) belongs to a new group of positive-strand rna viruses, presently classified as genus arterivirus [ ] , which also includes equine arteritis virus (eav), simian hemorrhagic fever virus, and porcine reproductive and respiratory syndrome virus (prrsv) [ , ] . expression of the viral genomes of - kb in infected cells is via the formation of 'coterminal nested sets of or subgenomic mrnas. the major structural proteins of these viruses are translated from subgenomic mrnas , , and . the ' three-quarters of the viral genome encodes two large proteins, la ( - amino acids) and lb ( - amino acids) which are translated from genomic rna. the orf lb protein is expressed via a frameshift mechanism involving a slippery sequence and a pseudoknot [ ] . the orf lb proteins of these viruses possess several common functional motifs, i.e. replicase, helicase, and zinc finger motifs, and probably represent the rna replicases of these viruses. the orf la protein of eav possesses an n-terminal papain-like cysteine proteinase (pcp) that autocatalytically cleaves off the n-terminal -kda end of the protein; (nonstructural protein-l; nsp- ) downstream of the catalytic pcp residues [ ] . the orf la proteins of ldv and prrsv possess two pcps. both are functionally active in cleaving off n-terminal products of about (nsp-la) and about kda (nsp-l[ ), respectively [ ] (see fig. ). in the case of eav, a kda product (nsp- ) is then removed by another cysteine proteinase cp [ ] . in addition, the orf la proteins of these viruses possess a serine proteinase (sp) motif in the c-terminal half of the molecule (see fig. ), and the sp has been suggested to be responsible for the further cleavage of the protein into functional units [ , ] . the function of the orf la proteins is unknown. however, hydrophobic moment analyses of the orf la proteins of ldv, eav, and prrsv have identified similar or potential transmembrane segments in their c-terminal segments of about amino acids [ ] which flank the sp motif, to segments on either side (see fig. ). the predicted structure is unique among virus-encoded proteins and implies some specific function of the orf la proteins in arterivirus replication. the only other proteins with or more transmembrane segments are membrane-associated transport proteins [ , , , ] . in the present study we provide strong evidence that the segment of ldv orf la protein with transmembrane segments - (see fig. ) becomes intimately associated with endoplasmic reticulum (er) membranes during synthesis and that none of its potential n-glycosylation sites becomes glycosylated. our approach has been to in vitro translate mrnas representing portions ofldv orf la in the absence and presence of microsomal membranes and then to examine the membrane association of the protein products and their susceptibility to degradation by proteinases. at the same time we have examined the efficiency of termination of the orf la protein and of frameshift/readthrough at the slippery sequence at the end of ldv-p orf la ( gcu uua aac uguuga... ; slippery sequence and orf la termination codon are underlined; [ ] ). plasmids pbsk-a , pbks ÷ - (g ), and pbks ÷ - carry overlapping segments of the ' end of ldv-p orf la (see fig. ; nt - , - and - , respectively; genbank accession number u ) and have been generated in previous studies [ ] . cdna - also encompasses the y-terminal end of orf lb including the putative pseudoknot that is postulated to play a role in frame-shifting between orf la and lb. cdna - was excised from its bluescript vector with restriction endonucleases bamhi and aatii, incubated with klenow dna polymerase in the presence of deoxynucleotide triphosphates to obtain blunt ends and ligated into the msci site of the pcite- vector (novagen, madison, wi) which supplied an aug initiation codon nucleotides upstream of the orf-la segment in pc . . the dnas of pbsk-a , pbsk÷ - and pc . were linearized and transcribed with t rna polymerases or with t rna polymerase in the case of pbsk-a as described previously [ ] . the integrity of the rna was verified by agarose gel electrophoresis (see later, fig. a ). the transcribed rnas were in vitro translated in a rabbit reticulocyte lysate system in the absence and presence of canine pancreatic microsomal membranes as also described previously [ , ] . for investigating their association with microsomal membranes, the products of translation were incubated and centrifuged under different buffer conditions - , , ] . the translation reaction mixtures were diluted about t -fold with tris-buffered sucrose ( ram sucrose, mm tris-hct, ph . ) or mm sodium carbonate, ph . . the mixtures were incubated on ice for h and then centrifuged for h at about x g at °c. when incubated in isotonic sucrose buffer, the microsomal membranes remain closed vesicles and all proteins associated with the membrane and those located within the vesicles will be located in the membrane pellet (p), whereas proteins synthesized on free ribosomes will be recovered in the supernatant (s). in contrast, when translation products are incubated in mm na÷-carbonate at ph . , the membrane vesicles are disrupted and only integral membrane proteins remain associated with the pelleted membranes, whereas secreted glycoproteins and peripheral proteins are recovered in the supernatant [ ] . after centrifugation, the proteins in the supernatant (s) were precipitated with trichloroacetic acid, washed with acetone, and air dried. the pelleted material (p) was washed once in sterile water. the s and p proteins were suspended in reducing sample buffer and analyzed by tricine sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page) [ ] using . %t: %c or . %t: %c gels (t refers to total percentage of acrylamide and bisacrylamide monomers and c refers to the percentage of crosslinker relative to t [ ] ) as described previously [ ] . only the orf la segment present in each transcript could yield a protein of significant size since the other two reading h-ames in each transcript did not encode any protein longer than amino acids. the main product of the a transcript whether translated in the absence or presence of microsomat membranes was a protein of about kda ( fig. a, lanes and ) . however, the presence of membranes enhanced translation of the a transcript and the product was almost exclusively recovered in the membrane pellet and thus associated with membranes, whereas the protein synthesized in the absence of membranes was mainly recovered in the s fraction. incubation in carbonate buffer, ph . , did not cause a significant release of the protein from the membranes (fig. b, lanes and ) . thus the protein product became integrated into the membranes during membrane-associated synthesis. translation must have been initiated at one of the three aug codons of orf la located close together at the ' end of the transcript, but which one is not clear. none of the three augs are in favorable context for translation [ ] with c rather than a purine at the - position, relative to the a(+ ) of the aug codon but the same is the case for the initiation codons of most of orfs - which function efficiently and specifically in translation initiation [ ] . the termination codon was provided by the vector so that the six terminal amino acids in the protein product were derived from vector sequences. initiation at one of the three aug codons would yield proteins with , or amino acids, respectively. another minor product of about -kda was consistently produced from the a transcript with properties similar to those of the about -kda protein. its nature is unknown. the results for the translation of the - transcript, which also encodes a protein sequence with potential transmembrane segments, were similar to those described for the a transcript. the main product of about kda was recovered in the s fraction when synthesized in the absence of membranes, whereas it was recovered in the p fraction when synthesized in the presence of membranes ( fig. a, lanes and ) . incubation in carbonate buffer, ph . , did not cause a significant dissociation of the protein from the membranes (fig. b, lanes and ) . translation was markedly enhanced by the presence of membranes. since the molecular weight of the product was the same whether the protein was syl~thesized in the absence or in the presence of membranes, none of three potential n-glycosylation sites in the predicted protein (see fig. ) became fig. . analysis of the membrane association of the in vitro translated proteins a , - , and - . the transcripts of the appropriate plasmids were in vitro translated in the absence and presence of pancreatic membranes (-or +, respectively). the reaction mixtures were further incubated in trissucrose, ph . (a), or carbonate buffer, ph . (b) and centrifuged. the proteins in the supernatant (s) and the pellet (p) were analyzed by tricine sds-page using . %t: % gels glycosylated. in contrast, we have shown previously [ , ] , that under the same experimental conditions the orf - glycoproteins of ldv-p become nglycosylated in vitro when synthesized in the presence of membranes. there were also membrane-associated proteins of about , , and kda synthesized from the - transcript (fig. a, lane ) . since the putative transmembrane segments fall in the middle of the expected product (see fig. ), the smaller products could have resulted from premature termination or initiation at more downstream aug codons. the former was probably the case, since there is only one additional in-frame aug codon that could yield a product with putative membrane segments and approximately the size ( kda) of one of the smaller products and it is in very unfavorable context for initiation. processing by the serine proteinase could also not play a role in generating the smaller products since the potential - product does not contain the complete sp motif. the primary findings concerning the a and - proteins were that they are, as predicted by their hydrophobicity profiles, integral membrane proteins and that they are not n-glycosylated. as also predicted by the hydrophobic moment analyses (fig. i) , the pc . product did not become membrane-associated. a transcript of pc . could potentially yield two protein products if a frame-shift occurred at the slippery sequence at nt - (see earlier and fig. ). one protein would be composed of amino acids ( . kda), nterminal amino acids encoded by the vector plus the amino acid long c-terminal end of the orf la protein. the second protein would possess an additional amino acids (about kda), representing the n-terminal end of the orf lb protein plus c-terminal amino acids encoded by the vector. both products were generated. since the kda product possessed twice as many met residues as the kda protein, namely six, the level of radioactivity in the two proteins indicates that the read-through product was synthesized in considerably lower amounts than the terminated orf la protein ( fig. a, lane ). since significant amounts of a kda protein were not produced, little, if any, cleavage of the kda orf lb protein segment from the kda read-through product occurred. the presence of membranes had no significant effect on the translation of the pc . transcript and the products were recovered in the soluble supernatant fraction whether synthesized in the presence or absence of membranes ( fig. a, lanes - ) . the predicted catalytic triad of the sp of the ldv-p orf la protein consists of his- , asp- , ser- [ , ] . his- and asp- are present in the c-terminal end of the a protein. ser- is encoded in cdna - . in order to examine the functionality of the sp and to further examine the membrane association of the orf la protein, we have joined a and - at a styi site in their overlapping segment (see fig. ) and in vitro transcribed/translated the construct. a was excised from pbsk-a using restriction endonucleases styi and ncoi releasing a segment of nt (nt - ). pbks ÷ - was cut with styi and the a segment ligated to it resulting in a clone which represented ldv nts - followed by ldv nts - . this clone was then digested with bamhi and ncol (removing ldv nt - from the ' end), blunt ended and ligated. the resulting construct pbks ÷ a / - possessed the r terminal aug codons of a and potentially encoded a protein of amino acids ( . kda), orf la amino acids - plus c-terminal amino acids encoded by the vector. it encompassed the sp-motif and putative transmembrane segments --- (see fig. ). transcription of the construct yielded a transcript of the appropriate size (fig. a) . translation of the transcript yielded in some experiments a protein of the expected size of about kda (see fig. ), but in many others, the main product had a size of about kda whether or not membranes were present (fig. b, lanes and ) . the reason for this difference is not known. the -kda protein could have resulted from processing of the -kda protein, but, if this was the case, processing must have occurred very rapidly since a time course experiment showed that maximum amounts of product were produced by min fig. . agarose gel electrophoretic analysis of the transcripts of pbks+a / - , pbsk a , pbks÷ - and pc . (a) and in vitro synthesis of protein a / - and analysis of its membrane association (b). the a / - proteins synthesized in the absence and presence of microsomal membranes were analyzed as described in the legend to fig. of incubation. the protein profile changed little, if at all, during the - min period of translation (data not shown) and there was no indication of the formation of an - kda protein that would have been expected to be formed in such a processing step (figs. b and ) . only a minor product of to kda was consistently produced (figs. b and ) . furthermore, the formation of the protein products was the same when all proteinase inhibitors ( gg pepstatin a, gg leupeptin and gg aprotonin per ml, and iam phenylmethylsulfonyl fluoride) were omitted from the translation reaction mixture (data not shown). thus very little, if any, processing by the sp was observed. regardless, when synthesized in the absence of membranes the protein products were recovered in the supernatant fraction, whereas when synthesized in the presence of pancreatic membranes they were recovered in the membrane fraction whether the reaction products were incubated in the buffered sucrose solution or in carbonate buffer, ph . , prior to centrifugation (fig. b, lanes and and and , respectively) . the results clearly indicate that the primary protein products become membrane associated when synthesized in the presence of membranes. furthermore, the lack of increase in size of the primary products when synthesized in the presence of membranes indicates that the proteins were not significantly glycosylated at any of three potential glycosylation sites. in order to further investigate the membrane association of the a / - protein we determined its proteinase resistance after synthesis in the presence of microsomal membranes. large portions of the protein seemed to be protected by membrane association from digestion by typsin, chymotrypsin, or proteinase k. the main digestion product was about kda but there were lower amounts of products with apparent molecular weights of about kda and kda k.s. faaberg and p. g. w. ptagemann fig. . sensitivity of the membraneassociated a / -t proteins to proteinase digestion. the products synthesized in vitro in the presence of membranes were incubated in trissucrose, ph . (a), or carbonate, ph . (b), and then digested for h on ice with trypsin, chymotrypsin or proteinase k (each at a concentration of . ~tg/ml), or without them (control) before analysis by tricine sds-page using a . %t: %c gel (fig. , lanes - ) . the same products were generated by proteinase digestion after incubation of the primary product in carbonate buffer, ph . (fig. b) . the lower recovery of some products after the carbonate treatment does not indicate additional protein digestion since a variable loss was also frequently observed with non-proteinase-treated proteins after carbonate, ph . , treatment, apparently resulting from sticking of the proteins to test tube walls [ ii. the smallest proteinase digestion products varied in size depending on the proteinase used (fig. ) ; it was the smallest after proteinase k digestion. the proteinase digestion products, except for the - -kda proteins, were larger than the segments making up the transmembrane segments - (about llkda) or -- (about kda). this finding indicates that portions of the a / - protein in addition to the transmembrane segments became proteinase resistant as a result of membrane association of the proteins. similar findings have been reported for coronavirus m proteins; protein segments upstream and downstream of their transmembrane segments seem to be resistant to chymotrypsin and protein k attack [ , ] . the same applies to the m/vp- and vp- p proteins of ldv [ ] . in contrast, the ldv proteins when synthesized in vitro in the absence of membranes were completely digested by the three proteinases under the same experimental conditions [ ] . the authenticity of the in vitro synthesized proteins as ldv proteins was confirmed by immunoprecipitation of the products by anti-ldv antibodies. both the about kda and - kda products of the a / - rna were precipitated by incubation with plasma from -month ldv-infected mice (imp) but not by normal mouse plasma (nmp), or monoclonal antibodies to ldv n/vp- or vp- (fig. , lanes - ) . the same applied to the about kda and fig. a , b. immunoprecipitation of in vitro synthesized products of the a / - transcript mouse plasma (imp), anti-vp- mab c . or anti-vp- pmab - as described previously [ ] kda products of the . rna (fig. , lanes - ) . the results are also of interest in that they demonstrate that mice mount antibody responses to the c-terminal half of the orf la protein. in conclusion, the present study shows that at least a large portion of the c-terminal half of the ldv orf la protein represents an integral membrane protein(s). the in vitro synthesis of the portion of the orf la protein with the putative transmembrane segments is enhanced by the presence of microsomal membranes and the protein(s) synthesized in the presence of the membranes becomes inserted into the membrane in a form that is not released by disruption of the membrane vesicles in a carbonate buffer, ph . , and that is largely protected from proteinase digestion. much larger portions of the protein seem to become proteinase resistant than strictly the portions making up the putative transmembrane segments, suggesting intimate association with the membrane. the membrane association of a large portion of the orf la protein suggests that the orf la protein or at least its c-terminal half is synthesized in vivo on rough er. one scenario suggests that the synthesis of the orf la protein starts on free ribosomes, that the n-terminal end is processed autocatalyticalty by the two paps releasing products of and kda, (fig. ; nsp-l~ and , respectively) [ - . this might be followed by the autocatalytic release of another protein (nsp- ) by the action of cysteine protease [ ] . one of the putative transmembrane segments then could function as a processed or uncleaved signal peptide so that the synthesis of the remainder of the orf la protein occurs on membrane vesicles, however, in a manner that prevents n-glycosylation. in contrast, all the n-glycosylation sites in the ectodomains of the orf and orf glycoproteins become glycosylated during membrane-associated in vitro synthesis and the oligosaccharide chains seem to become processed during traverse through golgi membranes in vivo [ , ] . nothing is known about the functions of the orf la protein. however, the integration of at least a large portion of the protein into er membranes suggests one possibility, namely that this process results in the formation of unique double-membrane vesicles that are invariably associated with the replication of all arteriviruses regardless of the nature of the host cell [ , , ] . electron micrographs suggest that these double membrane vesicles originate from rough er by protrusion and detachment [ ] . their formation is a very early event in ldv replication in macrophages and free nucleocapsids first appear among these vesicles before budding into single-membrane cisternae located generally next to golgi membranes [ , ] . these findings combined with the uniqueness of these structures suggests that these double membrane vesicles may play an important role in virus replication. the possibility that comes to mind is a role in the synthesis of viral genomic and subgenomic lnrnas since viral rna synthesis in general seems to be membrane associated. the function of membranes and their associated proteins in viral nucleic acid synthesis is not known, but they might supply some organizational component to the replication complex and/or facilitate rna folding as suggested for rna chaperones [ ] . in the case of the arteriviruses, the orf lb replicase protein is not an integrai membrane protein and probably supplies the catalytic rna replicase functions since it possesses helicase, replicase, and zinc binding motifs [ ] . our results as well as those for eav [ ] suggest that the orf lb protein is synthesized in much smaller amounts than the orf la protein which is typical for ribosomal frame shifting [ ] . our results are consistent with the hypothesis that the orf la and lb proteins provide structural and catalytic functions, respectively. ribosomal frame shifting on viral rnas structure and function ofcytochrome c oxidase revision of the taxonomy of the coronavirus, torovirus and arterivirus genera coronavirus ibv glycopolypeptides: locational studies using proteases and saponin, a membrane permeabilizer new variation on the translocation of proteins during early biogenesis of apolipoprotein b structure and function of mammalian facilitated sugar transporters processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two cysteine proteases equine arteritis virus is not a togavirus but belongs to a coronavirus-like superfamily analysis of membrane and surface protein sequencers with the hydrophobic moment plot disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity the envelope proteins of lactate dehydrogenaseelevating virus and their membrane topography open reading frame of lactate dehydrogenaseelevating virus encodes a soluble, non-structural highly glycosylated and antigenic protein isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum complete genomic sequence and phylogenetic analysis of the lactate dehydrogenaseelevating virus (ldv) the multidrug transporter, a double-edged sword rna chaperones and the rna folding problems molecular-sieve" chromatography of proteins on columns of cross-linked polyacrylamide the scanning model for translation: an update the high amnity na+/glucose cotransporter sequence ofgenome of lactate dehydrogenase-elevating virus: heterogeneity between strains p and c a former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains lactate dehydrogenase-elevating virus and related viruses lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the e glycoprotein of coronavirus mouse hepatitis virus a orf la protein ldv tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from to kda proteolytic processing of the replicase orf la protein of equine arteritis virus replication of lactate dehydrogenase-elevating virus in macrophages. . evidence for cytocidal replication electron microscopic studies on the morphogenesis of prrsv in infected cells -comparative studies we thank brent langley for competent secretarial help. during part of the work ksf was supported by usphs training grant ca . received december , key: cord- -n wnmq authors: nibert, max l.; manny, austin r.; debat, humberto j.; firth, andrew e.; bertini, laura; caruso, carla title: a barnavirus sequence mined from a transcriptome of the antarctic pearlwort colobanthus quitensis date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: n wnmq because so few viruses in the family barnaviridae have been reported, we searched for more of them in public sequence databases. here, we report the complete coding sequence of colobanthus quitensis associated barnavirus , mined from a transcriptome of the antarctic pearlwort colobanthus quitensis. the . -kb plus-strand sequence of this virus encompasses four main open reading frames (orfs), as expected for barnaviruses, including orfs for a protease-containing polyprotein, an rna-dependent rna polymerase whose translation appears to rely on − ribosomal frameshifting, and a capsid protein that is likely to be translated from a subgenomic rna. the possible derivation of this virus from a fungus associated with c. quitensis is discussed. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. the family barnaviridae is currently represented by one species, mushroom bacilliform virus, in genus barnavirus [ ] . the originally reported sequence for mushroom bacilliform virus (mbv) (genbank u . ; also nc_ . ) is from an australian strain of the cultivated button mushroom (basidiomycete) agaricus bisporus [ ] . a closely related sequence for mbv ( % nt sequence identity) (genbank ky . ) has been reported recently from a second strain of a. bisporus [ ] . in addition, the sequence of another apparent member of the genus barnavirus, rhizoctonia solani barnavirus (rsbv ; genbank kp . ), has been reported recently from the phytopathogenic basidiomycete rhizoctonia solani [ ] but has not yet been recognized by the ictv as a member of a separate species. mbv has a positive-sense rna genome that is ~ . kb long [ , , ] . the genome encompasses four main orfs (orf -orf ), respectively encoding a protein of unknown function (p ), a polyprotein that includes protease and vpg domains (p ), an rna-dependent rna polymerase (rdrp; p region), and a coat/capsid protein (cp; p ) [ , [ ] [ ] [ ] [ ] (fig. ). orf partially overlaps orf in the − frame and appears to be expressed as a p + fusion polyprotein dependent on programmed ribosomal frameshifting [ ] . orf does not overlap orf and is expressed instead from a subgenomic rna containing only orf [ , ] . the ~ . -kb genome of rsbv exhibits a coding organization closely related to that of mbv [ ] (fig. ; the presence of orf in rsbv is newly noted here). based on similar genome sizes, coding organizations, and p -and p -region sequence similarities, both mbv and rsbv appear to be most closely related to plant viruses in the unassigned genus sobemovirus [ , ] . sobemoviruses have isometric nonenveloped virions with regular t = icosahedral symmetry and a diameter of - nm [ ] . mbv, in contrast, has nonenveloped virions that exhibit "bacilliform" morphology, with typical dimensions of × nm [ ] and probable t = icosahedral symmetry at the virion ends [ ] . the name barnavirus reflects this bacilliform shape. this type of structure is unusual, but not unique; for example, bacilliform particles with probable t = symmetry are also formed by members of the unassigned genus ourmiavirus [ ] . because the sequences of so few barnaviruses had been reported to date, we decided to search for more of them in public databases. in particular for this report, we did a tblastn search of the transcriptome shotgun assembly (tsa) database at genbank, using the deduced p + fusion polyprotein sequence of mbv as query. these efforts yielded one hit with a strongly significant e-value, e− , suggesting that it represents a novel barnavirus. when this hit sequence (genbank gcib . ) was in turn used as query for a blastx search of the complete nonredundant protein sequences (nr) database at genbank, the top two hits were to mbv and rsbv (e-values e− , identities and %), supporting the preceding suggestion. notably, this apparent new barnavirus sequence derives from the transcriptome of a plant, specifically from leaves of the antarctic pearlwort colobanthus quitensis (kunth) bartl (bioproject prjna ) [ ] . based on the length ( nt) and coding organization of gcib . , relative to those of mbv, we concluded that this apparent new barnavirus sequence is complete or nearly complete at its positive-sense ´ end, but truncated within orf at its ´ end. importantly, the sequence reads from this transcriptome study are available as experiment srx [ ] in the ncbi sequence read archive (sra). we therefore used the terminal sequences of gcib . as queries for megablast searches of srx in an effort to obtain a more complete sequence for the apparent new barnavirus. in this manner, and in subsequent searches that progressively extended the sequence termini, we identified two reads that added nt to the positive-sense ′ sequence of gcib . and, more significantly, reads that combined to add nt to the ′ . the reading frame that includes orf is defined as frame in each genome. orf is defined by bracketing stop codons; the other orfs are defined as spanning from the first in-frame aug codon to the first downstream stop codon. in mbv, three small orfs with potential to encode proteins of - aa each [ ] are shown as rectangles with dashed outlines; a few similarly sized small orfs, with potential to encode proteins - aa each, are also found in cqabv and rsbv but are not located in the same genomic positions and are considered unlikely to be expressed. tm regions in encoded proteins, as predicted by sosui, are shown as diamonds. regions of similarity in encoded proteins to viral proteases (pro), rdrps, or cps, as determined by hhpred, are shown as thick lines within the respective orfs. known cleavage products of the orf -encoded polyprotein of representative sobemovirus sbmv are labeled. the orf spanning nt - in sbmv encodes the essential sobemovirus protein px [ ] , which may be analogous to barnavirus p based on the coding organizations of these viruses. the other ′-proximal orf in sbmv, spanning nt - , has been reported to encode a movement protein (p [ ] ), which is not expected to be present in the fungal barnaviruses sequence. as a result, the consensus sequence for the apparent new barnavirus reported here (genbank mg ) is nt long, appears to be coding complete for orfs - ( fig. ) , and was newly assembled from a total of individual reads (reads per position: mean, ; range, - ). we henceforth refer to this virus as "colobanthus quitensis associated barnavirus " (cqabv ). orf and orf are the two longer orfs in cqabv , as is also the case in mbv and rsbv (fig. ) . in cqabv , their region of overlap between stop codons spans nt (positions - ), with orf in the − frame relative to orf . a putative slippery sequence for − programmed ribosomal frameshifting is located within this region of overlap (see below) and is proposed to allow orf to be expressed as part of a p + fusion polyprotein. the deduced lengths of p and p + are aa and aa, respectively. p and the p region of p + are notable for a region spanning aa positions - with strong similarity to viral serine proteases (top p value, . % from hhpred analysis with defaults at https ://toolk it.tuebi ngen.mpg.de/#/tools /hhpre d [ ] ). the p region of p + , on the other hand, is notable for a region spanning approximately aa positions - with very strong similarity to viral rdrps (top p value, % from hhpred). results comparable to these were obtained for the respective mbv and rsbv proteins. orf and orf are the two shorter orfs in cqabv (fig. ) . p is notable for weak similarity to viral cps across its length (top p value, . % from hhpred), whereas p appears to lack significant similarity to other proteins (top p value, . % from hhpred). results comparable to these were again obtained for mbv and rsbv (including for the revised rsbv p sequence described below). there are no in-frame stop codons ′ to the proposed aug start codon of cqabv orf , and it is therefore possible that its orf translation initiates further upstream, either at a non-aug start codon or at an upstream aug if the current sequence remains ′-incomplete. orf , however, cannot have an upstream initiation site due to the presence of an in-frame stop codon just codons ′ to its proposed aug start codon. the positive-sense sequence of mbv has been previously noted to encompass three smaller orfs (orf -orf ) in addition to the four longer orfs described above [ , ] . whether these smaller orfs are expressed remains open to question. they are not conserved at similar positions in cqabv or rsbv (fig. ) , suggesting to us that they are probably not expressed. in the case of southern bean mosaic virus (sbmv) and other sobemoviruses, an n-terminal portion of the orf encoded polyprotein (called p here) is annotated in gen-bank (e.g., nc_ . ) as having membrane anchor function. using sosui for online transmembrane (tm) prediction (http://harri er.nagah ama-i-bio.ac.jp/sosui /sosui _submi t.html [ ] ), we found that sbmv p indeed has two tm regions predicted near its n-terminus. applying this analysis to cqabv p and mbv p , we obtained similar results: five and three tm regions, respectively, were predicted n-terminal to the protease homology region in each (fig. ) . similar results were also obtained for rsbv , but in its case, a downstream cluster of two tm regions was predicted in p , whereas an upstream cluster of two tm regions was predicted instead in p . no tm regions were predicted in cqabv p or mbv p . based on these findings, we predicted that the reported rsbv sequence contains an assembly error within its region of orf / orf overlap, which has caused the ′-terminal portions of these orfs to be artificially swapped. indeed, by accessing available sra data for rsbv (experiment srx ) and then reassembling the rsbv contig, we discovered that a single nt residue had been clearly omitted between nt positions and of the rsbv sequence reported in genbank kp . (fig. s ), causing the ´-terminal portions of orf and orf to be swapped as we had predicted. by incorporating this missing residue back into the rsbv sequence, not only was orf now shifted into the − frame relative to orf , as found in cqabv and mbv, but also five tm regions were now predicted via sosui in the n-terminal region of rsbv p , vs. none in p (fig. ) . to our knowledge, membrane association by n-terminal portions of the barnavirus p polyprotein has not been suggested previously. these membrane-associating portions of p seem likely to be involved in forming the rna replication compartments of barnaviruses, as known for many other positive-sense rna viruses [ ] . the slippery sequence for − programmed ribosomal frameshifting in the orf /orf overlap region of mbv has been proposed to be (g)_guu_uuu_c, where underlines indicate codon boundaries for orf and the parenthetical g was not initially included [ ] . however, the canonical motif for a − frameshift site is x_xxy_yyz [ ] , where z is any nucleotide except g, yyy is aaa or uuu, and xxx is normally any three identical nucleotides, though a number of exceptions-including ggu-also occur [ ] . thus, the proposed slippery sequence in mbv would have been better identified as g_guu_uuu, i.e., nudged upstream by one nt residue. notably, this g_guu_uuu sequence in mbv is also found at a similar position in rsbv , and the related sequence g_auu_uuu is found not only at a similar position in cqabv but also in some arteriviruses as a variant of the (normally g_guu_uuu) nsp n/nsp tf − /− frameshift site [ ] . given this degree of conservation (fig. ) , we have adopted this putative slippery sequence for deducing the p + fusion polyprotein sequence of cqabv . the revised consensus motif for the barnavirus slippery sequence is thus g_ruu_uuu. for efficient − frameshifting, a suitable slippery sequence is normally followed by an rna structure (stem-loop or pseudoknot), separated from the shift site by a -to -nt spacer sequence [ ] . a predicted long stem-loop has been identified in mbv, but beginning only nt downstream [ ] . this predicted stem-loop, however, includes a bulged residue near the middle of the stem, and the portion of the stem-loop after this residue is separated from the slippery sequence by nt. similarly, rsbv has a predicted stem-loop separated from the slippery sequence by nt, and cqabv has a predicted compact pseudoknot also separated from the slippery sequence by nt. these additional findings (fig. ) increase our confidence that the site for − programmed ribosomal frameshifting has been properly identified for these viruses. the preceding observations suggest that cqabv would be properly classified with mbv and rsbv in family barnaviridae. to address this further, we performed multiple sequence alignments and phylogenetic comparisons. maximum-likelihood trees for p , the p (rdrp) region of p + , or p showed cqabv , mbv, and rsbv clustering apart from the sobemoviruses, which form their own distinct cluster (figs. and s ) . these findings are also largely reflected in global pairwise comparison (needle) scores for these proteins, although the degree of sequence conservation between cqabv and either mbv or rsbv appears fairly low in this type of analysis, especially for proteins p , p and p (table s ). local pairwise comparisons with blastp, on the other hand, yield e-value scores that more clearly indicate the significant sequence similarities between the proteins of cqabv and those of mbv and/or rsbv (table s ) . we conclude from these results that it seems warranted at present to classify all three of these viruses in the family barnaviridae, genus barnavirus. there appears to be some question as to the specific origin of cqabv . within experiment srx , the sequence reads are reported under six different runs, which were in turn derived from six distinct sets of sampled leaves from a mixture of individual plants. only three of these six runs contain nearly all of the cqabv -matching reads (table s ), suggesting that-even within the samples from bioproject prjna -cqabv was largely or completely absent from some samples. individual reads from a second transcriptome study of antarctic pearlwort, bioproject prjna , are also available in the sra, under experiments srx and srx (tsa data from this study were not yet available at the time of this report). when we used the cqabv genome sequence as query for megablast searches of srx and srx , no significant hits were found (all e-values > ). thus, in this second study, cqabv appears to have been absent from all samples. one explanation for this variability in the presence of cqabv is that some of the sampled plants were infected with cqabv , whereas others were not. alternatively, the cqabv -positive samples might have included a symbiont or contaminant infected with cqabv , whereas the cqabv -negative samples did not. the possibility that cqabv was not derived directly from antarctic pearlwort but instead from an associated fungus, is especially intriguing, since both of the other barnaviruses reported to date, mbv and rsbv , have fungal origins. as a test of whether the bioproject prjna transcriptome might include a noteworthy fraction of fungal sequences, we performed a locally run diamond search [ ] to try to identify the top hit (parameter -top ) for each of the , contigs in this transcriptome. the results of this search were that , of the contigs registered a top hit with e-value ≤ e− , for , ( %) of which the top hit was from a plant (kingdom viridiplantae), consistent with this being a plant transcriptome. for , ( %), however, the top hit was instead from a fungus (kingdom fungi), with , ( %) and ( . %) from dikaryan phyla ascomycota and basidiomycota, respectively. in turn, the ascomycete hits were mostly from the classes leotiomycetes ( , ; %), dothideomycetes ( ; . %), eurotiomycetes ( ; . %), and sordariomycetes ( ; . %), and the basidiomycete hits were mostly from the classes agaricomycetes ( ; . %) and tremellomycetes ( ; . %). a locally run blastn search yielded similar results, with , of the contigs these results provide evidence that the prjna transcriptome indeed contains a noteworthy fraction of various fungal sequences, apparently representing a mixture of different dikaryan species. we thus speculate that cqabv was derived from one of these associated fungi, and not directly from antarctic pearlwort. this explains our inclusion of the word "associated" in the name for this new barnavirus. even more broadly, these findings indicate that the prjna transcriptome, like many others reported to ncbi and elsewhere, is probably better identified as a metatranscriptome because it was derived from samples comprising more than its single, primary target organism. unrooted radial phylogram. deduced protein sequences for the p (rdrp) region of p + of barnaviruses (black) and sobemoviruses (gray) were aligned using mafft . (g-ins-i) and then subjected to maximum-likelihood phylogenetic analyses using mod-elfinder, iq-tree, and ufboot [ , , ] as implemented with the "find best and apply" option at https ://www.hiv.lanl.gov/conte nt/ seque nce/iqtre e/iqtre e.html. the following were found to apply: best-fit model according to bic, lg+f+i+g ; model of rate het-erogeneity, invar+gamma with categories; proportion of invariable sites, . ; and gamma shape alpha, . . branch support values (from bootstrap replicates) are shown in %; branches with < % support have been collapsed to the preceding node. the scale bar indicates the average number of substitutions per alignment position. see table s for a summary of abbreviations and genbank numbers the mpi bioinformatics toolkit as an integrative platform for advanced protein sequence and structure analysis ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use expression of the rice yellow mottle virus p protein in vitro and in vivo and its involvement in virus spread mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal fast and sensitive protein alignment using diamond multiple viral infections in agaricus bisporus-characterisation of unique rna viruses and orfans identified by deep sequencing organelle-like membrane compartmentalization of positive-strand rna virus replication factories genomic resources development consortium genomic resources notes sosui: classification and secondary structure prediction system for membrane proteins modelfinder: fast model selection for accurate phylogenetic estimates an essential fifth coding orf in the sobemoviruses identification of diverse mycoviruses through metatranscriptomics characterization of the viromes of five major fungal plant pathogens ultrafast approximation for phylogenetic bootstrap iq-tree: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies virus taxonomy, ninth report of the international committee on taxonomy of viruses family barnaviridae identification of a subgenomic mrna encoding the capsid protein of mushroom bacilliform virus, a single-stranded rna mycovirus mushroom bacilliform virus rna: the initiation of translation at the ´end of the genome and identification of the vpg the nucleotide sequence and genome organization of mushroom bacilliform virus: a single-stranded rna virus of agaricus bisporus (lange) imbach purification and partial characterization of a bacilliform virus from agaricus bisporus: a single-stranded rna mycovirus virus taxonomy, ninth report of the international committee on taxonomy of viruses conflict of interest all six authors declare that they have no conflict of interest.ethical approval this article contains no studies with human participants or animals performed by any of the authors.open access this article is distributed under the terms of the creative commons attribution . international license (http://creat iveco mmons .org/licen ses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -ikco h k authors: moore, k. m.; jackwood, m. w.; hilt, d. a. title: identification of amino acids involved in a serotype and neutralization specific epitope with in the s subunit of avian infectious bronchitis virus date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: ikco h k localization of neutralizing, serotype specific epitopes of infectious bronchitis virus has been difficult because these epitopes are conformationally dependent. we identified amino acids involved in a serotype specific, conformationally dependent epitope by analysis of the s gene of monoclonal antibody-neutralization-resistant mutants. substitutions in the predicted amino acid sequence of these mutants were located at residues and/or . most of the substitutions at residue were from threonine to isoleucine, whereas the substitutions at residue were from arginine to proline, histidine, cysteine, or tryptophan. based on this data, it appears that aa residues at and on the s glycoprotein are involved in a virus neutralizing serotype specific epitope. avian infectious bronchitis virus (ibv), the etiologic agent of infectious bronchitis (ib), causes an upper respiratory tract disease in chickens. in addition to respiratory disease, ibv may cause damage to the reproductive tract, and some strains cause nephritis and urolithiasis. economic losses are due to poor weight gains in broilers, egg production losses, and decreased egg quality in layers and breeders [ ] . multiple ibv serotypes complicate vaccination programs due to a lack of cross protection between serotypes [ , , ] . infectious bronchitis virus, a member of the coronaviridae family, contains the following structural proteins ± a phosphorylated nucleocapsid protein, a membrane glycoprotein, a small membrane protein, and a spike glycoprotein. the spike glycoprotein is post-translationally cleaved into s and s subunits [ ] . the s subunit forms a globular structure anchored to the viral membrane by the s subunit [ ] . the s subunit is associated with virus neutralization, hemagglutination (ha), membrane fusion, and attachment [ , , , , ] . the total number of neutralizing epitopes on the s subunit of the spike glycoprotein is not known. however, based on data obtained using monoclonal antibodies (mab), there appears to be at least three to ®ve neutralizing, conformationally dependent epitopes on the s subunit in different ibv strains [ , , ] . one or more of these epitopes is a serotypic determinant [ ] . for production of molecularly engineered vaccines, it is important to localize areas of the s gene encoding these conformationally dependent epitopes. mab-neutralization-resistant (nr) mutants have been used to identify amino acids (aa) involved in conformationally dependent epitopes in the spike protein of ibv [ , ] . based on aa substitutions in mab-nr mutants, the s subunit was divided into three regions associated with neutralizing, conformationally dependent epitopes. these are designated i (residues ± ), ii (residues ± ), and iii (residues ± ) [ , ] . region i, corresponding to hypervarible region (hvr) i, was associated with a m strain-speci®c, neutralizing, and ha epitope due to a substitution at residue [ ] . other regions of aa variation, hvr ii and residues ± , were associated with neutralizing epitopes in regions ii and iii, respectively [ ] . the objective of this study was to identify aa involved in the serotype-speci®c, neutralizing epitope on the arkansas (ark) dip strain of ibv. this extends the work of kant et al. [ ] and cavanagh et al. [ ] by identifying speci®c aa involved in an epitope [ ] that is neutralizing and serotype speci®c. in this study, the ark dpi strain was used [ ] . the ark dpi strain was adapted to replicate in primary chicken embryo kidney cells (cekc) by serial passages (designated arkp ) until cytopathic effects (cpe) were observed. virus was propagated in developing speci®c pathogen free (spf) chicken embryos at to days of incubation, or on monolayers of primary cekc [ , ] . monolayers were grown in m culture medium (gibco brl) supplemented with % fetal bovine serum (gibco brl), % of f- nutrient mixture (gibco brl), % tryptose phosphate broth (gibco brl), . % of a . % sodium bicarbonate solution (gibco brl), mm hepes (gibco brl), mg/ml of gentamicin sulphate (gibco brl), and . mg/ml of fungizone (atlanta biologics, norcross, ga, usa). monolayers were maintained with the m culture media supplemented as above except % calf serum (gibco brl) was used. monoclonal antibody , previously described, neutralizes the ark serotype of ibv, and is speci®c for a conformationally dependent epitope on the s subunit [ ] . hemagglutination inhibition (hi) tests were conducted by procedures previously described using mab and the ark strain obtained from spafas (norwich, ct, usa) [ ] . the experimental design was based on the work reported by cavanagh et al. and kant et al. [ , ] . brie¯y, mab-nr mutants were selected by mixing virus x  plaque forming units (pfu)/ml) and mab (undiluted, ml neutralizes  pfu of virus) in equal volumes, and incubating at c for h [ ] . the chorioallantoic sac of -day-old developing chicken embryos was inoculated with . ml of the mixture, and incubated for h at c in a humidi®ed incubator. chorioallantoic¯uid was collected, and diluted : with growth medium. ten-fold serial dilutions of mab were mixed with an equal volume of chorioallantoic¯uid, and incubated at c for h. the mixture was inoculated on a monolayer of cekc, and incubated at c for h before the agar overlay was added. plaques were suspended in . ml media, and stored at À c. some mab-nr mutants were plaque-puri®ed twice before viral stocks were made in cekc. reverse transcriptase-polymerase chain reaction (rt-pcr) with primers mibvpcr and nibvpcr amplify a conserved region in ibv con®rming mab nr mutants as ibv [ ] . reverse transcriptase-polymerase chain reaction using primers s oligo h and s oligo h , and restriction fragment length polymorphism (rflp) of the s gene was used to serotype the mutants [ ] . the titer of each mab nr mutant and arkp was determined by limiting dilution in a cekc microtiter assay. ten-fold serial dilutions of the mab-nr mutants were inoculated ( . ml) onto cekc monolayers with . ml of m culture medium supplemented with % calf serum. the titers were determined by the reciprocal of the last dilution with cpe. for virus neutralization, mab was diluted in -fold serial dilutions, and added to equal volumes of tissue culture infectious dose (tcid ) of the mutants. the mixture was incubated for h at c, and mls were added to cekc in -well tissue culture plates. monolayers were observed for days for cpe. reverse transcriptase pcr with the primers s oligo h and s oligo h was used to amplify the s gene [ ] . for sequencing, pcr product from six or more reactions for each sample was combined and puri®ed using microcon columns (amicron inc., beverly, ma). double stranded dna was sequenced using the prism dyedeoxy terminator cycle sequencing kit as recommended by the manufacturer (applied biosystems, branchburg, nj, usa). the majority of automated sequencing was conducted at the usda southeastern poultry research facility (athens, ga, usa), while some was conducted at the molecular genetics instrumentation facility university of georgia, athens, ga, usa). oligo version . (national biosciences, inc., plymouth, mn, usa) was used to design sequencing primers synthesized at the molecular genetics instrumentation facility, to various regions within the s gene of arkp . nucleic acid and predicted aa sequences of the mutants were compared to arkp and ark dpi. analysis of the sequence data and secondary structure predictions were conducted using macdnasis prov . software (hitachi software engineering co., ltd., san bruno, ca, usa). one hundred twenty-four mab-nr mutants were plaque-puri®ed from cekc. of these mutants, were plaque-puri®ed again, and ampli®ed in cekc for characterization. all mutants were identi®ed as ibv by rt-pcr using the primers mibvpcr and nibvpcr. the expected . kilobase pcr product was observed in mutants after rt-pcr with primers s oligo h and s oligo h . restriction fragment length polymorphism patterns of all mutants using restriction endonucleases bstyi, haeiii, and xcmi were characteristic of the ark serotype [ ] . in the microtiter neutralization assay, mab at a dilution of  neutralized tcid of arkp , but neutralization was not observed for any of the mutants. hemagglutination inhibition was not observed for any of the mutants using mab . polymerase chain reaction products from the s gene of ark dpi, arkp and mab nr mutants were sequenced. after passages in cekc, comparison between ark dpi and arkp showed one point mutation at nucleotide translating to an aa substitution from asparagine to tyrosine at residue . sequence comparisons between the parent virus, arkp , and selected mutants, allowed for grouping of the mutants by type from a to h (fig. ) . type fig. . the s subunit is divided into three regions designated by koch et al. [ ] . lines represent s mutant types and the number of mutants each type is in parenthesis. numbers represent the aa substitutions observed in mab nr mutants in which and were the most frequently observed substitutions. (type a nr- ; type b nr- and nr- ; type c nr- , nr- , nr- , nr- , nr- , nr- ; type d = nr- ; type e nr- , type f nr- ; type g nr- ; type h nr- ) a consisted of one mutant containing one aa substitution at residue . type b consisted of two mutants containing a single aa substitution at residue . type c consisted of six mutants containing aa substitutions at both residues and . types d, e, and f consisted of one mutant each, containing aa substitutions at residues and along with other substitutions throughout the s subunit. types g and h consisted of one mutant each, containing an aa substitution at residue along with other substitutions throughout the s subunit. one mutant, nr- , was not grouped because no aa substitutions in the s subunit were observed. the most common aa substitutions were at residues and/or (table ) . transitions caused substitutions at residue replacing threonine (thr), uncharged polar side chain, with isoleucine (ile), nonpolar side chain. transversions or transitions resulted in aa substitutions at . transversions caused substitutions are residue replacing arginine (arg), charged polar side chain, with proline (pro), nonpolar side chain. transitions caused substitutions at residue replacing arg with histidine (his), charged side chain, or arg with cysteine (cys), uncharged polar side chain. two transitions occurred in nr- (type h) causing a substitution at residue in which arg was replaced with tryptophan (trp), nonpolar side chain. silent mutations were observed in several mutants at nucleotides , , , , , , . no predicted n-linked glycosylation sites (asn-x-thr/ser) were gained or removed. due to the conformationally dependent nature of the epitope examined in this study, changes in the secondary structure of mab-nr mutants may be important. the secondary structure for the s subunit was predicted by the robson method using dnasis prov . . comparisons between the parent, arkp , and mab-nr mutants resulted in four different secondary structures (data not shown). those conformational differences were observed between parent and mutant viruses that had either pro, cys, or trp at residue . none of the aa substitutions observed in mab-nr mutants caused addition or substraction of the predicted n-linked glycosylation sites (asn-x-thr/ ser) in the s subunit for the ark serotype. monoclonal antibody-nr mutants were isolated after mixing mab with arkp . the s gene was sequenced and compared to the parent virus, arkp . most of the predicted aa substitutions were observed at residues and . in previous research, aa substitutions in ibv mab-nr mutants were divided into three regions associated with neutralizing epitopes. these regions were designated as i (residues ± ) associated with the hvr i for the mass serotype and ha, ii (residues ± ) associated with the hvr ii for the mass serotype, and iii (residues ± ) associated with a neutralizing epitope [ ] (fig. ) . in our research, substitutions at residues and correspond to region iii designated by koch et al. [ ] . since mab is serotype-speci®c, did not inhibit ha, and the predicted aa substitutions were observed at and , we associate region iii with a serotype-speci®c, neutralizing epitope that is not involved in ha. some of the mab-nr mutants reported herein had more than two aa substitutions. koch et al. [ ] or [ ] also reported that multiple substitutions were observed in some of their mab mutants. predicted secondary structures, used to formulate general ideas about native protein structure, showed conformational differences between the parent virus and mutants containing either pro, cys, or try at residue (data not shown). changes in the predicted glycosylation sites on the spike glycoprotein, which has been reported to be important in virus attachment to host cells [ ] , were not observed. interestingly, nr- did not have any aa substitutions in the s subunit. similarly, in mouse hepatitis virus (mhv) mab-nr mutants selected with a mab speci®c for the s subunit did not have any substitutions in s , though some were observed in s . in mhv, no major conformational changes were observed, and it was suggested that residues in the s subunit interact with residues in the s subunit [ ] . further analysis of nr- may provide more information about interactions between s and s for ibv. in summary, we identi®ed that residues and are involved in a virusneutralizing, serotype-speci®c epitope on the s subunit of ibv using mab-nr mutants. localization of virus-neutralizing serotype-speci®c epitopes from other serotypes of ibv would contribute to our understanding of neutralizing epitopes on the s subunit, and possibly facilitate the construction of molecularly engineered vaccines. membrane and phospholipid binding by murine coronaviral nucleocapsid n protein coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides coronavirus ibv: structural characterization of the spike protein coronavirus ibv: removal of spike glycopolypeptide s by urea abolishes infectivity and haemagglutination but not attachment to cells amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes vaccination of -day-old broilers against infectious bronchitis by eye drop application or coarse spray and the effect of revaccination by spray serologic and crossprotection studies with several infectious bronchitis virus isolates from delmarvareared broiler chickens a laboratory manual for the isolation and identi®cation of avian pathogens single amino acid changes in the s subunit of the mhv surface glycoprotein confer resistance to neutralization by s subunit-speci®c monoclonal antibody cross-immunity in chickens using seven isolates of avian infectious bronchitis virus typing ®eld isolates of infectious bronchitis by the plaquereduction test location of antigenic sites de®ned by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes infectious bronchitis antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions epitopes of neutralizing antibodies are localized within three regions of the s spike protein of infectious bronchitis virus. world veterinary poultry association differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis monoclonal antibodies to the s spike and membrane proteins of avian infectious bronchitis coronavirus strain massachusetts m epitopes on the peplomer protein of infectious bronchitis virus strain m as de®ned by monoclonal antibodies the characterization of epitopes of the spike protein of a nephropathogenic strain of avian infectious bronchitis virus. world veterinary poultry association cross-protection studies with a holland strain (noblis h- ) of infectious bronchitis virus coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins we acknowledge dr. bruce s. seal and joyce s. bennett at the southeastern poultry research laboratory, usda, ars for their assistance with automated sequencing. we also acknowledge dr. pedro villegas and laura kelley for their assistance with virology. key: cord- -vz qvp authors: chitray, m.; de beer, t. a. p.; vosloo, w.; maree, f. f. title: genetic heterogeneity in the leader and p -coding regions of foot-and-mouth disease virus serotypes a and o in africa date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vz qvp genetic information regarding the leader (l) and complete capsid-coding (p ) region of fmd serotype a and o viruses prevalent on the african continent is lacking. here, we present the complete l-p sequences for eight serotype a and nine serotype o viruses recovered from fmdv outbreaks in east and west africa over the last years. phylogenetic analysis of the p and capsid-coding regions revealed that the african isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus. however, similar analysis of the l region revealed random groupings of isolates from serotypes o and a. comparisons between the phylogenetic trees derived from the structural coding regions and the l region pointed to a possibility of genetic recombination. the intertypic nucleotide and amino acid variation of all the isolates in this study supported results from previous studies where the externally located d was the most variable whilst the internally located a was the most conserved, which likely reflects the selective pressures on these proteins. amino acids identified previously as important for fmdv structure and functioning were found to be highly conserved. the information gained from this study will contribute to the construction of structurally designed fmdv vaccines in africa. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. foot-and-mouth disease (fmd) is a highly contagious disease that affects domestic and wild cloven-hoofed animals [ , ] . despite all the information accumulated over the years on many aspects of fmd basic biology, there is still a lack of information regarding fmd virus transmission, maintenance, virulence and host range. although fmd is referred to as a single disease [ ] , the causative agent of the disease, fmd virus (fmdv), consists of seven immunologically distinct serotypes [ , ] . the fmdv serotypes, i.e., a, o, c, asia and the south african territories (sat) types , and , have different global geographical distribution patterns [ - , , , , ] and are endemic in many countries. even on the african continent, the distribution of serotypes is variable, with the sat serotypes occurring in most regions of sub-saharan africa but a and o confined mostly to the central and northern parts of the region [ ] . mortality is usually low, but morbidity can reach % and therefore remains a major economic concern for livestock health in many developing countries and a continued threat to disease-free countries [ ] . the eradication and control of fmdv in africa is complex and difficult due to the role of wildlife in virus spread and maintenance [ ] and the presence of six of the seven serotypes, i.e., a, o, c, sat , sat and sat . serotype c has not been reported since [ ] . fmdv is a non-enveloped virus containing a singlestranded rna genome of positive polarity in the genus aphthovirus of the family picornaviridae [ , , ] . the large open reading frame (orf) of * , nt, which differs in length between the different serotypes [ ] , encodes a single polypeptide, which is co-and posttranslationally cleaved by viral proteases to give rise to the structural and non-structural proteins [ , , , ] . ten of the cleavage events are catalysed by the virally encoded c protease [ , , , ] . translation takes place from a single open reading frame by a cap-independent mechanism at the internal ribosome entry site (ires) [ ] , located in the ' untranslated region (utr). there are two different sites on the rna at which the initiation of protein synthesis occurs, resulting in the generation of two forms of l proteinase (l pro ), lb and the less abundant lab, where lb is the truncated version, which arises after the initiation of translation at the second aug start codon [ ] . lab and lb can cleave the l/p junction and ensure the proteolytic degradation of the cellular cap-binding protein complex (eif g), which results in the shutoff of host translation [ ] . the p region is the viral capsid precursor and consists of the proteins a (vp ), b (vp ), c (vp ) and d (vp ). the antigenicity of the viral particles is dependent on the amino acid (aa) residues that are exposed on the surface of the capsid [ , ] . furthermore, it has been shown that the external capsid proteins play a role in binding to the fmdv cell-surface receptors, i.e., the rgddependant integrins [ , , - , , ] and heparan sulphate proteoglycans (hspgs) [ , , ] . the genetic heterogeneity of the virus, which is due to the lack of a proofreading mechanism during virus replication, has resulted in the occurrence of extensive variability as well as different lineages and antigenic variants within a serotype that have established themselves in different geographical regions [reviewed in refs. - , , , , , , ] . this has resulted in the need for multiple vaccine strains required for each serotype to cover the antigenic diversity when using vaccination as a control option [ ] . in africa and countries bordering europe, the disease is mainly controlled using vaccination and restriction of animal movement. thus, it is imperative to obtain as much information as possible regarding the fmdv prevalent on the african continent to further our knowledge on fmd epidemiology, define genetic relationships of viruses causing outbreaks [ , ] and to enable better control strategies by successful vaccine development. genetic information regarding the leader (l) and complete capsid-coding (p ) region of serotype a and o viruses prevalent on the african continent is lacking, although the sat isolates have been broadly studied in the past [ ] [ ] [ ] ] . for this study, the l and p coding regions for eight fmdv a and nine fmdv o viruses isolated between and were successfully sequenced and analysed using phylogenetic analysis, examination of sequence variability, and identification of highly conserved genomic regions relating to previously identified fmdv functional and structural biological capabilities. non-conservative substitutions were mapped to the available o (o bfs) [ ] and a (a /hol/ ) [ ] capsid structures, and amino acid substitutions that may be involved in antigenic divergence were identified. the sub-saharan african isolates included in this study belong to different topotypes of fmdv serotypes a and o as defined by d sequencing and represent a broad geographical distribution of viruses within east and west africa. the nine fmdv serotype o isolates and eight serotype a isolates were obtained from the institute for animal health, pirbright laboratory, pirbright, united kingdom (table ) . for the purpose of analysis, a select few complete l and p fmdv sequences currently available in genbank were included (table ) . the fmdv type o viruses were passaged for a previous study and were used directly in this study for processing, whereas the fmdv a isolates were first propagated on ib-rs- cells (instituto biologico renal suino cell line, a pig kidney cell line) to obtain a high viral titer. the ib-rs- cells were maintained in rpmi medium (sigma) supplemented with % foetal calf serum (fcs; delta bioproducts) and x antibiotic-antimycotic ( , gibcoÒ), invitrogen). virus was added to prepared cells containing rpmi supplemented with % (v/v) fcs and antibiotic-antimycotic mixture and incubated at °c until complete cpe was attained (after h). clarified cell culture supernatant containing virus was stored at - °c until further use. chinese hamster ovary (cho) cells strain k (atcc ccl- ) were maintained in ham's f- medium (invitrogen) supplemented with % fcs. plaque assays were performed by infecting monolayer cells with the virus for h, followed by the addition of a -ml tragacanth overlay [ ] and staining with % (w/v) methylene blue [ ] . total viral rna was extracted using a modified guanidinium thiocyanate (guscn)-silica method [ ] . the viral rna template was reverse transcribed at °c for h using u of amv reverse transcriptase (promega) and the antisense p primer (wda; '-gaagggcccaggg ttggactc- ') [ ] as described previously [ ] . amplification of the l-p region was undertaken using the antisense p (wda) primer and the sense ncr primer ( '-taccaagcgacactcgggatct- ') followed by pcr reactions using long-template taq dna polymerase (roche) and thermal cycling conditions described by van rensburg et al. [ ] . pcr products of ca. , bp were excised from a % agarose gel and purified using a nucleospin Ò extract kit (macherey-nagel). purified pcr products were sequenced using a genome-walking approach with genome-specific oligonucleotides and an abi prism tm bigdyeÒ terminator cycle ready reaction kit v . (applied biosystems). sequences were analysed using an abi prism genetic analyser (applied biosystems). ambiguous nucleotides (nt) of the l-p sequences were resolved manually and assembled into a contig using the sequencher tm . dna sequence analysis software (gene codes corporation, ann arbor, mi, usa). a consensus sequence representing the most probable nt for each position of the sequence was obtained for each isolate. consensus sequences were translated in bioedit . . dna sequence analysis software [ ] , and the complete l-p nt and aa sequences were aligned using clustalx . . [ ] . hypervariable regions in the complete aa alignment were defined as a linear -aa region containing more than % variable residues. the phylogenetic analysis included the newly determined sequences as well as sequences of non-african serotype a and o isolates obtained from genbank (table ) . maximum-likelihood analysis of the aligned sequences was carried out in paup [ ] under the aikake information criterion. phylogenetic trees were constructed using the neighbour-joining (nj), minimum-evolution (me) and maximum-parsimony (mp) methods included in the mega . program [ ] for the l, a, b, c, d-coding regions separately as well as the full p -coding region. node reliability was estimated by bootstrap replications for nj, me and mp trees, whilst the nucleotide substitution model of kimura -parameter was employed for the nj and me trees and close-neighbour-interchange (cni) with search level in effect for the mp and me trees. mega . [ ] was utilised to determine the nt and aa variation. plots representing the aa variation, hydrophobicity and secondary structures for each protein were drawn using python (http://python.org) and the matplotlib package (http:// matplotlib.sourceforge.net). the number of different amino acids occurring at a specific position was used as a measure of variation, and the hydrophobicity scale of kyte and doolittle [ ] was used to measure relative aa hydrophobicity. the crystallographic protomers of the capsid proteins of o bfs ( fod) [ ] and a /hol/ (( zbe) [ ] were visualized and the surface-exposed residues identified with pymol v . rc pre (delano scientific llc). phylogenetic trees based on the p (fig. ) (fig. ) , the latter belonging to the middle east-south asia (me-sa) topotype based on d phylogeny [ ] . the exception is o/sar/ / , which was isolated in south africa in during an outbreak caused by illegal feeding of swill to pigs [ ] . (fig. ) . furthermore, these p groupings were also observed when me and mp phylogenetic models were utilised (not shown). clustering similar to that of the p region was observed for the separate gene regions, but with low bootstrap support except for b ( (fig. ) . the nt sequence differences in the p -coding region between members of each topotype were typically more than %, similar to the cutoff defined for a topotype [ ] . globally, fmdv serotype a exists in three geographically distinct topotypes, asia, africa and europe-south america (euro-sa), based on the genetic relationships of d sequences [ ] . using the sequence information of the african a isolates together with p sequences of serotype a viruses available in the genbank database, at least two separate clusters were observed for the type a viruses, i.e., non-african and african a isolates, supported by % bootstrap values for all phylogenetic methods used for the p (fig. ) , b, c and d gene regions (supplementary data, s -s ). two east african isolates, a/tan/ / and a/som/ / formed a well-supported subgroup for the p (fig. ) and d nj trees (supplementary data, s ). in addition, there was a consistently strong grouping for three west african isolates, a/nig/ / , a/civ/ / and a/sen/ / , in the p (fig. ) , b, c and d nj analyses (supplementary data, s -s ). the serotype a non-african and african viruses displayed similar genetic variability when compared to serotype o. the intratypic nt sequence variation in an alignment of the -nt p -coding region for type a was calculated to be . %, whilst the corresponding region ( - nt) of type o only revealed . % variable nucleotides. analysis of the a gene region resulted in phylogenetic groupings that differed from those of the p , b, c and d analyses. when performing phylogenetic analysis on the combined o and a dataset, the fmdv a and o isolates did not group strictly according to serotype (supplementary data, s ). for example, three non-african fmdv a strains, isolated from brazil and venezuela (a /agua-rulbos/iso , a /zulia/iso and a /brazil/ iso ), grouped with o viruses from the me-sa, sea and ea topotypes, but with low bootstrap support. as expected, the region encoding a was the most conserved, exhibiting . % variant nucleotides and was the only capsid-coding region with the highest average %ts/tv rate of . % ( table ). in contrast, d had the highest variability of . % and lowest average %ts/tv rate of . % ( table ) . the phylogenetic trees based on the l pro -coding region for the combined serotype o and a dataset had similar tree topologies for the a and o isolates, independent of the phylogenetic methods employed. the nj tree of the l procoding region (fig. ) showed that the viruses did not group strictly according to serotype, in contrast to those areas of fmdv hydrophobicity and aa variation are represented by blue and green lines, respectively. regions of variability or hypervariable sites were defined as sites on the p that had five or more variable aa residues within a window of residues based on the structural proteins. the non-african a and o isolates that form a part of the euro-sa lineage [ ] formed separate subgroupings in the l pro -coding sequence nj tree (fig. ) . the pan-asian isolates formed a separate grouping with high bootstrap support ( % the l pro aa sequence displayed significant variation for a functional protein: . % for the serotype a alignment and . % for the serotype o isolates (table ) . at least . % ( of aa) of the aa residues were variable in the alignment of the structural proteins (translated from the p region) of the serotype a isolates, whilst the (table ) . a systematic analysis of the capsid proteins revealed the variation not to be random but focused in local regions of hypervariability. the most variable capsid region, d, displayed the most regions of hypervariability. figure a shows the hypervariable regions of type o at aa positions - , - , - , - , - . at least seven discrete hypervariable regions ( - , - , - , - , - , - , - ) were identified in d of type a (fig. b) . the conserved n-terminal motif of b, dkkteettl-ledril-ttrnghttsttqssvg, described by carrillo et al. [ ] , was present in the african a and o sequences (results not shown). two hypervariable sites, residues - within the bb-bc loop and - in the be-bf loop, were mapped within b of type o (fig. c) . b of type a displayed the same two hypervariable regions, residues - and - , and a third hypervariable region, - (bh-bi loop; fig. d ). most of the c aa substitutions for type o were concentrated in one hypervariable region, i.e. - . a second region with significant variability worth mentioning was residues - , where three residue positions displayed high entropy and were located within a surface-exposed loop of c (fig. e) . the latter was situated in the b-b 'knob' of c and included the epitope site for serotype o [ ] . at least three hypervariable regions were identified in the type a alignment, i.e. residues - , - and - (fig. f) . the a protein of serotype o was most conserved, with only four variable residues and hypervariable regions that were not common for a (not shown). the amino acids that have previously been identified as critical for fmdv were compared to the complete aa sequence alignment of the african and non-african a and o isolates from this study and are summarized in supplementary data s , showing that the aa residues important for fmdv function are conserved. [ ] and fry et al. [ ] confirmed the importance of the r residue of c for hs binding and cell culture adaptation. fmdv plaque assays in cho-k cells (table ) confirmed that o/ken/ / was the only virus that was able to infect and replicate in this cell line. taking all of the serotype o capsid-sequence data together, of the o isolates had a his residue at position of c, and they might therefore require integrins to replicate in cell culture. amino acid sequence variation in relation to structure vaccines based on a /iraq/ , a/eri/ and o manisa are recommended for the control of fmd in africa [ ] . we examined the variation within the deduced amino acid sequences of the capsid proteins of the african o and a isolates and compared the surface-exposed regions with those of the three recommended vaccine strains. regions with high aa variability in an alignment of the capsid proteins were mapped onto the x-ray crystallographic structures of type a (a /hol/ ; qqp) [ ] and o (o bfs; fod) [ ] viruses. figure shows that the regions of variability were mostly located on surfaceexposed regions of the virion. not all of the aa side chains within a variable region were exposed on the surface. closer inspection of each aa position within a region of hypervariability indicated that positions with high variability had side chains exposed to the microenvironment of the virion. for serotype a viruses, most of the hypervariable regions outside the d bg-bh loop were concentrated around the -fold and -fold axes of the virion and the c-terminus of d (fig. ) and correlated to a large extent with residues previously found to be involved in escape from neutralization by monoclonal antibodies (table ) . furthermore, many of the putative epitopes were probably discontinuous. for example, there was close proximity of b residue and c residues - and - around the -fold pore of the virion (fig. ) . similarly the regions of variability for type o correlated strongly with epitopes previously identified with distribution around the -fold and -fold axes of the virion ( fig. ; table ). the data from the analysis of the complete capsid-coding region, p , as well as the individual capsid-coding regions indicated that very similar tree topologies existed for the different genomic regions when comparing the african a and o viruses with those from other regions of the world. in general, analysis of the entire structural protein-coding region improved bootstrap values relative to d analysis alone. the longer the capsid-coding region included in the analysis, the more accurate the relationship conclusion. this supports the view that sequencing of the entire capsidcoding region, rather than d alone, is desirable in molecular evolution studies. phylogeny based on the nj trees of the p , b, c and d sequences resulted in the grouping of viruses according to serotype. in addition, the a and o virus clusters could be further divided into separate groupings of the african and non-african a and o isolates, which were observed for the p , b, c and d nj, me and mp trees. the separate groupings of the african and non-african a viruses support previous findings for type a viruses. these could be grouped into three major restricted genotypes, i.e., euro-south america, asia and africa, based on d phylogeny (this study only included fmdv a viruses from euro-south america and africa) [ , , ] . similarly, based on d phylogeny, type o viruses were divided into three groups: those originating from asia, europe-south america and the far east [ , , , ] . the p phylogeny therefore supports the three major virus groups within serotype o. the eastern and western african o viruses were grouped together with the sea and me-sa lineages, together with the pan-asia strain [ , , ] , albeit as lineages restricted to geographic regions (east africa- , , , and west africa). furthermore, the phylogeny is indicative of the transboundary spread of fmdv in africa among the east african countries, uganda, kenya, somalia and tanzania, that are in close proximity to each other, which is also true for the west african countries, i.e. nigeria, ivory coast and senegal. the groupings also indicated that the east african and west african viruses fall into separate large groups. another well-supported grouping was observed for the p , b and c trees (all methodologies) for o/uga/ / , o/uga/ / and o/uga/ / , with a maximum of . % nt and . % aa substitutions in any pairwise alignment. this grouping most likely signifies that the outbreak strains re-emerged from older strains that have been maintained in the endemic area since the early s, i.e. from to ( years). there was a difference in the groupings for the a trees when compared to the p and other capsid-coding gene regions where three non-african a isolates clustered with the non-african o viruses (for all phylogenetic methodologies). the phylogenetic tree representing the region encoding the l protein differed from that of the structural proteins where sub-grouping according to serotype was much less apparent, which was consistent with previous findings for this region [ , ] . interestingly, certain a and o african viruses clustered together and also did not separate into geographical regions such as east and west africa as observed for the structural coding regions. for example, bootstrap support of % for the l-region nj tree was observed for the grouping of o/uga/ / , o/uga/ / , o/uga/ / & a/eth/ / , which was not observed with the a, b, c, d and p phylogenetic analysis. this suggests that the african viruses share similarities or are closely related when comparing the l sequences, irrespective of serotype. taking into account the extensive, uncontrolled movement of animals across the borders and the ease of virus spread and infection of multiple serotypes in one animal, the role of recombination events in the genetic diversification of fmdv cannot be excluded. although we did not perform a study on the occurrence of recombination, the similarities present between fmdv a and o l sequences could be due to the occurrence of intertypic recombination events [ , [ ] [ ] [ ] ] . due to the high mutation rates of fmdv, it is likely that even brief epidemics might result in the generation of substantial antigenic variability [ ] . however, the adaptive significance of this variation remains unclear [ ] . the antigenicity of fmdv is attributed to the aa residues that are exposed on the surface of the capsid [ ] . an important immunogenic determinant, the d g-h loop [ ] , exhibited a high degree of variation for the a and o isolates included in this study. consequently, aa changes in this region are most likely involved in the appearance of novel antigenic types. analyses of antigenic sites of picornaviruses have been carried out using neutralising monoclonal antibodies (mabs) to select and screen mab-resistant mutants. sequence analysis of these mutants resulted in the identification of five antigenic sites of serotype o virus, i.e., o kaufbeuren [ , ] , and six sites for the fmdv a viruses [ ] . alignments of the aa sequences of the african a and o viruses indicated that the regions of variability identified corresponded to the known antigenic sites, which points to the fact that the location of antigenic sites are structurally conserved for the african a and o viruses. in addition to these sites, other regions of variability were identified for both the fmdv o and a african isolates from the aa variability plots. these regions could potentially be antigenic determinants, which may be difficult to map by the classical methodology of mab-resistant escape mutants. we have recently shown that an approach combining sequence variation with structural data and antigenic variation results in the reasonably accurate identification of novel antigenic determinants on the virion surface [ ] . the aligned l pro aa sequences displayed marked variation in both the lab and lb regions (not shown); however, despite this variation, the aa residues identified as being critical for the l pro function were highly conserved, i.e., the residues c , h and d required for l pro catalytic activity, the e residue required for l pro autocatalysis, and two his residues (h and h ) important for cleavage of the translation initiation factor, eif g, [ , , , ] . a comparison of the l/p cleavage sequence at the c-terminus of the l protein and n-terminus of the a protein of the fmdv non-african a types revealed a sequence of r(q/w)klk*gagq (* indicates cleaved peptide bond), whereas the african a types included in this study had the sequence k(r)r(k)lk*gagq (results not shown). both the fmdv non-african and african o types revealed a sequence of (k/r)(k/r)l(k/r)*gagq (* indicates cleaved peptide bond) (results not shown). these observations compared well with the l pro / a junction previously described for serotypes a, o and c [ ] , where the residues k(r)r(k)lk(r) at the l pro c terminus and the gagq at the a n terminus were observed. these results suggest that for all the a and o types included in this study, the conserved sequence xxlk(r)*gagq (where x is either k or r) is sufficient for l/p cleavage by l pro . the degree of hydrophobicity/hydrophilicity of the loops connecting the b chains varied between the african a and o surface proteins. hydrophilic b-b loops tend to be exposed on the protein surface, sometimes protruding from the protein core, and are candidates for antibody binding [ ] . overall, the aa sequence variation observed for the fmdv a and o viruses included in this study showed that the a viruses exhibited more variation, possibly indicating that the a viruses evolved rapidly, which supports studies by bachrach [ ] and brooksby [ ] . additionally, tully and fares [ ] showed that among all of the fmdv serotypes, serotype a is the most divergent and that adaptive evolution has occurred in the c protease (involved in rna replication and processing of the polyprotein) and b (involved in membrane rearrangements), which supports the hypothesis of selection for faster replication in serotype a. neff et al. 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antigenic sites molecular epidemiology of footand-mouth disease virus pandemic strain of foot-and-mouth disease virus serotype o foot-and-mouth disease virus serotype a in egypt recent spread of a new strain (a-iran- ) of foot-and-mouth disease virus type a in the middle east foot-and-mouth disease virus leader proteinase: a papain-like enzyme requiring and acidic environment in the active site functional analysis of the internal translation initiation site of foot-and-mouth disease virus mega: molecular evolutionary genetics analysis version . . pennsylvania state university a simple method for displaying the hydropathic character of a protein the complete genome sequence of foot-and-mouth disease virus o/akesu/ strain and its some molecular characteristics structure of a major immunogenic site on footand-mouth disease virus mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease sat type viruses comparisons of the complete genomes of asian, african and european isolates of a recent foot-and-mouth disease virus type o pandemic strain (panasia) direct evaluation of the immunodominance of a major antigenic site of foot-and-mouth disease virus in a natural host genetic and antigenic analysis of type a foot-and-mouth disease viruses isolated in india during - role of rna structure and rna binding activity of foot-and-mouth disease virus c protein in vpg uridylylation and virus replication foot-and-mouth disease virus virulent for cattle utilizes the integrin avb as its receptor high efficiency utilization of the bovine integrin avb as a receptor for foot and mouth disease virus is dependant on the bovine b subunit the nucleotide sequence of foot-and-mouth disease virus o/fra/ / and comparison with its british parental strain o/ukg/ / the foot-and-mouth disease virus leader proteinase gene is not required for viral replication identification of the active-site residues of the l-proteinase of foot-and-mouth disease virus evidence of genetic recombination in footand-mouth disease virus sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus genetically engineered foot-and-mouth disease viruses with poly(c) tracts of two nucleotides are virulent in mice picornaviridae: the viruses and their replication tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle molecular epidemiology of serotype o foot-and-mouth disease virus isolated from cattle in ethiopia between - comparison of sat- foot-and-mouth disease virus isolates obtained from east africa between and with viruses from the rest of sub-saharan africa study of the genetic heterogeneity of sat- foot-and-mouth disease virus in sub-saharan africa with specific focus on east africa. onderstepoort identification of neutralising antigenic sites on vp and vp of type a foot-and-mouth disease virus, defined by neutralisationresistant variants foot-and-mouth disease type o viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes) molecular epidemiology of serotype o footand-mouth disease virus with emphasis on west and south africa retrospective genetic analysis of sat- type foot-and-mouth disease outbreaks in west africa ( - ) the structures of picornaviral proteinases control and eradication of foot-and-mouth disease structural and mutagenic analysis of foot-andmouth disease virus c protease reveals the role of the b-ribbon in proteolysis paup: phylogenetic analysis using parsimony (and other methods) antigenic sites on foot-and-mouth disease virus type a molecular phylogeny of leader proteinase gene of type a of foot-and-mouth disease virus from india infectious diseases of livestock with special reference to southern africa the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools shifts in the selection-drift balance drive the evolution and epidemiology of foot-and-mouth disease virus lymphocyte recognition elements on the vp protein of theiler's virus genetic heterogeneity in the foot-and-mouth disease virus leader and c proteinase a similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation review of the status and control of foot and mouth disease in sub-saharan africa sequencing and analysis for the full-length genome rna of foot-and-mouth disease virus china/ genetic heterogeneity of fmdv african types a and o acknowledgments this work was supported by the sa-uk collaboration initiative via the department of science and technology. the authors would like to express their gratitude to the personnel at the arc-ovi (tadp) for their contributions to virus isolation. we would like to thank dr. b. blignaut for assistance with plaque titrations. we also gratefully acknowledge dr. o. koekemoer, dr. m. van kleef and dr. n. singanallur for critical reading of the manuscript.open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord- - yh r w authors: choi, c. s.; joo, h. s.; molitor, t. w. title: replication of two porcine parvovirus isolates at non-permissive temperatures date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: yh r w previous studies have shown that replication in vitro of the porcine parvovirus (ppv) isolate, kbsh, was restricted at °c but not at °c. in contrast, replication of the kresse isolate was restricted at °c but not at °c. in this study, kresse and kbsh isolates were passaged up to ten times in swine testicle (st) cells at non-permissive temperatures, and at subsequent passage viral protein synthesis, viral dna synthesis, and progeny virus were evaluated. kbsh became adapted for replication at °c upon serial passages, displaying an appreciable increase in viral progeny, viral polypeptides, and viral dna concentration. this finding was also observed with kresse virus isolate continuously passaged at °c. neither isolate became adapted for replication at °c. in an attempt to examine the effect of in vitro passage at non-permissive temperatures on pathogenicity in swine, kbsh passaged times either at °c or °c was inoculated into swine fetuses. two of four fetuses inoculated with °c-passaged kbsh were dead and hemorrhagic or mummified. all four fetuses inoculated with °c-kbsh contained viral antigen and viral dna. in contrast, fetuses inoculated with °c-passaged kbsh, or with cell culture fluid were normal in appearance. viral antigen and viral dna were not demonstrated in fetuses inoculated with °c-kbsh or cell culture fluids. these findings suggest the possibility that the ability to replicate at °c is associated with virulence in swine fetuses. porcine parvovirus (ppv), a member of the autonomous parvoviruses, causes reproductive failure in susceptible pregnant sows [ , ] . a number of isolates of ppv have been made worldwide, some of which show obvious differences in pathogenicity [ , , ] . in a previous study, four ppv isolates, nadl- , nadl- , kbsh, and kresse, were compared for their replicative properties in vitro at either °c, °c, or °c to explain the differences observed in the replication of these isolates in swine [ ] . nadl- and nadl- are isolates pathogenic only to mid-term gestation fetuses [ , ] when inoculated in utero. these two isolates showed similar replication patterns at °c, °c, and °c. however, replication of kbsh, an isolate which is not pathogenic to swine fetuses [ ] , was restricted at °c, but not at °c or °c. the replication of kresse, an isolate which is pathogenic both to mid-and late-term fetuses as well as to young pigs [ , ] , was favored at °c and restricted at °c and °c. temperature sensitive (ts) mutants have been described for pseudorabies virus [ , ] , measles virus [ ] , coxsackie virus b [ ] , fowl plague virus [ ] , and parvoviruses, h- [ ] , kilham rat virus [ ] , and ppv [ , ] . h- parvovirus ts mutant synthesizes a nonsense capsid protein defective in hemagglutination and exhibits decreased synthesis of viral dna, but not r f -d n a [ ] . ts mutants isolated from nitrous acid-treated k r v [ ] show two distinct groups, one restricted in single stranded (ss) viral d n a and capsid protein production and the other restricted in accumulating rf-dna. low temperature adapted ts ppv mutants were shown to be effective when employed as modified live vaccines [ , ] . revertants of ts mutants have also been described and a possible mechanism for revertants has been suggested in various viruses [ , , , ] . in order to understand the relationship between permissive temperatures for virus replication and their pathogenicity in vivo, virus isolates were serially passaged in vitro at non-permissive temperatures. kresse and kbsh isolates were serially passaged at non-permissive temperatures, °c or °c. these passaged viruses were subsequently evaluated for their abilities to replicate in vitro in st cells and in vivo following in utero exposure to swine fetuses. an important consideration in this study is that the mean body temperature of swine is . °c in difference to . °c for humans. an established cell line, swine testicle (st) cells, was employed throughout this study for the propagation of ppv in vitro. conditions for the culture of st cells were followed as previously described [ ] . the isolates of ppv chosen for this study included kbsh, obtained from dr. p. tattersall (yale university, new haven, ct) and kresse from dr. j. kresse (nvsl, ames, ia). kbsh, previously passaged approximately times in kb cells at °c [ ] , had been propagated twice in st cells at °c in our laboratory before use in this study. kresse, originally isolated from skin lesions of young pigs [t ], was propagated twice in st cells and once in swine fetuses. using these isolates, stock virus was prepared in st cells at °c as previously described [ ] . kresse and kbsh isolates were passaged in st cells either at °c, °c, or °c and changes in virus progeny and virus antigen were evaluated. st cells seeded at x cells per c m flask were infected at an m.o.i, of . infected cells were harvested at days post-infection (pi) by freezing and thawing cycles. for subsequent passages, the virus inoculum was adjusted by hemagglutinating units (hau), hau per cm flask. at each passage level, infectious virus titers were determined by indirect immunofluorescence [ ] and titers were expressed as fluorescent focus unit (ffu) [ ] . in addition, viral protein and viral dna synthesis were determined at selected passages (see below). uninfected st cells were passaged up to times and served as controls. immunoprecipitation of s-methionine radiolabelled polypeptides from infected cell lysates was performed to evaluate viral protein synthesis following procedures previously discussed [ ] with one exception. st cells were infected with an m.o.i, of either at °c, °c, or °c for h. cells were then pulsed for an additional h with gci/ml of smethionine. the extended labelling times were necessary to observe the third structural protein of ppv, vp . antisera used for immunoprecipitation was from rabbits hyperimmuned with csc purified ppv ( . g/ml fraction), nadl- isolate [- ]. to evaluate ppv dna production, slot blot hybridization was performed on hirt extracted dna [ ] . ppv isolates were propagated in st cells either at °c, °c, or °c, for h, followed by dna extraction. extracted viral dna was placed onto nylon membranes contained within a manifold slot blot chamber (schleicher and schuell, keene, nh). the membrane was hybridized with a ppv specific radiolabelled probe following procedures described previously [ ] . to evaluate the influence of temperature adaptation on pathogenicity, °c and °c passaged kbsh were inoculated into swine fetuses. two sows, seropositive to ppv, at days and days of gestation, respectively, were subjected to laparotomies [ ] to expose fetuses for in utero virus inoculation. upon laparotomy it was learned that each sow had only fetuses. four fetuses from each sow were inoculated via the amniotic cavity with . ffu/ . ml of kbsh passaged at either °c or °c. the remaining fetuses were inoculated with uninfected cell culture extract, passaged times at °c without ppv infection. fourteen days later, the sows were euthanized and the uteri were collected. fetuses were observed for gross appearance and the age of death by measuring crown-rump (c-r) length. tissues from each fetus were individually collected and homogenized to a % (w/v) suspension in te buffer ( mm tris and mm edta ph . ). homogenized tissues were tested for ha antigen and viral dna [ ] . relative densities in hybridizations were analyzed by scanning densitometer (shimadzu scientific instrument, columbia, md) at a wavelength of nm. results were graphed comparing relative density to known concentrations of purified ppv rf-dna, as determined by spectrophotometry. the production of infectious virus for both kresse and k b s h either remained constant or increased at subsequent passage level, both at °c and °c, but not at °c ( fig. ). high infectious virus titers, x t o x ffu/ . ml, were maintained at °c through passages for kresse isolate replication (fig. a) . at °c, infectious virus titers increased from x ffu/ . ml at (fig. b) . at °c, low titers of infectious virus titers, x ffu/ . ml, were found at the st passage which increased to x ffu/ . ml at the th passage. again, at °c, infectious virus titers decreased from x ffu/ . ml at the st passage to x ~ ffu/ . ml at the th passage. infectious virus was not detected from uninfected st cells passaged up to times. viral polypeptides appeared as weaker bands at lower passage levels at nonpermissive temperatures, °c for kresse and °c for kbsh, compared to those at permissive temperatures. at subsequent passages, polypeptide bands of both isolates appeared more intense at non-permissive temperatures, °c for kresse and °c for k b s h ( fig. b and c) . this was especially true for vp which was more prominent in passages and indicating more complete full virus particle production. furthermore, bands corresponding to polypeptide vp were more intense at °c than at °c for both isolates. viral polypeptides were undetectable when viruses were passaged at °c, even with longer exposure times ( fig. a) . . radiolabelled s-polypeptides were immunoprecipitated with rabbit anti-ppv sera, electrophoresed, and exposed to x-ray film. numbers on the left hand margin represent molecular weight markers similar findings to polypeptide synthesis were observed for viral d n a synthesis (fig. ) . serial passage at °c for kresse and at °c for kbsh increased hybridization signals. hybridized with a ppv specific p-labelled probe followed by exposure to x-ray film [ ] and st cells [ ] all at °c before in vivo experiments. in this experiment we focused on whether serial passage of k b s h virus affected its ability to replicate in swine viruses. while all fetuses inoculated with °cpassaged k b s h isolate were n o r m a l in appearance, two fetuses (one f r o m each sow) inoculated with °c-passaged k b s h isolate were abnormal; one was slightly hemorrhagic, and another was m u m m i f i e d (table ). to ascertain the table ). all four fetuses inoculated with °c-passaged kbsh isolate exhibited viral antigen and viral dna in various tissues, such as spleen, kidney, liver, lung, and brain. however, no ha antigen or viral dna was demonstrated from tissues of fetuses inoculated with °c-passaged kbsh or cell culture fluid. kresse and kbsh isolates were serially passaged at non-permissive temperatures in an attempt to evaluate the effect of in vitro passages on the pattern of virus replication. this study was prompted by previous work that showed markedly different replication patterns in kresse and kbsh isolates at °c, °c, and °c [ ] . multiple passages of kresse isolate at °c and kbsh at °c yielded higher titers of infectious virus compared to those of non-passaged isolates. however, at °c, low titers of infectious virus were produced up to the th passage with kresse isolate, and decreased infectious virus titer was observed with the kbsh isolate. these findings indicate that two isolates ofppv passaged times became adapted for growth at °c or °c. neither isolate became adapted to growth at °c, following passages. this finding may be due to the slow doubling times of st cells at °c rather than an inability to replicate at such temperature. a possible selection mechanism is that in mixed phenotypes of a virus preparation, one phenotype becomes dependent on temperature. previous studies indicated that restricted replication at a non-permissive temperature may be due to a defect in virus assembly for kbsh, but replication of kresse appeared to be compromised at the initial stage of replication [ ] . since kbsh had been passaged more than times in kb cells [ ] and st cells at °c [ ] , and kresse had been passaged only times in st cells at °c, a possible explanation for temperature dependent replication of each isolate is that one phenotype which can grow at °c for kbsh or at °c for kresse became dominant in the virus preparation. it is possible that these virus preparations consist of two populations of mixed phenotypes. by passage at a non-permissive temperature, phenotypes which can grow at non-permissive temperatures might be generated and became dominant by natural selection. it is unlikely that reversion is caused by mutation because reversion occurred at low passages (less than times). in a previous report [ ] , kbsh was not pathogenic to swine fetuses when inoculated in utero with kbsh. however, it could not be excluded that kbsh upon blind passage in swine fetuses may cause fetal death. the role of di particles in selection mechanism for reversion remains unknown due to a paucity of information on di particles of ppv. analyses of viral polypeptides in this study, at either °c or °c, demonstrated that polypeptide synthesis of both isolates increased by serial passages at non-permissive temperatures. this was especially true with vp suggesting that more full virus particles were produced. alternatively, more effective protein cleavage may occur at °c. although polypeptides corresponding to vp were more intense at °c than at °c for both kresse and kbsh, progeny virus titers were comparable. in ppv, the ratio of empty to full virus particles in cell cultures are often greater than : in cell culture systems compared to : following experimental infection in animals [ ] . previous studies showed that inhibition of ppv replication by empty particles were dependent upon the concentration of empty particles present in virus preparation in celt cultures and in animals [- ]. comparable progeny virus titers, despite differences in the concentration of vp , may be due to higher concentrations of empty particles produced in proportion to the production of more full virus particles. kbsh appeared to gain the ability to replicate in swine fetuses following serial cell culture passages at °c evidenced by fetal death and virus detection in fetal tissues. this result can be supported by previous reports that the replication at near the body temperatures were correlated with virulence in poliovirus [ ] and mouse corona virus [ ] . compared to experimental fetal infection with nadl- or kresse isolate [ , ] , fetal abnormalities were less severe. one of the explanations for this may be the age of fetuses. joo et al. [ ] suggested that the most susceptible age to ppv infection was between and days of gestational age. the age of swine fetuses used in this study was between and days. it should be noted that in difference to nadl- , kresse isolate is highly pathogenic in late term swine fetuses [ ] . another possibility is that there was insufficient adaptation following passages to induce fetal death in late gestational age swine. further experiments with virus passaged greater than times and fetuses at various gestational ages will address this question. kbsh stock virus used in this study was not end-point cloned. because a markedly different growth profile was observed between kbsh and kresse [ ] , this study was focussed on the pathogenic changes of these isolates upon serial passages at nonpermissible temperatures. it may be valuable to characterize the mixed clones by end-point cloning for further study. in conclusion, kbsh and kresse isolates became adapted to °c or °c, respectively, by serial passages. the adaptation was evidenced by increased viral antigen, viral polypeptides, expecially vp , viral dna, and infectious virus. furthermore, kbsh isolate passaged at °c for times now showed the ability to replicate in swine fetuses, indicating that pathogenicity can be recovered by serial adaptation at the mean body temperature of pigs, °c. a temperature-sensitive nmtant of poliovirus type altered in capsid assembly partial characterization of temperature-sensitive mutants of pseudorabies virus pathogenicity of a skin isolate of porcine parvovirus in swine fetuses inhibition of porcine parvovirus infection by empty particles temperature dependent replication of porcine parvovirus isolates nuclear accumulation of measles virus nucleoprotein associated with a temperature-sensitive mutant parvovirus like particles associated with diarrhea in unweaned piglets isolation and characterization of a temperature-sensitive uncoating mutant of pseudorabies virus establishment of an attenuated strain of porcine parvovirus by serial passage at low temperature porcine parvovirus replication preliminary characterization of coxsackie virus b temperature-sensitive mutants parvovirus as contaminants of permanent cell lines. isolation from - porcine parvovirus: replication and inhibition of selected cellular functions of swine alveolar macrophages and peripheral blood lymphocytes establishment of the attenuated strain of porcine parvovirus and its biologicalimmunological characteristics observation of the pathogenicities of porcine parvovirus infection induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a is associated with restriction of viral replication in the brain parvovirus infection in pigs with necrotic and vesicleqike lesions reversion of the attenuated and temperature-sensitive phenotypes of the sabin type strain ofpoliovirus in vaccines pathogenesis of in utero infection: experimental infection of five-week-old fetuses with porcine parvovirus porcine parvovirus: virus purification and structural and antigenic properties of virus polypeptides porcine parvovirus dna: characterization of the genomic and replicative form dna of two virus isolates kbsh parvovirus. comparison to porcine parvovirus temperature-sensitive mutants of fowl plague virus defective in the intraceltular transport of hemagglutinin oronasal and intramuscular vaccination of swine with a modified live pordne parvovirus vaccine: multiplication and transmission of the vaccine virus the reversion of temperature-sensitive mutants of encephalomyocarditis virus upon serial passage in three continuous cell lines covalent associated of protein with replicative form dna of parvovirus h- replication process of the parvovirus h- . v. isolation and characterization of temperature-sensitive h- mutants defective in progeny dna synthesis isolation and characterization of thermosensitive mutants from kilham rat virus, a rodent parvovirus the authors thank dr. m. p. murtaugh for his critical review of the manuscript and the university of minnesota veterinary surgery team for the laparotomies. we are also grateful of s. russell for preparation of the manuscript. this work was supported in part of grants, crsr- - and crsr- - , from the u.s. department of agriculture. received january , key: cord- - lf j lo authors: anderson, kevin; bond, clifford w. title: structural and physiological properties of mengovirus: avirulent, hemagglutination-defective mutants express altered alpha ( d) proteins and are adsorption-defective date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: lf j lo structural and physiological properties of two mutants of mengovirus, and , were compared to those of wild-type virus to understand the molecular basis of changes exhibited in their biological function. two dimensional gel electrophoresis of wild-type and mutant structural proteins revealed alterations in the isoelectric character of the alpha ( d) protein of both mutant and . these data suggest that alterations in the alpha ( d) protein may be responsible for the phenotypic changes by the mutants. a delay in detectable virus-specified protein synthesis was exhibited in mutant-infected cells in comparison to wild-type. the amount of rna synthesized in mutant- and revertant-infected cells was less than that synthesized in wild-type infected cells. changes in virus-specified macro-molecular synthesis in mutant and revertant-infected cells reflected a decrease in the ability of the viruses to attach to cells. biological properties of two mengovirus mutants, and , were compared with those of wild-type virus ( ) . these mutants were defective in their ability to agglutinate erythroeytes, produced smmler plaques in cell culture, were avirulent in mice, but were not temperature-sensitive. these data suggested that changes in the structure of tile mutant viruses may be responsible for the expression of altered phenotypie traits. in this communication, the structurm proteins of the wfld-ty]pe and mutant, viruses were compared by sds-page, peptide mapping, and twodimensional gel electrophoresis to determine which of the mutant structural proteins differed from that of wild-type and the extent of homology among analogous proteins. the structural proteins of two revertants of mutant were also examined by two-dimensional gel electrophoresis to relate changes in biological function to structural differences in the analogous proteins of the mutants and wild-type mengovirus. in addition, the progression of events associated with wild-type, mutant, and revertant virus infection were examined to identify-factors responsible for physiological changes associated with mutant and revertant virus-specified macromolecular s:ymthesis. the growth and assay of wild-type, mutant and revertant viruses using bhk- cells have been described previously ( ) . mutant and revertant virus stocks used in the experiments described below were from the second passage of virus isolated by two successive rounds of plaque purification. purified virions were radiolabeled in vivo with [hc(u)]-l-amino acid mixture ( ~ci/ ml) [new england nuclear (nen), nec- ] were prepared and analyzed by sds-polyacrylamide gel electrophoresis as described previously ( ) . surface tyrosine residues of purified virions were labeled with [ ~i]-sodium iodide [(nen), nez- hi using a modification of the method described by millar and smith ( ) . pellets containing purified virions were resuspended in tn buffer [ mm tris-hc (ph . ), m~ nac ] and quantitated by absorbanee at nm as described ( ) . suspensions of purified virus in tn buffer containing . absorbance units ( nm), ~ci of lesi, and ~g of iodogen (pierce) were mixed in a total volume of . ml and agitated for l minutes. an equal volume of . ~m nai, . ~m -mercaptoethanol was added to the mixture and layered onto sephadex g- columns ( × mm) equilibrated with tn buffer. the excluded volumes were collected and centrifuged in an sw rotor at , rpm for minutes at °c. pellets were prepared for preparative sds-polyacrylamide gel eleetrophoresis (sds-page). virus-specified intracellular proteins were labeled with l-[ s]-methionine, immunoprecipitated and analyzed by sds-page. cells were mock-infected or infected with virus in suspension ( × eells/ml) at an moi of , adsorbed, plated into plastic mm dishes ( . × ceils/dish), and incubated at ° c. cells were pulse-labeled for minutes with ~ci/ml ~s-methionine in . ml methionine-free medium. the cells were washed twice with serum free medium and lysed with . ml b [ mm tris-hcl (ph . ), mm mgci~, . percent np , . percent sds, percent aprotinin, txg/ml ribonuelease a, ~g/ ml deoxyribonuelease] for minutes on ice. cellular lysates were centrifuged at × g for minute at ° c and the supernatant fluid was colleeted and stored at - ° c. b [ m~ tris (ph . ), mm nacl, m~i edta, . percent sodium azide, . percent np , percent aprotinin, . percent bovine sernm albumin]. twelve txl of mouse anti-mengovirus hyperimmune aseitic fluid ( ) was added to the diluted lysates ( . ml volume) and incubated at ° c for minutes. immune complexes were precipitated with t~l of percent fixed staphylococcus aureus (cowan strain) ( ) by incubation at ° c for minutes and pelleted by centrifugation for minute at × g. pellets were washed four times with b buffer at ° c and resuspended in t~l mm dithiothreitol (dtt), percent sds. after minutes of incubation at room temperature, the immunoprecipitated proteins were eluted and reduced by heating at ° c for minutes. bacteria were removed by eentrifugation and the supernatant fluids were prepared for sds-page. virus-specified intracellular rna was labeled with [ , -'~h]-uridine [(nen), net- ]. cells were infected in suspension with virus ( × ceus/ml) at an moi of , plated in plastic mm dishes ( . × cells/dish) following adsorption at ° c for minutes, and incubated at ° c. actinomyein d ( l~g/ml final concentration) was added to each dish at various time points and the cells were incubated at ° c for minutes. the medium was aspirated, replaced with . ml dme containing ~ci/mt '~h-uridine, and the monolayers were incubated at ° c for minutes. at the end of the labeling period, the cells were lysed at ° c for minutes with net buffer [ mm tris-hc (ph . ), mm nac , mm edta] supplemented with percent np . the lysate was centrifuged at × g for i minute. duplicate samples containing i~t of the supernatant fluid were spotted onto whatman gfc glass fiber filters and precipitated in ice cold percent trichloroacetic acid (tca). filters were placed in vials with scintillation fluid [ g / l , diphenyloxazoie (pp ) in xylene], and counted in a packard lsc cd liquid scintillation counter using the pre-set tritium channel ( ). to prepare virus particles containing radiolabeled i~na, cells were infected with virus at an m i of and were labeled at . hpi with ~ci/ml [ , - h]-uridine [(nen), net- ] or ~ci/m p-orthophosphate [(nen), nex- ] in medium containing . rag/ l monosodium phosphate. virus-infected cells were allowed to incubate at, ° c until lysis and virions were purified as described above. a~s-methionine labeled virions were suspended in sample buffer containing . m urea (bio-l~ad), percent. np- (particle data laboratories), percent bio-lyte / (bio-rad), and m~[ dtt as described ( ) and incubated at ° c for minutes. isoelectrie focusing (ief) was performed as described ( ). following equilibration, the tube gels were placed onto percent polyaerylamide slab gels and subjected to sds-page. the ph gradients for ief and nephge were generated from cm serial gel sections equilibrated in mm nac and the ph values were lowered by . ph unit~s to correct for measurement in the presence of urea ( ) . the isoclectric point (pi) value for each protein species was estimated from these gradients. bands corresponding to the structural proteins of mcngovirus were identified by exposure of dried preparative sds-page gels to x-ray film, excised, rehydrated in mm ammonium bicarbonate, . percent sds, and electroeluted as described ( , ) . protein samples were lyophilized and sds was removed as described ( ) . the proteins were dried, oxidized for minutes at ° c with . mt of fresh performie acid and lyophitized three times in distilled water. proteins were anmyzed for purity by sds-page prior to digestion. purified, oxidized proteins were resuspended in . ml of percent ammonium bicarbonate (ph . ) for digestion with n-mpha-p-tosyl-l-lysine ehloromethyl ketonetreated chymotrypsin (tlck-chymotrypsin) at ° c as described ( ), enzyme treated samples were frozen and lyophilized before peptide analysis by thin layer chromatography (tlc). two-dimensional peptide mapping was performed as previously described (ll). lyophilized samples were dissolved in electrophoresis buffer [butanol-pyridine-acetie acid-water ( : : : )] and -- pl was spotted onto cellulose tlc plates (e. merck). separation of peptides in the first dimension was perfomed by eleetrophoresis for minutes at v ( to ma). the tlc plates were air-dried and the peptides were separated in the second dimension by ascending chromatography in n-butanol-pyridineacetic acid-water ( : : : ) until the solvent front reached cm from the top. dried plates were sprayed with en hanee (nen) and exposed to preflashed kodak nag- x-ray film at - ° c. the number and percentage of wild-type, mutant, and revertant virus particles adsorbing to bhk- cells were determined as follows. cells were infected with purified ~ep_ labeled virus at a multiplicity of particles/cell. the p-labeled virus suspensions were allowed to adsorb to bhk- cell monolayers in plastic dishes ( mm diameter) for minutes at ° c. the cells were lysed with . ml b for minutes on ice. duplicate t~ samples were spotted onto whatman glass fiber filters and precipitated in ice-cold percent tca. the filters were placed in vials with scintillation fluid and counted in a packard cd liquid scintillation counter using the pre-set ~ p channel ( ) . the fraction of cell-associated virus particles was calculated by dividing the number of cell associated counts per minute (cpm) by the cpm of the input virus. the average number of virus particles adsorbed per cell was calculated by multiplying the fraction of cell-associated virus by the multiplicity of infection ( particles/cell). the structural proteins of purified c-labeled wild-type and m u t a n t mengovhnases were analyzed by s d s -p a g e on , a n d p e r c e n t polya e r y l a m i d e slab gels. a n a l y s e s of the structural proteins resolved on percent gels are shown (fig. ) . five structural proteins were resolved for each of the viruses: epsilon (lab), kd; alpha ( d), kd; b e t a ( b), kd; g a m m a ( c), kd; and delta (t a), . kd. no changes in the migration of the m u t a n t structurm proteins were o b s e r v e d in c o m p a r i s o n to those of the wild-type virus. in addition, the migration of the structural proteins of several h a + r e v e r t a n t s of m u t a n t was also similar to t h a t of wild-type (data n o t shown). although some v a r i a t i o n in the a m o u n t of delta ( a) p r o t e i n of the viruses w a s o b s e r v e d (fig. ) , this was not a result found consistently t h r o u g h o u t our analyses. s-methionine-labeled structural proteins of wild-type and m u t a n t viruses and were analyzed following digestion with t p c k -t r y p s i n b y r e v e r s e -p h a s e h p l c to further e x a m i n e the structure of the proteins by separating peptides in a gradient on the basis of charge, however, the spectra of methionine-eontmning peptides of the delta ( a), beta ( b), gamma ( c), and alpha ( d) proteins from the different viruses were identical (data not shown). although not all of the peptides generated by tpck-trypsin digestion were likely to contain methionine, the results suggested that the peptide compositions of the four strueturm proteins of the wild-type and mutant viruses were remarkably similar. hplc analyses of the tryptie peptide compositions of the four structural proteins of the wildtype and mutant viruses uniformly labeled with c-amino acid mixture by labeled strueturm proteins was inconclusive because the resulting ehromatograms contained many unresolvable, overlapping peaks (data not shown). to determine whether the arrangement of the structural proteins on the surface of mutants and was similar to that of wild-type, purified virions were labeled with r i and analyzed by sds-page (fig. ) . most of the tyrosine residues on the surface of the wild-type and mutant viruses were on the alpha ( d) protein as demonstrated by the extensive labeling of this protein. the beta ( b) protein was labeled to a much lesser extent. there was no detectable difference in the pattern of surface protein labeling among the three viruses suggesting that the arrangement of the structural proteins on the surfaces of the virions of the wild-type and mutant viruses was similar. to examine the structure of the surface proteins of the three viruses in greater detail, two-dimensional tlck-ehymotryptie peptide ehromato- although not all of the surface peptides generated by tlck-chymotrypsin are likely to be labeled, numerous peptides were labeled. these data suggest that the arrangement of the structural proteins on the surface of the three viruses was remarkably similar. the structural proteins of the wfld-ts~e, mutant, and revertant strains were analyzed by ph gradient gel electrophoresis to determine whether any changes were apparent in migration of the mutant and revertant structural proteins relative to those of the wild-type virus. the proteins were analyzed by ief and nephge two-dimensional gel electrophoresis in order to resolve both acidic and basic proteins. the two-dinlensional electrophoretie migration of the structural proteins of wild-type mengovirus is shown in fig. , panel a. the epsflon ( ab) protein had a pi of . . the alpha ( d) protein was separated into four major protein species, each with a distinct pi ( . , . , . , and . ). the beta ( b) protein was separated into two major protein species with pi values of . and . and a heterogenous species ranging from . to . the gamma ( c) protein was not well resolved by this technique and appears to be slightly acidic or neutral in charge. the delta ( a) protein does not appear in any of the two-dimensional ief gels. the two-dimensional electrophoretic migration of the structural proteins of mutant is shown in fig. , panel b . the etectrophoretic migration of the proteins was identical to that of the wild-type strain with one exception. only three alpha ( d) protein species were resolved. one of the protein species resolved in separation of the wild-type proteins, p i = . , was absent. however, this protein species was resolved in the wild-type/ mixed sample separation (fig. , panel d) . these data suggest that the absence of this particular protein species represents a phenotypie change or mutation in the alpha ( d) protein of mutant relative to the wild-type virus. in addition, the two-dimensional electrophoretic patterns of the structural proteins of the revertent viruses, -a and -d , were identical to those of mutant (data not shown). the two-dimensional electrophoretie migration of the structural proteins of mutant is shown in fig. , panel c. the eleetrophoretic migration of the proteins was identiem to t h a t of the wild-type strain with one exception. the pi of one of the four alpha ( d) protein species (pi = . ) was slightly more acidic t h a n tile analogous wild-type isoelectric species ( p i = . ). this species a p p e a r e d b r o a d e r and slightly lower in molecular weight t h a n the corresponding wild-type protein species. the wild-type/ mixed sample separation (fig. , panel e) shows a broad protein species in the region of the gel corresponding to tile apparent, change in the pis of the wild-type and alpha ( d) protein species. the altered migration of the m u t a n t isoeleetrie species was found consistently in two dimensional profiles of independently derived virion protein samples. these data suggest that the change in form and migration of this protein species represents a phenotypic change or mutation in the alpha ( d) protein of mutant relative to the wild-type virus. two-dimensionm nephge analyses confirmed the results obtained by ief analysis of the wild-type, mutant, and revertant structural proteins. the delta ( a) proteins of the wild-type and mutant viruses were resolved by this technique and migrated similarly as a single acidic protein species at ph . (data not, shown). the specific activities (cpm/particle) of the wild-type and mutants differed when bhk- cells were intbcted and labeled with s-methionine or c-amino acids under similar conditions (ani)e~son and bo~-d, unpublished data). therefore, to determine whether differences in the kinetics of virus-specified macromotecular synthesis exist among the viruses, virusspecified intraeellular protein and t~na synthesis were examined. virusinfected cells were pulse-labeled at hour intervals from to ll hpi with s-methionine for minutes. cytoplasmic lysates were immunoprecipitated and analyzed by sds-page (fig. ) . eight virus-specified intracellular proteins were resolved in wild-type and mutant strain-infected cells: a ( - a), b ( ), c ( ), d ( cd), abc, d ( cd), alpha ( d), and gamma ( c). the number and molecular weights of the proteins specified by the wild-type and mutant strains were identical. however, virus-specified protein synthesis was detected initimly at hpi for the wild-type strain and at hpi for mutant strains and . to determine whether changes in the kinetics of lgna synthesis could explain the delay in detectable protein synthesis observed in mutantinfected cells, virus-infected cells were treated with aetinomyein d, pulselabeled with h-uridine, lysed, and counted. the results are shown in fig. . the peak of rna synthesis for each virus was from to hpi. however, the amount of rna synthesized by the wild-type virus was -fold greater than that of the mutants and -fold greater than that of the revertants. since the magnitude of virus-specified rna synthesized in the mutant virusinfected cells was -fold less than that of wild-type virus, the beginning of virus-specified protein synthesis, as detected by immunoprecipitation (fig. ) , would appear to be delayed due to the fact that less rna would be available for translation. since the magnitude of virus-specified i%na synthesis in mutant virusinfected cells was -fold less than that of the wild-type virus, mterations in uncoating or adsorption of the m u t a n t viruses to cells m a y account for this difference. however, surface peptide analysis suggested t h a t the arrangem e n t of the strueturm proteins of the wild-type and m u t a n t virions was r e m a r k a b l y similar. therefore, it is possible t h a t uncoating of the virions would occur at a similar rate in infected ceils due to their similarity in structure. the average n u m b e r of wild-type, mutant, and revertaa~t virus particles adsorbing to bhk- cells was examined tbllowing incubation at ° c for minutes. u n d e r these conditions, the a m o u n t of virus adsorption to cells would approximate saturation. the results are shown in table . the average n u m b e r of virus particles adsorbing per cell clearly differed among the wild-type, mutant,, and r e v e r t a n t viruses. therefore, it is a p p a r e n t t h a t the mechanisms of adsorption of the m u t a n t and r e v e r t a n t viruses to cells is modified from t h a t of wild-type. the diftbrenee in the cellular binding affinities among the wild-type, mutant, and revei'~ant viruses would be an i m p o r t a n t factor contributing to the differences in the magnitude of viral rna and protein synthesis observed fbr these viruses. these data suggest t h a t fewer cells would be infected by the m u t a n t and r e v e r t a n t viruses and therefore, fewer virus-specified macromoleeules would be synthesized. kevln anderson and clifford w. bond: cells were infected at an moi of particles/cell and incubated at ° c for minutes b the average number of particles adsorbed/cell ° the percentage of adsorption of the mutants and revertants in comparison to wild-type mengovirus biologiem and structural properties of two mengo~drus :mutants have been compared to those of the parental wild-type strain. ~e s e mutants, and , exhibited alterations in agglutination of erythrocytes, virulence in mice, and plaque morphology ( ) . in addition, biological characterization of several ha + revertants of mutant isolated from the brains of mice infected intraeranieally indicated that agglutination and virulence may be linked traits and that the size of plaques produced by a pa~ieular mengovirus isolate may reflect its bindhag affinity to cells and virulence in mice. analysis of s-methionine-labeled structural proteins of wild-type and mutant viruses by sds-page and hplc revealed that extensive homology exists among these viruses. to further examine these viruses for structural differences, surface labeling of intact wild-type and mutant virus particles was done to compare the arrangement of the structural proteins which form the xdral capsids. previous studies have shown that surface iodination of intact potiovirus and mengovirus labels primarily the d (vp or alpha) protein of the respective virus eapsids ( , ) and the b (vp or beta) protein is also labeled, but to a much lesser extent than the d protein. we obtained similar data tbr the surface iodination of intact wild-type and mutant virus particles. in addition, no apparent differences were evident from the analysis of ehymotryptie peptides of i-labeled wild-type and mutant alpha ( d) proteins. these data suggest that arrangement of structural proteins on the surface of mutant eapsids did not differ from that of wild-type. therefore, the mutations may result in the expression of altered determinants that reflect differences in biologicm function and may not result in the masking of otherwise funetionm determinants due to altered arrangement of the capsid proteins. we compared the isoeleetrie character of the viral capsid proteins by two-dimensional gel electrophoresis. multiple species of the alpha ( d) and beta ( b) proteins were reprodueibly deteeted by this technique in both the presence (fig. ) and absence (data not shown) of sds prior to isoeleetrie focusing. previous studies have demonstrated multiple isoelectrie species for the major eapsid proteins of poliovirus [vp ( d), vp ( b), and vp ( c)] ( , ) and emc virus [alpha ( d) and beta ( b)] ( ). vriasen et al. ( ) also excluded the possibility that the multiple species are derived from differential binding of sds or the accumulation of mutants in the original virus stoek. these authors suggested that the multiple species may result from heterogeneity in the cleavage of structural protein precursors; although the sequenees of the amino-and earboxyl-termini of the major eapsid polypeptides of mengovirus were determined without mention of sequenee variations ( ) . we suggest that the charge heterogeneity of these molecules may reflect differences in the interaction of these proteins with the ampholines. the different protein species observed may represent different forms of the viral proteins associated with either intraeellular partieles or free extraeellular particles or extraeellular particles bound to or eluted from membranes, assuming that these particles share the same buoyant density in equilibrium gradients. therefore, the heterogeneity observed may reflect differenees in the manner of isolating virus partieles, whether intraeellularly or from lysed cells. differenees were deteeted among the alpha ( d) structural proteins of the wild-type and mutant viruses. since the mutants exhibited different alterations in the alpha ( d) protein, yet similar ehanges in biological activity, it is possible that alteration of this protein resulted in the observed phenotypie ehanges. the differenees in the number and migration of the alpha ( d) protein isoelectrie species are likely to be due to changes in uneharged amino acid residues affeeting the interaction of the altered species with the ampholines. changes in eharged amino acid residues would result in altered migration of all four isoeleetrie species. since the ehromatograms of surface-and metabolieally-labeled peptides were similar, we speculate that the alterations in the mutant alpha ( d) proteins may be assoeiated with a particular determinant on this protein. this determinant may serve as or be part of a funetional attachment site on the surface of virus partieles that determines their affinity for various cells. atomic resolution of the structure of another pieornavirus, human rhinovirus , revealed a large cleft on the ieosahedral faee that has been postulated to serve as the host eell receptor binding site ( ) . the large cleft separates the major part of five d (alpha) subunits from the other viral protein subunits. sinee the pieornaviruses share similar struetural eharaeteristies, by analogy it seems reasonable to speculate that mutations in the d (alpha) protein could affect the strueture of the deft, and thus, alter the binding affinities of the mutant and revertant viruses. alteration of the mengovirus host cell receptor binding site may lead to changes in affinity for erythrocytes as well as other cell types which may explain the lack of virulence of the mutants in mice. previous work by morishima et al. ( ) demonstrated that differences in binding affinities of closely related strains of emc and mengovirus to various cells reflects their difference in pathogenicity for mice. ha + revertants were isolated from the brains of mice infected intracranially with mutant ( ) . in addition to regaining agglutination activity, the revertants were also virulent for mice and exhibited a slight increase in plaque size. however, revertants required . to -fold more p f u to kill mice than the wild-type virus. since the isoelectric character of t/he proteins of the ha + revertants were identicm to that of mutant , this partial phenotypic reversion may have resulted from a change in an uncharged amino acid associated with a determinant, located on the alpha ( d) protein, which partially restores its biological activities. the partial reversion of this mutation may involve modification of the altered surface determinant, increasing the binding affinity of the virus for cells and restoring virulence, but would not result in reappearance of the isoelectric species absent in mutant and revertant alpha ( d) capsid proteins. alternatively, mutant may express more than one mutation since complete reversion to w i l d -t~e virulence and plaque size did not occur and the isoelectric profile of the alpha ( d) protein was the same as that of mutant . however, revertants were not isolated from mice infected intracranially with mutant , which shared biological properties as well as alpha ( d) protein alterations with mutant . therefore, the mutation observed in the alpha ( d) structural protein of mutant is apparently more stable than that of mutant . however, unlike mutant , expression of the mutation of mutant resulted in a more subtle, yet reproducible change in the isoelectric point of one of the four alpha ( d) protein species. changes in virus-specified macromolecular synthesis in mutant and revertant virus-infected cells can be explained by a decrease in the ability of these viruses to attach to cells. these data suggest that a smaller proportion of cells were productively infected with the mutant and revertant viruses in comparison to wild-type. therefore, the effective moi for the mutant and revertant viruses would be less than that of wild-type. this difference can be explained by changes in the cellular binding affinities; and the higher particle : p f u ratios exhibited by the mutant and revertants ( ), assuming that a greater proportion of noninfectious particles would lower the probability of these viruses to infect cells. this interference phenomenon would explain why p f u values obtained under dilute conditions could not be used to accurately predict the fraction of infected cells in high particle : cell infections. since fewer cells would be productively infected with mutant and revertant viruses, less virus-specified rna would be synthesized, although the peak hour of rna synthesis would be the same as that of wild-type. therefore, less rna would be available for translation; and protein synthesis, as detected by immunoprecipitation, would appear to be delayed in ceils infected with the mutant viruses. changes in the metabolic rates of mutant, virus-specified gna synthesis would predict different results than those presented here if tile fraction of infected cells were similar to ~t d -t y p e infections. collectively, our data suggest that the phenotypie changes expressed by the mutants may be due to mutations toeated exclusively within the alpha ( d) coding region of the genome. previous work by a(~ol et al. ( ) has indicated that the neurovirulenee of poliovirus maps to the ' end of the genome which includes the coding region of the eapsid proteins. consistent with these results, we have identified two different mutations in the alpha ( d) capsid protein, which maps to the ' end of tiffs region, of two avirulent mengovirus mutants. in addition, ko~tara et al. ( ) have stated that a molecular recombinant of the sabin vaccine strain of poliovirus containing vp ( d) and most of vp ( c) of the neurovirulent mahoney strain is virulent, but not as virulent as the mahoney strain and retain the small plaque morphology of the sabin strain. our data also indicate that changes in the alpha ( d) protein result in altered virulence of a pieornavirus. since revertants of mutant shared similar characteristics with the poliovirus recombinant, the plaque-forming ability of a particular virus isolate may be a characteristic that reflects as well as contributes to its virulence. future experiments which compare the nueleotide sequences of the nmtants and revertant structural proteins to that of the parental wild-type virus will be useful in determining the genetic basis for the altered biological properties exhibited by the mutant and revertant viruses. construction and properties of intertypie poliovirus reeombinants: first approximation mapping of the major determinants of neurovirulence biological properties ofmengovirus: characterization of avirulent, hemagglutination-defective mutants iodination of poliovirus capsid proteins liquid scintillation counting: elimination of spurious results due to static electricity relatedness of virion and intracellular proteins of the murine coronaviruses jhm and a two-dimensional electrophoretic analysis of encephalomyocarditis viral proteins identification of the initiation site of poliovirus protein synthesis two-dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines isoelectric points of polypeptides of standard poliovirus particles of different serological types and of empty capsids and dense particles of poliovirus type a micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction characterization of t antigens in polyoma-infected and transformed cells structure of the mengo virion. distribution of the capsid polypeptides with respect to the surface of the virus particle rapid isolation of antigens from cells with a staphylococcal protein a-antibody absorbent: parameters of the interaction of antigen-antibody complexes with protein a in vitro phenotypic markers of a poliovirus recombinant constructed from infectious cdna clones of the neurovirulent mahoney strain and the attenuated sabin strain protein iodination using iodogen genomic and receptor attachment differences between mengovirus and encephalomyocarditis virus two-dimensional polyacrylamide gel electrophoretic fraetionation structure of a human common cold virus and functional relationship to other picornaviruses on the structure and morphogenesis of picornaviruses systematic nomenclature for picornavirus proteins poliovirus replication proteins: rna sequence encoding p - b and the sites of proteolytie processing isoelectric points and conformations of proteins. i. effect of urea on the behavior of some proteins in isoelectric focusing gesolution of the major poliovirus eapsid proteins into doublets structure of the mengo virion. iv. amino-and carboxylterminal analysis of the major capsid polypeptides we thank drs. sandra ewald and andreas luder for their hetpfhl discussions and interest in our work and dr. andrew king for helpful comments on the manuscript. support for this work was obtained from three geseareh creativity development grants awarded to k. a. by the department of graduate studies, montana state universi~-and a grant from the montana heart association, inc. awarded to c.w.b. p~eceived march , key: cord- -tmvebf authors: stephenson, j. r.; ter meulen, v. title: a comparative analysis of measles virus rna by oligonucleotide fingerprinting date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: tmvebf isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. this comparison was based upon the electrophoretic analysis of t( ) oligonucleotides from single-stranded, full-length rna isolated from cytoplasmic nucleocapsids. although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. however, no group of oligonucleotides was observed which would allow a differentiation between viruses isolated from acute infections and those isolated from cns diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. measles virus is an ubiquitous and highly contagious agent which infects man and also primates in close contact with man. the disease usually occurs during childhood, and normally causes a relatively mild stereotyped infection. in a few cases, a more serious complication can arise which may include acute encephalitis. in addition to this disorder another cns disease (subaeute selerosing panencephalitis --sspe) has been associated with measles virus infection ( -- ) . this association has led to many studies on the possible biochemical and antigenic differences betweea measles and sspe viruses. several reports demonstrate differences in etectrophoretic mobility between the proteins of measles and sspe viruses, especially between the p and m proteins ( -- ) however the variation found between sspe and measles isolates was of the same order as that found among the measles isolates themselves ( ) . furthermore, little or no difference has been found in the antigenieity of these viruses when assayed by neutrali-zation, haemolysin inhibition, haemagglutination inhibition ( ) . moreover, all major virus-specific polypeptides from either measles of sspe viruses can be precipitated equally well by either homologous or heterologous r a b b i t sera ( ) . however, b y using short-term i m m u n i z a t i o n schedules with purified protein, antigenic differences were reported between the m protein of one sspe isolate a n d one measles isolate ( ) . i n addition, recent, studies using monoclonal antibodies against measles virus haemagglutinin have demonstrated antigenic differences between several measles and sspe isolates ( ) , b u t no characteristics, specific for sspe isolates were observed. i n addition a t t e m p t s have been made to analyse the sequence homology between the rnas of measles and sspe viruses. yi~h ( ) and hall a n d te]~ meulen ( ) reported only and per cent homology respectively between i~nas from measles and sspe isolates. as the methods employed in these previous studies could only detect gross differences in virus-specific products, or examined one protein or ~ small p a r t thereof, we have chosen in this study to analyse the oligonncteotides from a t digest of the complete genome. not only is this technique capable of detecting differences in r n a sequence throughout the genome, b u t is also capable of detecting differences as small as one base change. i n this report, a comparative analysis between isolates from acute measles cases, a case of acute measles encephalitis and several cases of sspe is described. the fusion inhibitor sv was obtained from bachem inc. torrance, california, u.s.a., and actinomycin d (am])) from serva, heidelberg, f.i£.g. p orthophosphate ( mci/ml), adenosine -a p triphosphate ( ci/mmol) and h uridine ( -- ci/mmol) were obtained from amersham-buchler, braunschwcig, f.r.g. neutral cellulose powder (ccllcx n-l) was purchased from bio-rad and further purified by the method of shilt and martin ( ) . bacterial alkaline phosphatase (bap) was purchased from worthington, polynucleotide kinase (from t xf- infected e. coli b) from p. l. biochemicals, and t ribonuclease and pancreatic ribonuclease a from sigma. xylene-cyanol ff(cf) and bromophenol blue (bpb) was obtained from chroma and trypan red was a gift from dr. j. j. skehel (nimr, mill hill, london). all isolates of measles and sspe viruses were grown on veto cell monolayers as described ( ) . as the lec isolate of sspe virus caused extensive celt fusion which resulted in premature cell death, vg/ml fusion inhibitor was added to the growth medium after the virus had been allowed to adsorb to the cell sheet. the hall isolate of sspe virus was kindly given by dr. f. b. wild, the mantooth isolate by dr. horta-barbose, and the braxator isolate was isolated from a case of acute measles encephalitis ( ). the "cm" and "joy" isolates from acute cases of measles were kindly given by dr. b. fields, harvard medical school, u.s.a. the virus stocks used in these experiments had been passaged as follows. the braxator and mantooth isolates had been passaged times in vero cells, the leg isolate times in vero cells and the hall isolate times. the cm and joy isolates were isolated in hela cells, passaged twice in cv~ cells and times in vero cells. cell monolayers were labelled with ~p orthophosphate as follows. cells ( x ) were infected at a m.o.i, of until per cent of the sheet was incorporated into syncytia ( hours post infection depending on the viral isolate). the cells were then in-cuba.ted in phosphate-free m e m c o n t a i n i n g i ixg/ml a m d a n d im h e p e s pi-i . ) for h o u r a n d t h e n in mi of fresh phosphate-free m e d i u m c o n t a i n i n g amd, h e p e s a n d m c i / m l of a p-orthophate for a f u r t h e r hours. t h e m e d i u m was t h e n decanted, cells w a s h e d once in saline a n d pelleted b y centrifugation for m i n u t e s at × g a n d o c. t h e cell pellet was homogenized with a d o u n c e h e m o g e n i s e r in t i m e s its v o l u m e of r s b ( m~ nac , m~i tris p h . , . mm mgci~), m a d e . per cent w i t h respect to n p a n d elarifed b y two cycles of centrifugation at × g a n d ° c. the s u p e r n a t e n t fluid was t h e n layered onto a step gradient c o n t a i n i n g a n d per cent sucrose in t k m ( m~ kc , l m~ mgci~ . per cent n p a n d m~ tris: pi-i . ) a n d centrifuged for hours a t , × g a n d ° c. a discreet b a n d was clearly visible a t t h e j u n c t i o n of t h e a n d p e r cent sucrose layers. this b a n d was n o t visible w h e n similar e x t r a c t s were m a d e from u n i n f e c t e d cells. t h e m a t e r i a l a t t h e interface of t h e two sucrose solution wass diluted. × in r s b plus . per cent n p a n d centrifuged on a similar g r a d i e n t a t , × g for hours at ° c. the m a t e r i a l a t t h e interface of t h e second gradient was diluted × in r s b plus . per cent n p a n d pelleted b y c e n t r i f u g a t i o n for h o u r s at , × g a n d ° c. t h e pellet was vigorously r e s u s p e n d e d in e x t r a c t i o n buffer ( mm nac , m?~ n a a c e t a t e p t { . , . mm e d t a ) a n d m a d e p e r cent w i t h respect to sds. ilg of g r a d i e n t purified s r n a from u n i n f e c t e d vero cells was a d d e d as carrier a n d r n a e x t r a c t e d as described ( ) . nucleoeapsid r n a p r e p a r e d b y this m e t h o d was a s s u m e d to be unc o n t a m i n a t e d b y messenger r n a as it dld n o t b i n d to oligo dt a n d was u n a b l e to s t i m u l a t e p r o t e i n synthesis in a cell-free p r o t e i n synthesizing s y s t e m from r a b b i t reticulocytes (data n o t shown). messenger izna from virus-infected a n d mock-infected cells was e x t r a c t e d b y p h e n o t / s d s a n d oligo dt c h r o m a t o g r a p h y as described ( ) . the final ethanolic precipitate from t h e i~na e x t r a c t i o n procedure was dried, r e s u s p e n d e d in gardient buffer ( r n~ lic , . m~ e d t a , . per cent [w/v] sds, m~ tris p h . ), a n d centrifuged for hours a t , × g a n d ° c on -- p e r cent linear sucrose gradients. this was acheived by allowing the rna to self-anneal and then separating free single s t r a n d s b y a modification of t h e m e t h o d of fi~a~'kli~- ( ) . a c o l u m n of purified cellulose powder ( . ml) was p r e p a r e d in a mt disposable syringe a n d was w a s h e d t h o r o u g l y w i t h a solution c o n t a i n i n g per cent absolute e t h a n o l a n d per c e n t s t e ( . was passed through the cellulose column and washed with ml of a solution containing per cent absolute ethanol/ per cent ste. the column was then washed with ml of a solution containing per cent absolute ethanol/ per cent ste. these fractions were pooled, precipitated at -- ° with a further volumes of absolute ethanol and designated "single stranded i~na". the column was finally washed with ml of ste, precipitated with absolute ethanol as above and designated "double stranded" lgna. single stranded s rna from intracellular nucleocapsids was lyophilysed and dissolved in ~l of buffer ( mm tris/hc p i t . ). then . ~ of t~ rnase ( . × units/ml) and ~zl bap ( . units/ml) was added and the solution incubated for rainutes at ° c. after the addition of y. of water, the digest was extracted three times with an equal volume of phenol and three times with an equal volume of diethyl ether. the finm aqueous phase was then lyophilysed. the dried rna was dissolved in ~ of a m~ solution of spermidine heated, for minutes at ° c and chilled rapidly on ice. the i~na solution was incubated for minutes at ° c after the addition of ~tl of × kinase buffer ( . m tris/hc p i l . , m~i mgc m~ dithiothreitol), al of [-a~p] atp (atp was lyophitysed and redissolved in the originm volume of water immediately before use), and ~xl of polynucleotide kinase ( unit/~l). the reaction was stopped by the addition of al of ammonium acetate ( ~) ~ sds ( per cent w/v) and tzl edta ( . ~). this mixture was extracted three times with an equal volume of phenol, three times with an equal volume of ether and precipitated at-- with . volumes of absolute alcohol after the addition of i ag of s earrier rna. the rna precipitate was dried and analysed by two dimensional gel electrophoresis as described ( ) . gels were dried under vacuum and exposed to x-ray film. as measles virus grows poorly in tissue culture and the virion has a pleomorphic structure, the isolation of r n a from purified virus is difficult. subsequently the yield of purified virion r n a is insufficient for adequate analysis. therefore it was decided to use intraeellular i~na from eytoplasm:ic nucleoeapsids for this study. as r n a from such a source is k n o w n to consist of a heterogenous p o p u l a t i o n ( , ) with both positive and negati~e strands of various sizes (s~eph~so~-a n d t~r meulen, unpublished data), further purification was necessary to ensure t h a t only full-length negative stranded s i~na was used in the final oligonueleotide analysis. initially, nueleocapsid r n a was analysed by eentrifugation on aqueous sucrose gradients as described in methods. i n fig. a, a t y p i e a l sedimentation profile is shown for nucleoeapsid r n a labelled with p in vivo. the m a j o r i t y of r n a in this sample sediments as a heterogeneously population of molecules below s, with only a small proportion sedimenting as an a p p a r e n t l y homogeneous peak at a b o u t s. the relative proportions of s a n d heterogeneous igna varied from isolate to isolate, b u t the general p a t t e r n shown in fig. a was always observed. no lgna species larger t h a n s were observed in nucteocapsids from infected cells. the s r n a was pooled, as shown in fig. a precipitated with ~g of s carrier i~na and volumes of absolute ethanol, and analysed on a second sucrose gradient. this sample sedimented as an a p p a r e n t l y homogeneous species at s (fig. l b ) , the sedimentation coefficient of this r n a species was indistinguishable whatever measles isolate was examined. w h e n the s r n a was further analysed under denaturing eonditons (fig. l e ) , heterogeneous low molecular weight i~na, with a sedimentation profile similar to t h a t of the subgenomie nueleoeapsid r n a seen in fig. a, is observed. as similar r n a was not a p p a r e n t of the previous aqueous gradient (fig. l b ) it. was assumed t h a t this i~na represented subgenomie positive stranded r n a which had hybridized to full length negative s t r a n d e d r n a during the extraetion and purification scheme. this subgenomic r n a was present in all preparations b u t varied in a m o u n t relative to the full-length s rna. as it was apparent t h a t the s i%na species contained positive sense r n a (see previous section), and t h a t other negative viruses, make full length positive sense strands ( ) it was necessary to purify single strands of one sense only, before analysis of their t oligonucleotides could be performed. if p-labelled s r n a from t w o sequential r n a gradients is mtowed to self-hybridize, and the various types of r n a separated by differential i%na precipitation, a.pproximately per cent of the radioactivity appears in the "single stranded" fraction and about per cent in the "double stranded" fraction, with less than per cent in the fraction containing small r n a species. this distribution of r n a species did vary from isolate to isolate, and to a lesser extent between experiments, with the highest proportion of double stranded r n a being per cent of the total and the lowest level being per cent. if the single stranded r n a all hybridizations were performed using radioactively labelled virus (lec) rna and unlabelled cellular rna as described in methods. the level of hybridization was calculated as that proportion of molecules which remained tca precipatable after digestion at °c for minutes with ~g/ml of pancreatic ribonuclease a and units/ml of t ribonuelease fig. a, b from such a. purification scheme is hybridized to unlabelled messenger r n a from infected cells (table ) , it can be shown t h a t at, least per cent, is protected from subsequent nuclease digestion and is thus assumed to be negative stranded. in order to compare the genomes of various measles isolates: unlabelled, single stranded, negative sense nueleocapsid i~na was prepared from infected cell cytoplasm and purified in parallel with similar radio-labelled material as described in the previous section. this procedure was adopted as previous a t t e m p t s to aualyse these species of r n a , labelled in vivo, were not successful, due to insufficient incorporation of isotope. the r n a was then digested with t endonuclease, labelled with -[~ a p] a t p and the resulting radioactive oligonucleotides analysed b y two dimensional electrophoresis as described in methods. autoradiograms of such two-dimensional gels with tt oligonucleotides from two acute measles isolates, one isolate of acute measles encephalitis and three s s p e isolates are shown in fig. . translucent tracings of these autoradiograms were m a d e (fig. ) and these tracings were then compared. only the oligonueleotides beneath the line drawn in figs. and were included in the analysis. i t was not possible to co-electrophorese the digests of two or more isolates for this comparison, as the large n u m b e r of oligonueleotides in one gel would preclude an accurate analysis of the p a t t e r n thus obtained. as seen in fig. , the a p p a r e n t m o l a r i t y of the larger oligonueleotides is not uniform. this is assumed to be either the result of the co-migration of or more oligonucleotides or an effect of the seconda~" structure of the oligonucleotides affecting the relative efficiency of the kinase reaction. similar observations have been noted when the genomes of several r n a viruses were analysed b y this method ( i). also the mobility and relative concentration of the smaller oligonueleotides differs in some aspects from t h a t obtained from in vivo labelled i~na molecules. i t is presumed t h a t this loss of small oligonucleotides from the digest occured during the ethanol precipitation step, and is also a result of the decrease in the concentration of bisaerylamide in the second dimension of p a g e , which was introduced in order to facilitate the drying of the gels. however, these observations were judged not to affect the v a l i d i t y of the conclusions as identical results were obtained from duplicate or triplicate preparations from each isolate; and identical p a t t e r n s were also obtained when r n a s from different passage numbers of the same isolate were compared. table summarizes the main differences and similarities between the various isolates examined. thus it can be demonstrated that all isolates examined show clear differences, even though they appear virtually identical when examined by classical serology. although oligonucleotides were common to all isolates examined, none were found to be specific for sspe isolates or for measles isolates. similarly none were found to be characteristic for all isolates from cases of encephalitis. if the total number of characteristic changes (i.e. the sum of the oligonueleotides shown to be specific for an isolate and the oligonucleotides specifically absent in an isolate) observed in individual isolates are compared, no clear distinctions can be made between isolates from acute cases of measles acute encephalitis or sspe. in addition, when the lec isolate of sspe was compared to either the edmonston strain of measles vaccine (j. ~. swephenso~ and v. ter meulen, unpublished observations) or to another isolate from acute measles, woodfolk ( ) , again no clear differences were found in the oligonucleotide maps to distinguish sspe isolates from measles isolates. although many attempts have been made by morphological, antigenic and biochemical techniques to distinguish sspe viruses from isolates derived from acute infections, no-one has been able to establish criteria specific for sspe viruses ( , , ) . as previous studies have relied either on detecting gross genetic, structural or antigenic changes, we have chosen to analyse the complete genome of various viral isolates by comparing oligonucleotides generated by digestion with t endonuetease. by using this technique, differences as small as one base change, occurring throughout, the genome of the virus can be detected. as it has proved difficult to obtain sufficient quantities of rna from purified virus, intracellular nueleocapsids have been used as a source of viral i~na. however, as such material can contain significant amounts of cellular ribosomal rna, messenger rna and dna, a purification scheme was devised to minimise the possibility of contamination from these cellular components. the nueteoeapsids were therefore purified by eentrifugation under conditions which ensured that all ribosomes, ribosomal subunits, messenger rnp, and nuclei were pelleted, while the viral nucleocapsids remained suspended at the sucrose interface. nueleocapsid rna prepared from such material contained no detectable amounts of ribosomal or transfer rna when centrifugated on a sucrose gradient and scanned at .d . in addition, when in vitro-labelled oligonucleotides from nucleoeapsid rna were compared to those oligonucleotides from s ribosomal rna, no similarities were observed (data not shown). thus indicating that no contaminating s ribosomal i~na could be detected in rna isolated from viral nueleocapsids. furthermore, s nucleocapsid rna did not bind to oligo dt cellulose nor was it capable of stimulating protein synthesis when added to a nuclease-trcated cell-free system from rabbit reticulocytes. a further complication of using infected cell material is that a significant proportion of the nucleocapsid rna consists of subgenomic species ( , ) . these species appear to have been successfully removed by sedimentation on sucrose gradients. also in infected cells, both positive and negative strands are necessary for the replication of paramyxoviruses ( ) , and can contaminate preparations of s i%na from infected cells. these positive strands have been removed b y allowing the r n a to self hybridize and then separating the excess single-strands b y differential ethanol precipitation. r n a p r e p a r e d b y this technique was shown to be at least. per cent measles-specific negative sense r n a b y hybridization to infected cell messenger i~na. w h e n single-stranded, full length, negative sense i%na from several measles isolates were compared b y analysis of the oligonucleotides generated from ~ t digest, a discreet family of oligonucleotides was generated from each isolate. this family of oligonucleotides was similar in number and distribution to those generated from negative-strand viruses with similar size genomes, such as vsv ( ) or spring viraemia of carp virus ( ) . no evidence of homopolymeres, such as the poly a or poly c tracts found in positive strand viruses like poliovirns, fmdv, coronavirus or r n a t u m o u r viruses was found. although all isolates a p p e a r to be virtually identical antigenically, when examined b y classical serology they have only about per cent or less of their specific t oligonueleotides in common. moreover, no oligonueleotides, specific for sspe isolates or measles isolates, were observed, and concomitantly, no oligonucleotides were characteristie of encephalitic isolates. w h e n the n u m b e r of detectable specific differences in all viral isolates was compared, all isolates whether from eases of acute measles, acute measles encephalitis or s s p e show similar differences from each other. therefore, if we assume t h a t all measles isolates have a common p a r e n t a l ancestor, viruses isolated from a chronic infection, i.e. s s p e do not a p p e a r to have m u t a t e d a t a faster rate t h a n those isolated from eases of acute measles. however, the techniques employed in these studies cannot analyse all the potential differences in the genomes of these viruses, and such studies m u s t await the future application of molecular cloning and edna sequencing to this question. subaeute selerosing paneneephalitis, a review subacute sclerosilig panencephmitis slow virus infections of the nervous system: virological, immunological and pathogenetic eonsiderations differences between the intra-eellular polypeptides of measles and subacut.e selerosing panencephalitis virus a comparison of polypeptides in measles and sspe virus strains intranuelear polypeptides of measles and subacute selerosing paneneephmitis virus-infected cultures measles virus and its biology antigenic relationship between measles and canine distemper virus. comparison of immune response in animals and humans to individual virus-specific polypeptides membrane protein of subaeute sclerosing panencephalitis and measles viruses antigenic characterization of measles and sspe virus haemagglutinin by monoelonat antibodies characterisation of virus-specific rnas from subacute sclerosing panencephalitis virus-infected cv- cells r n a homology between subaeute sclerosing paneneephalitis and measles virus chemiem linkage of nucleic acids to neutral and phosphorylated cellulose powders and isolation of specific sequences by affinity chromatography isolation of infectious measles virus in measles encephalitis characterisation of virus-specific messenger rnas from avian fibroblasts infected with fowl plaque virus purification and properties of the replicative intermediate of the r n a bacteriophage r genetic variation of neurotropic and non-neurotropie murine eoronaviruses persistent infection of cells in culture by measles virus. iii. comparison of virus-specific r n a synthesized in primary and persistent infection in hela cells biological and biochemical characterization of a latent subaeute sclerosing paneneephatitis virus infection in tissue cultures pius and minus strand leader rnas in negative strand virus-infected cells igonueleotide mapping of non-radioactive virus and messenger rna the virological state in subacute selerosing paneneephalitis subaeute sclerosing panencephalitis: characterization of the etiological agent and its relationship to the morbilliviruses. aspects of slow and persistent virus infection spring viremia of carp virus r n a and virion-associated transeriptase activity oligonueteotide fingerprints of r n a species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup we would like to thank magdalene pfohler for excellent technical assistance and jane brctt and helga kriesinger for typing the manuscript. this work was supported by the deutsche forschungsgemeinsehaft. authors' address: prof. dr. v. :reg meulen, institut f/ir virologie, universit/~t wfirzburg, versbacher strasse , d- w/irzburg, federal republic of germany.received october , key: cord- - wem u authors: wada, r.; fukunaga, y.; kondo, t.; kanemaru, t. title: ultrastructure and immuno-cytochemistry of bhk- cells infected with a modified bucyrus strain of equine arteritis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: wem u morphogenesis of a modified bucyrus strain of equine arteritis virus (eav) in bhk- cells was studied. bacillary tubules were first detected in the cytoplasm h after infection, and mature virions to nm in diameter, nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (rer) at h or later. they had isometrical cores and morphological subunits in the outer layer. budding occurred from the rer and the outer nuclear membrane, but not from the cell surface. structural linkage was detected between the tubule and the virus core. aberrant strands were occasionally demonstrated within the nucleus h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus. equine arteritis virus (eav) has been classified into the togaviridae family [ ] on the basis of the virion size and morphology [ ] . recently, however, the completely sequenced eav genome revealed that the virus is of the coronavirus superfamily [ ] . the eav virion was shown to be a spherical particle approximately nm in diameter, consisting of an icosahedral core of nm in diameter surruonded by an envelope [t, , , ] . because of difficulty of treating virions not only in vivo [ , , ] but also in vitro [ ] , few works have been published on the morphologic characterization of eav. in the present study a mutant virus showing higher growth on bhk- cells than the parental bucyrus strain of eav [ ] was examined for the fine structure, its morphogenesis and intracellular localization of viral antigen. confluent monolayers of bhk- cells grown in cm plastic dishes were rinsed with eagle's minimum essential medium (eagle's mem) and they were infected with a mutant virus, the r. wada et al. modified bucyrus (mb) strain of eav [ ] , at a multiplicity of infection of . after incubation at °c for h, the infected cultures were rinsed with eagle's mem and then received ml of eagle's mem with % fetal calf serum (maintenance medium). at to h of incubation at °c, samples were taken, and stored at- °c for the determination of virus yield and fixed for transmission electron microscopy (tem). the virus titer was determind by plaque assay, as described by fukunaga et al. [ ] . monolayer cultures of bhk- cells grown on coverslips in perti dishes were washed twice with . m phosphate-buffered saline (pbs), dried and fixed with aceton at- °c for rain. they were treated with mouse monoclonal antibody mb a specific for structural protein of eav [ ] , which was used as undiluted hybridoma supernatant. after washing times with pbs, the cultures were stained with fluorescent isothiocyanate conjugated goat ,/-globulin to mouse ~-globutin at °c for min. after washing with pbs and mouting in slowfade (molecular probes, inc., or, usa), the stained samples were examined under the fluorescent microscope. infected cells were washed twice with . m sodium cacodylate buffer (ph . ), and fixed at °c for h in % glutaraldehyde and . m cac in the same buffer. samples were then washed twice in the buffer solution, and postfixed at °c for min in the buffer with % osmium tetroxide. cells were then removed from the plastics using a rubber policeman, pelleted by centrifugation at rpm for min, embedded in % noble agar, dehydrated in an ascending ethanol series, and embedded in poly/bed (polyscience inc., pa, usa). ultrathin sections were made and stained with uranyl acetate ( %, w/v) and reynold's lead citrate. they were examined using a hitachi h- electron microscope at kv. for immunogold labeling sections were mounted on a -mesh nickel grid, which were dipped in pbs containing . % bovine serum albumin for min, and then incubated with monoclonal antibody mb a at room temperature for min. after washing in pbs, the grid was incubated with goat antibody to mouse igg, which was labeled with nm gold particles (biocell co., u.k.) at room temperature tbr min. after washing in pbs and rinsing in distilled water, the grid was counterstained with uranyl acetate and reynold's lead citrate. viral antigen was first detectable at h after infection and the growth reached a peak at h (fig. ) . virus antigen first appeared as fine granules in the cytoplasm of a few cells. specific fluorescence significantly increased to h after infection in the perinuclear region of the infected cells (fig. a) as well as within the nuclei at h or later (fig. b) . later, the distribution of viral antigen became more generalized within the cytoplasm, although there was some aggregations within the nuclei. no virus-specific fluorescence was seen in uninfected cells and those treated with normal mouse serum and without monoclonal antibody mb a . at to h after virus infection, the cells did not show any morphological changes. at to h an accumulation of ribosomes was shown and a few aberrant strands and short bacillary or tubular branching structures appeared in the cytoplasm. the tubules increased in number thereafter, especially in the vicinity of the nucleus (fig. a) . they were more than nm long and on average nm in width, ranging to rim. aberrant strands were observed frequently in the nucleus at t h or later after infection (fig. c) . virus particles first appeared at h after infection in the cisternae of the rough endoplasmic reticulum (rer) as well as in the perinuclear space. at h virions increased in number in a larger number of cells (fig. a) . they were usually spherical or oval to nm, nm average (n= ), in diameter, with isometrical cores . nm in diameter(n= ). the core had electron-lucent outer layer and dense center with uniformly high opacity (fig. b ) or electron lucency (figs. c, e). through cross-sections of the outer layer of the virion, a grape-like structure seemed to be subunits to nm in diameter around the core (fig. c) . some virions had an envelope with tiny surface projections (fig. b) . budding of the virus was occasionally observed at the cytoplasmic membrane (figs. d-f ). in some budding particles a structural linkage between the tubules and cores of the maturing virions was demonstrated (fig. g, h) . on the other hand, other small sized particles about nm in diameter, seemingly in a process maturation, were sometimes observed in the cytoplasmic vesicles (fig. i ). some of them had a very electron-lucent outer layer. maturation by budding across the cellular membrane could not be found. tubules (fig. a) and virus core (fig. b) in the cytoplasm as well as strands (fig. c) in the nucleus were labeled with immunogold staining. eav has been reported to be morphologically similar to arboviruses [ , , , ] and classified into the togaviridae family [ , , ] while morphogenesis of eav remains unclear. recently, however, den boon et al. [ ] reported that eav is evolutionally related to the coronavirus superfamily. a new virus family, the arteriviridae, which includes the lactate dehydrogenase-elevating virus, lelystad virus and simian hemorrhagic fever virus, has been proposed by plagemann et al. [ ] . eav are nm in diameter with - nm ring-like subunits on the surface, having a double membrane structure in an electron-dense core to nm in diameter and possessing an electron-lucent outer envelope [ , , [ ] [ ] [ ] ] . ringlike subunits may exist in the envelope [ , ] . they also have tiny spikes on their surface [ , , ] . those structural characteristics were commonly revealed in both purified virus samples [ , , ] and sections [ , , , ] . in the present study the mature virions of the mb strain of eav were to nm in diameter, nm on average, being significantly larger than those reported before. the outline of the mature virions in the rer cisternae was always indefinite, and some of them had distinct surface projections like coronavirus. the mature virions appeared to have subunits in the electron-lucent outer layer. each subunit was spherical to nm in diameter. these findings suggested that the ring-like subunits were viral capsid components produced inside of the envelope at the budding sites. other small sized particles with an approximate diameter of nm were sometimes observed in the cytoplasmic vesicles. they were similar to the typical eav particles with a thin outer layer around the core [ , ] . there might be possibilities that: ( ) the virus cores broke through a damaged endoplasmic membrane into the vesicles; ( ) the outer layer of the small particles was not properly stained; ( ) the small particles were incomplete virions without outer subunits. maturation and budding of the virus appears to occur at rer and perinuclear membrane, but not at the cell membrane. a large number of tubules, assumed to be specific structures related with the replication of eav in vivo [ ] and in vitro [ ] , were observed in bhk- cells infected with the mb strain of eav. in this study, a structural linkage was detected between the tubules and the virus core. these tubules reacted with immunogoldlabeled antibody to eav. similar tubules have been described also in infections with lactate dehydrogenase-elevating virus [ ] , mouse hepatitis virus [ ] and bluetongue virus [ ] , while larger number of tubules appeared in infection with the mb strain of eav. no ultrastructural or immuno-cytochemical change in the nucleus has been observed previously [ , , , ] . in this study, however, eav-specific ultrastructural and immunofluorescent findings were shown in the nucleus. by immunogold labeling of thin sections, antigen was detectable not only on the cytoplasmic tubules but also on strands in the nucleus. these findings suggested the same viral components were present in different forms in both sites. it could not be determined whether the strands in the nucleus were precursors of tubules or they represented in dead-end stage of the virus wandering in the nucleus, since no morphological transition was observed. electron microscopic characterization of equine arteritis virus equine arteritis virus: ferritin-tagging and determination of ribonucleic acid core electron-microscopic studies of tissues ofhorses infected by equine arteritis virus the pathogenesis ofbluetongue virus infection ofbovine blood cells in vitro: ultrastructural characterization pathology of maternal genital tract, placenta, and fetus in equine viral arteritis fluorescent and electron microscope studies of equine arteritis virus (abstr.) equine arteritis virus is not a togavirus but belongs to the coronavirus-like superfamily cell tropism and expression of mouse hepatitis viruses (mhv) in mouse spinal cord cultures the ultrastructure ofvascular lesions in equine viral arteritis classification and nomenclature of viruses. fifth report of the international committee on taxonomy of viruses complement-fixation reaction in equine viral arteritis morphogenesis of eav clinical and virological findings on experimental equine viral arteritis in horses studies on the substructure oftogaviruses. ii. analysis of equine arteritis, rubella, bovine viral diarrhea, and hog cholera viruses studies on equine arteritis virus structural proteins of equine arteritis virus immunofluorescent studies on the multiplication of equine arteritis virus in vero and e. derm (nbl- ) cells production and characterization of monoclonal antibodies against structural proteins of equine arteritis virus morphological studies on equine arteritis virus development ofa moditied virus strain and vaccine for equine viral arteritis lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus: a new group of positive-strand rna viruses replication of lactate dehydrogenase-elevating virus in macrophages. . evidence for cytocidalreplication classification and nomenclature of viruses. first report of the international committee on nomenclature of viruses the authors gratefully acknowledge t. yamakawa and s. shibata for their excellent technical assistance. authors' address: dr. r. wada, epizootic research station, equine research institute, the japan racing association, - , shiba, kokubunji-machi, shimotsuga-gun, tochigi - , japan.received december , key: cord- -fvf jn authors: kjeldsberg, elisabeth; hem, annelise title: detection of astroviruses in gut contents of nude and normal mice date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: fvf jn gut contents of nude and normal mice were examined by electron microscopy in association with an outbreak of diarrhea in a colony of nude mice. virus-like particles with a morphology consistent with previous descriptions of astroviruses of other species were demonstrated in a high percentage of the animals. gut contents of nude and normal mice were examined by electron microscopy in association with an outbreak of diarrhea in a colony of nude mice. virus-like particles with a morphology consistent with previous descriptions of astroviruses of other species were demonstrated in a high percentage of the animals. various viruses have been detected in recent years by electron microscopy (em) of stools and contents of the intestinal tract from both man and animals ( ) . the relationship of some of these viruses to gastroenteritis has been established or postulated. different viruses are causative agents of diarrhea in mice. the epizootic diarrhea of infant mice (edi~[) virus was demonstrated in by cheeve~ and muell~ ( ) . the morphology of this virus was described in by muck and zajac ( ) and it was later classified as a rotavirus. lethal intestinal virus of infant mice (livim) was first described by k~aft in ( ) . this virus could be distinguished from edim by its pathology and clinical signs, and by serological tests ( ) and it was later shown to belong to the coronavirus group ( , ). recently another coronavirus was isolated from an infant mouse in association with diarrhea and designated as diarrhea virus of infant mice (dvim) ( ) . astroviruses were first detected in human faeces in association wit, h gastroenteritis ( ) , and morphologically indistinguishable particles have been reported in diarrheal faeces of lambs ( ) , calves ( ) , birds ( ) and cats ( ) . astrovirus has not been demonstrated in mice so far. in this note we report the detection of astrovirus-like partieles in gut contents from nude mice, with and without clinical signs of illness, and from normal symptomless mice in association with an outbreak of diarrhea. gut contents of mice from different sources and of different strains and stocks were examined: . han:nmri nu/nu bred in isolators in the animal unit. . balb/e/nu/nu bom. . nih:bg/nu/nu bred in isolators in the animal unit. . nih:nihs/nu/nu bred in isolators in the animal unit. . bom:nml~i (normal controls). all the animals were caesarean derived and barrier maintained and of specified pathogen free quality when delivered from the breeder. the nude and thymus deficient mice were kept in a barrier unit. they were given irradiated altromin pelleted diet ad libitum. the water was acidified to ph . with hydrochloric acid. autoclaved woodshavings (hahnflock ) were used as bedding. the diarrhea episode started during a heat wave in the summer when the temperature in the anim m room varied between -- ° c and the relative humidity between -- per cent. the duration of the disease in the individual animal was generally protracted. when it was obvious that 'the animal was ill with diarrhea or wasting it was killed. the mice with normal haircoat were kept in a conventional animal room for -- days after arrival from the breeder before they were submitted to examination. only animals received from the same breeder were kept in this room. the age of the animals varied between weeks and months. both sexes were represented about evenly in nude and normal animals. the gut contents of the mice were scraped off and suspended to per cent v/v in phosphate buffered saline (pbs) ph . the suspension was shaken with a vortex mixer for one minute to disperse the material, left on the bench for minutes at room temperature and shaken by hand from time to time. the extract was centrifuged at g for five minutes and the supernatant was kept at -- ° c until required for use. in a few experiments gut contents were suspended to per cent v/v in pbs containing per cent w/v triton x- (pbs/tx- ) and disrupted with ten strokes of a glass homogenizer. the mixture was cleared at i × g for minutes, the pellet washed with a small volume of pbs/tx-i and the supernatants pooled. for electron microscopy the extracts from gut contents were treated as earlier described ( ). . ml extract was centrifuged for minutes at r.p.m, and the supernatant reeentrifuged for minutes at , r.p.m. in a sorvall rc -b centrifuge ss- rotor. the deposit was suspended in a few drops of distilled water and treated in a branson ultrasonic bath for minutes to disperse the virus particles. negative staining was performed by mixing equal volumes of virus suspension and per cent potassium phosphotungstate pit and layering the mixture on a formvar carbon coated mesh copper grid. after one minute excess fluid was removed with filtering paper, and the grid was air-dried. the specimen was examined in a jem b electron microscope at a magnification of , x using kv accelerating voltage. the magnification had been calibrated with a diffraction grating specimen. a sample was considered negative if no virus particles were observed within minutes examination. intestinal scrapings from nude mice with and without clinical signs of illnes and from normal symptomless mice were treated and examined symptomless or with symptoms other than diarrhea in the electron microscope as described above. particles resembling astrovirus were demonstrated in of the nude mice. the particles were roughly spherical in outline and had a diameter of about nm. the -- pointed star configuration, characteristic of astroviruses, was seen on some of the particles, which mostly appeared in aggregates (fig. a--c) . on some of the micrographs the virus particles showed a smooth outer edge (fig. a) . often, however, fiber-like structures which seemed to form bridges between the virus particles were seen (fig. b) . electron microscopy of pbs/tx- extracts of gut contents occasionally showed astrovirus particles penetrated with stain revealing an inner core structure -- nm in diameter (fig. c) . virus particles were demonstrated in both apparently healthy and diseased animals. the results of the examination of nude and normal mice are summarized in table . seventeen of the nude mice examined suffered from diarrhea while animals were killed for various reasons; i.e. abcesses, bite wounds, termination of experiments. astrovirus was demonstrated in ( per cent) of the animals with diarrhea and in ( per cent) from the control group. no attempt was made to quantify the amount of virus in the samples, but there appeared to be a higher number of virus particles in the samples from animals with diarrhea than from those without. small amounts of astrovirns-like particles were also demonstrated in of l normal mice showing no sign of illness. the morphology of the virus-like particles detected in gut contents of nude and normal mice corresponds to the previous description of astroviruses. the occurrence of the virus in association with diarrhea is consistent with the demonstration of astrovirnses in humans and other animal species in association with gastroenteritis. the failure to demonstrate virus in one of the animals with diarrhea may be due to low sensitivity of the technique. as the majority of astrovirns particles are usually aggregated ( ) a great deal is probably lost in the low speed centrifugation. demonstration of aggregates of virus-like structures in deposits after low speed centrifugation confirms this assumption. the presence of large aggregates of virus-like particles in the intestinal scraping suggests a multiplication of the astrovirus in epithelial cells of the intestinm tract, which would be in agreement with earlier findings in infected lambs ( ). demonstration of astrovirus in a high percentage of nude and normal mice without diarrheal symptoms might suggest that the animals are symptomless carriers of the virus and that the pathogenicity in the nude mice is enhanced by break-down of the climatic control or heavy experimental stress. this hypothesis needs further support. studies on the pathogenicity and characteristics of the mouse astrovirus and the relationship of the virus to diarrhea in these animals are in progress. lethal enteritis in infant mice caused by mouse hepatitis virus lethal intestinal virus of infant mice is mouse hepatitis virus epidemic diarrheal disease of suckling mice. i. manifestations, epidemiology and attempts to transmit the disease ultrastructure of the small intestine in astrovirusdnfeeted lambs purification and characterization of ovine astrovirus detection of astroviruses in feces of a eat with diarrhea comparison of solid-phase (immune electron microscopy, direct electron microscopy and enzyme-linked immunosorbent assay for detection of rotavirus in faecal samples an apparently new lethal virus disease of infant, mice epizootic diarrhea of infant mice and lethal intestinal virus infection of infant mice viruses in infantile gastroenteritis detection of astroviruses in turkey faeces by direct electron microcopy purification and characterization of epizootic diarrhea of infant mice virus detection and transmission of nm virus particles (astroviruses) in faeces of lambs with diarrhea morphnogicat and biological properties of a new eoronavirus associated with diarrhea in infant miee virus infections of the gastrointestinal tract isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves authors' address : dr. elisabeth kjeldsberg, national institute of public health key: cord- -ajf zig authors: ray, n. b.; power, c.; lynch, w. p.; ewalt, l. c.; lodmell, d. l. title: rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: ajf zig recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. as a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. infection, as determined by immunofluorescence, was detected in of the ( %) virus-glial cell combinations. replication of infectious virus, determined by infectivity assay, was detected in of the ( %) virus-cell combinations. these results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction. summary. recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. as a ®rst step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. infection, as determined by immuno¯uorescence, was detected in of the ( %) virus-glial cell combinations. replication of infectious virus, determined by infectivity assay, was detected in of the ( %) virus-cell combinations. these results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction. * rabies viruses have been well documented to primarily infect neurons [ , , ] . in addition, a number of studies have reported rabies viral antigens and virions in human [ , , ] and murine astrocytes [ , , ] , and in murine rami®ed microglial cells [ ] , begging the question of the role of these cells in viral replication and persistence, as well as pathogenesis. schneider [ ] reported that glial cells of the pia mater appear to be involved in replication, and matsumoto [ ] indicated that astrocytes support virus replication in vivo. tsiang also detected a minimal amount of virus when he infected a cloned mouse glioma cell line, but he did not detect virus when he attempted to infect primary glial cell cultures composed primarily of astrocytes [ ] . thus, it has not been clearly established whether rabies viruses productively infect microglia and astrocytes, leaving unanswered the question of whether these cells are susceptible to infection or simply absorb or phagocytose virus or cellular debris from infected neurons. evidence suggests that macrophages, which are related to microglial cells, and astrocytes play a critical role in many viral infections by supporting viral replication and sequestering viruses [ , , , , ] . we have previously reported evidence for rabies virus replication and persistence in murine bone marrow-derived macrophages and in human macrophage-like cells [ ] . it also has been shown that microglial cells and astrocytes infected by viruses, including vesicular stomatitis virus (vsv), may be involved in the disease process via secretion of cytokines and neurotoxins [ , , , ] , including nitric oxide (no) [ ] . in the present study, as an initial step toward evaluation of the potential involvement of these glial cells in rabies virus infections, we have directly examined the ability of different rabies virus strains and isolates to infect and replicate in primary cultures of microglia and astrocytes. four rabies viruses were used: ) the evelyn-rokitnicki-abelseth (era) strain, which had been adapted to tissue culture through multiple serial passages in cer cells [ ] ; ) a street isolate (srv) of bat origin [ ] that had been serially passaged six times intracranially (ic) in mice; ) an unpassaged isolate from a sheep brain; and ) an unpassaged isolate from a skunk brain. murine, feline, and human glial cultures were used to examine the infection of glial cells from different species. murine mixed glial cell cultures were prepared from brains of neonatal irw mice by the method of giulian and baker [ ] and incubated in rpmi- medium (gibco laboratories, grand island, ny) supplemented with % fetal calf serum (hyclone laboratories, logan, utah) (rpmi- ), as previously described [ ] . microglia that were shaken off mixed glial cultures were incubated in a : mixture of rpmi- medium and conditioned rpmi- medium that had been harvested from one week old mixed glial cultures. feline and human glial cultures were prepared in the same way with the exception that the cells were incubated in dulbecco's mem medium supplemented with % fetal calf serum. mixed glial cultures were prepared from neonatal and adult cat brains, and human brain tissue derived from week-old fetuses or adults during operative procedures for deep cns lesions requiring the removal of healthy tissues [ ] . human fetal astrocyte cell cultures were prepared from mixed glial cultures as described [ ] . microglia and astrocytes were identi®ed with speci®c reagents. microglia were identi®ed by their ability to endocytose¯uorescent-labeled dii-acldl [ ] as well as by immuno¯uorescent staining with speci®c reagents including rat-anti-mac- antibody for murine cells [ ] , rabbit anti-human ferritin antibody for feline microglia [ ] , and anti-human macrophage antibody cd [ ] for human microglia. murine microglia were also identi®ed by non-speci®c esterase staining and a phagocytosis assay [ ] . astrocytes of the three species were identi®ed with rabbit anti-bovine glial ®brillary acidic protein (gfap) antibody [ ] . all glial cultures were free of ®broblasts as determined by staining with anti-human ®bronectin antibody [ ] . rabies virus-infected cells were identi®ed by double immuno¯uorescent labeling as previously described [ ] . because infected cells did not deteriorate during the time course of these experiments, microglia containing viral antigens were considered to be infected and not to have phagocytosed and sequestered infected cell debris. we ®rst examined the ability of rabies viruses to infect and replicate in neonatal murine glial cultures that were greater than % microglia. infection of cells with either the tissue culture-adapted era strain, or mouse brainadapted virus resulted in substantial virus replication, evident by h postinfection and increasing in titer throughout the course of the study (up to h) (fig. a) . the infected cells remained phagocytic, but appeared to phagocytose fewer fluorescebrite (polysciences, inc., warrington, pa) beads per cell than uninfected cells ( fig. a) . infected cells also were detected by double immuno¯uorescent staining ( fig. b and c) . unpassaged street viruses also infected murine microglia (table ). in addition, double immuno¯uorescent staining revealed that the era strain infected astrocytes present in the mixed glial cultures (table and fig. ) . we next examined the ability of rabies viruses to infect and replicate in feline glial cells. both the neonatal and adult cultures were greater than % virus was assayed on cer cells using a¯uorescent focus assay [ ] microglia. the era strain of virus replicated well in adult microglia, with a greater than -fold increase in virus titer between and h. the mouse brain-passaged isolate replicated in neonatal microglia (greater than -fold increase in titer), but replication was not detected until h after infection. in contrast, this isolate did not infect adult feline microglia (fig. b) , a situation that may have been due to the state of activation and/or differentiation of the cells [ ] . using double immuno¯uorescent staining, it also was determined that the era strain infected the few astrocytes that were present in the microglia cultures (table ) . lastly, we examined the ability of the viruses to infect and replicate in cultures of human glial cells. the era strain and the mouse-passaged virus replicated well in adult glial cultures that were greater than % microglia, with a greater than -fold and a greater than -fold increase in titer, respectively, between and h postinfection (fig. c) . the era strain also replicated well in fetal glial cultures that were greater than % astrocytes, with a greater than -fold increase in viral titer between and h postinfection (fig. c) . double immuno¯uorescent staining revealed that unpassaged street virus isolates and the mouse passage isolate also infected the human fetal astrocytes (table ) . in summary, primary glial cell cultures from three different species were infected with several different rabies virus strains and isolates in of the ( %) virus-glial cell combinations tested. in addition, productive viral replication was detected in glial cells of all three species in of the ( %) combinations tested. the fact that mouse-passaged and unpassaged street viruses infected and replicated in these glial cells suggests that this was not a virus-cell passage-adaptation laboratory phenomenon. furthermore, the viral replication in primary microglia and astrocytes suggests that the previous detection of rabies viral antigens and virions within these cells may have been infectious virus rather than phagocytosed debris or absorbed virus from infected neurons [ ] . natural anatomical relations and cellular interactions within the central nervous system are known to be important for rabies virus infection of the arrow identi®es a cell that was infected, but did not stain with gfap glial cells in vivo [ ] , possibly making infection of relatively pure cultures of primary glial cells dif®cult. this situation may explain why others failed to detect rabies challenge virus strain (cvs) antigens in cultures of primary neonatal murine astrocytes to h postinoculation [ ] . we can only hypothesize on the role of glial cells in rabies virus infections. our data suggest that microglial cells and astrocytes support viral replication independent of neuronal infection, possibly contributing to viral spread or persistence of virus at the site of exposure in infections of extended incubation periods [ ] . glial cells may also be involved in rabies virus pathogenesis by affecting the physiological health of neurons via the release of cytokines or neurotoxins. support for this involvement has been shown recently in rabiesinfected mice and rats. in these animals, increased levels of inducible nitric oxide synthase (inos), which generates no in microglial cells and astrocytes [ ] , relate to or directly correlate with the neuronal dysfunction that underpins clinical rabies disease [ , ] . clearly, the infection of glial cells by rabies viruses and their potential role in viral replication, persistence or pathogenesis is intriguing and merits further investigation. rabies encephalitis: immunohistochemical investigations growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages on the cellular source and function of interleukin produced in the central nervous system in viral diseases diffuse microgliosis associated with cerebral atrophy in the acquired immunode®ciency syndrome slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages characterization of ameboid microglia isolated from developing mammalian brain ultrastructural location of major histocompatibility complex (mhc) class ii positive perivascular cells in histologically normal human brain human cytomegalovirus productively infects primary differentiated macrophages cell to cell transmission of virus in the central nervous system. ii. experimental rabies in mouse in vivo selection of lymphocytetropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection in vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases rabies encephaloradiculomyelitis. case report production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus in¯uence of cell type and virus upon lysis of rabies virus-infected cells by antibody and complement genetic control of resistance to rabies microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles electron microscope studies of rabies virus in mouse brain comparative pathogenesis of rabies and rabies-like viruses: infection of the central nervous system and centrifugal spread of virus to peripheral tissues infection of human fetal astrocytes with hiv- : viral tropism and the role of cell to cell contact in viral transmission distinct hiv- env sequences are associated with neurotropism and neurovirulence rabies virus replication im primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence the rabies pathogenesis in mice. ii. spread of virus in the cns use of a focal im-muno¯uorescence assay on live cells for quantitation of retroviruses. distinction of host-range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses unexplained rabies in three immigrants in the united states. a virologic investigation inducible nitric oxide synthase in the central nervous system paralysis of street rabies-infected mice is dependent on t lymphocytes activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus central nervous system lesions in human rabies. a study of twenty-four cases neurotropism of rabies virus. an in vitro study appearance of inducible nitric oxide synthase in the rat central nervous system after rabies virus infection and during experimental allergic encephalomyelitis but not after peripheral administration of endotoxin macrophage-and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune de®ciency syndrome rabies viruses ± pathogenesis and immunity cytokine-gene expression in measles-infected adult human glial cells we thank f. murphy, j, portis, s. priola, and k. hasenkrug for critical review of this manuscript. we also thank bob evans and gary hettrick for graphics art assistance and i. c. rodriguez for computer assistance. received october , key: cord- -xrv qs n authors: smith, c. b.; wei, l. s.; griffiths, marie title: mouse cytomegalovirus is infectious for rats and alters lymphocyte subsets and spleen cell proliferation date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: xrv qs n the smith strain of mouse cytomegalovirus (mcmv) was infectious for infant and mature da strain laboratory rats as judged by development of neutralizing antibodies and specific spleen cell proliferation on stimulation with mcmv antigen. an i. p. inoculum of ( ) pfu of mcmv was fatal for more than two-thirds of infant mice ( – days of age), and disseminated viral infection was documented by isolation of virus from body organs. in contrast, weanling and adult rats did not become ill as a result of infection with a larger inoculum of ( ) pfu. however, these older mcmv infected rats did show transient reversals of t helper/suppressor cell ratios and alterations of immune cell function as detected byin vitro spleen cell proliferation assays. seven days after mcmv infection, there was a generalized increase in( )h-thymidine incorporation by spleen cells in both resting (unstimulated) cultures and cultures exposed to mitogens (con a, pha, lps) and to mcmv antigen. at days, the spleen cell proliferation in the unstimulated cultures returned to normal but was depressed compared to controls in response to con a. these observations show that laboratory rats are susceptible to mcmv infection and that assymptomatic infection may occur and cause transient alterations in lymphocyte subsets and in their reactivity to mitogens. although natural cytomegalovirus infections have been described in many species of laboratory and domestic animals, it is generally believed that these viruses are quite species specific in terms of their infectivity and pathogenicity (rapp, ) . r, ecently, priscott and t¥ ~i~ell ( ) reported that the mouse cytomegalovirus (mcmv) was infectious by the intracranial route for newborn sprague-dawley rats. because acute mcmv infections of mice have been associated with transient alterations in immune functions (hamilton, ) , we assessed the infectivity and pathogenicity of mcmv for several strains of newborn and mature laboratory rats. in the following report, we describe a transient, effect of mcmv infections on rat t lymphoc~ helper/suppressor cell ratios and on spleen cell proliferation following stimulation with mitogens and mcmy antigen. the smith strain of mcmv was obtained from kelsey, who has maintained its pathogenicity by serial passage in swiss-webster mice (kelsey et at., ) . the virus pool was prepared by inoculating three week old swiss-webster mice intraperitoneally (i. p.) with pfu of salivary gland pool of virus and harvesting salivary glands from survivors days after inoculation. salivary glands were minced and disrupted by homogenization and a percent suspension was made in eagle's minimum essential medium (mem) with percent heat inactivated fetal bovine serum (gibco). the suspension was then clarified by centrifugation ( rpm, minutes) and stored at - °c. this pool contained . × s pfu/ml infectious virus by plaque assay on mouse embryo fibroblast cell cultures. the final inoculum was found to be free of contamination with mycopiasmas by culture and fluorescent antibody staining. all experimentally infected animals received . or . ml of a -i dilution of virus in mem by i.p. injection. da, wistar-firth (wf) and august n (aug) strain rats were obtained from our own breeding colonies. periodic testing of these colonies for antibodies (microbiological associates, bethesda, md) indicated that they were free of infections with the following spectific pathogens: rat parvoviruses h and kilham, sialodacryoadenitis virus, reo virus , rat corona virus, gdv-ii, sendal, lcm virus and mouse adenovirus. some animals had antibodies to pneumonia virus of mice. organs and tissues from rats sacrificed by cos were homogenized, freeze thawed once, and diluted : in mem before inoculation onto monolayers of mouse embryo fibroblasts (mef). cell cultures were maintained at ° c in mem witlh percent fetal bovine serum, penicillin units/ml and strepl~mycin t~g/ml. negative cell cultures were blind passed if tissue toxicity was apparent or after days if negative. mcmv was identified by the appearance of typical cytopathology. in some instances, spleen tissue slices were maintained in explant culture before passage to mefs according to the method of jor- dan and mar ( ) . the guinea pig complement assisted virus neutralizing antibody assay as described by stadler and ehrensberger ( ) was used. peripheral blood was obtained by retro-orbital bleeding and diluted with two parts pbs before layering over fieol-hypaque solution (s. g. . ). after centrifugation, the mononuelear cell-rich fraction was washed and resuspended in pbs containing percent fbs and . percent sodium azide to give a final concentration ofl cells/ml. this suspension contained percent lymphocytes and ~ percent monocytes and neutrophils. mouse monoclonal antibodies to rat t lymphocyte surface antigens ox , which identitles suppressor cells, and w / , which identifies helper cells (bhdeau et al, ) were obtained from accurate chemical (westbury, n.y.). both monoclonals were diluted / in pbs and reacted with equal volumes of cell suspensions for minutes at ° c. the cells were washed and then reacted with an equal volume of / dilution of fluorescein conjugated f(ab) fragment goat anti-mouse igg (cooper biomedical) for rain at ° c, the cells were washed and resuspended to a concentration of cells/ml in percent paraformaldehyde. the labeled cell suspensions were stored at ° c for no more than hours until counted in a fluorescence activated cell sorter (becton-diekenson facs iii). the results were calculated from a minimum of , cells counted for each monoclonal antibody. spleen cell suspensions were treated with tris-ammonium chloride buffer to lyse erythrocytes and then washed three times with rpmi- . cells were cultured in well flat bottomed microtiter plates and . × cells were added to each well in gpmi- supplemented with l-glutamine ( mm), mm hepes buffer, ~/ml penicillin, , mg/ml streptomycin, × - ram ~-mercaptoethanol and percent fresh rat serum (from same rat strain and matched according to sex). e. coli lipopolysaccharide (lps, sigma) was added at a concentration of . ~g/ml, phytohemagglutinin (pha, wellcome ltd.) was added at a concentration of ~g/ml and concanavalin a (con a, sigma) was added at a concentration of . ~g/ml to spleen cell cultures and plates were incubated for hours at ° c. antigen stimulation was with the inoculum strain of mcmv ( pfu/ml) passaged once in da rat embryo fibroblasts (ref), and heat inactivated at ° c/ minutes. similarly treated ref's served as controis. the incubation period for the antigen and lps stimulations was days. h-thymidine obtained from new england nuclear was added to each well ( ~ci) hours before termination of the culture. cells were harvested on filter discs with a beckman channel harvester and h-thymidine uptake was measured in a beckman liquid scintillation counter. each sample was tested in triplicate or quadruplicate and replicates were screened for aberrant values using the outlier test (grubbs, ) before calculating mean cpm uptake for each test. approximately percent of determinations were discarded by this method. statistical analysis for differences was done by the student's t-test with two degrees of freedom. mcmv was infectious and lethal for more than percent of one to seven day old da and wf strain rats inoculated i. p. with × l to × pfu of virus (table i) . this m(jrtality was greater than the percent mortality seen in four day old rats after injection of normal mouse salivary gland and greater than the percent two-week mortality of newborn da rats followed at the same time in our breeding colony. illness was first notable by four days and mortality was seldom detected after days. virus was isolated from internal organs of of rats that appeared moribund or died three to days after virus inoculation. kidneys and lungs were most often positive (> percent), while virus was grown from percent of spleens and only percent of livers. mortality was only percent in six week old animals inoculated with mcmv and none of the rats died that were inoculated at or more weeks of age even though they received higher titers of to . x pfu of virus. none of these animms appeared clinically ill, however, total body weights in the mcmv infected animals were transiently lower than those in control rats. virus was isolated from spleen and lung tissues of of six-week-old rats cultured to days after inoculation and from the lung and spleen tissues of / -week-old rats cultured four weeks after inoculation. virus could not be detected by culture in salivary gland or other body organs of older animals when cultured days aider inoculation. mcmv at an inoculum of or greater was infectious for all older animals as judged by the appearance at two to eight weeks after inoculation of serum neutralizing antibody at titers of / - / . control animals receiving heat inactivated ( °c, hour) mcmv inoeulum ( . x pfu virus antigen equivalence) did not develop detectable mcmv neutralizing antibodies. infectivity of mcmv for weanling and adult rats was also indicated by the appearance of high levels of spleen cell proliferation in response to stimulation with mcmv antigens (fig. ) . the proliferative responses of spleen cells to mitogens and mcmv antigen in mcmv infected as compared to control animals are presented in table and fig. and . these figures represent a compilation of findings from six separate experiments which utilized a total of mcmv infected and control rats. seven days after mcmv infection, a consistently greater proliferation of spleen cells from mcmv infected rats as compared to control animms was seen with m mitogens and also in unstimulated background cultures (table and fig. and ). these differences ranged from percent to percent of control values, and were highest for con a. when spleen cell proliferation response to mitogens was assessed , and days after mcmv infection, the acute ( day) stimulating effect of mcmv was gone. there was a transient inhibition ( percent of control, p < . ) of spleen cell responsiveness to con a at days following mcmv infection. the greatest differenee between mcmv infected and control animals was seen in response to mcmv antigen (fig. ) . this difference was greatest at days ( percent of control), and although it had declined to percent at days, the differenee was still statistically significant (p< . ) ( table ). the possibility that mouse salivary gland tissue rather than mcmv might be responsible for the observed changes was examined in a separate experiment. seven days after injection of normal mouse smivary gland tissue, there was a moderate ( percent) but not statistiemly significant, increase in the five day background proliferation response as compared to controls, while responses of spleen cells to con a and pha were percent and percent less than control values. mcmv intbction of da strain rats was associated with a reversal of lymphocyte t helper/suppressor cell ratios (table ) . prior to inoculation, th/ts ratios were in the range of . for both groups. in the mcmv infected rats, ratios fell to . and . at and days. there was a smaller decline in the th/ts ratio to the level of . - . in the animals receiving normal salivary gland. the greater decline in th/ts ratios in the mcmv infected animals was significantly different from that of controls at days. mcmv infection was associated with increases in total mononuclear cell counts at and days, suggesting that the reversed th/ts ratios were due to increases in total numbers of circulating suppressor cells. mouse cytomegalovirus (mcmv) was tbund to be infectious by the i. p. route for infants and adults of three strains of laboratory rats as judged by virus isolation, neutralizing antibody response and development of a high level of spleen cell reactivity to mcmv antigen. mortality following mcmv infection was more than percent in young infant rats, while weanling and adult rats survived the infection without apparent illness. this decrease in mortality as animals became weanlings has been described for mice infected with mcmv (boss and wiieelock, , mannini and medearis, ) . marked specificity of cytomegaloviruses for the host of origin has been a general characteristic of this group of viruses (rape, ). although cytomegaloviruses will occasionally grow in cell cultures derived from different species (kim and rare, ), infectivity and pathogenicity for other spepies is rare. mcmv appears to represent an exception to this rule. b~ugge-man et al. ( ) reported that mcmv would grow on rat cells and priscott and tyn~ell ( ) recently reported that the osborn strain of mcmv was infectious for infant sprague-dawley rats when given by the i. c. route, causing percent mortality. the greater mortality seen in our study was probably related to our use of a larger inoculum of virus ( vs ), although virus and rat strain differences are probably also important. our observations indicate that susceptability of laboratory rats to mcmv infection is not strain specific, and that infeetion can occur after i. p., as well as, i.e. inoculation. increased uptake of ~h-thymidine by spleen ceils t~om rats seven days after infection with mcmv was seen in background unstimulated cultures and in cultures stimulated with the mitogens pha, con a and lps. animms receiving injections of normal mouse salivary gland mso exhibited increased spleen cell uptake in resting unstimulated cultures, however, no appreciable differences were seen between spleen cells from salivary gland injected and eontrol animals in response to stimulation with mitogens or mcmv antigen. these results indicate that there was a general stimulatory effect, of mcmv infection that was in addition to a lesser effect seen in response to injection of salivary gland tissue. a similar immuno-stimulatory effect of mcmv infection of mice has been described by others. boss and wkeelook ( ) tested spleen cells collected four days after non-lethal infection of weanling or adult mice with mcmv and detected a moderate increase in uptake of h-thymidine by unstimulated cultures and a slight depression of response to con a. a larger increase (three-fold) in uptake of h-thymidine by unstimulated spleen cells collected - days after mcmv iiffeetion was reported by itowar~d et al. ( ) . the early (seven day) stimulatory effect of mcmv infection on rat spleen cells was generally-gone by days, and in one experiment, a depressive effeet was detected in response to stimulation by con a. these changes were smaller in magnitude than the depressive effect on the response to con a that boss and wheelook ( ) saw in mice following infection with mcmv. in an earlier study, howard et al. ( ) reported depressed lymphocyte proliferation in mixed lymphocyte cultures tbllowing mcmv infection of mice. cytomegalovirus infections of humans have also been associated with depression of lymphocyte reactivity to stimulation with the mitogens con a and pha (ri~ai~do et al., ) . the greatest differences between mcmv infected and control animals were seen in response of spleen cells to stimulation with mcmv antigen. this four-to six-fold increase is consistent with a vigorous immune response to eytomegalovirus infection. our failure to deteet a response to mcmv antigen in control rats injected with mouse salivary gland is further evidence for the specificity of the spleen cell response to mcmv antigen. the lymphocyte response to mcmv antigen was greatest seven days after infection, and a decline in responsiveness to mcmv antigen was seen after the first week, most notably at days. the possibility that active cmv infeetion may decrease the magnitude of the cell-mediated immune response to cmv antigens has been reported to occur in children and adults with cmv infection (reynolds et al., , lewn et al., . rats infected with mcmv consistently exhibited an acute reversal of lymphocyte t helper/suppressor ratios, the effect being greatest days after infection. human eytomegalovirus infections have been associated with similar reversals of th/ts ratios (rinaldo et al., ) and sell et al. ( ) found reversals of th/ts ratios in mice tested , , , and days after infection with mcmv. the general increase in total mononuelear cells observed following mcmv infection suggests that the reversal of th/ts ratios in the mcmv-rat model are due primarily to an increase in the ts population. the marked reversals seen following cmv infections in humans have been associated primarily with increases in ts lymphoeytes and to a lesser degree with decreases in total th cells (carney et al., ) . sell et al. ( ) observed that reversed th/ts ratios in mcmv infected mice were due to decreased th cell numbers on days and and due to increased ts cell numbers on day . it is tempting to try and correlate the observed changes in th/ts ratios with changes in lymphocyte reactivity to mitogens and antigens as has been done in the human cmv system (canxey et al., ). the early ( day) increase in reactivity of spleen cells following mcmv infection does not seem consistent with an increase in ts lymphocytes. this discrepancy could be related to the presence of circulating immunostimulating lymphokines shortly after infection and to a relatively delayed appearance of functional ts cells. our monoelonal antibodies to the ts cell subset measure surface antigen markers only and not functional activity. the suppression of responsiveness of spleen cells to con a at days correlates with an increase in funetionm ts cell activity at that time and is consistent with the observations of carney et al. ( ) in humans. rats injected with normal rat salivary gland tissue had changes in lymphocyte th/ts ratios that were similar in direction, but lesser in magnitude, to the changes we observed following mcmv infection. similar changes thought to be related to the non-specific effects of bleeding or stress were described by ross et al. ( ) . our observations indicate that several strains of laboratory rat, s are susceptible to mcmv infection and that infection is associated with changes in lymphocyte t helper/suppressor cell ratios and in spleen cell reactivity to mitogens. investigators studying immune functions in laboratory rats should be aware that their results can be altered by subclinical laboratory acquired mcmv infection. experimental mcmv infections in laboratory mice have been very useful in exploring the effect of viral infections on immune thnctions (hamilton, ) , and our observations suggest that, mcmyv infection of the laboratorw rat may also be a useful model for similar studies. correlation of survival from mufine cytomegalovirus infection with spleen cell responsiveness to concanavalin a two subsets of rat t lymphocyh'~ defined with monoe]onal antibodies isolation of a cytomegalovirus-like agent from wild rats analysis of t lymphocyte subsets in cytomegalovirus mononucleosis sample criterion for testing outlying observations cytomegaloviras and immunity cell-mediated immunity to murine cytomegalovirus cytomegatovirus-induced immune suppression. ii. cell-mediated immunity spontaneous activation of latent cytomegalovil"ds from murine spleen explants alteration of host defense mechanisms by murine cytomegaiovirus infection growth of murine eytomegmovirus in various cell lines immune response to herpes-virus antigens in adults with acute cytomegaloviral mononucleosis mouse salivary gland virus infections the isolation and partial characterization of a eytomegalovirus from the brown rat (rattus norvegicus) the biology of cytomegaloviruses specific cell-mediated immunity in children with congenital and neonatal cytomegalovirus infection and their mothers mechanisms of immunosuppression in eytomegalovirus mononucleosis lymphocyte subsets and natural killer cell responses during cytomegalovirus mononucleasis the effect of , -dimethylhydrazine (dmh) carcinogenesis on peripheral t cell subsets in the withstar fil%h rat t-lymphocyte subpopulations and function during murine cytomegalovirus infection microneutralization of cytomegalovirus author this work was supported by the veterans administration. key: cord- -lu noak authors: kempf, c.; michel, m. r.; kohler, u.; koblet, h. title: a novel method for the detection of early events in cell-cell fusion of semliki forest virus infected cells growing in monolayer cultures date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: lu noak semliki forest virus infected aedes albopictus cells were used to investigate virus induced cell-cell fusion. it was shown by a novel method that cell-cell fusion was completed within approximately minutes after triggering the fusion event by low ph. this method consists of fixing fusing cells with glutaraldehyde and microinjecting the highly fluroescent and rapidly diffusing dye lucifer yellow. in contrast, polykaryon formation, the usually used criterion to measure cell-cell fusion occurred only within minutes. furthermore, it was shown that potassium cyanide, a potent inhibitor of polykaryon formation in the described system, inhibits an early step of membrane-membrane fusion of neighbouring cells. , fusion of cell membranes is one of the most dramatic forms of membrane-membrane interactions ( ) . this fusion event is a characteristic feature of infections with enveloped viruses ( , and ). several enveloped viruses have been reported to cause fusion under certain conditions ( , and ) . we have previously described such a system consisting of semliki forest virus (sfv; an enveloped, positive strand rna virus) infected aedes albopictus cells ( ) . these sfv infected cells undergo celt-cell fusion at mildly acidic ph, leading to polykaryocytes. this process can be inhibited by a wide variety of chemicals ( , ) , which are useful tools for the elucidation of the molecular mechanisms underlying sfv induced cell-cell fusion. in contrast to fusion events occurring between viruses and artificial membranes, and viruses and biological membranes, onset of cell-cell fusion of cells growing in monolayer cultures is difficult to detect and to quantitate. the most commonly used method is phase contrast microscopy. however, it does not allow visualization of the initial event of cell-cell fusion. only the result in form of polykaryons can be clearly observed after approximately minutes. this handikap makes it very difficult to correlate biochemical processes with the progress of the actual fusion event. however, such correlations are mandatory for the elucidation of the fusion mechanism. in the following we introduce a novel method to monitor cell-cell fusion. furthermore, we show that potassium cyanide, a potent inhibitor of sfv induced polykaryon formation ( ) acts at an early stage in the fusion process. the aim of this investigation was to clarify the early events in cell-cell fusion and the action of potassium cyanide an inhibitor of oxidative phosphorylation-which inhibits sfv-induced polykaryon formation [ ] -on the fusion process. to this end aedes albopictus cells [clone c / , ( )] were infected with sfv. at hours post infection (pi) the growth medium (mitsuhashi-maramorosch medium, mm-medium) was exchanged against mmmedium of ph . at different times after lowering the ph ( to minutes) potassium cyanide was added at a final concentration of mm. the formation of polykaryocytes was examined minutes after lowering the ph to . the minutes correspond to the normal time required for sfv-induced fusion was observed at the times indicated above by means of phase contrast microscopy. the proportion of cells incorporated into polykaryocytes is indicated as described in table polykaryon formation to be clearly visible in the light microscope ( ) . as shown in table potassium cyanide inhibited the fusion process only when added during the tirst minutes. this result was compared with tile time course of sfv-indueed fusion which is summarized in table . as it can be seen, the first clear celt-cell fusions were observed after approximately minutes, when to per cent of the cells appeared to be integrated into polykaryoeytes. thus, these results indicate that potassium cyanide inhibits polykaryocyte formation at an early stage. however, the results presented so far do not show whether potassium cyanide inhibits the membrane-membrane fusion or the reorganization of the cellular membranes and the cytoplasms required to form a polykaryon. to clarify this uncertainty aedes cells were infected with sfv and hours pi the cells were exposed for various times to ph . then, the cells were flxed with . per cent glutaraldehyde for to minutes at ° c in isotonic phosphate buffered saline (pbs) at timely intervals after lowering the ph to . to remove excess glutaraldehyde the flxed cells were incubated at room temperature for minutes in pbs containing mn lysine. then the cells were washed three times with pbs. thereafter, single cells were microinjected with a per cent aqueous solution of lucifer yellow by means of the glasseapillary mieroinjection procedure using an eppendorf microinjector . distribution of the dye within the injected and neighbouring cells was observed with a nikon diaphot fluorescence microscope. photographs were taken using a asa ilford fihn. lucit~r yellow, a highly fluorescent dye, was reported to spread quickly throughout the injeeted cell without crossing the cell membrane ( ) . most importantly, the dye frequently spreads in vivo from the injected cell to nearby cells. this movement of dye from cell to cell has been termed dye coupling. dye coupling offers a method of recognizing functional connections between cells by morphological means ( ) . rl aus it could be reasonably assumed that microscopic membrane-membrane fusions-still invisible by phase contrast microscopyshould allow the dye to diffuse from the injected cell into neighbouring cells. that this assumption holds true is depicted in fig. a where a single, sfv infected cell was injected with lucifer yellow minutes after exposure to ph . it can be clearly seen that three cells had fused by this time. even discrete fusions could be detected as indicated by the arrows. with the phase contrast microscope it was impossible or questionable to come to the same conclusion ( fig. b) . the same experiment was carried out at different times after lowering the ph to . in each experiment to arbitrarily chosen, single cells were injected with the dye. the spreading into neighbouring cells was recorded. the degree of fusion was expressed as percentage of cells showing dye coupling versus the total number of fluorescent cells. this is depicted as a function of time in fig. . some degree of dye coupling was also detected at time zero of the exposure to ph . at time zero the average number of cells involved in a dye coupling event was . _+ . . this background could reflect dividing cells or other naturally occuring connections between cells. the number of cells in a "coupling" group significantly increased with time after exposure of the infected cells to ph ( fig. , inset) , thus showing that fusion occurred and progressed. in dye coupling, to per cent of cells involved in fusion was reached after approximately minutes when compared to minutes as juged by phase contrast microscopy. the data presented clearly show that microinjection of lucifer yellow is a useful tool to monitor cell-cell fusion at its earliest stage. in similar experiments m~ potassium cyanide was added concomitantly with the change of the ph from to . this treatment abolished the fusion process. even when the cells were incubated for minutes in presence of potassium cyanide and medium ofph the degree of cell-cell fusion remained at the control level as shown in fig. . also the average cell number per dye coupling event ( . _+ . ) did not exceed the value measured for the zero time point in absence of cyanide. the experiments with lucifer yellow clearly demonstrate that both the number of fusion events and the number of cells involved in one dye coupling event increase as a function of time. this result favors the conjecture that sfv-induced cell-cell fusion is not a strictly synchronised process. it can be assumed that fusion proceeds in a zipper-like fashion and that a postulated cascade of correlating biochemical processes would also be asynchronous. this could explain the fact that potassium cyanide can be added even minutes after triggering sfv-induced fusion in order to prevent a massive fusion as seen in phase contrast microscopy. however, a concomitant addition of potassium cyanide with the triggering by low ph totally abolished the fusion event. therefore, it can be concluded that potassium cyanide inhibits sfv-induced polykaryon formation at the level of membrane-membrane fusion. potassium cyanide is an inhibitor of oxidative phosphorylation and leads to depletion of cellular atp. for the following reasons it can be excluded that the results obtained in presence of the relative high concentration of potassium cyanide ( ram) represent an artefact: i) polykaryon formation occurred in presence of mm kcn when the drug was added to minutes after lowering the ph and proceeded from a microscopically invisible to a visible state (see table ); ii) furthermore, we have previously shown that syncytium formation of sfv infected aedes cells takes place also in presence of mm kcn if the infected cells are exposed to ph for a short time only ( ) . briefly, hours pi sfv-infected aedes cells were exposed for various ti~es ( , and minutes) to medium of ph containing mm kcn. then the medium was replaced with medium of pit also containing mm kcn. at exposure times of and minutes this treatment lead to a transient recovery of the intracellular atp, which was sufficient for polykaryon formation to occur. in contrast, longer exposures to ph in presence of kcn inhibited the fusion process. thus, the results reported herein strongly support the finding from our laboratory that sfv-induced celt-cell fusion is an event which requires a specific expenditure of energy ( ). this finding is in agreement with an earlier observation made by kada ( ) . it was reported that sendal virus-induced polynuclear cell tbrmation of ehrlich's ascites tumor cells was inhibited by the addition of dinitrophenol, a well known inhibitor of oxidative phosphorylation. ueber l~iesenzellbefunde in den gaumenmandeln, zugleich ein beitrag zur histopathologie der mandelver~nderungen im maserninkubationsstadium cytolytic effects of mumps virus in tissue cultures of epithelial cells isolation ofa singh's aedes albopictus cell clone sensitive to dengue and chikungunya viruses semliki forest virus-induced polykaryon formation is an atp-dependent event conformational changes at ph on the celt surface of semliki forest virus-infected aedes albopictus cells fusion of mphavirus infected mosquito cells. in: yunker ce (ed) arbovirus cultivation in arthropod cells in vitro analysis of giant polynuclear cell formation caused by hvj virus from ehrlich's ascites tumor cells. i. micx~seopic observation of giant polynuclear cell formation analysis of giant polynuclear cell formation caused by hvj virus from ehrlich's ascites tumor cells. iii. relationship between cell condition and fusion. reaction or cell degeneration reaction electron microscopic studies of a coronavirus membrane fusion lucifer dyes-highly fluorescent dyes for biological tracing cell fusion by semtiki forest, influenza, and vesicular stomatitis virus authors' address: dr. c. kempf, institute of hygiene and medical microbiology, university of bern, friedbuehlstrasse , ch- bern, switzerland.received january , key: cord- - stcx dd authors: mushegian, a. r.; koonin, e. v. title: cell-to-cell movement of plant viruses: insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: stcx dd cell-to-cell movement is a crucial step in plant virus infection. in many viruses, the movement function is secured by specific virus-encoded proteins. amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. this superfamily combines proteins of viruses belonging to all principal groups of positive-strand rna viruses, as well as single-stranded dna containing geminiviruses, double-stranded dna-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand rna genomes with two ambisense segments. in several groups of positive-strand rna viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative rna helicase. a distinct type of movement proteins with very high content of proline is found in tymoviruses. it is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. limited sequence similarities were observed between i) movement proteins of dianthoviruses and the mip family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and m protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. it is hypothesized that all movement proteins of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules. it is generally accepted that plant viruses exploit plasmodesmata to move from initially infected cells, which are usually negligible in amount, into neighbouring healthy cells. without cell-to-cell movement productive virus infection is not established. also it is known that specific movement proteins encoded by virus genomes are essential for cell-to-cell spread. frequently, a plant virus protein is referred to as movement protein if i) it is not a capsid protein and ii) disruption of the coding sequence of this protein abolishes infection in whole plants but has no effect on virus replication in protoplasts. putative movement proteins have been identified in this manner in about different plant virus groups (see [ , , ] for recent reviews). in some cases, additional virus proteins are also required for movement, e.g. coat protein [ , , ] or a specific segment in a replication protein [ ] , but these phenomena are not universal. on the other hand, a specific movement protein or at least movement domain is thought to be present in all viruses that are able to spread from cell to cell [ ] . apart from this genetic evidence for the movement function, the current knowledge of mechanisms which facilitate intercellular spread of plant virus genomes is insufficient. recently certain properties of some movement proteins, notably of the kda movement protein of tobacco mosaic virus (tmv), were characterized in vitro and in vivo and models for movement of plant viruses have been prompted by these studies [ , ] . additionally, movement proteins were subjects of comparative sequence analysis, which allowed to delineate common motifs in movement proteins from otherwise unrelated groups implying existence of common features among many movement proteins [ , ] . in this work, we sought to critically analyze currently available biochemical and ultrastructural data on movement proteins. we then investigated whether sequences of movement proteins could provide information on their function based on similarities with other viral or cellular proteins. fluorescent probes injected into a plant cell are either transported into neighbouring cells via plasmodesmata or retained in the initially injected cell. the crucial determinant for the exclusion of a particle from plasmodesmatal movement is its hydrodynamic (stokes) radius [ ] . studies of transgenic tobacco plants constitutively expressing tmv kda movement protein (mp + plants) revealed that certain molecules, which were too large to be transported in wild type plants, gained such an ability in mp + tobaccos. specifically, fitc-labelled dextran (mr . kda) moved from injected to neighbouring cells only in plants transformed with and expressing tmv kda protein but not in control mp-plant virus movement proteins plants [ ] . interestingly, plasmodesmata in mp + and mp-plants appeared to be structurally indistinguishable [ , ] . these data indicate that movement protein of tmv functionally modifies plasmodesmata. it is not understood yet whether these modifications and the changes required for moving virus genome through plasmodesmata are the same or different. virus nucleic acid would not be expected to exist in naked form in the cell; rather, it seems reasonable that, like all nucleic acids in eukaryotic cells, it is associated with cellular and/or virus proteins throughout its lifetime. while stokes radius of a kda dextran molecule is estimated to be about . nm [ , ] , ribonucleoproteins (rnps) are clearly larger, many of them having sizes within the range of - nm [ ] , which would exclude them from movement even in mp + plants described in [ ] . recently, plasmodesmatal permeability in nicotiana clevelandii leaves infected with tobacco rattle virus (trv) has been investigated [ ] . movement protein of trv, the kda protein, shares high sequence similarity with tmv movement protein, and both proteins are considered to be functionally very similar. simultaneous injection of virus and fluorescent tracers into cells of leaf trichomes allowed to observe movement of the labels under the conditions when the virus itself was known to move in the same cells. it has been shown that fitc-labelled dextran (mr . kda) moved from cell to cell only in trvinfected cells and synchronously with the virus. however, lucifer yellow-labelled dextran (mr kda) was excluded from plasmodesmatal movement even in infected cells. thus, certain molecules with the dimensions resembling the smallest of the known rnps did not move under conditions when virus rnps readily moved. apparently, either an extremely thin virus-specific rnp should be postulated (see below), or it might be concluded that slight increase in plasmodesmatal permeability induced by movement proteins hints at some important circumstances but is not sufficient to explain celt-to-cell movement of the virus genome. binding of movement proteins to single-stranded nucleic acids tmv kd movement protein has been overexpressed in e. coli, and purified protein has been shown to bind single-stranded (ss) dnas and rnas nonspecifically and cooperatively [ ] . a model has been proposed, in which movement protein binds genomic rna stoichiometrically to unfold and shape it into a thin complex [ , , ] . electron microscopy of such complexes formed in vitro revealed thin structures at the limit of microscope resolution; it was concluded that, as it was barely seen under em, the complex was thin enough to fit in plasmodesmata with size exclusion limit of nm [ ] . additionally, ss nucleic acid-binding properties have been reported for movement proteins of cauliflower mosaic caulimovirus (camv) [ ] , red clover necrotic mosaic dianthovirus (rcnmv) ( [ ] , d. cookmeyer, pers. comm.) and alfalfa mosaic virus (aimv) [- ] . interestingly, p , the movement protein of cauliflower mosaic virus, has been shown to preferentially bind rna rather than dna a.r. mushegian and e. v. koonin [ ] ; it was speculated that s transcript, a genome length virus replication intermediate, might be the form which is transported from cell to cell. these in vitro observations offer new insights into a mechanism of virus cell-to-cell movement. however, is should not be forgotten that virus rnas in vivo are associated with proteins, as revealed for tmv-specific rnas [ ] ; it is thought that such an association is maintained throughout the whole life of the rnps in the cell; a mechanism of stripping virus rna from these proteins has not yet been proposed. also, as binding of movement proteins to rna is thought not to be sequence-specific [ , ] , it could be anticipated that the majority of movement protein will bind to cellular rnas, which in the case of tmv infection are in excess over virus rnas at the time when kd protein is transiently expressed [ ] . the "ss nucleic acid-binding" model, therefore, has to be modified to deal with these difficulties. biochemical fractionation of infected cells revealed that in many virus-plant interactions movement proteins are found in cell wall-enriched fraction. immunogold labelling has confirmed these observations and demonstrated localization of tmv, a mv, camv, and rcmv movement proteins in plasmodesmatal channels or in cell wall near plasmodesmata [ , ] . movement protein of cowpea mosaic comovirus (cpmv), the kd/ kda protein, has been found to form tubular structures perpendicular to and intruding into plasma membrane and cell wall. these tubules have been found both in whole leaves and in protoplasts [- ] . virions have been observed inside the tubules [ ] . it has been speculated that the tubular inclusions are the structures required for movement. cucumber mosaic virus (cmv) movement requires the kda movement protein [ ] . this protein has not been found to associate with the cell wall or from tubules; instead, it was located in nucleoli of infected cells [- ] . observations on movement proteins of tmv and cpmv have given rise to the idea of two different types of virus movement, "tobamo-type" and "comotype". it has been proposed that the tobamo-type is characterized by movement protein localization in the plasmodesmatal area of the cell wall and by the virus infectious entity capable of cell-to-cell movement in the absence of the viral capsid protein. como-type of movement was thought to require both movement protein and coat protein and to involve formation of tubular structures protruding from cell wall into cytoplasm and formed by the movement protein [ , ] . complementation data argued, however, that there is certain compatibility among both types of movement as tobamoviruses were able to complement comovirus movement [ ] . when all available ultrastructural data are considered, the picture becomes not so clear-cut. it should be expected that rcmv, a comovirus closely related to cpmv, moves according to the como-type; instead, movement protein of rcmv was found exclusively in plasmodesmata of the infected plants [- ] which had been proposed to be a tobamo-type feature. camv movement protein is found in cell wall fraction and, more specifically, in plasmodesmata [ , ] ; this seemingly supports the attribution of caulimovirus movement to the tobamo-type. however, tubular structures of unknown composition have been observed in the cytoplasm of camv-infected cells [ ] and recently it has been shown that p , the movement protein of camv, forms tubular structures on the surface of inoculated protoplasts [ ] . these findings seem to be more consistent with the como-type action of the caulimovirus movement protein. the above discussion shows that it may be premature to draw too sharp a distinction between different types of movement based on ultrastructural observations. it also should be remembered that we see movement proteins at the sites where they are most easily observed and/or are retained for longer periods, but not necessarily at the sites of their essential activity. with full-length dna copies of numerous virus genomes available, attempts have been made to genetically dissect movement protein coding sequences. in most cases, lengthy deletions have been introduced, and effect of these deletions on the overall infectivity or individual properties of the movement proteins has been investigated. it has been shown that even small changes near the n-terminus of tmv kd movement protein abolish virus infectivity [ ] ; at the c-terminus, up to of the amino acid residues of this protein could be deleted without apparent effect on infectivity or cell wall localization of the movement protein. when c-terminal amino acids were deleted, virus moved slowly though movement protein was still found in plasmodesmata [- ] . deletion of cterminal residues ( to ) was lethal, and, at the same time, movement protein was no longer found in plasmodesmata. it has been noted that essential residues from to could comprise a domain with a specific function, or their deletion might simply cause misfolding of the protein i- ]. in another study, c-terminal and internal deletions have been introduced into tmv movement protein to determine location of ss nucleic acid-binding site(s) [- ] . initially, it was thought that residues to constitute rnabinding domain [ ] . later, this domain has been shown to rather decrease protein solubility [ ] . up to amino acids from the c-terminus of tmv movement protein could be eliminated without effect on ss nucleic acid binding capacity of the protein [- ]. in the remaining part, deletion of amino acids - abolished ss nucleic acid binding, but when the gene segment encoding these amino acids was fused to an unrelated sequence, the resulting himeric protein did not bind to ss nucleic acids [ ] . movement protein of a mv associates with cell wall upon infection and in transgenic tobacco plants; however, this did not happen when n-proximal amino acids ( - ) have been deleted [- ] . strains of tmv have been characterized that are temperature sensitive in a.r. mushegian and e. v. koonin: plant virus movement proteins cell-to-cell movement or break the host resistance which is based on plant genes restricting virus movement. involvement of mutations in kd movement protein cistron could be implied, and, indeed, in many cases nucleotide changes responsible tbr the altered phenotype were shown to be confined to the movement protein gene [ ] . many of these mutations were scattered around the middle one-third of kd movement protein and resulted in a change in polarity of the encoded amino acid [ ] . as no known function could be mapped to the area, these data are difficult to interpret. with representative sequences of plant virus genomes from many groups available, efforts were made to identify regions of similarity between known movement proteins and to predict movement functions for uncharacterized orfs. early observations made by this approach have been reviewed previously (e.g. [ ] ) and included strong similarity between tobamovirus and tobravirus movement proteins [ ] as well as less pronounced but significant local similarity between segments of tobamovirus and caulimovirus movement proteins [ ] . later, similarities between movement proteins of tricornaviruses and dianthoviruses were observed [ ] . in fact, the movement function for the respective proteins of tobraviruses, caulimoviruses and dianthoviruses has been implied by these observations and only subsequently confirmed by site-directed mutagenesis [ , , ] . more detailed analyses performed by melcher [ ] and by ourselves [ ] revealed longer (ca. amino acids) segments of sequence similarity between movement proteins from different virus groups. interestingly, one family of movement proteins included proteins encoded by viruses with positive-strand rna genomes, single-strand dna geminiviruses and by pararetroviruses with virion dna, namely caulimoviruses [ ] . in the time past after the publication of these analyses, further significant increase in the number of sequenced virus genomes was achieved and it has been argued that the current collection of such sequences may represent the majority of the existing virus groups [ , ] . with this in mind, we undertook a systematic comparison of the sequences of the movement proteins with each other and with the current sequence databases. using the blast program [ ] , statistically highly significant similarities were observed only between proteins of viruses belonging to a single group or two closely related groups. further comparisons were done using programs ma-caw [ a] and optal [ ] to generate multiple alignments according to the progressive alignment strategy [ ] , i.e. starting with pairs of most closely related sequences and proceeding to incorporate more and more distant ones. it was demonstrated that movement proteins of viruses belonging to groups contain a weak but confidently identified conserved motif consisting of approximately amino acid residues (fig. ) . this motif includes a nearly invariant aspartic acid residue (replaced by asparagine in dianthoviruses and ilarviruses) preceded by a region enriched in hydrophobic and nonpolar residues. in some of the movement proteins this region was predicted to form a transmembrane helix using the algorithm of rao and argos ( [ ] , data not shown; see also discussion below). remarkably, this conserved motif unites viruses with single-stranded dna genomes (geminiviruses of group ii), pararetroviruses with double-stranded dna genomes (caulimoviruses and badnaviruses), negative-strand rna viruses with ambisense genome segments (tospoviruses), and numerous groups of positive-strand rna viruses. among the latter, all the three major phylogenetically defined divisions [ , ] are represented, namely run-like viruses (tobamoviruses, tobraviruses, cucumoviruses, bromoviruses, ilarviruses, capilloviruses, furoviruses, raspberry bushy dwarf virus), picornalike viruses (comoviruses, parsnip yellow fleck virus, pea enation mosaic virus), and flavi-like viruses (dianthoviruses and tombusviruses). several groups of movement proteins showed significant similarity on longer sequence stretches. in fig. we show the amino acid sequence alignments for four of these groups, delineation of which included identification of new putative movement proteins. such new identifications are: i) kda protein of apple stem grooving virus, which is related to the putative movement proteins of the other capilloviruses, namely apple chlorotic leaf spot virus and potato virus t (fig. a) ; ii) kda protein of soil-borne wheat mosaic furovirus related to the movement proteins ofdianthoviruses (fig. b) ; iii) kda protein of pea enation mosaic virus and kda protein of tombusviruses related to the movement proteins of bromoviruses and cucumoviruses (fig. c) ; and iv) nsm protein of fig. . amino acid sequence alignments for groups of proteins including newly identified putative movement proteins. for each group of boundaries of the conserved segments were determined using the macaw program and the alignment itself was constructed hierarchically using the optal program. in the consensus u indicates a bulky aliphatic residue (i, l, v, or m), a indicates an aromatic residue (f, i , or w) & indicates a bulky hydrophobic residue (aliphatic or aromatic) and x indicates any residue. the conserved motif of the " k superfamily" is highlighted by bold typing, a putative movement proteins of capilloviruses. aclsv apple chlorotic leaf spot virus; pvt potato virus t; asgv apple stem grooving virus; b movement protein of red clover necrotic mosaic dianthovirus (rcnmv), putative movement proteins of raspberry bushy dwarf virus (rbd v) and soil-borne wheat mosaic furovirus (sb wmv); e movement proteins of cucumber mosaic cucumovirus (cmv) and brome mosaic bromovirus (bmv), putative movement proteins of pea enation mosaic virus (pemv) and cucumber necrosis tombusvirus (cnv); the similarity between kda proteins of tombusviruses and movement proteins of bromoviruses and cucumoviruses has been independently reported by u. melcher (pers. comm.); d putative movement proteins of caulimoviruses, badnaviruses, parsnip yellow fleck virus, and tomato spotted wilt tospovirus; asterisks show identical residues and colons show similar residues between the sequences of pyfv and fmv, and coymv and tswv tospoviruses and the n-terminal domain of the parsnip yellow fleck virus polyprotein related to the putative movement proteins of caulimoviruses and badnaviruses (fig. d; the similarity between caulimovirus movement proteins and n-terminal domains of badnavirus polyproteins was noted by u. melcher and cited in [ ] ). even among these smaller groups, the latter three brought together proteins of very different viruses. group iv) is particularly notable in that it compiles proteins of a positive-strand rna virus, negative-strand (ambisense) rna viruses, and dna-containing pararetroviruses. curiously, the array of domains, namely n-movement domain-capsid protein(s)-replicative domains-c in the polyproteins of such seemingly unrelated viruses as parsnip yellow fleck virus and the badnaviruses is very similar. a relevant observation from our previous work is the relationship between movement proteins of two component geminiviruses (bl /bc ) with those of tobamoviruses and tobraviruses [ ] . all the proteins containing the conserved motifs shown in fig. should be considered a single vast, and highly diverged superfamily of plant virus movement proteins. we would like to coin the nickname " k superfamily", after the most thoroughly studied movement protein of tobamoviruses, and also because most of the proteins of this superfamily have the size around this value (fig. a) . several groups of positive-strand rna viruses, namely potexviruses, carlaviruses, hordeiviruses, and a group including beet necrotic yellow vein virus, nicotiana velutina mosaic virus, and peanut clump virus, encompass the triple gene block that consists of two small membrane proteins and a putative rna helicase [ , ] . it has been demonstrated that disruption of any of the triple block genes abolished the cell-to-cell movement of hordeiviruses, potexviruses and bnyvv [ a, a, a]. the putative helicases in the triple block comprise a distinct group within superfamily i of dna and rna helicases and contain several unusual amino acid substitutions in the conserved helicase motifs [ , ] . unlike the genes coding for the proteins of the " k superfamily", the triple block is found only in positive-strand rna viruses. carmoviruses and necroviruses encode a pair of small proteins, one of which is predicted to be an integral membrane protein. it has been shown that disruption of either of these genes precluded cell-to-cell movement of turnip crinkle carmovirus [ ] . these two genes perhaps may be considered a "truncated triple block" although it was hard to demonstrate this at the level of sequence comparison due to the small size of the hydrophobic proteins. a third type of movement protein has been revealed in tymoviruses. a kda proline-rich protein that is encoded in the ' portion of the genomic rna and overlaps with the replicative polyprotein gene is essential for the movement of tymv in infected plants [ ] . this protein may have specific secondary and tertiary structure similar to that of other proline-rich filamentous proteins. interestingly, upon screening of the amino acid sequence databases with the sequence of the tymovirus movement protein the highest (albeit moderate) similarity was observed with the plant cell wall-associated protein extensin (data not shown). recently it has been shown that cell-to-cell movement of geminivirus msv is dependent on the small protein v [ ] . this protein and the related proteins of other one component geminiviruses did not show appreciable sequence similarity to movement proteins of other viruses. interestingly, however, alignment of their amino acid sequences revealed the conservation of a highly hydrophobic central domain terminating at an invariant aspartic acid residue and thus distantly resembling the conserved motif of the k superfamily (fig. ) . this domain was flanked by two proline-rich domains showing some similarity to extensins (fig. ) . thus, in a sense the movement proteins of one component geminiviruses combined the k superfamily and the tymovirus structural themes. for several virus groups, in which at least one member has been completely sequenced, the movement function has not been genetically mapped and identification of a putative movement proteins by amino acid sequence comparison was very uncertain if possible at all. among these, uncharacterized proteins of nepoviruses and closteroviruses showed a very limited sequence similarities to some members of the k superfamily, in particular to caulimovirus movement proteins [ ] . the observations discussed in the previous sections show that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins - , , ] . also, we were unable to notice any correlation between grouping of movement proteins and biological properties of the viruses encoding these proteins (e.g. host ranges). recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. this is clearly the preferential explanation for situations when related movement proteins are found in viruses with different type of genome nucleic acid (e.g. the striking similarity between the putative movement proteins of caulimo/badnaviruses fig. d) . in some cases recombinational transfer of movement protein genes appears very likely also between remote groups of positive-strand rna viruses (e.g. dianthoviruses and soil borne wheat mosaic furovirus; fig. c) . another equally important trend in the evolution of movement protein genes is the apparent high rate of mutational change leading to the very limited level of sequence conservation as featured above. even within compact groups of viruses that share common genome organization and functional properties (e.g. tobarnoviruses), the divergence between the movement protein sequences is much higher than that between the principal repticative domains although comparable to that between capsid proteins. it has been proposed that plasmodesmata are plant analogues for certain animal cell-cell contacts, namely gap junctions, based mainly on studies in permeability of both to fluorescent dye-labelled molecules [ , ] . details of protein organization of plasmodesmata are poorly understood, although a protein serologically related to a major gap junctions protein, connexin, was reportedly found in plasmodesmata and gene encoding this plant protein was cloned [ ] . in animal kingdom, gap junctions are important for electrical coupling of cells in various tissues [ ] . interestingly, permeability of both plasmodesmata and gap junctions for some low molecular weight tracer dyes is down-regulated by activation of protein kinase c [ ] . however, movement of macromolecules, in particular nucleoproteins, through gap junctions, has not been described. a case of movement of a nucleoprotein through a channel in a membrane is represented by nucleocytoplasmic trafficking of rnps (reviewed in [ , ] ). among the features of the nucleocytoplasmic export of cellular rnas revealed thus far, several might be relevant to the mechanisms of movement of plant virus genomes. in particular, it has been shown that at all stages of nucleocytoplasmic export, mrna is attached to specific protein frameworks, being transferred from nucleoskeleton to nuclear pore complex to cytoplasm [ ] ; specific proteins in mrnp are thought to chaperone association-dissociation of the mrna to and from these frameworks [ , ] . components of mrnp activate enzymes within the nuclear pore complex which are essential for energydependent translocation of rnp through nuclear pore; two of these enzymatic activities are atpase and rna helicase [ ] . it is tempting to speculate that mechanistic analogies may exist between the nucleocytoplasmic transport of cellular rnps and intracellular and/or intercellular movement of viral rnps. more specifically, the parallel between the rna helicase in the nuclear pore and the putative viral rna helicase in the triple block that is involved in virus movement (see above) certainly is provocative. it should be noted that plant viruses, which undergo some stages in the nucleus of the infected cells (i.e. pararetroviruses and geminiviruses), should be able to perform both nucleocytoplasmic movement and cell-to-cell movement of their genomes; one may wonder to what extent these two processes are similar and whether movement proteins are involved in both of them. comparison of the amino acid sequences of dianthovirus movement proteins with amino acid sequence databases revealed a marginally significant but provocative similarity with membrane proteins of the mip family [ ] . with the blast score of and probability of random matching of . , the similarity of the movement protein of rcnmv with tonoplast membrane protein tip from arabidopsis was the highest in the entire database except for the similarities with the homologous proteins of the other dianthoviruses. analysis of the multiple alignment of the dianthovirus movement proteins with a representative set of the mip proteins (fig. ) showed that the region of similarity included the conserved motif of the " k superfamily" described above and the characteristic conserved motif of the mip family [ ] . each of these motifs consisted of a putative transmembrane helix succeeded by a loop containing conserved amino acid residues (fig. ) . the drastic difference between the two types of proteins is that mip proteins contain six transmembrane helices whereas in the virus movement proteins the region shown in figs. and is the only such predicted helix. nevertheless, we believe that the observed sequence similarity may be functionally relevant indicating that the conserved motif of the " k superfamily" may mediate membrane interaction. for many proteins of this superfamily, membrane interaction is compatible with the observed localization in the cell wall [ ] . however, caution is due in the interpretation of these observations as the hydrophobicity of the conserved region in different movement proteins differed significantly and not for all of them a membrane-spanning helix was confidently predicted ( fig. b and fig. . a conserved sequence motif in the movement proteins of dianthoviruses and the mip family of membrane proteins. asterisks show identical residues and colons show similar residues in the sequences of the movement protein of rcnmv and tip . the consensus (for symbols see legend to fig. ) includes the conserved residues, with one possible exception in the mip-related proteins. the hydrophobic regions predicted to form transmembrane helices are underlined. the motif was extracted from an alignment generated using the macaw program in some of these proteins the conserved motif may be involved in hydrophobic interactions other than membrane spanning. as mentioned above, membrane localization also has been proposed for the triple block proteins and strongly predicted for the movement proteins of monopartite geminiviruses. the observations on the involvement of influenza virus m protein in nuclear export of virus ribonucleoproteins [- ] prompted us to directly compare the m sequence to those of plant virus movement proteins. moderate similarity was revealed between m and the movement proteins of bromoviruses and cucumoviruses (fig. ) . the region of similarity included the conserved motif of the " k superfamily", with the invariant aspartic acid residue and the preceding hydrophobic stretch. m protein chaperones newly formed influenza virus rnps from the nucleus to the cytoplasm; upon formation of virions, this protein is thought to provide the link between the rnp and the virion membrane (reviewed in [ ] ). both of these functions are apparently based on the ability of m to interact both with rnp and the membrane providing a provocative analogy with the plant virus movement proteins. very recently, a mutation in m protein gene resulting in impaired nucleocytoplasmic movement was mapped within the region of similarity to bromovirus and cucumovirus movement proteins [ ] . finally, our previous observations indicated a weak sequence similarity between a domain of cellular kda heat shock proteins with some of the movement proteins, specifically those of caulimoviruses [ ] . a common denominator for all these observations may be that the function of plant virus movement proteins is determined by domains mediating hydrophobic interactions. taking into account the limited sequence conservation and the absence of strictly invariant amino acid residues, it is very unlikely that these proteins (with the exception for the putative helicase in the triple block) have any enzymatic activity. among the known groups of plant viruses [ ] , movement proteins so far have been identified genetically or by sequence comparison for about percent. the variety of these proteins could be reduced to only three types, namely the " k superfamily", with the distantly similar proteins of monopartite geminiviruses; the triple block; and the proline-rich proteins of tymoviruses. obviously, future studies may lead to characterization of movement proteins unrelated to any of these groups. different types of movement proteins may affect different aspects in viruscell interaction leading to virus spread in planta. however, it appears likely that in many cases these proteins mediate hydrophobic interactions between viral and cellular components, in a general analogy with molecular chaperone action [ , ] . as discussed in the first part of this review, a large fraction of experimental data on plant virus movement proteins is very hard to be interpreted unequivocally. conceivably, this reflects the highly complex nature of virus-plant interaction. in this situation, it appears reasonable to focus further experiments on probing the specific functions fo conserved domains revealed by amino acid sequence comparison. hopefully, this approach will highlight new important aspects of plant virus lifestyle. recently, it has been shown that tmv movement protein expressed by transgenic tobacco behaves as an integral membrane protein [moore pj, fenczik ca, deom cm, beachy rn ( ) developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus. protoplasma : - ]. between nucleus and cytoplasm cauliflower mosaic virus gene i product detected in cell wall-enriched fraction basic local alignment search tool expression of a plant virus-coded transport function by different viral genomes dynamic continuity of cytoplasmic and membrane compartments between plant cells triple gene block proteins of white clover mosaic potexviurs are required for transport the tmv movement protein: role of the c-terminal amino acids in subcellular localization and function mutational analysis of cis-acting sequences and gene function in rna of cucumber mosaic virus an analysis of the complete nucleotide sequence of a sugarcane bacilliform virus genome infectious to banana and rice replication of maize streak virus mutants in maize protoplasts: evidence for a movement protein expression of orf- of turnip yellow mosaic virus is necessary for virus spread in plants nucleotide sequence analysis of the movement genes of resistance breaking strains of tomato mosaic virus a three-dimensional view of precursor messenger rna metabolism within the mammalian nucleus potato virus x as a vector for gene expression in plants the p movement protein of tobacco mosaic virus is a single-stranded nucleic acid binding protein gene i, a potential cell-to-cell movement locus of cauliflower mosaic virus, encodes an rna-binding protein how do plant virus nucleic acids move through intercellular connections visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids relationship of tobacco mosaic virus gene expression to movement within plant plant virus movement proteins increase in plasmodesmatal permeability during cell-to-cell spread of tobacco rattle virus from individually inoculated cells of urfs and orfs. a primer on how to analyze derived amino acid sequences the plant virus movement proteins informosome-like virus-specific ribonucleoprotein (vrnp) may be involved in the transport of tobacco mosaic virus infection an influenza virus temperature-sensitive mutant defective in the nuclear-cytoplasmic transport of the negative-sense viral rnas an n-proximal sequence of alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants classification and nomenclature of viruses. fifth report of the international committee on taxonomy of viruses efficient cell-tocell movement of beet necrotic yellow vein virus requires ' proximal genes located on rna a novel superfamily of nucleoside triphosphate-binding motif-containing proteins which are probably involved in duplex unwinding in dna and rna replication and recombination an atp-binding motif is the most conserved sequence in a highly diverged group of proteins involved in positive strand rna viral replication turnip crinkle virus genes required for rna replication and virus movement the complete nucleotide sequence of tobacco rattle virus rna- the sequence of carnation etched ring virus dna: comparison with cauliflower mosaic virus and retroviruses the phylogeny of rna-dependent rna polymerases of positivestrand rna viruses virus evolution: time for sturm und drang diverse groups of plant dna and rna viruses share related movement proteins that may possess chaperonelike activity evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences genes and proteins of the influenza viruses the subcellular localization of gene i product of cauliflower mosaic virus is consistent with a function associated with virus spread ultrastructural location of non-structural protein a of cucumber mosaic virus in infected tissue using monoclonal antibodies to a cloned chimeric fusion protein nuclear mrna export nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (m ) promotes export and inhibits import virus movement in infected plants translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography gap junction protein homologue in arabidopsis thaliana: evidence for connexins in plants similarities between putative transport proteins of plant viruses probable reassortment of genomic elements among elongated rna-containing plant viruses nuclear import-export: in search of signals and mechanisms detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected nicotiana clevetandii plants cooperative binding ofthe red clover necrotic mosaic movement protein to single stranded nucleic acids evolution of the mip family of integral membrane transport proteins cauliflower mosaic virus gene i product (p ) forms tubular structures which extend from the surface of infected protoplasts identification of barley stripe mosaic virus genes involved in viral rna replication and systemic spread a conformation preference parameter to predict helices in integral membrane proteins spray dc (eds) ( ) parallels in cell-to-cell junctions in plants and animals effects of deletions in the n-terminal basic arm of brome mosaic virus coat protein on rna packaging and systemic infection evidence for involvement of a nuclear envelope-associated rna helicase activity in nucleocytoplasmic rna transport a workbench for multiple alignments construction and analysis a mutation in cauliflower mosaic virus gene i interferes with virus movement but not virus replication deletion analysis of brome mosaic virus a protein: effects on rna replication and virus spread tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein cell-to-cell transport of cowpea mosaic virus requires both the / k proteins and the capsid proteins the roles of the red clover necrotic mosaic virus capsid and cell-to-cell movement proteins in systemic infection tobacco rattle virus rna- k gene product potentiates viral movement and also affects symptom production in tobacco evolution of rna viruses we appreciate helpful discussions with dr. v. v. dolja and dr. u. melcher. we would like to thank drs. d. cookmeyer, s. a. demler, r. kormelink, u. k. melcher, n. olszewski and yu. shirako for communicating data prior to publication. a. m. is grateful to dr. r. j. shepherd for constant support and encouragement. received june , key: cord- -g y ied authors: lee, s. k.; sung, h. w.; kwon, h. m. title: s glycoprotein gene analysis of infectious bronchitis viruses isolated in korea date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: g y ied fifteen isolates of infectious bronchitis virus (ibv) were obtained from the kidney, trachea, and cecal tonsil of ib suspected chickens between and years in korea. the s glycoprotein gene of ibv isolates were amplified by reverse transcriptase – polymerase chain reaction (rt-pcr) and analyzed by restriction fragment length polymorphism (rflp) analysis. fifteen korean ibv isolates were classified into groups by their rflp patterns using restriction enzymes, haeiii, bstyi, and xcmi. the rflp patterns for , , and of isolates corresponded to the patterns of ibv arkansas, connecticut, and massachusetts strains, respectively. ten of isolates generated unique km rflp pattern that was observed in the ibv km strain previously isolated in korea. to confirm genetic diversity in the s genes of ibv isolates, viral rnas of representative of ibv isolates were amplified, cloned, sequenced and compared with published sequences for non-korean ibv strains. korean ibv isolates showed amino acid sequence similarity between . % (k - and k - ) and . % (k - and k - ) with each other and they showed amino acid sequence similarity between . % (k - and ga ) and . % (k - and kb ) compared to non-korean ibv strains. by phylogenetic tree analysis, korean ibv field isolates were branched into five clusters in which clusters were differentiated from non-korean ibv strains. especially, korean ibv isolates k - , k - , k - and k - formed a separate cluster. it seems that ibvs continue to evolve and ibvs showing various genetic differences may cocirculate in korea. infectious bronchitis virus (ibv) is the etiological agent of infectious bronchitis (ib), which is an acute and highly contagious disease of the respiratory and sometimes the urogenital tracts of chickens causing tracheal rales, sneezing, coughing, a poor weight gain and reduced feed efficiency in broilers and a decline in egg production and egg shell quality in layers [ ] . ibv belongs to the family coronaviridae [ ] . it is a pleomorphic enveloped virus with club-shaped surface projections (spikes) on the surface of the virion and its genome consists of the single stranded positive-sense rna genome of approximately kilobases [ ] . the virion contains four major structural proteins: the spike (s) glycoprotein, the membrane (m) glycoprotein, the envelope (e) glycoprotein, and the nucleocapsid (n) protein [ , ] . the s glycoprotein of ibv is posttranslationally cleaved into n-terminal s and c-terminal s subunits [ , ] . the s glycoprotein forms the distal, bulbous part of the spike, and the s glycoprotein anchors the s glycoprotein to the viral membrane [ ] . the s subunit is known to contain regions that induce neutralizing, serotype-specific, and hemagglutination-inhibiting antibodies [ , , ] . a number of ibv serotypes and variants have been isolated and identified worldwide [ , ] . these antigenic ibv variants do not completely cross-protect [ ] . therefore, ib continues to be an economically important disease to the poultry industry although ibv vaccines have been used to prevent ib outbreaks worldwide. different serotypes and variants of ibv are thought to be generated by amino acid changes resulting from nucleotide insertions, deletions, or point mutations in the s subunit made by the viral polymerase [ , , ] . since ibv was first reported in in korea and nephropathogenic ibv was recognized in , various serotypes of ibv have been reported in korea. and these ibv isolates showed different patterns from each other and non-korean ibv isolates in reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism (rt-pcr-rflp) analysis [ ] . however, only massachusetts (mass) type live attenuated vaccines as well as inactivated oil-emulsion vaccines were used to control ib [ , , , ] . the purpose of the present study was to genetically characterize ibv strains isolated recently in korea. the s glycoprotein genes of the korean ibv strains were amplified by rt-pcr. amplified s genes were first classified by rflp analysis and then the representative strains were cloned, sequenced and compared to other non-korean published ibv sequences. by the rt-pcr-rflp and phylogenetic analysis, recent korean ibv isolates were classified into at least different groups at the genetic level in which one group included only korean ibv strains. field ibvs were isolated from kidney, trachea and cecal tonsil of ib suspected chickens using spf embryonated eggs between and according to the standard procedure [ ] . the history of these isolates is listed in table . ibv isolates were propagated in -dayold specific pathogen free embryonated eggs and identified as ibv by ibv-specific rt-pcr as described below [ ] . the harvested allantoic fluids were used to prepare viral rna. s gene of korean ibv isolates viral rna extraction and rt-pcr of s gene the viral rna was extracted from allantoic fluid as described previously [ ] . briefly, sodium dodecyl sulfate (final concentration, % wt/vol) and proteinase k (final concentration, µg/µl) were added to the allantoic fluid and the mixture was incubated for min at • c. viral rna was extracted with acid phenol : chloroform ( : , ph . , ambion, woodward, u.s.a.) and chloroform : isoamylalcohol (iaa) ( : ), and further purified using the rnaid kit (bio , carlsbard, u.s.a.). finally, the rna was resuspended in diethyl-pyrocarbonate (depc) treated water and stored at − • c until used in the reverse transcriptase (rt) reaction. amplification of the s gene by rt-pcr was performed using the forward s oligo ( tgaaaactgaacaaaaga ) and reverse s oligo ( ctaaactaacataagg gc ) primer pair [ ] . the rt reaction to synthesize cdna contained purified rna, pmol s oligo primer and rt premix accupower rt premix (rtase, stabilizer and tracking dye) (bioneer, korea). the mixture was incubated at • c for min, and then heated for min at • c to stop the reaction. for the pcr reaction, pmol each primer (s oligo and s oligo ) and cdna were added to accupower pcr premix (taq dna polymerase, each dntp, tris-hcl, kcl, mgcl , stabilizer and tracking dye) (bioneer, korea). the pcr was performed by cycles of denaturation at • c for sec, annealing at • c for sec, and polymerization at • c for sec. the final polymerization step was performed at • c for min. the pcr products were analyzed on a . % agarose gel. pcr products with the expected size (about bp) were excised from an agarose gel and purified using geneclean kit (bio ). the restriction enzymes haeiii, bstyi and xcmi were used to digest the pcr products of s gene as described by kwon et al [ , ] . the rflp patterns were observed after electrophoresis on a % agarose gel. nine representative ibv isolates (k - , k - , k - , k - , k - , k - , k - , k - , k - ) of isolates after rflp analysis were sequenced. pcr products were cut from % agarose gels and purified using geneclean (bio ). purified pcr products were cloned into the ta cloning vector (invitrogen, carlsbed, ca) and transformed into competent cells (top ) (invitrogen). cells carrying recombinant plasmid were selected on lb agar plates containing ampicillin and x-gal. plasmid dna for sequencing was prepared by plasmid maxi kit (qiagen, santa clarita, ca, u.s.a.). sequencing was performed with the m forward and m reverse primers. dna sequencing was performed using an abi prism dna analyzer (applied biosystems, foster city, ca, u.s.a.). nucleotide sequence data were compiled and analyzed using the clustal v method in megalign software (dnastar, inc. madison, wi). phylogenetic trees for s glycoprotein were generated using the maximum parsimony method with bootstrap replicates in a heuristic search with the paup . software program (sinauer associates inc., sunderland, ma, u.s.a.). the sequence data of s gene reported have been deposited in the genbank database (table ) . sequences used for comparison or phylogenetic analysis in this study were obtained from the following genbank database accession numbers: arkansas (m ), fifteen ibvs were isolated from trachea, kidney and cecal tonsil of broiler chickens (table ) . six, and of ibvs were isolated from trachea, kidney, and cecal tonsil respectively. the age of chickens with ib outbreaks was from to days. rt-pcr-rflp analysis was initially performed to classify ibv isolates. fifteen korean ibv isolates were classified into four groups by rflp analysis (fig. ) . the rflp pattern for isolate k - corresponded to a pattern for ibv connecticut strain. isolate k - showed both massachusetts and km rflp patterns. three isolates (k - , k - , and k - ) were identical to the ibv arkansas strain and isolates (k - , k - , k - , k - , k - , k - , k - , k - , k - and k - ) had same rflp pattern as the ibv km strain which was isolated in in korea [ ] . the whole s gene of representative of korean ibv isolates was sequenced to further characterize the isolates. the nucleotide and deduced amino acid sequences of those ibv isolates were determined and compared with the sequences of published non-korean ibv strains ( table , fig. ). korean ibv isolates had nucleotide sequence similarity between . % (k - and k - ) and . % (k - and k - ) with each other and they had nucleotide sequence similarity between . % (k - and de ) and . % (k - and h ) with non-korean ibv strains. korean ibv isolates had amino acid sequence similarity between . % (k - and k - ) and . % (k - and k - ) with each other and they had amino acid sequence similarity between . % (k - and ga ) and . % (k - and kb ) compared to non-korean ibv strains. the korean ibv k - isolate had an s amino acid sequence most similar to the mass strain ( . %) and . % similar to the h strain. korean ibv isolate k - had an s amino acid sequence most similar to h ( . %) and . % similar to mass . isolates k - and k - had amino acid sequences similar ( . %- . %) to the ark dpi strain. isolates k - , k - , k - and k - were . % to . % similar to one another, and they were . % to . % similar to eight published non-korean strains (ark dpi, beaudette, connecticut, d , gray, h , kb , and mass ). isolates k - had an s amino acid sequence . % to . % similar to other korean isolates and . % to . % similar to published non-korean strain (ark dpi, beaudette, connecticut, d , gray, h , kb , and mass ). the deduced amino acid sequences of korean ibv isolates were aligned with the sequences of published non-korean ibv strains (fig. ) . the most variations were observed between residues - , - , and - (numbering is in reference to mass strain). phylogenetic trees were constructed from the nucleotide and deduced amino acid sequences of the s glycoprotein genes of korean ibv isolates and non-korean ibv strains (fig. ) . nine korean ibv isolates were grouped into five distinct branches. the k - isolate formed the first branch in which mass was included. the k - isolate formed the second branch that related to non-s gene of korean ibv isolates fig. . phylogenetic relationship based on the deduced amino acid sequences of the s glycoprotein of the korean ibv field isolates (k - , k - , k - , k - , k - , k - , k - , k - , and k - ) and non-korean ibv strains generated by the maximum parsimony method with heuristic search and bootstrap replicates. the tree was rooted to a sequence of the ibv beaudette strain. the length of each branch represents the number of amino acid changes between sequences korean ibv isolates, kb , h , and d . the k - and k - isolates formed the third branch and the k - isolate formed the fourth branch. k - , k - , k - and k - isolates formed the fifth independent group. the spike glycoprotein of ibv is translated as a precursor protein (s ), and then cleaved into two subunits s and s [ , ] . the ibv spike glycoprotein cleavage recognition site for korean ibv isolates k - , k - , k - , k - , k - , k - and k - isolates had the sequence arg-arg-phe-arg-arg, which was found in the published ibv beaudette, d , h , kb , and mass strains [ , ] . whereas korean ibv isolates k - and k - isolates had the sequence arg-arg-ser-arg-arg which was found in the published ibv ark dpi and gray strains [ , ] . ib has been a continual problem in korea although both mass type live attenuated vaccine and inactivated vaccine were widely used to control the disease. fifteen korean ibv isolates were initially analyzed by rt-pcr-rflp and followed by nucleotide sequencing of the s glycoprotein gene in this study. korean ibv field isolates between and were characterized using rt-pcr-rflp analysis and pathogenicity testing but the sequences of those viruses were not reported [ ] . according to rt-pcr-rflp analysis, ibv field isolates were classified into five genotypes (i, ii, iii, iv, and v). six genotype i ibv isolates showed similar rflp patterns to mass type of ibv and only genotype ii ibv isolates were only isolated in . twenty-nine genotype iii ibv (km type) isolates showed distinct rflp patterns in pcr-rflp analysis using restriction enzymes haeiii, ecori, and bamhi [ ] . the km isolate is the representative genotype iii isolate. genotypes iv andv were newly isolated in . in this study, of field ibv isolates were classified as the km type and isolates were classified as arkansas and as connecticut types. in pathogenicity tests, isolate km caused % mortality, severe nephritis and renal urate deposition in the kidneys of infected chicks, but genotypes i, ii, iv and v only induced respiratory distress at to days after inoculation [ ] . the h vaccine could not protect chicks against challenge with the km isolate, genotype iii. therefore, the km type has seemed to be a major ibv circulating in korea. the korean ibv k - isolate showed both massachusetts and km type rflp patterns in pcr-rflp analysis. the detection and differentiation of two different kinds of viruses in a single sample is a significant advantage of rt-pcr-rflp analysis which other researchers have also reported [ ] . korean ibv isolates (k - , k - , k - , k - and k - ) sequenced among ibv isolates classified as km type by rflp analysis showed . % to . % nucleotide sequence similarity and . % to . % amino acid sequence similarity among themselves. it seemed that ibvs with differences in genetic composition existed in field condition although they showed identical rflp patterns. s gene of korean ibv isolates by alignment of the s glycoprotein of korean ibv isolates with published non-korean ibv strains, the most sequence variations were observed between residues - , - and - . regions between - and - showing high amino acid variations herein were similar to hypervariable region (hvr , residues - ) and hypervariable region (hvr , residues - ) of ibv [ , ] . the hvrs are associated with two separate viral neuralizing and conformationally dependent epitopes [ , ] . amino acid variation region - was similar to region iii ( - ) associated with a neutralizing epitope [ ] . virus neutralization tests will be needed to definitively classify korean ibv isolates into a distinct serotype although pcr-rflp pattern correlates with serotype [ ] . in the phylogenetic tree, korean ibv isolates formed five distinct clusters. korean ibv k - isolate was clustered into mass group although it was classified into the connecticut group by pcr-rflp analysis. korean ibv k - isolate was clustered into the same genotypic clade as non-korean ibv strains kb , h , and d that were an attenuated vaccine strains [ , , ] . therefore, k - isolate appears to be originated from a vaccine strain. korean ibv k - and k - isolates formed distinct clusters that were related to non-korean ibv ark , ark dpi, gray and jmk strains although k - and k - isolates were classified into the arkansas type by pcr-rflp analysis. two korean ibv isolates k - and k - had the same arg-arg-ser-arg-arg on spike glycoprotein cleavage recognition sites as the non-korean ibv strains ark dpi, gray, and jmk [ ] . spike glycoprotein cleavage recognition site does not appear to correlate with serotype or pathogenicity [ ] . korean ibv k - isolate formed a distinct cluster although it was classified into the km type with other several ibv isolates by pcr-rflp analysis. it showed . % to . % amino acid sequence similarity compared to other korean ibv isolates, which was lower similarity than other ibv isolates. it was noteworthy that k - isolate was related to non-korean ibv isolate de and ga in the phylogenetic tree although it showed . % (k - and ga ) to . % (k - and de ) amino acid sequence similarity. the ibv de strain was involved in ib outbreaks of broilers on the delmarva peninsula, u.s.a. in , and it was classified into the delaware variant (de var) type by virus-neutralization tests, cross-challenge tests and s- gene analysis [ ] . the ibv ga strain was isolated from broilers in georgia, u.s.a. and is similar to the ibv de strain [ ] . but it was genetically distinct from ibv de and shared very low antigenic relatedness with ibv de and other ibv isolates and thus was designated into a new serotype, georgia [ ] . it seems that the korean ibv k - isolate may be one of the korean ibv variants. further characterization by virus-neutralization tests and cross-challenge tests using several ibv strains will be needed. the korean ibv k - , k - , k - , and k - formed a distinct cluster that consists of only korean ibv isolates. they were classified into the ibv km type by pcr-rflp analysis, which has been isolated mainly in korea [ ] . the evolution of ibv is seemed to be influenced by a number of factors such as the use of multiple strains for vaccination, population density and host immune status [ ] . in addition, ibv has a high error rate during the genomic transcription and then, produces a quasispecies phenomenon where many different viral genotypes will cocirculate in the host, with each virus potentially having different levels of fitness for the host environment [ , ] . widespread uses of various vaccines made from heterologous ibvs in the field may play an important role in increasing the number of new genetic variants in korea. in conclusion, korean ibv field isolates were different from published non-korean ibv strains in nucleotide and amino acid sequences and were clustered into different groups from non-korean ibv isolates by phylogenetic tree analysis. and korean ibv isolates were clustered into at least groups in which groups were differentiated from non-korean ibv isolates. virus-neutralization test and cross-challenge tests using several ibv strains will be needed to further characterize the ibv isolates. completion of the sequence of the coronavirus avian infectious bronchitis virus molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates coronavirus ibv: further evidence that the surface projections are associated with two glycopolypeptides location of the amino acid differences in the s spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus coronavirus ibv: virus retaining spike glycopolypeptide s but not s is unable to induce virus-neutralizing of haemagglutination-inhibiting antibody, or induce chicken tracheal protection amino acids within hypervariable region i 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 propolypeptice of ibv strains beaudette and m infectious bronchitis the quasispecies (extremely s gene of korean ibv isolates heterogeneous) nature of viral rna genome population; biological relevance-a review variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens a laboratory manual for the isolation and identification of avian pathogens antigenic and s- genomic characterization of the delaware variant serotypes of infectious bronchitis virus cross-immunity in chickens using seven isolates of avian infectious bronchitis virus spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus further development and use of molecular serotype identification test for infectious bronchitis virus an outbreak of nephropathogenic infectious bronchitis in commercial pullets identification of recent infectious bronchitis virus isolates that are serologically different from current vaccine strains antigenic domains on the peplomer protein of avian infectious bronchitis virus; correlation with biological functions epitopes of neutralizing antibodies are localized within three regions of the s spike protein of infectious bronchitis virus. world veterinary poultry association sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus phylogeny of antigenic variants of avian coronavirus ibv molecular cloning and sequence comparison of the s glycoprotein of the gray and jmk strains of avian infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis the molecular biology of coronaviruses identification and analysis of the georgia serotype, a new serotype of infectious bronchitis virus sequencing and analysis of s gene of avian infectious bronchitis virus strain d epitopes on the peplomer protein of infectious bronchitis virus strain m as defined by monoclonal antibodies outbreaks of infectious bronchitis in korea epidemiology classification of infectious bronchitis virus isolated in korean between coronavirus proteins; biogenesis of avian infectious bronchitis virus virion proteins cloning and sequencing of genes encoding structural proteins of avian infectious virus evidence of natural recombination within the s gene of infectious bronchitis virus author's address: dr. hyuk moo kwon the authors thank dr. chang-won lee for his excellent assistance to make phylogenetic tree and dr. mark w. jackwood for editorial comments. this work was supported by korea research foundation grant (krf- - -g ), korea. key: cord- -gzts h y authors: fennestad, k. l. title: pathogenetic observations on pleural effusion disease in rabbits date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: gzts h y a pathogenetic study of pleural effusion disease (ped) in rabbits was made, using the virulent ped agent or virus (pedv) and an avirulent derivate of this isolate. independent of infective dose within the range examined, the virulent isolate caused fatal clinical disease, whereas the avirulent isolate caused subclinical infection. the two isolates differed in rapidity of initial spread of infection and in the maximum virus titres in serum, but they both resulted in a similar low level persisting viraemia. circulating virulent virus gradually became avirulent during the viraemia. avirulent infection induced protective immunity to virulent challenge during the first week after primary infection, but full clinical protection was not established until after the fourth week. the findings, corrobated with other closely comparable observations, suggest that the emergence of ped as an intercurrent mortality problem during rabbit passage of pathogenictreponema pallidum is the result of a specific selective pressure on a benign passenger virus. the expression of virulence of pedv appears to be dependent on length of interval between passages. pleural effusion disease (ped) is a rabbit infection probably caused by a host-specific, small, enveloped virus which measures -- nm by filtration ( ) . although known since the agent has not been convincingly demonstrated by tissue culture, electron microscopy or specific serological technique ( , i , ) . ped is considered to be a new-disease with characteristic clinical signs and pleural effusion as the typical post mortem finding ( , ) . the infection is characterized by a long lasting viraemia and formation of antibodies which appear to have only little neutralizing capacity ( , ) . however, there is clinical protection to re-infection after the fourth week ( ) . ped emerged during the sixties as a serious intereurrent mortality problem among rabbits inoculated with treponema pcdlidum ( , , , ) . subsequently, it was shown that. the causative agent was being passed from rabbit to rabbit as a passenger of the rabbit testieular suspensions of trepoheroes ( , ) . as such the infection has undoubtedly been spread from laboratory to laboratory, but the disease per se appears to be confined to this experimental situation ( ) . there is evidence to suggest that the serial propagation of treponemes in rabbits, as practised in a number of laboratories at intervals of to -- days, may have been instrumental in the emergence of disease. by such propagation a normally harmless virus may enter the passages and gradually acquire virulence through a selective pressure on a heterogeneous virus population during hundreds of passages. support for this contention is the enhancement of virulence of ped virus (pedv) observed in the "natural" disease and --after its isolation --by continued serial propagation in rabbits at intervals of --- days (fig. ) . however, there is also evidence which might suggest that increasing time of persistence of virulent virus in the individual rabbit host may operate in the reverse direction by selecting a population of predominantly avirulent pedv particles, which appear to be unable to cause disease unless undergoing new rabbit passages at short intervals (t, ). the present pathogenetic study on virulence, viral growth and the development of protection was carried out with the original pedv isolate and an avirulent progeny of this isolate obtained from a rabbit six months after experimental infection. conventional albino rabbits (new zealand-white type), aged -- months, were used, and all inoculations were made by the subcutaneous (se.) route in ml amounts. before use, these animals had been employed once for pyrogen testing of protein fractions of human blood. all rabbbits came from the same colony (sse:cph) which has been dosed since . the colony is considered free of pedv, since convincing observations to the contrary have never been made ( ) . the rabbits used for propagation of treponemes at statens seruminstitut sinee also came from this colony (ef. fig. ). virus isolates the highly virulent pedv was isolated in from a freeze-dried rabbit testicular suspension of t. pallidum ( ) . following isolation the virus was passed serially at short intervals through more than rabbits before preparing a virus stock. the stock consisted of pooled rabbit serum obtained hours after inoculation with blood or pleural fluid. i t will appear from fig. that the virulence of virus had not become "fixed" at time of preparation of the stock pool. the avirulent derivate of p e d v was obtained from blood of a healthy rabbit six months after experimentm neonatm infection with the above mentioned stock virus. this isolate was passed serimly through rabbits at weekly intervals by inoculation of . ml serum mixed with . ml pbs (ph . ). during these ahnost subctinicm passages no mortality occurred and typical clinical signs of p e d were seen in only two rabbits (passages nos. and ). the stock pool of the isolate consisted of serum from the l t h rabbit passage obtained hours after inoculation ( ) . both stocks were stored in ml miquots at -- o c until required for experiments. lack of in vitro methods necessitated the use of rabbits for demonstration and quantitation of pedv. for these purposes the previously described rabbit test was mortmity among several hundreds of rabbits annumly inoculated with treponemat suspensions at intervals of -- days. about p e d was recognized as a disease entity causing intercurrent death among the treponema-inoeulated rabbits, and in october the causative passenger virus was isolated. after isolation p e d v was initimly carried through passages in femme rabbits by sc. inoculation of blood or pleural fluid at intervals of -- days. the virus was then passed serimly through t male rabbits in the same manner, but at intervals of -- days. the black dots indicate the annual mortmity during the -year period of these passages. the intercurrent disease problem among the treponema-infected rabbits was solved by removing the passenger virus from the rabbit testicular suspensions of treponemes by p~ssing the contaminated treponemes through hamsters arid back to rabbits. this procedure was first used in , but due to a mistake not suecesfully accomplished until . until now ( ) these passages have remained free of p e d v k . l . fennes~ad : used ( ) . briefly, the inoculum to be examined was given sc. to a rabbit. fever together with uveitis or death with necropsy findings characteristic of ped, or both, was considered as evidence of p e d v in the inoculum. animals failing to show these signs were challenged days after inoculation with the highly virulent pedv. presence of clinical protection after challenge was considered as evidence of p e d v in the inoculum. the number of rabbit-infective doses (rid) per mi of the virus stocks was estimated by inoculating -fold dilutions in pbs, using ~ rabbits per dilution. the term r i d refers to a dose capable of producing typical signs of f e d and/or clinical protection against challenge. the highest dilution producing these responses was taken as endpoint titre of the virus. i n this way the stock of virulent f e d v was found to contain i~id per ml, while the stock of the avirulent derivate contained i~id per ml. uniform groups of rabbits were inoculated with decimal dilutions of the two stocks. during a period of days the rabbits were then observed twice daily for clinical signs of disease. dead animals were examined for gross lesions and bacteriologically in order to establish the cause of death ( ) . surviving rabbits failing to show the typical signs of f e d were challenged with i~td of p e d v and observed for a period of days. in a preliminary experiment six rabbits were inoculated with approximately r i d of fedv. two rabbits were killed for the study of infectivities of tissues and body fluids at hours, and days after inoculation. from each rabbit was obtained serum and heparinized blood. erythrocytes and buffy coat were washed -- times in pbs (ph . ). lung, thymus, liver, spleen, kidney, popliteal lymph nodes, and brain were frozen at -- ° c. a t per cent suspension (wt/vol) in hanks' solution was prepared from the frozen organs by grinding with sand. serial -fold dilutions of the preparations were made in pbs using one rabbit per dilution. in a succeeding experiment two groups of rabbits were inoculated, respectively, with p e d v and the avirulent derivate. these rabbits were followed by serum samples. endpoint virus titres of these samples were determined as for the virus stocks, using i -- rabbits per dilution. to observe the temporal development of clinical protection rabbits were inoculated with i~id of the avirulent derivate of pedv. groups of rabbits were then challenged with ~ i~id of f e d v at selected intervals after the primary infection. a control group of animals were inoculated with i~id of f e d v without primary infection. four groups of animals were examined for viraemia immediately prior to challenge, using . ml of serum mixed with . ml fbs as inoculum. and fever, together with uveitis. in assessing the infectivity of the two isolates all surviving rabbits failing to show clinical signs were challenged with pedv to determine whether or not the primary inoculation had induced protection (table ) . after inoculation with pedv of rabbits developed clinical disease and of rabbits died with pleural effusion. the severity of the clinical response did not appear to be dose dependent, but the incubation period, i.e. the time from inoculation until onset of fever, and the mean death time (mdt) appeared to be influenced by the dose. the i rabbits failing to showany clinical signs were fully susceptible to challenge. after inoculation with the avirulent derivate none of the rabbits showed typical clinical signs of ped, but ephemeral fever occurred in of rabbits receiving the highest dose. when challenged with pedv of the rabbits were clinically protected, indicating that the primary inoculation had induced clinical immunity. the remaining rabbits were fully susceptible to challenge. these data indicate a dose-independent difference in virulence between the two isolates. pedv infection resulted in clinical disease, whereas infection with the avirulent derivate almost exclusively resulted in a subclinical response. from the total incidence of typical clinical responses and the development of clinical protection the median infective doses (id ) per ml was calculated to be . for pedv and t - for the avirulent derivate. virus in blood did not appear to be cell associated, but it was demonstrable in serum and almost all tissues examined on days , and after inoculation with pedv ( table ). the highest concentration of virus, i.e. about rid was found in serum and lungs on p.i. day . in other experiments (data not shown) the same virus concentration was found in pleural fluid from animals dying about this time. pooled fluid from the anterior chamber of the eye examined at hours after inoculation, i.e. before uveitis became clinically detectable, had a titre of and on p.i. day , i.e. after the clinical uveitis had disappeared, the titre of this fluid was about rid. from p.i. days to there was a generm decrease in titre of all materim examined. the significance of the lower titres of various tissues as compared with serum is difficult to evaluate, particularly since no attempts were made to remove the blood by perfusion prior to the titrations of tissues and because of the method of quantitation. to examine viral growth and persistence of viraemia, serum was assayed for virus content at various times after rabbit infection with the two isolates ( figs. and ). after infection with rid of pedv, virus was demonstrable already at hours and then the titre increased rapidly to a maximum of about at hours p. i. inoculation of a -fold lower dose delayed the appearance of virus in serum until between and hours, but a titre of about was still reached at hours. this suggests that the dose of virus had no effects on the maximum titre attained. from p.i. day the titre slowly declined to about l s on p.i. day and on p.i. day it ranged from < to . the same range in titres was present during the following five months. one rabbit was consistently negative after p.i. day , and one was negative at and days after inoculation. fig. the results show that for comparable doses of the two isolates the virulent pedv became detectable earlier and reached a t -to -fold higher maximum titre than the avirulent isolate. the in vivo determination of virus concentration in serum at various times after infection offered an opportunity to compare virulence of circulating virus on primary rabbit inoculation (table ) . following infection with the virulent pedv, infectious serum diluted - to - almost invariably resulted in typical clinical disease provided that the serum was obtained during the first days of viraemia. serum from p.i. day diluted t - to - caused typical clinical response in two of six rabbits, but from day of viraemia serum diluted - to -a never resulted in clinical signs of ped. this suggests that the population of virulent pedv particles after p.i. day gradually was being replaced by a population of low or avirulent particles. after infection with the avirulent derivate, the isolates from serum obtained during the entire period of viraemia were demonstrable in serum dilution - to -a, but the primary rabbit inoculation of these dilutions never resulted in clinical signs of ped. the protective response was tested by sequential infection with the avirulent isolate and pedv. for this purpose groups of rabbits were inoculated with the avirulent isolate. ephemeral fever occurred on p.i. days -- (average . days) in of animals observed for -- days, but no other ciinical signs of disease were seen. four of the groups of rabbits were examined for presence of viraemia on p. i. days , , and and the proportion of animals with demonstrable viraemia was / , / , / , and / . at selected intervals after the primary infection, the various groups of rabbits were challenged with pedv. in addition, a control group received the same inoculum (fig. ) . no clinical signs of protection were present hours after chmlenge, i. e. at a time when viraemia after avirulent infection had not yet become detectable (fig. ) . six days after challenge there was clear evidence of protection, but typical clinical signs occurred in single animals challenged on days , and , indicating individual differences in immunogenic or protective potential. at days the only clinical sign of disease was transient fever in of rabbits and at all challenges later on there was a subclinical response. the results show that r i d of the avirulent virus induced full clinical protection against challenge with r i d of the virulent p e d v from the th-- th week after infection. the two isolates of the same origin tested in the study were selected for excessive, but not ultimate virulence and avirulence. the serial rabbit passages of the isolates before and after preparation of the virus stocks indicate stability of their virulence properties within a relative large number of passages. isolates of p e d v from other laboratories propagating contaminated treponemes in rabbits have varied widely in virulence and behaved similarly in serial passages, but concerning virulence they all appear to fall within the range observed in this study ( , ) . the difference in virulence was correlated with a difference in rapidity of the initial multiplication and in the maximum titres attained, but already after about three weeks the titre level in the two infections was similar. ped is accompanied by interferon production during the first days after infection and there is evidence to suggest that the interferon response also is correlated with the virulence of isolates ( , ) . from about one month after the virulent infection, circulating virus appeared to lose virulence, i.e. upon primary rabbit inoculation the various dilutions of serum induced mainly a subclinical response as observed after avirulent infection. a similar observation was made after infection of baby rabbits with virulent pedv ( ). in these experiments it was also observed that isolates from serum obtained on p.i. days and regained their virulence after -- serial transfers in rabbits, whereas isolates from p.i. day required from to more than serial passages before virulence appeared restored. these data suggest that throughout the course of infection the original virulent virus becomes avirulent and that the latter property becomes increasingly stable. an early change in virulence during residence in the host is suggested by other observations. thus serial passages of virulent pedv isolates preserved or enhanced virulence provided that the passages were carried out at intervals of -- days (mortahty: per cent,), whereas virulent isolates became avirulent when the passage interval was prolonged to days. passage at intervals of days resulted in a mortality of per cent ( , ) . this selection mechanism for virulence depending on the interval between passages may explain why ped was first recognized in laboratories using the t. pallidum immobilization (tpi) test. this test was introduced in ( ) and became widely popular from the fifties, particularly in europe. the test requires propagation of treponemes in rabbits at short intervals. ped emerged between and years later as a cause of intercurrent rabbit mortality in a number of tpi laboratories. this may suggest that the change in virulence by selection during passages was very slowly imposed, but besides the time of contamination of the treponemal suspensions, other factors such as the number of animals used per passage, passage interval, and the breed of rabbits have probably also been instrumental in the development of virulence. it has previously been shown that acute ped is followed by a rise of electrophoretic gamma globulin and that the concentration of serum igg in viraemic rabbits days after virulent infection is significantly higher than in uninfected controls ( , ) . failure of such antibodies to eliminate circulating virus is in accordance with the observation of poor protective value of non-infectious immune or hyperimmune sera in passive protection experiments ( ) . the development of clinical protection as seen in the present study may be explained by a cooperation between antibody and cellular factors. persisting viraemia is a characteristic of a number of infections caused by different viruses and it has been suggested that invasion of the lymphoreticular tissues is a common feature of such infections ( , ) . in the present study the titrations of various organs and tissues did not suggest any particular site of virus replication, but this does not exclude replication in the lymphoreticuiar tissues. pedv infection resembles lactate dehydrogenase-elevating virus (ldv) infection, which cause persisting viraemia in mice, at least with respect to discovery and laboratory transmission ( ) . both were first found as biological contaminants of commonly used biological preparations. ldv occurs as a contaminant of transplantable tumours without causing disease, and pedv occurs as a contaminant of treponemal suspensions causing intercurrent deaths or subclinical infections. ldv contaminated material may interfere with the interpretation of experimental results ( ) , and the same may well apply to pedv contaminated treponemal suspensions. ldv is probably larger than pedv and reaches a maximum plasma titre of l° which is considerably higher than that of pedv. this may perhaps explain why pedv, in contrast to ldv, has not yet been demonstrated by electron microscopy of serum. pleural effusion disease in rabbits. clinical and post mortem observations. acta path. microbiol, scand pteural effusion disease in rabbits. interferon in body fluids and tissues after experimental infection. acta path. microbiol, scand pleural effusion disease agent as passenger of treponema pallidum suspensions from rabbits pleural effusion disease in rabbits. observations on viraemia, immunity and transmissibility interferon in rabbit sera after inoculation with treponeraa patlidum suspensions contaminated with p e d virus pleural effusion disease in rabbits. properties of the aetiological agent fever after inoculation of rabbits with treponema pallidum screening out a virus-like agent from the testicular suspension of the nichols pathogenic t. pauidum. with observations on certain characteristics of the agent spontaneous deaths among rabbits inoculated with treponema pauidum less than weeks before intercurrent death of rabbits after inoculation with treponema pallidum in the netherlands. th iclas syrup general features of persistent virus infections immobilization of treponema pallidura in vitro by antibody produced in syphilitic infection coronavirus-like particles in laboratory rabbits -with different syndromes in the netherlands biological contaminants and scientific misinterpretations lactic dehydrogenase virus. (virology monographs rabbit cardiomyopathy associated with a virus antigenically related to human coronavirus strain e persistent, slow and latent viral infections entretien au laboratoire des souches de tr@pongmes pathog@nes authors' address: dr. k. l. fennestad, statens seruminstitut, amager boulevard , dk- copenhagen s., denmark.received june , i key: cord- - budstbg authors: takayama, n.; kirn, a. title: an improved method for titration of mouse hepatitis virus type in a mouse cell culture date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: budstbg plaque assay in dbt cells with deae-dextran and trypsin presents a titration system for mhv as sensitive as the ld( ) assay in mice. plaque assay of mouse hepatitis virus type (mttv ), a member of the genus coronavirus ( ) , was carried out in mouse embryo cells ( ) as well as mierofocus assay in mouse macrophages ( ) . however these methods were either not so sensitive as the ld assay in mice or else not convenient for routine use. in this paper we describe an improved method for the titration of mhv in the same continuous eeli-line that makes the growth of mhv possible ( ) . the cell-line sr-cdf -dbt (dbt), originating from a mouse brain tumor, was kindly supplied by dr. hirano (institute of medical sciences, university of tokyo, japan). dbt cells were grown in eagle's minimum essential medium (mem) supplemented with per cent calf serum (cs) and per cent tryptose phosphate broth (tpb). when dbt-monolayers were infected with mhv (american type collection strain previously submitted to three cycles of plaque purification in our laboratory), the formation of s}mcytia became visible at hours postinfeetion (p.i.) and increased in both size and number parallel to the virus titer. plaque assays were performed on dbt-monolayers in . cm-diameter petri dishes, following the method described by hii~ano et, al. ( ) . in dbt cells overlaid with me)/i containing per cent noble agar, per cent cs, per cent tpb, and : , neutral red, mhv -plaques of . -- . mm (mean value = . ram) in diameter could be counted at hours p.i. the plaques were clearly-defined and sometimes contained a deeply-stained area at their center. comparative titrations showed that the plaque assay gave tigers lower by about half a log than did the lds method in mice (table ) . to ira.prove the sensitivity of the plaque assay we examined the effect of diethylaminoethyl-dextran (deae-d) on the plaque formation and plaque diameter of mhv , as bradbullne and tyrrel] had reported that deae-d added to overlay agar increased the plaque number of human coronavirus ( ). when deae-d was added to the overlay medium, no significant increase in the mean plaque number was produced compared with that of cultures overlaid with a deae-d-free medium. when deae-d was given along with the virus suspension to dbt cells, a clear-cut increase in the plaque number was found (fig. ) . the most remarkable increase was observed in those cells which received a. ~zg/ml dose of deae-d. ~vith a tzg/mi dose of deae-d the mean plaque number was still times greater than that of the control. with regard to the plaque diameter, no significant difference was found between the control group and a n y of the treated groups. the effect of trypsin on m h v -p l a q u e formation in dbt-monolayers was also investigated. after infection, d b t cells were overlaid with mem containing per cent noble agar and trypsin at a concentration of , or [zg/ml. the dl t-dishes were incubated at °c for hours and plaques were stained with a second overlay medium consisting of mem with per cent noble agar, per cent cs, per cent t p b and : , neutral red at ° c for -- hours. whereas an enhancing effect, on the plaque formation of myxoviruses has been reported ( , ), no significant increase in the mean plaque n u m b e r of m h v was observed. however, the plaques were larger (mean value -- . mm in diameter; range = . -- . ram) and clearer t h a n those in the control group. the sensitivity of the plaque assay in dbt-monolayers was equal to t h a t of the lds assay in mice when ptg/ml of d e a e -d were added to the virus dilutions and the plaques could be counted without difficulty when the overlay m e d i u m contained [zg/ml of trypsin (table ) . as d b t is an established cell-line, it is far less troublesome in handling t h a n mouse macrophages and, through the plaque assay on d b t cells, virus titers are determined in a shorter period of time t h a n through the lds assay in mice which is widely used as a titration m e t h o d of m h v today. consequently, the assay method of m h v described above can a d v a n t a g e o u s l y replace not only plaque assay on mouse embryo cells or mouse maerophages but also the lds assay in mice. plaque formation by influenza viruses in the presence of trypsin the propagation of "coronaviruses" in tissue-culture plaque formation by a mouse hepatitis virus %eplication and plaque formation of mouse hepatitis virus (mttv- ) in mouse cell line dbt culture observations on the growth of mouse hepatitis virus (mitv- ) in mouse macrophages genetie recombination for antigenic markers of antigenieally differen$ strains of influenza b virus key: cord- -or azf g authors: huang, zheng; yao, dong; xiao, shan; yang, dong; ou, xinhua title: full-genome sequences of gii. [p ] recombinant norovirus strains from an outbreak in changsha, china date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: or azf g on march , school students suffered from vomiting, diarrhea, and abdominal pain after participating in a group activity at a commercial park. in this outbreak, multiple norovirus genotypes were observed, including gii. [p ], gii. [p ], and gii. [p ]. further, we determined the full-genome sequences of two strains of gii. [p ] recombinant noroviruses, which were nt long. phylogenetic analysis based on open reading frames (orfs) and revealed that these recombinants were related to stains of different genotypes from different countries. the full genome nucleotide sequences of the two isolates were . % and . % identical to those of strains from london and thailand, respectively. simplot analysis revealed the presence of a break point at nt in the orf region. the histo-blood group antigen binding sites were conserved in both recombinant viruses. our findings not only provide valuable genetic information about a recombinant norovirus but also contribute to our general understanding of the evolution, genetic diversity, and distribution of noroviruses. noroviruses are a major causative agent of acute gastroenteritis, with high prevalence across the globe [ ] . norovirus outbreaks in public spaces, such as kindergartens and primary or secondary schools, are generally associated with low hygiene levels and contaminated food or water [ ] [ ] [ ] . noroviruses belong to the family caliciviridae and have three open reading frames (orfs) in their genome. orf encodes a large nonstructural protein (ntpase, p , vpg, clpro and rdrp), orf encodes the major structural capsid protein (vp ), and orf encodes a minor structural protein (vp ) [ ] . based on the amino acid sequence of the capsid protein, noroviruses have been classified into genogroups (gi~gx) and approximately genotypes so far [ , ] . of the genogroups, gi, gii and giv can be found in human infections, with genogroup gii being the most common [ ] . the gii. [p ] genotype has been reported in many countries and is the prevalent strain in most outbreaks and human infections [ ] [ ] [ ] . previous reports have shown that gii. [p ] became the main epidemic genotype of norovirus in china, in [ ] [ ] [ ] [ ] . gii. [p ] and gii. [p ] have also been found in the coastal environment and in seafood in korea. these two genotypes share a close phylogenetic relationship with gii. [p ] . however, these genotypes are found infrequently in human infections, and this might be associated with a unique histo-blood group antigen (hbga) binding site involved in host susceptibility and the presence of decoy glycan receptors in the human gastrointestinal tract that prevents binding of the virus [ , ] . due to the high frequency of genetic recombination at the orf and orf junction, a number of recombinant strains have emerged under natural selection [ , , ] [ ] [ ] [ ] [ ] . although those genotypes have not caused severe outbreaks, studying these strains has helped us to obtain more information on the genetic diversity and gene constellation of noroviruses. here, we report two gii. [p ] recombinant strains from an outbreak in the city of changsha. in this outbreak, there were also two cases of coinfection with gi and gii. to better understand the genetic background and molecular characteristics of these viruses, we carried out a comprehensive analysis of the complete genome sequences of these noroviruses obtained by next-generation sequencing (ngs). the stool sample in this study was provided by the yuelu district center for disease prevention and control and tested by the changsha center for disease prevention and control (cscdc). the epidemiological and clinical information on laboratory-confirmed human cases of norovirus infection were collected by the cscdc staff and medical doctors at the hospital. the institutional review board reviewed and approved the use of those samples for this research (no. cscdc- - ). six stool samples were obtained from patients who suffered from fever, vomiting and diarrhea on april . the samples were stored at − °c until rna extraction. viral rna was extracted using a qiaamp rna mini kit (qiagen), and detected using a norovirus real-time pcr kit (jiangsu bioperfectus technologies, china) according to the manufacturer's instructions. then, the region corresponding to the orf and orf junction was amplified from norovirus-positive samples by reverse transcription polymerase chain reaction (rt-pcr) using norovirus type i and ii orf( + ) junction region gene amplification kits (biogerm, china). orf and junction sequences were obtained from the company (biogerm, china). the genotypes of the norovirus sequences were determined using the norovirus genotyping tool website (http://www.rivm.nl/ mpf/norov irus/typin gtool ) [ ] . the full genome was amplified from of norovirus-positive samples by rt-pcr using a superscript tm iii one- step rt-pcr system with a platinum tm taq high fidelity dna polymerase kit (thermo fisher, usa), performed in a geneamp pcr system (thermo fisher, usa) [ , ] . pcr products were purified using ampure xp beads (beckman, usa) according to the manufacturer's instructions and eluted using μl of nucleic-acid-free water. the purified nucleic acid sequences were quantitated by qubit . using a dsdna hs (high sensitivity) assay kit. a nextera xt dna library prep kit (illumina, usa) was used to construct a dna library with ng of input dna. then, the samples were sequenced using a miseq v reagent kit (illumina, usa) on a miseq platform (illumina, usa). the sequence data were analyzed using fastqc, cutadapt and virus identification pipeline (vip) software. sequences were assembled using spades- . . software. sequence alignments were performed with clustal w using molecular evolutionary genetic analysis software version (mega ). a phylogenetic tree based on fullgenome sequences was constructed by the maximumlikelihood method in mega ( bootstrap replicates). phylogenetic trees based on rdrp and vp sequences were constructed by the neighbor-joining method with the kimura two-parameter model in mega ( bootstrap replicates). the other complete and partial genome sequences were downloaded from ncbi. to identify break points in the genomes of the recombinant strains, their sequences were analyzed using simplot . . software. the analysis was conducted using -bp sequences from the orf and orf regions with a window size of nt and a step size of nt [ ] . amino acid sequences and hbga binding sites were analyzed using biological sequence alignment editor (bioedit . . ). the outbreak occurred in a senior high school after students participated in a group activity at a commercial park. on march , the students had lunch and dinner at the commercial park, and the first case of illness with nausea and vomiting was reported on march. in this outbreak, cases were reported, males and females, including one teacher. the infection cases were distributed in classes. the symptoms in students who suffered from gastroenteritis mainly included dizziness ( . %), nausea ( . %), vomiting ( . %), diarrhea ( . %) and abdominal pain ( . %). in all cases, the symptoms were resolved within - h, with no severe outcome. to identify the pathogens causing acute gastroenteritis in this outbreak, six stool specimens from patients, anal swab samples from the park staff, and food and drinking water samples were collected for norovirus testing. the results showed that the six stool specimens were gii positive, two of which exhibited a mixed infection with gi and gii. however, other samples from the commercial park were negative. the orf and orf junction was amplified from six samples by rt-pcr and sequenced, and the following genotypes were found: gii. to investigate the genetic relationship between the recombinants and other sequences available in the genbank database, full-genome sequences and orf - fragments of strains were analyzed by constructing a phylogenetic tree (fig. a) . a phylogenetic tree based on a fragment of the rdrp gene revealed that the recombinant strains belonged to the same branch as viruses from the united kingdom (mh ) and bhutan (mh ) and shared . %- . % sequence identity with mh ( fig. a) . the orf fragment of these strains belonged to the gii. genotype branch and shared . % sequence identity with mg (fig. b) . the complete genome sequences of the gii. [ ] . this site consists of eight residues located at the top of each p domain. the eight residues are as follows: n and w of the b loop; s , t , and s of the n loop; and n , n , and t of the t loop. none of these residues were mutated in the gii. [p ] recombinant strains in this study. however, other substitutions that might affect the structure of the binding site were also observed -n s, n s, and v q -as well as a v a substitution in the c-terminal amino acid sequence. the hbga binding sites were conserved in the gii. [p ] recombinant strains. t a and k r substitutions were found in the rdrp segment of these viruses. gi and gii noroviruses are the most important viral cause of non-bacterial gastroenteritis in humans globally. a large proportion of the population is infected with noroviruses via contact with contaminated food, water, and environment every year [ , , ] . various [ ] . hence, gii. [p ] and gii. [p ] genotypes are still evolving and spreading at a lower rate. although these genotypes might infect humans only sporadically, it is still useful to study the genetic diversity of these norovirus genotypes. due to the lack of in vitro cell culture systems and in vivo animal models for human noroviruses, there is still a lack of information on its pathogenesis and epidemiology. whole-genome sequencing of recombinant strains is important for vaccine development strategies and studies of viral evolution. hence, we determined the genome sequences of gii. [p ] recombinant strains isolated in the city of changsha, china. from the epidemiological information, although only visitors of the commercial park suffered from gastroenteritis, all food and water samples from the park were free of contamination, indicating that the source of the contamination might have been direct contact with infected persons or contaminated environments. based on the genetic and sequence data, we conclude that the outbreak involved multiple genotypes, including gii. [p ], gii. [p ], and gii. [p ] , which have been reported frequently in previous outbreaks [ ] . these results reveal that the prevalent genotypes continue to infect humans. phylogenetic analysis of the orf and orf segments showed that the recombinant strains belong to different branches. these results suggest that sequences in these recombinant strains might have originated in other countries neighboring china or that the strains might have evolved from local strains that have not been detected in the environment before. the high degree of sequence similarity between the two gii. [ ] . the break point identified in this study was located in orf segment, at the same position as reported previously in other recombinant strains [ ] . based on these findings, it is suggested that recombination between these genotypes might occur more easily than with other genotypes. although the genotypes gii. [p ] and gii. [p ] represent a new evolutionary lineage of norovirus selected by hbgas, the binding sites of the recombinant strains are still conserved [ ] . the role of substitutions in the b, n and t loops of the p domain, including n s, n s and v q, which are associated with adaptation of the hbga binding site, needs to be clarified in future research. thet a and k r substitutions in the orf sequence and the v a substitution in orf sequences may have no influence on the structure of the binding pocket. although the sporadic norovirus genotypes do not cause serious epidemic worldwide, unlike gii. [p ] and gii. [p ], due to these special binding sites, human infections are associated with severe vomiting and diarrhea. hence, it is important for us to enrich the database of norovirus genome sequences. although only a few samples were collected in this outbreak, we obtained the full genome of gii. [p ] recombinant strains that have rarely been reported in china. due to the high rate of genetic exchange in the norovirus genome, the virus can escape immune monitoring in individuals and acquire new host specificity [ ] . our research is important for understanding the diversity and wide distribution of noroviruses. evolution of norovirus genotypic and epidemiologic trends of norovirus outbreaks in the united states environmental surveillance for noroviruses in selected south african wastewaters outbreaks of norovirus and acute gastroenteritis associated with british columbia oysters structure(s), function(s), and inhibition of the rna-dependent rna polymerase of noroviruses advances in laboratory methods for detection and typing of norovirus updated classification of norovirus genogroups and genotypes proposal for a unified norovirus nomenclature and genotyping mechanisms of gii. norovirus evolution emergence and predominance of norovirus gii emergence of a new gii. norovirus variant in patients with acute gastroenteritis in gastroenteritis outbreaks caused by norovirus gii an outbreak of gastroenteritis associated with gii. norovirus-contaminated secondary water supply system in wuhan noroviruses recognize glycans with a terminal beta-galactose via an unconventional glycan binding site a unique human norovirus lineage with a distinct hbga binding interface human norovirus transmission and evolution in a changing world epidemiology of norovirus outbreaks reported to the public health emergency event surveillance system, china a norovirus intervariant gii. recombinant in victoria emergence of norovirus gii.p -gii. strains in patients with acute gastroenteritis in huzhou emergence of norovirus gii. variants among children with acute gastroenteritis in south korea analysis of uncommon norovirus recombinants from manaus, amazon region, brazil: gii.p /gii. , gii.p /gii. and gii novel recombinant gii.p _gii. and gii.p _gii. norovirus strains in italy full-genomic analysis of a human norovirus recombinant gii. / novel strain isolated from south korea full-genome sequence analysis of an uncommon norovirus genotype, gii. , from south korea detection of norovirus and rotavirus present in suspended and dissolved forms in drinking water sources emergence of gii. sydney norovirus in south korea during the winter of - distribution of norovirus and sapovirus genotypes with emergence of nov gii.p /gii. recombinant strains in chiang mai, thailand gii. in children with diarrhea occurrence of novel gii. and gii. norovirus variants in the coastal environment of south korea in norovirus-host interaction: implications for disease control and prevention publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we acknowledge all of the clinicians who collected data and samples.funding this study was supported by the hunan provincial health commission foundation (no. b ).norovirus strains from an outbreak in changsha, china the authors declare that they have no conflict of interest. key: cord- -llex yed authors: dea, s. a.; bilodeau, r.; martineau, g. p. title: isolation of encephalomyocarditis virus among stillborn and post-weaning pigs in quebec date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: llex yed encephalomyocarditis (emc) virus was isolated from aborted fetuses and lungs of suckling pigs from three quebec pig farms that experienced outbreaks of reproductive failure in sows and respiratory problems in suckling and post-weaning piglets. multifocal interstitial pneumonia and mild non-suppurative myocarditis and meningoencephalitis were the more significant histopathological lesions observed in piglets. vero cells were found to be more sensitive than bhk- cells and pig cell lines for primary isolation of emc virus. the quebec emc virus isolates were highly virulent for mice and were antigenically related to reference strain of emc virus as demonstrated by indirect immunofluorescence, seroneutralization and western immunoblotting. specific virus neutralization antibody titers up to : , were detected in samples of thoracic or abdominal fluids of the aborted fetuses. summary. encephalomyocarditis (emc) virus was isolated from aborted fetuses and lungs of suckling pigs from three quebec pig farms that experienced outbreaks of reproductive failure in sows and respiratory problems in suckling and post-weaning piglets. multifocal interstitial pneumonia and mild non-suppurative myocarditis and meningoencephalitis were the more significant histopathological lesions observed in piglets. vero cells were found to be more sensitive than bhk- cells and pig cell fines for primary isolation of emc virus. the quebec emc virus isolates were highly virulent for mice and were antigenically related to reference strain of emc virus as demonstrated by indirect immunofluorescence, seroneutralization and western immunoblotting. specific virus neutralization antibody titers up to : , were detected in samples of thoracic or abdominal fluids of the aborted fetuses. encephalomyocarditis (emc) virus, a member of the family picornaviridae, genus cardiovirus, causes a clinical disease with high mortality in young piglets [ , , ] . the virus has also been proposed as a cause of swine reproductive disorders in australia and in the united states [ i, - ] . heart lesions are consistently found in the aborted fetuses or dead piglets [- , , , ] . evidence for transplacental emc virus infection has been demonstrated in pregnant sows following natural or experimental exposure to the virus [ , ] . the existence of emc virus strains having different pathogenic properties for the swine fetus has also been reported [ ] . serological evidence of emc virus infection was reported in canadian swine herds and associated with sudden death in young piglets and reproductive problems [ , ] . final diagnosis based on the isolation of the virus from tissues of aborted fetuses, however, was not obtained. in the last two years, many piggeries in quebec have experienced successive outbreaks of a severe systemic disease affecting sows of all ages. the clinical signs included anorexia, hyperthermia that lasted up to days, late term abortion ( - days), often containing large mummified or partly autolysed fetuses, increase in weakborn pigs and neonatal deaths often associated with rapid abdominal breathing. following the episodes of reproductive disorders, severe respiratory problems affecting piglets of all ages have also occurred. this report describes the isolation of emc virus from the tissues of aborted fetuses and post-weaning piglets obtained from three different farms. nine piglets submitted were in relatively good body condition. they showed various degrees of respiratory distress and their body temperature ranged from to °c. three piglets exhibited a forced abdominal breathing pattern. at necropsy, variable degrees of congestion and edema of the lungs, along with increased firmness of lung tissue were the most significant and consistent lesions. lymph nodes were enlarged and congested. the heart was flaccid or apparently normal, and no macroscopic lesions were found on the other organs. histologically, lesions of multifocal serocellular and interstitial pneumonia were found in lungs from seven piglets. histiocytes, lymphocytes and a few neutrophils could be identified within the alveolar septa. increased numbers of alveolar macrophages, necrotic debris and lymphocytes were demonstrated within the alveolar spaces. mild multifocal nonsuppurative meningoencephalitis and myocarditis were also demonstrated in five piglets. no significant histopathological finding were observed in the other tissues. no inclusion bodies were evident. fluorescent antibody examination of tissues [ ] for porcine parvovirus (ppv), transmissible gastroenteritis virus (tgev), and swine influenza virus (siv) were negative. routine bacteriological examination of multiple tissues from the necropsied piglets and from aborted fetuses yielded no pathogens. growth of non-haemolytic escherichia coli was obtained in one case and was considered to be of no significance. fetal (pooled tissues) and piglet tissues including the lungs, spleen, tonsils and heart were examined for viruses. ten per cent homogenates were prepared in . m phosphate buffered saline (ph . ), using a sorvall omnimixer, and clarified by centrifugation at , x g for min at °c. aliquots ( . ml) of clarified supernatant fluids were inoculated into the allantoic cavity of -day old embryonating chicken eggs [ ] and onto confluent cell monolayers. continuous monkey kidney (vero), baby hamster kidney (bhk- ), porcine kidney (pk- ), porcine fallopian tube (pft) and swine testicles (st) cell lines were used for virus isolation. the cell cultures were prepared in cm -tissue culture flasks using culture media described elsewhere [ ] [ ] [ ] . after an adsorption period of h, the infected cell monolayers were rinsed twice with pbs and reincubated at °c in culture medium without fetal bovine serum. cultures were monitored daily for appearance of cytopathic effects (cpe). subpassages were done at day intervals, depending on the extent of cpe. well-defined cpe, characterized by cell rounding and pyknosis, were ob-served after one or two passages in vero cells, and were apparent within to days of infection ( fig. a) . the cytopathic changes were demonstrated only after inoculation with homogenates prepared from the lungs of two of the piglets and after inoculation with pooled tissues from one aborted fetus. after three successive passages, complete degeneration of the cell monolayers was obtained within to h after inoculation. no cpe were observed in cell cultures inoculated with homogenates of the spleen, heart, tonsils and brain of the necropsied piglets. picornavirus-like particles were observed by negative stained electron microscopy (em) in the concentrated ( x ) supernatant fluids of degenerated cell cultures (fig. b) . no cpe could be demonstrated following two successive passages of the clinical specimens in bhk- , but the three virus isolates that first propagated in vero cells could be easily adapted in bhk- cells. both cell lines were of comparable sensitivity in subsequent passages as evaluated by the relative extent of cpe and infectivity titers. with both cell lines, infectivity titers reached to . tcids / . ml after three to four successive passages. evidence of viral replication in pig cell lines (pft, pk- , st) was not obtained after weekly blind passages, as suggested by the absence of cpe, absence of hemagglutinating (ha) activity in supernatants when tested against chicken, rat and guinea pig erythrocytes, and absence of viral particles in cell extracts examined by em. no viral particles could be observed by em in the allantoic fluids of embryonating chicken eggs after two successive passages of the clinical samples, and no hemagglutinating activity was demonstrated with chicken erythrocytes. this seems to eliminate siv as the etiological agent involved in these cases. reference emc virus against which identification was made was purchased from the american type culture collection, rockville, maryland (atcc-vr ). emc virus stock was produced by four successive passages in either vero or bhk- cells. the virus was harvested by freezing and thawing infected cells three times and removing cellular debris by centrifugation at , x g for minutes. virus purification was performed by differential and isopycnic ultracentrifugation on cesium chloride gradients as previously described [ ] . after an overnight centrifugation at , x g, fractions of the continuous gradient corresponding to buoyant densities of . to . g/ml were collected and used for rabbit immunization following a protocol described elsewhere [ ] . specificity of the rabbit anti-emc virus hyperimmune serum was confirmed by seroneutralization and indirect immunofluorescence [ , . when tested against a nominal virus dose of tcids , a seroneutralization titer of : , was determined to the homologous virus. at the fourth passage of the virus in vero cells, maximal cytoplasmic fluorescence was obtained to h post-inoculation using antiserum dilutions up to : , . neutralization tests to identify isolates from diagnostic specimens were conducted as one-way tests. equal amounts of constant antiserum dilution ( : ) and one tenth dilutions of supernatant fluids from virus-infected vero cells were mixed and incubated h at °c. then, the various mixtures ( . ml) were each inoculated to wells of a microtiter plate containing vero cells. end points of infectivity were determined on day and neutralization indices calculated. preliminary testing showed : dilution of the rabbit anti-emc virus hyperimmune serum to have neutralizing indices greater than log for the homologous atcc-strain. neutralizing indices of to log were obtained against the three virus isolates recovered from the clinical cases, thus confirming their identity. sds-page and western-immunoblotting analyses were done to further confirm the serological identity of the viruses isolated from vero cells. for polyacrylamide gel electrophoresis, clarified infected cell culture fluids ( ml) were ultracentrifuged at , x g (l ultracentrifuge, rotor sw , beckman, calif.) for two hours through a cushion of % sucrose (w/v) solution. the viral pellets were disrupted in pl of double strength laemmli sample buffer containing per cent -mercaptoethanol, boiled for min, and clarified at , x g for min before electrophoresis in per cent sds-polyacrylamide slab gels, as previously described [ ] . western immunoblotting assays were also done as previously described [ ] . under the conditions used, five major polypeptide species with estimated mr of , (vp ), , (vp ), , (vp ), , (vp ) and - , (vp ) were consistently demonstrated after electrophoresis of purified atcc strains of emc virus. these four polypeptides were immunochemically stained with homologous rabbit antiserum (fig. , lanes to ) . identical polypeptide profiles were obtained for the three quebec isolates of emc virus. the four fig. . western immunoblotting of emc virus structural proteins. - csc gradientpurified atcc-strain of emc virus ( and ) or extracellular virus pelleted through a cushion of % sucrose (w/v) solution ( ) was solubilized in sample buffer and electrophoresed in per cent polyacrylamide gel. viral proteins were then electrophoretically transferred to nitrocellulose membranes and incubated with : , dilution of rabbit anti-emc virus (atcc strain) hyperimmune serum. immunoblots were revealed as described previously [ ] . [ ] [ ] [ ] immunoblots of quebec emc virus isolates q. - ( ), q. - ( ), and q. - ( ) . estimated molecular weights of viral structural proteins are indicated in thousands. positions of molecular weight standards are on the left major polypeptides of the quebec isolates cross-reacted with the four homologous proteins of the atcc-strain (fig. , lanes to ) . the pathogenicity of quebec emc virus isolates was investigated in mice. groups of five cd- mice, approximately g weight, were inoculated intraperitoneally with . ml of infected cell culture supernatants ( to tcids / . ml) and observed at least twice daily. in cases of the three quebec emc virus isolates, % mortality was observed within h post-inoculation. death was sudden or after signs of illness including ruffled coat and convulsive periods. concurrent investigations of similar outbreaks of reproductive failure in five other large intensive piggeries located in the same geographical region did not result in the isolation of emc virus. however, of sows with stillborn and mummified piglets had serum neutralization titres of emc virus that varied from : to greater than t : . high virus neutralization titres ranging from : to : , were also detected in to samples of fetal fluids submitted from these farms. no significant antibody titres were detected either in sera from the sows or in fetal fluids to tgev, ppv, and siv (serotype hswl n ) using microtiter neutralization or hemagglutination-inhibition assays [ , , ] . many infectious agents have been associated with reproductive failure in swine [ ] . ppv, siv, and leptospira are the most common causes of swine reproductive failure in canada, but a large proportion of mummified or stillborn pigs still remain undiagnosed. [ ] . in the last two years, multiple episodes of undiagnosed reproductive failure in sows with severe systemic clinical manifestations have also been reported in intensive piggeries from more than states in the u.s.a. [ , , tl] and a nearly identical syndrome was noted in a saskatchewan herd, where researchers proposed a possible involvement of a porcine cytomegalovirus~ [ ] . emc virus was also isolated from aborted fetuses in minnesota and iowa [ ] . although the clinical signs in pregnant sows and unweaned piglets observed in quebec piggeries were compatible to what has been reported in these states and in saskatchewan, the histopathology was different. necropsied fetuses did not show typical gross and microscopic heart lesions previously reported from experimental and natural infection of newborn piglets with emc virus [ ] [ ] [ ] . the demonstration of emc virus associated to reproductive and respiratory problems in swine supports the hypothetical existence of emc virus strains with different pathogenic properties, as previously demonstrated by different authors [t ] . although the quebec emc virus isolates were serologically indistinguishable to the reference atcc-emc virus, they were more virulent for newborn mice. the absence of consistent heart lesions in the aborted fetuses and in piglets with consistent findings of various degrees of pneumonia suggested that the quebec isolates may be biologically distinct. encephalomyocarditis infection of pigs. an outbreak in new south wales viral susceptibility of a cell line derived from the pig oviduct cultivation of a porcine adenovirus in ]porcine thyroid cell cultures parvovirus-like particles associated with diarrhea in unweaned piglets identification of the structural proteins of turkey enteric coronavirus encephalomyocarditis virus infection in florida, - encephalomyocarditis virus infection and pig disease in queensland detection of antibody to encephalomyocarditis virus in mummified or stillborn pigs pathogenesis of porcine parvovirus infection: pathology and immunofluorescence in the foetus keffaber kk (i ) reproductive failure of unknown etiology serologic, virologic, and histopathologic observations of encephalomyocarditis virus infection in mummified and stillborn pigs pathogenic properties of encephalomyocarditis virus isolates in swine fetuses an association between encephalomyocarditis virus infection and reproductive failure in pigs encephalomyocarditis virus from stillborn pigs reproductive failure in pigs caused by encephalomyocarditis virus classification and nomenclature of viruses an epizootic of swine influenza in quebec epizootic infection of a minimal disease swine herd with a herpesvirus the nucleotide and deduced sequences of the encephalomyocarditis viral polyprotein coding region antibodies to encephalomyocarditis virus in aborted and stillborn pigs encephatomyocarditis virus outbreak among suckling pigs interstitial pneumonia in piglets associated with porcine parvovirus the authors thank m. roger dubuc and luc heroux for technical assistance. this work was partly supported by grants received from agriculture canada (entente auxiliaire sur le d~veloppement agro-alimentaire) and the minist~re de l'agriculture, des p~cheries et de l'alimentation du qu bec (recherche et d veloppement). key: cord- - ixegm authors: liu, s. w.; zhang, q. x.; chen, j. d.; han, z. x.; liu, x.; feng, l.; shao, y. h.; rong, j. g.; kong, x. g.; tong, g. z. title: genetic diversity of avian infectious bronchitis coronavirus strains isolated in china between and date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ixegm twenty-six avian infectious bronchitis (ib) viruses (ibv) were isolated from outbreaks in chickens in china between and . they were characterized by comparison with twenty-six chinese reference strains and five other ibv strains. chinese ibvs, which were mainly nephropathogenic, were placed into seven genotypes. fourteen chinese ibv isolates were placed in genotype i, having small evolutionary distances from each other. genotype ii included strains that were isolated in the s in china. genotype iii consisted of eight chinese isolates that showed close relationship with korean ibv isolates. another eight ibv isolates clustered in genotype iv and showed larger evolutionary distances. the massachusetts serotype was present in china in s and was in a separate genotype. two isolates, hn and ck/ch/lhn/ i, which might be a reisolation of vaccine strains, clustered into genotype vi. four chinese ibv isolates formed another genotype and showed larger evolutionary distances from other chinese ibv genotypes (genotype vii). ibvs in same genotypes showed more than % amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than %. the results showed that ibvs in china came from genetic changes both in ibv populations that existed before the advent of vaccination and in the viruses that were introduced through live vaccines. ibvs showing various genetic differences are cocirculating in china. infectious bronchitis virus (ibv) is a highly infectious and contagious pathogen of chickens worldwide [ ] . the primary tissue of ibv infection is the respiratory tract, though some isolates replicate in the kidney and oviduct, resulting in nephritis and reduced egg production. generally, infectious bronchitis (ib) has been controlled with serotype-specific vaccines, but outbreaks of ib still occur, because vaccines offer little cross-protection between serologically distinct viruses [ ] . a high mutation frequency and rna recombination leads to the emergence of new viruses capable of causing disease in vaccinated chickens [ , , ] . although many countries share some common antigenic types, ibv strains within a geographic region are unique and distinct [ , , - , , ] . the identification of the circulating ibv field strains is extremely important for the selection of vaccine strains for the corresponding geographical region. ibv is the type species of the genus coronavirus in the family coronaviridae, order nidovirales [ ] . it is a pleomorphic enveloped virus and has a singlestranded rna genome, approximately kb in length, of positive polarity that specifies the production of three major structural proteins: the phosphorylated nucleocapsid (n) protein, the membrane (m) glycoprotein, and the spike (s) glycoprotein. the s glycoprotein of ibv, located on the outside of all virions, is responsible for fusion (virus envelope to cell membrane and cell membrane to cell membrane) and is translated as a precursor protein (s ), then cleaved into a carboxy-terminal s subunit (approximately amino acid residues), which anchors s in the virus envelope, and an amino-terminal s subunit (approximately residues), believed to largely form the distal bulbous part of s [ , ] . the s subunit of spike glycoprotein of ibv is responsible for inducing neutralizing and serotype-specific antibodies in chickens, and mutations in the antigenically important spike glycoprotein s subunit leads to the emergence and proliferation of variant serotypes [ ] associated with disease outbreaks. serotypic evolution in ibv is associated primarily with the sequences of the s glycoprotein, and the genetic diversity of ibv is mainly monitored by analysis of the s gene [ , , , , , , ] . ibv strains have been isolated and identified since in china. the outbreaks of ib have been ongoing, and ib continues to be an economically important disease to the poultry industry, although vaccines based on massachusetts (mass) strains such as h and h have been used for many years. however, the epidemiological analysis of ibv isolates in china has not been thorough except for with a few strains [ , , ] . the relationships between chinese ibv isolates and foreign ibv isolates, especially korean, taiwanese and japanese ibv isolates, are not known. the focus of this study was to determine the molecular typing of the spike glycoprotein s subunit of ibv isolated between the years and in china. this will determine the ibv type(s) which are necessary for understanding the epidemiology and evolution of ibvs, as well as isolation of the virus, which is important for improved vaccination. twenty-six field ibvs were isolated from kidney, preventriculus, or oviduct of ib-suspected broilers or layers using specified pathogen-free (spf) embryonated eggs between (table ) were pooled and % w/v tissue suspensions were made in . % phosphate-buffered saline containing u penicillin and µg streptomycin/ml. after h at • c, µl supernatant from the suspensions was inoculated into the allantonic cavity of -to -day-old embryos. three to eggs were used for each sample. the inoculated eggs were incubated at • c and candled daily. two to blind passages were performed until the characteristic embryo changes such as the dwarfing, stunting, or curling of embryos were observed between and days after inoculation [ ] . all allantoic fluids were harvested and tested for the presence of ibv using electron microscopy. samples of allantoic fluids were submitted for electron microscopy. briefly, after low-speed centrifugation at g for min (allegra tm r centrifuge; beckman), the supernatant of the . ml allantoic fluids were centrifuged at g for min. the resulting pellet was resuspended in a minimal volume of deionized water and examined by negative contrast electron microscope (jem- , ex). genomic rna was extracted from virus-inoculated allantoic fluid with trizol reagent (invitrogen) following the manufacturer's instructions. the first-strand cdna was synthesized according the procedures of a previous report [ ] using s oligo [ ] and genomic antisense ibv- oligonucleotide, -atacaaaatctgccataa- . ibv- was designed based on a comparison and alignment of the genbank sequences of several known chinese ibv strains and situated in the downstream of s oligo which had nt overlapped between them. the pcr profiles involved an initial denaturation for min at • c followed by cycles of denaturation at • c for min, annealing at • c for min, and polymerization at • c for min. the final polymerization step was performed at • c for min. owing to genetic variations among ibv isolates, it is difficult to design pcr primers that can be used to detect all ibv isolates. therefore, three genome-sense oligonucleotides, s oligo [ ] , s uni [ ] , or ibv- , -tattgattagagatgttggg- , which was selected from conserved areas by aligning several known chinese ibv sequences from genbank, were used with s oligo [ ] or ibv- as antisense primer ( table ). the pcr products were analyzed on a . % agarose gel and were sequenced directly. in addition, pcr products were also sequenced after cloning into the pmd -t vector (takara). each region was sequenced at least three times from two pcr products from different rt reactions. the nucleotide and amino acid sequences of the s protein gene of the twenty-six ibv isolates were assembled, aligned, and compared with reference ibv strains using the megalign program in dnastar. phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences of the s protein gene was performed by the clustal v method using dnastar software [ ] . thirty-one reference strains were selected for molecular his-arg-arg-arg-arg dq ck/ch/lgd/ i s oligo + ibv- his-arg-arg-arg-arg dq ck/ch/ldl/ i ibv- + s oligo arg-arg-thr-gly-arg dq ck/ch/lln/ i s oligo + ibv- his-arg-arg-arg-arg dq ck/ch/ldl/ i ibv- + s oligo arg-arg-thr-gly-arg dq ck/ch/lhlj/ i s oligo + ibv- ibv- + s oligo arg-arg-thr-gly-arg dq ck/ch/lxj/ i s oligo + ibv- his-arg-arg-arg-arg dq ck/ch/lhlj/ i s oligo + s oligo his-arg-arg-arg-arg dq ck/ch/lshh/ i s uni + s oligo his-arg-his-arg-arg dq ck/ch/lshh/ ii s uni + s oligo his-arg-his-arg-arg dq ck/ch/lgd/ i s oligo + ibv- arg-arg-phe-arg-arg dq ck/ch/lah/ i s oligo + ibv- arg-arg-his-ser-arg dq ck/ch/lsd/ i s oligo + ibv- his-arg-arg-arg-arg dq ck/ch/ljl/ i s oligo + ibv- his-arg-arg-arg-arg dq ck/ch/lhlj/ v s oligo + ibv- his-arg-arg-arg-arg dq ck/ch/ldl/ ii s oligo + ibv- arg-arg-tyr-arg-arg dq ck/ch/lgd/ ii s oligo + ibv- arg-arg-phe-arg-arg dq ck/ch/lgd/ iii s oligo + ibv- arg-arg-leu-arg-arg dq ck/ch/lhlj/ xi s oligo + ibv- his-arg-arg-arg-arg dq a oligonucleotides used for amplifying s protein gene b arg arginine, phe phenylalanine, his histidine, thr threonine, gly glycine, ser serine, tyr tyrosine, leu leucine analysis. of these, twenty-six were chinese ibv strains from the genbank database, and they represented most of the chinese ibv field isolates available through genbank or other publications. a total of fifty-two chinese ibv field isolates, including our twenty-six isolates, were chosen to give a representation based on geographic distribution, year of isolation, and phylogenetic position. in addition, two ibv strains, / and t / , representing tw i and tw ii ibv isolates in taiwan [ ] , were selected. a korean ibv isolate, k - , was also selected. this ibv strain belonged to genotype iii of korean ibv strains, and this genotype was a major type of ibv in korea. jp , a japanese ibv strain, was also selected, and its s protein gene was compared with chinese ibv isolates. ibv strains from the above geographically different areas were selected because we were interested in knowing whether the ibv isolates in china were introduced from neighboring countries and continents or whether they arose by mutations of circulating chinese ibv strains. furthermore, the s protein gene of the h vaccine strain was selected and compared in this study because the vaccine was widely used for many years on poultry farms in china. the entire coding region of the s protein gene of these strains was chosen for analysis. the fifty-seven ibv strains, including our twenty-six isolates, were molecularly analyzed. the twenty-six ibv isolates in this study and their accession numbers are listed in table . the ibv reference strains and their accession numbers are listed in table . twenty-six ibv strains were isolated from flocks that were suspected of ibv infection. the isolates were from flocks in different parts of china (fig. ) that showed clinical ib and had to % mortality. the nephritis observed in all flocks was characterized by enlarged and pale kidneys, frequently with urate deposits in the tubules, and severe dehydration and weight loss. typical signs, including dwarfing and death of the embryo, were observed in different passages when each isolate was inoculated into embryos (table ) . diagnoses based on electron microscopy examination showed all isolates had typical coronavirus morphology and were free of other agents such as newcastle disease virus (ndv) (results not shown). to assess the genetic relatedness among the ibv strains, a phylogenetic tree was performed with s protein genes. the results are shown in fig. . the fiftyseven ibv strains were separated into seven genotypes (i to vii) by phylogenetic analysis of the s protein genes (fig. ) . genotype i consisted of fourteen chinese strains having small evolutionary distances from each other as shown in the rooted tree (fig. ) . genotype ii included strains that were isolated in the s in china. most of the chinese ibv isolates included in genotype iii were also isolated in the s, except tl/ch/ldt / and ck/ch/lgd/ i, which were both isolated in guangdong province in from teal [ ] and layer hens, respectively. the korean ibv isolate, k - , which belonged to genotype iii of korean ibv strains [ ] , was closely related to those isolates in genotype iii. six of eight ibv isolates displayed in genotype iv were isolated after , and most of them came from southern china. furthermore, isolates included in genotype iv showed larger evolutionary distances (fig. ). ten chinese ibv isolates formed the genotype v in which h was included, and none of our twenty-six isolates were grouped under this genotype. the isolates hn and ck/ch/lhn/ i, both isolated in henan province in and , respectively, together with a japanese isolate, jp , were grouped into genotype vi. our three ibv isolates recovered in dalian, china, between and , were grouped into genotype vii. a chinese ibv isolate, j , which was isolated from the proventricular tissues of infected chickens [ ] , was also placed in genotype vii. two ibv isolates, / and t / , belonging to tw i and tw ii, formed a unique genotype. phylogenetic relationships, based on the sequence of the s subunit of the s protein gene, of our twenty-six isolates and thirty-one reference strains (the first nt, starting at the aug translation initiation codon, of the s protein genes) using the megalign program dnastar with the clustal v method [ ] . our ibv isolates are in bold type the spike glycoprotein of ibv is translated as a precursor protein (s ) and then cleaved into two subunits s and s [ , ] . cleavage site motifs of the fifty-seven ibv strains are listed in table and table , and twelve different cleavage site sequences were observed. the most common cleavage recognition site observed ( of viruses) was arg-arg-phe-arg-arg. viruses with this cleavage recognition site are the h vaccine strain, one korean strain, k - , taiwan isolates / and t / , ten chinese mass-type isolates, and ten other chinese isolates included in genotype iii (six strains) and iv (four strains). the second most common site was his-arg-arg-arg-arg. viruses with this cleavage recognition site include twelve isolates in genotype i and three in genotype ii. this recognition site was unique for virus isolates in china. the third most common site was arg-arg-thr-gly-arg. viruses with this cleavage recognition site were placed in genotype vii, which included our three isolates (ck/ch/ldl/ i, ck/ch/ldl/ i, and ck/ch/ldl/ i) and isolate j . this cleavage recognition site was also unique to viruses in china. the jx/ / , ck/ch/lah/ i, and sc viruses had a cleavage recognition site, arg-arg-his-arg-arg, as did d [ ] . chinese ibv isolates hn and ck/ch/lhn/ i, which were grouped in genotype vi, had a cleavage recognition site, arg-arg-ser-arg-arg, which was the most common site reported by jackwood [ ] , who had compared the cleavage recognition sites of fifty-five ibv isolates to determine if the site sequence correlates with host cell range, serotype, geographic origin, and pathogenicity. the ck/ch/lshh/ i and ck/ch/lshh/ ii viruses had a unique cleavage recognition site, his-arg-his-arg-arg, as did isolates bj and bjy, arg-arg-thr-arg-arg, ck/ch/lah/ i, arg-arg-his-ser-arg, ck/ch/ldl/ ii, arg-arg-tyr-arg-arg, ck/ch/lgd/ iii, arg-arg-leu-arg-arg, bjs, his-arg-thr-lys-arg, and japanese strain, jp , arg-arg-phe-lys-arg. the complete nucleotide and predicted amino acid sequences of the s protein of the fifty-seven ibv strains were determined and compared. except for isolates in genotype v, which included the mass-type strains, none of the chinese ibv isolates examined in this study shared more than % amino acid similarity in the s protein with the h vaccine strain. the s protein genes, which varied from . to . % among the strains examined, indicated that point mutations, deletions, and insertions contribute to the evolution of ibv. ibvs in same genotypes showed more than % amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than %, with the exceptions of isolate bjs (genotype ii) and isolates in genotype iv, bjq (genotype iii) and isolates in genotype v, isolates between genotypes i and iii, which showed amino acid similarities of - . %, . - %, and . - . %, respectively. the overall predicted amino acid sequence comparisons of the entire s protein of fifty-seven ibv strains reflected that most of the sequence variations were concentrated in three regions. the first included residues - , corresponding to the s protein of the h vaccine strain, in which the hypervariable region (hvr ) is located [ , , ] . the second contained amino acid sequences between residues - , which encompasses the hypervariable region (hvr ) [ , , ] . the last included residues - , in which the hypervariable region (hvr ) a amino acid abbreviations: s serine, d aspartic acid, g glycine, a alanine, n asparagine, h histidine, e glutamic acid, t threonine, y tyrosine, p proline, q glutamine, v valine, i isoleucine, k lysine, r arginine, m methionine; b positions of residues in deduced amino acid sequences of the s protein of the h vaccine strain; c positions of residues in deduced amino acid sequences of the s protein of the h vaccine strain between which the residue(s) of other ibvs was (were) inserted; d missing amino acid residues is present [ ] . furthermore, almost all of the chinese ibv isolates contained deletions and insertions except for those of the mass-type ibv, which were included in genotype v in this study, and had amino acid sequences similar to those of the h strain ( table ). the deletions and insertions, which occurred in the predicted amino acid sequences of the s proteins of fifty-seven ibv strains in this study, were correlated with the genotypes of s protein genes, as shown in table . in addition, the korean strain, k - , shared most of the motifs of deletions and insertions with chinese ibv field isolates in genotype iii. in , winterfield and hitchner reported a nephrosis condition associated with ib in the united states, and cumming reported an ib outbreak causing severe kidney lesion in chickens in australia [ , ] . since this time, various nephropathogenic strains of ibv have been identified throughout the world [ ] . in china, ib with nephritis was first reported in and several nephropathogenic ibv strains have been isolated in different parts of china since then [ , , ] . of the twenty-six ibv isolates in this study, one was isolated from atrophic oviduct of a diseased layer hen, six from swollen proventricular tissues of infected chickens, and the rest from swollen kidneys of ib-suspected chickens. although seven ibv strains were isolated from tissues other than kidney, the gross lesions of kidney in these diseased chickens were also obvious. based on the fact that these ibv strains were isolated from to in china, we considered that ib was prevalent all the while in china, although vaccines based on mass-type strains such as h and h have been used for many years on poultry farms, and nephropathogenic ibv was the major type of ibv circulating in china. although the genetic basis of ibv pathogenicity is not known, the s protein gene of ibv has serotype-specific and neutralization-specific epitopes, and serotypic evolution and the genetic diversity of ibv is mainly monitored by analysis of the s gene [ , , , , , , ] . in the present study, phylogenetic analysis of s genes showed that chinese ibv isolates were grouped into seven genotypes (fig. ) . ibvs isolated ten years ago were included in the same genotype with the strains isolated recently (for example, ck/ch/lsc/ i and ck/ch/ lgd/ i in genotype iii), indicating that this genotype may be indigenous and has been prevalent in china for at least ten years. serotype differences among the genetically distinct ibvs generally correlated with variations in the hvr of the s protein gene [ , ] and differences of as little as % between s sequences of ibv could result in poor cross-protection offered by currently used vaccines [ ] . the low identities (< %) of amino acid sequences between chinese ibv isolates and h , except for those of the mass-type ibv, which were included in genotype v in this study, may account for the prevalence of the viruses during the past ten years in spite of the extensive use of mass-type vaccines in the field in china. hence, developing vaccines from local strains is necessary for ibv control in china. although the number of basic residues around the spike glycoprotein cleavage recognition site of ibv does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with the envelope glycoproteins of orthomyxoviruses and paramyxoviruses, the sequences of cleavage recognition sites was correlated with geographic distribution of the viruses [ ] . nine spike glycoprotein cleavage recognition site sequences were found in viruses of genotypes i to iv, in which six were unique to isolates in china, indicating genetically distinct evolution from viruses in other countries by cleavage recognition site analysis. however, a korean ibv strain, k - , shared the same cleavage recognition site sequence, arg-arg-phe-arg-arg, with ten chinese isolates included in subgenotypes iii (six strains) and iv (four strains), ten chinese mass-type isolates (genotype v), and two taiwan isolates, / and t / . furthermore, k - and chinese isolates in genotype iii shared more than % amino acid identities and they were also grouped into the same genotype (fig. ) . k - was clustered into genotype iii of korean ibv strains, which was the major type of ibv circulating in korea, and isolates in this genotype induced % mortality in -day-old chicks as well as severe renal urate deposition on the kidneys [ ] . this was similar to chinese isolates in genotype i [ , ] . based on these facts, ibvs between chinese genotype iii and korean genotype iii had a close relationship, as did ndv [ ] , owing to the increased trade of agricultural products including poultry between two countries. unlike k - , isolates / and t / , which represented tw i and tw ii strains, respectively [ ] , were clustered into a separate branch that was separated from the chinese genotypes, indicating that they had different origins. ten chinese ibv strains were classified into the mass serotype (genotype v by phylogenetic analysis). as in china, mass-type ibvs were also present in other asian countries, such as korea [ , ] , japan [ , ] , and taiwan [ , ] , although mass-type vaccines were commonly used in these countries or continents. however, chinese mass-type strains were all isolated in the s and were not the major ibv type circulating in recent years in china. molecular studies have shown that a new serotype or variant can emerge as a result of only a few changes in the amino acid composition in the s part of the virus spike protein, with the majority of the virus genome remaining unchanged [ ] . this could be due to immunologic pressure caused by the widespread use of vaccines, to recombination as a consequence of mixed infections, or to the decrease of dominant serotypes as a result of vaccination, allowing other field strains to emerge. to this study, the mass-type viruses may have come from the vaccine strains by point mutation, although the possibility that some of them were reisolations of vaccine strains cannot be excluded. two strains, hn and ck/ch/lhn/ i, both isolated in henan province in china, together with a japanese strain, jp , constituted a "novel" genotype vi (fig. ) . these two chinese isolates did not show close similarity to any of the s protein sequences of other chinese ibv isolates available through genbank or publications. interestingly, blast searches revealed significant similarity ( %) of s protein genes between isolate hn and a vaccine strain, jaas (ay ), which was from australia and used in china to control ibv. the isolate ck/ch/lhn/ i shared % similarity in the s protein gene with another ibv vaccine strain, jilin (ay ), which was also used in china. when ck/ch/lhn/ i was inoculated experimentally into -day-old spf chickens, no disease signs were apparent (s. liu et al., unpub. data) . the spreading of a virus from one area or country to another could be due, at least in part, to its improper introduction by the trading of birds or by the use of attenuated vaccines. to our knowledge, no other ibv strains related to hn or ck/ch/lhn i were isolated in recent years in china, and considering the pathogenicity and genetically close relationship between the two isolates and the corresponding vaccine strains, we speculated that the two isolates would be reisolations of vaccine strains. isolate j , which was very similar to q and t , was genetically distinct from most of the chinese ibv isolates [ ] . in this study, our three isolates (ck/ch/ldl/ i, ck/ch/ldl/ i, and ck/ch/ldl/ i) were clustered into the same group (genotype vii) with j . similar to j , ck/ch/ldl/ i and ck/ch/ldl/ i were isolated in swollen proventriculars tissues of infected chickens, whereas ck/ch/ldl/ i was isolated from atrophic oviduct of a diseased layer hen. it was found that the gross lesions of the kidney in these diseased chickens were also obvious, as with isolate / [ ] . / was isolated in taiwan and was very similar to the j strain by comparison of s protein genes [ ] . the diversity of the pathogenicity of ibv strains was expected; although the primary tissue of ibv infection is the respiratory tract, some isolates can grow in nonrespiratory organs such as the kidney, the female reproductive tract, intestine, and spleen of chickens [ , , , ] . with the exception of the massachusetts strain, a very interesting aspect of ibv epidemiology, as far as it is possible to know, is the presence and the spreading of the various ibv serotypes in different continents. about emergent serotypes in north america did not spread to other continents. similarly, the european, australian, and asiatic serotypes apparently did not spread elsewhere. in china, ibv epidemiology is more complicated. besides genotypes i to iv and vii, the mass-type ibv and ibv closely related to australian classical strains were also present, indicating ibvs showing various genetic differences were cocirculating in china. molecular analysis of the /b serotype infectious bronchitis virus in great britain emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in india completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus molecular characterization of infectious bronchitis virus isolates foreign to the united states and comparison with united states isolates severe acute 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strains isolates in japan nephropathogenic avian infectious bronchitis viruses. world's sequence comparison of avian infectious bronchitis virus s glycoproteins of the florida serotype and five variant isolates from georgia and california emergence of subtype strains of the arkansas serotype of infectious bronchitis virus in delmarva broiler chickens local antibody production in the oviduct and gut of hens infected with a variant strain of infectious bronchitis virus genetic diversity of avian infectious bronchitis virus california variants isolated between and based on the s subunit of the spike glycoprotein complete nucleotide sequences of s and n genes of infectious bronchitis virus isolated in japan and taiwan molecular characterization of three new avian infectious bronchitis virus (ibv) strains isolated in quebec epidemiological classification of infectious bronchitis virus isolated in korea between evidence of natural recombination within the s gene of infectious bronchitis virus evolutionary implications of genetic variations in the s gene of infectious bronchitis virus etiology of an infectious nephritis-nephrosis syndrome of chickens antigenic and immunogenic characterization of infectious bronchitis virus strains isolated in china between characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia rapid detection and identification of avian infectious bronchitis virus author's address: dr. xiangang kong we would like to thank dr. j. j. giambrone, department of poultry science,auburn university, auburn, u.s.a., for his helpful comments in reviewing the manuscript. key: cord- -rmnjs t authors: welch, siao-kun wan; saif, linda j. title: monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rmnjs t twelve hybridomas secreting monoclonal antibodies (mabs) against miller virulent strain of transmissible gastroenteritis virus (tgev) were generated and characterized. in a cell culture immunofluorescence (ccif) assay, three mabs directed against peplomer protein (e ) had perinuclear fluorescence and four unclassified mabs showed cell membrane fluorescence. six of these seven mabs neutralized both attenuated and virulent tgev, and the seventh (an unclassified mab) neutralized only the latter virus. two mabs able to bind the cell membrane of infected cells had low neutralizing antibody titers ( to ) but were able to distinguish between virulent and attenuated tgev ( - to -fold differences in neutralizing titers). two e -specific mabs had higher neutralizing antibody titers ( to , ) and showed - to -fold differences in titers against the attenuated and virulent tgev strains. five mabs which were specific for nucleocapsid (n) protein had cytoplasmic, particulate fluorescence in ccif, and did not neutralize tgev. comparison of ccif antibody titers of mabs to the virulent and attenuated strains of tgev indicated that differences existed in titers of most e and all n-specific mabs, with titers consistently higher against virulent tgev (homologous strain). hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate of tgev immunoprecipitated the major structural proteins of both the attenuated and virulent tgev strains. relative mol. wt. differences in the e and e proteins between the two virus strains were revealed using either the hyperimmune pig sera or mabs. in addition to the k n protein, a k protein was coimmunoprecipitated by the hyperimmune sera and mabs, but mainly from lysates of attenuated tgev. miller virulent strain of transmissible gastroenteritis virus (tgev) were generated and characterized. in a cell culture immunofluorescence (ccif) assay, three mabs directed against peplomer protein (e ) had perinuclear fluorescence and four unclassified mabs showed cell membrane fluorescence. six of these seven mabs neutralized both attenuated and virulent tgev, and the seventh (an unclassified mab) neutralized only the latter virus. two mabs able to bind the cell membrane of infected cells had low neutralizing antibody titers ( to ) but were able to distinguish between virulent and attenuated tgev ( -to -fold differences in neutralizing titers). two e -specific mabs had higher neutralizing antibody titers ( to , ) and showed -to -fold differences in titers against the attenuated and virulent tgev strains. five mabs which were specific for nucleocapsid (n) protein had cytoplasmic, particulate fluorescence in ccif, and did not neutralize tgev. comparison of ccif antibody titers of mabs to the virulent and attenuated strains of tgev indicated that differences existed in titers of most e and all n-specific mabs, with titers consistently higher against virulent tgev (homologous strain). hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate oftgev immunoprecipitated the major structural proteins of both the attenuated and virulent tgev strains. relative mot. wt. differences in the e and e proteins between the two virus strains were revealed using either the hyperimmune pig sera or mabs. in addition to the k n protein, a k protein was coimmunoprecipitated by the hyperimmune sera and mabs, but mainly from lysates of attenuated tgev. transmissible gastroenteritis virus (tgev) belongs to the genus coronavirus of the family coronaviridae [ ] . it causes enteric disease in swine, producing a usually fatal diarrhea in seronegative piglets less than weeks old [ ] . although there is only one known serotype, both attenuated and virulent strains of tgev have been described [ ] . despite development of inactivated or live attenuated vaccines, no safe, effective, practical prophylaxis is yet available [ ] . thus a need exists to further define the protective antigens of tgev and to evaluate possible antigenic differences between attenuated and virulent strains of tgev. transmissible gastroenteritis virus possesses club-shaped peplomers and is enveloped and pleomorphic, with a diameter of - nm [ ] . analysis of tgev revealed three major structural and two minor nonstructural proteins [ ] . the glycosylated peplomer protein (e ) has an apparent molecular weight (mol. wt.) of - k and elicits neutralizing antibodies [ ] . the nucleocapsid protein (n), associated with the rna genome, is phosphorylated and has a mol. wt. of - k [ ] . the matrix or transmembranous protein [e ] is also glycosylated and has a tool. wt. of - k [ ] . the two minor proteins whose functions are unknown have mol. wts. of k and . k. recently, a k intracellular protein was identified in cells infected with the purdue strain of tgev [ ] : its function is also unknown. in two previous studies, monoclonal antibodies (mabs) to the structural proteins of the attenuated (purdue) strain of tgev were described [ , ] . there are no published reports describing mabs to a virulent strain of tgev. in the present study, a panel of mabs specific for structural proteins of the virulent (miller) strain of tgev was produced. these mabs were further characterized in various comparative assays including cell culture immunofluorescence, virus neutralization, and radioimmunoprecipitation for reactivity against the miller virulent and purdue attenuated strains of tgev. primary porcine kidney (ppk) cells were used for propagation of tgev and a swine testicle (st) cell line was used in various assays (described below). both ppk and st cells were maintained in eagle's minimum essential medium (mem) (gibco, grand island, ny) supplemented with % fetal bovine serum (fbs) (gibco) and pg/ml of gentamicin (schering veterinary, kenilworth, ny). for virus neutralization (vn), cell culture immunofluorescence (ccif), and radioimmunoprecipitation assays, the purdue attenuated strain (p ) and low cell culture-passaged miller (m ) virulent strain of tgev were used. both virus stocks were prepared in ppk cells. viruses were harvested h post-infection by three cycles of freezing and thawing, and the viruses stored in aliquots at -- °c. the miller virulent strain (m c) of tgev has been maintained by five serial passages in gnotobiotic pigs and represents the reference challenge strain of tgev [ ] . the field zy isolate of tgev was obtained during an outbreak of tgev in from a day-old diarrheic pig. both the m c and m strains of tgev, and the recent field zy isolate of tgev produced clinical signs and lesions typical of virulent tgev in gnotobiotic pigs, including vomiting, diarrhea and villous atrophy. pools of intestinal contents from m c infected gnotobiotic piglets were purified as described in the following section. intestinal contents containing m c tgev or tgev negative intestinal contents (from noninfected gnotobiotic pigs) were diluted t : in tris-cac buffer . m tris-hc , . m nac , and mm caci ), ph . and sonicated for rain (biosonik iii, bronwill, rochester, ny) on ice. crude suspensions were clarified by low speed centrifugation at , x g for rain and supernatant fluids were layered onto discontinuous sucrose gradients of %, %, and % in tris-cac buffer. after centrifugation at , x g for h, the light-scattering bands at %/ % and %/ % interphases were collected separately and diluted : in tris-cac buffer. sucrose was removed by pelleting virus at , x g for h and virus pellets were resuspended in tris-cac buffer. for each fraction, virus integrity was assessed by immune electron microscopy (iem) [ ] and virus titers were determined by ccif (reciprocal of the endpoint dilution showing immunofluorescing cells). the fractions ( %/ % and %/ % interphases) with intact viral particles and virus titers > l s were used to immunize balb/c mice. confluent st cell monolayers in -well plates were infected with either m or p strains of tgev at a multiplicity of infection (m.o.i.) of . pfu/cell in eagle's mem. after incubation at °c for h, monolayers were rinsed with phosphate buffered saline (pbs), ph . and fixed with % acetone. the fixed cells were used for ccif immediately. one hundred microliter per well of undiluted cell culture fluids from fusion plates or from limiting dilution plates or serial two-fold dilutions of ascites were added. the plates were incubated at °c in a humid incubator for h, and then rinsed with pbs for minutes. goat anti-mouse igg + iga + igm conjugated to fluorescein isothiocyanate (fitc) (kirkegaard & perry, gaithersburg, ma) at a : dilution was added to each well. after h incubation at °c, plates were rinsed once with pbs, ph . and once with pbs, ph . for rain. following addition of a drop of mounting medium ( % glycerol in pbs, ph . ), cells were examined for immunofluorescence (indicative of the presence of tgev antibodies) using a fluorescence microscope (olympus im, japan). to screen hybridomas for virus neutralizing (vn) antibodies to tgev, a cytopathic effect (cpe) reduction assay was performed. briefly, gl of hybridoma cell culture fluids were transferred to each well of a -weu plate. an equal volume of tcids / gl of m or p strain of tgev was added. the mixture was incubated at °c for h and gl of cells/ml of an st cell suspension in eagle's mem supplemented with % fbs was added to each well. the plates were incubated at °c for h and neutralizing activity was determined by the absence of cpe. a plaque reduction assay was performed using -day old st cell monolayers in sixwell plates to determine virus neutralizing antibody titers. equal volumes of m or p strains of tgev containing to plaque forming units (pfu) in gl were added to serial fourfold dilutions of heat inactivated ( °c, rain) ascites fluids and incubated at °c for h. then, gl of inoculum were added to duplicate wells followed by an additional h incubation at °c. to each well, ml of . % noble agar and . % (of . % stock) neutral red in eagle's mem were added. the virus neutralizing antibody titers were expressed as the reciprocal of the highest sample dilution which produced an % reduction in plaques when compared to the virus control wells. seronegative gnotobiotic pigs were used for production of tgev hyperimmune sera. two pigs were inoculated with m c virulent tgev, one pig with zy isolate tgev and pig with p attenuated tgev. each pig was inoculated orally with - ml of tgev and subsequently hyperimmunized (both intramuscularly and subcutaneously), one time with virus mixed with an equal volume of freund's complete adjuvant at two weeks post-oral inoculation, and three times with virus mixed with freund's incomplete adjuvant, at weekly intervals. sera were collected days after the last injection. hybridomas were produced using modifications of procedures described previously [ ] . to obtain hybridomas secreting tgev specific mabs, female balb/c mice were hyperimmunized five times at weekly intervals with semi-purified m c strain of tgev [approx. fluorescent focus units (ffu)/mt]. spleen cells from these mice were fused with sp / myeloma cells at a ratio of : in the presence of % polyethylene glycol (mw , sigma, st. louis, mo). hybridomas secreting mabs to tgev were detected by vn and ccif tests two times at day intervals. selected clones which were positive by either test were subcloned at least two times by limiting dilution [ ] using conditioned medium which was prepared as follows: thymocytes and spleen cells from female balb/c mice were prepared using x cells/ ml and maintained in rpmi supplemented with % fbs at °c for days. the supernatant medium was then removed and stored at °c for use in limiting dilution. the isotype and subisotype of mabs were determined by using the ouchterlony immunodiffusion technique [ ] . monoclonal antibodies produced in culture medium were precipitated with % ammonium sulfate and resuspended to x the initial concentration. monospecific antiserum to each isotype or subisotype was purchased commercially (icn immunobiologicals, lisle, il). ascites fluids containing mabs were produced in pristane ( , , , -tetramethyl pentadecane, aldrich chemical co., milwaukee, wi) primed-mice as described previously [ ] . mice were primed at least to days prior to use. approximately to x t hybridoma cells were injected intraperitoneally. ascites was produced and the fluids harvested - days post-injection. tests were performed using modifications of procedures described by wesley and woods [ ] . briefly, seven-day old confluent st cell monolayers grown in cm ~ flasks were infected with m tgev at an m.o.i, of . pfu/cell or p tgev at an m.o.i, of . pfu/ cell. mock infections using tissue culture media were done concurrently for each virus. at h post inoculation (pi) mock and tgev infected monolayers were washed with and maintained in methionine-deprived eagle's mem supplemented with gg/ml of gentamicin. labeling experiments were repeated using l ~tg/ml of actinomycin d added at h pi for studying the effect of actinomycin d on viral protein synthesis and for reducing the amount of labeled host cell proteins. at h, pi, ~tci/ml of l- s-methionine (amersham, arlington heights, il) was added to each flask containing mi of methionine-deprived medium. the flasks were incubated at °c for an additional h with gentle agitation. the flasks were then rinsed with pbs (ph . ) containing mm phenylmethylsulphonyl fluoride (pmsf, sigma), and the cell monotayers were lysed in ml lysis buffer . m nac , . m kc , . ram mgci , ram tris-hc , ph . , % triton x- , and units/ml aprotinin). cells were lysed at room temperature for rain and cell debris and particulate material were removed by centrifugation at , x g for rain. the supernatant was stored in ~tl aliquots at -- °c. the rip assay was performed using modifications of methods described by wesley and woods [ ] . cell lysates were preabsorbed with pooled normal mouse sera bound onto protein-a sepharose (pharmacia, sweden) at °c for h. undiluted mouse ascites fluids ( gl) or hyperimmune porcine sera ( ~tl) were absorbed onto protein-a sepharose and then reacted with unlabeled, uninfected st cell lysate for h at °c. this treated asc~tes or sera bound to protein-a sepharose was mixed with to gl of preabsorbed cell lysate at °c for h and then at °c overnight. the sepharose was pelleted in a microfuge and washed four times with lysis buffer and one time with . m tris-hc (ph . ) buffer. the immune complexes bound to sepharose were pelleted and t gl of laemmli's sample buffer [ ] was added. the mixtures were heated at °c for min and the sepharose was pelleted in a microfuge. the resulting supernatants were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) in % stacking and % ( : -acrytamide : bis) running gels. the gels were treated with en hancer (new england nuclear, boston, ma), dried and exposed to x-ray films (kodak xrp- ) at -- °c for days [ ] . molecular weight standards used were c-methylated myosin ( , ), phosphorylase-b ( , ), bovine serum albumin ( , ), ovalbumin ( , ), carbonic anhydrase ( , ), and lysozyme ( , ) (amersham). negative controls including a mab against osu porcine rotavirus, sp / cell-induced ascites, and tge negative porcine and mouse sera were also analysed by rip. a panel of twelve m a b s against the virulent strain of t g e v was generated and their reactivities against virulent and attenuated t g e v characterized (table ) . their isotype and subisotype specificity was also determined ( table ) . protein profiles of p t and m infected cell lysates are shown in fig. . viral specific proteins (e , e , and n) were evident for both p and m infected cell lysates. n o differences in protein profiles were observed with or without the presence of actinomycin d (data not shown). a unique approx. k species of protein was revealed only in the p infected cell lysates (fig. ) . seven neutralizing and five non-neutralizing m a b s were selected for characterization of their viral protein specificity by r i p of s-methionine labeled p and m t g e v infected cell lysates (table ) . a faint b a n d in the k (e ) region was evident with m a b s d , a , h , and d (in fig. a represented by d ), but because of the low intensity of this reaction and t- fig. c represented by h ) . the k protein band was consistently more intense in immunoprecipitates of the p than of the m strain of tgev. a and processed for rip. resulting immune complexes were analysed on % sds-polyacrylamide gels and the gel autoradiographs were exposed for days. p p infected lysate; m m infected lysate; c mock infected control. molecular weight markers are shown in the far left lane. o origin, bb bromophenol blue dye marker, e membrane protein, e peplomer protein, n nucleocapsid protein band (possibly actin) with a mol. wt. of k as consistently resolved in immunoprecipitation of mock and tgev infected cell lysates ( fig. a, b, c) . tge viral specific proteins (e , e , and n) were not immunoprecipitated by negative control sera or ascites fluid (e.g., mabs against porcine osu rotavirus, sp / cells induced ascites fluid and tgev negative mouse and porcine sera) (data not shown). results of rip of p or m tge viral proteins using the four hyperimmune sera are shown in fig. . the viral proteins immunoprecipitated by these sera were compared with those recognized by the mabs. the three major tge viral proteins (e , e , n) from m or p infected cell lysates were immunoprecipitated by each of the hyperimmune sera prepared against the three tge strains (virulent, attenuated and field isolate). the relative mol. wt. of the e protein was consistently higher and the tool. wt. of the e protein was consistently lower for the m strain of tgev than for p tgev. sera produced against all three strains oftgev immunoprecipitated the k protein from p infected cell lysates: this viral protein was undetectable or of low intensity in lysates from m -infected cells (fig. ) . seven of the selected hybridornas produced tge virus neutralizing antibodies (three e -specific and four unclassified mabs) ( table ). four to -fold differences in neutralizing antibody titers against attenuated and virulent tgev were observed for unclassified mabs h and d , and h and e specific mab e ( table ). monoclonal antibody h did not neutralize the p attenuated strain of tgev (titer < ); whereas it has a titer of against the m virulent strain of tgev. monoclonal antibodies d and e (anti-e ) had to -fold higher neutralizing antibody titers against m than against the p strain of tge virus. both p and m tgev produced plaques larger than those in virus control wells (data not shown) in the presence of mab h and d in plaque reduction assays. only mabs h and c had higher neutralizing antibody titers ( to -fold) against p than against m tgev ( table ) . none of the anti-n mabs neutralized tgev. all twelve mabs reacted with tgev in a ccif test and five hybridomas produced tgev antibodies which were detected only by ccif. the antibody titers determined by ccif against both p and m tgev are summarized in table . using ccif, four mabs whose protein specificity was undetermined, showed cell membrane fluorescence (fig. a) . three mabs which reacted with e protein showed faint diffuse perinuclear fluorescence (fig. b) . those mabs which reacted with n protein produced bright particulate cytoplasmic fluorescence (fig. c ). all mabs except the four unclassified mabs had higher ccif titers against homologous (m ) tgev than heterologous (p ) tgev; fold differences were observed for mabs h , e , and f (anti-e and n, respectively) and -fold differences were noted for mab h (anti-n). no differences in fluorescence staining patterns of mabs directed against the same proteins were observed when either p or m was used to infect st cells. however differences in fluorescence intensity were observed using either p or m as the test antigen (data not shown). monoclonal antibody e and f produced stronger immunofluorescence with m infected st cells than p t infected st cells at all dilutions tested. monoclonal antibody g , a , and h at lower dilutions ( : to : , ) induced similar fluorescence intensity with either p or m infected st cells, but brightness diminished after : , to : , dilutions for p infected st cells. monoclonal antibodies against the virulent strain of tgev were generated, and viral protein specificities were determined for all but four mabs. anti-e mabs recognized e proteins with tool. wt. of - k in rip, as reported similarly by others for mabs to p tge [ , ] . anti-n mabs immunoprecipitated a k protein derived from both p and m infected cell lysates, and a k protein was co-immunoprecipitated mainly from p infected cell lysates. the same finding was noted when three of the four hyperimmune porcine sera were tested. this unique k protein has not been reported by other siao-kun wan welch and linda j. saif researchers. one explanation may be that the percentage of cross-linker in our gel system was different from others; therefore, the resolution for the k protein was better. our results suggest that although this k protein occurs in both p and m infected cells and was detected by hyperimmune sera and mabs produced against virulent tgev, it accumulates more in the p infected cells. the nature of this extra species recognized by n-specific mabs is unclear. it may be a precursor or cleavage product of the viral n protein. more conclusive explanations may be obtained if the complete gene coding sequence for the n protein becomes available, or the kinetics for n protein synthesis are explored. in addition, a minor k band detected in p infected cell lysates, but not in m cell lysates ( hpi), may be similar to the k protein described in p cell lysates by wesley and woods [ ] or to the k protein described by hu et al. [ ] . whether this k protein is an intrinsic protein for only purdue attenuated tgev or a cleavage product of e is not yet known. nevertheless, this protein was not immunoprecipitated by the mabs or the four hyperimmune swine sera. the four mabs which had low neutralizing antibody activity, showed high background and low intensity reactions in rip and were reported as unclassified mabs which may possibly be e -specific. additional tests are required to confirm their protein specificities using either more extensive absorption methods or a solid phase immunoisolation technique [ ] . epitopes which elicited tgev neutralizing antibodies resided on the e and e proteins [ ] . in contrast, anti-e mabs produced by jimenez et al. [ ] did not neutralize tgev. anti-e mabs prepared against a mixture of virulent miller and purdue attenuated strains of tgev had virus neutralizing antibody titers only in the presence of guinea pig, rabbit or swine complement [ ] . it is possible some anti-e mabs may inactivate tgev infectivity via complement-mediated virolysis. interestingly, all four unclassified mabs (which may react with e protein) produced in the present study had low to moderate virus neutralizing antibody titers, but the neutralizing activities were not enhanced in the presence of hemolytic units/ml of swine complement (data not shown). by comparison, woods et al. [ ] reported that their e -specific tgev mabs had vn activity only in the presence of to hemolytic units/ml of swine complement. the distinctive immunofluorescence patterns associated with the different tgev protein specificities, suggest that a panel of such mabs may be useful for studies of the morphogenesis of tgev. immunofluorescence patterns in tgev-infected cells reacted with e -specific mabs suggest that e proteins are produced mainly in the cell perinuclear regions. anti-n mabs produced bright cytplasmic particulate immunofluorescence in tgev-infected st cells suggesting that n proteins are abundant in cytoplasm and often in aggregates reflected by the particulate nature of the fluorescence. in the case of the four unclassified mabs, fewer immunofluorescent tgev-infected st cells were evident (although all cells were infected with the same m.o.i.) and fluorescence on the cell surface was more distinct around cell to cell junctions. laude et al. [ ] demonstrated similar fluorescence patterns. however the membrane fluorescence was shown in paraformaldehyde-fixed cells reacted with an anti-e mab and a fluorescence pattern for anti-e mabs was not identified. biochemical and biophysical differences among field isolates and cell cultureattenuated tgev have been reported [ , ] , but serologic differences were not identified [ , ] . using mabs to purdue tgev in competitive assays, delmas et al. [ ] demonstrated that the neutralization mediating determinants were highly conserved among tgev strains. furthermore, jimenez et al. [ ] showed that neutralizing epitopes on attenuated tge virus were conformational and highly conserved. our data suggest differences exist between a virulent and attenuated strain of tgev, perhaps in the density of certain epitopes expressed on the viruses, or the proteins they elicit in infected cells. first, neutralizing antibody titers of unclassified mabs h and d and anti-e mab e were , and -fold higher respectively, to m than p tgev. second, anti-e mab h had a -fold higher neutralizing antibody titer to p than to m . third, all anti-n and anti-e mabs had to -fold higher ccif titers to m than p . furthermore, fluorescence intensity was stronger against the m strain than p strain of tgev. finally, differences in the relative mol. wt. of e and e proteins and in the quantity of the k protein between the virulent and attenuated tgev strains, imply that phenotypic variation between the strains may exist. this has not been reported previously. further studies are needed to define and compare tgev epitopes on attenuated, virulent and field isolates of tgev. although major epitopes mediating neutralization were associated with e proteins and these were conserved, nonneutralizing mabs against e , e , or n proteins may be unique for certain isolates and could thus be used for differentiation of tgev strains as shown previously by laude et al. [ ] . it is important to continue characterizing critical epitopes of the virulent strain of tgev for future development of possible subunit or r d n a vaccines. monoclonal antibodies to tgev described in this study, as well as additional mabs, should also aid in development of better diagnostic reagents for detection and possible differentiation of field and vaccine strains of tgev. antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus a film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gel antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein comparison of properties between virulent and attenuated strains of transmissible gastroenteritis virus the polypeptide structure of transmissible gastroenteritis virus / ) antigenicity of structural components from porcine transmissible gastroenteritis virus in vitro differentiation and ph sensitivity of field and cell culture-attenuated strains of transmissible gastroenteritis virus cloning and expression of the surface glycoprotein gp of porcine transmissible gastroenteritis virus critical epitopes in transmissible gastroenteritis virus neutralization continuous cultures of fused cells secreting antibody of predefined specificity cleavage of structural proteins during the assembly of the head of bacteriophage t antigenic structure of transmissible gastroenteritis virus. i. properties of monoclonal antibodies directed against virion proteins immunologic discrepancy between intestinal and cell culture viruses of transmissible gastroenteritis of swine neutralization of a transmissible gastroenteritis virus of swine by colostral antibodies elicited by intestine and cell culturepropagated virus immunoglobulin-producing hybrid cell lines structure of swine transmissible gastroenteritis virus examined by negative staining immunodiffusion and immunoelectrophoresis. in: weir dm (ed) handbook of experimental immunology transmissible gastroenteritis immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique identification of a , molecular weight antigenic polypeptide in transmissible gastroenteritis virus-infected cells neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies key: cord- - ui lqc authors: laurin, marc-andré; dastor, margaux; l’homme, yvan title: detection and genetic characterization of a novel pig astrovirus: relationship to other astroviruses date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ui lqc emerging viruses represent a continuous threat to human health and to farmed animals, as evidenced on multiple occasions by outbreaks of influenza, henipavirus and sars. knowledge about the diversity of viromes present in reservoir species can lead to a better understanding of the origin of emerging pathogens. in this study, we extend the knowledge of astrovirus diversity in pigs by reporting the genetic characterization of an unknown astrovirus lineage. phylogenetic analyses provided evidence that this porcine astrovirus lineage is unique and does not appear to share a recent common ancestor with any known mamastrovirus. the data reported in this study extend the number of porcine astrovirus lineages to a total of five, all of which most likely represent distinct species of different origins. the family astroviridae consists of small ( - nm) , non-enveloped, single-stranded positive-sense rna viruses of approximately kb in length. these viruses generally exhibit a distinctive five-or six-pointed star-shape appearance when viewed using electron microscopy. the astv genome is arranged into three open reading frames (orfs) designated orf a, orf b and orf . orf a and orf b are situated at the end of the genome and encode non-structural polyproteins, including a protease and an rna-dependent rna polymerase (rdrp). orf , situated at the end of the genome, encodes the structural capsid protein and is transcribed as a subgenomic mrna [ , ] . the family astroviridae is separated into two genera; viruses of the genus mamastrovirus infect mammals, and viruses of the genus avastrovirus are found in avian hosts [ ] . mamastroviruses appear to have a broad host range, since they have been isolated from numerous host species, including humans, mink, sheep, pigs, rats, marine mammals, dogs, cheetahs, roe deer, cattle and bats [ , , , , , [ ] [ ] [ ] ] . the list of susceptible species is likely to continue growing as more species are investigated. astroviruses are generally associated with either mild or severe enteric disease symptoms such as diarrhea and vomiting in a number of mammalian species [ ] . human astrovirus (huastv) serotypes - , which are the most extensively studied astroviruses, are known to be a common cause of diarrhea in young children, the elderly and the immunocompromised [ , , , ] . in addition, a number of genetically distinct strains of huastv have recently been identified, characterized and proposed to represent novel astv species based on genetic distance criteria [ , ] . porcine astvs (poastv) were first detected by em in the feces of a diarrheic piglet [ ] and later isolated in culture [ ] . molecular characterization of the orf gene from this isolate followed some years later [ ] . in the last five years or so, different research groups have successfully used pcr approaches to investigate the presence and diversity of porcine astroviruses [ , , ] . collectively, these studies have unveiled an impressive genetic diversity among poastv strains. this diversity suggests different origins for the various poastv lineages and, presumably, a number of transpecies transmission events between susceptible mammalian hosts [ , ] . in the present study, we report the identification and characterization of unforeseen and divergent poastv strains. phylogenetic analysis of the end of this strain suggests that a novel and possibly fifth lineage of astv exists in swine and heightens concerns about the role of pigs as a potential source of emerging astrovirus infections. a total of fecal samples originating from the individual cecal content of slaughtered adult pigs in canada were collected in - . viral rna was extracted as described previously and stored at - °c until needed [ ] . the general strategy employed for obtaining poastv sequences is summarized in fig. . briefly, primers panav-f (forward), panav-f (forward) and panav-r (reverse) were originally designed by chu and colleagues based on a conserved region situated in the rna-dependent rna polymerase (rdrp) gene of astvs [ ] . pcr conditions using these primers were as described by luo and colleagues [ ] . pcr products were analyzed on a qiaxcel instrument (qiagen, mississauga, ontario, canada). amplicons of approximately bp were then cloned using a topo . (t/a) cloning kit (invitrogen, mississauga, ontario, canada) and sequenced in both directions using the big dye v . chemistry on a xl instrument from applied biosystems (foster city, ca). based on the nucleotide sequences of the rdrp fragments from the four poastv strains, specific forward primers were designed for race-pcr. primers cc fe ( -atcgctatgtcctgttgccttcag- ) and cc f ( -tcttaatggtccagatggctggga- ) were used in combination with primers (qt), (qo) and (qi) with poastv strain cc as described previously [ , ] . despite numerous attempts, we were unable to amplify the end from the genomes of strains a , d and d , and hence, the race-pcr primer sequences are not shown. race-pcr products from strain cc were deposited on % agarose gels and stained with sybr safe (invitrogen), and fragments of approximately kb were purified on mini spin columns (qiagen). gel-purified amplicons were cloned using a pcr topo . (t/a) cloning kit (invitrogen), and a minimum of three clones were selected and amplified in liquid broth, followed by plasmid dna extraction using an alkaline lysis method (qiaprep spin miniprep, qiagen). each clone was then completely sequenced in both directions using a primer walking strategy. sequence editing, assembly and analysis were performed using bioedit version . . . (http://www. mbio.ncsu.edu/bioedit/bioedit.html). multiple sequence alignments were constructed with clustal w (version . ). phylogenetic analysis was conducted using the neighbor-joining (nj) method with p-distances for nucleotides and a poisson distance correction calculation for amino acids using the molecular evolutionary genetic analysis (mega . ) software with default settings, except that all missing data or gaps were completely ignored. confidence values at the nodes were obtained by performing , bootstrap analyses. we have previously reported the detection and characterization in canadian pigs of novel and previously unknown astvs belongs to the genus mamastrovirus using a ''broad-range'' pcr strategy [ ] . we hence applied the same strategy to screen a collection of fecal samples from pigs slaughtered in - . a total of samples generated amplicons of the expected size, which were cloned and sequenced. putative amino acid sequences from all strains revealed the presence of the characteristic ''ygdd'' motif located near the c-terminal region of the rdrp (not shown). this motif has been reported for all astrovirus polymerases characterized thus far. blast analysis revealed that most sequences were, in fact, closely related ([ % nucleotide identity) to known porcine astv sequences previously characterized by luo and colleagues [ ] . however, a group of four related sequences revealed only approximately % nucleotide identity to bat and human astv sequences in the database, suggesting that these sequences were divergent from known poastv strains and possibly novel. pairwise identity comparisons of the partial rdrp nucleotide sequences revealed that the four strains were - % identical to each other. phylogenetic analysis of these sequences, in addition to prototypical animal astv strains, confirmed their relatedness and grouped them in a unique lineage on a divergent branch distantly related to other known mamastroviruses (poastv in fig. a) . to permit more in-depth genomic investigation and strengthen their taxonomic grouping, we performed race-pcr on these four poastv samples. we were able to amplify and characterize a nt-long sequence from strain poastv cc only. it is unclear why race-pcr failed with the other samples. the amplified region from strain poastv cc included the end of the rdrp gene, the complete capsid gene, and the utr (fig. ) . the conserved motif situated at the junction between orf b and orf , which is thought to represent a regulatory element serving as a promoter for subgenomic rna transcription, was present in strain poastv cc : tttgggggggaggaccaaaaagagacgatggc (following the convention of huastv, which places the initiation atg codon, underlined, for orf immediately upstream of the orf b stop codon) [ ] . the orf start codon of strain cc , underlined, appeared in an optimal kozak context for translation initiation (rnnaugg, where r = a/g and n = a/t/g/c) [ ] . the orf gene is predicted to encode a capsid protein amino acids long, which is comparable to most astvs. the length of the utr is nucleotides. the highly conserved stemloop-ii-like motif (s m) present in the utrs of most mamastroviruses is also present in strain poastv cc (not shown). this motif has been suggested to have an important function in viral rna replication [ ] . phylogenetic analysis of the complete capsid coding region of poastv cc confirms that this strain forms a distinct lineage in the family mamastroviridae, including previously identified porcine astroviruses from different continents (fig. b ). in addition, the tree topology revealed by our analysis is largely in agreement with previous studies [ - , , , ] . pairwise amino acid comparisons reveal that strain poastv cc shares only between % and % aa identity in the complete orf with prototypical astv strains. since phylogenetic analysis ' step -pan astv primers step - ' race-pcr placed the cc strain in a unique lineage, we named this strain poastv , as there now appear to be five distinct lineages of astvs in swine [ , , , ] , and this name maintains the continuity in the nomenclature of poastv strains. the genetic distance between poastv cc and the other strains is comparable to distances between members of established astv species, suggesting that cc possibly represents a new, fifth poastv species. until recently, a relatively small number of astroviruses from very few hosts were known [ ] . however, in the last few years, thanks in part to metagenomic analyses and broad-range pcr, a number of studies have revealed a wide array of divergent astv strains in a growing number of mammalian species [ , , , , , [ ] [ ] [ ] . the present work extends current knowledge about these agents and underscores the vast diversity and divergence of astrovirus strains harbored by swine [ , , ] . poastvs now appear in five distinct lineages of the astv evolutionary tree, which suggests different ancestral origins and numerous interspecies transmissions involving many mammalian species, including humans. as additional mammalian species are screened for the presence of astvs, a clearer picture of the historical transmission path used by these viruses between different hosts might emerge and shed new light on the zoonotic potential of these viruses. emerging pathogens represent a constant threat to human health. since the majority of these pathogens arise from animal reservoirs, a more thorough knowledge about the presence and diversity of viruses in animal species such as pigs could lead to a better characterization of the risk posed by such agents. in addition, a better understanding and appreciation of the virome present in wild and domestic animals could lead to early identification of the source of an emerging outbreak and therefore to faster and more targeted interventions to control and limit the spread of a disease. the discovery of novel poastv strains described here provides an example of how diverse these viruses are in this domestic reservoir species. existence of multiple outbreaks of viral gastroenteritis among infants in a day care center in japan characterization of an outbreak of astroviral diarrhea in a group of cheetahs (acinonyx jubatus) detection by electron microscopy of caliciviruses, astroviruses and rotavirus-like particles in the faeces of piglets with diarrhoea detection of novel astroviruses in urban brown rats and previously known astroviruses in humans novel astroviruses in insectivorous bats a prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children human stool contains a previously unrecognized diversity of novel astroviruses complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea detection of newly described astrovirus mlb in stool samples from children identification of a novel astrovirus (astrovirus va ) associated with an outbreak of acute gastroenteritis environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit an outbreak of gastroenteritis in a home for the elderly associated with astrovirus type and human calicivirus isolation and partial characterization of a novel porcine astrovirus comparison of capsid sequences from human and animal astroviruses multiple novel astrovirus species in human stool structural features in eukaryotic mrnas that modulate the initiation of translation multiple novel and prevalent astroviruses in pigs virus taxonomy. eighth report of the international committee on taxonomy of viruses identification of a novel astrovirus in a domestic pig in hungary characterization of phylogenetically diverse astroviruses of marine mammals end cdna amplification using classic race cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney osterhaus ad ( ) identification and characterization of deer astroviruses genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea re-discovery and genomic characterization of bovine astroviruses isolation and characterization of canine astrovirus in china detection of diverse astroviruses from bats in china acknowledgment this work was supported by the science division of the canadian food inspection agency (cfia). key: cord- - xglujqk authors: chasey, d.; alexander, d. j. title: morphogenesis of avian infectious bronchitis virus in primary chick kidney cells date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: xglujqk primary chick kidney cells were infected with avian infectious bronchitis virus (ibv) and examined by electron microscopy. virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles. virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles. studies on the morphogenesis of coronaviruses have sho~ that replication takes place in the cytoplasm of the host cell by a budding process from either the membranes of the endoplasmie retieulum or cytoplasmic vacuoles ( ). this method of development has been demonstrated for human coronaviruses in wi- cells ( , ) and human embryo lung cells ( ) , and avian infectious bronchitis virus (ibv) in ehorioatlantoie membrane cells ( ) , chick embryo fibroblasts ( ) , vero cells ( ) and the trachea of infected fowls ( ) . the budding mechanisms of virus particles in infected cells have been recorded in detail but little is known of the modes of entry and release although it has been generally reported that infection and release occur by viropexis and cell lysis respectively. in the present study we have examined the morphogenesis of two ibv strains in primary chick kidney (ck) cells with particular emphasis on aspects of entry and release. t h e b e a u d e t t e strain of i b v was used as described previously ( ) . tissue culture a d a p t e d 't' s t r a i n of i b v was o b t a i n e d from c. d. braeewell, central v e t e r i n a r y l a b o r a t o r y , w e y b r i d g e , u.k. a n d h a d been passaged six times in ck cells. a stock was g r o w n a n d this virus was used for infecting m o n o t a y e r tissue cultures. virus infectivity was e s t i m a t e d as plaque forming units (pfu) as described ( ) . p r i m a r y c k cells were p r e p a r e d from -week-old chicks as described ( ) a n d t h e m e d i u m was m a i n t a i n e d a t pi-i . -- . w i t h rn i t e p e s buffer. cells were grown on . cm~ surfaces in ml plastic culture bottles a n d inoculated a t conflueney w i t h ' -- p f u of virus per cell. cultures that. were to be e x a m i n e d at, times up to one h o u r after infection were inoculated with a p p r o x i m a t e l y p f u of virus per cell. a f t e r a n a d s o r p t i o n period of one hour, t h e inoculum was r e m o v e d a n d t h e monolayers were w a s h e d prior to overlay a n d i n c u b a t i o n a t ° c. a t specified times after i n o c u l a t i o n cell cultures, including u n i n f e c t e d controls, were p r e p a r e d for electron microscopy. t h e m e d i u m was r e m o v e d from t h e cells a n d t h e monolayers were rinsed briefly in per cent g l u t a r a l d e h y d e in . n sodium p h o s p h a t e buffer p h . (spb). f u r t h e r g l u t a r a l d e h y d e was a d d e d a n d t h e ceils fixed for -- m i n u t e s a t room t e m p e r a t u r e before washing in s p b a n d post-fixation in per cent o s m i u m tetroxide for minutes. a f t e r d e h y d r a t i o n in g r a d e d e t h a n o l sohations, t h e monolayers were covered w i t h araldite a n d left for a p p r o x i m a t e l y one hour. the m i x t u r e of resin a n d residual alcohol was replaced w i t h fresh araldite which was allowed to h a r d e n . t h e araldite blocks c o n t a i n i n g t h e m o n o l a y e r s were finally prised from t h e plastic s u b s t r a t e a n d small chips were r e -e m b e d d e d in capsules. t h i n sections of t h e s a n d w i c h e d monolayers were cut on a n l k b u l t r a t o m e , using glass knives, a n d s t a i n e d in p e r cent m e t h a n o l i e u r a n y l a c e t a t e for ck cells were inoculated with approximately pfu of ibv strain beaudette per cell and examined , and minutes later. the inoculum, which was concentrated by centrifugation, contained large amounts of cell debris visible with virus particles outside the cells. one hour after inoculation particles and cell debris were also seen within the cells in cytoplasmic vacuoles (fig. la) . virus particles appeared to enter ck cells by one of two me~hods, phagocytosis or micropinocytosis, both of which may be termed viropexis. phagocytosis occurred with the engulfment of virus particles and cell debris by surface processes to produce phagocy.tic vacuoles (fig. a--d) . entry by micropinocytosis occurred with the formation of small invaginations, each containing a single virion, which were distinctive in that the membrane enveloping the virus appeared thickened (fig. a) . one hour after inoculation small virus-containing vesicles with thickened membranes could be seen within the cells (]pig. b). the uncoating of ingested virions and subsequent core replication was not evident in our preparations. six to eight hours after infection with strain beaudette, virus was visible within the cisternae of the endoplasmic reticulum. virus particles could not be seen in large numbers until about ten hours after inoculation by which time extracellular particles were also observed. evidence was obtained which showed that virus particles were formed by budding from the membranes of the smooth endoplasmic reticulum into the cisternae (fig. ) . electron microscopy of cells infected with strain beaudette indicated that virus release occurred by at least two distinct mechanisms. a small number of virus particles were enveloped individually by thickened portions of the endoptasmic reticutum which formed rounded vesicles (fig. a, b) . these vesicles, with a diameter in the range -- nm, resembled those associated with incoming particles and often exhibited small exterior spicules. vesicles with thickened membranes were seen in infected cells throughout the cytoplasm both empty and containing virus particles, and empty vesicles were also present in uninfected cells. the observations of vesicles at cell boundaries suggested that, after formation in the endoplasmic reticulum, the vesicles moved to the cell surface where they could be seen fused with the plasma membrane (fig. c, d) . since this mechanism is the reverse of micropinocytosis it was important to establish in which direction the vesicles at the cell surface were moving in order to distinguish outgoing particles from already released progeny virus re-entering the monolayer in a secondary cycle of infection. that infection or re-infection of cells occurred for a considerable time (at least ten hours) after inoculation was evident from the continued phagocytosis of virus particles (fig. ) . as a rule it was not possible to judge the direction of movement for vesicles opening onto the exposed cell surface (i. e. away from the substrate) since the large amount of extracellul~r virus seen close to the surface prevented unambiguous interpretation. however, virus release by reverse micropinocytosis could be demonstrated for thickened vesicles opening into regions of the monolayer in which different cells were in close contact. in this ease virus particles could be seen to have been effectively trapped in the narrow (fig. a, b) . the majority of virus particles remained within the endoplasmic retieulum and were seen later in infection in large cytoplasmic vacuoles, many of which were apparently formed by dilation of the eisternae. the size of the vacuoles was not related to the number of enclosed virus particles and many vacuoles were empty (fig. ) ; control cells often contained large numbers of empty vacuoles. there was evidence to suggest that the -¢acuoles fused with each other (fig. ) but with strain beaudette there was no observation of fusion with the plasma membranes. the overall density of the cytoplasm increased during the last stages of infection, cells fused, and individual organelles tended to lose their identity fig. . l~elease of virus, strain beaudette, by reverse micropinocytosis a) a thickened vesicle (arrowed) enclosing a virus particle close to the cell surface. i hours after inoculation. the confined extraeellular space already contains released particles. note the 'spicules' on the vesicle membrane × , . b) virus particle released (arrowed) from a vesicle into a confined extracellular space, hours after inoculation; × , in a granular mass; the nuclei were fragmented and appeared as small axeas of densely stained material, while regions of endoplasmic reticulum remained electron transparent. the cell masses rounded-up and detached from the substrate prior to lysis and, from the beginning of the stage at which cells were seen dying, large numbers of extracellular virus particles were present in the sections. disintegrating cells were observed and progeny virus within the cycoplasmic vacuoles was seen to be released b y m e m b r a n e rupture. virus particles released into confined extracellular spaces tended ~o lie in dose-packed arrays with hexagonal configuration. virus particles were seen in the process of budding from internal membranes but, unlike strain beaudette, appeared to form predominantly within pre-existing cytoplasmic vacuoles (fig. ) . however, because of the difficulty in tracing membrane continuity in thin sections it was not always possible to distinguish between vacuoles and endoptasmic retieulum. the majority of progeny virus particles appeared to be released by cell lysis. virus release by reverse micropinocytosis was not observed with 't' strain but there was evidence that the large virus-containing cytoplasmic vacuoles could fuse with the plasma membranes and release virus particles before gross degenerative changes had taken place in the cytoplasm (fig. ) . virus particles of both strains were circular in cross-section with diameters of -- nm although some ]urger particles were also observed. individum virions consisted of an outer envelope, approximately nm thick, surrounding a pale central region. areas of more densely stained material were often seen at the periphery of the central region close to the envelope and apparently 'empty' particles were also present. many virus particles, especially those seen outside the cells after release, possessed well-defined surface projections or spikes. these could be seen clearly on both strains of virus, and were particularly distinctive on 't' strain particles (fig. a, b) , but the number of spikes visible varied considerably from one particle to another. each spike was -- n m in length and approximately n m across with a small thickened knob at the distal end. although projections were most numerous and clearly visible on released virions they could also be identified on particles still in the process of budding; generally no more than one or two spikes were seen attached to the incomplete envelope (figs. % ) . fig. . morphology of ibv particles a) extracellular t strain particles hours after inoculation. note the well-defined surface projections × , . b) extracellular ssrain beaudette virus hours after inoculation also exhibiting surface spikes x , . e) a strain t virus particle budding into a cytoplasmic vacuole hours after infection. a surface spike can be seen (arrowed) on the immature particle; × , the general features of i b v development in primary ck cells observed in this study are consistent with those reported in previous investigations of eoronavirus morphogenesis ( ). i n common with other i b v studies the intraeellular inclusions seen in ceils infected ~dth mouse hepatitis or human respiratory viruses ( , ) were not observed in our material. the i b v particles seen in this investigation showed morphological characteristics similar to those already described for the eoronaviruses ( ). a distinctive feature, however, was the presence on m a n y particles of well-defined surface projections which have not previously been clearly observed in sections of cells infected with i b v ( ) or other eoronaviruses. the generai shape of the individual spikes was similar to that seen in negatively-stained preparations ( , ) . the presence of spikes on budding particles was also established and this observation demonstrates the early attachment of at least some of these subunits during virus formation. the apparent absence of surface projections on m a n y particles, particularly those within large vacuoles, could represent fnndamental differences due to different methods of maturation or, alternatively, be merely an artifact of specimen preparation. further information was obtained concerning the mode of virus entry and release. the electron micrographs indicate that ibv was able to enter ck cells by two distinct mechanisms both of which may be classified as viropexis. although we did not observe any morphological evidence of uncoating, no other means of entry was detected and we assume that viropexis initiates infection. the mechanism by which the majority of particles entered the cells involved engulfment of the virus, and cell debris present in the inoculum, by surface processes or pseudopodia to form phagocytic vacuoles. the second method involved the uptake of individual particles by micropinocytosis. the thickened membrane forming the enveloping invagination can be identified with the modified or 'coated' merebrane characteristic of the transport vesicles present in many cell types ( , ) . virus entry by micropinocytosis and subsequent association with coated vesicles has not been described previously for coronaviruses but has been observed in l cells infected with a rhabdovirus, vesicular stomatitis virus ( ) , and in hela cells infected with type adcnovirus ( ). the budding of particles from the endoptasmie reticuium and the release of progeny virus took place by processes which may be general for all eoronaviruses ( ). however, the envelopment of virus particles by coated vesicles and their subsequent release from the cells by reverse mieropinocytosis are phenomena not previously described for any virus. this mechanism may be peculiar to those members of the coronaviru s group which bud into the cisternae of the endopl~smic reticulum since the particles are formed within the membranes from which the coated transport vesicles are produced (t ). in this respect our failure to observe any association of 't' strain virus particles with coated vesicles may be significant since this strain was seen to bud predominantly into cytoplasmic vacuoles. the method of release of virus particles by fusion of vacuoles with the cell plasma membranes seen in 't' strain infected cells appears similar to the process described for coronaviruscs from human respiratory infections ( ) . one of the more conspicuous aspects of the morphogenesis of ibv in ck cells was the large number of virus particles seen in the cytoplasmic vacuoles and outside the cells late in infection. although infectious particle release was not measured in the present study, alexander and colli~s (i) have demonstrated release of infectious virus, measured as pfu, from ck cells infected with approximately pfu of ibv strain beaudette per cell. the maximum titre of released virus was reached -- hours after inoculation, at a time when light microscopy of the infected cells revealed cytopathie effect in the form of small syncytia, but no obvious cell death or lysis had occurred. in their study the maximum titre represented a total infectious particle production of -- pfu per cell (als~xa~der, unpublished). this tow total and the failure to detect an increase in the rate of infectious particle release late in infection would suggest that the majority of particles seen in our thin sections were non-infectious. similarly, observations on human coronaviruses grown in tissue culture have shown a lack of correlation between the infectivity titres and the large numbers of particles seen by electron microscopy ( ) . while it is tempting, in the ease of strain beaudette, to correlate the number of released infectious particles with the frequency of virus release from coated vesicles in our work, clearly further study is required to show more exactly the relationships between the different methods of particle release and the production of infectious virus. effect of pt-i on the growth and eytopathogenicity of avian infectious bronchitis virus in chick kidney cells the biology of large i~na viruses morphogensis of avian infectious bronchitis virus and a related human virus (strain e) antigenic relationships between strains of infectious bronchitis virus as shown by the plaque reduction test in chicken kidney cells early events in the interaction of adenoviruses with hela eelis. i. penetration of type and intraeellular release of the dna genome l%eplication of avian infectious bronchitis virus in african green monkey kidney cell line vero an electron microscope study of the development of a mouse hepatitis virus in tissue culture cells growth and intracellular development of a new respiratory virus isolation from man of "avian infect.ious bronchitis virus-like" viruses (coronaviruses) sit~filar to e virus, with some epiderniological observations morphogenesis of avian infectious bronchitis in chicken embryo fibroblasts uitrastructure of animal viruses and bacteriophages: an atlas electron microscopic studies of coronavirus specialized sites on the cell surface for protein uptake viropexis of vesicular stomatitis virus by l ceils an electron-microscope study of the trachea of the fowl infected with avian infectious bronchitis virus a simplified lead citrate stain for use in electron microscopy v~;e thank judy luddingto~l, mike collins, and nick chettle for their excellent assistance. t~eceived march , key: cord- -hhge sl authors: remond, m.; boireau, p.; lebreton, f. title: partial dna cloning and sequencing of a canine parvovirus vaccine strain: application of nucleic acid hybridization to the diagnosis of canine parvovirus disease date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: hhge sl the cloning and sequencing of aneco ri-pst i fragment derived from the replicative form of a canine parvovirus (cpv) vaccine strain are reported. the variability of the ′ end of ns protein gene in the genome is confirmed by comparison with previously determined dna sequences. a nucleotide deletion was also observed in this vaccine strain. in order to improve cpv diagnosis, radioactively labelled rna or dna and biotin labelled dna obtained by random priming of the recombinant plasmid were used as probes mainly on gut or stool samples from naturally infected dogs. results of filter hybridization correlated well with histopathological diagnosis of parvovirus infection and with hemagglutination tests performed on dog faeces. we propose that nucleic acid hybridization may be an alternative diagnostic method to ascertain the presence of cpv, especially in frozen samples. canine parvovirus (cpv) like other parvoviruses contains a linear kilobase (kb) single stranded dna (ss dna). the viral genome encodes two nonoverlapping transcription units ]. when replication occurs, dna is converted into double stranded replicative forms (rf) . restriction sites have been mapped [ ] on the genome and the nucleotide sequence has been determined - , ] . canine parvovirus, in spite of vaccination, remains an important cause of disease and is often implicated in fatal disease in young puppies. cpv diagnosis is best achieved with hemagglutination test on faeces or by the detection of histological changes in gut mucosa , , , ] . alternatively, virus can be isolated in cell culture from various organs, but this method is reported to be much less sensitive because of the high lytic properties of the intestinal content. so, even if tests are available cpv diagnosis may be difficult in some cases. a sensitive cpv diagnosis test based on viral nucleic acid hybridization has been developed. the eco ri-pst i restriction fragment of the cpv replicative form dna has been cloned into the multiple cloning site of the pt t u plasmid. viral nucleic acid hybridization was realized using radioactively labelled dna or rna probes and also a biotin labelled dna probe. the biotin labelled probe was found to be fold less sensitive. the cloned dna was sequenced and compared to the previously described cpv sequences. short deletion and point mutations were observed, which emphasised the high variability of c terminal region of the non structural (ns ) protein gene. cells used for virus propagation were freshly seeded crandell feline kidney cells maintained with eagles's minimum essential medium supplemented with % foetal bovine serum. the cpv strain used in the present study was derived from the carmichael strain (cpvb) partially sequenced at passage [ , ] . it was obtained from a commercial vaccine at passage and six additional passages in cell culture were performed before dna cloning. passage and passage were further designated as cpv-b and cpvb . feline panleukopenia virus (fplv), porcine parvovirus, mink aleutian disease virus (gorham strain) and derzsy goose virus (kindly supplied by v. marius, laboratoire central de recherches avicole et porcine, ploufragan) were used to test the probe specificity. organs and faeces were collected from diseased puppies with parvovirus-related symptoms. some of them were kindly supplied by a. moraiuon, veterinary school, maisons-alfort. organs from kittens with panleukopenia-like symptoms were also collected and included in this study. histological analysis or hemagglutination test on faeces were performed on an aliquot of each sample. samples were then stored at - °c before being processed. the replicative form of cpv-b dna was extracted by a modified hirt procedure as described by mcmaster et al. [ , ] . viral single stranded dna was either prepared from purified viral particles [ ] or directly from infected cell supernatants. virus was then treated with . % sds and proteinase k ( ~tg/ml) for h at °c, followed by a phenol chloroform extraction and ethanol precipitation. viral dna was extracted from organs and faeces of diseased animals as described by orth [ ] . gut, spleen and faeces were minced and left in lysis buffer ( mm tris ph , mm nac , mm edta, sds . %) for h at room temperature. proteinase k was added ( .tg/ml) and samples were incubated for h at °c. after clarification, the nac concentration of the supernatant was adjusted to m and mixtures were kept on ice overnight. the supernatant was centrifuged for h at , rpm and treated with an equal volume of phenol for h at room temperature. it was then treated by chloroform and precipitated by ethanol. the dna pellet was suspended in te with rnase ( lag/ml) for min at °c, and gg of this treated dna were spotted onto a nitrocellulose filter after denaturation by sodium hydroxide. partial dna cloning and sequencing of a canine parvovirus vaccine strain dna cloning dna was digested with ecori and psti (boehringer) and ligated into the pt t u plasmid (pharmacia). eseherichia coli nm was transformed with the recombined plasmid as described by hanahan [ ] . recombinants were identified by in situ hybridization of white colony replicas with a radioactive labelled viral probe obtained from purified virions. the hybridization procedure was performed as described by maniatis etal. [ ] . recombinant plasmid dna was also further analyzed by agar gel electrophoresis after digestion with eco ri and pst i or with hind iii. dna sequencing m dideoxynucleotide sequencing was carried out as already described [ ] . for direct sequencing of denatured plasmid dna, we used synthetic primers (biosearch apparatus) [ ] with the sequenase kit (usb). sequence data were analyzed by using the microgenie (beckman, ) and pc gene (intellegenetics, ) computer programs. hybridization were carried out overnight at c in % formamide, x ssc ( x ssc: mm sodium chloride, mm sodium citrate), x denhardt's solution (denhardt's solution: % polyvinyl pyrrolidone, % ficoll, % bovine serum albumin), . % sds and gg/ml of sonicated calf thymus dna (sigma). % dextran sulfate was also added for biotinylated probe. filters were washed twice in . % ssc, . % sds for h at °c. radioactive filters were exposed to x-ray film (kodak xar) at - °c for h. biotin labelled probe was detected with streptavidin-alkaline phosphatase conjugate (brl). after m. remond etal. incubation with a luminescent substrate, ppd ( methoxy - -phosphate phenyl spiro - -dioxetane . ' adamantan), as recommended by the supplier (photogene-brl), light emission was detected by autoradiography for rain on x-ray film. eight recombinant clones containing cpv sequences were selected by colony hybridization, six clones contained an insert of the kb; as expected the other two contained . kb and . kb inserts. all clones hybridized with purified viral partial dna cloning and sequencing of a canine parvovirus vaccine strain it is noteworthy that sequence analysis of hind iii fragment allowed differentiation between the four cpv strains a see [ ] b reversions c consistent differences between cpv-b and the other cp¥ strains d unique difference between cpv-n and cpv parrish del deletion nc non coding nd not done in brackets, amino acids probe after restriction enzyme digestion and southern transfer. as predicted from previously sequenced cpv [ ] , digestion of the six kb recombinant plasmids with hind ill yielded the predicted bp fragment (fig. ) . the sequence of both strands of cloned dna was determined for three different plasmids after subcloning into the m phage. the resulting nucleotide sequence represented about % of the cpv genome. as expected, it included parts of two major open reading frames (orfs) which are in the same phase corresponding to the ' end of nsi gene and the first nucleotides of the vp /vp gene (fig. ) [ ] . comparison of this sequence with that of three other cpv strains [ , , ] and one fplv strain [ ] revealed point mutations between the different isolates (table ) . furthermore, a nucleotides deletion, located in the ns gene, and extending from nucleotide to nucleotide of the norden cpvs strain (cpv-n) sequence [ ] was observed in the cloned dna. to elucidate whether the deletion observed was originally in the vaccine or was generated by cell culture passages in our laboratory, dna sequence was determined on cpv-b after pcr amplification of a bp fragment including the deleted region and the two h i n d i i i restriction sites (fig. ) . pcr amplification products were then cleaved with hind iii, cloned in pt t u and sequenced. the presence of the deletion in the original vaccine was thus confirmed. consequently, the modified ns gene did not alter viral replication in cell culture as cpv-b could be multiplied without loss of infectivity. application of p labelled dna or rna probe for routine diagnosis dna from various organs of dogs and cats was extracted, spotted onto nitrocellulose and assayed with probes. samples came from animals which died of parvovirus infection as could be deduced from histological examinations, expected for three of them which were classified as "suspect"and one gut sample taken from a puppy suffering from distemper which was chosen as a negative control. out of gut samples from diseased dogs, were positive with dna or rna probes. two spleen extracts were tested: only one hybridized. four faecal extracts which showed specific hemagglutinating properties hybridized strongly and the fifth one from a suspect dog was negative. two samples for panleukopenia diagnosis, one from the gut and the other, a mixture of spleen and kidney, did not hybridize. results presented with the dna probe in fig. were identical with the rna probe (data not shown). twofold dilutions of purified cpv-dna were spotted in duplicate and assayed respectively with p labelled dna probe and biotin labelled probe in order to determine the sensitivity of the non-radioactive probe. results are shown in the vaccine strain used in this study was derived from the carmichael strain (fig. ) . it underwent additional passages in cell culture (cpv-b ) before being cloned and sequenced. eco ri and pst i sites were chosen because they are perfectly conserved among different isolates of cpv and fplv [ ] . moreover, the eco ri-pst i fragment covered partially non-structural protein genes which present a strong homology between some members of the family parvoviridae, allowing its use for diagnosis of related diseases [- ] . comparison with the previously published sequence of cpv-b strain [ ] showed a high rate of point mutations and one deletion which could have been generated by cell culture passages. this result emphasises the high variability of the cpv genome and sequence comparison was further investigated on three other cpv strains [ , , ] and one fplv strain [ ] . point mutations were observed between the different cpv sequences and between cpv-b and fplv (table ) . reversions appeared in cpv-b strain; the nucleotidic changes were only observed in cpv-b at passage , compared to fplv and no longer existed in the partially sequenced dna of cpv-b . by contrast, no changes were observed in regulatory regions (polyadenylation sites, p and p promoters, potential splicing sites). most of the coding mutations appeared in the c terminal part of ns ( table ) and this is in contrast with the results published for minute virus of mice [ ] . the short deletion observed in cpv-b and cpv-b was also located in this part of the ns gene. short deletions often appeared in the noncoding part of the genome but were never described in the parvovirus ns gene. this deletion, located bases downstream of the p promoter did not m. remond et al. r i f a f h g w n y v k v c h a i c c v l n r q g g k r n t v l f h et s k l t n f s l p d t r t c k i f a f h g w n y v k v c h a i c c v l n r q g g k r n t v l f h e t s k l a n f s m a s t r t c r i f a e h g w n y i k v c h a i c c v l n r q g g k r n t v l f h k p s m l p t f n i s n t r t c k i f s m h n w n y i k c c h a i t c v l n r q g g k r n t i l f h d n t k l t n f d l a n s r t c q i f r m h g w n w i k v c h a i a c v l n r q g g k r n t v l f h f e q v m ---c i k d n k i v k l l l c q n y d p l l v g q h v l k w i d k k c g k k n t l w f y a p h y d ---c -q g n l v f k l l n l q g y n p w q v g h w l v m m l s k k t g k r n s t l f y fpfni~pnknilwweecimt alter the reading frame and defined a genetic marker for this vaccine strain. the n terminal part of the ns gene appeared more conserved in the analyzed sequences. furthermore, the alignment of ns protein sequences from different parvovirus allowed identification of a highly conserved sequence already de-scribed as a homologous domain of proteins which used purines nucleotides (fig. ) [ ] . the kb insert has been used as probe on cpv and other parvoviruses available in the laboratory for cross-hybridization in order to measure to what extent the probe could be useful for other parvoviruses. results were~as expected [ , ] . cpv and fplv are closely related but the sensitivity of the probe for fplv is one hundred fold lower as measured by the difference in titer of the infectious particles detected. the sensitivity of the dna cpv probe on homologous dna is comparable to that reported for other viruses: herpesvirus [ ] , adenovirus [ ] and, rotavirus [ ] . probes for viruses in the family parvoviridae have only been reported for the human b parvovirus [ , ] with higher sensitivity in terms of quantity of target dna detected but the number of infectious particles detected is of the same order. in fact, there is a discrepancy between the number of infectious particles detected ( s ccids ) and the quantity of purified dna spotted and detected: . ng dna correspond to s viral particles. this may be explained by the production of defective dna genomes and/or a greater number of rf copies in the supernatants of infected cell culture which cannot be measured in terms of infectivity. rna probes have been developped to overcome any background due to hybridization of the plasmid with bacteria present in stools and gut contents but we did not encounter such difficulty. although rna probes have been shown to offer up to tenfold more sensitivity than dna probes, at least on rna viruses such as enterovirus [ ] and on dna viruses such as b parvovirus [ ] , the results obtained in our study did not demonstrate any difference in the sensitivity and specificity of the two types of probes. data presented on clinical samples demonstrated that cpv nucleic acid probes were effective for diagnosis of parvovirus disease even on specimens which had been stored under unappropriate conditions. we plan to simplify dna extraction and to use this probe in combination with pcr to improve the sensitivity of the test. non-structural protein of parvoviruses: homology to purine nucleotide using proteins and early proteins of papovaviruses dna sequence of the lymphotropic variant of minute virus of mice, ravin(i) and comparison with the dna sequence of the fibrotropic prototype strain the complete dna sequence of minute virus of mice, an autonomous parvovirus detection of b parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe nucleotide sequence of the glycoprotein s gene of bovine enteric coronavirus and comparison with the s proteins of two mouse hepatitis virus strains cloning and sequence of dna encoding structural proteins of autonomous parvovirus feline panleukopenia virus hemagglutination by canine parvovirus: serologic studies and diagnostic applications a modified live canine parvovirus strain with novel plaque characteristics. i. viral attenuation and dog response complete nucleotide sequence and genome organization of bovine parvovirus detection of group b rotavirus in fecal specimens by dot hybridization with a cloned cdna probe a technique for radiolabelling dna restriction endonuclease fragments to high specific activity dna hybridization for diagnosis of enteric adenovirus infection from directly spotted human fecal specimens techniques for transformation ore. coli selective extraction of polyoma dna from infected mouse cell cultures int~rat des sondes arnc (ribosondes) synth&is~es in vitro dans la d~tection des enterovirus par hybridation mol culaire mapping of porcine parvovirus dna and development of a diagnostic dna probe optimal conditions for supercoil dna sequencing with escherichia coli dna polymerase i large fragment diagnosis of pseudorabies virus infection in pigs with specific dna probes molecular cloning. a laboratory manual nucleotide sequence of feline panleukopenia virus: comparison with canine parvovirus identifies host-specific differences characterization of an immunosuppressive parvovirus related to the minute virus of mice comparison of canine parvovirus with mink enteritis virus by restriction site mapping antigenic relationships among autonomous parvoviruses lesions of spontaneous canine viral enteritis characterization of a new type of human papillomavirus that causes skin warts infectious, process of the parvovirus h : correlation of protein content, particle density and viral infectivity embl data bank access number histopathologic evidence for parvovirus infection in dogs porcine parvovirus: dna sequence and genome organization nucleotide sequence and genome organization of canine parvovirus parvovirus genome: nucleotide sequence of h and mapping of its genes by hybrid-arrested translation nucleotide sequence of the coat protein gene ofcanine parvovirus dna sequence comparison between two tissuespecific variants of the autonomous parvovirus, minute virus of mice rapid detection of human parvovirus b dna by dot-hybridization and the polymerase chain reaction nucleotide sequence and genome organization of human parvovirus b isolated from the serum of a child during aplastic crisis characteristics and taxonomy of parvoviridae authors' address: p. boireau, centre national d'etudes v t rinaires et alimentaires, laboratoires central de recherches v t~rinaires, rue pierre curie, f- we thank dr. f. fontaine and dr. l. grosgean for critical review of the manuscript, and v. del moral for excellent secretarial help. key: cord- - kizif authors: deng, guangcun; bi, jianmin; kong, fuli; li, xuezhu; xu, qiang; dong, jun; zhang, miaojie; zhao, lihong; luan, zhihua; lv, nana; qiao, jian title: acute respiratory distress syndrome induced by h n virus in mice date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: kizif h n avian influenza viruses have repeatedly caused infections in swine and humans in some countries. the purpose of the present study was to evaluate the pulmonary pathology caused by h n viral infection in mice. six- to eight-week-old balb/c mice were infected intranasally with × ( ) mid( ) of a/chicken/hebei/ / (h n ) virus. clinical signs, pathological changes and viral replication in lungs, arterial blood gas, and cytokines in bronchoalveolar lavage fluid (balf) were observed at different time points after infection. a control group was infected intranasally with noninfectious allantoic fluid. h n -infected mice exhibited severe respiratory syndrome, with a mortality rate of %. gross observations showed that infected lungs were highly edematous. major histopathological changes in infected lungs included diffuse pneumonia and alveolar damage, with neutrophil-dominant inflammatory cellular infiltration, interstitial and alveolar edema, hemorrhage, and severe bronchiolitis/peribronchiolitis. in addition, h n viral infection resulted in severe progressive hypoxemia, lymphopenia, and a significant increase in neutrophils, tumor necrosis factor-α and interleukin- in balf. the features described above satisfy the criteria for acute respiratory distress syndrome (ards). our data show that h n viral infection resulted in ards in mice, and this may facilitate studies of the pathogenesis of future potential h n disease in humans. h n avian influenza viruses have become highly prevalent in poultry in many eurasian countries since the early s [ , ] . although these viruses generally cause only mild to moderate disease, they have been associated with severe morbidity and mortality in poultry as a result of coinfection with other pathogens [ ] . since , h n viruses have been isolated from pigs and humans with influenza-like illness in hong kong and mainland china [ - , , , , , , ] . these findings indicate that the h n avian influenza virus can also cross species barriers and expand its host range from avians to mammalians. to date, h n virus has usually caused relatively mild clinical signs in humans [ , ] . however, recent studies have shown that infection of swine with h n viruses causes significant morbidity and mortality [ ] . most of the diseased pigs showed typical respiratory signs, including fever, nasal and ocular discharge, coughing and dyspnoea. in some cases, paralysis associated with fatal disease was also observed [ ] . pigs are believed to serve as intermediate hosts for adaptation of avian influenza viruses that infect humans, and it has been shown that some of the h n influenza viruses currently circulating in china have molecular features that allow them to preferentially bind to human a- , -neuacgal receptors [ , ] . the recurring presence of h n infections in pigs and humans has raised concerns about the possibility that h n viruses are capable of evolving into pandemic strains [ , , ] . previous studies revealed that h n viruses were able to infect mice without adaptation, and this resulted in different levels of lethality and kinetics of replication [ , ] . more recently, it was demonstrated that an h n virus showed enhanced replication and efficient transmission by direct contact in a ferret model [ ] . it is well known that influenza viruses mainly cause pulmonary infection in animals and humans, but little information is available regarding h n viral infection in lungs in mammals. therefore, it is urgent to evaluate the pathology of pulmonary infection caused by currently circulating h n viruses in an appropriate animal model. here, an h n avian virus with high lethality for mice, isolated recently from northern china, was used to address this in a mouse model. the results suggest that h n viral infection induces a typical acute respiratory distress syndrome (ards) in mice that resembles the common features of ards [ , ] . our data may facilitate studies of the pathogenesis of future potential avian h n disease in humans. the h n virus used in this study was isolated from chickens in hebei province of northern china in and was identified by means of hemagglutination inhibition and neuraminidase inhibition tests. the isolate was designated as a/chicken/hebei/ / (h n ) (ck/hb/ / ). the complete genome sequences of the virus are available from genbank under accession numbers fj -fj . our previous studies showed that this isolate was a reassortant virus containing a/chicken/beijing/ / -virus-like ha, na, and ns genes, an a/quail/hongkong/g / -like m gene, and a/chicken/shanghai/f/ -like rnp genes. we analyzed its pathogenicity in chickens and mice in detail and found that it replicated efficiently in chicken lungs but did not cause obvious clinical signs in specificpathogen-free (spf) chickens. however, the mice exhibited high mortality rates and severe lung injury when inoculated with ck/hb/ / virus without prior adaptation (data not shown). the virus was propagated in the allantoic cavities of -day-old embryonated spf chicken eggs at °c for h and stored at - °c for use in all of the experiments described herein. the % mouse infectious dose (mid ), % mouse lethal dose (mld ) and % egg infectious dose (eid ) of ck/hb/ / were determined by serial titration of virus in -day-old embryonated spf chicken eggs at °c. titers were calculated by the method of reed and muench as described previously [ ] . all manipulations were performed under bsl- ? laboratory conditions. animal experiments were conducted according to established guidelines and approved by the animal care committee of china agricultural university (beijing, people's republic of china). to assess the pathogenicity of h n virus in mice, six-to eight-week-old female spf balb/c mice, purchased from beijing laboratory animal research center (beijing, people's republic of china), were housed in microisolator cages ventilated under negative pressure with hepa-filtered air. during the experiment, mice had access to food and water ad libitum. a pilot experiment indicated that a dose of mid of ck/hb/ / h n virus was optimal, because the course of the disease was prolonged and the infected mice presented obvious signs of respiratory illness. therefore, in the present study, the mice were lightly anesthetized with diethyl ether and then inoculated intranasally ( ll) with mid of ck/hb/ / h n virus diluted in sterile saline. mock-infected control animals were inoculated intranasally ( ll) with an equivalent dilution of noninfectious allantoic fluid. two types of experiments were carried out in this study. the first experiment was to investigate clinical signs, gross lesions and mortality rates of h n -infected mice over a -day time period. in this experiment, mice were divided randomly into two groups of mice each. the h n -infected group was inoculated with ck/hb/ / virus, and the control group received the noninfectious allantoic fluid, as described above. the animals' general behavior and clinical signs, including food intake, body weight, inactivity, anal temperature (measured with an infrared thermometer) and mortality, were monitored daily in each group for days. in the second experiment, we studied the features of ards induced in mice by h n viral infection. mice were divided randomly into two groups with mice in each group. since some infected mice died between day and day postinfection (p.i.), larger groups ( per group) of mice were used. ten mice of each group were chosen randomly, weighed and euthanized on days , , , , , , and p.i., and the following parameters were observed: lung injury was assessed by testing lung water content and histopathology. arterial blood gas, white blood cell counts, tumor necrosis factor (tnf)-a and interleukin (il)- levels in bronchoalveolar lavage fluid (balf), and viral titers in the lungs were measured at different times. lung histopathology and assessment of lung water content three mice per group were weighed and sacrificed on days , , , , , , and p.i. the whole lungs were removed. the left lobes of the lungs were fixed in buffered % formalin and embedded in paraffin for histopathological evaluation. five-micrometer-thick sections were stained with hematoxylin-eosin for light microscopy. the upper parts of the right lung lobes were used to determine the lung wet weight:body weight ratio and lung wet:dry weight ratio. the remaining lobes of the right lung were stored at - °c until needed for determining the lung virus titer. to assess lung water content, the lung wet weight:body weight ratio and lung wet:dry weight ratio were determined by weighing the right lung before and after oven desiccation at °c, and this was used as an indicator of lung edema [ ] . lung wet:dry weight ratio = weight of the whole wet lung/weight of the whole dry lung; lung wet weight:body weight ratio (%) = weight of the whole wet lung/body weight %. virus titration was performed as described previously [ ] . lungs, kidneys, brains, livers, spleens, and hearts were collected and homogenized in cold phosphate-buffered saline on days , , , , , , and p.i. clarified homogenates were titrated for viral infectivity in embryonated chicken eggs from initial dilutions of : . viral titers were expressed as mean log eid per milliliter ± standard deviation ( mid is about eid ). arterial blood gas analysis and peripheral blood leukocyte counts after blood sample collection, blood gas analysis was conducted as described by fagan et al. [ ] . briefly, arterial blood samples ( . ml) were collected in a heparinized syringe by percutaneous left ventricular sampling of lightly anesthetized mice spontaneously breathing room air. blood gas analysis was immediately conducted with an il ph/blood gas/electrolytes analyzer (instrumentation laboratory, lexington, ma). heparinized blood samples ( ll) were used for leukocyte counts at various time points. cell numbers for three individual mice were determined in triplicate by counting with a hemocytometer. for differential counts, two blood smears from each mouse were stained with wright stain, and the number of lymphocytes was determined. at least cells were counted for each slide at a magnification of , [ ] . neutrophil counts and measurement of tnf-a and il- in balf according to the protocol described by majeski et al. [ ] and nick et al. [ ] , bronchoalveolar lavage was performed immediately following sacrifice of the animal by cervical dislocation on the days indicated. in brief, the lungs were lavaged twice in situ with the chest cavity opened by midline incision with a total volume of . ml of saline ( °c) inserted through an endotracheal tube. the rate of recovery of balf was not less than % for all of the animals tested. after the amount of fluid recovered was recorded, an aliquot of balf was diluted : with . % crystal violet dye and . % acetic acid for leukocyte staining and erythrocyte hemolysis. the number of leukocytes in balf was counted with a hemacytometer under a microscope. the remaining balf was centrifuged ( g, min). neutrophil differential counts were determined by wright staining of a spun sample, on the basis of morphological criteria, under a light microscope with evaluation of at least cells per slide. all slides were counted twice by different observers blinded to the status of the animal. the supernatant for cytokine analysis was immediately frozen and stored at - °c. the concentrations of tnf-a and il- were determined in balf and serum, using elisa kits (sigma, st. louis, mo). all data are expressed as means ± sd. statistical analysis was performed with the spss statistical software package for windows, version . (spss inc., chicago, il). differences between groups were examined for statistical significance by two-tailed student t test. a p-value less than . was considered statistically significant. overall, infected mice presented a relatively acute clinical process. some mice showed inactivity, altered gait, ruffled fur, inappetence, and an average weight loss of . % on day p.i. by day p.i., most mice presented severe signs of respiratory disease, including visual signs of labored respiration and respiratory distress, and exhibited more severe inappetence, emaciation, and . % weight loss. sixty percent of the mice ( of ) died between days and p.i. gross observation showed the infected lungs to be highly edematous, with profuse areas of hemorrhage (fig. a) . retention of gas in the stomach was occasionally found in infected mice. surviving mice began to recover on day p.i. no obvious gross lesions were observed in the hearts, livers, brains or kidneys in infected mice. the infected mice displayed a similar histopathological pattern with severe diffuse pneumonia, characterized by inflammatory cellular infiltration, interstitial and alveolar edema and hemorrhage, as shown in fig. . diffuse pneumonia with severe alveolar damage was found in the whole lung (fig. b) on day . figure b -h showed the kinetic observations of lung lesions of h n -virus-infected mice. on day p.i., lung lesions were characterized by edema around the small blood vessels (fig. c, d, solid arrows) , thickening of the alveolar wall, and dropout of mucous epithelium adhering to the surface of bronchioles (fig. c, d, open arrows) . on days - p.i., as shown in fig. e , f, severe edema and neutrophil-dominant inflammatory cellular infiltration could be seen around small blood vessels (solid arrows) as well as diffuse pneumonia with fig. g, h) . severe peribronchiolitis (solid arrows in fig. g, h) , edema around blood vessels and severe hemorrhage were found (fig. g, h) . in addition, prominent dropout of bronchial epithelium and a great number of neutrophils, fibrin, and suppurative exudates infiltrating the bronchioles were also observed (solid arrows in fig. g, h) . in comparison, lungs from control mice had no apparent histological changes. kinetic observation of h n viral replication in mouse tissues indicated that the viruses in the lung replicated more efficiently than those in other tissues. viral infection resulted in high titers of virus in the lungs on days - p.i. (fig. ) . the peak viral titer was observed on day p.i., reaching . log eid /ml. however, viral titers dropped to a relatively low level on day p.i. viruses were also isolated with lower titers from livers, kidneys, spleens, and hearts on days - p.i., but was not isolated from brains (data not shown). lung water content: edema fig. a shows the dramatic increased lung wet:dry weight ratios on days - p.i. (p \ . ) after h n viral infection. a similar change in lung wet weight:body weight ratios was also observed in infected lung, shown in fig. b , with the peak value nearly fourfold that of the control on day p.i. table shows the time courses of arterial blood gas parameters in mice. the partial pressure of arterial oxygen (pao ), saturation of arterial oxygen (sao ), and ph value were slightly decreased, while partial pressure of arterial carbon dioxide (paco ) was increased in infected mice on day p.i. subsequently, when most of the infected mice presented apparent clinical signs of respiratory distress at day p.i., pao and sao also dramatically decreased as compared with the controls (p \ . ). figure a shows that the number of leukocytes in peripheral blood progressively decreased on days - p.i. in infected mice compared with those in control mice. the lowest value was seen on day p.i. differential blood counts (fig. b) revealed that the lymphocytes of infected mice dropped by about % on day p.i. from day p.i. onwards, both leukocytes and lymphocytes gradually increased in survived mice. white blood cell summary and differential counts in balf figure shows the time course of white blood cell (wbc) summary and neutrophil counts in balf on days , , , , , , and p.i. the number of wbcs in infected mice increased gradually from day p.i. and reached its peak, with about fourfold that of the control group, by day p.i. neutrophils in balf increased dramatically from days - p.i., and the peak was approximately -fold greater than that of the control group on day p.i. concentrations of tnf-a and il- in balf and serum were measured at different time points after h n infection. as shown in fig. a , levels of tnf-a in infected mice significantly increased on days - p.i. in balf (p \ . ) compared with those of the controls. tnf-a levels also rose significantly in serum on days - p.i. (fig. b) , but this alteration was not as significant as that in balf. il- levels increased slightly on days - p.i. in serum but increased dramatically in balf on days - p.i. and reached a peak on day p.i., with about . fold that of control mice. previous studies have revealed that h n viruses demonstrate different levels of lethality and kinetics of replication inflammatory cellular infiltration, interstitial and alveolar edema, hemorrhage, and severe bronchiolitis/peribronchiolitis. additionally, arterial blood gas analysis demonstrated that the pao decreased dramatically and the paco increased significantly when disease was exacerbated. this indicated that most of the infected mice developed progressive and severe hypoxemia consistent with the time course of clinical signs and pulmonary lesions for ards. the features described above satisfy the criteria of ards [ , [ ] [ ] [ ] ] and show that h n viral infection resulted in the prominent ards in mice. in comparison with ards in mice with h n viral infection [ ] , h n viral ards in mice shows a shorter course of disease. most of the h n virus-infected mice presented overt signs and died between days and p.i., while death of mice with h n viral infection occurred between days and p.i. besides this, the infected lungs are more edematous after h n viral infection. observation of viral replication showed that the h n viruses replicated efficiently in the lung. viral infection resulted in high titers of viruses on days - p.i., with the peak viral titer on day p.i. this was consistent with the time course of the severity of h n viral respiratory disease in mice, indicating that high viral replication may be important for h n viral disease pathogenesis. it is believed that tnf-a and il- may play an important role in the development of ards [ , , ] . in most studies, cytokines in patients with ards have been described as an inflammatory 'cascade' or 'network', and thus their actions were not easily manipulated [ ] . h n virus induced high levels of tnf-a and il- in balf and serum in the mouse ards model [ ] . in this regard, our finding that h n viral infection resulted in significantly increased tnf-a and il- in balf is similar to that of the previous study of h n virus infection. the role of these cytokines in lung inflammation during h n viral infection remains to be investigated. a secondary intense neutrophil-predominant host inflammatory response is usually considered an important feature of ards. the inflammatory response triggered by direct or indirect insults to the lung involves the recruitment of blood leukocytes, the activation of tissue macrophages, and the production of a series of different mediators such as cytokines, chemokines and oxygen radicals [ , , , , , , ] . lung injury may be a direct consequence of this inflammatory response. in h n -virus-infected mice, we observed that circulating leukocytes dramatically decreased in the blood and that a great number of inflammatory cells infiltrated the lungs. in addition, the neutrophils in balf increased approximately -fold compared to control mice on day p.i. these results suggested that a great number of leukocytes, especially neutrophils, were recruited from the bloodstream and sequestrated mainly in the lungs, and this might be involved in the host inflammation response and severe pulmonary lesions induced by h n viral infection. some studies have shown that h n viruses could cause upper respiratory tract illnesses in humans [ ] . subsequent studies have revealed that h n viruses can also infect pigs and cause typical respiratory signs with significant morbidity and mortality [ , ] . further investigation demonstrated that currently circulating h n influenza viruses in china continued to evolve and generate multiple genotypes [ , , ] , raising the possibility that the h n virus might increase pathogenicity and transmissibility in humans and would be a potential threat to the human population. our findings highlight the serious potential threat of h n for human health. in summary, our data show that h n viral infection induced typical ards in mice, which might facilitate studies of the pathogenesis of future potential avian h n disease in humans. evaluation of pathogenic potential of avian influenza virus serotype h n in chickens acute respiratory distress syndrome: a historical perspective role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome human infection with an avian h n influenza a virus in hong kong in antigenic and genetic characterization of h n swine influenza viruses in china swine infection with h n influenza viruses in china in the pulmonary circulation of homozygous or heterozygous enos-null mice is hyperresponsive to mild hypoxia molecular characterization of h n influenza viruses: were they the donors of the ''internal'' genes of h n viruses in hong kong? characterization of the pathogenicity of members of the newly established h n influenza virus lineages in asia infections and the inflammatory response in acute respiratory distress syndrome sequence analysis of the hemagglutinin gene of h n korean avian influenza viruses and assessment of the pathogenic potential of isolate ms evolution of h n influenza viruses from domestic poultry in mainland china avian-to-human transmission of h n subtype influenza a viruses: relationship between h n and h n human isolates respiratory reovirus /l induction of intraluminal fibrosis, a model of bronchiolitis obliterans organizing pneumonia, is dependent on t lymphocytes h n influenza a viruses from poultry in asia have human virus-like receptor specificity the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h n -a review role of p mitogenactivated protein kinase in a murine model of pulmonary inflammation cocirculation of avian h n and contemporary ''human'' h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? human infection with influenza h n prone position in acute respiratory distress syndrome pulmonary and extrapulmonary acute respiratory distress syndrome are different chemokines in acute respiratory distress syndrome a simple method of estimating fifty percent endpoints characterization of a human h n influenza virus isolated in hong kong bronchoalveolar and systemic cytokine profiles in patients with ards, severe pneumonia and cardiogenic pulmonary oedema genetic analysis of four porcine avian influenza viruses isolated from shandong depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans replication and transmission of h n influenza viruses in ferrets: evaluation of pandemic potential acute lung injury and the acute respiratory distress syndrome: a clinical review characterization of a pathogenic h n influenza a virus isolated from central china in isolation and identification of swine influenza recombinant a/swine/shandong/ / (h n ) virus acute respiratory distress syndrome induced by avian influenza a (h n ) virus in mice key: cord- -mntcor d authors: oka, tomoichiro; yamamoto, seiji p.; iritani, nobuhiro; sato, shigenori; tatsumi, chika; mita, tetsuo; yahiro, shunsuke; shibata, shinichiro; wu, fang-tzy; takagi, hirotaka title: polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: mntcor d sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. human sapoviruses are currently assigned to genotypes (gi. - , gii. - , giv. , and gv. - ) based on the sequence of the region encoding the major structural protein. in this study, we evaluated polymerase chain reaction (pcr) assays using published and newly designed/modified primers and showed that four pcr assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic dna or cdna prepared from human sapovirus-positive fecal specimens. these assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses. for the initial reactivity test, double-stranded dna fragments (gblocks® gene fragments) including the sequence targeted by the pcr primers (approximately bp each) of the human sapovirus genotypes gi. (genbank accession number x ), gi. (ab ), gi. (ab ), gi. (aj ), gi. (dq ), gi. (aj ), gi. (ab ), gii. (aj ), gii. (ay ), gii. (ab ), gii. (ab ), gii. (lc ), gii. (ay ), gii. (ab ), gii. (kx ), giv. (dq ), gv. (dq ), and gv. (ab ) were synthesized (integrated dna technologies, coralville, ia), and . × copies of these fragments were used. to confirm the reactivity against clinical specimens, random-hexamer-primed cdna synthesized from stool suspensions positive for sapoviruses (gi. - , gii. - and gii. and gii. , giv. , and gv. - ) as well as stool suspensions positive for noroviruses (gi, gii), astrovirus types i and iv, and rotavirus groups a and c and dna purified from stool suspensions positive for adenovirus type / that had been used in a previous study and stored at − °c [ ] , were tested. pcr assays were performed using µl of a reaction mixture containing µl of synthetic double-stranded dna or µl of cdna or dna from clinical specimens, µl of kapa g fast hotstart readymix with dye (kapa biosystems, wilmington, ma), and pmol of each primer. pcr amplification was performed using a geneamp pcr system (applied biosystems, foster city, ca) under the following conditions: initial denaturation at °c for min, followed by cycles of °c for s, °c or °c for s, and °c for s, and a final extension at °c for min. the pcr reaction time was approximately . h. seven µl of each pcr reaction mixture was analyzed by electrophoresis in a % agarose gel containing mm tris-acetate, mm edta and . µg of ethidium bromide per ml, and the gel image was captured using a fas v gel documentation system (nippon genetics, tokyo, japan). eleven pcr assays with different primer combinations were performed using a synthetic dna panel. as shown in fig. a (left panels), seven of the pcr assays, including those using the primers sv-f and sv-r ; sv-f and sv-r ; slv and slv ; sav f, sav- f, sav- f, sv-r , and sv-r ; sav rfwd and sv-r ; sv-f , sv-f , sv-r , and sv-r ; and sv-f and sv-r [ ] [ ] [ ] [ ] , either did not amplify all of the human sapovirus genotypes tested or generated pcr products with unusual sizes (i.e., gi. sapovirus with the sv-f and sv-r primer set) under the test conditions. in contrast, four assays including the primer combinations i) sv-f , sv-f , sv-g -r, sv-g -r, sv-g -r, and sv-g -r [ ] , ii) m f-sav rfwd, m r-sv-g -r, m r-sv-g -r, m r-sv-g -r, and m r-sv-g -r, iii) m f-husav-f , m f-husav-f , m f-husav-f , and m r-husav- r, and iv) m f-husav- f and m r-husav- r amplified all the tested human sapovirus genotypes (fig. a , right panels). as shown in fig. b , these four pcr assays gave similar results when cdna prepared from sapovirus-positive clinical specimens ranging from approximately to copies/reaction (ct value range: - /reaction determined by real-time rt-pcr) representing the human sapovirus genotypes (gi. - , gii. - and gii. and gii. , giv. , and gv. - ) was used [ ] . samples with ct values > (approximately to copies/reaction) were only slightly detectable or difficult to detect, depending on the assay used. no cross-reactivity was observed with specimens that were positive for norovirus gi and gii, human astrovirus types i and iv, rotavirus a and c, or adenovirus type / in the four pcr assays (data not shown). the locations of the primers used for the four pcr assays are shown in fig. . the pcr assay including six primers (sv-f , sv-f , sv-g -r, sv-g -r, sv-g -r, and sv-g -r) reported by okada et al. was originally designed to generate pcr products of different lengths, depending on the genogroup: gi ( bp), gii ( bp), giv ( bp), and gv ( bp) [ ] (fig. a , right panel, fig. b, fig. ). this assay has been used in several studies to detect sapoviruses in fecal specimens [ ] [ ] [ ] . in the current study, we confirmed that these primer combinations detected all human sapovirus genotypes (gi. - , gii. - , giv. , and gv. - ) using cdna and/or synthetic dna ( table ) . the pcr assay including five primers (m f-sav rfwd, m r-sv-g -r, m r-sv-g -r, m r-sv-g -r, and m r-sv-g -r) also generated pcr products of different sizes depending on the genogroup: gi ( bp), gii ( bp), giv ( bp), and gv ( bp) (fig. a , right panel, fig. b, and fig. ) . the assay including four primers (m f-husav-f , m f-husav-f , m f-husav-f , and m r-husav- r) amplified similar-sized pcr products (approximately bp) independently of the genogroup and genotype (fig. a, right panel, and fig. b) . the target regions of husav-f , -f , and -f recently designed as forward primers in a broadly reactive real-time pcr for human sapoviruses [ ] are similar to those of sv-f and sv-f (fig. ) , and the sapovirus-specific sequence of m r-husav r ( ′-ccccanccngcvhacat- ′) ( [ ] are shown in parentheses. the primer combinations and pcr product size(s) are indicated at the top and right side, respectively, of each gel image. the annealing temperature is indicated in parentheses after the primer names. m, dna size marker bp dna ladder (new england biolabs); nc, negative control without template cac at- ′) and svr-ds ( ′-ccccamccmgcmma-cat- ′) primers reported by sano et al. [ ] . the assay including two primers (m f-husav- f and m r-husav- r) generated similar-sized pcr products (approximately bp) for all of the sapovirus genotypes tested (fig. a, right panel, and fig. b) . the sapovirus-specific sequence of m f-husav- f was identical to that of rfwd [ ] except for nt (fig. and table ), and they shared the same complementary sequence in the reverse primers (husav-r, and sav r, respectively) used in real-time pcr assays for detection of human sapoviruses [ , ] . in primer-dependent pcr assays, the selection of broadly reactive primer sets is important for detecting genetically diverse human sapoviruses. in this study, seven out of primer combinations, including widely used primer sets, did not detect all of the sapovirus genotypes tested (fig. a and b) . this would cause a bias towards the detected strains or cause several genotypes to be missed when used for surveillance. we recently reported an improved broad-range real-time rt-pcr assay that detected all of the currently identified human sapovirus genotypes [ ] . this assay can be used as a screening tool, but the amplicon size (approximately bp) is too short for further genetic characterization by sequence analysis. selection and combination of the human-sapovirus-targeting, broad-range rt-pcr assays described in this report can be used to confirm real-time rt-pcr results and an alternative screening tool to identify genetically diverse human sapoviruses in samples. an assay that distinguishes genogroups based on pcr product size will be useful in cases in which a sample includes sapoviruses of different genogroups. new sapovirus strains assigned as gii.na were reported recently [ ] . based on their nucleotide sequences, the primers used in the four broadly reactive pcr assays would probably also recognize this new candidate genotype ( fig. and table ), although experimental confirmation is required. funding this work was supported by a grant from the research program on emerging and re-emerging infectious diseases from the japan agency for medical research and development (amed) (jp fk and jp fk ). the funding body had no role in study design, analysis and interpretation of data, or the decision to submit the article for publication. conflict of interest there are no conflicts of interest among the authors. the ethics committee of the national institute of infectious diseases deemed that human subject regulations did not apply to this study because it only used extracted and stored virus nucleic acids (receipt number ). nt-ctgctatcctgccaccaggtg cacaggggcagtcacgagtaa- nt-tttccttggggctatccacc- nt-agcgca atgtttgctgggtgggg - nt-ctgccatcttgccacccggag tacaggggccgtcagcaacaa- nt-ctttcttggtgccatccatc- nt-agtgcc atgtttgctggctgggg - nt-tcgcagtgctgcctccaggtg aactggggcagtcaccagcaa- nt-gttcctgggcgcaatccacc comprehensive review of human sapoviruses children attending day care centers are a year-round reservoir of gastrointestinal viruses a foodborne outbreak of sapovirus linked to catered box lunches in japan characterization of sapoviruses detected in gastroenteritis outbreaks and identification of asymptomatic adults with high viral load detection of human sapovirus by real-time reverse transcription-polymerase chain reaction human sapovirus classification based on complete capsid nucleotide sequences genetic diversity of human sapovirus across the americas viral metagenomics reveals sapoviruses of different genogroups in stool samples from children with acute gastroenteritis in jiangsu investigation of a food-borne outbreak of gastroenteritis in a school canteen revealed a variant of sapovirus genogroup v not detected by standard pcr complete genome sequence of a novel gv. sapovirus strain, ngy- , detected from a suspected foodborne gastroenteritis outbreak broadly reactive real-time reverse transcription-polymerase chain reaction assay for the detection of human sapovirus genotypes molecular epidemiology and phylogenetic analysis of sapporo-like viruses the detection of human sapoviruses with universal and genogroup-specific primers detection of norovirus (gi, gii), sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex pcr detection and genetic analysis of human sapoviruses in river water in japan surveillance of pathogens in outpatients with gastroenteritis and characterization of sapovirus strains between epidemiology and genotype analysis of sapovirus associated with gastroenteritis outbreaks in a confirmation of sapovirus re-infection gastroenteritis cases with different genogroups and genetic shifts in the evolving sapovirus genotypes quantification and genotyping of human sapoviruses in the llobregat river catchment near-complete human sapovirus genome sequences from kenya key: cord- - lry ys authors: smith, a. l.; winograd, deborah f.; burrage, t. g. title: comparative biological characterization of mouse adenovirus strains fl and k and seroprevalence in laboratory rodents date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: lry ys the growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. the fl strain of mouse adenovirus grew in both l murine fibroblasts and in cmt- murine rectal carcinoma cells, whereas the k strain grew only in cmt- cells. the bulk of the fl progeny virus was released from the host cells. k virus was largely cell-associated. both virus strains were stable at ° c in liquid medium. the k strain was completely inactivated after – minutes at ° c, whereas fl infectivity was still detected after two hours at this temperature. both virus strains were stable in the dessicated state for days, although fl viability was more dependent on the presence of protein in the virus diluent. seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony. laboratory mice reportedly may naturally sustain infection with either of two distinct mouse adenovirus strains. the fl strain (mad-fl) was isolated by hartley and rowe ( ) as a contaminant of a friend leukemia virus stock. this virus has been used to develop mouse models of virusinduced myocarditis, endocarditis and addison's disease ( , , , ) . l~'lad-fl replicates in primary and continuous cells of murine origin and produces a fatal disease in suckling mice inoculated by any of several routes ( ) . experimentally infected adult mice usually do not exhibit clinical signs ( ) . the k strain (mad-k ) was isolated by hasmmoto et al. ( ) from the feces of clinically normal mice. until recently, the in vitro replication of this strain has reportedly been limited to primary mouse kidney cells. we have successfully grown this virus strain and mad-fl in cmt- cells, a continuous line derived from a chemically induced murine rectal carcinoma ( , ) . mice inoculated intracerebrally, intraperitonemly, intramuscularly, subcutaneously or orally reportedly localize mad-k in the intestine ( ) . virus is not present in urine or nasal tissue but is excreted for long periods in the feces. in a recent review ( ) , the statement was made that "... mouse adenovirus has been essentially eliminated as an ambient indigenous agent ..." however, there is at least one published report suggesting that this may not be the ease ( ) . mouse adenovirus-like inclusions were found in the intestines of apparently healthy mice submitted to necropsy for routine diagnostic evaluation; however, using mad-fl as the test antigen, seroeonversion to mouse adenovirus could not be confirmed. similarly, a breeding colony of mice at the yale school of medicine experienced high infant mortality with associated intestinal inclusions suggestive of adenovirus infection (unpublished data). we were also unable to document seroconversion of mice in this facility using mad-fl as the detecting antigen. another controversy surrounding mouse adenovirus relates to the antigenie relationship between mad-fl and mad-k . using antisera made in mice or guinea pigs, van d~ vee>t and mes ( ) found the two virus strains to be antigenicmly distinct by both the complement tixation (cf) and neutralization tests. in contrast, wmand st al. ( ) found that antiserum to mad-k neutralized both homologous virus and mad-fl. antiserum to mad-fl contained only a low level of neutralizing antibody to mad-k . using both cf and indirect immunofluoreseence (ifa) tests, we have found that. mad-fl antiserum from mice exposed once to the virus reacts equally well with mad-fl and mad-k antigens, whereas similarly prepared antiserum to mad-k reacts only with homologous antigen ( ) . the source of these discrepancies is not immediately apparent; however, our results may explain why seroconversion to mouse adenovirus is not observed among mice thought to be infected with the virus. mad-fl has traditionally been used as the test antigen in serologic tests performed in u.s. laboratories. the inability of antibody to mad-k or an mad-k -like virus to react with mad-fl antigen would explain the apparent lack of seroconversion among infected mice. in this report, we compare the kinetics of growth of mad-fl and mad-k in two continuous cell lines of murine origin and the stability of the two viru's strains' in both liquid and desiccated states. we provide further docu, mentation of the unilateral antigenic relationship between mad-fl and mad-k . in addition, we report data whieh suggest that the host range of mouse adenovirus or related viruses is not limited to the laboratory mouse. finally, we provide serologic evidence that an outbreak of disease in a mouse breeding colony which sustained high infant mortality was associated with the introduction of a mouse adenovirus strain closely related to mad-k . the fl strain of mouse adenovirus was obtained from the amelican type culture collection (atcc, rockville, md) and passaged twice in l mouse fibroblasts. this stock had a titer of l s.° tcids per ml in both l cells and cmt- cells. the k strain of mouse adenovirus was kindly supplied by dr. k. hashimoto (tokai university school of medicine, isehara, japan) and was passaged twice in primary suckling mouse kidney cells and tbur times in cmt- cells. this stock had a titer of . tcid~ per ml in cmt- cells. l mouse fibroblasts were obtained from atcc and grown in minimal essential medium containing earle's salts and percent heat-inactivated fetal bovine serum (fbs). cmt- cells ( ) were obtained from atcc and grown in percent dulbecco's modified eagle's medium (dmem), percent liebowitz l medium and percent heat-inactivated fbs. both cell lines were transferred once weekly, the l cells by scraping and the cmt- cells by treatment with . percent trypsin mixed in equal parts with . ~i edta. both cell lines were maintained at °c in a percent c . atmosphere. cells growing in cm ~ culture flasks were infected at a multiplicity of infection of . after a one hour absorption at ° c, the flasks were washed extensively and ml of medium were added to each flask. cells and fluid were immediately collected from one set of two flasks. a similar procedure was followed at the intervals shown in the results. at late time intervals, after c~%opathic effect was observed, supernatant fluids were clarified by centrifugation, and the cell pellet was pooled with cells remaining on the vessel surface. cells were subjected to three cycles of freezing and thawing to release intracellular virus. titrations were performed with cells grown in -well cluster dishes and inoculated with txl per well. cytopathic effect (cpe) in mad-fl titrations was evmuatcd daily from days to post-inoculation. cpe in mad-k titrations was evaluated daily from days to post-inoculation. titers were calculated by the method of re~d and muenc~ ( ) based on cpe observed in four replicates per virus dilution and were adjusted to reflect the titer per em flask. electron microscopy cmt- cells growing in cm flasks were infected with mad-fl or mad-k at multiplicities of infection of . sixty hours later, the medium was removed and the cells were scraped and pelleted at x g. the cells were resuspended in a solution containing percent glutaraldehyde in . rv~ eaeodylate buffer (pit . ) and fixed for one hour at ° c. the cells were postflxed for minutes with percent osmium tetroxide using . percent tannic acid as a mordant. the inclusion of tannic acid in the protocol enhanced visualization of the fiber protein ( ). following an overnight incubation at ° c with . percent magnesium uranyt acetate, the cells were processed for embedding in epon resin. sections were cut with a diamond knife, stained with uranyl aeetate and lead citrate and were photographed with a philips electron microscope. to test the stabitit~ ~ of the viruses in liquid medium, ~[ad-fl and mad-k were adjusted to s.° tcids per ml in dmem containing percent fbs and aliquoted in t~ volumes. after incubation at ° or ° c for the time periods detailed in the results, . mt of ice cold dmem containing percent fbs was added to each of two vials per strain. one hundred ~l volumes of this material and serial ten-fold dilutions thereof were added to cmt- cells growing in -well cluster dishes (three wells per dilution). virus titers were calculated as described above for the growth curves. to test viability after drying, mad-fl and mad-k were adjusted to ,° tcids per ml in dmem containing a) no fbs; b) percent fbs; e) percent fbs; or percent fbs. these preparations were added in ~tl volumes to several wells of -well cluster dishes. the drops were air dried in a laminar flow safety cabinet. after , , , , or days, tll of cold dmem containing percent fbs were added to three wells per preparation. after thorough mixing, ~i of this material and serial ten-fold dilutions were added to wells of -well cluster dishes containing cmt- cells (three wells per dilution). titers were calculated as described above. for indirect immunofluorescenee tests, serum samples which had been stored at - ° c were diluted : and were added to wells of teflon-coated slides (cel-line associates, newfield, nj) containing bivalent (mad-fl and mad-k ) antigen. the antigen was prepared by mixing l cells which had been infected for days with mad-fl with cmt- cells which had been infected for days with mad-k . after a minutes incubation, the slides were washed with agitation through two changes of phosphate buffered saline (pbs). after air drying, the cells were exposed to fluorescein isothiocyanate-conjugated anti-mouse or anti-rat immunoglobulin (antibodies, inc., davis, ca) containing . percent evan's blue eounterstain (l~oboz surgical instrument co., washington, dc). after another minutes incubation, the slides were again washed with pbs, dipped in distilled, demineralized water, mounted with pbs : glycerol ( : ) and examined with an olympus bh halogen-illuminated transmitted light fluorescence microscope. cells which had been exposed to serum containing antibody stained apple-green (nuclear and cytoplasmic). cells which had been exposed to serum which did not contain antibody were stained reddish brown. selected positive sera were then tested in parallel against monovalent mad-fl or mad-k antigen. for neutralization tests, fifty-d] of serum diluted : were mixed in transfer plates (dynatech) with ~tl of tissue culture fluid diluted to contain tcids of virus. these mixtures and the virus titrations were incubated at ° c for one hour prior to the addition to cmt- cells growing in -well cluster dishes. each serum sample was tested in duplicate wells and was considered to contain neutralizing antibody only if it inhibited the development of cytopathic effect in both test wells. mad-fl neutralization tests were held for days, and ~d-k tests were held for days. some sera which were positive at : were tested at dilutions of : through : . serum titers are expressed as the reciprocal of the dilution which completely inhibited the development of cytopathic effect in both rephcate wells. multiparous cf mice (charles r, iver breeding laboratories, kingston, ny) were inoculated intraperitoneally with tcids of either mad-fl or mad-k emulsified with freund's complete adjuvant (total inoculum per mouse was . ml). each mouse was simultaneously exposed orally to tcidso of virus. the virus was in the form of an l (mad-fl) or cmt- (mad-k ) infected cell lysate. the mice were exsanguinated by cardiac puncture twenty-eight days later ( mice per virus). serum to be used for imnmno-fluorescence staining was diluted : in sterile pbs and ml were absorbed for hours at ° c with a lysate of l s uninfected l cells (mad-fl antibody) or cmt- cells (sla_d-k antibody). two-fold sei al dilutions of serum ( : to : , ) were tested. neutralizing antibody assays were performed with unabsorbed sara {dilutions of i : i to : ). mouse and rat sara were collected between and as part. of a quality assurance monitoring program. animals were killed with co and exsanguinated immediately upon arrival in this facility. sera from six commercial mouse suppliers and two intramural, barrier-maintained mouse breeding colonies were tested (total number-- ). sera from six commercial rat suppliers and one intramural, barrier-maintained rat breeding colony were also tested (total number = ). selected sara which contained mad antibody as determined by ifa were tested by neutralization. ( ) for mad-fl grown in cells cultured from outbred infant mouse kidneys, more than percent of the progeny virus was released from the infected cells. the ultrastructure of cmt- cells infected with mad-fl was similar to that previously reported by wi~and et al. ( ) . virions appeared to be assembled near electron dense regions in the nucleus (fig. a) . cmt- cells which did not contain visible viral inclusions at this late stage of infection often had large numbers of virions bound to membrane blebs via the fiber proteins ( fig. b) . virions were - nm in their greatest dimension and had fibers approximately nm long projecting from their vertices. these fibers terminated in a knob (fig. b) . moto ( ) . virions occurred as paracrystalline arrays in the nucleus (fig. a) . extracellular virions could be observed on invaginations of uninfected cmt- cell membranes (fig. b) . these virions were - nm in diameter and had nm fibers projecting from their vertices (fig. b) . both mad-fl and mad-k were extremely stable at ° c in dmem containing percent fbs (data not shown). neither virus was inactivated to any appreciable extent after hours, maintaining titers of per ml. infectious mad-fl was detected after u hour incubation at ° c (table ) , mthough the average tiger was reduced by - log tcids per ml from the starting materim. in contrast, mad-k lost . - . log~ tcids per mi during the first minutes at ° c and was reduced to undeteetable levels between and minutes at this incubation temperature (table ) . the stability of the two mad strains under dried conditions at room temperature was studied as an experimental approximation of the condition of the viruses after excretion from the natural rodent host. both virus strains (diluted to contain tcids per ml) were quite stable in this situation. mad-fl viability was promoted by the presence of protein in the form of fbs. at days, the titer in percent fbs was . _+ . log tcid~ per ml, whereas the titer in the absence of fbs was . + . loglo tcidso per ml, a reduction of . percent from the starting material. by days, mad-k viability was relatively unaffected by protein concentration in the virus diluent,, varying from . log tcid~ per ml in the absence of fbs to we have previously reported that antibody to mad-fl reacts equally well ~dth homologous and heterologous mad antigens in cf and ifa tests ( ) . in contrast, antibody to mad-k reacts only with the homologous antigen in these tests. antisera prepared in mice immunized once by the intraperitoneal and oral routes were tested in both ifa and neutralizing antibody assays ( table ). the mad-fl antiserum reacted to equivalent titer in the ifa test, with m_ad-fl and i~[ad-k antigens; however, the neutralizing antibody titer of this serum was four-fold higher with homologous antigen than with heterologous antigen. at the dilutions used, the antiserum prepared against mad-k did not react in either test with mad-fl. sera from mice obtained from six commercial suppliers were tested, and mice from only one of these suppliers had serum antibody to mad (table ) . sera from mice housed in two intramural, barrier-maintained facilities were mso free of antibody to mad. only three mouse sera ofl which were tested reacted by ifa with mad bivalent antigen. these sera reacted with mad-k antigen and not with mad-fl antigen when tested on monovalent sera were tested at a i : dilution by indirect immunofluoreseenee (ifa) and neutralization (challenge virus dose was tcids ). results are expressed as the number positive / number tested slides. they also contained neutralizing antibody which was directed against mad-k and not against mad-fl. the seroprevalenee of adenovirus was much higher in rats than in mice (table ). rats from four of six suppliers had serum antibody which reacted sera were tested at:, a : dilution by indirect immunofluorescenee (ifa) and neutralization (challenge virus dose was tcidso). results are expressed as the number positive / number tested u one rat senlm neutralized both mad-fl and mad-k ° the two sera which neutralized mad-k did not neutralize mad-fl with mad antigen by ifa. an intramural, barrier-maintained breeding colony of wag rij rats was free of antibody to mad between and . selected rat sera which were ifa positive on bivalent mad slides were tested on monovalent slides and by neutralization. in contrast to the findings with mouse sera, rat sera reacted with a) mad-k only (source d), b) mad-fl only (some sera from source c), or c) with both mad-fl and mad-k antigens (source b). despite the apparent low seroprevalence of mad infection among commercial mice, there have been occasional reports of mice with histologically evident lesions which were presumed to be associated with mad. in mid-september of , high infant mortality was noted among akr mice in a large intramural breeding colony. a tentative diagnosis of adenovirus infection was made based on the finding of intranuclear inclusions in the intestinal tracts of affected mice. the colony remained seronegative to adenovirus as determined by cf testing with mad-fl antigen. retrospective immunofluorescence serology for mad antibody revealed that, prior to the outbreak of clinical disease, the colony had been free of antibody to either mad strain (data not shown). however, in september of , of ll sera tested contained antibody to ~d-k , but not mad-fl, as determined by ifa. this colony has maintained a very high seroprevalence of mad through october, . eleven sera collected in were tested for the presence of mad neutralizing antibody, and all contained antibody which neutralized mad-k , not mad-fl. pooled intestines from three affected mice were f~ozen at - °c in september, . these intestines were homogenized ( percent w/v) in dmem and five cf mice were inoculated by the combined intraperitoneal and oral routes with the clarified suspension. after days, these mice and five control mice were exsanguinated, and the resulting sera were tested for the presence of antibody to any of eleven common murine viruses. sera from inoculated mice contained antibody to mad-k as determined both by ifa and neutralization [ of mice], theiler's murine encephalomyelitis virus (tmev, picornavirus) [ of mice], and epizootic diarrhea of infant mice (edim) virus (rotavh~as) [ of mice]. sera from all five control mice also contained antibody to edim virus but not to any other tested agents. as reported by wma~d et al. ( ) for growth of mad-fl in primary mouse kidney cultures, we have found that the majority of infectious progeny virions (between and percent) are released fl~om infected l or cmt- cells. in contrast, mad-k is much more cell associated with only about percent of the virus released into the tissue culture fluid. ultrastructural studies confirmed this finding to the extent that extracellular virions were more likely to be found in mad-fl-infected cmt- cultures than in mad-k -infected cultures. both mad-fl and ivl~d-k were found to be resistant to inactivation by heating at ° c, maintaining full viability for at least hours. mad-fl maintained some viability after heating at ° c for hours, whereas mad-k was inactivated after a to minutes exposure to this temperature. both strains of mad were stable for extended periods in the desiccated state. mad-fl viability was more dependent on the presence of protein in the diluent than was mad-k viability. we have presented neutralizing antibody data which confirm our earlier finding that antibody to mad-k reacts only with homologous antigen. as stated earlier, we cannot explain the divergent results reported by different investigators ( , , ) . however, in the original report of the isolation and identification of mad-k , hashilvioto eta[. ( ) stated that they had preliminary data indicating that anti-mad-k sera prepared in rabbits or mice did not neutralize mad-fl. we are not aware of any subsequent report from these investigators amplit~ring this observation. from the laboratory animal perspective, the most important finding presented in this paper is the seroprevalence of mad, both strains fl and k , in laboratory rats. occasional cf antibody titers have been reported in rats, but these have not been correlated with disease, pathology or virus isolations ( ) . there has been one report of intranuclear inclusions and typical adenovirus particles in the intestines ofimmunosuppressed rats ( ) , but the responsible agent was not isolated. that adenovirus infection of rats has not been recognized before with any regularity may be due to the test (complement flxation) which has traditionally been used for antibody detection and to the antigen (mad-fl) which has been used in that test. the complement flxation test has been found to be very insensitive for the detection of antibody to many rodent viruses, most notably mouse hepatitis virus ( ) . v~re have previously reported that antibody titers to mad as determined by the indirect immunofluorescence test are approximately -fold higher than titers determined from the complement fixation test ( ) . since the pattern of reactivity of rat sera differed substantially from that of mouse sera, it may be hypothesized that the virus(es) infecting rats are antigenically related to, but distinguishable from, the known mouse adenovirus strains. future efforts will be directed toward isolation of the agent(s) infecting rats for antigenic and biological comparison with the two known strains of mouse adenovirus. we have found, however, that two week-old rats are not susceptible to mad-k after a route of exposure (oral) which simulates the presumed natural situation (a. l. smith and s. w. barthold, unpublished results). we have provided serologic evidence linking an outbreak of clinical disease in a breeding colony of mice to infection with mad-k or a very closely related virus. one observation remains puzzling. mad-k has never been associated with lethal infection in the experimental situation, irrespective of route of inoculation. several possibilities must be considered to explain the mortality observed in the outbreak at yale. one is that the infecting agent, while antigenically very closely related to mad-k , was biologically different. a second relates to the microbiological status of the affected colony at the time of the outbreak. in , mice from this colony had serum antibody to mouse hepatitis virus (mhv), reovirus type , edim virus, tmev and minute virus of mice (mvm). the role of edim virus in this epizootic cannot be evaluated, since the mice used for the antibody production test had already been infected with this agent. tmev may have played a role, but since only of the inoculated mice made antibody to this agent, it is assumed that the virus was present in the intestinal homogenate at very low concentration. also, since tmev was enzootic at the time of the outbreak of clinical disease, it should not be considered as the primary cause of disease. however, the possibility that intercurrent infection with one or more of these viruses or a particularly virulent bacterial pathogen exacerbated the clinical course of the infection cannot be excluded. adenovirus endocarditis in mice adenovirus myoearditis in mice. an electron microscopic study demonstration of murine adenovirus fiber protein in embedded cells using tannic acid and protein a gold immunolabelling a cell line from an induced carcinoma of mouse rectum a new mouse virus apparently related to tile adcnovirus group an adenovirus from the feces of mice. i. isolation and identification pathogenesis of experimentally produced mouse adenovirus infection in mice experimental adenovirus infection of the mouse adrenal gland. ii. electron microscopic observations viral diseases a naturally occurring intestinal mouse adenovirus infection associated with negative serologic findings serological relationship between mouse adenovii~s strains fl and k experimental adenovirus infection of the mouse adrenal gland. i. light microscopic observations mouse adenovirus a simple method of estimating fifty percent endpoints an immunofluoreseenee test for detection of serum antibody to rodent coronaviruses electron microscope study of experimental enteric adenovirus infection in mice serological classification of two mouse adenovirnses latent adenoviral infection of rats: intranuclear inclusions induced by treatment with a cancer chemotherapeutic agent biological and biophysical characteristics of mouse adenovirus, strain fl section of comparative medicine this research was supported by grant l%r from the division of research resources, national institutes of health, bethesda, md. the authors thank k. p. ~h for help with assembly of the manuscript. key: cord- - p ma authors: duan, x.; nauwynck, h. j.; pensaert, m. b. title: effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: p ma in this study, the susceptibility of porcine peripheral blood monocytes (bmo), peritoneal macrophages (pmφ) and alveolar macrophages (amφ) to prrsv was examined. to test the effect of differentiation and activation on their susceptibility, amφ and bmo were aged, cultivated in either adhesion or suspension and treated with bacterial lipopolysaccharide (lps) and phorbol myristate acetate (pma). it was found that freshly isolated pmφ and bmo were non-permissive to prrsv. pmφ remained refractory but a few bmo became susceptible after day cultivation. amφ were permissive with a significant increase of their susceptibility after one day cultivation. in a binding assay, it was demonstrated that the attachment of biotinylated prrsv to amf is much more efficient than to pmφ and bmo. two monoclonal antibodies (mabs) d and d which block prrsv infection of amφ and are directed against a candidate receptor for prrsv only reacted with the cell membrane of amφ. pma treatment of amφ blocked prrsv replication in the cells in a dose-dependent manner. the blocking effect of pma decreased after h continuous pre-treatment and diminished after h continuous pre-treatment. pma treatment did not affect the binding of prrsv and mab d and d to amφ. direct or indirect treatment of amφ and bmo with lps or cultivation in suspension did not significantly affect their susceptibility. these results provide clear evidence that prrsv has a strongly restricted tropism for only some sub-populations of porcine monocytes/macrophages and that some specific states of differentiation and activation of monocytes/macrophages considerably affect their susceptibility. porcine reproductive and respiratory syndrome virus (prrsv) resembles lactate dehydrogenase-elevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv), three other members of the arterivirus group with regard to morphology, genetic organisation and structural proteins [ , ] . one peculiar common characteristic of these viruses is that they have a strong tropism for monocytes/macrophages. of many procine cell systems tested, only porcine alveolar macrophages (amf) support replication of prrsv [ , , , ] . replication of prrsv in some cultivated porcine peripheral blood monocytes (bmo) has also been reported [ ] . unexpectedly it was also found that prrsv replicate in two non-porcine cell lines: an established cell line from monkey kidney, marc- [ ] , and a proprietary cell line, cl [ ] . similarly, the replication of ldv and shfv is also highly restricted to primary cultures of host macrophages in vitro. eav forms an exception as it replicates in many different cell types [ ] . prrsv also shows a strict cell speci®city``in vivo''. cells of the macrophage lineage have previously been identi®ed as the predominately and consistently infected cell type in prrsv infected pigs [ , , , ] . furthermore, several observations indicated that prrsv only replicates in some sub-populations of monocytes/macrophages. it was found that amf even during the period of the highest virus titres in the lungs after natural or experimental infection, only a low percentage of levaged amf carries prrsv antigens [ , ] . also, immature macrophages and macrophages progenitor stem cells are not or less susceptible to a prrsv infection. this was particularly evident for bone marrow cells, where replication of prrsv was not detected in experimentally prrsv infected pigs [ ] . similar evidence comes from``in vitro'' experiments in which different sub-populations of alveolar macrophages fractionated by density gradient centrifugation were shown to have different susceptibilities to a prrsv infection [ ] . such heterogeneity may be a re¯ection of the sate of differentiation and/or activation of the macrophages [ ] . a number of factors such as ageing, some cytokines and a few chemical products have been found to be able to differentiate and activate mononuclear phagocytes [ ] . bacterial lipopolysaccharide (lps) and phorbol myristate acetate (pma) are two frequently used stimulants. both lps and pma have a strong, rapid and easily reproducible activating capacity. their structure and sites of activation in the cells have been extensively studied [ , ] . lps is a structural component of the outer membrane of gram-negative bacteria. it activates monocytes and macrophages and stimulates them to produce certain factors, including tnf-a, il- , and prostaglandin e [ , ] . studying the effect of lps treatment on the susceptibility of amf to prrsv may give new insights in the pathogenesis of dual infection with prrsv and bacteria. pma has effects on monocyte/macrophages, including dramatic changes in cell shape, spreading, endocytosis, and release of lytic mediators and regulators [ , , ] . pma also stimulate premonocytic cells of some continuous cell lines to differentiate into a more mature monocyte/macrophage phenotype [ ] . because of those remarkable properties, pma has been used to investigate the relationship between cell activation/differentiation and viral replication with some viruses [ , ] . in this study, it was examined if porcine monocyte/macrophage lineage cells isolated from peritoneal cavity, lungs and peripheral blood are susceptible to prrsv and if their susceptibility was related to the degree of virus attachment. furthermore, the effect of differentiation and activation of monocytes/macrophages by ageing, adhesion and treatment with lps and pma on their susceptibility to prrsv was evaluated. two prrsv isolates were used: the lelystad strain of prrsv (kindly provided by dr. wensvoort) and a belgian isolate of prrsv designated v . a lelystad virus stock of the thirteenth passage grown in porcine amf with a titre of . tcid /ml was used in this study. the v was adapted to marc- cells, puri®ed and biotinylated as earlier described [ ] . brie¯y, a ®fth passage of v was ®rst clari®ed by centrifugation at  g for min, then precipitated at  g for h in a beckman t rotor at c. the pellets were resuspended in / of original volume in tne buffer ( mm nacl, mm edta, mm tris-hcl, ph . ) and centrifuged on a . to . m discontinuous sucrose gradient in a sw rotor at  g for h. after centrifugation, the virus band was harvested and its purity was determined with a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the titre of the resulting virus preparation was to  tcid /mg as determined on marc- cells. the number of virus particles, as determined by negative staining electron microscopy, was to  /mg virus protein. puri®ed prrsv was labelled with biotin by using a protein biotinylation kit (amersham, buckinghamshire, uk). the virus was pelleted and re-suspended in biotinylation buffer ( mm na co , ph . ) at a protein concentration of mg/ml. after a brief sonication ml biotin reagent was added per mg of viral protein. the mixture was shaken for h at c and the reaction was terminated by addition of tris-hcl (ph . ) to a ®nal concentration of mm. biotinylated virus was collected after purifying on a sephadex g- column and diluted in pbs at a concentration of . mg/ml. after biotinylation, the titre of prrsv was reduced by %. biotinylated virions were stored at À c. a swine myeloid cell speci®c monoclonal antibody (mab), . . , was used to determine the percentage of porcine monocyte/macrophage cells [ ] . mabs against prrsv nucleocapsid protein, wbe and wbe ± were used for immuno¯uorescenece [ ] . two monoclonal antibodies (mab), d and d , which have been raised against amf and which are able to block prrsv infection of amf [ ] , were used for membrane immuno¯uorescence staining to test their reactivity with various cells. isotype matched irrelevant mabs e and g directed against suid herpesvirus type [ ] , were used as negative controls. porcine alveolar macrophages (amf) amf were obtained from -to -weeks old conventional belgian landrace pigs from a prrsv negative herd according to the method previously described by wensvoort et al. [ ] . brie¯y, the lungs were lavaged with ml cold phosphate buffered saline solution without calcium and magnesium (pbs). the lavaged cells were collected by centrifugation at  g for min at c. after two washings with cold pbs, a cell smear was made and stained with hemacolar reagents (diagnostica merck, darmstadt, germany) and the percentage of neutrophils was determined. the cells were stained with . . by membrane immuno¯uorescence. the percentage of cells from the monocytes/macrophages lineage was estimated by subtracting the percentage of neutrophils from the . . positive cells. by doing so, more than % of lung lavage cells were found to be monocyte/ macrophage lineage cells. pmf were isolated by lavaging the peritoneal cavity of four -to -weeks old conventional belgian landrace pigs originating from a prrsv negative herd with ml cold pbs. after centrifugation at  g for min at c and two washings with pbs, the cells were collected and the percentage of monocyte/macrophage lineage cells was determined with the technique described above. more than % of lavaged peritoneal cells were characterised as monocytes/macrophages. bmo were separated and cultivated as previously described [ ] . brie¯y, peripheral blood was obtained from four to weeks old conventional belgian landrace pigs originating from a prrsv negative herd and peripheral blood mononuclear cells (pbmc) were isolated from blood by ficoll-paque (pharmacia, uppsala, sweden) density gradient centrifugation according to the method recommended by the manufacturer.  pbmc were layered on a polystyrene cell culture dish (corning glass works, corning, ny, usa) which was coated with ml autologous plasma. after h incubation at c, non-adherent cells were removed by three washings with pbs. adherent cells representing enriched monocytes were harvested by gently¯ushing, the number of cells were counted and the percentage of monocytes was estimated with mab . . . more than % of cells were found to be monocyte/macrophage lineage cells. the viability of all cells used was b as assessed by % nigrosin staining. amf, pmf and bmo were brought in -well cell culture plates (nalge nunc international, roskilde, denmark) at a concentration of cells per well. the medium used for cultivation was rpmi supplemented with % of foetal calf serum. amf, bmo and pmf were inoculated by replacing the medium with ml stock solution containing . tcid prrsv. after incubation at c for h, three washings with pbs were performed. then, the cells were refed with medium and further incubated at c with % co . to evaluate the effect of maturation of monocytes/macrophages on their susceptibility to prrsv, freshly isolated amf, pmf and bmo from ®ve donors were seeded in -well tissue culture plates at a concentration of cells/ml/well and further incubated in rpmi medium plus % of foetal bovine serum at c with % co . after and h incubation, the cells were inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. to test if the adhesion of bmo and amf affect their susceptibility to prrsv, freshly isolated amf and bmo from ®ve donors were cultivated either in suspension in te¯on inserts (poly labo, strasbourg, france) or attached to polystyrene in -well cell culture plates at a concentration of cells/well in rpmi medium plus % of foetal bovine serum at c with % co . after h cultivation, the cells were inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and hours after inoculation. the effect of pma treatment on prrsv replication in bmo was examined by treating one day cultivated bmo with ng/ml phorbol -myristate -acetate (pma) (sigma-aldrich vertriebs gmbh, deisenbofen, germany) for h before prrsv inoculation. after pma treatment, the cells were washed three times, then inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. in order to examine the effect of duration of pma pre-treatment of amf on prrsv replication, amf were treated with ng/ml pma for different time periods prior to prrsv inoculation. after pma treatment, the cells were washed three times then inoculated with prrsv. intracellular virus titre was determined at h after inoculation. to test the effect of pma treatment during virus replication in amf, the amf were treated with ng/ml pma for h at different times after prrsv inoculation. intracellular virus titre was determined at h after inoculation. to estimate the dose dependent effect of pma treatment of amf on the prrsv replication, one day cultivated amf grown in -well-plates were treated with different concentrations of pma for h after h prrsv inoculation. the prrsv positive wells were determined by ®xing and staining the plate using an immunoperoxidase monolayer assay (impa) as earlier described [ ] after h inoculation. direct and indirect lps treatments were performed to evaluate the effect induced directly by lps or indirectly by lps-induced cytokines. for direct treatment, bacto lipopolysaccharide (lps) of e. coli :b (difco laboratories, detroit, michigen, usa) was used at a concentration of mg/ml medium. for indirect treatment, % of the supernatant from h lps treated porcine amf was used. after and h treatment, the cells were washed three times with pbs and inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. pma and lps had no negative effect on the viability of the treated cells as assessed by nigrosin staining after each treatment. a cytotoxicity bioassay with pk cells for detecting tnf [ ] and a proliferation assay on d (n )m cells for determining il- concentrations [ ] were performed to asses the ef®cacy of the lps treatment. for determining the titre of extracellular virus, medium from prrsv infected cells was collected and centrifuged at  g for min. ml of tenfold dilutions of the supernatants were inoculated in -well microtiter plates (nalge nunc, roskilde, denmark) previously seeded with amf. for determining the titre of intracellular virus, amf, pmf and bmo were harvested by¯ushing, washing, washed twice with pbs and lysed by freeze-thawing. ml of tenfold dilutions were inoculated in -well microtiter plates previously seeded with cultivated amf. inoculated -well plates were ®xed h after inoculation and stained using an immunoperoxidase monolayer assay (ipma) as earlier described [ ] . macrophages and monocytes were detached by thorough¯ushing of the bottom of polystyrene dishes and washed once with pbs. cell smears were made using a cytocentrifuge at  g for min. the smears were ®xed in acetone for min at À c. a streptavidin-biotin immuno¯uorescence technique was performed. brie¯y, the smears were pre-incubated with : diluted sheep serum to block non-speci®c staining. the smears were ®rst incubated with a mixture of mabs wbe and wbe ± , then with biotinylated sheep-anti-mouse immunoglobulin and ®nally with streptavidin-¯uorescein isothiocyanate (fitc)-conjugate (amersham, buckinghamshire, uk). the smears were washed three times with pbs between each incubation. to con®rm the speci®city of the staining, two mouse ascites¯uids containing irrelevant mabs against suid herpesvirus type of the same isotype as wbe and wbe ± were used as negative controls [ ] . positive cells were counted using a leica dm rbe¯uorescence microscope. the membrane reactivity of biotinylated prrsv, mabs d and d to pbmc, bmo, pmf and pma-treated ( ng/ml pma for h) and -untreated amf were evaluated by ā ow cytometer facscalibur (becton dickinson)¯ow cytometer.  cells were washed three times with cold pbs and incubated with ml of % paraformaldehyde at room temperature for min. after washing once with cold pbs, the ®xed cells were ®rst incubated with biotinylated prrsv ( mg) or mabs ( ng/ml of d , d or e ), then : diluted streptavidin-¯uorescein isothiocyanate (fitc)-conjugate or goat antimouse igg fitc (amersham, buckinghamshire, uk) for h on ice. the cells were washed three times after each incubation. the mean¯uorescence intensity of each sample was measured on the¯ow cytometer. relative mean¯uorescence measurements were corrected for auto¯uorescence of control cells. all data were statistically analysed by the student's t-test. prrsv productively replicated in amf. table shows the virus titres and percentage of prrsv positive cells in freshly isolated and one day cultivated amf at , and h after prrsv inoculation. the virus titres and percentage of viral antigen positive cells in freshly isolated porcine amf at and h after inoculation were respectively to log tcid and to times lower than those of one day cultivated ones, which is signi®cantly different (p` . ). a productive replication of prrsv was not detected in freshly isolated bmo obtained from four pigs. however, when the bmo were inoculated after one day of cultivation, x to . tcid per cells virus and . to . % viral antigen positive cells were detected at hours and hours post inoculation, respectively ( table ) . productive replication of prrsv was not found in freshly isolated pmf obtained from ®ve pigs. after one day cultivation, pmf remained refractory to prrsv infection. as shown in table , no statistically signi®cant differences in both viral antigen expression and virus titres were observed between amf and bmo cultivated in suspension in te¯on inserts and those attached to polystyrene dishes. the ef®cacy of activation after lps treatment was shown by the presence of tnf-a (titre: biological units per ml for the supernatant of amf and biological units per ml for that of bmo) and il- (titre: biological units per ml for the supernatant of amf and biological units per ml for that of bmo) in the supernatant of amf and bmo after h treatment with lps. however, no signi®cant differences in both viral antigen expression and virus titres were found between untreated and the directly or indirectly lps treated amf (table ) . no virus replication was detected in the bmo directly or indirectly treated with lps (data not shown). no virus replication was detected in the bmo treated with pma. when amf were pre-treated with pma for a short time ( to h), they became resistant to prrsv infection (fig. ) . after h continuous exposure to pma, amf gradually regained their susceptibility to prrsv and after h continuous pre-treatment, virus production reached the level similar to that of untreated cultures. the effect of a h treatment of amf with pma at different time points after a prrsv inoculation on the virus replication is shown in fig. . simultaneous virus inoculation and pma treatment for h reduced the virus production to a level of . tcid per cells. replication of prrsv was completely blocked when amf had been inoculated for h before pma treatment. the longer the interval between inoculation and treatment, the higher the virus titre. the blocking effect was concentration dependent as shown in fig. . concentrations as low as  À mg/ml pma completely inhibited prrsv infection. the membrane reactivity of bmo, amf and pmf to biotinylated prrsv and anti-amf monoclonal antibodies d and d as shown in fig. , the binding of biotinylated prrsv to bmo, amf and pmf was demonstrated by¯ow cytometry with a signi®cantly higher uorescence intensity in amf. the binding of biotinylated prrsv to the amf was not affected by pma treatment. after staining with anti-porcine amf monoclonal antibodies d and d , the membrane¯uorescence was detected only on amf and not on pbmc, bmo, and pmf (fig. ) . the results of this study show that only some subsets of cells from the porcine monocyte/macrophage lineage are susceptible to prrsv and that the speci®c differentiation and activation state may considerably affect their susceptibility to prrsv infection in vitro. cells of the porcine monocyte/macrophage lineage from different anatomic locations are clearly heterogeneous in their permissiveness for prrsv infection. prrsv replication was detected in porcine amf, while freshly isolated bmo and pmf were completely resistant. these results are in agreement with earlier``in vivo'' experiments, in which the replication of prrsv was found in alveolar macrophages but not in peripheral blood mononuclear cells [ ] . in the present study, very low virus titres and few infected cells were detected in cultivated bmo. this is in contrast with the observations of voicu et al. [ ] , who found much higher virus titres in cultivated bmo. this variation may be due to differences in the genotype/phenotype of the donor animals, differences in prrsv isolates and differences in techniques for the isolation of bmo. genetic, antigenic and pathogenic variations among prrsv isolates in the usa and europe have been reported [ , ] . even with amf and cl , two commonly used cell systems for prrsv isolation, nearly one third of american prrsv isolates grown in one cell type failed to grow in the other one [ ] . the experiments also revealed that amf show some restriction to a prrsv infection when they were freshly isolated, even though they are relatively the most sensitive cell type for prrsv isolation [ , ] . the susceptibility clearly increases after one day cultivation. a similar increase of permissiveness to prrsv infection was observed in a very small number of cultivated bmo. these results suggest that the state of monocyte/macrophage differentiation plays an important role in determining their susceptibilty to prrsv. similar enhancing effects of differentiation on virus replication have been observed with other viruses such as the respiratory syncytial virus (rsv), parain¯uenza virus- , african swine fever virus, cytomegalovirus or herpes simplex virus (hsv), human immunode®ciency virus type (hiv- ), visna-maedi virus and pseudorabies virus in monocytes/macrophages [ , ] . since bmo are precursors of tissue macrophages and readily mature into macrophages when cultivated in vitro [ ] , it is logical to predict that some bmo may mature into susceptible macrophages similar to alveolar macrophages by cultivation in vitro. resident pmf, which represent another population of well-differentiated tissue macrophages from a restricted lineage, were completely resistant to a prrsv infection. in this study, treatment of amf with pma was associated with a clear reduction of the replication of prrsv and the blocking effect of pma was transitory. when amf were continuously pre-treated for to h, the virus replication was almost completely blocked. the cells regained full susceptibility after h of continuous treatment. this rather peculiar ®nding can be explained by the fact that, while the activating capacity of monocytes and macrophages by pma reaches a maximum after h exposure, a prolonged exposure to pma for more than h causes macrophages and monocytes to become refractory to pma stimulation [ ] . when monocyte/macrophage lineage cells are treated with pma, both enhanced or decreased viral replication has been observed with other viruses. for example, an increased virus replication with herpes simplex virus has been noticed upon cell differentiation [ ] whereas a down-regulation of viral replication was observed with feline immunode®ciency virus [ ] . the mechanism through which pma inhibits prrsv infection of amf is unclear. in contrast to the pma treatment, lps treatment of porcine amf and bmo did not in¯uence the susceptibility to prrsv infection. the biological properties of pma are generally considered to be due to the activation of protein kinase c, an enzyme involved in many cellular responses. although the activation of protein kinase c is also an essential process for lps induced activation of the macrophage, the signal pathways used by pma and lps in macrophages are quite different [ ] . therefore, it seems that pma modulated inhibition of prrsv infection might be mediated by a speci®c pma-signalling pathway in porcine amf. recent observations suggest that prrsv enters amf through a process of receptor-mediated endocytosis (unpubl. data). pma treatment does not change the binding activity of prrsv and monoclonal antibody against the putative prrsv receptor(s) to amf. therefore, the blocking effect of pma does not act through down-regulating the expression of the virus receptor(s) on the cells. mononuclear phagocyte differentiation is a process of normal cellular development and homeostasis and re¯ects a permanently altered expression of a cell's genetic potential, while macrophage activation is the process that causes reversible changes in macrophage phenotype and functions [ ] . the differentiation may induce some factors that are essential for virus replication, such as receptors or transcription factors, while activation may up-or downregulate the expression of these factors in cells. inef®cient binding of biotinylated prrsv to pmf, bmo and pbmc suggests that these cells lack the more speci®c binding sites that are expressed on the membrane of amf. different binding activity of mab d and d , which block prrsv infection of amf and recognise a candidate receptor on the cell, to different monocytes/macrophages provide further evidence that the restriction of prrsv replication in some subsets of monocytes/macrophages is due to a reduced expression of virus receptor(s). comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome 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and europe diagnosis of porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav in vitro induction of cytologic functional differentiation of the immature human monocyte cell line u- with phorbol myristate acetate replication of virulent aujeszky's disease virus (adv) in different blood mononuclear cell subpopulations virus production and viral antigen expression in porcine blood monocytes inoculated with pseudorabies virus effect of speci®c antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types preparation and characterization of monoclonal antibodies reactive with porcine pbl lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses pathological, ultrastructural, and immunohistochemical changes by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (pears)) chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs mechanism generating functionally heterogeneous macrophages: chaos revisited lps induces selective translocation of protein kinase c-beta in lps-responsive mouse macrophages, but not in lps-nonresponsive mouse macrophages interaction of porcine reproductive and respiratory syndrome virus with swine monocytes mystery swine disease in the netherlands: the isolation of lelystad virus antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mechanism of intrinsic macrophage-virus interaction``in vitro isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome we thank c. bracke for technical assistance. we are grateful to dr. g. wensvoort for the supply of the lelystad isolate of prrsv and dr. t. w. drew for the gift of monoclonal antibodies wbe and wbe ± . received january , key: cord- -q ogrem authors: barthold, s. w.; smith, abigail l. title: viremic dissemination of mouse hepatitis virus-jhm following intranasal inoculation of mice date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: q ogrem using a sensitive infant mouse bioassay to detect infectious virus, the pattern of mouse hepatitis virus (mhv) jhm dissemination in blood and other tissues was examined during the first days following intranasal inoculation. mhv replicated in nasal turbinates of both susceptible balb and resistant sjl mice from days through , but balb mice had higher titers on days and . viremia was detectable on days through in balb mice, but only on days and in sjl mice. transient virus replication occurred in the lungs of both mouse genotypes at and days, then ceased. this correlated with more consistently demonstrable virus in blood collected from the left atrium of the heart, compared to jugular vein, portal vein and right atrial blood. virus was associated equally with the plasma and cellular fractions of blood on day , but was primarily in the buffy coat of the cellular fraction on day . interferon-α/β was detected in serum and spleen, but not liver or brain of balb mice or in any tissue of sjl mice. balb serum and spleen interferon was first detected at h, peaked between and h, and was undetectable by h. the distribution of virus in nose, cervical, axillary and mesenteric lymph nodes, spleen, peyer's patch, thymus, bone marrow and liver was examined at , , and days. the resulting pattern suggested lymphatic spread of virus to cervical lymph node and mesenteric lymph node as pathways of dissemination in addition to viremia. mouse hepatitis virus (mhv) is a highly contagious and prevalent coronavirus of laboratory mice, with numerous related strains that partially differ antigenically, genetically and biologically [ , ] . like coronaviruses of other species, mhv strains display primary tropism for upper respiratory or enteric mucosa [ , ] . in susceptible mice inoculated with respiratory-type mhv strains by the s.w. barthold and abigail l. smith intranasal (i.n.) route, virus spreads by direct extension from the nose to the brain [ , ] and to other target organs such as liver and lymphoid tissue by a presumed viremic course. viremia is suspected, since lesions and antigen are distributed in a vascular pattern [ , , , ] . mhv viremia has been very difficult to demonstrate, since cell culture systems are relatively insensitive for detection of infectious virus in tissues. despite evidence that mhv disseminates in a vascular distribution and infects blood-associated tissues such as bone marrow, lyrnphoid organs and vascular endothelium, viremia following i.n. mhv inoculation of immunocompetent mice has only recently been demonstrated by using an infant mouse infectivity assay. under these circumstances, infectious virus could be detected in blood as early as h after i.n. inoculation [ ] . the purpose of the present study was to examine the early kinetics of viremia as the mechanism of mhv dissemination in genetically susceptible and resistant mice inoculated with a moderately virulent, respiratory-type strain of mhv. the sequential appearance of mhv in respiratory tissues and blood was initially examined in genetically susceptible balb/cbyj (balb) and resistant sjl/j (sjl) mice following i.n. inoculation with moderately virulent mhv strain jhm. previous studies have shown that both of these mouse strains develop disseminated infections between days and after i.n. inoculation, but virus titers and disease are significantly greater in balb mice compared to sjl mice [ ] . groups of randomly selected mice of each genotype were killed on days , , , , and after inoculation. infectious virus in nasal turbinates and lung on clays , , , and , and blood on days , , , , and was titrated. sera were tested for detectable mhv antibody. to determine the relative rate of infection and mhv titers in each blood compartment, sets of blood samples were obtained from jugular vein, portal vein, left cardiac atrium and right cardiac atrium in a group of balb mice at h after i.n. inoculation. viremic blood samples from additional balb mice on days and were pooled and differentially separated into cellular (erythrocyte and buffy coat) and plasma fractions. mhv titers were determined in whole blood, cellular and plasma fractions. the association of interferon-a/~ with viremia was explored initially by analyzing pooled samples of nasal turbinate, blood, liver, brain, or spleen obtained from mice of each genotype on days (controls), , , , and after i.n. inoculation. based on findings from this experiment, interferon was assayed in individual samples of serum and spleen from balb mice per interval at , , , , , , , , and h after i.n. inoculation. finally, as another means of examining the routes of mhv dissemination, the sequential appearance of mhv in nose, cervical lymph nodes, axillary lymph nodes, ruesenteric lymph nodes, spleen, peyer's patches, thymus, bone marrow, and liver was examined in groups of balb mice at , , and clays after i.n. inoculation. certified virus-free, - week old balb and sjl mice were purchased from the jackson laboratory, bar harbor, me and pregnant outbred cri:cd br (cd ) mice were purchased from charles river laboratories, portage, mi, shipped in filtered boxes, then transferred upon arrival into autoclaved micro-isolator containment cages (lab products, mouse hepatitis virus viremia maywood, nj) containing wood shavings, food (prolab animal diet, agway, syracuse, ny) and water. cages and mice were manipulated within a class ii biological containment cabinet to preclude inadvertant exposure to adventitious murine viruses. randomly selected mice were killed with carbon dioxide gas at specific intervals, and tissues were collected aseptically and frozen at - °c until tested for virus. nasal turbinates were collected with forceps after removal of the dorsal nasal bones with a razor blade. blood was collected in non-heparinized glass syringes and placed in vials containing edta as an anticoagulant. peyer's patches consisted of intestinal mucosa containing gut associated lymphoid tissue. blood was fractionated by centrifugation. plasma was drawn off from the cellular fraction in separate aliquots. in additional samples, buffy coat was removed from erythrocytes, then erythrocytes were washed and centrifuged twice in saline: virus mhv-jhm was obtained and maintained as previously described [ ] . mice were inoculated i.n. with gl of cell-free culture fluid containing approximately ~ tcids of mhv-jhm. virus was detected in tissues by intracerebral inoculation of neonatal cd mice with % (w/v) tissue homogenates or whole blood in . ml volume. virus titers were determined by similar inoculation of infant mice with serial -fold dilutions of tissue homogenates, and expressed as log lds /g, as previously described [ ] . interferon-a/ was assayed in tissues by a cytopathic effect reduction assay, using mouse l cell monolayers challenged with median infectious doses of vesicular stomatitis virus (indiana serotype), as previously described [ , ] . international units (iu) were determined with reference to a niaid, who international reference standard (g - - ). test tissues were triturated as % (w/v) homogenates, acidified to ph . overnight and neutralized prior to assay. antibody to mhv-jhm was determined by enzyme immunoassay, using formalin-fixed mhv-jhm-infected c - cells as antigen and horseradish peroxidase-conjugated goat anti-mouse igg (biorad, richmond, ca) as described [ ] . differences in proportions were determind by ~ analysis and virus titers were compared with student's paired or unpaired t tests. mhv was detected in nasal turbinates of both balb and sjl mice during the first days after i.n. inoculation, but significantly higher titers were found in balb mice compared to sjl mice on days (p ~< . ) and (p ~< . ) (fig. t) . viremia was detectable in balb mice on days , , , , and , but only on days and in sjl mice (fig. ) . remarkably, mhv titers in blood on days and were equivalent between genotypes. mhv was detected in of balb (table ) . mhv lung titers at days were significantly higher (p ~< . ) in balb compared to sjl mice. viremia was most consistently detected in blood samples taken from the left atrium compared to jugular vein, portal vein and right atrium at days after i.n. inoculation (table ). cellular and plasma fractions of balb blood contained equivalent titers of mhv on day , but titers in plasma dropped significantly on day relative to day (p ~< . ) ( table ) . on day , the cellular fraction contained significantly more virus as the plasma (p ~< . ). in of the blood samples tested on day , virus could only be detected in the cellular table . mhv-jhm titers (log intracerebral lds /g) and rate of lung infection at intervais after intranasal inoculation of balb and sjl mice genotype interval after inoculation (days) interferon-a/ was detected in pooled serum and spleen, but not nasal turbinate, liver or brain, of balb mice and was not detected in any tissues of sjl mice. levels in serum and spleen of balb mice peaked at the day interval. based on these findings, interferon levels were assayed in serum and spleen of individual balb mice (table ). interferon titers were much higher in spleen compared to blood. in both tissues, interferon was first detectable at h and peaked between and h. none was detectable by h. mhv antibody was not detectable in sera of balb or sjl mice through day . at day after i.n. inoculation, infectious virus was detectable in nasal turbinate of all mice, in cervical lymph node in of mice, in spleen and peyer's patch of a few others, but not other sites (table ) . thus, cervical lymph node sustained significantly higher rate of early infection compared to more distant axillary lymph node (p <~ . ), suggesting lymphatic spread of virus. by days, mhv was present in nose, cervical lymph node, mesenteric lymph node and spleen of all mice; axillary lymph node, peyer's patch and liver of over half of the mice; bone marrow of a few mice and not in thymus. both cervical lymph node and mesenteric lymph node had higher rates of infection compared to axillary a number positive/number tested lymph node (p ~< . ), suggesting lymphatic spread through both the head and gut. by days, most tissues were infected in high prevalence, except thymus, which was positive in only of mice. hepatitis and inflammation of other internal organs are important features of respiratory mhv infection in laboratory mice and are responsible for the name "hepatitis virus" that has been given to this group of murine agents. we now know that some mhv strains are strictly entertropic and seldom cause hepatitis regardless of the age or immune status of the host, while others, which replicate initially in nasal mucosa, can cause hepatitis if the virus is sufficiently virulent or the host is susceptible [- ]. the polytropic, generalized nature of disease caused by these latter mhv strains in susceptible mice following i.n. inoculation is suggestive of viremic dissemination, but demonstration of mhv viremia has been inconsistent and usually following artificial routes of inoculation of immune-impaired mice with highly virulent strains of mhv. viremia was detected with c - cell culture in c bl/ mice on day and after intracerebral (i.c.) inoculation with mhv-a , but not following intraperitoneal (i.p.), i.n., or intragastric inoculation [- ] . viremia was demonstrable with dbt cell culture in athymic balb mice on day after i.n. inoculation with wild-type mhv [- ] . swiss mice inoculated i.n. with mhv-s at days of age had viremia detected in dbt cells on day , but not days , , or . viremia could not be detected in older mice [ ] . much greater success was achieved in detecting and quantifying viremia as early as h and through days after i.p. inoculation of ddd and cdf mice with virulent mhv- , using dbt cell culture [ ] . highly virulent mhv- could be detected and quantified in the serum of day old a mice on days , , and after i.p. inoculation, using a mouse infectivity bioassay [ ] . likewise, virus titers in blood were monitored at several intervals up to hours after intravenous inoculation of adult swiss mice with mhv- , using a mouse bioassay [ ] . on the other hand, we and others [ , ] have failed to detect viremia with cell culture assays following i.n. inoculation of adult mice with mhv-jhm. these inconsistencies are no doubt due to difficulties of growing nonadapted mhv in cell culture systems and their relative insensitivity for detecting infectious virus. we have found that the most sensitive means of detecting infectious mhv in tissues is the infant mouse bioassay and we have previously shown viremia following i.n. inoculation of young adult, immunocompetent mice with moderately virulent mhv [ using the infant mouse bioassay that was employed in the current study. the current results suggest that viremia is a very early event following i.n. inoculation, and can be detected within h. furthermore, lung appears to be an important early target for virus replication, and probably contributes to secondary viremia. mhv has been shown to replicate in pulmonary capillary endothelium and interstitium, with minimal infection of airway epithelium [ , , ] . virus titers in cardiac blood samples were highest in the left atrial samples, compared to the right atrial, jugular or portal samples, supporting the pulmonary origin of some of the virus in the circulating blood. fractionation studies of cardiac blood samples demonstrated infectious virus in both the plasma and cellular fractions on day , but virus had largely cleared from plasma by day , when it was associated with the buffy coat cells (leukocytes). mhv has a well known tropism for lymphoid tissue, as well as hematopoietic elements in bone marrow and spleen. the mechanism of plasma clearance by day was not determined, but it was preceded by a drop in virus titer on day , which was apparent in both balb and sjl mice. this biphasic pattern would suggest the influence of antibody or interferon, but antibody was not detected and serum interferon was present at low concentrations only in balb mice. in a previous study, antibody was detectable on day , but not on day , in balb mice and only of sjl mice tested on day had detectable antibody [ ] . the sequential distribution of mhv in parenchymal tissues suggests that virus dissemination also occurs through lymphatic drainage of the head, since cervical lymph nodes became infected earlier than other lymphoid tissues. this is not a significant event in mhv dissemination, since generalized involvement of virtually all target tissues has taken place by days. the higher rate of mesenteric lymph node infection relative to axillary lymph node at days suggests enteric entry as well. mhv-jhm, like most respiratory mhv strains, does not replicate to a very great extent in intestinal mucosa, but has a marked tropism for gut associated lymphoid tissue (galt) [ ] . the current study suggests early infection of galt from the intestinal lumen, then mesenteric lymph nodes following i.n. inoculation. as previously demonstrated, this study confirms that lymphoid tissue in general is an important target for mhv. involvement of the thymus in mhv-jhm infection has been reported in mice inoculated intracerebrally [ ] , but appears to be relatively rare following i.n. inoculation. interferon has been detected in serum, liver, spleen, and peritoneal exudate cells of mice infected with various mhv strains [ , , , , ] . correlation of interferon levels with susceptibility has been variable. serum interferon concentrations were low in mhv-s infected, immature mice and elevated in adult mice [ ] . others [ , ] have shown that resistant mouse genotypes (ddd, a/j) produce less serum and tissue interferon than susceptible genotypes (cdf , c bl) when infected with mhv- or mhv- . no differences in serum interferon concentrations were detected between susceptible (c h, balb) and resistant (ddd, cf ) mice infected with mhv- or mhv-jhm [ , ] , although peak interferon titers occurred later in susceptible balb compared to resistant cf mice [ ] . in the current study, interferon was found in spleen and serum of mhv-jhm-infected balb mice, but not nose, liver or brain and was not detectable in any tissue of sjl mice. since all of these tissues were infected in both genotypes, as demonstrated previously [ ] , interferon had no correlation with presence, clearance or absence of infectious mhv. these data, and those of others, indicate that serum or tissue interferon responses of mice to mhv vary greatly with mouse genotype. furthermore, mhv strains vary in their sensitivity to interferon in vitro [- , ] . sjl mice are well known to possess remarkable resistance to mhv disease. they have been shown to lack a functional cellular receptor for mhv-a in at least some target tissues [ , ] . we have previously demonstrated that sjl mice develop very mild infections of liver, spleen and lymphoid tissue compared to balb mice, when inoculated by the i.n, route [ ] . the current study demonstrates that sjl mice support virus replication in their nasal turbinates and also develop a demonstrable viremia, albeit intermittent. thus, their resistance to mhv-jhm-induced disease is not a reflection of their absolute resistance to infection or ability to support virus replication. mouse hepatitis virus biology and epizootiology olfactory neural pathway in mouse hepatitis virus nasoencephalitis susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species mouse hepatitis virus strain-related patterns of tissue tropism in suckling mice response of genetically susceptible and resistant mice to intranasat inoculation with mouse hepatitis viurs jhm genetic resistance of mhv correlates with absence of virus-binding activity on target tissues pulmonary vascular lesions in nude mice persistently infected with mouse hepatitis virus responses of mice susceptible or resistant to lethal infection with mouse hepatitis virus, strain jhm, after exposure by a natural route nasoencephalopathy of mice infected intranasally with a mouse hepatitis virus, jhm strain infection and involution of mouse thymus by mhv- effects of cortisone on interferon response of germfree mice to mouse hepatitis virus mouse hepatitis virus viremia mhv-a pathogenesis in mice immunopathology of mouse hepatitis virus type infection. i. role of humoral and cell-mediated immunity in resistance mechanisms the fate of murine hepatitis virus (mhv- ) after intravenous injection into susceptible mice convenient assay for interferons activation of natural killer cells and induction of interferon after injection of mouse hepatitis virus type in mice two enzyme immunoassays for the detection of antibody to rodent coronoaviruses pathogenesis of mouse hepatitis virus infection. the role of nasal epithelial cells as a primary target of lowvirulence virus difference in response to mouse hepatitis virus among susceptible mouse strains difference in sensitivity to interferon among mouse hepatitis viruses with high and low virulence for mice factors involved in the age-dependent resistance of mice infected with low virulence mouse hepatitis virus the role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (mhv- ) the biology and pathogenesis of coronaviruses purification of the -kilodalton glycoprotein receptor for mhv (mhv)-a from mouse liver and identification of a nonfunctional, homologous protein in mhv-resistant sjl/j mice t-lymphocyte.dependent difference in susceptibility between ddd and c h mice to mouse hepatitis virus, mhv- this work was supported by grant rr from the national center for research resources, national institutes of health, bethesda, maryland. the assistance of d. s. beck and d. winograd is gratefully acknowledged. received april , key: cord- -vgq gjpx authors: hou, yuxuan; peng, cheng; yu, meng; li, yan; han, zhenggang; li, fang; wang, lin-fa; shi, zhengli title: angiotensin-converting enzyme (ace ) proteins of different bat species confer variable susceptibility to sars-cov entry date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vgq gjpx the discovery of sars-like coronavirus in bats suggests that bats could be the natural reservoir of sars-cov. however, previous studies indicated the angiotensin-converting enzyme (ace ) protein, a known sars-cov receptor, from a horseshoe bat was unable to act as a functional receptor for sars-cov. here, we extended our previous study to ace molecules from seven additional bat species and tested their interactions with human sars-cov spike protein using both hiv-based pseudotype and live sars-cov infection assays. the results show that ace s of myotis daubentoni and rhinolophus sinicus support viral entry mediated by the sars-cov s protein, albeit with different efficiency in comparison to that of the human ace . further, the alteration of several key residues either decreased or enhanced bat ace receptor efficiency, as predicted from a structural modeling study of the different bat ace molecules. these data suggest that m. daubentoni and r. sinicus are likely to be susceptible to sars-cov and may be candidates as the natural host of the sars-cov progenitor viruses. furthermore, our current study also demonstrates that the genetic diversity of ace among bats is greater than that observed among known sars-cov susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of sars-cov or its progenitor viruses. this calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of sars-cov. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. severe acute respiratory syndrome coronavirus (sars-cov) is the aetiological agent responsible for the sars outbreaks during - , which had a huge global impact on public health, travel and the world economy [ , ] . the host range of sars-cov is largely determined by the specific and high-affinity interactions between a defined receptor-binding domain (rbd) on the sars-cov spike protein and its host receptor, angiontensin-converting enzyme (ace ) [ , , ] . it has been hypothesized that sars-cov was harbored in its natural reservoir, bats, and was transmitted directly or indirectly from bats to palm civets and then to humans [ ] . however, although the genetically related sars-like coronavirus (sl-cov) has been identified in horseshoe bats of the genus rhinolophus [ , , , ] , its spike protein was not able to use the human ace (hace ) protein as a receptor [ ] . close examination of the crystal structure of human sars-cov rbd complexed with hace suggests that truncations in the receptor-binding motif (rbm) region of sl-cov spike protein abolish its hace -binding ability [ , ] , and hence the sl-cov found recently in horseshoe bats is unlikely to be the direct ancestor of human sars-cov. also, it has been shown that the human sars-cov spike protein and its closely related civet sars-cov spike protein were not able to use a horseshoe bat (r. pearsoni) ace as a receptor [ ] , highlighting a critical missing link in the bat-to-civet/human transmission chain of sars-cov. there are at least three plausible scenarios to explain the origin of sars-cov. first, some unknown intermediate hosts were responsible for the adaptation and transmission of sars-cov from bats to civets or humans. this is the most popular theory of sars-cov transmission at the present time [ ] . second, there is an sl-cov with a very close relationship to the outbreak sars-cov strains in a non-bat animal host that is capable of direct transmission from reservoir host to human or civet. third, ace from yet to be identified bat species may function as an efficient receptor, and these bats could be the direct reservoir of human or civet sars-cov. unraveling which scenario is most likely to have occurred during the - sars epidemic is critical for our understanding of the dynamics of the outbreak and will play a key role in helping us to prevent future outbreaks. to this end, we have extended our studies to include ace molecules from different bat species and examined their interaction with the human sars-cov spike protein. our results show that there is great genetic diversity among bat ace molecules, especially at the key residues known to be important for interacting with the viral spike protein, and that ace s of myotis daubentoni and rhinolophus sinicus from hubei province can support viral entry. hela cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (gibco, usa). rabbit polyclonal antibodies against ace of r. pearsoni (rpace ) were generated using r. pearsoni ace protein expressed in escherichia coli at the wuhan institute of virology following standard procedures. bats were sampled from their natural habitats in hubei, guangxi, guizhou, henan and yunnan provinces in china as described previously [ ] . bat identification was initially determined in the field by morphology and later confirmed in the laboratory by sequencing the mitochondrial cytochrome b gene from samples of blood cells or rectal tissue as described previously [ ] . total rna was extracted from bat rectal tissue using trizol reagent (invitrogen, usa) and treating with rnase-free dnase i at °c for min. first-strand cdna was synthesized from total rna by reverse transcription with random hexamers. full-length bat ace fragments were amplified using the forward primer baf ( -cttgg taccatgtcaggctcttyctgg- ) and the reverse primer bar ( -ccgctcgagctaaaab[g/t/c]ga v[g/a/c]gtctgaacatcatc- ). the pcr mixture ( ll) contained . ll cdna, . mm mgcl and . lm of each primer, and the fragments were amplified using the following parameters: °c for min, cycles of °c for s, °c for s and °c for min, with a final elongation step at °c for min. all bat ace s were cloned into pcdna . with kpni and xhoi, and this was verified by sequencing. for samples in which full-length ace amplification was unsuccessful, the n-terminal region ( - , bp) was amplified using the forward primer baf and the reverse primer rmr ( -ttagctccatttcttagcaggtag g- ). chimeric ace was constructed by combining the n-terminal region of bat ace with the c-terminal portion of human ace at the unique bamhi site ( , - , bp). the chimera was subsequently cloned into pcdna . with kpni and xhoi and sequenced as above. construction of bat ace mutants ace from m. daubentoni was chosen to generate a series of ace mutants using a quikchange ii site-directed mutagenesis kit (stratagene, usa). the altered amino acid codon for each mutant is indicated as follows: i t, n k, k e, and h y. mutants were confirmed by sequencing. all bat ace s were submitted to genbank (ef , gq -gq ). sequence alignment was performed using clustalx version . [ ] and corrected manually. a phylogenetic tree based on amino acid (aa) sequences was constructed using the neighbor-joining (nj) method in mega version . . [ ] . lysates of hela cells expressing human ace or bat ace were separated on a - % sds-page gel, followed by transfer to a polyvinylidene difluoride (pvdf) membrane using a semi-dry protein transfer apparatus (bio-rad, usa). the membrane was probed with a rabbit polyclonal antibody against the bat ace protein ( : ) at room temperature for h, followed by incubation with alkaline-phosphatase-conjugated goat anti-rabbit igg ( : , ) (chemicon, australia). the probed proteins were visualized using nbt and bcip color development (promega, usa). an hiv- -luciferase pseudotype virus carrying the sars-cov bj s protein, hiv/bj -s, was prepared as described previously [ ] . hela cells were seeded onto -well plates for h and then transfected with . lg recombinant plasmid containing bat or human ace using . ll lipofectamine (invitrogen, usa) according to the manufacturer's protocol. at h post-transfection, ll medium containing hiv/bj -s was added to each well. at - h postinfection, unadsorbed viruses were removed, and fresh medium was added. the infection was monitored by measuring luciferase activity, expressed from the reporter gene carried by the pseudovirus, using a luciferase assay kit (promega, usa). cells were lysed at h postinfection by adding ll lysis buffer provided with the kit, and ll of the resulting lysates were tested for luciferase activity by the addition of ll luciferase substrate in a turner designs td- / luminometer. each infection experiment was conducted in triplicate, and all experiments were repeated three times. live sars-cov infection was carried out under bsl conditions at the australian animal health laboratory (aahl) as described previously [ , ] . briefly, h after transfection, the time at which expression of the ace receptor on the hela cell surface is optimal, tcid of virus was added to the cells for infection. the cells were fixed h later by treatment with % methanol for min and washed five times with pbst. the primary antibody, chicken anti-sars-cov s (produced against the recombinant s protein expressed in e. coli at aahl), at a : dilution in % bsa/pbs, was added and incubated with the cells for h at room temperature. an fitc anti-chicken conjugate (chemicon, australia) at : , in % bsa/pbs was added after washing the cells five times and incubated with the cells for h. infection was monitored by immunofluorescent microscopic analysis. cloning and expression of ace genes from different bat species ace genes from seven bat species were amplified and cloned (fig. , sfig. ) . full-length genes were obtained from rhinolophus ferrumequinum from hubei province (rf-hb), r. macrotis from hubei province (rm-hb), r. pearsoni from guangxi (rp-gx), r. pusillus from hubei province (rpu-hb), r. sinicus from guangxi province (rs-gx) and r. sinicus from hubei province (rs-hb). for the following bat species, amplification of the full-length coding region was not successful, and instead,the n-terminal region was cloned in frame with the c-terminal region of the human ace gene to form a chimeric fulllength ace molecule: r. pearsoni from guizhou province (rp-gz), myotis daubentonii bat from yunnan province (md-yn) and hipposideros pratti bat from henan province (hp-hn) . the full-length sequences of bat ace are identical in size to that of hace ( aa in total). sequence comparison showed that bat ace s are closely related to ace s of other mammals and have an aa sequence identity of - % to human and civet ace . the aa identity of ace from different bat families ranges from to %, and within the genus rhinolophus, the sequence identity increases to - %. the major sequence variation among bat ace s is located in the n-terminal region, which has been identified in structural studies as the sars-cov-binding region [ , ] . a phylogenetic tree was constructed based on the sequences of bat ace (sfig. ) using the mega package [ ] . all ace genes were cloned into a eukaryotic expression vector and used to transfect hela cells. western blot analysis showed that all ace s were expressed efficiently and at very similar levels and were recognized by a rabbit anti-bat ace antibody with an apparent molecular weight of approximately - kda (fig. c) . to examine the susceptibility of different bat ace molecules to sars-cov entry, the hiv/bj -s pseudovirus system was used to infect hela cells transiently expressing bat ace or human ace genes. among the bat ace s, only mdace (mdace ) and rs-hb ace demonstrated significant pseudovirus infection, as deduced from the significantly higher level of luciferase activity in comparison to background activity in the negative control (fig. a) . although such assays are not to be viewed as an absolute quantification of receptor activity, it is nevertheless worth noting that mdace -mediated infection seemed to be more efficient than with rs-hb ace . in the same context, it is clear that the bat ace s were less efficient overall than the human ace in this particular assay system. the biological significance of this observation remains to be determined. the functionality of mdace and rs-hb ace as sars-cov entry receptors was further confirmed by infection with live virus. as shown in fig. b , both bat ace proteins could clearly support sars-cov infection. no attempt was made to quantify infection efficiency in this study due to difficulties encountered in conducting experiments under bsl conditions. homologous structural modeling of human sars-cov rbd complexed with mdace supports mdace as a receptor for human sars-cov s protein. the crystal structure of human sars-cov rbd complexed with hace shows that two salt bridges at the sars-cov-hace interface, between hace lys and glu and between hace lys and hace glu , are both buried in a hydrophobic environment and contribute critically to the sars-cov-hace interactions (fig. a , c) [ ] . disturbance of the formation of either of these salt bridges weakens sars-cov-hace binding. the lys -glu salt bridge at the sars-cov-hace interface becomes an asn -lys hydrogen bond at the sars-cov-md-ynace interface (fig. b) , which possibly weakens virus-receptor binding but still is largely compatible with the virus-receptor interface. thr on hace supports the lys -gu salt bridge through hydrophobic interactions with tyr (fig. a) ; ile on mdace supports the asn -lys hydrogen bond more efficiently than thr through tighter hydrophobic interactions with tyr (fig. b) . moreover, tyr on hace supports the lys -glu salt bridge (fig. c) ; his on mdace supports the same salt bridge less efficiently than tyr (fig. d) . overall, mdace is an efficient receptor for sars-cov, despite the fact that its receptor activity is lower than that of hace . compared with mdace , rs-hb ace contains glu and glu , which are not compatible with each other due to their same negative charges, which disfavor . the alignment was generated using clustalx v . . in black are single, fully conserved residues. in gray are strongly conserved residues. in light gray are weakly conserved residues. asterisks indicate residues that interact directly with the receptor-binding domain of the sars-cov s protein virus-receptor binding. however, rs-hb ace also contains thr and tyr , both of which support sars-cov entry by contributing favorably to the hydrophobic interactions at the virus-receptor interface. thus, rs-hb is a low-efficiency receptor for sars-cov. all of the other bat ace molecules contain combinations of the aforementioned key residues that are completely incompatible with virus-receptor interactions. more specifically, they either contain same-charged residues at the and positions, which repel each other, or contain his and lys , which disfavor sars-cov binding (fig. ) . in particular, lys on some of these bat ace molecules is incompatible with certain hydrophobic residues, such as leu and phe , on sars-cov rbd (fig. a, b) . therefore, these bat ace molecules are not receptors for sars-cov. to confirm the above homologous structural analysis, we carried out site-directed mutagenesis on mdace . our results show that mutations e k, k e, and i t all dramatically decrease the receptor activity of mdace , whereas mutation h y greatly increases its receptor activity (fig. a) . therefore, our mutagenesis data further confirmed that key residues in ace determine the receptor activity of mdace . our finding that m. daubentoni and r. sinicus could support sars-cov infection has important implications in relation to the origin of sars-cov. since all lines of investigation have indicated that ace -binding affinity is among the important determinants for sars-cov host range, our data would suggest that m. daubentonii and r. sinicus have the potential to serve as the direct reservoirs for human sars-cov or its highly related civet sars-cov. to further investigate the potential of m. daubentonii and r. sinicus as reservoirs for sars-cov, more efforts will have to be directed toward widening the surveillance of bats in these families and in different geographical locations. another important finding of our current study is the great genetic diversity of bat ace proteins, which is in contrast to the genetically homogenous hace [ ] . sequence variations of bat ace , especially in positions that are critical to sars-cov binding, such as residues , , , and , suggest that, in addition to the md-yn and rs-hb ace s, there may be many other bats with an ace protein that makes them susceptible to sars-cov entry. this again highlights the need for more field surveillance and molecular characterization of different bat ace proteins until the true reservoir host of sars-cov is identified and its spillover mechanisms and transmission pathways are fully characterized. interactions. a critical salt bridge between hace lys and glu and the hydrophobic residues surrounding it, based on the experimentally determined crystal structure of sars-cov rbd complexed with hace (pdb ajf). b homologous structural modeling of the hydrogen bond between mdace asn and lys . the modeling was done in the program o [ ] . c critical salt bridge between hace lys and glu and the hydrophobic residues surrounding it, based on the structure of sars-cov rbd complexed with hace . d homologous structural modeling of the salt bridge between mdaace lys and sars-cov glu and the hydrophobic residues surrounding it. structural illustrations were prepared using the program povscript [ ] evolutionary relationships between bat coronaviruses and their hosts povscript?: a program for model and data visualization using persistence of vision raytracing improved methods for building protein models in electron density maps and the location of errors in these models a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats structure of sars coronavirus spike receptor-binding domain complexed with receptor structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections bats are natural reservoirs of sars-like coronaviruses receptor and viral determinants of sars-coronavirus adaptation to human ace animal origins of the severe acute respiratory syndrome coronavirus: insight from ace -s-protein interactions coronavirus as a possible cause of severe acute respiratory syndrome full-length genome sequences of two sars-like coronaviruses in horseshoe bats and genetic variation analysis difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin mega : molecular evolutionary genetics analysis (mega) software version . the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools determination and application of immunodominant regions of sars coronavirus spike and nucleocapsid proteins recognized by sera from different animal species intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans bats as reservoir of sars-cov progenitor virus key: cord- -bjjfkfgg authors: mcelligott, susan; collins, p. j.; sleator, roy d.; martella, vito; decaro, nicola; buonavoglia, canio; o’shea, helen title: detection and genetic characterization of canine parvoviruses and coronaviruses in southern ireland date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: bjjfkfgg canine parvovirus (cpv) and canine coronavirus (ccov) are considered the main pathogens responsible for acute gastroenteritis in dogs. from a collection of samples, seven cpv strains and three ccov strains were identified in symptomatic irish dogs. samples were screened for the viruses using polymerase chain reaction (pcr) and typed via dna sequence analysis. three cpv strains were characterized as cpv- a, while four others were characterized as cpv- b. to date, cpv- c remains unreported in ireland. two ccov strains were characterized as ccov-ii and one as ccov-i. in the case of one sample, ph / /ire, a mixed infection with cpv and ccov was detected. viruses with a large genome of - kb in length. enteric canine coronavirus typically causes mild enteritis in dogs, with more severe clinical signs observed in young animals [ ] . the single-stranded genome of cpv makes the virus more susceptible to modifications, and error-prone replication can lead to significant dna sequence variation and greater potential for evolution of these viruses, when measured over defined periods, or during growth of cpv through serial passage in cell culture [ ] . it is presumed that cpv originated from feline panleukopenia virus via genetic mutations and evolution [ ] . shortly after its emergence, cpv- was entirely replaced by a new type, designated cpv- a, which possessed the ability to infect both cats and dogs [ ] . in the mid-eighties, another type, designated as cpv- b, began to emerge. cpv- b is distinguished from cpv- a by a single amino acid (aa) substitution, asparagine (asn) to aspartic acid (asp), at position of the major antigenic site of the vp capsid protein [ ] . in , a novel cpv type, called cpv- c, was detected in italy, and this is now progressively replacing other cpv types [ ] . cpv- c has since been detected throughout parts of europe, including spain [ ] , germany [ ] , portugal [ ] , the united kingdom [ ] , as well as korea [ ] , the united states [ ] , south america [ ] and vietnam [ ] . cpv- c is distinguishable from cpv- a or b by substitution of glutamic acid (glu) in place of asn or asp, respectively, at the th aa residue of the vp protein. therefore, due to the positioning of these aa substitutions in an antigenic site, it is possible to differentiate between types by employing monoclonal antibodies [ ] . alternatively, the nucleotide sequence required to substitute glu at aa position created a novel mboii restriction site (gaaga), which is unique to cpv- c, and therefore, cpv- c can be distinguished from cpv- a/ b by restriction enzyme analysis [ ] . infection with cpv- c has been reported to result in clinical symptoms similar to those exhibited by dogs infected with cpv- a and b [ ] . some studies reported milder symptoms in infected animals [ ] , while other reports found evidence of more severe clinical symptoms and higher rates of mortality, even in vaccinated dogs [ , , ] . ccov was first discovered as an enteropathogen in [ ] . enteric ccov exists in two closely related forms, the original form, which was isolated in the s, is named ccov-ii. a new ccov strain, which was identified in italy in , is designated as ccov-i. ccov-i and ii share a high nucleotide homology in the viral genome but are highly divergent in the spike protein gene [ ] . the strains are so named according to genetic similarities observed between ccov-i and ii, and feline coronavirus (fcov)-i and ii, respectively [ ] . as coronaviruses contain large rna genomes, they are highly susceptible to frequent mutation as a result of the high error rate of rna polymerase, which results in the accumulation of several base substitutions per round of replication [ ] . coronaviruses can accumulate small insertions and deletions in their genomes, which promotes their evolution [ ] . they also undergo a high rate of homologous rna recombination, mediated by a ''copy-choice'' mechanism based on sequence homology surrounding the recombination sites [ ] . as a result of all of these mechanisms, coronaviruses can mutate rapidly, leading to new genotypes (ccov-i), biotypes (pantropic ccov) and host variants (canine respiratory coronavirus). following the emergence of severe acute respiratory syndrome (sars) in humans, the continual epidemiological surveillance on covs has been reinforced, as they are considered to be potential agents of direct and indirect zoonoses [ ] . infection can occur with a single ccov strain, or both strains may be present simultaneously [ ] . ccov infection generally has a low mortality; however, a recently characterized strain detected in italy (cb/ ) resulted in a fatal disease as a consequence of systemic spread of the virus [ ] . deaths have also been caused by the synergistic effect produced when a mixed infection occurs with other canine enteric viruses. mixed infections have been reported between ccov and cpv- , canine distemper virus and canine adenovirus type i [ , , ] . mixed infections have also been reported between cpv- and calicivirus [ , ] . dual infection results in illnesses that are usually more severe than either virus can produce alone [ ] . synergistic mechanisms are common with other gastroenteritis viruses such as rotavirus. studies have demonstrated this effect with calves co-infected with group a rotavirus and group c rotavirus, or with group a rotavirus and escherichia coli [ , ] . the original cpv- strain is still employed in many commercial vaccines. however, this strain was entirely replaced by its variant types (ccov-i and ii) shortly after its emergence. there is concern that these current vaccines may fail to protect pups against the cpv- variants [ , ] . a plan to use current strains in vaccine formulations has led to a cpv- b-based vaccine being licensed in europe [ ] . currently, all canine vaccines used to protect against ccovs are based on ccov-ii. however, it has been shown that the level of cross-reactivity between ccov-i and ii is limited [ ] . recombinant ccovs have been reported that were derived from ccov-ii and transmissible gastroenteritis virus of swine (tgev), related in the n terminal domain of the s protein. antigenic differences have also been found between tgev-like ccovs and reference ccov strains [ ] . as a result of this viral evolution, the efficacy of prophylaxis programs in place to protect dogs against ccov challenges may need to be assessed. to date, there is no epidemiological information on the circulation of coronaviruses and parvoviruses in ireland. this study was undertaken to investigate the prevalence of these viruses in a wide spectrum of irish dogs, both with and without clinical symptoms, and to assess the efficacy of vaccination programs currently in place to protect against these viruses. a total of faecal samples were collected from both symptomatic and asymptomatic dogs in and (table a ). the samples were collected from dogs of all ages ( months to years) from a wide spectrum of groups, including the irish society for prevention of cruelty to animals (ispca), the guide dogs association, racing greyhounds, hunting hounds and domestic dogs presenting in veterinary practices, which represent the major groups of canines in ireland. total nucleic acids were extracted from the samples by a standard phenol-chloroform method with ethanol precipitation. the extracted nucleic acids were resuspended in ll of sterile depc-h o and stored at - °c prior to use. samples were screened for parvovirus 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. [ ] . the reaction was carried out on an mj researcher ptc- thermocycler (gmi inc, minnesota, usa). reaction conditions were as follows: °c for min, followed by cycles of °c for s, °c for min and °c for min, with a final extension at °c for min. positive and negative controls were included in the reaction. samples were subjected to screening for the presence of canine coronavirus (ccov) by rt-pcr amplification of the polymerase gene, using primers described by stephenson et al. [ ] and an enhanced avian reverse transcriptase kit (sigma-aldrich). reaction conditions were as follows: °c for min, and °c for min, followed by cycles of °c for min, °c for min and °c for min, with a final extension at °c for min. all samples that tested positive for the presence of the cov conserved polymerase gene were subjected to rt-pcr amplification of an n gene segment in order to specifically detect canine coronavirus. a segment of the s gene was amplified for application in sequence and phylogenetic analysis, using primers and reaction conditions as described by erles and brownlie [ ] . pcr products were run on a . % agarose gel following ethidium bromide staining and visualized using uv light transillumination. all samples that tested positive for the vp gene segment of cpv- via pcr amplification were subjected to rflp analysis of this pcr product. for restriction enzyme digests, ll of each pcr amplicon was digested with five units of the restriction enzyme mboii (fermentas, germany) and visualized on a . % agarose gel to determine the cleavage pattern of the nucleic acid. pcr amplicons were purified using a qiaquick pcr purification kit (qiagen ltd, west sussex, england) and sequenced using a commercial service (mwg-biotech, ebersberg, germany). nucleotide sequences were submitted to the genbank database, and their accession numbers are displayed in tables b and c. nucleotide and amino acid sequence alignment was performed using the clustalw application with bioedit sequence alignment editor [ ] . preliminary analysis was accomplished by comparison with sequences available in the database using the webbased program blast (http://www.ncbi.nlm.nih.gov/ blast). phylogenetic analysis was conducted using the mega program [ ] . two phylogenetic trees, based on the partial cpv vp ( figure ) and ccov s ( figure ) gene segments, were constructed by the maximum-parsimony ( replicates) and maximum -ikelihood ( replicates) method, respectively, supplying a statistical support with bootstrapping, using cpv- and ccov reference strains obtained from the genbank database, as displayed in tables and , respectively. the sequences of the vp gene segments analysed, in the cpv- viruses under study, were deposited in genbank under the following accession numbers: cg / /ire, gq ; ph / /ire, gq ; ph / /ire, gq ; ph / /ire, gq ; bc / /ire, gq ; bc / /ire, gu . the sequences of the s gene segments analysed, in the ccov viruses under study, were deposited in genbank under the following accession numbers: ph / /ire, gu ; avc / /ire, gu . following pcr amplification of a segment of the vp gene, a single band of -bp was observed in seven samples (table b) as well as the positive control when visualized on a . % agarose gel. samples from clinically healthy dogs were used as negative controls, and none of these produced an amplified product. out of the seven positive parvovirus samples, one sample (ph / /ire) also tested positive for the presence of ccov. a total of three out of samples (table c) tested positive for the presence of the cov polymerase gene. subsequently, the presence of canine-specific cov in these three samples was confirmed by the successful amplification of a -bp segment of the n gene. rflp analysis indicated that none of the cpv strains were variant type c, as the pcr amplicon was not digested and remained as a single band when run on an agarose gel. sequence analysis confirmed these findings. the n terminus of the s gene was amplified in all isolates using a set of three primers that were designed to differentiate between ccov-i and ccov-ii [ ] . amplification of the s gene of ccov-i produced a band of bp, whereas amplification of the s gene of ccov-ii produced a band of bp. strain ph / /ire was characterized as ccov-i, whereas strains avc / /ire and avc / /ire were characterized as ccov-ii. sequencing of the seven cpv strains revealed that three strains, ph / /ire, bc / /ire and bc / /ire, possessed the aa asn at position of the vp protein. therefore, these strains were characterized as cpv- a. strains cg / /ire, ph / /ire, ph / /ire and bc / /ire possessed the aa asp at position and were therefore characterized as cpv- b. in addition, the sequence of the s gene of the three ccov strains, ph / /ire, avc / /ire and avc / /ire, was obtained. however, the sequence of strain avc / /ire was not exploitable for further analysis. following alignment of ph / /ire and avc / /ire with other ccov-i and ii reference strains, the pcr typing results were confirmed. this twelve-month epidemiological study was conducted in southern ireland from october to october . two hundred fifty samples were collected in total, fig. phylogenetic tree based on partial s gene nucleotide sequences of ccovs described in this study and reference strains. evolutionary history was assessed using the maximum-likelihood method based on the tamura-nei model [ ] . sequence alignments were performed using muscle [ ] . the tree with the highest log likelihood (- . ) is shown. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( , replicates) is shown next to the branches. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved nucleotide sequences. all ambiguous positions were removed for each sequence pair table a ). the primers f and r were used to target a vp gene segment of cpv. cov detection was performed by pcr amplification of the conserved region of the polymerase gene, which allows the detection of all three groups of coronavirus, and subsequently, canine-specific cov presence was confirmed via pcr amplification of the n gene. in total, out of samples, seven samples tested positive for the presence of cpv, three samples tested positive for the presence of ccov, and a dual infection was detected in one sample. dogs infected with cpv and ccov may shed the virus once clinical symptoms have ceased. in addition, recovered dogs may serve as asymptomatic carriers and shed the virus periodically [ , ] . this is an important mechanism for the continued circulation and persistence of cpv and ccov in the environment. however, in this study, both viruses were isolated exclusively from dogs that presented with clinical symptoms. the viruses were isolated from samples sourced in veterinary clinics (n= ) and the ispca (n= ), from dogs aged two months to years, several of which had been vaccinated previously. out of samples sourced from symptomatic dogs, seven were positive for the presence of cpv ( . %). all dogs presented with haemorrhagic enteritis, while three also presented with vomiting. additionally, one dog presented with pyrexia, and another case resulted in death (table b) . three ccov were detected in samples from symptomatic dogs ( . %), one of which was present in combination with cpv, and this mixed infection produced severe haemorrhagic gastroenteritis (table c) . rflp analysis was initially used to diagnose cpv- c infection in vitro, as the nucleotide sequence required to substitute glu at aa position created a new mboii restriction site (gaaga), which is unique to this strain [ ] . this restriction site was not detected in any of the current isolates. subsequently, the vp gene segment was sequenced in order to confirm the rflp results, to determine which amino acid each isolate contained at position and thus reveal if isolates were cpv- a or cpv- b. three isolates possessed the aa asn at position of the vp protein and were therefore characterised as cpv- a. all other isolates possessed the aa asp at position and thus were characterised as cpv- b. there does not appear to be any correlation between cpv type and symptom severity. a variety of phylogenetic trees were constructed from this information. however, it can be seen in figure that the vp gene has a low variability, and thus is not particularly informative from a phylogenetic view. typically, a % maximum identity is displayed between the vp gene of cpv- strains. the phylogenetic tree prepared using the maximum-parsimony method proved to give the best possible resolution (figure ). cpv strains are subtyped according to a single amino acid in a strategic location, which involves few changes in the nucleotide sequence. as a result, when comparing cpv nucleotide sequence data, the cpv subgroups a, b and c are not segregated into three distinct groups, as there are several other nucleotide differences at other less antigenically significant locations on the vp gene, which must also be taken into consideration. the ccov strains identified were differentiated using a typing pcr, which was based on the variable nature of the s gene [ ] . the spike protein is the major antigenic determinant of covs. ccov-i and ii share a high nucleotide homology in the viral genome but are highly divergent in the spike protein gene [ ] . the strain ph / /ire was characterized as ccov-i, with strains avc / /ire and avc / /ire being characterized as ccov-ii. ccov-i was first discovered in italy in [ ] . since then, it has been reported in other countries, such as the united kingdom, greece, portugal, belgium, romania, sweden and slovenia [ ] . this is the first report of ccov-i and ii in ireland. in order to confirm pcr typing results, the sequence of the s gene of the three ccov samples was determined. subsequent sequence analysis confirmed that ccov-ii was more prevalent than ccov-i, which is in accordance with other published findings [ ] . although the low numbers of positive samples in this study lack statistical support, it considering that the single-stranded nature of cpv renders it more susceptible to mutation than a doublestranded virus, with a mutation rate similar to that observed in rapidly evolving rna viruses [ ] , it is imperative to continuously monitor the genotypes of the virus present in the population to ensure any new types that have evolved are detected. the latest cpv variant type to emerge, cpv- c, has been shown to produce a more pathogenic clinical outcome in some cases, whereas others have a lower severity and mortality rate [ ] . as the aa that determines the type is located in a major antigenic site, a mutation that affects the type may have serious implications for vaccine efficacy and the antibody response generated in pups. a number of commercially licensed vaccines are available in ireland and are given to pups at six weeks and again four weeks later, followed by an annual vaccination booster. it has been shown that the vaccines based on the original cpv- isolate protect against cpv- variant type challenges; however, a plan to use current strains in vaccine formulations has led to a cpv- b-based vaccine being licensed in europe [ ] . a number of commercially licensed vaccines are available in ireland and are given to pups at a young age to protect against ccov-related enteritis. currently, all vaccines are based on ccov-ii. however, it has been shown that the level of cross-reactivity between ccov-i and ii is limited [ ] . a lack of cross-reactivity has also been reported between the original ccov-ii strain, designated ccov-iia, and a more recently described ccov-ii strain that is closely related to tgev in the ' end of the spike gene, designated ccov-iib [ ] . as a result of rna recombination, insertions, deletions and a high susceptibility to frequent mutation, coronaviruses can mutate rapidly, leading to new genotypes (ccov-i), biotypes (pantropic ccov) and host variants (canine respiratory coronavirus), all of which may present possible difficulties regarding successful vaccination of dogs. such dilemmas have been encountered with vaccination against other rna viruses, such as influenza virus and rotaviruses. the evolution of these viruses is well documented to play an important role in the continuous requirement to assess the efficiency of the vaccines and vaccinal updates. for example, the h haemagluttinin protein (the major antigenic determinant) exhibits a rate of accumulation of amino acid substitutions of approximately - per year [ ] . similarly, human rotavirus strains, detected over an -year period from to in palermo, italy, displayed a rate of accumulation of amino acid substitutions of approximately - per year [ ] . in this study, viruses were isolated from vaccinated and unvaccinated pups. it is possible, in the cases where vaccination had been carried out, that maternal antibody had interfered with the immune response of the pup at time of vaccination. a suspected lack of efficacy of vaccines based on the original cpv- strain against the new cpv variants in circulation has been suggested previously after worldwide cpv outbreaks were observed in regularly vaccinated dogs [ , ] . as a result of this study, it can be concluded that although it appears that the viral evolution of cpv type is not yet a significant problem for the canine population in ireland, vaccine efficacy may need to be re-evaluated by the animal healthcare industry, as it is a possibility that the new cpv- c strain will eventually emerge into the population as a result of importing dogs from other parts of the world. this study provides evidence that both cpv and ccov are causative agents of gastroenteritis in southern ireland in symptomatic dogs, with no evidence of viral shedding in asymptomatic dogs. it has also been demonstrated that dual infection of ccov along with cpv is of significant concern regarding animal health and well-being. continued surveillance would be beneficial to evaluate better vaccine efficacy, to understand the underlying mechanisms of vaccine breakthroughs and to implement successful prophylactic measures. infectious diseases of the dog and cat ( th ed) isolation and immunisation studies of a canine parvo-like virus from dogs with hemorrhagic enteritis an update on canine coronaviruses: viral evolution and pathobiology the three-dimensional structure 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in the control region of mitochondrial dna in humans and chimpanzees muscle: a multiple sequence alignment method with reduced time and space complexity acknowledgements this work was funded by the irish research council for science, engineering and technology (ircset). we are grateful to all those who participated in the study to collect research samples: veterinary surgeons bill cashman, pat o'doherty, conor goold and peter hill, as well as the staff at the guide dogs association and the cspca. we also thank patrick o'donoghue and conn o'shea for their help organizing the sample collection of hunting dogs. a cpv-positive control was kindly provided by professor canio buonavoglia from the department of animal health and wellbeing in bari, italy. key: cord- -ywzpwlrb authors: ikeda, t.; shimokata, k.; daikoku, t.; fukatsu, t.; tsutsui, y.; nishiyama, y. title: pathogenesis of cytomegalovirus-associated pneumonitis in icr mice: possible involvement of superoxide radicals date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ywzpwlrb we have studied the pathogenesis of murine cytomegalovirus (mcmv) pneumonitis in immunocompetent icr mice and in mice treated with cyclophosphamide (cp). intranasal infection of immunocompetent mice with mcmv resulted in transient and self-limited pulmonary lesions. when mice were given mg/kg of cp one day before virus infection, transient splenic atrophy and subsequent splenic hypertrophy were induced, and the lesions in the lung were markedly augmented in their number and size although there was no significant enhancement of the virus growth. the augmentation coincided with the period of splenic hypertrophy. a marked increase in the number of pulmonary lesions was also induced in mice given mg/kg of cp every days following the initial dose of mg/kg. in these mice, however, continuous splenic atrophy and augmented replication of mcmv in the lung were observed. when the activity of xanthine oxidase (xo) in lung tissue homogenates was measured, the activity was found to significantly increase after intranasal infection with mcmv irrespective of cp administration and there was a good correlation between the elevation of xo activity and the degree of pathological changes in the lung. in addition, we found that the administration of allopurinol, a specific inhibitor of xo and superoxide dismutase, a superoxide radical scavenger, reduced the number of the pulmonary lesions. these results suggest that superoxide radicals are involved in the pathogenesis of mcmv-associated pneumonitis in icr mice. infection with human cytomegalovirus (hcmv) is an important cause of morbidity and mortality in severely immunocompromised patients, particularly in those who have had allogeneic bone marrow or solid organ transplantation, or who have the acquired immunodeficiency syndrome (aids), and hcmvassociated interstitial pneumonitis is now a major cause of death in those patients. however, the pathogenesis of hcmv pneumonitis is not completely understood. on clinical setting, graft-versus-host disease (gvhd) is often associated with hcmv-associated interstitial pneumonitis after bone marrow transplantation; no cases of hcmv pneumonitis were found among recipients of syngeneic bone marrow from identical twins in which no gvhd occurred [ ] . hcmv pneumonitis after allogeneic bone marrow transplantation have not been successfully treated with ganciclovir ( -[ -hydroxy-l-(hydroxymethyl)ethoxymethyl]guanine) alone despite its excellent antiviral activity, but better therapeutic effects of ganciclovir were obtained when the drug was given in combination with anti-hcmv immunogtobulin [ , ] . in murine models of cmv pneumonitis, immunosuppression is often required to produce pathological changes [ ] . however, immunosuppression is not mandatory [ , , ] . on the contrary, immunosuppression inhibited the formation of pulmonary lesions in an animal model [ ] . from these studies, it is suggested that cmv pneumonitis is an immunopathological condition [- ]. similar observations have been made in murine models of influenza virus pneumonitis, it has been suggested that the lethal effect of influenza infection is determined by immunopathological consequences of the host rather than the direct cytopathic effect of viral replication [ ] . furthermore, recent studies have shown that the enhanced production of superoxide radicals by xanthine oxidase (xo) plays a key role in the pathogenesis of influenza virus-induced pulmonary infection [ , . in this study, we have established a model of murine cytomegalovirus (mcmv) pneumonitis in icr mice, measured xo activity in tung tissues, and evaluated the effect of superoxide dismutase (sod) and allopurinol in this model. the degree of the pathological changes expressed as the number of foci correlated well with xo activity but not with virus titers, and both sod and allopurinot administration suppressed the formation of pulmonary lesions. we suggest that superoxide radicals generated by xo are involved in the pathogenesis of mcmv-associated pneumonitis in mice. specific pathogen free male icr mice ( -weeks-old) were purchased from japan slc (shizuoka, japan) and used throughout this study. infected mice were maintained in groups of to in isolator units, and given water and food (oriental yeast co., ltd., japan) ad libitum. the smith strain of mcmv has been kindly provided by dr. y. minamishima (department of microbiology, miyazaki medical college, miyazaki, japan). the virus was maintained superoxide radicals in mcmv pneumonitis by serial passage in icr mice and was harvested as a % (wt/vol) homogenate of salivary gland tissue. since it has been reported that such a salivary gland tissue homogenate itself induces a non-specific bronchiolitis [ , ] , virus stocks were prepared from tissue culture supernatants after a single passage in mouse embryo fibroblasts. the stocks, which usually contained ~ x pfu of mcmv per ml, were stored at - °c until use. for infection mice were anesthetized with mg/kg body weight of , , -tribromoethanol (avertin; aldrich chemical co., milwaukee, wi, u.s.a.) and . ml of virus stock ( x t pfu) was instilled into the nose. control mice were inoculated in parallel with an equivalent volume of culture medium. lungs and other organs were excised from lethally anesthetized mice. homogenates of these organs ( % wt/vol) were prepared in eagle's minimal essential medium (mem) containing % fetal calf serum (fcs) and were stored at - °c until virus assay. mouse embryo fibroblasts were prepared from embryos of late-term pregnant icr mice by trypsinization, and were maintained in mem containing % fcs. infectivity of mcmv was titrated by plaquing on secondary mouse embryo fibroblasts in duplicate mm dishes (falcon labware, becton-dickinson & co., oxnard, ca, u.s.a.). after a h adsorption period at °c, the cultures were overlaid with ml of . % agarose in mem containing % fcs. the monolayers were fixed with % formalin days after infection and stained with crystal violet, and the number of plaques were counted by using a dissecting microscope at x magnification. cyclophosphamide (cp) (sigma chemical co., st. louis, mo, u.s.a.) was prepared as a mg/ml solution in phosphate-buffered saline (pbs) and given intraperitoneally. lung tissues excised from mice were immediately homogenized with ml of ice-cold pbs containing mm edta, mm phenylmethanesulfonyl fluoride (pmsf), and mm dithiothreitol (dtt) in potter-elvehjem homogenizer. the homogenates were centrifuged at , g for rain at °c. the supernatants were assayed for xo activity according to the method described by avis et al. [ ] . the conversion of xanthine to uric acid was followed at wave length of nm in a ml reaction volume containing . mm xanthine (katayama chemical co., osaka, japan) in . m pbs, ph . for min at °c. one unit of enzyme activity is defined as the amount of enzyme that caused the formation of gmol of uric acid per minute using an extinction coefficient difference at nm of . x t c mt m- [ ] . for histologic evaluation of tissues, lungs were fixed with % buffered formalin. lungs and hearts were taken en bloc from lethally anesthetized animals and were perfused with the buffered formalin via the pulmonary artery. then the lungs were gently inflated by intratracheal instillation of the fixative. the paraffin-embedded sections were stained with hematoxylin and eosin (h & e). to estimate the magnitude of pathologic changes, inflammatory foci per section were counted under light microscope at x magnification. some of the sections were immunohistochemically analyzed using a monoclonal antibody to a nuclear antigen of mcmv-infected cells, as described previously [ ] . this monoclonal antibody, when tested in cultured mouse fibroblasts, reacted with the nuclear antigen h after infection and also reacted with nuclear inclusions in a late phase of infection. the sections were treated with . % periodic acid in distilled water at room temperature for rain to inactivate endogenous peroxidase, washed with pbs three times, and then incubated with the monoclonal antibody at room temperature for rain. after washing with pbs, the sections were incubated with peroxidase-conjugated antimouse igg (cappel lab., cochranville, pa, u.s.a.) for min at room temperature. then, the diaminobenzidine reaction was performed as described previously [ ] . recombinant human sod was provided by research laboratories, pharmaceutical group, nippon kayaku co., ltd., tokyo, japan. sod was prepared as a ku/mi solution in normal saline. mice were intravenously given , u/body of sod every h. allopurinol (tanabe seiyaku co., ltd., osaka, japan) was prepared as a mg/ml solution in normal saline. mice were intraperitoneally given mg/body of allopurinol every h. control mice were treated with an equivalent volume of vehicle (normal saline). values are expressed as mean ± standard error (se). student's t-test was used for statistical analysis. significant difference is defined as p < . . replication of m c m v and pathological changes in the lung of normal mice experiments were performed to determine the susceptibility of icr mice to m c m v infection. mice were intranasally infected with m c m v smith strain at a dose of x pfu. viruses were readily recovered from a number of major organs including spleen, liver and salivary gland (data not shown), but mortality was not observed. as shown in fig. b, panel a, virus titers in lung tissues already reached the plateau by day , and decreased gradually after day , but significant amounts of infectious virus were detectable even after days after infection. light microscopic observation of lung tissues showed focal peribronchiolar mononuclear cell infiltration with occasional extension into the interstitium of alveolar septae. these inflammatory foci first appeared on day , reached the maximum in number between day and day , and then disappeared in spite of the presence of substantial titers of m c m v (fig. c, panel a) . in order to modulate the immune system, we used two kinds of cp administration regimens similar to that described by shantey et al. [ ] ; one group of mice (cp mice) was administrated mg/kg of cp one day before virus inoculation and the other group of mice (cpn mice) was administrated rag/ kg of cp every four days after an initial dose of mg/kg one day before virus infection. as reported previously [ ] , a single dose of cp ( mg/kg) resulted in marked splenic atrophy on day but induced marked hypertrophy on day ( fig. a, panel b) . there was no significant difference in the virus growth between mcmv-infected mice and mcmv-infected cp mice (fig. b, panels a and b) . however, a single dose of cp before mcmv infection resulted in a marked increase in the number of foci in the lung (fig. c, panel b) . no mortality was seen. on the other hand, mcmv-infected cpn mice exhibited a marked increase in mcmv titers in the lung on and after day ( fig. b, panel c) . in this regimen of cp administration spleen remained atrophic (fig. a, panel c) . about a half of mice were killed by day and mortality reached % by day . the number of foci on day in mcmv-infected cpn mice was similar to that of mice infected with mcmv alone, but markedly increased thereafter ( fig. c, panels a and c) . it was also noted that there was a marked increase in the lung index (lung weight/body weight ratio) ( fig. d, panel c) . histopathology of the lung lungs were taken from mice for histological examination on day , , , and . neither tissue damage nor mcmv-antigen positive cells were seen in the tissues from mock-infected mice (fig. a) or cp-treated mock-infected mice. inflammatory foci were detectable on day in all of the three groups of mcmvinfected mice irrespective of cp administration. as described above, the number of foci in the lung was much larger in mcmv-infected cp mice and mcmvinfected cpn mice than in mcmv-infected mice. there was essentially no histological difference between mcmv-infected cp mice and mcmv-infected mice, except that the sizes of the foci in mcmv-infected cp mice were larger than those of mcmv-infected mice. the lesions consisted of peribronchiolar mononuclear cell infiltration with nuclear debris and fibrin (fig. b and c) . in the foci, a few mcmv-antigen positive cells were detected by immunohistochemistry (fig. d) , but there were few mcmv-antigen positive cells in the other areas of the lung. mcmv-infected cpn mice showed different histopathological abnormalities. the number of mcmv-antigen positive cells in the foci was significantly larger in mcmv-infected cpn mice than in mcmv-infected mice or mcmvinfected cp mice (fig. b) . the foci consisted of much less cellular components and much more fibrin when compared with those in mcmv-infected cp mice (figs. c and a) . in mcmv-infected cpn mice, mcmv antigens were frequently detected in alveolar macrophages, bronchial epithelial cells (fig. c) and vascular endothelial cells (fig. d) . in order to elucidate the role of superoxide radicals in the pathogenesis of mcmv-associated pneumonitis, we measured xo activity in the lung tissue homogenates. as shown in fig. e, panel a, intranasal infection with mcmv in normal mice resulted in an increase in the level of xo activity. the activity reached the maximum level ( . + . mu/g tissue) on day , and then rapidly decreased. in mcmv-infected cp mice, however, the increase in xo activity persisted until day although the overall time-course of this elevation of xo activity was similar to that of mcmv-infected mice (fig. e, panel b) . in mcmv-infected cpn mice, xo activity continued to increase until day , and the activity ( . ± . mu/g tissue) on day' was significantly higher than that of mcmv-infected mice ( . + . mu/g tissue) or mcmv-infected cp mice ( . ± . mu/g tissue) (fig. e, panels a -c) . the time-course well correlated with the development of pathological changes in the lung (fig. c and e) . the xo activity in the sera of mcmv-infected mice was also elevated on day and day (data not shown). the above results suggest that superoxide radicals produced by xo may be involved in the pathogenesis of mcmv pneumonitis in our mouse model. we therefore studied the effects of sod and allopurinol on the formation of tbcal inflammatory lesions in mcmv-infected cp mice, and found that sod administration significantly reduced the number of foci in mcmv-infected cp mice (table ) without significant reduction of virus titers in the lung (data not shown). allopurinol also tended to reduce the number of foci. we a mice were administrated mg/kg of cp one day before infection, intranasally infected with x pfu of mcmv, and treated either allopurinot or sod. drug treatment was started at h after infection. at days after infection, mice were sacrificed and lungs were removed for the histological examination. the histologic evaluation of lungs was done by counting the number of inflammatory foci b allopurinol dissolved in . ml of normal saline was given intraperitoneally every h. control mice were treated with vehicle (normal saline) in the same manner ° u of sod dissolved in . ml of normal saline was given intravenously every h. control mice were treated with vehicle in the same manner d p = . compared with saline control, n = e p < . compared with saline control, n = activity in the later period [ , ] . in addition, it has been shown that nk cells are involved in the pathogenesis of mcmv interstitial pneumonitis [ ] . therefore, the enhanced pathological changes observed in mcmv-.infected cp mice could be due to augmented nk cell and cytotoxic t cell activities against mcmv infection. on the other hand, a marked increase in the number of loci was also observed in mcmv-infected cpn mice, but histopathological features of the lesions were different from those observed in mcmv-infected cp mice. the foci in the cpn mice were characterized by lesser extent of mononuclear cell infiltration and much more content of fibrin. since the spleens of cpn mice remained atrophic, the immunological potential is considered to be kept suppressed during the period of observation. thus it seems that severe pneumonitis in mcmv-infected cpn mice was the consequence of enhanced virus replication in the lung rather than augmented immune response. recent studies have shown that oxygen-derived free radicals may play important roles in the pathogenesis of pulmonary diseases associated with inflammation, including pulmonary toxicity [ ] , the adult respiratory distress syndrome (ards) [ , ] , pulmonary emphysema [ ] and idiopathic pulmonary fibrosis [ , ] . it has been further demonstrated that the activity of xo, which generates superoxide radicals, increases in plasma of ards [ ] and in the serum and lung of influenza-associated pneumonitis [ , ] , suggesting that xo mediates lung injury through the production of toxic oxygen metabolites. in the present study, we found that xo activity in the lung significantly increased after intranasal infection with mcmv, and that there was a good correlation between the elevation of xo activity and the development of pathological changes in lung tissues. moreover, allopurinol, a specific inhibitor of xo, and sod, a specific superoxide radical scavenger were found to reduce the number of the focal lesions in mcmv-infected cp mice. these results suggest that superoxide radicals are involved in the pathogenesis of mcmv-associated pneumonitis in icr mice. viral infection itself or host immune response to virus-infected cells may raise the level of superoxide radicals in the microenvironment around the infected cells, probably by accelerating the conversion of xanthine dehydrogenase to xo. since superoxide radicals can directly cause cellular injury [ ] and also act to produce chemotactic substances that attract additional leukocytes to the site of inflammation [ ] , a rise of the level of the superoxide radicals in the microenvironment could induce further production of superoxide radicals by the secondary cellular injury. although the precise role of superoxide radicals in the pathogenesis of mcmv pneumonitis is still unknown, such a process seems to be involved in the formation of pulmonary lesions in mcmv-infected mice. oxygen free radicals including superoxide radicals are highly reactive, short lived and exist only at low concentrations, and hence their detection in vivo is generally very dit~cult. up to now, we have been unsuccessful in identifying the cells or the sites producing superoxide radicals. however, it is known that metabolically activated inflammatory cells release a considerable amount of the t. ikeda etal. radicals into the suspending m e d i u m [ ] and the level of xo activity increases in activated leukocytes and macrophages [ ] . besides, capillary endothelial cells, which are a c o m m o n target of c m v infection, are shown to contain xanthine dehydrogenase in high concentrations [ ] . these cells could be cogent candidates for superoxide-producing cells. further studies will be required to clarify this point. dependence on o 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gancictovir and high-dose intravenous immune globulin regulation of delayed-type hypersensitivity iii. effect of cyclophosphamide on the suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice plasma xanthine oxidase activity in patients with adult respiratory distress syndrome superoxide radicals in mcmv pneumonitis is cytomegalovirus interstitial pneumonitis in transplant recipients an immunopathological condition? lancet ii effect of cyclophosphamide on infections in mice caused by virulent and avirulent strains of influenza virus significance of xanthine oxidase in capillary endothelial cells interstitial pneumonia and subclinical infection after intranasal inoculation of murine cytomegalovirus studies on milk xanthine oxidase. some spectral and kinetic properties superoxide and inflammation: a mechanism for the anti-inflammatory activity of superoxide dismutase oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated sod the role of natural killer cells and antibodydependent cell-mediated cytotoxicity during murine cytomegalovirus infection role of cytotoxic t lymphocytes in murine cytomegalovirus infection involvement of natural killer cells in the pathogenesis of murine cytomegalovirus interstitial pneumonitis and the immune response to infection treatment of cytomegalovirus pneumonia with ganciclovir and intravenous cytomegalovirus immunoglobulin in patients with bone marrow transplants transfer to cyclophosphamide-treated mice of natural killer (nk) cells and in vivo natural reactivity against tumors murine cytomegalovirus pneumonia. description of a model and investigation of pathogenesis effects of immunosuppression with cyclophosphamide on acute murine cytomegalovirus infection and virus-augmented natural killer cell activity the pathogenesis of pneumonitis due to murine cytomegatovirus free radicals in medicine. ii. involvement in human disease differential effects of cyclophosphamide on the b and t cell compartments of adult mice oxygen radical production by alveolar inflammatory cells in idiopathic pulmonary fibrosis neutrophils and the adult respiratory distress syndrome murine cytomegalovirus infection of cultured mouse embryos superoxide radicals in mcmv pneumonitis polymorphonuclear leucocytes and macrophages in mice in three pathological situations selective depletion of lymphoid tissue by cyclophosphamide we thank professors h. saito and k. maeno for their encouragement throughout this work and t. tsuruguchi and e. iwata for their technical assistance. authors' address: dr. k. shimokata, first department of medicine, nagoya university school of medicine, tsuruma-cho, showa-ku, nagoya , japan.received march , t key: cord- -rj mzqj authors: gerna, g.; campanini, g.; rovida, f.; sarasini, a.; lilleri, d.; paolucci, s.; marchi, a.; baldanti, f.; revello, m. g. title: changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: rj mzqj from through , pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or rt-pcr during three consecutive winter-spring seasons. on the whole, ( %) patients were detected as positive for one or more respiratory viruses. the most widely circulating virus was human respiratory syncytial virus (hrsv) infecting % of positive patients, followed by human metapneumovirus (hmpv) found in % of patients, and then by influenza virus type a, human parainfluenzaviruses and coinfections. significant variations in the circulation rate of hrsv, hmpv and influenzavirus type a were observed during the individual seasons. in addition, the circulation rates of the different types of hmpv changed yearly. in – and – hmpv circulated at a significant lower proportion than hrsv, while in – the circulation rates of the two viruses were closer. in conclusion, the hmpv subtypes circulated yearly in northern italy flanking hrsv as major respiratory pathogens in the infantile patient population. young children are among patients most severely affected by hmpv infections [ , , ] . however, several epidemiological features of hmpv infections remain to be investigated. in particular, differences in the circulation rate of hmpv strains as well as different hmpv types and subtypes during different seasons, and the relative pathogenicity of hmpv with respect to human respiratory synctial virus (hrsv) remain to be defined. in this study, we examined: i) the circulation rate of hmpv among the other respiratory viruses during consecutive winter-spring seasons; ii) the relative circulation of the types and the subtypes of hmpv during the same -year period; iii) the relative impact of hmpv as compared to hrsv in determining admission to the hospital of infants with acute respiratory infections. from december through november , infants and young children were admitted to hospital because of an acute respiratory virus infection. all patients had fever, cough, and lower respiratory tract involvement. nasopharyngeal aspirates (npa) were collected upon admission and then divided into aliquots: one was used for molecular assays (rt-pcr), the second for direct fluorescent antibody (dfa) staining of respiratory cells, the third for short-term and long-term virus isolation in cell cultures, and the fourth was frozen as a back-up sample [ ] . specimens were examined for influenzaviruses a and b, parainfluenzaviruses (hpiv) to , hrsv, and human adenoviruses (hadv) by dfa and virus isolation in cell cultures. in addition, hcovs, group i ( e-like) and ii (oc -like), and hmpv types a and b were sought by rt-pcr. typing of hmpv strains was achieved by sequencing and phylogenetic analysis. similarly, grouping of hcovs was achieved by sequencing. the study was approved by the irccs policlinico san matteo ethics committee. the simultaneous detection of respiratory viruses in the same npa was referred to as coinfection. rapid virus recovery was achieved in cell cultures by inoculating npa samples onto shell vials of a mixture of a and mv lu ( : ) cells [ ] , and shell vials of both llc-mk and mdck cells, which were stained with mabs at and, if required, days p.i. conventional virus recovery was obtained following inoculation of tubes of llc-mk and mdck cells, which were stained with mabs days p.i. or, if required, during the following weeks. a pool of fluorescein-labeled mabs to conventional respiratory viruses (influenzavirus a and b, hpiv - , hrsv, and hadv), as well as the single fluorescein-conjugated mabs included in the pool (purchased from chemicon international, inc., temecula, ca) were used for both dfa and virus identification in cell cultures. following rt reaction, pcr assays for the identification of hcovs and hmpv strains were optimised to detect at least input plasmid copies. to this purpose, primers for hcov (groups i and ii) were selected from a published protocol [ a] , whereas primers for hmpv types a and b (genes n and f) were originally designed from genbank published virus sequences (table ) . amplification products were cloned in pcr . plasmid vector (ta cloning kit, invitrogen, carlsbad, ca) and quantitative standards were obtained following titration of insert-containing plasmids [ ] . thus, defined amounts of target sequences could be amplified in parallel with clinical samples. nucleic acids were extracted using nuclisens ® iso kit (biomerieux, lyon, france). rt and pcr reactions were performed as reported [ ] . pcr products were examined on % agarose gel. classification of hmpv strains of this study into types a and b, and subtypes a -a , and b -b , was achieved by phylogenetic analysis of fragments of both genes f and n of hmpv strains, respectively. viral sequences of the amplified gene f (nt to ), and gene n (nt to ) fragments of hmpv isolates as well as reference strains were aligned with the clustal w program version . , whereas sequence similarity comparisons were carried out with the megalign program (dnastar inc., madison, wi). the philips (njplot) program was used to construct phylogenetic trees with nucleotide sequences by means of the neighbour-joining method from the same distance matrices. bootstrap support was determined by resamplings of the sequences. statistical differences in the circulation rate of respiratory virus infections among the consecutive seasons were determined by the chi square test. the relative distribution of different respiratory viruses causing severe infections requiring admission to the hospital of infants and young children in three consecutive winter-spring seasons from through is reported in table within the aliquot of patients found positive for some respiratory virus, no difference in the circulation rate was observed for respiratory virus infections caused by influenzavirus b, hpivs, hadvs, hcovs, and coinfections along the three years studied. on the contrary, significant differences in the circulation rate table were found for influenzavirus a, hrsv, and hmpv (table ) . however, while hrsv and hmpv infections predominated during the first months of age, the other infections were evenly distributed during the first years of age. (table ) . surprisingly, in the group of coinfections, simultaneous infection of the same patient by hmpv and hrsv was observed in a single case in - ( table ) . the circulation rate of hmpv reached its peak (fig. ) . due to the high similarity of the pathology caused in newborns and infants, the circulation rates of hmpv and hrsv were analysed by comparing the relevant number of infected patients admitted to hospital in the winter-spring (december through may) and summer-fall (june through november) seasons of each year. both viruses were nearly absent during the summer-fall seasons, whereas they were highly circulating, at a different proportion, during the winter-spring seasons. although the incidence of hrsv infections in pediatric patients admitted to the an epidemiological survey on the circulation rate of the most important respiratory viruses responsible for admission to the hospital of infants and young children in a defined geographical area during the -year period from through was conducted using npa samples as clinical specimens. in order to contain costs, on the basis of recently acquired results showing a comparable sensitivity of the molecular (rt-pcr) and the immunological (monoclonals) diagnostic approaches [ ] , influenzaviruses a and b, hpiv - , hrsv, and hadvs were detected by monoclonal antibodies, while hcovs and hmpvs were detected by rt-pcr. while no significant difference was observed in the circulation rate of the majority of respiratory viruses, a significant difference in the circulation of influenzavirus a, hrsv and hmpv, was found during the consecutive winterspring seasons. apart from influenzaviruses a, which are known to circulate differently in different years, hmpv and hrsv circulated at a stable rate in the first seasons examined. on the contrary, the relative proportion of patients infected by hrsv decreased sharply in the most recent season, while that of patients infected by hmpv increased significantly. as a consequence, the number of young patients admitted to the hospital because of lower respiratory tract infections caused by hmpv increased accordingly. yearly variation in the circulation rate of hmpv has been recently reported [ , , , ] . besides a striking variation in the overall circulation rate of hmpv, a significant variation in the circulation of the hmpv subtypes was observed during the study period. whether different hmpv types and subtypes may be differentiated only on the basis of nucleotide sequence (phylogenetic analysis) or may represent true serotypes or subserotypes remains to be determined. in fact, while ferret immune sera have been reported to identify two distinct serotypes (a and b) showing a ≥ fold dilution difference between homologous and heterologous neutralizing antibody titer [ ] , several primate as well as hamster immune sera did not show any discriminating capacity [ ] . similarly, in our experience, as already reported by others [ ] , guinea pig immune sera were not able to identify two distinct serotypes. thus, at the moment, the terms types and subtypes indicate genotypes and subgenotypes rather than serotypes and subserotypes. in a recent study by our group, using a fragment of gene n (nt to ), it was possible to identify amino acid substitutions at positions , , , , , , , and , that might be used to differentiate type a from type b strains [ ] . monoclonal antibodies reactive with epitopes containing these differential amino acids might discriminate between the two hmpv types. the simultaneous circulation of hrsv and hmpv in the same period of the year (winter-spring season) and in the same patient population (infants and young children), along with a comparable degree of pathogenicity, could hypothetically facilitate coinfections by these two members of the paramyxovirinae subfamily. in a recent population-based prospective multicenter study of the children requiring intensive care conducted in germany over years, % had hmpv infections, and % of these children were infected with hmpv in combination with hrsv, whereas hmpv was not detected in a large series of hrsv-positive children not requiring intensive care support. this data support the hypothesis that coinfections with hrsv and hmpv are more severe than infections with either hrsv or hmpv alone, at least in children less than years of age [ ] . in our study, outside the intensive care unit, surprisingly a single case of coinfection by hmpv and hrsv was found, thus suggesting that this coinfection is very infrequent in the immunocompetent host. with reference to the italian pediatric population admitted to hospital during the winter-spring season, the following conclusions can be drawn from our epidemiological study: i) hmpv strains circulate at a different rate in different years; ii) changes in the prevalence of hmpv types and subtypes occur yearly; iii) the prevalence of hmpv versus hrsv infections may also change significantly in different years. virological features and clinical manifestations associated with human pneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups human metapneumovirus infections in hospitalized children global genetic diversity of human metapneumovirus fusion gene human metapneumovirus infection in the canadian population human metapneumovirus detection in patients with severe acute respiratory syndrome evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children human metapneumovirus infections in young and elderly adults evidence of human metapneumovirus in children in argentina mink lung cells and mixed mink lung and a cells for rapid detection of influenza virus and other respiratory viruses prospective study of human metapneumovirus infection in children less than years of age human metapneumovirus associated with respiratory tract infections in a -year study of nasal swabs from infants in italy children with respiratory disease associated with metapneumovirus in hong kong identification of severe acute respiratory syndrome in canada monoclonal antibodies versus reverse transcription-pcr for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital detection and pathogenicity of human metapneumovirus respiratory infections in pediatric italian patients during a winter-spring season the two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (f) protein is a major contributor to this antigenic relatedness analysis of the genomic sequence of a human pneumovirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease antigenic and genetic variability of human metapneumoviruses prevalence and clinical symptoms of human metapneumovirus infection in hospitalised patients identification of a new human coronavirus human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children author's address: prof. giuseppe gerna, servizio di virologia, irccs policlinico san matteo this work was partially supported by grants from ministero della salute, ricerca finalizzata irccs policlinico san matteo (convenzione n. ), and ricerca corrente (research code ). we thank daniela sartori for preparing the manuscript and linda d'arrigo for revision of the english. we are also indebted to dr. massimo fabbi for preparing guinea pig hyperimmune sera. key: cord- - cfphi authors: carter, m. j. title: transcription of feline calicivirus rna date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: cfphi we report here the cloning and ′ sequence determination of feline calicivirus strain f . subcloning the ′ terminus enabled the production of strand specific probes for rna synthesis. we extend the number of virus specific rnas detected intracellularly to , and show that numbers – are represented as negative strands which may serve as templates in the synthesis of these rnas. the family caliciviridae are non-enveloped viruses of distinctive morphology previously classified with the family picornaviridae [ ] . like the picornaviruses, caliciviruses are positive-stranded and their genomes bear a covalently attached protein (vpg) at the ' end [ ] . however, caliciviruses differ from members of the picornavirus group in that rna molecules smaller than the genome are synthesized intracellularly [ ] . in this respect the caliciviruses may have more in common with the corona-or togaviruses. ehresmann and schaffer have studied cells infected with san miguel sealion virus (smsv) and reported such species in addition to genomic rna; a s subgenomic single stranded molecule, two double-stranded molecules each consisting of two strands of genomic or subgenomic rna respectively and a heterogenous partially doublestranded form [ ] . black et al. [ ] obtained broadly similar results working with vesicular exanthema of swine virus (vesv) and indicated that a third single-stranded subgenomic rna molecule ( s) was present. all the singlestranded molecules were polyadenylated and lacked cap structures at their ' termini [ ] . more recently, neill and mengeling have cloned fcv rna and used these cdna molecules to identify intracellular virus-specific rna [ ] . in this way these workers have identified three intracellular rnas ( . , . , and . kb) as well as the genome ( . kb) and shown that these form a nested set of ' co-terminal molecules similar to that observed in coronavirus infected cells. we have been working in a similar manner and have also cloned rna extracted from purified feline calicivirus (fcv) particles. we have used this to probe fcv-infected cells for the synthesis of virus specific rna and confirm and extend the observations of neill and mengeling. subcloning of the virus ' end into a single stranded vector has allowed us to examine the occurrence of positive and negative sense rna separately. sequence analysis has shown a small open reading frame (orf) at the ' end, which suggested that the nested set could include small, as yet undetected mrnas. feline calicivirus strain f was obtained from prof. o. jarrett, university of glasgow veterinary school and grown in the feline kidney cell line crfk (flow laboratories, rickmansworth), in which it produces widespread cpe within h. fcv was plaque-purified by three rounds of single plaque selection in these cells and a working stock of virus ( . x i s pfu/ml) was prepared. virus particles were purified for cloning experiments as follows: six confluent roller bottles of crfk cells were each infected with ml of virus stock. growth was allowed to proceed for h when the bulk of the cell sheet had been destroyed. cell debris was removed by sedimentation at , g for rain and then at , g for rain before virus particles were pelleted at , g for . h. the virus pellets were then thoroughly resuspended in nt buffer ( ram nac , ram tris-hc ph . ) and the volume adjusted to ml. the virus suspension was then overlayered onto four discontinuous csc gradients each consisting of .sml of csc ( . g/cm ) and . ml of csc solution of density . g/cm . gradients were formed in sw tubes (ultraclear, beckman ltd., high wycombe) and spun at , rpm for rain at °c. under these conditions virus sedimented to the interface between the two csc solutions although a distinct band was often not visible. the interface regions (in about ml per tube) were then pooled and adjusted to a density of . g/cm with csc . this virus suspension was then centrifuged overnight at , rpm in the sw rotor and allowed to decelerate without the brake. under these conditions a tight band was normally visible by scattered light in the central region of the tube and could be removed with a syringe. the band was then diluted with nt buffer and repelleted at , rpm for l h before being resuspended in . ml of nt buffer. this material contained one protein band corresponding to the virus capsid on coomassie blue-stained sds-polyacrylamide gels. rna was extracted from this purified virus for cloning procedures. double-stranded cdna synthesis for cloning was performed using the procedure of gubler and hoffman [ ] employing oligo-dt as primer for the reverse transcription of gg of virus rna. cdna molecules were cloned by the homopolymer tailing method. approximately c residues were added per end using terminal deoxynucleotidyl transferase (gibco brl). radioactive dctp was used for this step in order to increase the specific activity of the product. prepared cdna molecules were hybridized with otigo dg-tailed. psti cut plasmid vector pbr and introduced into escherichia coli dh alpha f' (gibco brl) rendered competent by the method of hanahan [- ] . transformed bacteria were selected on media containing tetracycline and screened by colony hybridization [ ] . rna was purified from crfk cell cytoplasmic extracts for the preparation of probes with which to select recombinant colonies. polyadenylated species were selected using poly-u sepharose chromatography, edna probes were produced by reverse transcription of gg of cellular a + rna and omission of the unlabeled dctp to increase specific activity. actinomycin d was included at gg/ml to prevent labelling of any sequences derived from contaminating host cell dna. this protocol led to the production of short transcripts and so to ensure complete sequence representation the otigo dt primer was replaced with random hexanucleotide primers (pharmacia, milton keynes u.k.). a probe specific for the ' end of the virus, i.e., enriched for sequences immediately adjacent to the poly a tail, was prepared using this procedure with oligo dt as primer. any clones to be sequenced were re-cloned into the bacteriophage vectors m mp and mpl after digestion with appropriate restriction enzymes. single-stranded template was prepared and sequenced using a sequenase v . sequencing kit (cambridge bioscience, u.k.) exactly as recommended by the manufacturers. all sequence data presented here were confirmed by sequencing both strands. data was analysed using the pc gene software package supplied by genofit sa (geneva, switzerland). r n a extracted from virus particles showed some degradation but a sharp band of - kb was visible, c d n a synthesis from this r n a led to a broad size range of products. cloning from this material resulted in the production of many clones which were predominantly small in size (approx. kb). accordingly the larger molecules of ds c d n a were eluted from the gel and cloned separately. this resulted in the production of clones all greater than . kb in size, the largest of which p f c . was found to be . kb in size. this clone was excised fi'om the vector as two psti fragments . and . kb in length. restriction mapping of this clone led to the m a p shown in fig. . similar analysis showed that all the other clones tested could be aligned on this map and most formed a nested set extending from the right hand end. those larger than bases possessed the ecori site and those larger than . kb contained the central the ecori site appears to be common to this clone and also that derived previously [ ] , but the central psti site is found only in pfc . . the sinai site is present in both clones but is differently located. both hindiii sites found by n~ill and mengeling are absent in pfc . which possesses two bamhi sites, neither of which are in the same position as the single site found previously [ ] psti site as well. this suggested that all clones were derived from a common priming site, most likely the ' end of the virus. accordingly a probe was prepared which was enriched in ' end sequences as described. this was than hybridized to a psti restriction digest of clone pfc . separated on an agarose gel and blotted to nitrocellulose. the result (fig. ) indicated that the right hand end of the map presented (i.e., the . kb fragment) hybridized most strongly to this probe, also indicating that this constituted the ' end of the virus. finally this inference was confirmed by subcloning the terminal ecori/psti fragment into m and determining its sequence (fig. a) , this showed a poly a stretch preceded by a short run ofpoly c which is exactly that structure expected for cdna clones derived by this method from an mrna ' end. furthermore, an open reading frame extended towards the ' end terminating some residues from the poly a tail. a search of known nucleotide sequences as contained in the embl database (release no. ) failed to reveal any significant homology between this sequence and any already determined. sequence determination from this ecori site toward the ' end in the virus rna sense encountered a group of closely-spaced termination codons in all frames. this was confirmed by sequencing additional clones covering this region and overlapping the ecori site. however, an orf was observed beyond these termination signals which extended towards the ' end of the clone (fig. b) e c o r ctattggtaa agctcagcag attgaattgg acaaagctgc acttggtcaa cagcgtgaat ctggatttag ggttgaccct tactcataca caaaccaaaa cttttatgac gatcaattaa the probes used in this case were prepared from the ecori/psti restriction fragment corresponding to the ' end of the fcv genome and subcloned into m . single-stranded sense-specific probes were made by klenow enzyme transcription of the purified ssdna templates. both virion (assumed positive) and complementary (assumed negative) senses were prepared and used separately. incorporation of radioactivity was similar in both cases and acid precipitable radioactivity was equalized in each hybridization. the results of this analysis are given in fig. . rna sizes were determined using prokaryote and eukaryote rrna markers whose length is known from the sequence of their genes [ , , , ] . a different pattern of intracellular rna was observed using probes of each sense. the virus-sense probe detected the genome and a . kb virion-sense rna at early times. both of these were found in the first sample taken at rain p.i. the amounts of these species remained fairly steady until h p.i. when a marked accumulation was observed. other rnas appeared at this time and increased . cytoplasmic extracts were prepared by disruption in mm tris (ph . ), mm nac , . mm mgc containing % np , . % sodium deoxycholate, and rnasein. nuclei were removed by sedimentation and rnas were extracted by proteinase k and phenol, denatured with formamide, and analysed in duplicate on a . % mops-buffered agarose gel containing . m formaldehyde. separated species were transferred to nitrocellulose filters by capillary blotting. filters were then cut in half and each half hybridized with one of the sense-specific probes described above. a positive stranded rna; b negative sense rna transcription of feline calicivirus rna in amount as the infection progressed. eight separate rnas were detected in this analysis but many were faint. at all times the genome and the . kb rnas were the most abundant species as reported [ ] . however, previous analysis has revealed only two mrnas in addition to these dense bands giving a total of rna species. the experiments described here have identified novel bands. all bands are numbered in order of decreasing size in fig. (genome is assigned as no. ). bands reported by neilt and mengeling correspond to no. , , , and which are the most readily discernible. novel bands reported in this study are no. and - . sizes of these rnas, derived as the average of multiple determinations, are given in table . the antisense probe detected the genomesized rna after min and this preceded the large increase in the virion-sense form of this band. negative sense . kb rna was not clearly detected until min p.i. and thus lagged behind the appearance of the positive form. however, although the positive form of this rna was present earlier, it did not increase dramatically in abundance until after the appearance of the negative sense. exposure times required for the negative sense blots were longer than those for the positive sense indicating that positive rna predominated in the cells. the genome and the . kb mrna (no. ) are known to be synthesized in double-stranded forms and therefore their detection by both probes was expected. however, the negative strand-specific probe also showed hybridization to subgenomic messages - , but this was not observed until later in infection than the detection of the positive forms of these molecules. negative sense forms of rnas - were not detected at any time. the possibility that probes of each sense recognize both positive and negative sense rna is discounted since only one probe reacted with rna extracted from purified virus (not shown). furthermore, whilst both positive and negative genome-sized rna can be detected at early times, negative sense . kb rna was not observed before h p.i. even though large amounts of its positive form were present during this period. recently fcv mrnas have been found to form a ' co-terminal nested set in common with other virus families [ , ] . in coronaviruses, successive orfs from the ' end are exposed by an unknown mechanism and translated from the next smallest member of the series. such a mechanism could explain our finding of a small orf at the ' end of the fcv genome and would predict that a small mrna should be detectable inside fcv-infected cells formed by activation of this orf. a candidate band was observed in our blotting experiments (band , bp). the sequence of fcv determined in the region of this orf was compared with the known junction sites for members of the family coronaviridae [ ] . the short sequence aaacuuu which is found on the ' side of coronavirus-junction sites and precedes coding information [ ] was found in fcv, flanked by an extra a and u. however, unlike coronavirusjunctions, it is not followed by a suitable initiation codon and it occurs well downstream from the termination codons separating the orfs. it therefore seems unlikely that a mechanism identical to that of the coronaviruses is involved and sequence determination from the intracellular molecules is required to characterize any processing which may occur. this investigation of intracellular rna synthesis has shown that the first intracellular virus rnas detected are the positive and negative sense genomesized molecules and the positive sense . kb rna. both positive forms were present in approximately the same amount. the negative forms of these two species clearly differ in amount, and that of the . kb molecule is barely detectable until min p.i. we have recently shown that the sensitivity of the blotting method in our hands approaches that of a single copy per cell [ ] . much of the positive sense genome detected in these experiments at early times could result from infecting virus whose multiplicity ( pfu per cell) greatly exceeds this limit. however, this explanation cannot apply to the negative form of this molecule, nor to the positive . kb rna. neither of these are part of the virion [ ] . it is therefore possible that the first molecule transcribed from the incoming genome may be a full length complementary rna. the . kb positive sense molecule could be transcribed from this and subsequently synthesized in its negative form. this model would explain the apparent differences in ratios observed. we consider it likely that all the subgenomic mrnas (with the exception of - ) may be made in a similar manner and are synthesized first as positive strands. these may then be replicated independently via negative stranded forms. this would provide a powerful amplification system and account for the rapid accumulation of all positive sense rnas which commenced in the second hour post infection onward. this is at the same time as the amplification of protein synthesis we have recently reported [ ] . a similar mechanism for rna transcription has been observed in coronaviruses, toroviruses, and arteriviruses during the preparation of this report [ , , ] . this suggests that the replication strategies of these virus groups may be very similar in principle despite the apparent lack of similarity in sequence at a presumed junction site in the genome of fcv. neill and mengeling [ ] observed only three subgenomic rna bands; however, these workers used a larger probe which did not correspond to the exact ' end of the virus and lacked the terminal ecori fragment. we have found that the use of larger probes increases the background in the central region of the blots which could have masked minor r n a species. the small r n a (no. ) was clearly absent in the previous analysis [ ] but this r n a is only marginally larger than the ' ecori fragment we have used as a probe and which was absent from the probes used previously. the overlap between this r n a and the probe used by neill and mengeling [ ] would thus have been small and consequently it may not have been recognized. there is considerable antigenic variation within the one serotype of fcv and it is likely that a spectrum of antigenic types may exist [ ] . the restriction maps generated for the ' end of fcv strain cf / fiv [ ] and strain f (this report) also differ and this suggests that these differences will be represented at sequence level. the sizes of the rnas reported here also differ from those reported by neill and mengeling. to some extent this may be accounted for by the different nucleic acid markers employed (ssrna or ssdna) in the two studies. however, strain variation in polypeptide size is known and r n a sizes may therefore also differ. comparative sequence analysis and investigation of any strain-specific pathogenesis is urgently required. the structure and replication of calicivirus rna complete nucleotide sequence of a s ribosomal rna gene from escherichia cotl complete nucleotide sequence of a s ribosomal rna gene from escherichia coli a model for vesicular exanthema virus, the prototype of the calicivirus group feline calicivirus protein synthesis investigated by western blotting is a persistent adenovirus infection involved in coeliac disease rna synthesized in calicivirus-infected cells is atypical of picornaviruses calicivirus intracellular rna: fractionation of - s rna and lack of typical '-methylated caps on s and s san miguei sea lion virus rnas colony hybridization: a method for the identification of cloned dnas that contain a specific gene a simple and very efficient method for generating cdna libraries transcription of feline caticivirus rna techniques for transformation of e. coli the complete nucleotide sequence of mouse s rrna gene. implications for the process of size increase of the large subunit rrna in higher eukaryotes further characterization of the virus-specific rnas in feline calicivirus infected cells serological relationship among feline caliciviruses complete nucleotide sequence of mouse s rrna gene: comparison with other homologs coronavirus transcription: subgenomicmouse hepatitis virus replicative intermediates function in rna synthesis soergel me ( ) a protein, vpg, covalently linked to s calicivirus rna coronavirus subgenomic minus strand rnas and the potential for mrna replicons the biology of coronaviruses a ' co-terminal nested set of independently transcribed mrnas is generated during berne virus replication coronavirus mrna synthesis involves fusion of non-contiguous sequences the initial cloning studies described here were supported by a grant from the thrasher research fund, u.s.a. and the subsequent investigation of rna synthesis by the wellcome trust, u.k. i thank margaret willcocks for excellent technical assistance. received april , key: cord- -b xb f authors: hulst, marcel; kerstens, hinri; de wit, agnes; smits, mari; van der meulen, jan; niewold, theo title: early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: b xb f germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at and hours post infection (two piglets per time point). ifn-gamma mrna expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. rna pools prepared from two infected piglets were used to compare whole mucosal gene expression at and hpi to expression in uninfected germ-free piglets (n = ) using a porcine intestinal cdna microarray. microarray analysis identified down-regulated and up-regulated genes. northern blot analysis of a selected group of genes confirmed the data of the microarray. genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. furthermore, up-regulation was observed for ifn-γ induced guanylate binding protein , a protein that effectively inhibited vsv and emcv replication in vitro (arch virol : – , ). this protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. with an estimated death rate of more than , per year, mainly affecting children less than years of age in developing countries, rotavirus is recognized as one of the major infectious diseases of the gastrointestinal tract [ ] . rotaviruses are members of the family reoviridae, viruses with segmented double-stranded rna genomes [ ] . in the small intestine, mature enterocytes near the top of the villi are the primary target cells for virus replication [ ] . replication in these cells provokes numerous intraand extracellular pathological changes that inevitably lead to disruption of the absorptive and digestive functions of the small intestine, and consequently, to malabsorption and diarrhea. these changes include destruction of enterocyte brush borders, enterocyte vacuolization, loss and destruction of enterocytes, villus blunting and atrophy, thinning of the intestinal wall, and crypt hyperplasia (for comprehensive reviews, see [ , ] ). however, the nature and severity of histopathological alterations in vivo can be quite different depending on the species and virulence of the rotavirus strain. there is no clear correlation between these alterations and manifestation of clinical symptoms. a systemic inflammatory response can be absent, and rotavirus infections can be asymptomatic [ , ] . this suggests that the interplay between host and viral factors is important for determining the course of this disease. for electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. instance, rotavirus nsp acts as an enterotoxin that induces diarrhea in mice in the absence of rotavirus replication [ ] . nsp affects ca + and electrolyte homeostasis in an auto-and paracrine fashion in both rotavirus-infected and uninfected intestinal cells [ ] . nsp increases ca + permeability of the er and plasma membrane, resulting in an increased ca + concentration in the cytosol ([ca + ] cyt ), causing derailment of numerous ca + -dependent cellular processes [ ] . in uninfected enterocytes and crypt cells this rise in [ca + ] cyt is induced by binding of exogenous nsp to an apical receptor that modulates the plc-ip pathway [ ] . the higher [ca + ] cyt triggers laminal secretion of peptides and amines by uninfected enterocytes, and luminal cland h o secretion by crypt cells [ , ] . in infected enterocytes, the rise in [ca + ] cyt is believed to be independent of plc modulation [ ] , and this rise perturbs cytoskeleton and tight junction integrity, which ultimately leads to cell lysis [ , , , ] . in vitro studies with cell lines, mainly derived from colon, have contributed significantly toward understanding the pathogenesis of rotavirus on a molecular level. however, the intestinal mucosa consists of a diversity of specialized cell types in different states of differentiation. presumably, all these different types of cells respond differently to environmental changes, and accordingly to changes in their neighboring cells. therefore, the regulation of genes responsible for these complex phenotypic responses in vivo may not be detected by challenging single types of cultured cells with rotavirus. to address this issue, we studied the early transcriptional response in jejunal mucosa of -week-old, just-weaned piglets after oral infection with virulent group a rotavirus. to assign measured responses exclusively to rotavirus, we performed these experiments in germ-free piglets. differential expression patterns of uninfected versus infected jejunum were recorded and h before severe diarrhea was expected, using a homemade pig intestinal cdna microarray [ ] . the biological significance of elevated or reduced expression of these genes for rotavirus pathogenesis is discussed. seven germ-free piglets (groot yorkshire [cofok large white]) were obtained by caesarean section and housed in isolators, fed with sterilized condensed milk till the age of days and thereafter with pelleted feed (sterilized by x-ray radiation) and water ad lib. on day , three of the seven piglets were transported to the necropsy room and served as uninfected control piglets. the four remaining pigs were orally infected with virus suspension diluted in a total volume of ml pbs and containing rotavirus particles (as determined by negative-stain semi-quantitative electron microscopy) of strain rv [ ] . the virus suspension was prepared from the contents of the small and large intestine of a rotavirus-infected gnotobiotic piglet [ ] . the above applied oral dose caused severe diarrhea from hpi (hours post infection) in -week-old gnotobiotic piglets [ ] . infected piglets were housed in their isolators under the same conditions as described above for another period of (two piglets) or h (two piglets) before they were transported to the necropsy room. immediately after arrival in the necropsy room, ml of edta blood for hematological analysis was collected from the jugular vein. subsequently, animals were killed by barbiturate overdose and their intestines were taken out. the jejunum was opened and rinsed with cold saline, and cm of mucosa in the middle of the jejunum was scraped off with a glass slide, frozen in liquid nitrogen, and kept at - °c until rna and dna extraction. an adjacent part of the collected jejunum was fixed in % formaldehyde and used to determine the villus height and crypt depth. villus and crypt dimensions were determined on hematoxylin-eosinstained -lm tissue sections [ ] . during the experiment, fecal samples were collected at , and hpi from the rectum for determination of the percent dry matter [ ] . fecal samples were tested for the presence or absence of rotavirus by elisa [ ] . the germ-free status of each piglet was confirmed by analyzing throat saliva and feces samples, collected on days , and , and on the day of slaughter, for the presence of microorganisms. isolation of rna and dna from g of frozen mucosal scrapings, total rna (dnasefree) was isolated using trizol Ò reagent (invitrogen) as described recently [ ] . the yield per gram of tissue and the purity of the rna were calculated from measurement of the extinction at and nm. the integrity of all rna samples was checked by analyzing lg of rna on a denaturizing % (w/v) agarose gel. after ethidium bromide staining, the gel was scanned to calculate the s/ s peak ratio (volume s over volume s) for each preparation. rna with a ratio [ was considered of adequate quality to be used for real-time pcr and microarray analysis. a part of the isolated rna was used to prepare rna pools for microarray analysis. a control pool was prepared by mixing equal amounts of rna isolated from the jejunum of the three uninfected piglets (n = ). the same was done for the two infected piglets slaughtered at h and for the two piglets slaughtered at h. after gentle homogenization in lysis buffer, dna was extracted from . g of frozen mucosal scrapings, and lg of purified dna was analyzed on a . % agarose gel [ ] . the relative concentrations of interferon-gamma (ifn-c) and ornithine decarboxylase antizyme (oaz ) mrna in all rna samples was determined by real-time pcr. two hundred ng of total rna was reverse transcribed in a standard rt reaction using superscript ii reverse transcriptase (invitrogen) and pd(n) primers. ifn-c cdna in these rt reactions was quantified using labeled light-cycler probes (roche diagnostics) as described [ ] and expressed as pg/ll control plasmid. a -mer forward primer ( -gacccgacgcttgcttcatg- ) and a mer reverse primer ( -gagtgagcgtttatttgcac- ), generating a cdna fragment homolog to nucleotide - of the human oaz mrna reference sequence (gi: ), were used to quantify oaz cdna using cybergreen as label in a standard lightcycler reaction. the relative concentration of oaz mrna was calculated by extrapolation on a standard curve prepared from dilutions of an rt reaction prepared from a reference rna sample [ ] . the quantity of s rrna in each rna sample was determined using the above described rt reactions by real-time pcr [ ] and used to normalize the ifn-c and oaz concentrations. the quantity of s rrna showed no essential differences among all individual rna samples (average concentration ± sd; . ± . lg/ll of control plasmid). the same collection of pig probes (ests) used in earlier studies [ , ] were spotted in triplicate on corning ul-tragaps slides. briefly, this collection consisted of , probes prepared from jejunal mucosal scrapings collected from -week-( ) and -week-( , ) old pigs, probes coding for porcine cytokines (ifn-c, tnf-a, gmcsf, il- , , , , and ) and lung surfactant proteins sftpa and sftpd, and marc and marc probes (porcine ests) homolog to trefoils, collectins, defensins, and glycosyltransferases [ ] . a list of the probes already sequenced/ annotated is accessible on the website of arch virol (gene list hulst et al. pdf). dual-color (cy -cy ) hybridization of slides was performed using the rna micromax tsa labeling and detection kit (perkinelmer) as described earlier [ ] . messenger rna levels in both infected pools ( and hpi) were independently compared to the expression levels in the control (uninfected) pool. for each comparison, a dye swap was performed. in addition, a control hybridization experiment was performed in which a microarray slide was simultaneously hybridized with cy labeled control rna and cy -labeled control rna. scanning of slides, processing of raw images, creation of data reports, data-normalization and statistical analysis were performed as described by niewold et al. [ ] with minor modifications. briefly, probes were considered to be differentially expressed when at least four of the six data points (spots) on both dye swap slides hybridized with a ratio of . -fold (m = [log (cy /cy )] \ - . or [ . ) or more ( . is considered significant according to the manufacturer of the tsa kit) and were identified by significant analysis of microarrays (sam) [ ] with a median false discovery rate (fdr or q value) of \ %. equal amounts of total rna ( lg) were separated on a denaturizing % (w/v) agarose gel. after several washes with rnase-free water, the gel was blotted on hybond-n membranes (amersham), and blots were hybridized with p-labeled dna fragments homolog to the mrna in question, in the same manner as was described in an earlier study [ ] . after post-hybridization washes, the blots were scanned using a storm phosphor-imager (molecular dynamics, sunnyvale, california, usa). four -week-old germ-free piglets were orally infected with a dose of rotavirus that caused severe diarrhea from hpi in -week-old gnotobiotic piglets [ ] . for practical reasons, three uninfected germ-free piglets were slaughtered at the zero time point (mock, see table ). in order to isolate highquality rna from jejunal mucosal scrapings, infected piglets were slaughtered and hpi. thus, and h before severe diarrhea would have been induced. in three of the four infected animals, rotavirus was detected in their feces. determination of the percent dry matter showed that only the fecal samples collected hpi (piglets and ) had a significantly lower (pasty) consistency [ ] . this indicated that not all of the piglets developed diarrhea before hpi, and that the two piglets slaughtered hpi did not develop the severe form of diarrhea normally observed at hpi [ ] . in jejunal tissue sections prepared from these two -h piglets, villus length was decreased to two-thirds of the average length measured in corresponding sections prepared from the three control piglets. no significant differences in crypt depths were observed between infected and control animals. for both piglets that were slaughtered at h, these results indicated that the orally applied rotavirus reached the transcriptional response to rotavirus jejunum and induced the desired limited (not severe) pathological symptoms. in addition, the lower concentration of lymphocytes in the blood indicated that the animals were effectively infected with rotavirus [ ] . in the feces of piglet , slaughtered at h, no rotavirus could be detected. moreover, only a small decrease in villus length ( %) was observed for this piglet and its -h replicate. to find additional evidence whether the jejunum of piglet was effectively challenged with rotavirus, the level of ifn-c mrna in jejunal mucosal scrapings was measured by real-time pcr (fig. ). in rna samples isolated from the three uninfected pigs, hardly any ifn-c mrna could be detected. in contrast, the ifn-c mrna levels in scrapings of all infected piglets were up to -fold higher than in uninfected piglets, whereas oaz mrna levels were nearly equal for all rna samples. this indicated that the jejunum of all piglets, including piglet , responded to the orally applied rotavirus challenge. despite the loss of cells from the tip of the villi, the amount of rna (table ) isolated from all infected piglets was comparable to that of uninfected piglets. on an ethidium-bromide-stained agarose gel, no degradation of rna was visible for any of the extracted rna samples (see also fig. a ). in addition, s/ s peak ratios were [ for all these samples (table ). these results showed that scrapings collected from all piglets yielded high-quality rna suitable for real-time pcr and microarray analysis. no random (necrotic) and/or fragmentized (apoptotic) dna was visible after gel electrophoreses of dna samples extracted from any of the piglets, indicating that the majority of cells imbedded in the epithelial layers of all the piglets were not apoptotic or necrotic (results not shown). mid-jejunal mucosal gene expression analysis was performed using a homemade pig cdna small intestinal microarray [ ] . in two separate hybridization experiments, mrna expression levels in an uninfected rna pool (n = ) were compared to expression levels in rna pools prepared from -and -h infected piglets (both n = ). for both comparisons, dye swaps were performed. probes that hybridized differentially with a ratio (fc; infected over uninfected) of . or [ . in both slides of the dye-swap and that were identified with the lowest possible false-discovery rate (fdr; based on sam, [ ] ), i.e., . % for the -h comparison and . % for the -h comparison, were selected for further analysis. raising of the fdr to a maximum of % did not identify additional probes with a fc \ . or [ . in either comparison. selected probes were sequenced and annotated after blastn or blastx analysis (when not yet annotated). for each differential expressed probe, the mean fc calculated from the two dye-swap slides is presented in table . when cy -and cy -labeled cdna was prepared from the same uninfected rna pool and simultaneously hybridized on the array, none of the probes that hybridized differentially in the -and -h comparisons hybridized differentially (results not shown). six out of the nine probes that hybridized with a fc of . -fold or more in the -h comparison (panel ''higher in infected'') also hybridized significantly more strongly in the -h comparison. for three of these probes (r -r ), the ratio of differential expression further increased with time. in contrast, only one probe (r ) hybridized significantly less strongly at both time points. based on literature search and data mining, a tentative function was assigned for the genes identified by blast analysis (see table ). in addition, the fc (infected over uninfected) of genes which were also found to be regulated in a previous study by cuadras et al. [ ] , i.e., h after infection of human intestinal epithelial caco- cells with rotavirus live virus vaccine rvv, is provided in parentheses in table after the annotations. probes coding for ifn-c, tnf-a, gm-csf, and il- , , , , and were spotted on the array. however, the fluorescence intensity was not higher than the background threshold for any of these probes, indicating that mrna concentrations of these cytokines (including ifn-c) in infected and uninfected mucosal scrapings were too low to detect by microarray analysis, probably due to the relatively low percentage of cytokine-producing (immune) cells present in intestinal mucosa [ ] . northern blots (nb) loaded with equal amounts of rna from each of the piglets were hybridized with p -labeled cdna probes homologous to six differentially expressed mrnas and to three mrnas that were not identified as differentially expressed. for all nine mrnas, the length of the transcript(s) detected on blots were comparable to the length of porcine or human mrna reference sequences posted in the ncbi databank or reported in the literature. in accordance with array data, nb analysis showed that expression levels of gbp- , krt , sat, mgam, and casp mrnas were significantly higher in both -hinfected piglets than in uninfected piglets. for all of these mrnas, hybridization intensities for piglet were nearly equal to that of piglet . gbp- , krt , sat, and mgam mrna expression was also up-regulated in infected piglet , slaughtered at hpi. this indicated that the response to rotavirus infection in this -h piglet was comparable to the response observed in both -h piglets. however, no significant up-regulation of these mrnas was observed in the other piglet slaughtered at h ( ), indicating that this piglet responded differently to the rotavirus infection than its -h replicate and the two piglets slaughtered at h. in accordance with array data, nb analysis showed that the expression level of ifabp mrna was significantly lower in -h-infected piglets than in uninfected piglets. for mrnas that showed no significant differential expression on the arrays (glutathione-s-transferase, calbindin-d, and aldolase-b), no large differences in hybridization intensities were observed between uninfected piglets and the two piglets slaughtered hpi and one of the piglets slaughtered hpi ( ). however, significantly lower hybridization intensities were observed for calbindin-d and aldolase-b mrnas for piglet than for its -h replicate. using microarray analysis, we detected a set of genes that are differently expressed in rotavirus-infected jejunal mucosa compared to uninfected mucosa. for nine mrnas, expression levels in individual piglets were analyzed by nb. these analysis confirmed the array data. in addition, nb analysis showed that one piglet slaughtered at hpi responded quite similarly to rotavirus infection as both h piglets did, whereas its -h replicate did not, despite the fact that this latter piglet also showed an ifn-c mrna response. in addition, out of the genes differentially expressed at hpi also reacted at hpi. these results indicated that three out of four infected piglets responded quite analogously. because we used a limited number of germ-free piglets per time point and measured responses in a mixed population of cells, we imposed stringent criteria for selection of genes ([ . -fold up-or down-regulation and a false-discovery ratio of less than %). using this approach, we minimized the chance of selecting genes hybridizing differentially solely due to inter-animal variation in gene expression and/or cell composition. however, such stringent selection criteria could have excluded the detection of more rotavirus-regulated genes, especially of genes regulated exclusively in specific types of cells that are present in low quantities in the jejunal mucosa. the different responsiveness of one of the -h piglets, however, obliged us to interpret our overall results carefully, especially, concerning the five genes that reacted solely at hpi (txn, frk, dppc- , if , and actb; see table ). nevertheless, data mining revealed relevant relationships between these five genes, h response genes, and processes known to be important for rotavirus pathogenesis. cuardras et al. [ ] measured the transcriptional response in the human enterocyte cell line caco- , , , and hpi with rhesus rotavirus live vaccine. four genes up-regulated in our experiments (gbp- , sat, mgam, ppa ) were also up-regulated hpi in caco- cells. recently, aich et al. [ ] profiled the transcriptional response in surgically prepared jejunal loops from -dayold colostrum-deprived calves after h of perfusion with bovine rotavirus (brv). several genes for which we detected more than . -fold up-or down-regulation (txn, nadh , sglt , actb, sat, casp , and ppa ) were also present on the cdna array they used (ncbi geo acc. number gpl ). none of these genes showed a differential expression of twofold or more in their study. the different route of administration and virulence of the strain used, the digestive differences between the jejunum of omnivores and herbivores, and, in the case of the study of cuardras et al., the various specialized cell-types present in the jejunum of living animals versus cultured colon-derived caco- cells are probably responsible for the poor correspondence between these three studies. based on relevant literature and functional information in databanks, we assigned a function and a possible type(s) of cell(s) responsible for expression for most of the genes on our list (fig. ) . in this hypothetical model, information from existing models dealing with the pathogenesis of rotavirus infection [ , , ] and the development and maintenance of the small intestinal epithelium [ ] were used to fit in our data. possible functions of these genes in relation to processes and pathways known to be important for rotavirus pathogenesis are discussed below. measurements of villus length indicated that considerable numbers of epithelial cells were lost from the tip of the villi, including (infected) mature enterocytes. in part, down-regulation of genes involved in transport of ions and nutrients over the membranes of mature enterocytes, like meprin a, sglt , and ifabp , may be a direct result of this loss. in another part, replication of rotavirus in enterocytes imbedded in the epithelial layer could have down-regulated transcription of these genes. this may also be the case for other down-regulated genes from our list, especially for genes detected only at hpi (r -r , table ). in addition to down-regulation of sglt , ifabp , and meprin a, we observed up-regulation of two other genes that may affect the absorptive and digestive function of the intestine: a gene coding for a protein carrying a ca + -permeable cation channel cd /ige fc receptor subunit b domain (ms a ) and a gene homolog to a bacterial oligo/dipeptide permease (dppc- ). it is tempting to link up-regulation of ms a directly to nsp induced enhancement of ca + permeability of the plasma and er membranes in intestinal epithelial cells [ , ] . likewise, up-regulation of the dppc- homolog may be related to enhanced laminal secretion of peptides and amines by uninfected epithelial cells, a process believed to be triggered by raised [ca + ] cyt [ ] . characterization of these porcine transcripts/proteins is needed to provide further insight in the role of these genes in rotavirus pathogenesis. the same applies for the tmem b gene. this gene showed the highest level of up-regulation. so far, only a duf protein domain with unknown function has been predicted in tmem b. recently, it was reported that rotavirus infection in infant mice induced apoptosis in vivo [ ] . although dna analysis showed that the majority of cells present in infected mucosal scrapings were not apoptotic, we observed upregulation of the apoptosis effector protein casp . this suggests that programmed cell death in the epithelial layer was stimulated by rotavirus infection. nb analysis detected a considerable level of casp mrna expression in uninfected mucosal scrapings. this constitutive expression of casp is, most likely, related to the process of maintenance of the absorptive status of the intestinal epithelial layer. a process in which mature enterocytes continually die due to apoptosis and are replaced by differentiating cells migrating from surrounding crypts to the tip of the villi [ ] . in contrast to mature enterocytes, an in vivo study in mice showed that goblet cells are largely spared from apoptosis in rotavirus-infected mice [ ] . moreover, migration of goblet cells from the crypt to the tips of the villi was stimulated in these mice. we found up-regulation of the goblet cell marker gene krt [ ] and the lower and mid-villus immature enterocyte marker gene mgam [ ] . this could indicate that transcriptional activity in both of these cell types was promoted. stimulation of apoptosis in rotavirus-infected enterocytes and higher proliferation/differentiating activity in goblet cells and immature enterocytes could be a coordinated response of the jejunum to remove infected enterocytes and overlay villus tips with fresh enterocytes, goblet cells, and mucus layer. we did not detect genes on our array that were directly associated with cell-cycle progression/arrest. however, down-regulation of the nuclear kinase frk (an antagonist of cell proliferation) and tms f may be associated indirectly with this process. in humans, tms f is strongly homologous to tm sf , a protein that reduced the ability of the crypt cell line ht to proliferate [ ] . several other genes that may play a role in cell death and repair were regulated. sat was up-regulated, and txn and prr were down-regulated. for this later protein, reduced expression in cells was correlated with increased sensitivity to taxane-induced cell death, a caspase-independent process characterized by the polymerization of tubulins to extraordinarily stable microtubules [ , ] . txn is the major carrier of redox potential in cells, and it is crucial for the defence of cells against oxidative-stressmediated apoptosis. txn also regulates gene expression by increasing binding of redox-sensitive transcription factors like p [ ] , nf-jb [ ] , and the nrf- /polyamine- table . information about the paneth cell marker thoc is provided in reference [ ] transcriptional response to rotavirus inhibited sat expression [ ] . therefore, up-regulation of sat gene expression at hpi may be directly related to down-regulation of txn at hpi. sat is a rate-limiting enzyme in spermine/spermidine metabolism. acetylation of these polyamines by sat promoted their degradation and excretion [ ] . recently, it was reported that depletion of polyamines suppresses apoptosis in normal intestinal epithelial cells by akt-kinase-mediated inhibition of casp activity [ ] . nb analysis showed that the increase in sat mrna expression coincided with the goblet cell marker krt . therefore, it would be interesting to determine whether sat expression in goblet cells can be stimulated by rotavirus infection and whether it plays a role in protecting these cells from apoptosis [ ] . interestingly, it was recently demonstrated that rna viruses can directly modulate polyamine metabolism by regulation of sat transcription and splicing [ ] . a most interesting gene that we found more than tenfold up-regulated codes for an as yet not-well-characterized hypothetical human protein (loc ) carrying a phospholipase a inhibitor domain (pla -inh). the magnitude and kinetics of up-regulation of this gene corresponded exactly with gcnt , suggesting that this gene was expressed in the same types of cells as gcnt , most likely mucus-producing goblet cells and/or differentiating (immature) enterocytes. the amino acid sequence translated from our pla -inh est showed an overall amino acid identity of %, and all cysteines aligned perfectly with cysteines of the human loc protein and of other proteins that bear a typical pla -inh domain. pla s comprise a diverse family of cytosolic and secreted enzymes that hydrolyze membrane phospholipids to free fatty acids. they play an important role in many exogenous and intracellular processes, ranging from fatty acid metabolism and lysis of membranes to the synthesis of arachidonic acid (a-acid), an essential precursor for the production of inflammatory mediators such as eicosanoids. secreted pla s are calcium-dependent enzymes. cytosolic pla s (cpla ) can also be calcium-independent. a moderate increase in [ca + ] cyt mediated translocation of calcium-dependent cpla to intracellular membranes where it hydrolyses phospholipids to a-acid [ ] . a similar effect was observed after activation of the ms a calcium-permeable cation channel (up-regulated here at hpi, see above) on the surface of mast cells [ ] . perhaps, enhanced expression of a pla -inh in our study may be a countermeasure of specific intestinal epithelial cells to normalize and/or inhibit pla enzyme activity in response to extra-and intracellular changes in [ca + ] evoked by rotavirus replication, either to protect specific cells from extra-and intracellular membrane-damage or to negatively regulate a-acid production. with respect to the latter process, we observed down-regulation of the enzymes cyp c and acsl , which both utilize a-acid acid as substrate. interestingly, the capsid protein of parvovirus possesses pla activity [ ] , and hmcv particles carry a cell-derived pla activity [ ] . for both viruses, pla activity appeared to be essential for infectivity. our results showed that ifn-c mrna expression in the jejunum of infected piglets peaked around hpi and tended to decline beyond this time point (see fig. ). this suggests that ifn-c was produced for a short period. recently, aich et al. [ ] measured the mrna expression levels of several cytokines in jejunal loops perfused for h with brv. however, they observed no ifn-c mrna response. because our orally administered rotavirus needed time to reach the jejunum, our h infection period represents a shorter period than h of perfusion. after h of perfusion, expression of ifn-c mrna may have dropped to normal levels. interestingly, they did detect a rotavirus-induced il- (alias ifn-b ) mrna response at hpi [ ] . recent studies showed that the interplay between ifn-gamma and il- controls the influx and clearance of neutrophils and, subsequently, the transition to a more sustainable influx of mononuclear cells during acute inflammation [ , ] . therefore, it would be interesting to study which immune cells produce ifn-c and il- and whether an orchestrated action of these cells regulates an influx of vital immune cells in the jejunum after rotavirus infection. the ifn-c-inducible gbp- gene was up-regulated hpi. overexpression of gbp- and gbp- in hela cells and nih t cells abrogated the cytopathogenic effect mediated by vsv and emcv, respectively, by an unknown mechanism [ , ] . furthermore, it was shown that expression of murine gbp- in nih t cells neutralized the cytotoxic effect of the taxane drug paclitaxel [ ] . this drug specifically stimulates polymerization of tubulins to extraordinarily stable microtubules. these stable microtubules interfere with the function of normal microtubule filaments and inevitably induce cell death [ ] . the reduced expression of prr we observed (discussed above) may be an indication that the formation of extraordinarily stable microtubules in intestinal epithelial cells actually takes place in response to rotavirus infection. in fact, several studies have shown that rotavirus infection induces disorganization of the cytoskeleton network and microtubule filaments in enterocytes [ , , ] . moreover, we also observed the up-regulation of the ifn-a/b-inducible gene ifi , a cytosolic protein associated with microtubular structures, and the cytoskeleton gene actb. therefore, enhanced expression of gbp- in specific intestinal epithelial cells could contribute to a cellular mechanism(s) that impairs and/or prevents disorganization of the microtubule filaments. further in vivo studies are needed to determine whether the genes identified in this study are representative for an intestine with a normal microflora. if so, more focussed studies involving in situ hybridization and immuno-histology may specify where along the crypt-villus axis and in which type of epithelial (or immune) cells elevated or reduced expression of these genes is induced. comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and bovine coronavirus human cytomegalovirus carries a cell-derived phospholipase a required for infectivity interferon-induced guanylate binding protein- (gbp- ) mediates an antiviral effect against vesicular 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regulate cell density-dependent proliferation a viral phospholipase a is required for parvovirus infectivity akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase- after polyamine depletion keratin serine phosphorylation is a stress and intestinal goblet cell marker acknowledgments the authors would like to thank arie hoogendoorn for his assistance in the animal experiment.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -lq ah x authors: mayo, m. a.; haenni, a.-l. title: report from the (th) and the (th) meetings of the executive committee of the international committee on taxomony of viruses date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: lq ah x nan a. taxonomic proposals yet to be voted on by ictv (i) (b) new taxonomy for the reverse-transcribing viruses, retrotransposons and retrons. formal proposals were discussed for the classification of all taxa containing viruses that replicate by using a reverse transcription step (pararetroviruses; families retroviridae, hepadnaviridae, caulimoviridae, pseudoviridae and metaviridae) and also the classification of retrotransposons and retrons. the ec felt that the idea of grouping the reverse-transcribing elements has already been accepted by the ictv as this has received broad support from those who work with such elements. however, the detail of the proposal involved the use of the taxon sub-order, which is not a recognized virus taxon. workers in the field had not been supportive of creating an order to contain retrotransposons and retrons, largely because of there being no lateral transfer of these latter elements. also it was noted that there was a question of whether or not ictv should be concerned with these elements. the proposals were therefore referred back to the proposers for amendment (proposal numbers . - f. ). at ec there were extensive discussions of proposals to revise the statutes under which ictv operates and to revise the international code for virus classification and nomenclature (icvcn). the proposals arose in part from papers published in vdn [ , ] . in response to the proposal that ictv should provide for each approved species a latinized binomial name to link its common name(s), the 'type' description and the classification, the ec felt that there was little support in the virology community for using latinized names. the use of binomial name forms is at present being considered by various study groups (sgs). it was proposed that the icvcn be modified so as to become congruent with the other codes of nomenclature. however, ec members could see no reason to link virus nomenclature to that of organisms such as animals, plants and microbes. moreover, significant disadvantages were identified for virologists if the rules in icvcn were to be changed to be like those contained in other codes of nomenclature. in particular, and in contrast to practice under other codes of nomenclature, the icvcn states that there is no rule of priority in virology. this greatly simplifies the nomenclature of virus taxa. the changes proposed further called for the publication of draft minutes of ec meetings. members thought that this would be unhelpful (necessitating pre-draft minutes). the publication of vdn reports after ec meetings together with the public exposure of taxonomic proposals on the ictv website were felt to meet the criterion of transparency. the changes proposed further stipulated that ictv should keep an official list of virus names that were used previously but are no longer in current taxonomy. the lack of such a list was felt to be a weakness of current procedures. the ec will work towards creating such lists but without creating a new rule to make it an obligation to include old virus names in the lists. the proposition that ictv keep synonyms in all major languages was felt not to be feasible. it was also stated that taxon names cannot be translated and that any translation attempted becomes a vernacular name and thus not the responsibility of ictv. it was proposed that ictv use the internet more for communication. the ec already recognizes this as a desirable aim and this will be developed along with other changes to the web-based operation of ictv. it was felt not to be useful to specify a deadline. furthermore, it was proposed that all taxonomic information relevant to the decisions of the ictv be made available via the internet in a database under the guidance of the virus database sc. the ec thought that the message board on ictv net where comments on proposals are posted goes a long way towards achieving this. it was stated that a database is being developed so as to have accessible all currently approved virus taxa. this development should satisfy the spirit of the proposed amendment. the proposal that ictv be responsible for the classification and nomenclature of viruses at all taxonomic levels was felt to be likely to lead to an overwhelming task for the sgs of ictv and therefore to be unworkable. the proposal that taxonomic groupings indicated by virus names shall be based, if possible, on evolutionary analyses reflects current ictv objectives. the proposition that ictv should maintain a public record of the characteristics that distinguish each approved taxon from related ones or from those with which it might be confused, is one of the aims of the development of the ictv database (ictvdb). the ec did not approve any of the proposals to change the statutes or icvcn. however, many of the points raised by the proposed changes are being addressed, either by current practice or by various developments that are currently in progress. most notably, it is expected that the development of the ictvdb will address many concerns. it was decided that a working group be set up to review the code in a comprehensive fashion taking into account the proposals on the table. as a means of promoting contact between ictv and its national members and to ensure that lists of members are up-to-date, it was proposed that the statutes be modified such that societies would be obliged to either renew national members or replace them. in effect, national members would have a -year tenure that could be renewed indefinitely. this was accepted unanimously by the ec and the ictv in plenary session. it was subsequently also approved by the virology division. it has been agreed with the ec that ( ) in order to propose a species, at least one entry of an isolate description should be made; ( ) data entry into the ictvdb could be made at the isolate level, the first taxonomic level being the species; ( ) an isolate is the only real data entry; ( ) the virus database sc will work with the national center for biotechnology information (ncbi) to link virus genome descriptions with isolate data entered into the ictvdb; and ( ) a simple two-page data entry process will be developed and supplied to ncbi to facilitate the submission of isolate data into the ictvdb and its linking with genbank entries. discussions have taken place with the american society for microbiology about the long-term support of ictvdb and funding of ictv ec activities. bulk data entry processes will be developed for entry from private databases and collections of isolates, resulting in the accessing of information on nearly , virus isolates, including the world reference collection of foot-and-mouth disease viruses, major plant virus databases, and some very significant collections of arboviruses. there is agreement that the next arbovirus catalogue will be developed with the assistance of the ictvdb provided there is sufficient arbovirus information and entry. the ictvdb has moved from the australian national university to columbia university. the ictvdb has the copyright (in the name of the ictv) in order to protect the information for the ictv and virologists. an opinion poll conducted at the paris icv showed that about % of those who voted were in favour of adopting a non-latinized virus binomial (nlvb) system for constructing the names of virus species. in this system, each species name would end with the genus name rather than the word "virus". however it was recognized that if applied universally this system would result in some strange names, and it was acknowledged that some sections of the virology community (as represented by sg opinion) are firmly against nlvb. the advantages of nlvb were summarized as follows:- • it conveys a sense of the affiliation of the species. • it would allow more flexibility in naming, as for example with latin binomial names for higher organisms. • it would make clear the distinction between species names and virus names. the disadvantages were summarized as follows:- • it would necessitate many name changes and thus conflict with the stability principle. • it would create difficulties for the ictvdb. • species in a family but unassigned to a genus could not have a genus name in the species name. • it would necessitate formal name changes when species are moved from one genus to another or if an unassigned species were assigned to a genus. • it would produce odd names because of curious genus names in use, in particular when these contain numerals. the president stated that he had decided to listen to opinion rather than be an advocate for the system and suggested that any adoption be on a case-by-case basis. the picornaviridae sg had suggested that the use of nlvb for new names be strongly encouraged but that existing names not be changed, but the ec felt that any changeover to nlvb should then apply to all species in a genus. at the end of the discussion, a vote showed that ec opinion was evenly divided between those in favour of nlvb and those not in favour, and that of those in favour, half were strongly in favour and half were only moderately supportive. the use of greek characters in species and genus names can be a major complication because of difficulty in dealing with the characters electronically. there was a general feeling that this problem should be addressed and that it would be legitimate to convert the affected names to names consisting entirely of roman characters. the proposition was passed to the chairs of the prokaryote virus sc and the invertebrate virus sc for them to consult the affected sc members and/or sgs. a paper was presented outlining ideas for the use of standard suffixes on vernacular names to signify the taxonomic level of the viruses being described. the system is used widely at the moment for viruses in a particular taxon, usually genus, but no distinction is made between "potyvirus" as a member of the genus potyvirus and "potyvirus" as a member of the family potyviridae. a comprehensive scheme was presented that includes distinctive endings for all taxa including those containing viroids. the ec thought that it would be useful to promote the simplest scheme ('-virad' for members of an order, '-virid' for members of a family, '-virin' for members of a subfamily and '-virus' for members of a genus) but that it would be better to omit endings for as yet unused taxa and for the viroid taxa, at least until it was clear that virologists welcomed the scheme. a paper will be prepared in the near future for publication in vdn to present the suggestions in detail. a proposal was discussed that there should be a fixed degree of sequence relatedness that would indicate that two viruses were either members of the same species, or belong to different species in the same genus, or belong to species in different genera. these values would then be applied to all classification decisions. there was little support for this idea as all ec members agreed that genera differed in the detail of species demarcation criteria. it was pointed out that fixing these lists of criteria was an important task for individual sgs. it was proposed that the category "tentative species" be eliminated from taxonomic usage because a virus can either be a member of a species or it cannot. the tentative assignment had been a source of confusion in the ictv reports and for ncbi. however, it was pointed out that at least for some virologists the idea of identifying a tentative species as a first step towards recognizing a species was well understood. most ec members agreed with the proposal, and sc chairs were asked to ask their sgs to take note and consider what to do with the tentative species in their lists. at ec , it has been agreed that considering proposals for taxonomic change was not well served by being part of the plenary session. the use of postal balloting has allowed members more time to consider proposals, and proposals are now available for inspection and for comment on the ictv web site. therefore it was decided that in normal circumstances all voting on taxonomic proposals will be done by postal/electronic balloting. at ec it was decided that the current statutory definition of who is entitled to vote on taxonomic proposals was unsatisfactory as sg chairs were not entitled to vote. it was agreed to propose changes to statutes such that all members of any sc become entitled to vote. this proposal was subsequently approved by the ictv and then by virology division prior to the plenary session at the san francisco icv. a proposal from david mindell that ictv adopt a statement explicitly linking taxonomy and evolution was discussed. it was pointed out that a statement about the objectives of ictv and how and when evolution can be linked with taxonomy is already in the introduction to the th report. in the light of the successful workshop held at the american society for virology meeting in montreal in , it was agreed that a symposium every to years reviewing virus evolution and the state of virus taxonomy would be valuable. ec members agreed to encourage people to organize satellite symposia that would promote interest in taxonomic matters. virus nomenclature; continuing topicality viral nomenclature, where next? authors' addresses: dr. m. a. mayo, scottish crop research institute e-mail: mmayo@scri.sari.ac.uk parc d'innovation, boulevard sébastian brandt, illkirch, france. -redaktion: sachsenplatz - key: cord- -qote nx authors: vassão, r. c.; sant' anna, o. a.; pereira, c. a. title: a genetic analysis of macrophage activation and specific antibodies in relation to the resistance of heterogeneous mouse populations to mhv infection date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: qote nx the genetically selected high antibody responder mice (h(iii)) are susceptible and the low antibody responder mice (l(iii)) are resistant to the experimental infection with mouse hepatitis virus (mhv ). the mortality rates of the f( ) hybrids and of the f( ) segregants showed the codominance of the susceptible and resistant characters. the direct individual intrapopulation correlation between the induction of antiviral state in macrophages activated by ifn gamma and the resistance to the virus infection, showed that an antiviral state could be induced in resistant mouse macrophages, whereas in susceptible mouse macrophages no restriction of virus replication could be observed. a direct inter- and intrapopulation correlation of pre-existing antibody titres against mhv with the mortality and a direct interpopulation correlation of those titres with the mean survival time of susceptible animals was shown. the data indicate, among the mechanisms of resistance against the virus infection, a role of ifn gamma macrophage-activation and of antibodies against mhv which may delay the mean survival time in susceptible animals. the mouse hepatitis virus (mhv) strains of coronavirus are responsible for well-known epizootics of enteritis that occur endemically in most mouse colonies. most of the animals in these colonies were found to have antibodies against different types of mhv, such as mhv , which have been isolated from a variety of mouse strains under diverse conditions [ , , , , ] . the mhv was isolated by dick et al. [ ] and has been used as a model of viral infection in which resistance varies according to the genetic background of the mouse strain [- , , , , , ] . the resistance pattern of mouse strains has been shown to be not directly linked to the presence of antibodies in sera of animals from contaminated colonies [ ] . the virus replication in target cells, the antiviral state induced by interferon (ifn) and the expression of a monokine with procoagulant activity (pca) have been implicated in the resistance/susceptibility of the genetic homogeneous or heterogeneous mouse populations to the mhv infection [t, , , , , , , . we have shown that resistance to mhv infection can be a consequence of a t-cell-dependent mechanism, in which the production of ifn gamma and the sensitivity of macrophages to ifn gamma play an essential role [ , , ] . the genetically defined high (h) and low (l) responder selected mice and their segregants and hybrids have been proved to be a useful tool for studying the intervention of multiple alleles in the polygenic control of infectious diseases such as salmonella typhimurium and rabies virus infection [ , ] . moreover, the analysis performed in the f heterogeneous population allows the elucidation of the major specific and nonspecific traits implicated in resistance/susceptibility to a given pathogen, as well as that of the environmental and genetic factors that play in these characters. mice selected for high (h) and low (l) responsiveness, were obtained by bidirectional selective breeding, and the effect of the polygenic regulation of responsiveness to selection antigens is essentially multi-specific, i.e., selected genes regulate the antibody response to many complex immunogens unrelated to those used during the selective breeding, the high or low states resulting from distinct mechanisms, including the regulatory role of macrophages [ ] . the hm and lm mice, although showing no mhv in their tissues, were chronically infected by a mhv strain, and had in their sera distinct levels of antibodies against mhv . h m mice were shown to be fully susceptible and lm mice fully resistant to the experimental infection with mhv . one of the mechanisms suggested to be involved in the resistance against this virus infection was the ability of macrophages to develop an antiviral state following ifn gamma activation [ , . the present study was undertaken in an attempt to investigate whether the sensitivity ofmacrophages to the induction of an antiviral state and the antibody responsiveness characters, correlated individually with the resistance to mhv and were influenced at least partially by the same genetic control. therefore the investigation was directed towards a co-inherited expression of the two traits, aiming to elucidate the complexity of the biological factors that intervene in resistance to infection. high (hm) and low (lm) antibody responder mice from selection ill [ ] , from the laboratorio de imunogenetica, instituto butantan, were used in the experiments. mice of both sexes were analysed at months of age. interline reciprocal crosses of (h m x ln~)f hybrids, f segregants and backcrosses (bchh~ and bclh , were analysed as well. virus mhv originally obtained from dr. j. l. virelizier, institut pasteur, paris, france, was cloned by limiting dilution. one plaque was selected and amplified on l cells to serve as the inoculum for future stocks - ] to limit spontaneous mutations. the stocks were always titrated by plaque assay on l cells as previously described [ ] . aliquots containing x l s plaque forming units per milliliter (pfu/ml) were stored at - °c and used in all experiments. for the study of the resistance to virus infection, the animals were subcutaneously inoculated with pfu of mhv , and mortality was recorded daily for days. the peritoneal exudates were collected by peritoneal lavage with ml of rpmi, and centrifugated at g for minutes. the peritoneal exudate cells were re-suspended at a concentration of x cells/ml of rpmi containing % of fetal calf serum (fcs) and cultured on -well plates ( gl/well). the cells were incubated for h at °c in % co and washed three times with medium after vigorous shaking to remove nonadherent cells. ninety percent of the cells were macrophages as determined by their ability to take up zymosan particles. peritoneal macrophages were treated with u/ml of murine recombinant ifn gamma (holland biotechnology, leiden, holland). twenty-four hours later, activated or nonactivated cultures were infected with mhv at a multiplicity of infection (m.o.i.) of . , in order to study the inhibition of mhv replication. the supernatants of cell cultures were collected at h after infection and tested for the virus titre by plaque assay [ ] . for the study of the individual correlation between in vivo resistance and in vitro inhibition of virus replication by ifn gamma, peritoneal macrophages from hm, lm and f segregants were collected without sacrificing the animals, which were infected with mhv a week later. the macrophages were treated with ifn gamma, infected with mhv and the supernatants titrated, as described above. mice from the colony were bled from the retro-orbital venous plexus and the individual anti-mhv antibody titres in their sera, reported as log , were expressed by the rec]procal of the highest serum dilution producing a % inhibition of cytopathic effect induced by mhv on l cells [ ] . the results in table show the resistance/susceptibility of mice to m h v infection, and indicate that the macrophages from lm or resistant f or f mice were sensitive to the induction of an a n t i -m h v state by i f n gamma. however, under the same conditions, no a n t i -m h v effect could be induced in macrophages from the susceptible hin parental line nor from the susceptible r.c. vass~o et al. cultured macrophages were individually collected, treated for h with ifn gamma ( u/mt) before infection with . m.o.i, of mhv . virus titres were determined in the supernatants collected h after the infection. one week later, the same animals were infected subcutaneously with p f u of mhv , observed for days and the percent (~o) of mortality recorded. the mhv titres are the average +_ standard deviation fi or f mice. the in vitro treatment with i f n gamma, which induced an anti-mhv effect only in resistant mouse macrophages, correlated with the in vivo resistance observed after mhv infection. figure shows the linear regression analysis of individual values of the induction of antiviral state in macrophages of resistant and susceptible f phenotypes, indicating a significant direct correlation between the resistance and the induction of an antiviral state in the macrophages. the figure shows the linear regression analysis of the correlation between the mean survival time (mst) and the antibody titres against mhv in groups which exhibited mortality. it indicates a significant direct correlation (r = . , p < . ) between the mean survival time of susceptible mice infected with mhv and the anti-mhv antibody titres. the genetic studies based on the segregation analysis of resistant or susceptible inbred mouse lines to mhv infection showed that these characters are under the control of two major loci [ , ] . more recently, a comparative study performed in genetically homogeneous and heterogeneous mouse populations confirmed these results, and demonstrated the codominance of the susceptibility and resistance characters. the same genes were shown to be present in the isogenic balb/c (susceptible) and a/j (resistant) lines, as well as in genetically selected hh~ (susceptible) and l m (resistant) antibody responder mice [ ] . the use of hm and lm mice made it possible to analyse the relationship between distinct traits in segregation studies, and demonstrated a direct interpopulation correlation between antibody production to unrelated antigen and mortality to mhv infection [ ] . the present studies go deep into the intra-and interpopulation analysis of the correlation between resistance to infection and antiviral macrophage action induced by ifn gamma or specific anti-mhv antibody production. by studying, individually, the mortality of the parental mouse lines, their f~ hybrids, f segregants and backcrosses, the anti-mhv activity of their macrophages after ifn gamma activation, as well as the titre of antibodies against mhv , we were able to show: i) a direct individual correlation between the resistance and the ability of macrophages to partially inhibit mhv replication after ifn gamma activation (table and fig. ) , ii) an inter-and intrapopulation inverse correlation between the mortality and the antibody titres against mhv (fig. ) , iii) an interpopulation direct correlation between the mean survival time of hn~, f~, f and bchm susceptible animals and the antibody titres against mhv (fig. ) . the genetic analysis performed in this study, demonstrates that the resistance to mhv experimental infection and the ability of ifn gammaactivated macrophages from resistant mice to display an antiviral activity, are submitted to at least partially common genetic control. this brings further support to our previous suggestion that one of the main mechanisms involved in the resistance of mice to the mhv infection is based on the ability of their macrophages to restrict the virus replication upon ifn gamma activation [ , . although the participation of antibodies in the resistance to mhv infection remains unclear, the data here shown indicate their possible role in resistance mechanisms and in prolongating the mean survival time of susceptible animals. adult mouse hepatocytes in primary monotayer culture express genetic resistance to mouse hepatitis virus type a comparative study of resistance to mhv infection in genetically homogeneous and heterogeneous mouse populations a virus related to that causing hepatitis in mice (mhv) susceptibility/resistance in mouse hepatitis virus strain and macrophage procoagulant activity are linked and controlled by two non-h- linked genes genetically determined resistance to mouse hepatitis virus type is expressed in hematopoietic donor cells in radiation chimeras new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice immunopathology of mouse hepatitis virus type infection. i. role of humoral and cell-mediated immunity in resistance mechanisms induction of monocyte procoagulant activity by murine hepatitis virus type (mhv ) parallels disease susceptibility in mice genetic study of mouse sensitivity to mhv infection. influence of the h- complex a major role of macrophage activation by interferon gamma during mouse hepatitis virus type infection. i. genetically dependent resistance a major role of macrophage activation by interferon gamma during mouse hepatitis virus type infection. ii. age dependent resistance acquired immunity of a/j mice to mouse hepatitis virus infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma in vivo depletion of interferon gamma leads to susceptibility of a/j mice to mouse hepatitis virus infection temperature sensitive mutants of mouse hepatitis virus type (mhv ): isolation, biochemical and genetic characterization virus specificity of the antiviral state induced by ifn gamma correlates with resistance to mhv infection gennari m (t ) rabies virus resistance of heterogeneous mouse populations to mhv infection immunity in genetically selected high and low responder lines of mice interaction between mouse hepatitis viruses and primary cultures of kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection in mice salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection selective breeding of mice for antibody responsiveness to flagellar and somatic antigens of salmonellae isolation of a routine hepatitis virus from swiss mice treated with antilymphocyte serum resistance of genetically selected mice to mhv infection is not dependent on the h release by macrophages role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type infection of susceptible and resistant strains of mice we thank d. mouton for critically reading the manuscript, f. souberbielle for editorial assistance and m.l. silva and a.c. barbosa for technical assistance. this work was supported in part by grants from the fapesp and cnpq. c.a. pereira and o.a. sant'anna are recipients of cnpq research fellowships, key: cord- -ido mh authors: nagy, Éva; lomniczi, b. title: polypeptide patterns of infectious bronchitis virus serotypes fall into two categories date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ido mh molecular weights of six major polypeptides of infectious bronchitis virus (ibv) are: . , ; . , ; . , ; . , ; . , or , , and . , dalton. according to the mobility of protein the polypeptide patterns of ibv serotypes fall into two main categories. medical institute, helsinki; strain l e d (connecticut serotype) and strain pv (massachusetts serotype) were isolated in h u n g a r y ( ) . virus strains were propagated in -day-old embryonated eggs and purified at ° c as follows. virus was clarified at , × g for minutes and the supernatant pelleted at , × g for hours in a type rotor of beckman l - b ultracentrifuge. the pellet was gently resuspended in n t e buffer (nac i ram, tris-hc] p h . mm and e d t a mm). after further clarification the virus suspension was pelleted through per cent (w/v) sucrose made up in nte-buffer in a sw- rotor with , × g for hour. r, esuspended and clarified virus was subjected to a velocity gradient centrifugation for i hour into to per cent (w/v) sucrose. the band (or double band) containing at least half of the virus applied was further purified on a to per cent (w/v) sucrose gradien~ at , × g for hours. equilibrium centrifugation alone resulted in virus preparations of insufficient purity as described ( ) . virus peak was recovered b y sedimentation and taken up in sample buffer ( mi~{ tris-hc p h . , per cent glycerol and . per cent bromphenol blue). virus was dissolved in per cent sodium dodecyl sulphate (sds) and , per cent -mercaptoethanol in a boifing water bath for minutes. samples were run in per cent slab gels ( : . b y weight acrylamide bis-acrylamide) using a discontinuous buffer system ( ) for hours at volt. gels were fixed in a mixture of methanol, acetic acid and water ( : : ) and stained in . per cent coomassie blue. molecular weights were calculated by using the following markers: myoglobin ( , ), ovalbumin ( , ), bovine serum albumin ( , ), newcastle disease virus ( ) and sindbis virus proteins ( ) . all ibv strains examined showed one of two protein patterns with polypeptides having apparent molecular weights in the range of , and , dalton (fig. ) . six major polypeptides were observed in all strains: vp (virion polypeptide of , dalton), vp , vp , vp , vp or vp (depending on the serotype) and vp . minor polypeptides were also noted (vp , vp ) but their viral origin is uncertain. the viral origin of vp is also doubtful for two reasons: a) there was a tendency of reduction of the relative amount of vp when velocity gradient ccntrifugation was combined with equilibrium gradient purification, h) this protein is equally present in preparations of egg-grown sindbis virus which have been purified only by pelleting the virus under a per cent sucrose layer, which also suggest that it may be a cellular contaminant (fig. ) . according to the variation in the mobility of a "broad" polypeptide at least two basic polypeptide patterns were recognized (fig. ) . pattern m (named after massachusetts) includes strains from two serotypes, massachusetts and georgia (se ), and here the "broad" protein has an apparent molecular weight of , dalton. four strains (beaudette, m , pv, and lerida) belonging to the massachusetts serotype all showed identical m-pattern (pv is not shown). pattern c (named after connecticut) is characterized by a smaller "broad" protein of an apparent molecular weight of , dalton, and includes the five remaining serotypes studied: connecticut, delaware (gray), iowa , iowa and australia t. ]~va na.gy and b. lo~iozi: three connecticut strains (one prototype strain from weybridge, another from helsinki and led) also exhibited identical c-pattern. apart from variation in the mobility of the "broad" protein it was found that in strain gray and led vp was prominent relative to the other strains. whether this suggests a closer relationship of the two strains, or an artifact, is presently unknown. it is to be noted, that there was no difference between the polypeptide patterns of heated and unheated samples. the polypeptide patterns presented here classify ibv scrotypes into at least two main categories but no known biological properties common in serotypes belonging to a particular pattern can so far be identified. our results seem to be at variance with those of formerly published investigations ( ) in two respects: a) molecular weights of the majority of the polypeptides in that study fall in the range of , to , dalton, although using harsher reducing conditions resulted in polypeptides smaller than , dalton; b) no significant difference was found between the protein pattern of strain beaudette and connecticut. the polypeptide patterns of ibv presented here, however, show a partial similarity to that published by colli~s et el. ( ) , and a remarkable similarity to that obtained after a different purification method by lascer and i-iowagd ( ) . the similarity with pattern obtained in the latter works resides in the presence of smaller than , dalton proteins in the virions of ibv. the polypeptide composition of avian infectious bronchitis virus heterogeneity of infectious bronchitis virus grown in eggs antigenic variation in strains of avian infectious bronchitis virus serological comparison of strains of infectious bronchitis virus using plaque-purified isolants cleavage of structural proteins during the assembly of bacteriophage t characterization of infectious bronchitis virus serologic differences between strains of infectious bronchitis virus from new zemand, australia, and the united states isolation of the virus of infectious bronchitis genome of infectious bronchitis virus the polypeptide composition of avian infectious bronchitis virus particles coronaviruses: a comparative review characterization of the strueturm proteins of different strains of newcastle disease virus reproduction of togaviruses presence of infectious polyadenylated i%na in the eoronavirus avian bronchitis virus the evaluation of akr leukemia virus purity: the requirement for both velocity and isopycnie eentrifugation methods the skillful technical assistance of mrs. sz. sz esy is appreciated.authors' address: dr. ]~va nagy, budapest, p.f. , hungary. %eeeived february , key: cord- -vekok k authors: dewerchin, h. l.; cornelissen, e.; nauwynck, h. j. title: replication of feline coronaviruses in peripheral blood monocytes date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vekok k feline infectious peritonitis virus (fipv) (coronaviridae) causes the most lethal viral infection in cats: fip. the related feline enteric coronavirus (fecv) causes mild enteritis. why these feline coronaviruses manifest so differently in vivo is not known. in this study, infection kinetics (titres and antigen expression) of fipv - , and fecv - , were determined in peripheral blood monocytes from donor cats and compared to those in crandell feline kidney (crfk) cells. the infection kinetics in monocytes were host dependent. monocytes from cat were resistant to both fipv- and fecv-infection. monocytes from the other cats could initially be infected by both fipv and fecv but fipv infection was sustained in monocytes of only one cat. fecv-infection was never sustained and viral production was up to times lower than in fipv-infected monocytes. in crfk cells, fipv and fecv infection kinetics did not differ. in monocytes of a larger cat population (n = ) the infection patterns were also found. considering all investigated cats, / were not susceptible for fipv and fecv. the rest could be infected with fecv and fipv but / cats had monocytes that only sustained fipv infection and / sustained neither fipv nor fecv infection. two coronaviruses are described in cats: feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv). these feline coronaviruses are spread world-wide and infect cats and other members of the family felidae. an infection with fecv is usually subclinical, except in young kittens where it may cause mild to severe diarrhoea [ ] . in contrast, fipv infection causes a chronic and very often fatal pleuritis/peritonitis. it is the most important cause of death of infectious origin in cats. two forms of fip exist: the effusive or wet form with the typical effusions in body cavities and the less common non-effusive or dry form [ ] . characteristic lesions of both forms are granulomas on the surface of target tissues. despite the large biological differences, more than % of the genome is identical in fipv and fecv isolates from the same environment [ ] . therefore, it has been proposed that fipv arises from fecv by mutation but the exact mutation and the inducing factors have not yet been clarified [ , ] . the main difference between fecv and fipv is the invasive nature of fipv. fecv replicates mainly locally, in enterocytes of the intestine, whereas fipv also infects blood monocytes and spreads systemically [ , ] . the reason for this pathogenic difference is not understood. after infiltration of infected monocytes in the perivascular tissue, the infected monocytes and surrounding cells release numerous chemotactic and vasoactive factors [ , , ] . this leads to vasodilatation and increased vascular permeability and attraction of new monocytes to the area, which can be infected in turn. the outcome of the inflammatory reaction is a characteristic vasculitis which causes the venules to leak large amounts of protein rich plasma into the body cavity. the release of progeny virus also leads to the formation of virus-antibody-complement complexes which are concentrated around the small venules in the target organs [ ] . these complexes further activate inflammation. although the difference between fipv and fecv is very clear in vivo, it is not in vitro. the first in vitro characterisation of fipv strain - and fecv strain - was done by mckeirnan et al. [ ] in crandell feline kidney (crfk) cells. they found similar growth curves for fipv and fecv. the replication of fipv and fecv was also studied in peritoneal macrophages [ ] . it was reported that fecv infected fewer macrophages and reached lower production titres than fipv. the in vivo relevance of these infection studies is most likely higher than those performed in a continuous cell line. but, until now, the fipv and fecv replication cycles have never been studied in the in vivo target/carrier cell of fipv: the feline blood monocyte. in the present study, we present the in vitro replication kinetics of fipv and fecv in the target cell of fipv, the blood monocyte. it was found that the replication kinetics were dependent on the origin of the cells. no differences between fipv and fecv were found in crfk cells. viruses a third passage of fipv strain - and fecv strain - on crfk cells was used [ ] . fecv strain - was obtained from the american type culture collection (atcc) and fipv strain - was kindly provided by dr. egberink (utrecht university, the netherlands). polyclonal antibodies originating from cats infected with fipv - were kindly provided by dr. egberink (utrecht university, the netherlands). these antibodies were purified and biotinylated according to manufacturer's instructions (amersham bioscience, buckinghamshire, uk). the monoclonal antibodies (mab) - - , f - , e - , recognising respectively the s-, m-and n-protein, were kindly provided by dr. hohdatsu (kitasato university, japan). a monocyte marker, dh b, recognising cd a was purchased from veterinary medical research and development (pullman, washington, usa). three cats of a non-specific breed from a fcov free closed household were used as blood donors for the extensive infection kinetics study. seventeen stray cats brought to the clinic of small animals in the faculty of veterinary medicine (ghent university) and spf cats were used for a study on the distribution of the infection kinetics patterns. the sex and felv, fiv and fcov status of the cats are listed in table . six ml blood was collected on heparin ( u/ml) (leo, zaventem, belgium) from the vena jugularis and blood mononuclear cells were separated on ficoll-paque (pharmacia biotech ab, uppsala, sweden) following manufacturer's instructions. mononuclear cells were resuspended in rpmi- (gibco brl, merelbeke, belgium) medium containing % fetal bovine serum (fbs), . mg/ml glutamine, u/ml penicillin, . mg/ml streptomycin, . mg/ml kanamycin, u/ml heparin, mm sodium pyruvate, and % non-essential aminoacids × (gibco brl). afterwards, cells were seeded in a -well dish with cell culture coating (nunc a/s, roskilde, denmark) at a concentration of × cells/ml and cultivated at • c with % co . non-adherent cells were removed by washing the dishes two times with rpmi- at and h after seeding. the adherent cells consisted for ± % of monocytes (as assessed by fluorescent staining with the monocyte marker dh b). crfk cells and monocytes were inoculated with fipv strain - or fecv strain - at a multiplicity of infection (m.o.i.) of . after h incubation at • c with % co , cells were washed times with rpmi- and further incubated in medium. at different time points post inoculation, culture medium was harvested and centrifuged at × g for min. the supernatants were used for determination of extracellular virus titres. the cells were removed from the well by scraping and added to the pellet for determination of intracellular virus titre. virus was released from the cells by freeze-thaw cycles. the samples were stored at − • c until titration. both intra-and extracellular virus titres were assessed by a % tissue culture infective dose assay using crfk cells. the fifty percent end-point was calculated according to the method of reed and muench [ ] . a virus inactivation curve was determined by keeping cell free virus in medium at • c with % co . samples were taken at different time points and stored at − • c until titration. a m: male, f: female b tested on plasma samples with snap ® fiv antibody/felv antigen combo test (idexx) c ipma antibody titer three independent assays were carried out and the inactivation curve was calculated by linear regression. at different time points post inoculation, cells seeded on glass coverslips, were fixed with % formaldehyde. surface-expressed viral proteins were labelled with biotinylated anti-fipv polyclonal cat antibodies and streptavidin-fitc (molecular probes, eugene, oregon, usa). after permeabilisation with . % triton x- (sigma-aldrich gmbh, steinheim, germany), cytoplasmic viral proteins were stained with a mixture of monoclonal antibodies ( - - , f - and e - ) and with goat anti-mouse-texas red (molecular probes). finally, the glass coverslips were mounted on microscope slides using glycerin-pbs solution ( . : . , vol/vol) with . % , -diazabicyclo( , , )octane (janssen chimica, beerse, belgium) and analysed by fluorescence microscopy. for the stray cats and spf cats, only cytoplasmic viral proteins were stained with fitc labelled anti-fipv antibodies (vmrd inc, pullman, washington, usa). the samples were stained to visualise the cytoplasmic and the surface-expressed viral proteins as described above and examined with a leica tcs sp laser scanning spectral confocal system (leica microsystems gmbh, wetzlar, germany) linked to a dm irb inverted microscope (leica microsystems). argon and helium/neon laser lights were used to excite fitc ( nm line) and texas-red ( nm line) fluorochromes. the images were obtained and processed with leica confocal software. all experiments were repeated or more times. the "area under the curve" was calculated for each experiment. triplicate assays were compared using a mann-withney u test. statistical analysis were performed with spss . (spss inc. chicago, illinois, usa). the growth curves of fipv and fecv in crfk cells are given in fig. . production of progeny virus started between and hpi and increased strongly until hpi. between and hpi there was only a slight increase of virus titres to reach a maximum of . log tcid / cells at hpi. there was no significant difference between the growth curves of fipv and fecv. the growth curves of fipv and fecv in monocytes varied between the different donor cats. figures a and a show that the production of fipv started between and h post inoculation for both cat and . between and h post inoculation there was a slight increase in virus titre for cat whereas the curve from cat (figs. c and c) . the growth curves of cat for fecv showed a low-level production ( fig. b and d) . the growth curves of cat for fecv began with a slight titre increase, similar to the fipv growth curve, but then the virus titre decreased with a slope comparable to the inactivation curve ( fig. b and d) . these findings suggest that monocytes could be infected by fecv but that the cells did not sustain a productive infection. figure shows that the growth curves for cat followed the inactivation curve, suggesting that there was no progeny virus produced. (fig. , lane ) . some showed a larger amount of surface-expressed viral proteins (fig. , lane ) . the antigen expression kinetics varied between the donor cats. figure e and f show the fipv and fecv cytoplasmic expression kinetics for cat . the percentage of fipv infected cells with cytoplasmic expression increased till hpi. the infection of monocytes with fecv initiated in the same manner but at hpi the curve started to decline. the cytoplasmic expression in monocytes of cat is shown in fig. e and f. infection with fipv or fecv led to the same expression kinetics. after an increase till or h post inoculation the percentage of cells with viral expression decreased rapidly. the number of fecv infected monocytes was lower than the fipv-infected monocytes. the fipv and fecv surface expression, for both cat and , followed the same curve as the cytoplasmic (fig. g and h; fig. g and h) . the results of cat were quite different from cat and . here, viral antigen positive monocytes were not found. knowing the total production of infectious progeny virus and the number of infected cells, it can be calculated that fipv-infected monocytes from both cat and have produced approximately infectious viruses per infected cell at h post inoculation. fecv-infected monocytes from cat produced times less progeny virus at h post inoculation whereas the fecv-infected monocytes from cat produced the same amount of progeny virus as the fip-infected monocytes. in order to clarify the prevalence of the patterns of viral replication observed in this study in a bigger cat population, the antigen expression kinetics were studied in stray cats and spf cats for both fipv and fecv. the antigen expression was visualised at , and hours post inoculation. the results are presented in fig. . the different expression kinetics that were found in monocytes from the closed household cats were also seen in monocytes from the stray cats and the spf cats. within this population of cats, the monocytes isolated from cats showed a continuous increase in viral antigen positive cells during a hour time span after inoculation with fipv. when these monocytes were inoculated with fecv, the number of viral antigen positive cells increased until hours post inoculation and then diminished. in monocytes from cats, the percentages of both fipv-and fecv-infected cells increased until hpi and then decreased. the monocytes from cats were resistant to infection. in this study, in vitro infection kinetics of fipv (strain - ) and fecv (strain - ) were established in peripheral blood monocytes from cats ( cats of a closed household, stray cats and spf cats). it is the first time that infection studies were performed in peripheral blood monocytes, the host/carrier cell of fipv. three distinct patterns were found in the infection studies. monocytes from cats were not infected by either strain (first pattern). the reason for the insusceptibility of these cells is not yet clear. virus particles were detected in the cells shortly after inoculation of the cells but no production of viral antigens was observed using polyclonal antibodies (data not shown). thus, it seems that new viral proteins were not formed. this suggests that the block of infection is located after entry of the virus but before (or at) the translation step. in vivo, resistance to fipv infection has been observed in experimental inoculations. after inoculation with a lethal dose of fipv, a varying part of the cats (depending on experiment - %) showed no clinical signs and some of them remained seronegative [ , ] . this was also seen in control groups of vaccination trials (no vaccination, only fipv challenged) [ , ] . resistance to fcov infection has also been suggested to occur in natural infections in the field [ ] . a small percentage of cats in fcov endemic households had no shedding, remained seronegative or had a low antibody titre over a time period of years. it would be most interesting to investigate the correlation between in vitro and in vivo resistance to fcov. this might give perspectives for selection of cats insusceptible for fip. monocytes from cats showed an increase of fipv antigen positive cells till hpi whereas the amount of fecv antigen positive cells dropped after hpi. this shows that the fipv infection was sustained whereas the fecv infection was not sustained (second pattern). monocytes from cats did not sustain both fipv and fecv infection since the number of viral antigen positive cells dropped after or hpi (third pattern). the drop in antigen positive cells after or hours post inoculation may be explained by the fact that the infected cells died due to infection and were washed away during the staining. however, the same kinetics were found with staining in suspension, a technique which prevents cell loss (data not shown). another explanation is that monocytes stopped producing viral proteins and assembling new virions. the extracellular virus titres showed indeed that (almost) no new progeny virus was produced between and hours post inoculation. some graphs show differences in virus titres between experiments (with the same virus and with monocytes from the same donor cat) of up to log units. these differences are intrinsic to working with primary cells and are reported in viral infection studies with porcine and equine monocytes as well [ , ] . although fecv initially infects monocytes, the infection is never sustained. this implicates that fecv might reach the blood circulation in vivo. in several studies, healthy cats from fcov endemic households were investigated [ , , , , , ] . in such households, where the fcov was most likely fecv, a part of these healthy cats were viraemic for fcov. fcov was detected both in plasma and in monocytes. therefore, it may be hypothesised that when fecv reaches the blood circulation, the lack of sustainability and long-term production of progeny virus (the total virus production was up to times lower in fecvinfected monocytes) may be the reason for the lack of disease progress. this might form the basis for the difference with fipv since fipv infection is sustained and reaches higher titres. however, the non-sustained fecv infection, might also be attributed to the virus strain that was used. although fecv - is a reference strain, it may act differently from other fecv strains due to its deletion in the b orf [ ] . it has been described that loss of the ab orfs results in loss in virulence [ ] . it could be that this loss in virulence is translated in loss of the ability to replicate efficiently in monocytes. thus, whether the hampered replication of strain - in monocytes is a universal property of fecv strains or only of b deleted/mutated strains, remains to be determined. the different fipv infection kinetics depending on the cat from which the monocytes were isolated suggests that cellular factors, influenced by genetic background and/or differentiation/activation status, are very important in determining the outcome of a fipv infection. in an infection kinetics study where another cell type, feline peritoneal macrophages, was used, different results in the antigen expression kinetics were obtained [ ] . the number of fecv infected peritoneal macrophages was lower than the number of fipv infected peritoneal macrophages throughout the infection kinetics. since the viral antigen kinetics was only performed till hours post inoculation, a possible drop in antigen expression, like reported here, could not be evaluated. in contrast, our results suggest that fipv and fecv can initially infect the same amount of cells but at h post inoculation, differences in sustainability of the infection are prominent. since the same viruses were used as in our study, the different results are most probably due to cellular factors and/or a different differentiation status of the cells. differences in susceptibility depending on the differentiation and/or activation status of the monocytes/macrophages has been reported for different viruses such as porcine reproductive and respiratory syndrome virus, caprine arthritis-encephalitis virus, suid herpes virus , herpes simplex virus, human immunodeficiency virus type and maedi-visna virus [ , , ] . the differences in activation status might explain the discrepancy between our results and those of stoddart and scott [ ] . what this variation in susceptibility and sustainability means for the pathogenesis of fecv and fipv in vivo, remains to be elucidated. in an inoculation study using fipv - , different patterns of disease progression were detected, based upon survival time: progressors (rapid, intermediate and delayed) and survivors (prolonged and long-term) [ ] . with natural in vivo fcov infection, different clinical outcomes (besides resistance to fcov) have been described: persistent carrier, transiently infection and development of fip [ ] . it is not clear what the viral and host factors are that determine the different clinical outcomes. since in the inoculation study the same strain (fipv- - ) was used and considering the fact that in the field cats are often infected with the same strain of fcov, it is likely that genomic variation between cats contributes to a different clinical outcome. a genetic background was also suggested during a field study with pure-bred cats, in which it was shown that susceptibility to fip is indeed inheritable [ ] . a possible explanation for the different disease progression is the possibility of the cats to develop an efficient t-cell response [ ] . however, it could also be that the susceptibility of the monocytes to fipv plays a role, considering the results presented here. it would be interesting to investigate if cats that show a different outcome to an experimental or natural infection also show different infection kinetics in vitro. this might be important since a correlation between in vitro and in vivo infection kinetics would allow easy screening and selection. in this study, it was shown that viral proteins can be expressed on the surface of fcov infected cells. however, only a part of the infected cells showed surfaceexpressed viral antigens. on hpi, % of the infected crfk cells and % of the infected monocytes showed surface-expressed viral antigens. s-and mproteins, but no n-proteins were found on the cell surface of both crfk cells and monocytes using specific monoclonal antibodies (data not shown). this indicates that the observed surface expression does not represent virus particles. possible explanations for the observed differences in amount of surface-expressed viral antigens could be the retention of a part of the viral proteins or spontaneous internalisation of the surface-expressed viral antigens. retention of viral proteins has been described for porcine coronavirus [ ] . spontaneous internalisation of viral proteins has been described for suid herpes virus [ ] . the presence of viral antigens on the cell surface can be of importance for the recognition and elimination of infected cells by the immune system. binding of virus-specific antibodies to viral proteins present on the surface, makes infected cells recognisable for the classical complement pathway, phagocytes and natural killer cells, which will lead to lysis of the infected cell [ ] . interestingly, not all fipv-and fecv-infected monocytes/macrophages showed surface expression. absence of viral proteins on the cell surface has been described for other viruses, such as human cytomegalovirus and equine herpesvirus as a strategy to avoid recognition by the antibody-dependent immune responses [ , ] . why only half of the infected cells showed surface expression and whether the cells without surface expression are indeed less susceptible towards antibody-dependent complement mediated lysis, remains to be elucidated. in fip research, the crfk cell line is often used to perform in vitro experiments. the results of this study reveal that the crfk cell line is not the best suitable in vitro model for the study of fipv and fecv replication at a cellular level. firstly, the course of infection of fipv and fecv is similar in crfk cells, whereas in monocytes there is a clear difference (as there is in vivo). secondly, a high percentage of infected cells can be reached in crfk cells (up to % of the inoculated cells) with each cell producing and releasing a relatively small amount of infectious virus (< viruses/cell). in monocytes on the other hand, less than % of the cells can be infected, but a single fipv-infected monocyte releases up to new infectious viruses. thirdly, crfk cells showed surface expression in almost all infected cells, in contrast to monocytes, which showed surface expression in only half of the infected cells. in conclusion, it can be stated that fcov infection kinetics in vitro are strongly dependent on cellular factors. monocytes from some cats cannot be infected. if monocytes are susceptible to fcov infection, then both fipv and fecv can infect them. however, fecv infections are never sustained and production of viral antigens and progeny virus ceases at h post inoculation. sustainability of a fipv infection depends on the origin of the host cells. fipv production in susceptible monocytes was always to times higher than fecv production. what this variation in susceptibility and sustainability implicates for the development and pathogenesis of fip and/or fecv in vivo, remains to be elucidated. use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats persistence and transmission of natural type i feline coronavirus infection natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease effects of origin and state of differentiation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) fip, easy to diagnose? vet q a novel mechanism for persistence of human cytomegalovirus in macrophages the inheritance of susceptibility to feline infectious peritonitis in purebred catteries il- activity in feline infectious peritonitis detection of interleukin in ascites from cats with feline infectious peritonitis 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 live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis viral interactions with the immune system 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 isolation and characterization of feline c and evidence for the immune complex pathogenesis of feline 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infected with a feline enteric coronavirus a simple method of estimating fifty percent endpoints a novel sorting signal for intracellular localization is present in the s protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus independent evaluation of a modified live fipv vaccine under experimental conditions (cornell experience) a mrna pcr for the diagnosis of feline infectious peritonitis intrinsic resistance of feline infectious peritoneal macrophages to coronavirus infection correlates with in vivo virulence replication of equine herpesvirus type in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation absence of viral antigens on the surface of equine herpesvirus- -infected peripheral blood mononuclear cells: a strategy to avoid complement-mediated lysis internalization of pseudorabies virus glycoprotein b is mediated by an interaction between the yqrl motif in its cytoplasmic domain and the clathrin-associated ap- adaptor complex a comparison of the genomes of fecvs and fipvs: what they tell us about the relationships between feline coronaviruses and their evolution feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses genomic organization and expression of the end of the canine and feline enteric coronaviruses fcov replication in monocytes evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity pathogenesis of feline infectious peritonitis: nature and development of viraemia pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence increased plasma levels of leukotriene b and prostaglandin e in cats experimentally inoculated with feline infectious peritonitis virus macrophage-virus interactions author's address we are grateful to dr. hohdatsu and dr. egberinck for supplying antibodies. we thank chantal vanmaercke for excellent technical assistance, myriam hesta and kris gommeren for their help with handling the cats, h. favoreel, s. van gucht and p. delputte for critical reading of this manuscript. we also thank the clinic of small animals of the faculty of veterinary science for their co-operation. h. l. dewerchin was supported by the institute for the promotion of key: cord- -cdas cx authors: morozov, i.; meng, x. -j.; paul, p. s. title: sequence analysis of open reading frames (orfs) to of a u.s. isolate of porcine reproductive and respiratory syndrome virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: cdas cx the sequence of orfs to of a u.s. isolate of porcine reproductive and respiratory syndrome virus (prrsv), atcc vr , was determined by analysis of a cdna λ library. the cdna clones containing prrsv specific sequences were selected using a vr orf specific pcr probe and sequenced. the orfs , and overlapped each other and encoded polypeptides with predicted m(r) of . kda (orf ), . kda (orf ) and . kda (orf ), respectively. no overlap was found between orfs and , and instead there was a bp sequence which separated these two orfs. the nucleic acid homology with corresponding orfs of the european prrsv isolate lelystad virus (lv) was % for orf , % for orf and % for orf . comparison of the orf sequences of vr with that of another u.s. isolate mn- b revealed only % amino acid sequence homology and the presence of deletions in the orf of mn- b. our results further strengthen the observation that there is sequence variation between us and european prrsv isolates. analysis of a cdna )~ library. the cdna clones containing prrsv specific sequences were selected using a vr orf specific pcr probe and sequenced. the orfs , and overlapped each other and encoded polypeptides with predicted m r of . kda (orf ), . kda (orf ) and . kda (orf ), respectively. no overlap was found between orfs and , and instead there was a bp sequence which separated these two orfs. the nucleic acid homology with corresponding orfs of the european prrsv isolate lelystad virus (lv) was % for orf , % for orf and % for orf . comparison of the orf sequences of vr with that of another u.s. isolate mn-lb revealed only % amino acid sequence homology and the presence of deletions in the orf of mn-lb. our results further strengthen the observation that there is sequence variation between us and european prrsv isolates. porcine reproductive and respiratory syndrome virus (prrsv) belongs to the newly proposed virus family arteriviridae, which also includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv) and simian hemorrhagic fever virus (shfv). porcine reproductive and respiratory syndrome (prrs) was first described in the u.s. in [ ] . a similar disease referred to as porcine epidemic abortion and respiratory syndrome (pears) was then reported in europe [ ] . prrsv was first isolated in europe and is believed to be widespread in swine population around the world [ , , ] . all european isolates of prrsv are antigenically and genetically related, whereas there are antigenic variations between us and european isolates as well as among us isolates [ , , ] . the complete nucleotide sequence of the genome of lv has been determined [ ] , but until recently limited information was available about the molecular structure of the genome of north american isolates of prrsv [ ] [ ] [ ] [ ] . we have previously reported the cloning and sequencing of the orfs to ofa u.s. isolate of prrsv vr of high virulence [ ] . the ' end of the genome of the vr and the other u.s. prrsv isolates showed a striking difference when compared to the european isolates [ ] . in this study, we report on the cloning and sequencing of the orfs - of the u.s. isolate vr . for sequencing and characterization of the viral genome of vr a cdna ;~ library was constructed. the crl cells were infected with vr virus at a m.o.i. of . and the total rna from infected cells was isolated at h post infection by using a guanidinium thiocyanate method [ ] . polyadenylated rna was enriched, reverse transcribed and cloned into the )~ zap vector using the uni-zap cdna cloning kit (stratagene, la jolla, ca). a pcr probe generated by orf specific primers dp ( 'gctttgctgtcctccaag ') and dp ( 'gatgcctgacacattgcc ') [ ] were used to screen the library. plaques that hybridized with the probe were isolated and purified. the phagemids containing viral cdna inserts were rescued by in vitro excision using exassist helper phage and e. coli solr cells (stratagene, lajolla, ca). several recombinant phagemids with virus specific cdna inserts with sizes ranging from . to . kb were selected and sequenced by sanger's dideoxynucleotide chain termination method [ ] with an automated dna sequencer (applied biosystems, foster city, ca). universal, reverse and specific internal primers were used to determine the sequence. at least independent clones representing sequence of the orfs to were sequenced. the sequencing data was assembled and analyzed using mac vector (international biotechnologies, inc., ct) and geneworks (intelligenetics, ca) computer programs. the nucleotide sequence reported in this paper has been deposited in the genbank with the accession number u . analysis of the nucleotide sequence identified three partially overlapping orfs. the orf extended from nucleotide to , orf from to , and orf from to . there was an overlap of bp between orfs and , and bp between orfs and . surprisingly, no overlap was found between orfs and . the start codon oforf was located bp downstream of the stop codon of orf . however, the atg start codon of orf and tga stop codon of orf overlapped by only t bp in lv [ , ] . the sequence at the region of the orf and orf junction of lv is atatga. we sequenced the corresponding region of additional independent clones of vr and in all cases the sequence of this region of vr was atttga. the point mutation from a to t in vr and probably some other unidentified changes in this region of vr made the orf atg start codon bp downstream of the stop codon of orf , and a bp non-coding region appeared in the orf and junction of vr . the characteristics oforfs to of vr are summarized in table . the orf encodes a amino acid polypeptide with a predicted size of . kda. the carboxy and amino terminus of the predicted protein are hydrophobic (data not shown) and there are two potential n-glycosylation sites in the orf protein. orf encodes a protein of amino acids and contains potential n-glycosylation sites. the amino terminus of the orf protein is extremely hydrophobic. orf encoded a amino acid protein with a predicted size of . kda. the amino and carboxy termini and regions within the protein are highly hydrophobic. comparison of the nucleotide sequences of vr and lv showed extensive variations. nucleotide sequence identity between vr and lv is % for orf , % for orf and % for orf . alignment of the predicted amino acid sequences of orfs - of vr and lv is presented in fig. . amino acid identity between vr and lv is % for orf , % for orf and % for orf . we also compared the sequence ofvr orf with that of mn-lb, another us isolate of prrsv [ ]. the orf of vr is bp longer and shares an % nucleotide sequence homology with mn-ib. the amino acid homology between the orf of vr and mn-lb is % (fig. lc) . several deletions were found in the orf of mn-lb compared to vr . the orfs and of prrsv are predicted to encode the viral membrane glycoprotein and the viral nucleocapsid protein, respectively [ , ] . analysis of predicted amino acid sequences encoded by orfs - of lv, ldv and eav showed that all of these proteins share features of membrane associated proteins [ , , , ] . the eav orf product was identified as the main envelope glycoprotein ~sequence for vr orfs - is presented in the study, orfs was reported by meng et al. [ ] and lv orfs - was reported by meulenberg et al. [ ] bdistance in nucleotides between proposed junction motif and aug start codon of downstream orf [ ] . our data indicates that the proteins encoded by orfs ~, of vr possess characteristics similar to those of lv and probably are envelope or membrane associated glycoproteins because of their hydrophobicity and presence of potential glycosylation sites. further work is necessary to determine the roles of these proteins. the variability found in the orf sequence between the two u.s. isolates correlate with the findings that orf protein of the mn-lb expressed in e. coli reacted with only % of prrsv positive sera by western blot analysis [ ] . a nested set of subgenomic mrna is formed during replication of prrsv and other members of the arterivirus group [ , , , , ] . all subgenomic mrnas contain a common leader sequence derived from the ' noncoding region of the viral genome. the site of the leader-mrna junction is similar and located upstream of the start codon of each orf. the consensus leader-mrna junction sequence of the six subgenomic mrnas of lv was determined to be (u/a)(c/u/a)(a/ g)acc [ ] . similar sequences were also found as leader-mrna junction regions for ldv [ ] . the potential leader-mrnajunction motifs oforfs to of vr was proposed and compared with those of lv ( table ). the last four nucleotides of the motif for orfs , , , , and in lv are aacc, and for orf is gacc. the aacc motif has been found upstream of orfs and of vr [ ] as well as orfs and . there are two potential junction regions for orf , bp and bp upstream of the orf start codon, respectively (table ) . no aacc motif was found upstream ofvr orfs and . however, the sequences uugacc and cagacc upstream of orf , uugacc and gagacc upstream of orf , may be the leader-mrna junction regions for the mrnas and of vr . multiple potential leader-mrna junction sites suggest that polymorphism of subgenomic mrnas may exist among prrsv isolates. experiments to determine the exact locations ofleader-mrnajunction regions are now in progress. the sequence variations observed in this study between a u.s. and a european prrsv isolate, as well as between two north american prrsv isolates, indicates the heterogenetic nature of prrsv isolates and the need for further characterization of additional prrsv isolates. whether this genetic variation between vr and lv reflects the observed difference in virulence needs to be further studied. comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) sequences of ' end of genome and of ' end of open reading frame la of lactate dehydrogenase-elevating virus and common junction motifs between ' leader and bodies of seven subgenomic mrnas 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 equine arterifis virus is not a togavirus but belongs to the coronavirus-like superfamily structural proteins of equine artefitis virus complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-etevating virus (ldv) cloning, expression, and sequence analysis of the orf gene of porcine reproductive and respiratory syndrome virus mn-lb identification of major differences in the nucleocapsid protein genes of a quebec strain and european strains of porcine reproductive and respiratory syndrome virus molecular cloning and nucleotide sequencing of the '-terminal genomic rna of porcine reproductive and respiratory syndrome virus phylogenetic analysis 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 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 differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies porcine reproductive and respiratory syndrome: an overview molecular cloning: a laboratory manual dna sequencing with chain terminating inhibitors 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 virus mystery swine disease in the netherlands: the isolation of lelystad virus this work was supported by a grant no. - from the national research initiative competitive grants program of the u.s. department of agriculture, and in part by a grant from the solvay animal health, inc., mendota heights, mn. the authors would like to thank drs. pat halbur and melissa lure for their expert help throughout this project, and dr. harold hills at the nucleic acid facility, iowa state university for his assistance in sequence analysis. received december , key: cord- -fmnro kw authors: hoshino, y.; zimmer, j. f.; moise, n. s.; scott, f. w. title: detection of astroviruses in feces of a cat with diarrhea date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: fmnro kw astroviruses were detected by electron microscopy in the feces from a month old kitten with diarrhea. the mean diameter of the viral particles was . nm, and they showed characteristic five- or six-pointed star-shaped surface configurations. the clinical disease manifested by the cat and the observed morphology of the viral particles are consistent with previous reports on astroviruses of other species. astroviruses were detected by electron microscopy in the feces from a month old kitten with diarrhea. the mean diameter of the viral particles was . nm, and they showed characteristic five-or six-pointed star-shaped surface configurations. the clinical disease manifested by the cat and the observed morphology of the viral particles are consistent with previous reports on astroviruses of other species. . viral gastroenteritis is one of the leading causes of morbidity and mortality in neonatal and young animals, birds and man throughout the world ( , , , , ) . the development of methods for identification of viruses in fecal samples by direct electron microscopy (em) has led to the discovery of a number of previously unrecognized enteric viruses, including astroviruses. astroviruses are isometric, approximately nm in diameter viral particles which, when negatively stained, give the impression of a white, five-or six-pointed star superimposed on the particle. astroviruses have been detected in the feces of humans ( , , , , t , , ) , calves ( ) , lambs ( ) and turkeys ( ) with diarrhea. since attempts to propagate the mammalian and avian astroviruses in cell cultures have been unsuccessful, their size and morphology as determined by em are the primary criteria by which they are identified. viral antigen was detected by indirect immunofluorescent staining within the cytoplasm of cultured cells infected with fecal material of human ( , ) and bovine ( ) astroviruses. however, these infections were abortive in y. hos~l-¢o, j. f. zi~iet~, n. s. molse, and f. w. scott: that no specific fluorescence was seen in passaged celt cultures, t~ecently, t-ierrii~g et al. ( ) reported that ovine astrovirus has a single-stranded rna genome which resembles that of the picornaviruses and ealiciviruses but the polypeptide composition is unlike that of either of these groups. we report here the detection by direct. em of astroviruses in feces from a month old kitten with diarrhea. an intact female -month-old kitten was presented to the small animal hospital at the new york state college of veterinary medicine, cornell university, on october , . the chief complaint was that the kitten had had diarrhea, characterized as loose and watery, for approximately one week. a tentative diagnosis of parasite infestation and a diet-induced diarrhea was made, and the cat was vaccinated for feline panleukopenia, rhinotracheitis, and eaticivirus. the cat was returned five days later (october ) with the report that the diarrhea had continued and that the cat had been anorectie for three days. the diarrhea was reportedly more severe than on the initial visit. physical examination revealed slight dehydration, poor body condition, gas-distended loops of small intestine, and a green, watery diarrhea. a fecal sample was submitted for em examination for viral particles. two days later (october ), the kitten was hospitalized because of more severe dehydration and diarrhea. the results of a hemogram were normal, but the kitten was mildly acidotic and hypokalemic. during the subsequent nine days of hospitalization, the kitten's feces improved in consistency. the cat was discharged on october . re-examinations one week and months after discharge indicated that the kitten improved steadily and made an uneventful recovery. the feces had become normal, and the appetite was good. fecal sample was examined by em for ~ ral particles using the procedures reported previously ( ) . a percent (w/v) fecal suspension was prepared in eagle's minimum essential medium (mem) with antibiotics and without serum. this suspension was centrifuged at , × g for minutes (crude fecal suspension), and the supernatant was then ultra.centrifuged at , × g for minutes (clarified fecal suspension). one to drops of crude fecal suspension were mixed with drops of distilled water, one drop of . percent bovine serum albumin and three to four drops of percent phosphotungstie acid adjusted to ph . . this mixture was applied to a carbon-parlodion-coated copper grid with an all-glass nebulizer and examined in a philips electron microscope at kv. direct em examination demonstrated the presence of astrovirus-like particles, mostly in aggregates, in the fecal suspension (fig. ) . these viral particles were roughly spherical in outline, ranged in size from . nm to . nm in diameter and h ad a mean diameter of . nm. the surface structure had the appearance of characteristic five-or six-pointed stars ( fig. , arrows). this star-shaped configuration was not always apparent on all particles. bridging structures between virus particles were frequently seen (fig. t, arrowheads) . these structures have been observed on human ( ) and ovine ( ) astroviruses. it is speculated that these bridging structures may be surface fibers similar to those on adenoviruses. attempts at viral isolation with cell cultures were carried out as previously reported ( ) . confluent monolayer cultures ( × mm culture tubes) of first transfer cells of feline kidney and an established fetal rhesus monkey kidney celt line (ma ) were inoculated with . ml of clarified fecal suspension. after one hour adsorption at ° c, cell cultures were washed once with phosphate buffered saline, p h . , fed with . ml of maintenance medium (eagle's mem supplemented with . percent laetalburnin hydrolysate, . percent sodium! bicarbonate, antibiotics and no serum), and incubated at ° c. three blind passages were made at one week intervals. attempts to propagate this feline astrovirus in cell cultures have been unsuccessful. no calicivirus or other eytopathogenic virus was isolated in cell cultures. experimental transmission experiments with mammalian astroviruses indicate that. they induce mild, constitutional symptoms ( , , , ) . studies on the pathogenesis of astrovirus infection in lambs have shown the site of virus multiplication to be the mature villous epithelial cells of the small intestine ( , ) . there is a clinical impression that astrovirus-associated gastroenteritis in man and animals is generally milder than that associated with rotaviruses. asymptomatie excretion of astrovirus particles has been reported in man ( , ), calves ( ) and lambs ( ) . studies on the pathogenicity of feline astrovirns have been hampered by a shortage of material. as this is the first report of the identification of a feline astrovirus and since no serological test. is available, it is unknown how widespread and how significant this virus is. since eats frequently have close contact with humans, it will be important to study the relationship between feline and human astrovirnses. astrovirus-associated gastroenteritis in children the role of viruses in acute diarrhoeal disease viral intestinal infections of animals and man. comp. i m m u n . microbiol purification and characterization of ovine astrovirus viral gastroenteritis coronavirus-tike particles in the feces of normm cats isolation and characterization of feline rotavirus small spherical viruses in faeces from gastroenteritis patients astrovirus-associated gastroenteritis in a children's ward astrovirus infection in volunteers astroviruses detected by immunofluorescence (letter) ). : viruses in infantile gastroenteritis (letter) -n~ particles in feces in infantile gastroenteritis (letter) comparison of the features of astroviruses and caliciviruses seen in samples of feces by electron microscopy viruses in the stools detection of astroviruses in turkey faeces by direct electron microscopy the tecumseh study. x i . occurrence of acute enteric illness in the community coronavirus et "astrovirus" observ@s d ans ies selles d'enfants atteints de gastroentdrites recent advances in viral gastroenteritis detection and transmission of -nm virus particles (astroviruses) in faeces of lambs with diarrhoea pathogenesis of diarrhoea caused b y astrovirus infections ultrastructure of the small intestine in astrovirus isolation of small viruses resembling astroviruses and ealiciviruses from acute enteritis of calves key: cord- -rhkrmftn authors: hoshino, y.; wyatt, r. g.; scott, f. w.; appel, m. j. title: isolation and characterization of a canine rotavirus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rhkrmftn canine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. the virus was subsequently propagated in cell cultures; it was chracterized and compared with rotaviruses from other species. replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, ma , under overlays of carboxymethyl cellulose or agarose. intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected ma cells. by plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (cu- ) and simian (rhesus mmu and sa- ) rotaviruses. the canine rotavirus had a one-way antigenic relationship with feline (taka), bovine (ncdv), and porcine (osu) rotaviruses. t~otaviruses are among the m.ost important causative agents of acute neonatal gastroenteritis in various mammalian and avian species. rotaviruses have been detected in the feces of man, monkey, horse, cattle, bison, antelope, sheep, goat, deer, pig, cat, rabbit, guinea pig, mouse, chicken, turkey, duck and parrot ( , , , , , , , , ) . recently the worldwide appearance of parvovirus enteritis in dogs has caused an intensified search for infectious agents in dog feces. subsequently, reports of visualization of rotavirus particles in canine feces, and in two cases cultivation porcine rotavirus (osu) by dr. e. h. bohl, simian rotavirus (sa- ) by dr. h. mmherbe, and simian rotavirus (rhesus mmu ) by dr. n. j. schmidt. the ds- strain of human rotavirus is a reassortant virus between a temperature-sensitive mutant of a cultivatable bovine rotavirus (uk strain) and a noncultivatable human rotavirus (ds- strain) (t ). all rotaviruses were plaque-purified prior to use. hyperimmune rabbit sera to canine and feline rotaviruses were prepared by intramuscular (i.m.) inoculation of . ml of purified virus mixed . ml freund's incomplete adjuvant followed by two i.m. re-inoculations of . ml purified virus without adjuvant on days and . rabbits were bled one week after the last injection. all other hyperimmune antisera used were prepared in guinea pigs ( ) . plastic six-well plates (costar, cambridge, mass.) with confluent ma cell monolayers were washed three times with leibovitz l- medium (m. a. bioproducts, walkersville, md.) supplemented with . percent gelatin-glutamine and antibiotics (l- /gel) and inoculated with . ml of virus samples (pretreated for minutes at ° c with trypsin at a final concentration of i fxg/ml). the virus inoeulum was allowed to adsorb for minutes at ° c. excess inoculum was then removed, plates were washed once with l-tb/gel and ml of the overlay medium was applied to each well. the overlay medium was either: a) eagle's basal medium, . percent carboxymethyl cellulose (cmc, hercules, inc., wilmington, del.), antibiotics, tzg deae-dextran/ ml (pharmacia fine chemicals, piscataway, n. j.) and . izg × crystalline trypsin/ ml, or b) eagle's minimum essential medium (mem) containing . percent agarose, glutamine, antibiotics, ~g deae-dextran/ml, and . ~zg × crystalline trypsin/ml. vc'ith the use of cmc overlay, plates were stained with formalin/crystal violet solution after days of incubation in a humidified co incubator. with the use of agarose overlay, plates were applied a second overlay containing neutral red ( . mg/ml) after to days of incubation. plaques were then counted -- days later. plaque reduction neutralization tests were performed by mixing equal volumes of fourfold dilutions of sera (previously heat-inactivated at ° c for minutes) and the virus suspension diluted to contain approximately plaque-forming units (pfu) * per welt. after an incubation of minutes at, ° c, ml aliquots of the virus-serum mixtures were inoculated onto the cell monolayers of -well plates. two wells were used for each dilution. after minutes of adsportion at ° c, the inoculum was removed and the cell monolayers were washed once with l- /gel, an overlay medium was applied, the cultures were incubated, stained and read as described above. the neutralizing titer was expressed as the reciprocal of the calculated serum dilution causing a percent reduction in plaque counts ( ) . neutralization test criteria of significance were arbitrarily established on the basis of enterovirus standard system ( , , ) . if the difference between homologous and heterotogous neutralizing titers was tess than -fold, the serological relationship was considered to be significant; if the difference between titers was -fold or greater, viruses were considered to be significantly different. monolayers of ma cells grown either on glass slides (lab-tek chamber slides, lab-tek products, napperville, ill.) or on coverslips in leighton tubes infected with viruses, washed with pbs, fixed with acetone at -- °c for minutes, airdried at room temperature, and then stored at -- °c until used. the fixed celt cultures were stained by the indirect immunofluorescent methods using rabbit or canine sera and the corresponding fluorescein isothiocyanate (fitc)-conjugated antigammaglobulin sofa or protein a-fitc conjugate (pharmacia fine chemicals, piscataway, n. j.). the slides were then eounterstained with a : dilution of percent evans blue in pbs for t minutes and washed twice with pbs. after air -drying, slides were mounted with percent glycerol in distilled water, and examined for specific flourescence with an epifluorescence uv microscope (ernst leitz, ltd., midland, ontario, canada). in negatively stained preparations of the fecal sample, both double-shelled and single-shelled particles were observed (fig. ). the canine rotavirus particles were indistinguishable in morphology from those of known rotaviruses of other species. double-shelled particles had sharply defined margins forming a continuous covering over the main capsid, which is characteristic for rotaviruses ( , ) . smooth particles were approximately nm in diameter and rough pai~icles were approximately nm in diameter. complete particles showed a characteristic "spoke-like" arrangement of inner capsomeres surrounded by an outer layer, which gave a honeycomb-like appearance on the surface of the virion. the first cythopathie effect (cpe) was observed i day after inoculation (dpi). many cells adhered to the glass wall by only a single process ("flagging" cpe), which is a characteristic cytopathic effect produced in vitro by rotaviruses ( ) and reoviruses ( ) . as incubation progressed, these cells detached from the glass wall and floated free in the medium. the rotavirus isolate (cu-i) could be passaged in ma cells without the aid of trypsin. viruses were serially passaged more than ten times in ma cells. virus particles were demonstrated by em in cell culture lysates in all passages. canine rotavirus (cud) induced intracytoplasmic inclusion bodies in mat cells. inclusions were first noticed hours post-infection. as incubation progressed, infected cells detached from the glass wall as noted above, however, inclusions were detectable in cells attached to the glass wall dpi. inclusions were of different sizes and shapes (fig. ) . reproducible and clear-cut plaques were formed d p i under the overlays of civic or agarose in the wesenee of trypsin (fig. ) . although canine rotavirus readily replicated and produced cpe in monolayer cultures of ma cells in the absence of trypsin, detectable plaques were not formed even d p i under the overlays of cmc or agarose without trypsin. the antigenic relationship between canine and other mammalian rotaviruses was studied by plaque reduction neutralzation assay (table ) . a two-way relationship between canine and simian (rhesus mmu and sa- ) rotaviruses was found, indicating that these viruses were similar, if not identical, by this assay. in contrast, canine rotavirus (cu-t) was distinct from two human rotavirus strains (wa and ds- ). there was a one-way antigenic relationship between the canine and the feline, bovine and porcine rotaviruses; in each instance, serum. to the non-canine strain demonstrated the broader reactivity. virus-specific immunofluoreseence was demonstrated with rabbit immune serum but not with preimmunization serum in the cytoplasm of ma cells infected with canine rotavirus (cu- ). in some infected cells, the areas of immunofluorescence were small and scattered in the cytoplasm (fig. a) , while in other cases, the areas coaleseed to from large diffuse fluorescent foei (fig. b) . a crossreactive antigen(s) was demonstrated in the celts infected with canine rotavirus by indirect immunofluorescenee test (ift) using hyperimmune anti-bovine rotavirus serum. canine rotavirus antigen was not stained by hyperimmune sera against reovirus type i, ii, and iii by ift; neither did hyperimmune serum to rotavirus stain reovirus antigens. fluorescence was not detected in the uninfected control ma cells. infected cell lysates were treated with trypsin (final concentration of trypsin at fxg/ml), incubated for hour at ° c and then directly assayed by plaquing. the pfu infectivity was enhanced approximately -fold. in mcnulty et al. ( ) reported that antibody to rotavirus was detected by indirect immunofluorescent test (ift) in of dogs ( percent) from the belfast area in northern ireland. takahasm et al. ( ) in reported that of dogs ( . percent) in aomori prefecture of japan had antibody against calf rotavirus as determined by complement fixation test (cft). since both ift and cft detect only group-specific antibodies, and rotaviruses from one species can infect another species ( , , , , , , , , ) , it was not clear from these studies whether the antibodies in the dogs resulted from infection with distinctly canine rotavirus or with human or some other rotavirus. evgster and sidwa ( ) reported in that rotavirus was visualized by em in feces from a -week-old puppy with diarrhea. virus isolation and characterization were not reported. in , e~-gla~d and posto~ ( ) reported visualization by em and subsequent isolation in mdck cells of a rotavirus from a dog with fatal neonatal diarrhea. they also reported that experimental inoculation of two -month-old beagles with purified intestinal contents did not result in clinical signs of illness or virus shedding. in , fulto~ et al. ( ) isolated a rotavirus from a newborn dog that died after having clinical signs of diarrhea. they reported that their rotavirus isolate, "designated lsu -- , may be a specific canine rotavirus or a rotavirus from another species". the results of the present study confirm the existence of a canine rotavirus. this canine rotavirus (isolated in new york state) was found to be similar, if not identical, serologically by plaque reduction neutralization assay to simian rotaviruses isolated in california ( ) and south africa (table ). however, they are different in rna eleetropherotype, in hemagglutination pattern and in plaque size (hostiino, y., kalica, a. t~. et al., unpublished results). in addition, the relatedness of two rotaviruses based on serotype does not always correlate with the relatedness based on genotype ( , i-ioshino, y. et al., unpublished results). further biological and biochemical studies, including hybridization analysis, on both canine and rhesus rotaviruses, are in progress. whether these findings are unique to this specific isolate (cud st~rmn) of canine rotavirus or this is a common characteristic of any canine rotavirus isolate is being investigated in our laboratory. recently we isolated by cloning two small variants of feline rotavirus which had a two-way antigenic relationship with the cu- strain of canine rotavirus. further studies on plaque variation of rotaviruses are underway. no antigenic relationship was found between this canine rotavirus and two human rotaviruses by plaque reduction neutralization assay (table ) , although we have observed that there is a one-way antigenic relationship between this isolate of canine rotavirus and another strain of human rotavirus (hosm•o, y. et al., unpublished results). since dogs, as pets, have close contact with humans, it will be interesting to examine further the serological relationships between canine and human rotaviruses, and to look for evidence of interspecies transmission. the negatively stained canine rotavirus particles were indistinguishable in morphology from those of known rotaviruses of other species. the spoke-like arrangement of inner capsomeres of double-shelled particles, with a sharply defined outer capsid margin, differs from reovirus which has a featureless layer located either over or among capsomeres of the main capsid ( ) , and from orbivirus which has a fuzzy indistinct layer covering the main eapsid ( ) . rotaviruses are, in general, difficult to isolate and to grow in cell cultures using conventional techniques. however, certain strains may be serially propagated in cell cultures if the virus inuculum is treated with proteolytic enzymes or if proteolytic enzymes are incorporated into the medium after infection ( , , , , , , , , , , ) . engi, a~d and poston ( ) reported that rotavirus observed in feces by e?¢[ from a diarrheal dog grew in cell cultures with the aid of trypsin, but did not grow in cell cultures without trypsin treatment. our isolate, canine rotavirus cu- , readily replicated in cell cultures without trypsin treatment. both double-shelled and single-shelled virus particles were demonstrated by em in cell culture lysates in all passages. it has been reported that both simian and bovine rotaviruses induced intracytoplasmic inclusoin bodies which were small with clearly defined round or oval outlines, and they did not coalesce to from large bodies such as those seen in cells infected with reovirus ( , ) . canine rotavirus, cud, produced intracytoplasmic inclusion bodies in different sizes and shapes. some were small and multiple as have been reported for those induced by bovine and simian rotaviruses and some were large and coalesced like those induced by reoviruses. the size and shape of inclusion bodies may depend upon each virus-cell culture system. we observed large inclusions in bovine rotavirus/ma cell and simian rotavirus/ma cell systems (unpublished observation). further serological studies will determine antibody prevalence to canine rotavirus and establish whether or not infection with this strain is associated with disease. if canine rotavirus (cu- ) is found to be of low virulence, it may be evaluated as a potential vaccine strain for use in homologous or even hetero]ogous hosts. the potential use of a heterologous bovine rotavirus for possible use as a vaccine for humans has been suggested previously ( , ) . the effect of trypsin on the growth of rotaviruses rotavirus isolation and cultivation in the presence of trypsin rotaviral enteritis in turkey poults transmission of human rotaviruses to gnotobiotic piglets production of high-titer bovine rotavirus with trypsin committee on the echo viruses: enteric cytopathogenic human orphan (echo) viruses propagation of bovine rotavirus by dogs electron microscopic identification and subsequent isolation of a rotavirus from a dog with fatal neonatal diarrhea rotavirus (reovirus-like) infection of neonatal ruminants in a zoo nursery rotaviruses in diarrheic feces of a dog viral intestinal infections of animals and man tile rotaviruses use of transcription probes to genotype rotaviruses isolation of a rotavirus from a newborn dog with diarrhea proteolytic enhancement of rotavirus infectivity: biologic mechanism rescue of noncultivatable human rotavirus by gene reassortment during mixed infection with ts mutants of a cultivatable bovine rotavirus viral gastroenteritis coronavirus-like [)articles in the feces of normal cats isolation and characterization of feline rotavirus studies on the effect of chymotrypsin on reovirions rotavirus infection in commercial laying hens rhinoviruses: a numbering system diagnostic procedures for viral, rickettsial and chlamydial infections approaches to immunization of infants and young children against gastroenteritis due to rotaviruses a morphological study on human rotavirus simian virus sa- and the related agent infection in avian species antibody to rotavirus in dogs and cats. vet. %ec. t} isolation and cell culture propagation of rotaviruses from turkeys and chickens viruses and diarrhoea in dogs cell culture propagation of neonatal calf diarrhea (scours) virus diarrhea in gnotobiotic calves caused by the rotavirus-like agent of human infantile gastroenteritis classification of human enteroviruses propagation of infantile gastroenteritis virus (orbi-group) in conventional and germ-free piglets canine viral enteritis: prevalence of parvo-, corona-, and rotavirus infections in dogs in the netherlands morphology and stability of infantile gastroenteritis virus: comparison with reovirus and bluetongue virus canine viral enteritis: %ecent developments simian rotavirus sa- plaque formation in the presence of trypsin proteolytic enzymes and rotavirus sa- plaque formation electron microscopy detection and characterization of viral particles in dog stools washington enhancement of antigen incorporation and infectivity of cell culture by human rotavirus a plaque assay for the simian rotavirus sa-ii human rotavirus in lambs: infection and passive protection antigenic comparisons of two new rotaviruses from rhesus monkeys antibody to rotavirus in various animal species. n'at cell culture propagation of porcine rotavirus (reovirusdike agent) diarrhea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis human rotavirus in young dogs propagation of human rotavirus in young dogs structure of the bluetongue virus capsid cell culture studies of a neonatal calf diarrhea virus l~otaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus human rotavirus type : cultivation in vitro methods of virus culture in vivo and in vitro we thank dr. a. z. kapikian for his valuable advice and criticism. received december , key: cord- - w n fy authors: sekiguchi, k.; sugita, s.; fukunaga, y.; kondo, t.; wada, r.; kamada, m.; yamaguchi, s. title: detection of equine arteritis virus (eav) by polymerase chain reaction (pcr) and differentiation of eav strains by restriction enzyme analysis of pcr products date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: w n fy a polymerase chain reaction (pcr) based assay capable of detecting and differentiating seven strains of equine arteritis virus (eav) from around the world was developed. the primers for the pcr were chosen from the orf gene encoding the unglycosylated membrane protein (m). viral rna from cell culture fluids infected with each of the seven eav strains and rna from the live vaccine, arvac, was detected by pcr using four sets of primers. the sensitivity of detection was increased from to times by performing nested pcr enabling the detection of rna at a level of . – pfu. differentiation among the virus strains and the live vaccine was achieved by cutting the pcr-amplified products from three sets of primers with six restriction endonucleases. using this procedure it was possible to distinguish among the seven eav strains used. a potymerase chain reaction (pcr) based assay capable of detecting and differentiating seven strains of equine arteritis virus (eav) from around the world was developed. the primers for the pcr were chosen from the orf gene encoding the unglycosylated membrane protein (m). viral rna from cell culture fluids infected with each of the seven eav strains and rna from the live vaccine, arvac, was detected by pcr using four sets of primers. the sensitivity of detection was increased from to times by performing nested pcr enabling the detection of rna at a level of . - pfu. differentiation among the virus strains and the live vaccine was achieved by cutting the pcr-amplified products from three sets of primers with six restriction endonucleases. using this procedure it was possible to distinguish among the seven eav strains used. the equine arteritis virus (eav) has been classified as a member of the togaviridae family [ , ] . however, recently eav has been found to resemble the coronavirus and torovirus in its genome organization and gene expression strategy [ ] , yet differing from them in virion genome size and virion morphology. therefore eav may be a member of a recently proposed new family of viruses, the arteriviridae, consisting of the lactate dehydrogenase-elevating virus, porcine reproductive and respiratory syndrome virus, and simian hemorrhagic fever virus [ , ] . the clinical signs of equine viral arteritis are very variable and subclinical infections are the most common sequel [ ] . to date a single serotype has been recognized, and no major antigenic variation has been demonstrated among eav isolated from disparatechronological and geographic origins [ ] . murphy et al. showed high levels of genomic heterogeneity among isolates of eav through oligonucleotide fingerprint comparisons [ , ] . we determined the nucleotide sequence of o rf , which encodes the unglycosylated membrane protein (m), from several strains from around the world, and showed many genetic variations among the strains in spite of limited antigenic variations [ ] . the objective of this study was to detect each of the eav strains using pcr, and to differentiate the strains through restriction enzyme analysis of their pcr products, based on the m protein nucleotide sequence data of the strains, together with those of the bucyrus [ ] and modified bucyrus strain [ . eav strains used in this study were as follows: bucyrus (ohio) [ ] , modified bucyrus [ , ] , red mile (kentucky) [t ], ky-a (kentucky) [ ] , wroclaw- (poland) [ ] , bibuna (switzerland) [ ] and vienna (austria) [ ] . in addition, a modified live virus vaccine (arvac) was used. all strains of eav were propagated in an rk rabbit kidney cell line. rna was extracted from gl of culture fluid with gl of rnazol b (biotecx laboratories, inc.). rna was precipitated with isopropanol, using gl of ethachinmate (nippon gene co., japan) as a carrier. the rna pellet was dissolved in ill of te buffer ( mm tris-hc , ph . , mm edta). six primers were selected from the coding region for the m protein. the structure of the eav genome, the location of the primer pairs and the restriction sites used are shown in fig. . upper primer; m , '-ctgaggtatgggagc- these mismatches were single internal mismatches, which probably had no significant effect on the pcr product yield. the mismatches in primer m , m , m and m were c:a (primer: template), a:c, c:t, and g:t, respectively. these mismatches were efficiently amplified even when located at the ' end of the primer [ ] . the sensitivity of pcr for detecting eav was determined by using serial ten-fold dilutions of infected cell fluid with a modified bucyrus strain ( x pfu/ml). the first round pcr using the m -m primer pair generated a faint though visible bp product at a dilution level of - , corresponding to about pfu (fig. a) . when the pcr product obtained from the first round pcr was reamplified with one of the three sets of inner primers m -m , m -m , or m -m , all of the respective bp, bp and bp products were detected at a . dilution level of cell fluid (fig. b) . the results showed that reamplification with the nested or hemi-nested primers increased sensitivity by at least - times, enabling us to detect at a level of . - pfu. however, amplified dna was not detected when the first round pcr product was amplified using an equine herpesvirus (ehv)- gc primer, even though a bp product of ehv was produced using the primer in the presence of an ehv genome (fig. b) . the genome of ehv and equine influenza virus yielded no amplification band using the eav primer pairs. each of the pcr products amplified using the m -m primer was digested with each of the four restriction enzymes; xba i, mva i, mbo ii, and alu i. digestion of pcr products from the bucyrus, modified bucyrus, live vaccine, and wroclaw- strains with xba i gave rise to fragments of about bp and bp (fig. a) . products from red mile and ky-a digested with mva i produced fragments of about bp and bp (fig. b) . digestion of pcr products with mbo ii gave rise to fragments of about bp and bp in all strains except for the wroclaw- and vienna strains (fig. c) . the pcr products from all strains were cleaved by digestion with alu i into two fragments of bp and bp. in bibuna strain a bp fragment was further cleaved into bp and bp fragments, allowing for three fragments of bp, bp and bp to be produced (fig. d) . of the pcr products using m -m primer only pcr products derived from modified bucyrus and the live vaccine were digested by hha i, to give fragments of about t bp and bp (fig. e) . pcr products from ky-a and red mile strains were shown to share the same fragment patterns by the above restriction enzymes. (fig. f) . the results of the digestion of pcr products using m -m , m -m , and m -m , with the appropriate restriction enzymes, are summarized in table . the bucyrus, modified bucyrus and live vaccine, the red mile and ky-a , and the wroclaw- , bibuna and vienna strains were differentiated by the digestion of pcr products using the m i -m primer with the four restriction enzymes xbai, mva i, mbo ii and alui. the modified bucyrus strain and live vaccine were discriminated from the others by the digestion of their pcr products using the m -m primer with hha i. ava ii digestion of pcr products using m -m enabled us to distinguish between the red mile and ky-a strains. in pcr based detection systems for eav, chirnside et al. selected primer from the leader sequence, polymerase (orf l-b) and nucleocapsid gene (orf ) [ ] , and bel~tk et al. chose from the nucleocapsid gene [ ] . we selected oligonucleotide primers from the o r f gene because it has been shown to be an area with a higher sequence homology than o r f among the members of the arterivirus group [ , , ] . we designed primer pairs so that there would be no mismatches or at most, a single base pair mismatch, and ensured that the area of amplification product contained the strain specific restriction site for the endonuclease. [ ] . there were some differences in the m gene nucleotide sequence of kentuncky and wroclaw- isolates from our data. if the restriction fragment length polymorphism (rflp) patterns determined by the restriction enzymes are assessed according to their nucleotide sequences, pleurat fluid, maff ireland, / , nea v , kentucky , and wroclaw- isolates should show the same rflp patterns as the bucyrus isolate. these isolates exhibited a - % nucleotide identity with bucyrus isolate [ ] . however, our rflp patterns were exactly as predicted from our nucleotide sequence data. in this paper we showed the rflp patterns of single amplified pcr products from seven disparate strains using three sets of primers (fig. ) . the same restriction enzyme patterns were also obtained by cutting the second round pcr products with restriction enzymes (data not shown). nested pcr and the restriction enzyme analysis of product can allow for the detection and identification of virus in small amounts of clinical samples. the live vaccine was provided after repeated passages of the modified bucyrus strain in tissue culture. rna from the live vaccine showed the same behavior in restriction enzyme digestion of pcr products as did the rna from the modified bucyrus virus. the restriction site ofhha i in the m -m amplified area seems to be unique to the live vaccine, arvac, and to the modified bucyrus strains, according to our study and to reports by chirnside et al. [ ] . fukunaga et al. demonstrated some serological differences between the modified bucyrus strain and the bucyrus, ky-a , and wroclaw- strains using a serum cross neutralization test [ ] . the differentiation between horses vaccinated with live vaccine and horses infected with wild strain should be reliable through rflp analysis by using restriction sites from other orfs, such as orf , , and , in combination with serological differentiation using the neutralization. the application of pcr for eav detection in clinical samples is presently under investigation. evaluation of a nested pcr assay for the detection of equine arteritis virus infection the virology of equine arteritis reverse transcription and cdna amplification by the polymerase chain reaction of equine arteritis virus (eav) comparison of m and n gene sequences distinguishes variation amongst equine arteritis virus isolates molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily isolation of a filterable agent causing arteritis of horses and abortion by mares. its differentiation from the equine abortion (influenza) virus immunization against equine viral arteritis using modified live virus propagated in cell cultures of rabbit kidney use of the serum neutralisation test for equine viral arteritis with different virus strains identification and antigenic comparison of equine arteritis virus: isolated from an outbreak of epidemic abortion of mares effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type model studies development of a modified virus strain and vaccine for equine viral arteritis studies of an epizootic of equine viral arteritis in racehorses halbur pg (t ) molecular cloning and nucleotide sequencing ofthe '-terminal genomic rna of the 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 preparing for equine arteritis detection and differentiation of eav strains by pcr t analysis ofgenetic variation among strains of equine arteritis virus genomic variability among globally distributed isolates of equine arteritis virus lactate dehydrogenase-elevating virns, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses molecular evolution of equine arteritis virus sequence analysis of modified bucyrus strain of equine arteritis virus key: cord- -qht q l authors: takano, tomomi; azuma, natsuko; satoh, miyuki; toda, ayako; hashida, yoshikiyo; satoh, ryoichi; hohdatsu, tsutomu title: neutrophil survival factors (tnf-alpha, gm-csf, and g-csf) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: qht q l feline infectious peritonitis (fip) is a feline coronavirus (fcov)-induced fatal disease of domestic and wild cats. the infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of fip. this study aimed to investigate the reason for the lesions containing neutrophils in cats with fip. neutrophils of cats with fip were cultured, and changes in the cell survival rate were assessed. in addition, the presence or absence of neutrophil survival factors was investigated in specimens collected from cats with fip. furthermore, it was investigated whether macrophages, one of the target cells of fipv infection, produce neutrophil survival factors (tnf-alpha, gm-csf, and g-csf). we showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. these observations suggest that sustained production of neutrophil survival factors by macrophages during fcov infection is sufficient for neutrophil survival and contributes to development of granulomatous lesions. feline coronavirus (fcov) belongs to group i of the family coronaviridae. fcov consists of three major proteins: nucleocapsid (n) protein, membrane (m) protein and peplomer spike (s) protein [ ] . fcov is classified into serotypes i and ii according to the amino acid sequence of its s protein [ , ] . both serotypes consist of two biotypes: feline infectious peritonitis (fip) virus (fipv) and feline enteric coronavirus (fecv). thus, there are types i and ii fecv and fipv in fcov. fecv is asymptomatic in cats, but fipv causes fip. it has been proposed that fipv arises from fecv by mutation [ , , ] , but the exact mutation and inducing factors have not yet been clarified. fip is a fatal disease, characterized by vasculitis associated with granulomatous inflammation containing b cells, neutrophils and macrophages. the infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of fip [ ] . macrophages/monocytes play an important role in the pathogenesis of fip. it has been reported that the difference in the proliferation of macrophages/monocytes is related to the difference in pathogenicity between fecv and fipv [ , ] . the possibility of feline vascular endothelial cell injury caused by metalloproteinase- and tnf-alpha produced by fipv-infected monocytes has also been reported [ ] . we reported that virus replication in macrophages induced tnf-alpha production, and the tnf-alpha produced was involved in aggravation of the fip pathology: tnf-alpha produced by fipv-infected macrophages was involved in lymphopenia and an increase in the level of the cellular receptor of type ii fipv, aminopeptidase n [ ] . tnf-alpha reportedly inhibits neutrophil apoptosis [ ] , suggesting its involvement in the infiltration of neutrophils into granulomatous lesions in cats with fip, but this has not yet been clarified. it is also unclear whether fipv-infected macrophages/monocytes produce factors other than tnfalpha that are related to the survival of neutrophils. in this study, we investigated the reason for the granulomatous lesions containing neutrophils in cats with fip. neutrophils of cats with fip were cultured, and changes in the survival rate were examined. the presence or absence of neutrophil survival factors in specimens from cats with fip was also investigated. furthermore, whether macrophages, one of the target cells of fipv, produce neutrophil survival factors was assessed. type ii fipv strain - ( tcid /ml) was administered orally to -to -month-old spf cats. nine cats that developed fip symptoms (fip cats), such as fever, weight loss, peritoneal or pleural effusion, dyspnea, ocular lesions, and neural symptoms, and nine -to -month-old spf cats administered a medium as mock infection controls were used in this study. fip diagnoses were confirmed upon postmortem examination, revealing peritoneal and pleural effusions and granulomatous lesions in major organs. all experiments were performed in accordance with the guidelines for animal experiments of kitasato university. felis catus whole fetus- (fcwf- ) cells were grown in eagle's minimum essential medium containing % l- medium, % fetal calf serum (fcs), u/ml penicillin, and lg/ml streptomycin. feline neutrophils and alveolar macrophages were maintained in rpmi growth medium supplemented with % fcs, u/ml penicillin, lg/ml streptomycin, and lm -mercaptoethanol. type ii fipv strain - was grown in fcwf- cells at °c. fipv strain - was supplied by dr. m. c. horzinek of state university utrecht, the netherlands. mab - - (igg a) used in the present study recognizes the s protein of type ii fipv, as demonstrated by immunoblotting. it has been reported that mab - - has virusneutralizing activity in assays carried out in fcwf- and crfk cells, but an enhancing activity in feline macrophage cultures, depending on the reaction conditions [ ] . blood collected from spf and fip cats using a heparinized syringe was centrifuged at , rpm for min, and the supernatant was used as a plasma sample. ascites fluid was collected from fip cats using a heparinized syringe and centrifuged at , rpm for min, and the supernatant was collected. heparinized blood ( ml) from spf and fip cats was diluted in twofold steps with phosphate-buffered saline (pbs) and subjected to ficoll-hypaque density gradient centrifugation at , rpm for min. after the removal of peripheral blood mononuclear cells and supernatant by aspiration from the top layer, the pellets were mixed with an equal volume of saline containing % dextran for granulocyte separation and allowed to stand for min at °c. the top clear layer was centrifuged at g for min, and the pellet was mixed with ml of . % nacl for min to eliminate contaminating erythrocytes and then mixed with ml of . % nacl. the cells were washed three times with pbs and resuspended with growth medium. cell purity was assessed to be more than % neutrophils by the examination of a smear stained with wright/giemsa solutions. to examine the effect of specimens from cats on the survival rate of neutrophils, feline neutrophils ( cells/ ll) were seeded into -well plates and cultured in the presence of fip-cat-derived ascites fluid (final concentration of : ), plasma (final concentration of : ), and spf-cat-derived plasma (final concentration of : ) for h. prior to and after incubation, ll of wst- solution (wst- cell proliferation assay kit; kishida chemical co., ltd, japan) was added. wst- is a tetrazolium salt that reacts with mitochondrial dehydrogenases, forming the formazan dye. expansion of viable cell numbers results in an increase in the activity of the mitochondrial dehydrogenases in the cells, corresponding to an increase in formazan dye metabolism. after the cells were returned to the incubator for h, the absorbance of the formazan produced was measured at nm with a -well spectrophotometric plate reader, as described by the manufacturer. the percent viability was calculated using the following formula: cell viability (%) = (after incubation od/prior to incubation od) . feline alveolar macrophages were obtained from spf and fip cats by broncho-alveolar lavage with hank's balanced salt solution (hbss) as described previously by hohdatsu et al. [ ] . rna isolation and cdna preparation rna isolation and cdna preparation were performed employing the method of takano et al. [ ] . determination of levels of feline gapdh mrna, tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n gene expression cdna was amplified by pcr using primers specific for feline gapdh mrna, tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes. the primer sequences are shown in table . pcr was performed using the method of takano et al. [ ] . the band density was quantified under appropriate uv exposure by video densitometry using scion image software (scion corporation, usa). tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes were quantitatively analyzed in terms of the relative density value compared to the mrna for the housekeeping gene gapdh. confluent fcwf- cell monolayers in -well multi-plates were inoculated with ll of the sample dilutions. after virus adsorption at °c, the cells were washed with hbss, and ml of growth medium containing . % carboxymethyl cellulose was added to each well. the cultures were incubated at °c for days, fixed in % buffered formalin, and stained with % crystal violet. inoculation of feline alveolar macrophages with fipv viral suspension (fipv strain - , tcid / . ml) and mab - - solution were mixed in an equal volume ratio and allowed to react at °c for h, and . ml of this reaction solution was used to inoculate feline alveolar macrophages ( cells) cultured in each well of -well multi-plates. as controls, medium alone and virus suspension alone were added to feline alveolar macrophages. after virus adsorption at °c for h, the cells were washed with hbss and ml of growth medium. the cells and culture supernatant were collected every h thereafter. the cells were used for measurement of the tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes. tnfalpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes were quantitatively analyzed in terms of the relative density value compared to the mrna for the housekeeping gene gapdh. the culture supernatant was employed for determination of the virus titer. data were analyzed by student's t test. the data in fig. a , b were also analyzed using the mann-whitney test. p values \ . were considered to indicate a significant difference between compared groups. the neutrophil counts in peripheral blood of fip cats were examined and compared with those of uninfected spf cats. the count of neutrophils at the time of blood sampling is shown in fig. . the blood neutrophil counts in spf and fip cats were , ± , ll - (mean ± sd) and , ± , ll - , and median values were , and , ll - , respectively (p \ . ). to investigate the cause of increases in the neutrophil counts in fip cats, neutrophils were isolated from spf and fip cats, and the survival rates after -h culture were compared. the survival rate of neutrophils from fip cats was increased, but not significantly (p = . ), compared to that of spf cats (fig. ) . the survival rate of neutrophils in the presence of specimens from fip cats was significantly higher than those of neutrophils cultured with spf cat-derived plasma and medium (fig. ) . tnf-alpha, gm-csf, and g-csf mrna and fcov n gene expression levels in macrophages of spf and fip cats tnf-alpha, gm-csf, and g-csf mrna and fcov n gene expression levels were increased in alveolar macrophages derived from fip cats (fig. ) . the virus titer was significantly higher in the culture supernatant of macrophages infected with a mixture of fipv and mab - - than in that of macrophages cultured with medium and fipv alone. the neutrophil survival rates were significantly increased in the presence of the culture supernatant of macrophages infected with the mixture of fipv and mab - - compared to those in the presence of other supernatants (fig. ) . when spf-cat-derived alveolar macrophages were infected with a mixture of fipv and mab - - , the intracellular tnf-alpha, gm-csf, and g-csf mrna levels increased (fig. ). neutrophils are important for host defense against pathogens. viral infection generally reduces the number of neutrophils. the transfer of peripheral blood neutrophils to marginal tissues, destruction of neutrophils due to excess antibody production, and direct cell death caused by viral infection are considered to be the causes of neutropenia [ , , , ] . in human immunodeficiency virus and canine parvovirus infections, a reduced neutrophil count has been suggested to allow severe bacterial infection [ , ] . feline immunodeficiency virus and feline panleucopenia virus infections also affect stromal cells in the bone marrow, thus leading to a decreased production of neutrophils [ , ] . in contrast, severe acute respiratory syndrome (sars) coronavirus, which belongs to the same family as fipv, can also cause neutrophilia [ ] . neutrophilia has been also reported in cats with fip [ ] . paltrinieri [ ] reported that cytokines accelerate the delivery of neutrophils to the inflamed lesions, thereby prolonging the lifespan of circulating neutrophils. he also reported that the apoptosis of neutrophils is delayed by cytokines, thus increasing the life of neutrophils in lesions. it is likely that neutrophilia in cats with fip is associated with the infiltration of neutrophils into granulomatous lesions. however, there seems to be no established theory to explain the infiltration of neutrophils into granulomatous lesions in cats with fip. in humans, the lifespan of neutrophils is short. when neutrophils are isolated from peripheral blood and cultured in vitro, apoptosis is induced, and about half of the cells die within h [ ] . when neutrophils isolated from peripheral blood of spf cats were cultured for h, more than % died, suggesting that feline neutrophils also die due to apoptosis, similar to human neutrophils. furthermore, coculture with specimens from fip cats increased the survival rate of neutrophils, suggesting that the production of factors involved in the survival of neutrophils is enhanced in fip cats, and these factors act on neutrophils and prolong their lifespan. the tnf-alpha, gm-csf, and g-csf mrna levels were increased in macrophages of fip cats. these cytokine mrna levels were also elevated in macrophages infected with fipv and mab - - , clarifying the presence of neutrophil survival factors in the macrophage culture supernatant. it was suggested that: ( ) fipv-infected macrophages release tnf-alpha, gm-csf, and g-csf in response to virus replication, and ( ) these cytokines act on neutrophils and prolong their survival. we previously reported that fipv-infected macrophages produced tnfalpha and b-cell differentiation/survival factors, and these factors may have been involved in lymphopenia and hypergammaglobulinemia [ ] [ ] [ ] . kipar et al. [ ] suggested that fipv-infected monocytes are involved in feline vascular endothelial cell injury. cytokines and chemical mediators produced by fipv-infected macrophages/monocytes may play an important role in the pathogenesis of fip in cats. fig. the culture supernatant of fipv-infected macrophages promotes neutrophil survival. spf-cat-derived alveolar macrophages ( cells) were cultured with medium alone, fipv, or fipv and mab - - . the culture supernatant was collected after h. the virus titer in the culture supernatant was measured by the plaque assay method (left). the neutrophils of spf cats ( ml - ) were cultured at °c for h in the presence of each culture supernatant (final dilution of : ), and cell viability was assessed by the wst- assay (right). n = . nd not detected fig. relationship between tnf-alpha, gm-csf, and g-csf mrna expression and fipv replication in macrophages. spf-catderived alveolar macrophages ( cells) were cultured with medium alone, fipv, or fipv and mab - - . the cells were collected at h (as a control) and h. the intracellular tnf-alpha, gm-csf, and g-csf mrna expression levels were measured by rt-pcr. tnf-alpha, gm-csf, and g-csf mrna were quantitatively analyzed in terms of the relative density value to mrna for the housekeeping gene gapdh. n = neutrophil survival factors produced by macrophages in cats tnf-alpha, gm-csf, and g-csf are cytokines inhibiting the apoptosis of neutrophils [ , , , ] . these cytokines are secreted by macrophages and t cells. when these cytokines act on neutrophils, the intracellular apoptosis-inhibitory protein level increases, whereas the apoptosis-promoting protein level decreases, prolonging the survival of neutrophils [ , ] . in inflammatory diseases, such as cystic fibrosis and kawasaki disease, the pathological condition is aggravated as the survival time of neutrophils is prolonged [ , ] . moreover, the pathological aggravation of inflammatory diseases is associated with protease and reactive oxygen species produced by neutrophils [ , ] . these mediators may also be produced in excess and aggravate the granulomatous lesions in fip cats. we are now progressing with studies on the expression of apoptosis-inhibitory and -promoting proteins in neutrophils of fip cats. the enhancement of cathepsin b production in macrophages by elastase produced by neutrophils has been reported [ ] , while fipv reportedly requires cathepsin b to invade cells [ ] , suggesting that neutrophils are also involved in a viral replication enhancement mechanism, different from antibody-dependent enhancement (ade), in fipv infection. addie et al. [ ] reported that fcov re-infection of anti-fcov antibody-positive domestic cats might not result in the development of ade. the involvement of neutrophils in the enhancement of fipv production in macrophages and the influence of macrophages on neutrophil survival should be investigated further. virus production and tnf-alpha, gm-csf, and g-csf mrna expression were markedly enhanced in macrophages infected with fipv and mab - - compared to macrophages infected with the virus alone. we used immunofluorescence to detect fipv antigen: - % of cells were positive when spf cat-derived alveolar macrophages were infected with fipv and mab - - , whereas - % of cells were positive when alveolar macrophages were infected with fipv [ , ] . it seems that the increased number of virus-infected macrophages leads to the overexpression of neutrophil survival-factor-associated mrna in macrophages. in cats with fip, virus can be detected in macrophages of granulomatous lesions. to elucidate the mechanism of the infiltration of neutrophils into granulomatous lesions, we used alveolar macrophages, which can be readily collected in appropriate numbers. in addition, alveolar macrophages can be cultured without activation treatment, unlike monocytes. in this study, we showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. these observations suggest that sustained production of neutrophil survival factors by macrophages during fcov infection is sufficient for neutrophil survival and contributes to the development of granulomatous lesions. these findings may be important for elucidating the pathogenesis of fip. risk of feline infectious peritonitis in cats naturally infected with feline coronavirus gamma interferon/interleukin balance in tissue lymphocytes correlates with down modulation of mucosal feline immunodeficiency virus infection immune and idiopathic neutropenia replication of feline 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patients with severe acute respiratory syndrome: retrospective analysis neutrophil survival factors produced by macrophages in cats acknowledgments this work was supported by grant for encouragement of young scientists (no. e ) from the school of veterinary medicine, kitasato university. key: cord- -jozdnhgy authors: snodgrass, d. r.; angus, k. w.; gray, e. w.; menzies, j. d.; paul, g. title: pathogenesis of diarrhoea caused by astrovirus infections in lambs date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: jozdnhgy experimental infection of -day-old gnotobiotic lambs with lamb astrovirus produced mild diarrhoea after an incubation period of about hours. no other clinical symptoms developed. infection was studied by immunofluorescent and histological examination of tissues from the lambs. astroviruses infected only mature villus epithelial cells and subepithelial macrophages in the small intestine, where they produced partial villus atrophy. infected enterocytes were replaced with cuboidal cells from the crypts, and the lesion gradually healed by days after infection. no serological relationship was detected by immunofluorescence between lamb astrovirus antigen in gut sections and antisera to either calf or human astrovirus. viruses of -- nm diameter with a circular outline and stellate surface structure have been observed in faeces from diarrhoeic children ( , ) , lambs ( ) and calves ( ) . the name astrovirus has been suggested for these morphologically distinctive viruses ( ) and will be used in this paper. little is known of the pathogenic potential of these viruses. after oral inoculation they produced mild diarrhoea in lambs ( ) , and partial villus atrophy in calves ( ) and inconsistently caused diarrhoea in adult human volunteers ( ) . this paper reports findings made on the pathogenesis of astrovirns infections in gnotobiotic lambs. six gnotobiotic lambs were infected orally when approximately hours old with intestinal contents from the third passage of lamb astrovirus in gnotobiotic lambs ( ) . these intestinal contents, while the sixth (that killed hours after infection) received . ml of ulfiltered bacteria-free contents. the inoeulum appeared by electron microscopic examination to contain many fewer astroviruses after filtration. one lamb was killed at each of the following hours after infection (p.i.): , , , , and . five gnotobiotie lambs killed between and hours of age served as controls for the histology. six gnotobiotie lambs between and hours of age were controls for laetase estimations. lambs were deeply anaesthetised with sodium pentobarbitone or halothane. segments were obtained from jejunum (about cm distal to the duodeno-jejunal flexure); from midgut; and from ileum (about cm proximal to the ileo-caecm junction, from an area free of peyer's patches). the lambs were then killed by exsanguination, and tissues collected from caecum, colon, kidney, liver and lung. tissues for histological examination were fixed in per cent formalsaline and processed as described previously ( ) . additional small ( mm a) blocks of intestine were fixed in per cent glutaratdehyde in phosphate buffer (pit . ) and processed to armdite. sections bm thick were cut and stained by per cent giemsa, at ° c. villus heights and crypt depths in he-stained sections were measured by ocular micrometer on ten vertically-cut, full length villi and crypts at each site of small intestine. additional portions of all tissues were frozen in a co -ethanol freezing mixture. lengths of small intestine and colon were filled with embedding medium (tissue-tek ii, lab-tek products) prior to freezing, to aid proper orientation of villi. frozen tissues were mounted on microtome chucks and ~m sections cut on a cryostat. an antiserum to lamb astrovirus was prepared as follows :--a gnotobiotic lamb was infected orally with astrovirus, and reinfected days later. after a further days the lamb was given by intramuscular inoculation an astrovirus preparation partially purified by differential centrifugation of intestinal contents. blood was collected for serum preparation days after final inoculation. tissue sections were treated with this antiserum, followed by fluoreseein-conjugated rabbit anti-sheep globulin. control sections were treated with gnotobiotic lamb antiserum to lamb rotavirus, followed by the conjugated anti sheep globulin. for purposes of serological comparison, astrovirus-containing gut sections were stained with calf antiserum to calf astrovirus (kindly supplied by dr. j. c. bridger, compton, berkshire) or human antiserum to human astrovirus (kindly supplied by dr. j. b. kurtz, churchill hospital, oxford), followed by the appropriate fluoresceinconjugated globulins. additional portions of tissue from the three sites of small intestine were collected and stored at -- ° c. laetase estimations used the methods of dahlqvist ( ). faeces samples were collected daily from all lambs, and suspensions examined by electron microscopy for the presence of astrovirus ( ) . the l~mbs killed at , and hours p.i. did not develop diarrhoea. the other three infected lambs developed diarrhoea -- hours after infection, faeces changing from firm and dark brown in character to very loose and yellow. voluntary milk intake remained normal. astrovirus was not observed in faeces from the lambs killed t and hours p.i., but was first seen in the faeces of all other infected lambs between and hours p.i. at necropsy, astrovirus was detected in intestinal contents of all iambs except that killed hours p.i. the control lambs remained clinically normal, and passed firm brown faeces throughout the duration of the experiment. no virus particles were detected in their faeces. specific immunofluorescent staining was detected only in scattered epithelial and subepithelial cells on small intestinal villi (fig. ) . the immunofluorescenee was usually fine and stippled in appearance (fig. b) . infected cells were present between and hours p.i. (table ). fewer infected celts were present in jejunum than in midgut or posterior ileum. the greatest number of infected cells was present early in infection, during the incubation period from to hours p.i. the infected enterocytes were generally scattered through the apical half of the villi. occasional infected cells were observed in the villus lamina propria..no specific reaction was observed when the rotavirus antiserum was used. tissues other than small intestine showed no specific reaction. no immunofluoreseenee was observed with lamb astrovirus-infeeted intestinal sections and the antisera to either calf or human astrovirus. the proximal intestine was unchanged throughout the experiment. no morphological alterations were seen in the midgut at hours p.i. many enterocytes in the ileum at hours p.i. contained large ovoid vacuoles apical to the nucleus, although the villi were long and slender. changes were first observed at hours p.i. in the midgut. here the villi were long and their lateral margins contained many clefts or mieroerypts (fig. ) , compared with control villi (fig. ) . the enteroeytes lining the lower one-third of the villi appeared normal, but those of the apical two-thirds had rounded margins and were cuboidal rather than columnar. many enterocytes contained large single basal vacuoles and multiple small apicm vacuoles (fig. ) . the apical vacuoles often impinged upon and indented the nucleus, which consequently appeared collapsed or pyknotic. both apical and basal vacuoles contained pleomorphie schiff-negative bodies which stained deep mahagony-red by pollak's trichrome method. similar intra-eytoplasmie bodies were also seen in some enteroeytes and subepithelial maerophages, usually close to the nucleus. these bodies were most clearly demonstrated in araldite s~etions (fig. ) , and have been shown subsequently (gray, e. w., in preparation) to contain arrays of astrovirus partieles. the lamina propria of affected villi contained moderate numbers of maerophages with abundant cytoplasm. goblet cell numbers were comparable to controls. the ileum was unaffected at this stage; single clear apical vacuoles were present in many of the enterocytes. by hours p.i., villi in midgut and ileum were obviously shorter and more spatulate than those in equivalent control sites, or at earlier stages of the infection. at hours p.i., villi in the midgut and ileum were short and blunt with crenated epithelium, and crypts which were elongated contained numerous mitotic figures. the lamina propria contained infiltrates of macrophages, lymphoeytes and neutrophils, as well as eosinophils in similar numbers to the control iambs. none of the enteroeytes in the midgut contained the multiple apical vacuoles and granular bodies seen at hours p.i. ij[owevcr, single apical vacuoles were seen in the ileum of the infected iambs at and hours p.i. by hours p.i. the villi in the midgut site were long and slender and indistinguishable from normal intestine, but those in the ileum were stunted and lined by a erenated, partly euboidal epithelium. at days p.i., however, all three intestinal sites were morphologically normal. large basal vacuoles were seen in m i d g u t enterocytes of control lambs at and hours of age, but apical vacuoles were confined to the ileum of control lambs. these findings are in accord with those m a d e in normal calves ( , ) and piglets ( , , ) . the vacuolated cells m a y be absorptive with a m a r k e d pinoeytotic c a p a c i t y ( ) . a few neutrophils were present in b o t h caecal and colonic mucosa of the lambs killed hours p.i., b u t not in a n y other lamb. no changes were found in a n y of the other tissues at a n y stage. the villus heights and crypt depths at the three sampling sites in the five control iambs did not v a r y significantly with age. n o r m a l measurements for villi and crypts at each site were therefore obtained b y pooling observations for all five lambs. measurements from individual infected lambs were compared with these n o r m a l values ( table ) . the length of the villi in j e j u n u m did not differ significantly from normal. villus a t r o p h y was observed at and hours p.i. in midgut, and from to hours p.i. in ileum. the crypts in all three sites showed a progressive elongation t h r o u g h o u t the experiments. the most m a r k e d changes were observed in ileum, and are illustrated in figure . lactase levels in the control lambs were ¢. ± . , . - . , and . - . units/g tissue for the proximal, mid, and distal small intestinal sites respectively. in midgut of infected lambs, observations were consistently below those of the controls, fmling to a minimum of . units/g tissue at hours p.i. :no consistent change was observed in laetase concentrations in proximal and distal sites. these experiments confirmed the ability of lamb astrovirus to multiply in the intestinal tract of gnotobiotic lambs, and to cause diarrhoea. the site of multiplication was shown by immunofluorescence and electron microscopy (gray, e. w., in preparation) to be the small intestine, and these same techniques failed to find evidence of astrovirus infection in any other site. immunofluorescence showed less evidence of virus multiplication in jejunum than in other levels of small intestine, and this was reflected in the absence of histological change. crypt hypertrophy was, however, as marked in jejunmn as at more distal sites, but the stimulus for this is not known. lamb rotaviruses have similarly been found to cause least damage in the jejunum ( ) . damage produced by astrovirus infection could be demonstrated by histopathology and estimations of lactase, and was consistently associated with a mild transient diarrhoea. a sequence of events with initial epithelial cell infection and destruction leading to partial villus atrophy, reclothing of the villi with relatively immature cells, and eventual healing of the lesion, can be postulated. this effect is similar to that of other viral infections of villus epithelial cells, particularly rotavirus ( , ) and coronavirus ( , , ) infections. in the lamb, astrovirus infection is at each stage less severe than rotavirus infection ( , ) , with fewer enterocytes infected, a lesser degree of villus atrophy and a milder clinical disease. the astroviruses of lambs, calves, and man have not been shown to be serologically related by immunofluorescence. further serological and biochemical studies are necessary to investigate the relationships between these viruses. this study has confirmed that lamb astrovirus is a pathogen of the small intestine of lambs. however, the only information available on the epidemiology of any of the astroviruses is that antibody to bovine astrovirus was detected in cattle in or herds ( ) , so no attempt can be made as yet to define their role in causing diarrhoea under natural conditions. the excellent technical assistance of ~¢iessrs j. redmond and n. inglis is gratefully acknowledged. method for assay of intestinal disaccharidases gnotobiotie piglets experimentally infected with neonatal calf diarrhoea reovirus-like agent (rotavirus) intestinal infection of neonatal dogs with canine coronavirus -- : studies by virologic, histologic histochemieal and immunofluoreseent techniques astrovirus associated gastroenteritis in a children's ward astrovirus infection in volunteers .: nm particles in faeces in infantile gastroenteritis stool viruses in babies in glasgow pathology of neonatal calf diarrhoea induced by a reo]ike virus scanning electron, light and transmission electron microscopy of intestine of gnotobiotie calf scanning electron, light and immunefluorescent microscopy of intestine of gnotobiotic calf infected with calf diarrheal coronavirus vacuolated villous epithelium of the small intestine of young pigs pathological changes in the small intestine of neonatal pigs infected with a pig reovirus-like agent (rotavirus) transmissible gastroenteritis of swine: virus-intestinal cell interactions a survey of rotavirus associated with gastroenteritis in aboriginal children in western australia detection and transmission of nm virus particles (astroviruses) in faeces of lambs with darrhoea rotavirus infection in lambs: pathogenesis and pathology experimental rotavirus infection in lambs pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs isolation of small viruses, astrovirus-like and calicivirus-like, from acute enteritis of calves authors' address: dr. d. r. snodgnass, animal diseases %esearch association, moredun institute, gilmerton/toad, edinburgh, scotland. %eeeived december , key: cord- -h ppa authors: plagemann, p. g. w. title: hepatitis c virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: h ppa hepcv is the major cause of nanb pt hepatitis and is also implicated as the cause in a large proportion of sporadic cases of nanbh. chronic infection with hepcv has also been linked to the development of hepatocellular carcinoma. chimpanzees and marmosets are the only animals found to be experimentally infectable and the virus has not been propagated in any cell culture system. hepcv is an enveloped virus with a diameter of – nm and a -kb positive-stranded rna genome. its genome organization resembles that of the flaviviruses and pestiviruses. a ′-untranslated segment of nucleotides precedes a continuous orf of / nucleotides which is followed by a nucleotides long ′-non-coding segment. further work is required to resolve the question of whether the genomic rna possesses a ′-poly(u) or poly(a) tail. the genome also carries an internal poly(a) segment towards the ′-end of its orf. genomic rna is probably translated into a single polyprotein of / amino acids which is processed into functional proteins. the viral proteins have not been identified, but on the basis of the predicted amino acid sequences, hydrophobicity plots, location of potential glycosylation sites and similarities of these properties to those of pesti- and flaviviruses, the following genome organization has been predicted. the predicted viral structural proteins, a nucleocapsid protein and two envelope glycoproteins are located at the aminoterminal end of the polyprotein. they are followed by a highly hydrophobic protein and proteins that exhibit proteinase, helicase and replicase domains and thus are probably involved in rna replication and protein processing. the replicase domain is located close to the carboxy terminus of the polyprotein. although the overall nucleotide and amino acid homologies between hepcv and pestiviruses are low, a number of similarities exist that point to a closer ancestral relationship to the latter than the flaviviruses. first, the ′-untranslated segment of the hepcv genome resembles that of the pestivirus genomes in size and presence of several short orfs and it contains several segments with high nucleotide homology. second, the two putative envelope glycoproteins of hepcv resemble two of the three putative envelope glycoproteins of the pestiviruses. because its genome organization and predicted virion structure closely resemble those of the flaviviruses and pestiviruses, hepcv has been proposed to be placed in the familyflaviviridae. it has been suggested to be classified as a new third genus in this family because it is only remotely related to the pestiviruses and flaviviruses in nucleotide sequence of its genome and the amino acid sequences of the predicted viral proteins. on the basis of genomic sequence information, an immunoprobe has been devised for screening blood supplies and donors for anti-hepcv antibodies to a non-structural protein of hepcv. the immuno-assay exhibits a high efficiency in detecting infected donors, though one caveat is that antibodies to the test antigen develop in infected individuals only between and months post infection. on the other hand, viral rna can be detected in plasma by reverse transcription and amplification of the cdna by pcr within a few days post infection. thus the latter technique may become more important in the detection of hepcv in blood and tissues once the technique becomes more widely established as a diagnostic tool. the untranslated ′-segment has been found to be highly conserved in the genomes of different hepcv isolates from various parts of the world. the replicase domain is also highly conserved, but considerable amino acid and nucleotide differences exist in other segments of the long orfs of various hepcv isolates. divergence among different isolates is particularly great (up to %) in the segment encoding the two putative envelope glycoproteins and the upstream hydrophobic protein. the variability in envelope glycoproteins needs to be considered in the development of immuno-probes and of vaccines for hepcv. segment of the hepcv genome resembles that of the pestivirus genomes in size and presence of several short orfs and it contains several segments with high nucleotide homology. second, the two putative envelope glycoproteins of hepcv resemble two of the three putative envelope glycoproteins of the pestiviruses. because its genome organization and predicted virion structure closely resemble those of the flaviviruses and pestiviruses, hepcv has been proposed to be placed in the family flaviviridae. it has been suggested to be classified as a new third genus in this family because it is only remotely related to the pestiviruses and flaviviruses in nucleotide sequence of its genome and the amino acid sequences of the predicted viral proteins. on the basis of genomic sequence information, an immunoprobe has been devised for screening blood supplies and donors for anti-hepcv antibodies to a non-structural protein of hepcv. the immuno-assay exhibits a high efficiency in detecting infected donors, though one caveat is that antibodies to the test antigen develop in infected individuals only between and months post infection. on the other hand, viral rna can be detected in plasma by reverse transcription and amplification of the cdna by pcr within a few days post infection. thus the latter technique may become more important in the detection of hepcv in blood and tissues once the technique becomes more widely established as a diagnostic tool. the untranslated '-segment has been found to be highly conserved in the genomes of different hepcv isolates from various parts of the world. the replicase domain is also highly conserved, but considerable amino acid and nucleotide differences exist in other segments of the long orfs of various hepcv isolates. divergence among different isolates is particularly great (up to %) in the segment encoding the two putative envelope glycoproteins and the upstream hydrophobic protein. the variability in envelope glycoproteins needs to be considered in the development of immuno-probes and of vaccines for hepcv. hepatitis c virus (hepcv) is a new pesti-flavi-like virus which has been discovered only relatively recently as the main cause of non a, non b hepatitis (nanbh). the abbrevation hepcv has been suggested for hepatitis c virus [ ] instead of hcv used previously in most communications in order to distinguish it from hog cholera virus and human cytomegalovirus which also have been generally abbreviated hcv. the abbreviation hocv has been suggested for hog cholera virus [ ] . the present review will only highlight main aspects of hepcv pathogenesis and molecular properties, especially as they relate to pestiviruses [for review of pestiviruses, see ] . for more detailed information the reader is referred to recent reviews by choo etal. [ ] , bradley et al. [ ] and choo [ ] . post-transfusion (pt) hepatitis is still a relatively common consequence of blood transfusion [ ] . from studies before the incidence in the u.s.a. was estimated as - % [ , ] . the application of diagnostic tests for hepatitis b virus (hbv) and hepatitis a virus (hav) during the 's indicated that most of pt hepatitis was not caused by these viruses but rather by another virus(es) termed nanbh virus. recent estimates are that to > % of pt-nanbh presently observed in the u.s.a., europe and japan is caused by hepcv [ ] , which amounts to t , to , cases per annum in the u.s.a. alone [ , ] . in addition, intravenous drug addicts and hemophiliacs are at high risk of acquiring hepcv infections and hepcv has also been implicated as the cause of - % of sporadic cases of nanbh [ , . another recently discovered hepatitis virus, hepatitis e virus (hev), is a calicivirus-like virus mainly associated with enterically transmitted hepatitis [ ] . acute infections with hepcv seem to be less severe than those caused by hbv or hav but a greater proportion of hepcv than hbv infections (about %) are likely to progress to a persistent chronic state and it is estimated that about % of the chronic carriers develop cirrhosis of the liver [ , , ] . furthermore, chronic infections with hepcv have been linked to the development of hepatocellular carcinoma in europe, japan and the u.s.a. [ , - (see below) . no definitive information is presently available on the mechanism of nonparenteral transmission of hepcv. no vector or alternate hosts have been identified. intrafamilial and sexual transmission have been implicated [ , t , ] . little is also presently known about the pathogenesis of hepcv. attempts to infect various cell culture systems or small laboratory animals have been unsuccessful. however, hepcv can be readily transmitted by parental injection to chimpanzees which have served for years as primary model system for nanbh [see , , ] . chimpanzees also developed hepatitis after injection of factor viii concentrate and factor ix complex that had been implicated in the transmission of nanbh to human recipients and nanbh was serially transmitted by parental injection from chimpanzee to chimpanzee [see , - . the clinical disease in chimpanzees mimics that observed in humans, but may vary greatly between individual humans or chimpanzees. infections often remain asymptomatic. when acute illness develops, it generally occurs - weeks p.i. and consists of a range of symptoms including fever, chills, headache, weakness and jaundice. after parenteral or non-parenteral transmission hepcv infections seem to be mainly localized in the liver since tissue damage seems limited to this tissue [ - . liver injury is associated with characteris~ic alterations in the cytoplasm of infected or affected hepatocytes in both humans and chimpanzees [reviewed in ]. these include dense reticular inclusion bodies, convoluted membranes de-rived from proliferated smooth endoplasmic reticulum, bundles of granular microtubules and characteristic tubular structures, which initially earned hepcv the name tubule-forming agent [ ] . also many endothelial cells were found to contain peculiar bundles of hexagonal-packed microtubules, which, when sectioned, resembled arrays of nm particles. some of these ultrastructural changes; however, are not unique to hepcv infections; similar changes have been observed in cells infected with other rna viruses [reviewed in ] and may represent general responses of cells to cytotoxic effects of these viruses. monoclonal antibodies have been generated that specifically interact with tubular aggregates in liver biopsies of nanbh and hepatitis delta virus-infected chimpanzees [ , ] . it seems likely that the ultrastructural changes and antigen formation are mediated by interferon formed in the course of host cell-specific responses to infection by these viruses [ , ] . the mechanism of liver injury is not fully understood and factors other than cytocidal replication of hepcv in hepatocytes may be involved. liver injury is usually associated with an inflammatory reaction and marked increases in alanine aminotransferase (alt) activity in the circulation, which are general features reflecting liver injury. elevated plasma alt activity serves as general indicator of liver injury and is measured by blood banks in a "surrogate" assay to screen blood donors for potential infection by nanbh viruses [ , , ] . the recent development of assays for antibodies to a hepcv polypeptide (c - ) that is part of the viral non-structural proteins (see below) has allowed studies on the humoral antibody response of hepcv-infected patients and chimpanzees. a recent study of stored blood samples from prospective studies of u.s.a. patients with transfusion-associated chronic hepatitis documented by liver biopsies indicated that the time course of changes in plasma alt activity and of the formation of anti-hepcv antibodies can vary considerably between patients [ ] . a number of common features, however, were observed. in agreement with earlier studies, the onset of acute infection generally occurred between and weeks after transfusion and was associated with increases in plasma alt activity of fold or more as well as with increased serum bilirubin levels. all of the patients developed anti-c - hepcv antibodies but only at an average of about weeks after transfusion. however, high antibody levels persisted in all but one patient for at least - years. serum alt activity levels varied greatly between patients during the course of the chronic infection and a significant elevation was not always prevalent. similarly, % of donor sera tested contained significant anti-hepcv antibodies, but of the anti-hepcv antibody positive sera only % exhibited elevated alt activity. in another prospective study in taiwan, % of chronic pt-nanbh patients developed anti-c - hepcv antibodies - weeks after transfusion [ ] . comparative results have been reported from studies in the u.s.a., japan, italy, germany, greece and holland [summarized in , ] . of a total of patients with chronic pt-nanbh about % were seropositive for hepcv antibodies. the prevalence of serum anti-hepcv antibodies in patients with sporadic nanbh, hemophiliacs and intravenous drug abusers was only slightly lower, whereas the prevalence among blood donors with normal serum alt activity ranged from . to . %. in two experimentally infected chimpanzees anti-c - hepcv antibodies became detectable only and weeks p.i., respectively, whereas ultrastructural changes in the liver became apparent during the first week p.i. and infectionrelated increases in serum alt activity occurred between and weeks p.i. [ ] . ultrastructural abnormalties in the liver and serum anti-c - antibodies persisted in one of the chimpanzees for at least months p.i. but disappeared in the other one, and serum alt activity returned to near normal in both. the overall results have suggested that screening of donors for anti-hepcv antibodies could prevent the majority of cases of pt-nanbh, including many that might be missed by the assay of surrogate markers [ , , ] . the long latent period between hepcv infection and development of anti-c - antibodies in both humans and chimpanzees ( - weeks), however, indicates that a considerable time period exists when hepcv can be transmitted but not detected by the immunological test. of interest is the question of whether the long latent period may reflect a late antibody response to the non-structural proteins measured by the anti-c - immune assay. perhaps antibodies to the structural proteins of hepcv (see below) are formed more rapidly. indeed, it has been reported that antibodies to the hepcv core protein were detected by western blotting in a higher proportion of sera from patients with chronic nanbh ( %) than antibodies to the c - antigen ( %) [ ] . in another recent study, % and % of sera from chronic nanbh patients were positive for antibodies to hepc virus core protein and c antigen, respectively [ ] . furthermore, in one nanbh pt patient, anti-core protein antibodies became first detectable in serum weeks after transfusion, whereas antibodies to the c antigen were first detected weeks after transfusion [ ] . it also has become apparent that hepcv rna becomes detectable by polymerase chain reaction (pcr) procedures much sooner after infection than anti-hepcv antibodies and that hepcv rna might be detectable in the absence of detectable levels of serum antibodies. the procedure involves centrifugation of plasma at , rpm in a beckmann ultracentrifuge for h [ ] , extraction of rna from the pelleted material, reverse transcription of the rna using an appropriate "antisense" oligonucleotide primer to a hepcv genomic sequence and amplification of the product after addition of an appropriate "sense" oligonucleotide primer [ , ] . the pcr product is then detected by southern blot analysis with the use of a labeled hepcv cdna that lies between the two primers used [ ] . or, if so desired, the pcr product is cloned and sequenced [ , , , ] . using primers to published sequences (see below) hepcv rna was detectable in serum of two experimentmly infected chimpanzees days p.i. [ ] . it was also detected in serum of one patient with acute pt nanbh, who was seronegative for anti-c - antibodies, in serum from one chronically infected chimpanzee [ ] , and in livers or sera of patients with chronic nanbh [ , , , , ] . in this context, it is of interest that only - % of patients with acute and resolving hepcv infections develop anti-c - antibodies. the question arises as to what factors are involved in the resolution of the infection and elimination of the virus. perhaps the latter are mediated by antibodies to viral proteins not represented by the c - antigen or by cellular immune responses. since the only hepcv titration assay available at present is an expensive endpoint dilution assay in chimpanzees or marmosets [see , ] , only limited information is available on hepcv viremia in infected humans and chimpanzees over the course of the disease. nevertheless, all data indicate that serum hepcv titers are generally low; i.e., < chimpanzee infectious doses (cid)/ml. however, titers of up to cid/ml of serum have been observed in plasma of selected chimpanzees during periods of exacerbated chronic phase disease [ ] . also, liver tissue from one of the animals contained about t cid/g of tissue [ ] and an unusual titer of at least cid/ml has been observed in serum from one patient with acute nanbh (strain h) [ ] . in a recent study in japan, % of patients with hepatocellular carcinoma who lacked serum antibodies to hbv were found to be seropositive for the hepcv c t - antigen compared to an incidence of - % in control groups [ ] . a similar incidence of serum anti-c - hepcv antibodies ( %) has been reported for another group of patients with hepatocellular carcinoma in japan [ ] . although these and similar results reported from the u.s.a. and europe implicate chronic hepcv infections as the cause of hepatocellular carcinoma, potential mechanisms of this effect remain to be elucidated. since viruses, like hepcv, lack oncogenes and do not replicate via dna intermediates, indirect effects are indicated. perhaps extensive stimulation of hepatocyte proliferation in the course of chronic liver injury plays a role. recent randomized, controlled trials have indicated that treatment with - units of recombinant interferon a b (intron a, schering-plough, kenilworth, nj) thrice weekly is effective in controlling nanbh in many patients [ , ] . both decreases in serum alt activity and improved histological features of the liver were observed. however, interferon treatment does not seem to result in termination of the infection since most patients relapsed when treatment was discontinued after months. only limited information is presently available on the structure and biochemical properties of hepcv. filtration studies have shown that the diameter of infectious virions is < nm [ ] or between and nm [ ] . electron microscopic examinations revealed togavirus-like particles with diameters of - nm in sera of chimpanzees and humans with chronic nanbh and similar particles were observed in cytoplasmic cisternae of hepatocytes of infected chimpanzees but the origin and identity of these particles has not been ascertained [ ] . the virus-like particles detected by electron microscopy exhibited a buoyant density in sucrose density gradients of . - . g/cm [ ] . in contrast, a buoyant density of . % . g/cm was observed in sucrose density gradient centrifugations in which the gradient fractions were assayed for infectious hepcv by titration in chimpanzees (d. w. bradley, pers. comm.). from other sedimentation analyses it has been concluded that the sedimentation coefficient of hepcv is < - s [ , ] hepcv is an enveloped virus since its infectivity is destroyed by extraction with chloroform [ , ] . infectivity is also abolished by heating at °c for min [ ] . the sequence of the hepcv rna genome has been determined via sequencing of cdnas [ , , , ] . initially a random-primed expression cdna library in x gttl was prepared from rna extracted from infectious material concentrated from a large volume of high titer plasma from a chimpanzee (see above) with a chronic nanbh infection. the animal represented the second chimpanzee passage of the virus present in a human factor viii concentrate [ , ] . the cdna library was screened with serum from a chronic nanbh patient as a potential source of antiviral antibodies [ ] . a single positive clone ( - - ; see fig. ) was identified among a total of about clones. using the - - clone, a larger overlapping cdna clone ( ) was isolated. clone cdna did not hybridize to human or chimpanzee cell dna in southern hybridization analyses, but it hybridized to rna extracted from the liver of nanbh virus- [ ] infected chimpanzees [ ] . in northern hybridization analyses, clone cdna hybridized to oligo(dt)-selected rna in the to kb range. the results indicated that the putative positive strand viral rna isolated from infectious plasma had a size of at least kb. the results also suggested that hepcv rna contains a poly(a) sequence(s) but it is unclear whether its binding to igo(dt) is due to an internal or a '-terminal poly(a) sequence [ , , ] (see below). three additional cdna clones were isolated from the original cdna library using clone - - cdna as a probe and found to encode a continuous orf [ ] . a reconstructed clone (c ) encoding this orf (see fig. i ) was fused to a human superoxide dismutase (sod) cdna (c - ), which facilitates the efficient expression of foreign proteins in yeast, and expressed as a fusion protein containing amino acids in yeast. the sod/hepcv polypeptide produced in yeast has been used to develop a radioimmune assay (ria) and an elisa for the assay of anti-hepcv antibodies [ ] . commercial elisa kits are available from ortho diagnostic systems (raritan, ny) and abbott diagnostics (chicago, il). as discussed already, the assays have been used extensively already in prospective studies and in screening blood donors for serum anti-c hepcv antibodies. by primer extension analyses a sequence of a total of of the hepcv genome was initially determined which encodes a continuous orf of amino acids [ ] . this sequence is located towards the '-end of the hepcv genome (see fig. ) and exhibits certain similarities to those of flaviviruses and pestiviruses (see below). recently, sequence analyses of the '-region, presumably encoding the structural proteins, as well as of the '-end of the original chimpanzee hepcv isolate (variously referred to as prototype hepcv or hepcv-pt, hepcv- or hepcv-uc) have been completed [ , , ] . a sequence of a total of nucleotides has been reported for the hepcv- genome which encodes a continuous orf of amino acids ( table ). the translation initiation codon starts nucleotides from the reported '-terminus. recently, consensus sequences have also been reported for two hepcv genomes isolated from pooled plasma of nine japanese nanbh patients (referred to as hepcv-j) [ ] and of fifty japaneses blood donors with elevated plasma alt activity (referred to as hepcv-kb) [ ] . in both studies virus particles were collected from plasma by ultracentrifugation, rna was extracted from pelleted material and reverse transcribed and cdna libraries prepared in k~ gttl. in one study, the viral rna was reverse transcribed using appropriate antisense oligonucleotides as primers or the viral rna was polyadenylated and reverse transcribed using oligo(dt) as primer [ ] . in the other study, viral rna was reverse transcribed using random hexanucleotide primers [ ] . the nucleotides long sequence of hepcv-j rna contains '-and '-terminal untranslated segments of and nucleotides in length, respectively (table ) . the genome of hepcv-kb is nucleotides long and possesses and nucleotides long '-and '-terminal untranslated segments ( table ). the '-untranslated segment of hepcv- is and nucleotides longer than those reported for hepcv-j and -kb, respectively. it may represent the true '-terminus of the hepcv genome since several independent clones had the same terminal sequence [ ] . the y-terminus can potentially form a hairpin structure with a calculated ag value of - . kcal [ ] . the '-untranslated segment resembles those of pestiviruses, rather than those of flaviviruses, in its greater length and presence of short orfs [ ] . the hepcv '-untranslated segment encodes three orfs of - amino acids [ ] but no functions have been identified for these orfs. in addition~ it exhibits considerable nucleotide homology ( - %) with the '-untranslated segments of pestivirus genomes [ , , ] . in fact, four blocks of , , , and nucleotides show sequence identity of > % [ ] . whether the y-ends of these genomes are capped is unknown. the '-untranslated segments of hepcv-.j and -kb rna are nucleotides longer than that reported for hepcv- and contain a '-aggcca- ' repeat separated by au as well as a terminal string of us (table ). this sequence probably represents the actual '-terminus of the viral genome since the same '-terminal sequence, except for the displacement of one c has been reported for hepcv-kb and -j rna (see table ). however, some uncertainties still exist concerning the true '-terminus since for both reported genomes the 'terminal sequence has been derived from a single cdna. these results may suggest that the genomic rna of hepcv does not possess a '-poly(a) tail, but rather a poly(u) tail. if this conclusion is correct, it follows that the complementary negative strand will possess a '-poly(a) tail. interestingly, the genomic rna also possesses an a-rich segment in the most 'end of its orf (nucleotides - ) [ ] . this segment is '-aaaaaaaaaacaaa- ' for hepcv- and '-aaagaaaaaccaaa- ' for hepcv-j and -kb. whether these a-rich segments have a function in hepcv replication is unclear at present. nevertheless, they could account for a weak binding of both positive and negative strands of hepcv rna to oligo(dt) as has been observed for the genomic rna [ ] . on the other hand, han et al. [ ] suggested that at least some hepcv rna molecules may be polyadenylated because cdnas to the '-end of the viral genome could be generated by reverse transcription of rna extracted from high titer plasma of an infected chimpanzee using an unrelated oligonucleotide with a '-oligo(dt) tail as primer. amplification of the product by pcr yielded three types of cdnas, all of which contained "-poly(a) tails. one cdna encompassed the nucleotide '-non-coding segment of hepcv- rna plus a poly(a) tail. in addition to the sequences of the hepcv- , -j, and -kb genomes, the 'terminal end nucleotides of a human hepcv isolate derived from another batch of plasma of healthy japanese carriers have been determined [ , [ ] [ ] [ ] . a non-coding segment of nucleotides is followed by an orf of amino acids. it overlaps with a amino acid-long sequence (amino acids - ) that has been determined for six different hepcv isolates, four from the u.s.a. and two from italy [ ] . the gene products of hepcv have not been identified, but on the basis of the amino acid sequences of the long orfs of the various hepcv isolates, the hydrophobicity profiles of the predicted proteins, the location of potential glycosylation sites and similarities in these properties to the proteins encoded by the pestivirus and flavivirus genomes, certain predictions have been made. the first amino acids of the n-terminal end are thought to represent the nucleocapsid protein (c; fig. ). the formation of a kda (p ) protein has been demonstrated in mammalian cells transfected with this cdna sequence under the control of a foreign promoter [ , , ] and in transfected bacteria [ ] . furthermore, the putative nucleocapsid protein of hepcv exhibits a similar high content of basic amino acids as the nucleocapsid proteins of flaviviruses and pestiviruses and lacks n-glycosylation sites [- , , , ] . the next amino acids in the hepcv genome are thought to represent an envelope glycoprotein (e ; fig. ) because the potential protein is mostly hydrophobic and contains six possible n-glycosylation sites. the hydrophathic plot of this domain resembles that of gp of the pestiviruses [ , ] . the hepcv genome seems to encode a second adjacent glycoprotein, about amino acids in length, which seems to correspond in location to the major structural glycoprotein of pestiviruses (gp ) and the non-structural ns glycoprotein of the flaviviruses (see fig. ). the two predicted hepcv glycoprotein orfs are preceded by putative signal peptide sequences [- , ] . other preliminary studies indicate that hepcv may possess at least three structural proteins, the - kda basic nucleocapsid protein (c) and two glycoproteins, a hydrophobic kda protein (e ) and a kda glycoprotein (e /ns ) [ , ] . this arrangement basically resembles that of the pestiviruses rather than that of the flaviviruses, except that the hepcv genome does not encode a protein that corresponds to gp of the pestiviruses and therefore is shorter [for review of pestiviruses, see ] . another interpretation of the reported hepcv genomic sequences is that only the first amino acids make up the nucleocapsid protein, that the next approximately amino acids represent a matrix (m) protein, that e is the single envelope glycoprotein of hepcv and that e /ns is a non-structural protein [ ] . this genome organization would be equivalent to that of the flaviviruses, except that the pre-m segment would be deleted. the question of which of these interpretations is correct can only be resolved by isolation and characterization of the structural and non-structural proteins of the virus. considerable similarities also exist between hepcv and flaviviruses and pestivirus genomes in the location of specific motifs generally associated with the genomes of positive-strand rna viruses (see fig. ). the replicase motif characterized by the consensus gdd sequence is located close to the '-end of the hepcv orf comparable in its location to the ns and p proteins of flaviviruses and pestiviruses, respectively [ , , , ] (see fig. ). a serine proteinase motif (gxsgxp) [ ] is found in the hepcv genome at the same relative position as in the genomes of flaviviruses (ns ) and pestiviruses (p ; see fig. ) [ ] . immediately downstream from the putative proteinase sequence are consensus sequences in the genomes of the three viruses that are typical for the proposed superfamily of helicase proteins [ , ] . the hepcv genome also encodes a highly hydrophobic protein sequence equivalent to ns of the flaviviruses and the amino terminal end of p of the pestiviruses, respectively [ , , ] (see fig. ). overall, however, the amino acid sequence of hepcv- is only distantly related to those of the flaviviruses and pestiviruses [ , , , ] . because of these differences but otherwise great similarities in genome organization and viron properties, hepcv has been suggested to be classified as a new genus within the family flaviviridae [ , , ] . the y-untranslated segment is highly conserved. for example, the -nucleotides '-segment upstream of the continuous orf of hepcv- , -kb, and -j differ from each other by only one or two nucleotides [ , , ] . similarly, a maximum divergence of - nucleotides was observed between the r-untranslated segment of hepcv- and those of other isolates from the u.s.a., australia, italy, japan, korea, argentina, south africa, and taiwan [ ] . the high degree of conservation of the '-segment suggests that it provides important regulatory functions. the '-terminal segment seems somewhat less conserved. although only one nucleotide differs in the '-untranslated segments of hepcv-kb and -j [ , ] , the nucleotides t-segment of hepcv- differs from those of the japanese isolates by at least % [ , ] . considerable sequence differences also exist between various hepcv isolates in the long orf, but not all parts vary equally. for example, the orfs of the two japanese isolates hepcv-kb and -j exhibit . % amino acid identity, but the amino acid identity is only . and . % for the chimpanzee isolate hepcv- and hepcv-kb and hepcv-j, respectively. (sequences were analyzed using the molecular biology information resource suite of programs (mbir; baylor college of medicine, houston, tx). multiple alignments were produced with implementation of the klotz and blanten method [ a] . protein sequences were further analyzed using "homology" [ a] with the dayhoff scoring matrix.) however, even though the overall percentage of amino acid differences between the hepcv- rf and those of the two japanese isolates are about the same, a considerable number of different amino acids of the latter are involved indicating a rather complex relationship between the reported genomic sequences. amino acid differences between the three strains are particularly prevalent in a segment encompassing amino acids - , that is the segment representing e /e /ns , and the amino terminal portion of the adjacent downstream protein (see fig. ), which seem to contain areas with higher than average variability (see below). in this segment amino acid divergence amounts to . , . , and . % for hepcv-kb and -j, hepcv- and -kb, and hepcv- and -j, respectively. in contrast a replicase domain of amino acids is the same for hepcv-kb and -j, and that of hepcv- differs only by two amino acids (see fig. in [ ] ). the overall nucleotide sequence homology of the coding segment is . % between hepcv-kb and -j, . % between hepcv- and -j and . % between hepcv- and -kb. in addition, specific sequences of various hepcv isolates have been amplified by pcr, cloned and sequenced. the putative nucleocapsid protein sequence derived from pcr products of rna extracted from healthy japanese carrier plasma exhibited a . % amino acid identity with the sequence determined for the original chimpanzee hepcv isolate (hepcv- ) but only a . % identity at the nucleotide level [ ] [ ] [ ] . furthermore, only - % amino acid and nucleotide identity was observed for a nucleotides sequence encoding part of the putative envelope proteins (fig. ) of the two hepcv isolates [ ] [ ] [ ] . by comparing the nucleotide and amino acid sequences of four hepcv isolates from the u.s.a. and two from italy (amino acids - ), a moderately variable domain of approximately amino acids in the e region and a hypervariable domain (region v) of approximately amino acids in the junction between e and e /ns (see fig. ) have been identified [ ] . although the overall divergence between the six isolates was only % for nucleotides and % for amino acids, the hypervariable region accounted for up to % of the observed amino acid differences between the isolates. the significance of the apparent variabilities in the glycoprotein coding sequences is unclear. it could be a consequence of immune selection and needs to be considered in the future development of hepcv vaccines. considerable differences were also reported for the non-structural protein sequences of various other hepcv isolates in some studies but not in others. cdna clones ( bp) generated by pcr from rna extracted from japanese patients with chronic n a n b h exhibited only - % nucleotide identity with hepcv- [ ] which resembles the difference between hepcv- and -kb/j. furthermore, the hepcv r n a extracted from these patients was not detectahle by pcr using certain primers based on the sequence of hepcv- [ . other c d n a clones to the non-structural region generated by pcr from rna extracted from japanese blood donors, however, exhibited less divergence from the prototype hepcv- . sequencing of selective genome segments by the cdna/pcr method has shown that the predicted amino acid sequences of most japanese hepcv isolates diverge from those of hepcv-j by < %, but that some japanese isolates are more homologous to hepcv- than hepcv-j [ , ] . combined the results indicate that numerous variants of hepcv are present in various parts of the world and that some of these variants, such as hepcv-j, might represent subtypes of hepcv- [ , ] . further work is required to establish the prevalence of various variants in 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circulating antibodies to a major etiologic virus of human non-a, non-b hepatitis a cdna fragment of hepatitis c virus isolated from an implicated donor of post-transfusion non-a, non-b hepatitis in japan definition and identification of homology domains hepatitis c virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups detection of antibody against antigen expressed by molecularly cloned hepatitis c virus cdna: application to diagnosis and blood screening for posttransfusion hepatitis plagemann pgw (t ) the pestiviruses a structural protein of hepatitis c virus expressed in e. coli facilitates accurate detection of hepatitis c virus detection of hepatitis c virus rna in sera and liver tissues of non-a, non-b hepatitis patients using polymerase chain reaction lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus hepatitis b virus, hepatitis non-a, non-b virus and hepatitis delta virus in lyophilized anti-hemophilic factor: relative sensitivity to heat hepatitis c virus p . g . w . plagemann: hepatitis c virus inl?ction is associated with the development of hepatocellular carcinoma cytoplasmic antigen in hepatocytes of chimpanzees infected with non-a, non-b hepatitis virus or hepatitis delta virus: relationship to interferon production of antibody associated with non-a, non-b hepatitis in a chimpanzee lymphoblastoid cell line established by in vitro transformation with epstein-barr virus early events in hepatitis c virus infection of chimpanzees okayama h (t ) structure and organization of the hepatitis c virus genome isolated from human carriers hepatitis c viral cdna clones isolated from a healthy carrier donor implicated in posttransfusion non-a, non-b hepatitis nucleotide sequence of core and envelope genes of the hepatitis c virus genome derived directly from human healthy carriers the putative nucleocapsid and envelope protein genes of hepatitis c virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers hepatitis c virus in a prospective study of posttransfusion non-a, non-b hepatitis in taiwan detection of hepatitis c viral sequences in non-a, non-b hepatitis variable and hypervariable domains are found in the regions of hcv corresponding to the flavivirus envelope and ns proteins and the pestivirus envelope glycoproteins i thank dr. daniel w. bradley for providing preprints of publications from his laboratory and helpful discussion and comments, and joy laiti for competent secretarial assistance. received june , key: cord- -qci khki authors: lima, william gustavo; brito, júlio césar moreira; overhage, joerg; nizer, waleska stephanie da cruz title: the potential of drug repositioning as a short-term strategy for the control and treatment of covid- (sars-cov- ): a systematic review date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: qci khki the novel human coronavirus (sars-cov- ), the causative agent of covid- , has quickly become a threat to the public health and economy worldwide. despite the severity of some cases, there are no current pathogen-specific antivirals available to treat the disease. therefore, many studies have focused on the evaluation of the anti-sars-cov- activity of clinically available drugs. here, we conducted a systematic review to describe the drug repositioning strategy against sars-cov- and to discuss the clinical impact of this approach in the current pandemic context. the systematic review was performed on march , , using pubmed/medline, scopus, cochrane library, and biblioteca virtual de saúde (bvs). the data were summarized in tables and critically analyzed. after the database search, relevant studies were identified as eligible for the review. among the drugs reported in these studies, showed some evidence of antiviral activity. antivirals, especially antiretrovirals, are the main class of therapeutic agents evaluated against covid- . moreover, studies have reported the anti-sars-cov- activity of antitumor ( %; / ), antimalarial ( %, / ), and antibacterial ( %; / ) agents. additionally, seven pharmacological agents (chloroquine, tetrandrine, umifenovir (arbidol), carrimycin, damageprevir, lopinavir/ritonavir) are in phase iv of clinical trials. due to the evidence of the anti-sars-cov- activity of various clinically available agents, drug repositioning stands out as a promising strategy for a short-term response in the fight against the novel coronavirus. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. in late , a cluster of pneumonia cases reported in wuhan (china) was associated with a novel coronavirus, initially called the novel coronavirus ( -ncov) [ ] . posteriorly, the sequence of the -ncov genome revealed high similarity to sars-cov, the causative agent of the epidemic of severe and acute respiratory syndrome (sars) between and in asia. then, the international committee on taxonomy of viruses (ictv) renamed -ncov as sars-cov- , and the world health organization (who) defined that this pathogen causes the coronavirus disease of (covid- ) [ ] [ ] [ ] [ ] . sars-cov- is responsible for a respiratory infection that can progress to severe pneumonia. covid- has an estimated mortality rate of approximately - . %, which increases with age and the presence of comorbidities (e.g., hypertension, cardiac insufficiency, diabetes, and asthma). by april , , the novel coronavirus had affected , , people and caused more than , deaths worldwide [ ] . the public health calamity caused by covid- has led to the exhaustion of health systems worldwide, forcing countries to adopt extreme measures, such as the closure of their land borders and initiating social distancing policies to slow down the spread of the disease [ ] . currently, laboratories and medical teams worldwide have focused on the repurposing of food and drug administration (fda)-approved drugs to treat the most severe cases of covid- , since there are no specific chemotherapeutic agents to treat this infection [ ] . indeed, drug repositioning might be a short-term alternative to fight this disease. since the efficacy, safety, and toxicity of these drugs are already well known, the initial phases of clinical trials could be skipped, which would reduce the cost and duration of the process [ ] . in general, drug repurposing is a cheaper, faster, and accessible way to make drugs available to the clinic [ , ] . in this context, several clinical and preclinical studies have searched for new pharmacological alternatives against covid- in clinically available drugs. however, current studies remain decentralized, and no recent review has been able to summarize the available evidence of the anti-sars-cov- activity of these fda-approved drugs. thus, in this systematic review, we aim to describe the drug repositioning strategy against sars-cov- and its clinical impact in the current context of the covid- pandemic. we performed a systematic review according to the cochrane handbook [ ] . the search and selection of articles, as well as extraction, analysis, and interpretation of data, were conducted according to the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [ ] . pubmed/medline, scopus, cochrane library, and biblioteca virtual de saúde (bvs) were searched for articles investigating the antiviral activity of clinically available drugs published until march , . we aimed to select articles describing clinical and pre-clinical tests (in vitro, in vivo, and in silico) to include the largest amount of data in this review. additionally, we searched the clinicatrial.gov website to identify ongoing trials with potential candidates for the drug repositioning strategy against sars-cov- . indexed keywords from medical subject headings (mesh) were used to build search strategies. the terms "antiviral agents" or "drug repositioning" or "drug repurposing" were combined with the keywords "covid- " or "sars-cov- " or " -ncov" by the use of boolean and between the terms, as in the example: "drug repositioning" and "covid- ". all details about the search strategies can be found in the supplementary file. to avoid losing any possible study, the reference list of all included studies and relevant reviews regarding this topic were also screened. two authors (w.g.l. and j.c.m.b.) independently screened the databases and extracted relevant information following the prisma flowchart. the degree of agreement between the evaluators was determined by the kappa coefficient (performed with a % confidence interval) [ ] . discrepancies about the relevance of the sources were resolved by a third researcher (w.s.c.n.). finally, all data of interest were summarized in tables (tables , , and ) for further critical analysis and interpretation. as shown in figure , we identified articles during the initial search ( from pubmed/medline, two from biblioteca virtual de saúde, and one from scopus). after the exclusion of repeated records and the selection of articles by the inclusion criteria ( fig. ) , relevant studies were preselected. of these, were excluded following the criteria described in figure , and four were selected for extraction of variables of interest [ ] [ ] [ ] [ ] . the reference lists of included articles were analyzed, and eight new studies were identified [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , totaling papers [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the degree of agreement between the two authors was considered substantial (kappa = . ) [ ] . all selected studies were published in , and they describe a total of drugs that showed some evidence of antiviral activity against sars-cov- ( fig. and tables , , and ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as shown in figure a , different classes of drugs showed a potential therapeutic effect against covid- . antivirals, especially antiretrovirals, were the most frequently studied class of therapeutic agents ( %; / ). however, the activity of antitumor ( %; / ), antimalarial ( %, / ), antibacterial ( %; / ), anticoagulant ( . %; / ), anti-inflammatory ( . %; / ), phosphodiesterase (pde)-inhibiting ( . %; / ), anti-rheumatic ( . %; / ), sedative-hypnotic ( . %; / ), and anti-venous insufficiency ( . %; / ) agents was also investigated. other classes of drugs have also been studied against sars-cov- (i.e., anthelmintic, antiallergic, antiemetic, antiepileptic, antifungal, antihypertensive, antipsychotic, anti-hemolyticuremic syndrome, anti-angioedema, lipid-lowering, immunomodulatory, and anti-pulmonary-hypertension drugs); however, only one agent of each class was evaluated ( / , . %) ( fig. a) . most of the drugs with potential activity against covid- were identified by molecular docking ( %; / ) ( fig. b ) [ ] [ ] [ ] [ ] [ ] using the main protease ( clpro) of sars-cov- as the molecular target (table ) . moreover, % ( / ) (fig. b ) of the drugs showed an anti-covid- effect in clinical trials (table ) , and % of them showed evidence of action in clinical practice ( %; / ) [ ] [ ] [ ] [ ] (table ) . additionally, % ( / ) [ , ] ( fig. b ) of the drugs studied showed antiviral activity in vitro. for instance, remdesivir showed the highest activity against sars-cov- , inhibiting % of the virus at a concentration of . μm [ ] , while ribavirin showed a less pronounced effect ( . μm) [ ] (table ) . therefore, the antiviral activity of only compounds ( %; / ) has been experimentally evaluated. the remaining reported drugs were identified only by theoretical methods (in silico) and need proof of antiviral efficacy in further studies (table ) . regarding the clinical trials of new therapeutic options against covid- , most drugs are in phase ii ( %; / ) or iii ( %; / ) (table ). only seven drugs (chloroquine, tetrandrine, umifenovir (arbidol), carrimycin, table clinical evidence of potential candidates for drug repositioning against covid- (sars-cov- ) *lopinavir ( mg) + ritonavir ( mg), q h, orally; associated with umifenovir ( mg), q h, orally. the duration of antiviral treatment was - days **solution containing umifenovir ( . %), lopinavir + ritonavir ( . %) and interferon ( . %) administered through inhalation ***in this study, patients were assigned to receive lopinavir/ritonavir ( mg and mg, orally), and patients were assigned to the standard of care (oxygen supplementation, noninvasive and invasive ventilation, antibiotic agents, vasopressor support, renal-replacement therapy, and extracorporeal membrane oxygenation) # in these studies, the therapeutic scheme used was not clearly defined treatment with lopinavir/ritonavir did not improve the clinical condition of patients compared to the standard of care*** (table ). interestingly, two of these agents in advanced clinical studies, umifenovir (arbidol) and the association lopinavir/ritonavir, have demonstrated previous evidence of action. wang et al. [ ] reported that the use of arbidol in combination with lopinavir/ritonavir inhibits the aggravation of pneumonia caused by sars-cov- and promotes a virus-negative conversion in patients from china. arbidol has also shown a potent in vitro effect against sars-cov- , inhibiting the virus up to times compared to the untreated control at concentrations ranging from to μm [ ] . the novel coronavirus (sars-cov- ), the causative agent of covid- , has quickly become a threat to the public health and economy worldwide [ , ] . recent clinical reports have shown that sars-cov- causes mild, selflimiting respiratory tract illness as well as severe progressive pneumonia, which can progress to multiorgan failure and death [ ] . despite the severity of some cases, there are no current pathogen-specific antivirals available to treat this disease [ ] . therefore, many studies have focused on the evaluation of the anti-sars-cov- activity of clinically available drugs [ ] . after the analysis of the selected studies, we identified molecules with potential antiviral activity against sars-cov- . of these, only six drugs (lopinavir/ritonavir, umifenovir (arbidol), remdesivir, chloroquine, and hydroxychloroquine) have shown promising results in preclinical trials and have clinically lessened the symptoms of covid- . since the other fda-approved drugs reported in this review showed weak activity against covid- , we have chosen to discuss only the most promising molecules. lopinavir and ritonavir are antiretrovirals widely used as a combination drug to treat human immunodeficiency virus (hiv) infections. lopinavir is an hiv type aspartate protease inhibitor, and ritonavir increases its plasma half-life through the inhibition of cytochrome p [ ] . previous studies showed that lopinavir inhibits the -chymotrypsinlike protease, which is involved in viral replication and is highly conserved among members of different viral species [ ] . lopinavir inhibited the replication of mers-cov and sars-cov in huh cells (human liver strain) in a dosedependent manner, with an ec of . μm for both viruses [ ] . moreover, lopinavir/ritonavir significantly reduced the mortality rate of mice infected with mers-cov, suggesting a strong in vivo antiviral effect [ ] . little is known about the activity of lopinavir/ritonavir against sars-cov- and its effectiveness (table ) . for instance, wang et al. [ ] showed that the use of lopinavir/ ritonavir considerably improved the clinical condition of patients with covid- . however, arbidol, a broad-spectrum antiviral with activity against several enveloped and non-enveloped viruses (e.g., influenza virus, adenovirus, avian coronavirus, and hepatitis b and c viruses) [ ] , was also administered to the patients (n = ). the combination of lopinavir/ritonavir with arbidol makes it difficult to associate the observed antiviral effect to lopinavir/ritonavir or to its synergistic effect with arbidol. in contrast, mo et al. [ ] showed that the clinical improvement of patients with covid- was more associated with supportive measures, such as mechanical ventilation and oxygen supplementation, than with the use of lopinavir/ritonavir. likewise, a study of hospitalized adult patients with confirmed sars-cov- infection showed that the use of lopinavir/ritonavir does not increase the effectiveness of the standard treatment [ ] . these findings confirm that lopinavir/ritonavir has low clinical efficacy and is not a therapeutic option to treat covid- . however, the efficacy related to the use of these antiretrovirals as a combination therapy with other drugs should be elucidated in future studies. arbidol (umifenovir) is an anti-influenza agent that has been used in china and russia for many years. it interacts with the viral hemagglutinin (ha) and inhibits the fusion of the viral particle with the plasma membrane [ ] . this drug inhibited crucial stages of the sars-cov- replication cycle in vitro in a concentration ranging from to μm [ ] . also, a study has indicated that arbidol significantly lessened pneumonia associated with covid- [ ] . these results have stimulated the initiation of clinical trials with this medicine (table ) . for instance, a chinese group of the ruijin hospital is currently conducting a phase iv study to evaluate the efficacy and safety of arbidol hydrochloride tablets in treating pneumonia in patients with covid- (clinicaltrial.gov, nct ). previous in vitro studies have indicated that arbidol shows significant activity against other coronaviruses. herein, a research group showed that this compound negatively affects the early stages of viral replication of sars-cov at a concentration of μg/ml. this study also highlighted the stronger antiviral activity of arbidol mesylate compared to arbidol as an alkaline preparation [ ] . these preliminary results indicate that arbidol is a promising candidate for drug repositioning against sars-cov- . however, further studies are required to assess the difference in the effectiveness of arbidol and arbidol mesylate in patients with covid- . remdesivir is a broad-spectrum prodrug developed to treat infections caused by ebola virus and marburg virus. its active ingredient (gs- ) decreases the production of rna by interfering with viral rna polymerase and evading proofreading by viral exonuclease [ ] . recent studies have shown that remdesivir inhibits the replication of sars-cov and mers-cov in human lung cells [ ] . wang et al. [ ] showed that remdesivir has potent activity against sars-cov- in kidney cells, with an ec of . μm. additionally, this drug induced the clinical remission of the symptoms of covid- in the first reported case in the usa (table ) [ ] . due to these promising results, a phase iii clinical trial has evaluated the antiviral activity of remdesivir and its safety in patients with severe covid- . this study is assessing the ability of remdesivir to normalize body temperature and oxygen saturation in hospitalized adult patients (clinicaltrial.gov, nct ). therefore, the results of this clinical trial can guide the prescription of remdesivir as an anti-sars-cov- agent in the future. chloroquine and hydroxychloroquine are antimalarial agents widely used to treat rheumatic diseases and have also shown promising activity against covid- [ ] . the first evidence of their antiviral effect against coronavirus was reported in when keyaerts and collaborators showed that chloroquine inhibited the in vitro replication of sars-cov on vero cells e . in this study, chloroquine negatively affected sars-cov- at a concentration of μm [ ] . interestingly, chloroquine inhibited the virus when the cells were treated with the drug before or after exposure to sars-cov, suggesting prophylactic and therapeutic effects [ ] . this drug also affects the entry and replication of sars-cov- , with an ec of . μm [ ] . moreover, hydroxychloroquine, a less toxic derivative of chloroquine, is also able to inhibit the entry and replication of sars-cov- with an ec of . μm ( table ) [ ] . additionally, the preclinical results have suggested that this antimalarial blocks the transport of sars-cov- from endosomes to endolysosomes, which is a process required to release the viral genome [ , ] . the promising in vitro results of chloroquine and hydroxychloroquine have motivated the initiation of clinical studies of these substances ( table ) . one of these studies evaluates the prophylactic effect of the oral use of chloroquine in , patients against sars-cov- (clinicaltrial. gov, nct ). additionally, several clinical trials have evaluated the prophylactic use of hydroxychloroquine (clinicaltrial.gov, nct , and nct ), its therapeutic use in monotherapy (clinicaltrial.gov, nct ), or the effect of its combination with other antivirals (clinicaltrial.gov, nct , nct and nct , and nct and nct ) against covid- . the rapid popularization of these preliminary results has led to a massive and irrational demand for chloroquine and hydroxychloroquine. in brazil, for example, after the first reports of the clinical effectiveness of hydroxychloroquine against covid- , this drug quickly sold out in pharmacies, which has compromised patients who use this drug continually to treat autoimmune diseases. this irrational demand caused the agência nacional de vigilância sanitária (anvisa) to include these two drugs in the list of controlled substances to prevent their use to treat covid- without prescription or proof of effectiveness. this situation highlights the importance of controlling the release of preliminary results, especially in the panic scenario created by the pandemic. although several studies have identified clinically available agents that are active against sars-cov- infections, supportive therapy remains essential. for instance, mechanical ventilation and oxygen supplementation have been critical to the survival of patients with severe covid- . mo et al. [ ] showed that that oxygen supplementation and noninvasive or invasive ventilation have generated similar results to the use of an antiretroviral with known in vitro activity against sars-cov- . additionally, wang et al. [ ] showed that the clinical recovery of patients with covid- is more associated with supportive therapies than with the use of antiviral agents. however, the identification of available drugs with anti-sars-cov- activity and their use in association with supportive therapies should be considered. also, the development of faster diagnostic tools to test for covid- might be crucial, since some of the candidates for drug repositioning must be administered early in the course of infection. thus, better testing methodologies could lead to the early administration of drugs and improve the treatment of covid- . the rapid spread of sars-cov- worldwide has put pressure on research centers to develop effective therapies and vaccines for the treatment of sars-cov- . drug repositioning is a promising short-term strategy in the fight against the novel coronavirus. however, supportive measures are essential mainly for severe covid- patients, and the implementation of drug repositioning should be done only if the efficacy of the drugs have been proven. although lopinavir/ ritonavir had low anti-sars-cov- activity, arbidol, remdesivir, and chloroquine/hydroxychloroquine showed promising effects against this coronavirus. therefore, the outcomes of the ongoing clinical trials are urgently needed to evaluate the best treatment options for covid- . furthermore, additional studies of the antiviral activity of molecules that have shown a promising in silico effect may increase the therapeutic arsenal against the novel coronavirus. a novel coronavirus from patients with pneumonia in china sars and mers: recent insights into emerging coronaviruses host factors in coronavirus replication epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study coronavirus infections-more than just the common cold coronavirus cases the global macroeconomic impacts of covid- : seven scenarios new uses for old drugs drug repositioning: 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sars-cov- ( -ncov) c-like protease ( clpro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates nelfinavir was predicted to be a potential inhibitor of -ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation therapeutic drugs targeting -ncov main protease by high-throughput screening potential inhibitors for -ncov coronavirus m protease from clinically approved medicines predicting commercially available antiviral drugs that may act on the novel coronavirus ( -ncov), wuhan, china through a drugtarget interaction deep learning model evolution of ct manifestations in a patient eecovered from novel coronavirus ( -ncov) pneumonia in wuhan li lanjuan's team: abidol and darunavir can effectively inhibit coronavirus going global-travel and the novel coronavirus lopinavir/ritonavir: a review of its use in the management of hiv infection screening of an fda-approved compound library identifies four smallmolecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset arbidol: a broad-spectrum antiviral compound that blocks viral fusion arbidol as a broadspectrum antiviral: an update antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures therapeutic strategies to target the ebola virus life cycle coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine chloroquine is a potent inhibitor of sars coronavirus infection and spread publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank ufmg/pharmacy school-ppgcf for the key: cord- -zolowuhj authors: yu, peifa; wang, yining; li, yunlong; li, yang; miao, zhijiang; peppelenbosch, maikel p.; pan, qiuwei title: ’-fluoro- ’-deoxycytidine inhibits murine norovirus replication and synergizes mpa, ribavirin and t date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: zolowuhj noroviruses are the main causative agents of acute viral gastroenteritis worldwide. however, no vaccine or specific antiviral treatment is available, imposing a heavy global health burden. the nucleoside analogue ’-fluoro- ’-deoxycytidine ( ’-fdc) has been reported to have broad antiviral activity. here, we report that ’-fdc significantly inhibits murine norovirus replication in macrophages. this effect was partially reversed by exogenous supplementation of cytidine triphosphate. the combination of ’-fdc with mycophenolic acid, ribavirin or favipiravir (t ) exerts synergistic antiviral effects. these results indicate that ’-fdc is a potential candidate for antiviral drug development against norovirus infection. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. human norovirus (hunv) is a non-enveloped, positive single-stranded rna virus [ ] . recently, noroviruses have been classified into at least genogroups (gi-gx) on the basis of the amino acid sequence diversity of the viral vp protein [ ] . viruses in the gi, gii, giv, gviii and gix genogroups can infect humans and are a major cause of acute epidemic viral gastroenteritis worldwide [ ] . it is estimated that noroviruses are responsible for million gastroenteritis cases per year [ ] and , deaths in children under years of age in developing countries [ ] . although norovirus gastroenteritis is usually self-limiting, it has been recognized as an emerging burden in immunocompromised populations, particularly transplant recipients [ , ] . however, research into hunv infection has been hampered by the lack of availability of robust experimental models sustaining viral infection. murine norovirus (mnv), which is capable of replicating in both cell culture and small-animal models, shares similar traits with hunv in structural and genetic features and has thus been widely used as a surrogate model [ , ] . to date, no vaccine or specific antiviral treatment is available, and clinical management is restricted to supportive care and oral rehydration. thus, the development of specific antiviral drugs for norovirus infection is urgently needed. potential inhibitors of noroviruses have been identified, and some of these have demonstrated efficacy in experimental models. ribavirin has been extensively studied and exhibits broad antiviral activity against multiple viruses, including hepatitis c virus (hcv) [ ] , hemorrhagic fever virus [ ] , hepatitis e virus [ , ] , and norovirus [ ] . in a clinical study, ribavirin treatment resulted in complete viral clearance in a subset of norovirus-infected patients, but treatment failure occurred in two cases [ ] . we demonstrated previously that mycophenolic acid (mpa), a potent inhibitor of imp dehydrogenase (impdh), can inhibit norovirus replication in cell culture [ ] . favipiravir, also known as t- , has been approved for the treatment of influenza in japan and has been repositioned to treat patients with ebola virus infection [ , ] . it has been shown to be effective against noroviruses, but the treatment can induce mutagenesis in mice and in patients, challenging the application of favipiravir for treating chronic norovirus infection [ ] . recently, '-fluoro- '-deoxycytidine ( '-fdc), also known as '-deoxy- '-fluorocytidine, has been reported to exert broad antiviral activity against hcv, lassa virus, crimean-congo hemorrhagic fever virus, and bunyaviruses [ ] [ ] [ ] [ ] . given the success of '-fdc against the abovementioned viruses, we aimed to investigate the potential antiviral activity of this compound against mnv replication. '-fluoro- '-deoxycytidine was purchased from biosynth carbosynth and dissolved in dimethyl sulfoxide (dmso, sigma, zwijndrecht, the netherlands). mpa (sigma), ribavirin (bio-connect bv), t (biovision), cytidine triphosphate (ctp; sigma), guanosine triphosphate (gtp; sigma), human ifn-α (thermo scientific, the netherlands) and jak inhibitor (santa cruz biotechnology, usa) were used. a rabbit polyclonal antiserum against mnv ns / [ ] was kindly provided by prof. vernon k. ward (school of biomedical sciences, university of otago, new zealand). β-actin antibody (#sc- ) was purchased from santa cruz biotechnology. irdye® cw-conjugated goat anti-rabbit and goat anti-mouse iggs (li-cor bioscience, lincoln, usa) were used as secondary antibodies, as appropriate. raw . and j a. were cultured in dulbecco's modified eagle's medium (dmem; lonza verviers, belgium) supplemented with % (vol/vol) heat-inactivated fetal calf serum (fcs; hyclone, logan, ut, usa) and μg of streptomycin, and iu of penicillin per ml. the murine norovirus strain mnv- (mnv- .cw ), the acutely cleared strain mnv cw , and the persistent strain mnv cr were produced by consecutively inoculating the virus (kindly provided by prof. herbert virgin, department of pathology and immunology, washington university school of medicine) onto raw . cells [ ] . human huh hepatocellular carcinoma cells harboring a genotype hunv replicon (hg ) were kindly provided by dr. kyeong-ok chang (kansas state university) [ ] . a neomycin resistance gene was engineered into orf , conferring hg resistance to neomycin. gentamicin (g ; gibco) was added to hg culture medium at . mg/ml for selection before experimentation. mnv was quantified using a % tissue culture infectious dose (tcid ) assay. briefly, tenfold dilutions of mnv were inoculated onto raw . cells grown in a -well tissue culture plate at , cells/well. the plate was incubated at °c for another days, and each well was examined under a light microscope for a cytopathic effect (cpe). the tcid was calculated by using the reed-muench method. the antiviral assay was initiated by inoculating raw . or j a. cells with mnv at a multiplicity of infection (moi) of . after h of infection, cells were washed twice with phosphate-buffered saline (pbs) to remove free virus particles and then treated with the indicated compounds. for combination assays, raw . cells were infected with the virus for h, and the medium was replaced with medium containing '-fdc, mpa, ribavirin, or t , alone or in combination, at the indicated concentrations. after h of treatment, total rna, protein and the supernatant samples were collected and further analyzed by qrt-pcr, western blot and tcid assay, respectively. total rna was isolated using a macherey nucleospin rna ii kit (bioke, leiden, the netherlands) and quantified using a nanodrop nd- spectrophotometer (wilmington, de, usa). cdna was synthesized from ng of rna using a cdna synthesis kit (takara bio, inc., shiga, japan). the cdna of all target genes was quantified by sybr-greenbased (applied biosystems) real-time pcr on a stepone-plustm system (thermo fisher scientific lifesciences) according to the manufacturer's instructions. human glyceraldehyde- -phosphate dehydrogenase (gapdh) and murine the % cytotoxic concentration (cc ) (n = ) and % inhibitory concentration (ic ) (n = - ) against viral replication were calculated using graphpad prism software. data were normalized to the untreated control (set as ). **, p < . . β-actin was used as a loading control. for immunoblot results (e and f), the band intensity of the ns / protein in each lane was quantified using odyssey software, and the quantification results were normalized to β-actin expression (control, set as ) (n = ) . data were normalized to the untreated control (set as ). *, p < . ; **, p < . ; ns, not significant. β-actin was used as a loading control. for immunoblot results (b and d), the band intensity of the ns / protein in each lane was quantified using odyssey software, and the quantification results were normalized to β-actin expression (control, set as ) gapdh genes were used as reference genes to normalize gene expression. the relative expression of the target gene was calculated as -ΔΔct, where ΔΔct = Δct sample -Δct control (Δct = ct [target gene] -ct [gapdh] cultured cells were lysed in laemmli sample buffer containing . m dtt, heated for min at °c, and loaded onto a % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) gel. after electrophoresis, the proteins were electrophoretically transferred to a polyvinylidene difluoride (pvdf) membrane (pore size, . μm; invitrogen) for h with an electric current of ma. subsequently, the membrane was blocked with a mixture of . ml of blocking buffer (odyssey) and . ml of pbs containing . % tween for h, followed by overnight incubation with primary antibodies ( : ) at °c. the membrane was washed three times and then incubated with irdye-conjugated secondary antibody ( : ) for h. after washing three times, protein bands were detected using an odyssey . infrared imaging system (li-cor biosciences). raw . or j a. cells infected with mnv- at an moi of for h, and the culture medium was replaced by medium containing different concentrations of '-fdc in an -well chamber (cat. no. ; ibidi gmbh) for h. the cells were fixed with % paraformaldehyde in pbs, permeablized with . % triton x- , blocked with % skim milk for h, reacted with rabbit polyclonal antiserum against mnv ns / , and stained with ', -diamidino- -phenylindole (dapi). secondary antibody anti-rabbit igg (h+l), f(ab') fragment (alexa fluor® conjugate) was used. imaging was performed on a leica sp confocal microscopy using a x oil objective. the % inhibitory concentration (ic ) value and % cytotoxic concentration (cc ) were calculated using the formula y¼bottom þ (top-bottom)/ ( þ ^((logic -x)*hillslope)) using graphpad prism software (graphpad prism ; graphpad software inc., la jolla, ca, usa). cells were seeded into -well tissue culture plates, and cell viability was assessed by adding mm -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide (mtt) (sigma, zwijndrecht, the netherlands). after h, the medium was replaced with μl of dmso and was incubated at °c for min. the absorbance at nm was recorded using a microplate absorbance reader (bio-rad, ca, usa). data are presented as the mean ± sem. comparisons between groups were performed using the mann-whitney test in graphpad prism . (graphpad software inc., la jolla, ca, usa). differences were considered significant at a p-value less than . . to test the potential anti-norovirus activity of '-fdc, we used the murine norovirus as a surrogate model. we found that '-fdc significantly decreased the viral rna and ns / protein expression of mnv- in raw . cells, a murine macrophage cell line that is susceptible to mnv propagation ( fig. a and b) . the inhibitory effect of this compound was confirmed, with decreased viral ns / expression observable by confocal fluorescence microscopy (fig. c) . moreover, the viral titer was found to decrease after treatment with µm '-fdc (fig. d) . to further examine the antiviral effects, another murine macrophage cell line, j a. , was used, and a similar inhibitory effect of '-fdc on mnv- replication was observed, with decreased viral rna and ns / protein expression ( supplementary fig. ). with '-fdc emerging as a potential anti-mnv candidate, we further evaluated its antiviral effect on two other mnv strains with distinct biological characteristics, the acutely cleared strain mnv cw and the persistent strain mnv cr . notably, '-fdc inhibited viral rna replication and protein expression of both viral strains ( fig. e and f). in addition, the ic value of '-fdc against mnv- replication in raw . cells was . µm (fig. g) , and the cc of '-fdc in raw . cells was . mm (fig. g ). moreover, we tested the antiviral effect of '-fdc on hunv by using hg cells harboring an hunv replicon and found moderate inhibition of viral replication (supplementary fig. ) . nucleoside analogues have been reported to induce an antiviral interferon response [ , ] . interferon-stimulated genes (isgs) are considered the ultimate effectors against viral infection, but we found that '-fdc treatment did not significantly increase isg expression (supplementary fig. a) , and inhibition of viral rna production by '-fdc was not affected by treatment with a jak inhibitor ( supplementary fig. b) , suggesting that the antiviral effect of '-fdc does not require isg induction. theoretically, nucleoside analogues exert potential antiviral activity because they bind to the viral rna polymerase active site to impede viral replication. since '-fdc is an analogue of cytidine and fluorine is isosteric with a hydroxyl group, chemical conversion of '-fdc to the corresponding '-fdc-triphosphate (fdctp) results in a compound with antiviral activity against hcv, possibly targeting the viral ns b enzyme [ ] . thus, we performed a competition assay by using ctp and gtp, which showed that ctp partially reversed the inhibitory effects of '-fdc on mnv replication, as reflected in viral rna and protein levels ( fig. a and b) as well as the viral titers (fig. d) . in contrast, no significant effect of gtp on '-fdc-mediated inhibition of viral replication was observed ( fig. c and d) . interestingly, we found that both ctp and gtp decreased the viral rna level and ns / protein expression ( fig. a-d) . it has been shown that mnv infection can induce viperin transcription in raw . cells [ ] , and viperin can convert ctp into '-deoxy- ', '-didehydro-ctp (ddhctp), which acts as a chain terminator of rna-dependent rna-polymerases and inhibits replication of zika virus [ ] . moreover, exogenous ctp/gtp might complete with the endogenous ctp/gtp for mnv replication [ ] . these results suggest a potential mechanism of action of '-fdc against mnv, and it needs to be investigated whether '-fdc exerts anti-mnv activity by targeting the viral replicase. since mpa, ribavirin, and t have been reported to have anti-norovirus activity, a combined treatment using '-fdc together with these compounds might be envisaged. to achieve better antiviral efficacy, we evaluated the combined antiviral effects of '-fdc with mpa by mathematical modeling using macsynergy [ ] . surprisingly, the results showed a moderate synergistic antiviral effect ( . µm %), which is greater than either '-fdc or mpa alone (fig. a) . similar synergistic antiviral effects were observed when combining '-fdc with ribavirin ( . µm %) or t ( . µm %) ( fig. b and c) . to confirm the predicted synergistic antiviral effects, we measured viral protein expression and viral titers by using high concentrations of the antivirals without major cytotoxicity (fig. f ). as shown in fig. d and e, the viral ns / protein expression and viral titers were further decreased when the antivirals were used in combination, supporting the synergistic antiviral effects of '-fdc with mpa, ribavirin, or t against mnv replication. despite their wide clinical application, the potential side effects or unintended off-target effects of nucleoside analogues should be considered. induction of mutagenesis by t treatment in patients has raised questions for treating chronic norovirus infections [ ] . previous studies have reported that '-fdc exhibits delayed toxicity after prolonged exposure, and no adverse clinical effects were observed in rats and woodchucks after days of treatment [ ] . several derivatives of '-fdc have shown promise as anti-hcv drugs with progress to clinical trials [ , ] . however, due to their potential mitochondrial toxicity, the long-term adverse effects of treatment with '-deoxynucleoside analogues remains a concern [ ] . thus, although '-fdc is an interesting antiviral compound, its potential adverse effects as well as its combination with other compounds should be carefully evaluated in future studies. in conclusion, '-fdc exerts potent anti-mnv effects in macrophages. importantly, '-fdc acts synergistically with the well-known antivirals, including mpa, ribavirin, and t . although further studies are still required for evaluation of the antiviral effects of '-fdc or its derivatives against hunv infection in robust models, our results suggest that '-fdc can serve as a potential backbone for anti-norovirus drug design. the combined effect of '-fdc and mpa on viral replication was analyzed by using qrt-pcr assay (n = ) and mathematical modeling using macsynergy. (b) raw . cells were infected with mnv- at an moi of for h and then left untreated or treated with '-fdc and ribavirin with the indicated concentrations for h, alone or in combination. the combined effect of '-fdc and ribavirin on viral replication was analyzed using a qrt-pcr assay (n = - ) and mathematical modeling using macsynergy. (c) raw . cells were infected with mnv- at an moi of for h and then left untreated or treated at the '-fdc and t with indicated concentrations for h, alone or in combination. the combined effect of '-fdc and t on viral replication was analyzed using a qrt-pcr assay (n = ) and mathematical modeling using macsynergy. the three-dimensional surface plot represents the differences (within % confidence interval) between actual experimental effects and theoretical additive effects of the combination at various concentrations of the two compounds. raw . cells were infected with mnv- at an moi of for h and then left untreated or treated with '-fdc and mpa, ribavirin, or t at the indicated concentrations for h, alone or in combination. (d) the viral rna level and ns / protein expression, and (e) viral titers were analyzed by qrt-pcr (n = ; data were derived from a, b and c), western blotting, and tcid (n = ) assays, respectively. (f) raw . cells were left untreated or treated with '-fdc ( µm), mpa ( µm), ribavirin ( µm), t ( µg/ml) or combinations thereof for h. cytotoxicity was determined by mtt assay (n = ). *, p < . . β-actin was used as a loading control. for immunoblot results (d), the band intensity of the ns / protein in each lane was quantified using odyssey software, and the quantification results were normalized to β-actin expression (control, set as ) ◂ advances in norovirus biology updated classification of norovirus genogroups and genotypes global economic burden of norovirus gastroenteritis systematic literature review of role of noroviruses in sporadic gastroenteritis norovirus gastroenteritis in immunocompromised patients noroviruses as a cause of diarrhea in immunocompromised pediatric hematopoietic stem cell and solid organ transplant recipients replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages murine norovirus: a model system to study norovirus biology and pathogenesis sofosbuvir plus ribavirin for treating egyptian patients with hepatitis c genotype molecular approaches for the treatment of hemorrhagic fever virus infections sofosbuvir inhibits hepatitis e virus replication in vitro and results in an additive effect when combined with ribavirin nucleoside analogue '-c-methylcytidine inhibits hepatitis e virus replication but antagonizes ribavirin nitazoxanide inhibits human norovirus replication and synergizes with ribavirin by activation of cellular antiviral response the role of chronic norovirus infection in the enteropathy associated with common variable immunodeficiency inhibition of calcineurin or imp dehydrogenase exerts moderate to potent antiviral activity against norovirus replication pharmaceutical and food safety bureau. ministry of health experimental treatment with favipiravir for ebola virus disease (the jiki trial): a historically controlled, single-arm proof-ofconcept trial in guinea mutagenesis in norovirus in response to favipiravir treatment ′-fluoro- ′-deoxycytidine is a broad-spectrum inhibitor of bunyaviruses in vitro and in phleboviral disease mouse models inhibition of the subgenomic hepatitis c virus replicon in huh- cells by ′-deoxy- ′-fluorocytidine lassa and ebola virus inhibitors identified using minigenome and recombinant virus reporter systems identification of ′-deoxy- ′-fluorocytidine as a potent inhibitor of crimean-congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus murine norovirus replication induces g /g cell cycle arrest in asynchronously growing cells replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages stable expression of a norwalk virus rna replicon in a human hepatoma cell line inhibiting pyrimidine biosynthesis impairs ebola virus replication through depletion of nucleoside pools and activation of innate immune responses norovirusmediated modification of the translational landscape via virus and host-induced cleavage of translation initiation factors a naturally occurring antiviral ribonucleotide encoded by the human genome biochemical evaluation of the inhibition properties of favipiravir and ′-c-methylcytidine triphosphates against human and mouse norovirus rna polymerases a three-dimensional model to analyze drug-drug interactions synthesis and pharmacokinetics of valopicitabine (nm ), an efficient prodrug of the potent anti-hcv agent '-c-methylcytidine propel: a randomized trial of mericitabine plus peginterferon alpha- a/ribavirin therapy in treatment-naïve hcv genotype / patients sensitivity of mitochondrial transcription and resistance of rna polymerase ii dependent nuclear transcription to antiviral ribonucleosides human and animal rights statement this article does not contain any studies with human participants or animals performed by any of the authors and is in compliance with ethical standards for research.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -r b tjm authors: kaiser, c. j.; radsak, k. title: inhibition by monensin of human cytomegalovirus dna replication date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: r b tjm monensin, at concentrations which depended on the multiplicity of infection, was found to prevent dna replication of human cytomegalovirus (hcmv) as well as production of viral progeny in human foreskin fibroblasts. the drug did not affect dna replication of herpes simplex virus. inhibition of consecutive hcmv dna synthesis was also observed following delayed addition of the drug within – hours postinfection, but was fully reversible upon its removal. viral replication proceeded, however, without impairment in cultures treated with monensin prior to infection. induction of viral dna polymerase activity was not impeded by the inhibitor. analysis of protein- and glycoprotein synthesis revealed that monensin interfered with the production of a number of hcmv-specific polypeptides. furthermore, evidence was obtained that the drug may hinder intracellular transport of a kd glycopolypeptide. the ionophore monensin, an antibiotic from streptomyces cinnamonensis, has found wide application recently for the study of the biosynthesis of viral glyeoproteins ( , , , ) . although there is evidence for its multiple sites of action ( ) this compound apparently does not interfere, like e.g. tunieamyein, with the primary steps of glyeosylation but with glyeoprotein transport within the golgi apparatus and thus with the later stages of their processing ( ). for enveloped viruses this mode of action implicates a significant reduction of release of progeny ~us from infected cells ( , ) . in addition, assembly may be affected of those viruses which bud from the outer plasma membran ( , ) . with regard to the herpesviruses which receive their envelope at the nuclear and inner cytoplasmic membranes (ll) most authors agree that monensin mainly affects viral egress whereas viral morphogenesis is hardly impaired ( , ) . furthermore, the expression in the presence of monensin of herpes simplex virusinduced surface membrane antigens as targets for immunolysis is either reduced or abolished ( , ) while it appears to be unimpaired in bovine herpes virus-infected cells ( ) . as compared with other members of the human herpesvirus group human c:(comegalovirus (hcmv) presents several characteristic properties, e.g. pronounced species specificity, a very long replication cycle ( , ) , and a particular dependence on host cell functions ( , ) . yet another property which distinguishes the hcmv host cell system is the sensitivity of virusinduced dna replication to inhibitors of glycosylation, such as -deoxy-dglucose and tunicamycin ( , ) which are ineffective, on the other hand, on dna replication in herpes simplex virus (hsv)-infected cells ( ) . evidence was forwarded in this context tbr the hcmv system for an "early" virusinduced chromatin-associated glycoprotein as the target for the inhibitors ( ) . in this investigation these initial observations are verified and extended by the use of monensin which was found also to prevent hcmv dna replication. furthermore, our experiments suggest that the drug may affect, intracellular transport of a vires-induced glycoprotein. human foreskin fibroblasts (hff; generously donated by dr. b. fleckenstein, erlangen, federal republic of germany) were used between the th and th passage for all experiments as well as for virus propagation ( ) . the monolayers were cultivated in plastic flasks of various sizes (nunc, wiesbaden, federal republic of germany; , , and cm corresponding to . × l , × and . × cells) in eagle's minimum essential medium with earle's salt solution (mem, gibco, eggenstein, federal republic of germany) supplemented with units penicillin plus ~g gentamycin/ml ~nd per cent fetal calf serum (fcs). all experiments were performed with cultures p~rtially altested by serum deprivation ( . per cent fcs for hours), beginning on a.verage days after the cultures re~ched confluency ( , ) . the towne strain of hcmv ( ) was propagated on confluent hff at a multiplicity of infection (moi) of . using per cent fcs. the kos strain of herpes simplex virus type (hsv- ; ) was propagated in the same cells in serum-free medium. virus stocks of hcmv and hsv- were prepared as described previously (¢, ) . for purification hcmv was collected from the cell-free culture medium and subjected to centrifugation on gradients of -- per cent sucrose ( ) . experimental infections of hff were performed with stock virus at the desired mot a.s indicated in results. under the conditions used about. -- and per cent of the hcmv-or hsv-infected cells, respectively, contained viral antigen ( ) . virus titers were determined by the endpoint dilution method combined with indirect immunofluorescence for viral antigen ( , ) . for pulse labelling of dna, h-thymidine (sp.act. ci/mmol) was used at gci/ml culture medium. protein and glyeoprotein labelling was performed with s-methionine (sp.act. ci/mmol) and sh-mannose (sp.aet. ci/mmol) or h-glucoseamine (sp.act. ci/mmol), using ~ci and ~ci/ml of culture medium which was either depleted of methionine or, in the case of tritiated sugars, in which glucose was replaced by sodium pyruvate ( , ) . labelling of glyeoproteins of purified hcmv in vitro with ~h-borohydride (sp.act. ci/mmol) was carried out as described by lutjokone~ et al. ( ) . all radiochemicals were purchased from amersham buehler (frankfurt, federal republic of germany). dna was extracted from labelled cells following lysis with per cent sodium dodecyl sulphate (sds) by phenol/chloroform/isoamyl alcohol treatment ( , ) . the dna content of the samples was estimated by the method of giles and myegs ( ) . separation of viral and host cell dna was achieved by isopycnic centrifugation in neutrm csc (mean density . g/ml) where hff dna bands at a density ofl. g/ml, hcmv dna at . g/ml and hsv- dna at . g/ml ( ) . to determine dna polymerase activity hff ( × cells) were subjected to the desired treatment, washed twice with cold phosphate buffered saline (pbs) and harvested in pbs by scraping at hours p.i. prior to solubilization in . ml tnt buffer ( mm tris-hc , ph . , ram nac and . per cent triton x- ) and sonication at maximum setting for × seconds with a branson sonifier. following sedimentation of the insoluble material at × g the supernatant cellular extract was examined for dna polymerase activity. the basic assay contained in ~ ( , ) : ~g of bovine serum albumin (bsa), mm tris-hc , ph . , m~ mgc , . m~a dithiothreitol (dtr), ~g of activated calf thymus dna ( ), . mm of datp, dctp, dgtp, ~m h-~tp as the labelled substrate (sp.act. ct/min/pmol), and d of cellular extract corresponding to about gg of protein. kc and/or inhibitors were supplemented at the concentrations indicated in t~esults. incubations were performed in duplicate assays for minutes at ° c prior to determination of acid insoluble radioactivity (see below). sds-page for separation of polypeptides was performed according to lxm~li ( ) as described by gallwltz et al. ( ) using either total cellular or cytoplasmic and nuclear extracts. for preparation of extracts hff ( . × v cells) were washed twice with cold pbs, harvested by scraping in pbs and solubilized in ml [i~t supplemented with . per cent sodium deoxyeholate (doc). total extracts were obtained by subsequent sonication and sedimentation of the insoluble material (see above). to prepare subcellular extracts sedimentation of nuclei followed directly after treatment with tnt-doc buffer prior to an additional wash in ml of the same buffer. the combined supernatants were pooled representing the cytoplasmic extract. the nuclear pellet was resuspended in ml tnt-doc buffer and subjected to sonification followed by sedimentation of the insoluble material. the supernatant was used as the nuclear extract. after eleetrophoresis the slab gels were subjected to fixation, staining ( ) and fluorography according to the procedure of bo~er and laskey ( ) using rotifluoreszint d (roth, karlsrube, federal republic of germany) for impregnation. the latter steps were omitted when further analysis included immunoblotting. immunobtotting for immunoblotting ( , ) , electrotransfer of polypeptides from slab gels after sds-page to nitrocellulose sheets (ba , schleicher & schiill, dussel, federal republic of germany) was performed at v and ma in a chamber constructed in our laboratory ( ) . the transfer buffer consisted of mm tris-hcl, ph . , m~ glycine and per cent methanol. indirect immunostaining was carried out at dilutions between : to : of anti-hcmv hyperimmune serum (biotest pharlna, dreieieh, federal republic of germany) or human recoltvatescent sera, tested for the presence ofhcmv-specific igg by standard enzyme-linked immunosorbent assays (elisa), as the first antibody, and horseradish peroxidase-conjugated rabbit antihuman igg (dakopatts, hamburg, federal republic of germany) as the second antibody at a dilution of : . , '-diaminobenzidine was used as the substrate. triehloroacetie acid (tca) precipitable radioactivity of labelled maeromolecules was determined after transfer of aliquots to glass fiber filters (gf/c, schteieher & schtill, dassel, federal republic of germany) which were successively washed with , per cent tca, ethanol and ether ( ) followed by counting in a toluene-based scintillation cocktail (quickszint, roth, karlsruhe, federal republic of germany). protein content in cellular extracts was estimated by the method of lowl~y et al. ( ) . phosphonoacetic acid (paa) and monensin were purchased from sigma, deisenhofen, federal republic of germany, tunieamycin from calbiochem, la jolla, u.s.a. in a typicm experiment for the determination of infected cell dna synthesis in the presence of monensin, parallel cultures of hff ( × cells) were infected by hcmv (moi of ) and pulse labelled with att-thymidine ( ~ci/ml) during the late phase of the consecutive infectious cycle ( ) . prior to infection hff were subjected to serum starvation ( , ) to avoid cellular stimulation in addition to that by hcmv. under these conditions concentrations above . ~m effectively abolished virus-mediated induction of precursor incorporation (table , exp. ). when a higher moi was used, the drug concentration had to be raised (table , exps. and ) to obtain a comparable suppression of h-thymidine uptake. for all subsequent experiments an moi of was chosen. in contrast to this, herpes simplex virus (hsv)-induced dna synthesis proved to be resistant to monensin even at relatively high concentrations ( which also prevents hcmv-mediated induction of ah-thymidine ineorporation but fails to inhibit that triggered by hsv ( ) . serum-mediated induction of preeursor uptake, on the other hand, was again sensitive to the action of monensin (table , exp. ), whereas arrested hff were not inhibited (not shown). subsequent analysis of infeeted eell dna by isopyenie eentrifugation in neutral csc revealed that inhibition of the homv-host cell system in the presence of monensin was indeed due to prevention of thymidine incorporation into viral dna ( fig. a and b) . labelling of hsv dna, on the other hand, was not affected by the drug (fig. c and d) . as expected from these observations, the drug also prevented the production of intra-as well as extraeellular hcmv progeny (not shown). to examine whether pretreated cells support, viral replication, confluent serum-starved hff were exposed to various concentrations of monensin prior to virus infection and subsequently pulse labelled with h-thymidine during the interval of expected viral dna synthesis ( ; fig. a) . pretreatment with drug concentrations up to . t ~ had no inhibitory effect on conseeutive precursor uptake under our conditions. likewise, anmysis of the dna showed that synthesis of viral dna was essentially unimpaired, as compared with the control (fig. a, w/o and monpre) . pretreatment with higher drug concentrations (e.g. tx~) which induced signs of beginning eytotoxieity, led to a reduced h-thymidine incorporation into viral dna (not shown). in a further experimental setup cultures were infected and subsequently kept in the presence of monensin ( . tim) for hours. at this time the drug was removed the cultures refed with fresh medium without inhibitor and subjected to three sequential pulses with tritiated thymidine for hour intervals each, to monitor dna synthesis (fig. b) . this protocol again did not prevent subsequent recovery of viral dna replication within - hours after removal, an observation which was based on the comparative anmysis by isopycnie centrifugation in csc of dna samples from untreated and drug-treated infected cultures labelled during corresponding pulse intervals (fig. b, w/o and monint) . the following experiments served to determine differences in sensitivity of hcmv-induced dna synthesis to the drug during the infectious cycle. for this purpose serum-starved hff were infected as described above, drug addition ( . tzm), however, followed only delayed at , , , and hours p.i. prior to pulse labelling from - hours p.i. (fig. ) . dna extracted from a hcmv-infeeted hff which were left untreated (w/o) or exposed to monensin (monpre; . txm) prior to infection and pulse labelled ( txci tt-thymidine/ml) from - hours p.i. ; and from b hcmv-infeeted hff which were left untreated (w/o) or exposed to monensin (monint; . txm) from. -- hours p.i. pulse labelling with tritiated thymidine ( txci/mi) was from - hours (w/o) and -- hours p.i. (monint). and untreated infected parallel cultures were labelled during the same interval as a control (pig. w/o). this experimentm approach showed that drug addition until hours p.i, was effective in complete abolishment of conseeutive viral dna synthesis (fig. a) . when added before hours p.i. monensin reduced (fig. b and e) , drug addition at later times did not impair precursor incorporation into viral dna (fig. d and e) . to exclude the obvious possibility of suppression by monensin of induction of the viral dna polymerase, cell extracts were prepared from drugtreated infected hff at hours p.i. and their activities compared with those of appropriate control extracts ( table ) . under the conditions used monensin did not impede induction of enzyme activity showing the characteristic properties with regard to in vitro paa-sensitivity and salt stimulation. in addition, the drug did not inhibit dna polymerase activity in the in vitro assay (table ). from the observed suppression ofhcmv dna synthesis one should also expect an impairment in the presence of monensin of expression of (late) ~al pol)teptides. to prove this conjecture parallel cultures ( × cells) of infected hff were subjected to drug treatment and pulse labelled with smethionine (fig. a) or with tritiated sugars (fig. ) from - hours p.i, at the concentrations used monensin prevented the virus-mediated increase in precursor uptake, i.e. the specific radioactivities of the protein samples from drug-treated infected cells equaled those from uninfected cells in the case of s-methionine-labelled extracts (approximately cpm s/ixg protein), and were decreased to about and per cent, respectively, of those of the control cells following labelling with tritiated sugars ( and epm h/txg protein for ah-mannose-and h-glueosamine-labelled extracts, respectively). analysis by sds-page and fluorography revealed for the methionine-labelled extracts that monensin inhibited synthesis of the most prominent polypeptides (fig. a, lanes b and e) of and kilo-daltons (kd) which are assumed to represent known major viral proteins ( , , ) . as compared to the cultures exposed to paa (fig. a, lane e; ) monensin treatment also prevented labelling of polypeptides of about and kd (fig. a, lanes e and e) . these observations were essentimly verified a n d e x t e n d e d by immunoblotting of the s a m p l e s with h u m a n hcmv-specific r e e o n v a l e s e e n t sera (fig. b) . the latter technique showed in addition suppression b y the drug of two polyi)eptides in the r a n g e of kd a n d a further two of about and kd, respectively (fig. b , lane e). of the three viral polypeptides recognized in the molecular weight r a n g e of a p p r o x i m a t e l y / kd ( , a n d kd; fig. analysis of the extracts after labelling with tritiated sugars (fig. ) showed t h a t m o n e n s i n interfered with glueosamine-but not m a n n o s e incorporation into a k d p r o d u c t ( fig. a a n d b, lanes b -d ) . u p t a k e was largely reduced, on the other hand, with both p r e c u r s o r s into a kd glyeopolypeptide (fig. a and b, lanes b and d) . interestingly, the m a i n glyeop r o t e i n of the h-borohydride labelled purified virus p r e p a r a t i o n m i g r a t e d (fig. c, lane c) . furthermore, drug treatment abohshed precursor uptake into the kd glycoprotein but resulted in induction of a glycosylated polypeptide of about / kd (fig. a and b , lane d). tunicamycin-resistant incorporation was obvious only in the very high molecular weight range after labelhng with ah-glucosamine (fig. b , lane e). the observation that sensitivity of hcmv dna synthesis to delayed addition of monensin is restricted to the initial phase of the infectious cycle supports the speculation that intracellutar transport of a glycopotypeptide may be involved. it was thus attempted to prove this assumption by the following experimental approach: hcmv-infected hff were labelled with tritiated sugars for hours p.i. (fig. a, lane c) and subsequently subjected to a chase in the presence of monensin until hours p.i. prior to cell fractionation (fig. , lane d) . uninfected as well as infected cultures chased without the inhibitor were included as controls (fig. a, lanes b and d) . analysis of the cytoplasmic and nuclear extracts by sds-page and fluorography (fig. a) showed that cytoplasmic extracts from hcmv-infected cells chased in the presence of the drug retained a glycopolypeptide in the range of kd (fig. a, lane e, cy) which was apparently chased into the nuclear fraction in the absence of the inhibitor (fig. a, lane d, nu) . immunoblotting (fig. b) with hcmat-specific antisera of the extracts from chased cultures again revealed decreased amounts in the presence of monensin of viral polypeptides of and kd (see above), but did not support the assumption from the parallel tluorogram (fig. a ) that monensin affected the intracehular transport of a i kd virus-specific product. the use of inhibitors for the analysis of complex biological systems is often hampered by adverse side effects. this possibility has to be considered allthemore when results are obtained which do not feature an immediate consequence of the known mechanism of the inhibitor action. in the case of the drug used here several observations argue against its toxicity at the concentrations used, e.g. by h'reversible damage of cellular polypeptide synthesis: i) neither treatment of cultures prior to infection, intermediary exposure of infected cells nor addition of monensin during the infectious cycle prevented resumption or progress of consecutive hcmv dna synthesis. in addition, monensin showed no inhibitory effect on viral dna synthesis in hsv-infected hff. ii) analysis of the irrfiuence of monensin on viral polypeptide synthesis as determined by precursor incorporation and immunblotting favors a relatively selective ettect which is particularly obvious when a protocol of delayed drug addition was used (e.g. fig. b) . furthermore, monensin effeeted specific and consistent changes of glycoprotein synthesis in infected cells which indeed reflected its action on posttranslational protein modification, e.g. relative resistance of h-mannose incorporation as compared to that of h-glucosamine. of the main virus-induced glyeoproteins (gp) lahetled by h-mannose, i.e. gp , and , monensin altowed incorporation only into gp whereas labelling of gp and is abolished, and a new strongiy labelled polypeptide of about / kd is induced. (minor bands of lower molecular weight appearing after sugar labelling are not considered here). by hglucosamine incorporation additional gp were revealed in the range of - kd. monensin prevented appearance of radioactive bands at the high molecular weight position ( - kd) as well as in the and kd position, but again allowed some uptake into a glycopolypeptide of / kd. in view of pereira's elaborate analysis of the polymorphism of hcmv-speeific glycoproteins ( ) which was performed also using reducing conditions for electrophoretic analysis, gp and / whose synthesis appears unimpaired in the presence of monensin, may represent partially processed immature forms of gpa (a~, a ) and gpb (b ), respectively. the recent repots of ras~vsse~ et al. ( ) and b~tt and auger ( ) support the view that maturation of the gpa complex involves cleavage of complexed precursors. monensin may be an experimental aid to define the influence of posttranslational protein modification on fmal gpa processing. our observations (figs. and ) suggest that a major gp of kd is made "early" after infection in the absence of viral dna synthesis. its relationship to the main viral envelope gp which migrates slightly faster under our conditions remains to be determined. taken together, the interference by monensin with hcmv-induced glycopolypeptide-as well as polypeptide synthesis (fig. ) appears to be much more pronounced than those described for hsv-infected cultures ( ) . as for the particular inhibitory effect of monensin on viral dna synthesis which distinguishes hcmv from hsv, the underlying mechanism is difficult to assess. previous observations on prevention of hcmv dna synthesis by glycosylation inhibitors ( , ) , as well as the findings described here suggest that glyeosylated products participate in the control of viral dna synthesis. our observation that monensin impedes synthesis of several virus-specific polypeptides including structural proteins may thus reflect a secondary effect of the drug. it appears pertinent in this context to define the specificity (host-or virus-specific) of the kd gp whose intraeellular distribution is affected by monensin. analysis of glyeoprotein processing in serum-stimulated hff whose dna synthesis is equally sensitive to the action of monensin is hoped to shed light on this question. a film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels synthesis and processing of the envelope gp - complex of human cytomegmovirus physiological state of embryonic human lung cells affect their response to human cytomegalovirus nonproductive infection of guinea pig cells with human cy-tomegalovirus growth characteristics of cytomegalovirus in human fibroblasts with the demonstration of protein synthesis early in viral replication hcmv envelope antigens induce both humorm and cellular immunity in guinea pig restricted growth of human cytomegalovirus in uv-irradiated wi- human fibroblasts translation of hela cell histone messenger .rna in cell-free protein synthesizing systems from rabbit reticulocytes, hela cells and wheat germ an improved diphenylamine method for the estimation of deoxyribonucleic acid induction of alpha-type dna polymerases in human eytomegalovirus-infected wi- cells ultrastructural study on the sequence of human cytomegalovirus infection in human diploid cells vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores monensin inhibits the processing of herpes simplex vil~s glycoproteins, their transport to the cell surface, and the egress of virions from infected ceils the effect of monensin on virion production and protein secretion in pseudorabies virus-infected cells monensin and fccp inhibit the intraeellular transport of alphavirus membrane glycoproteins cleavage of structural proteins during the assembly of the head of bacteriophage t protein measurement with the fotin phenol reagent effect of -deoxy-d-glucose on herpesvirus-induced inhibition of cellular dna synthesis surface labelling of semliki forest virus glyeoproteins using gmactose oxidase influence of cell cycle on the efficiency of transfection with purified human cytomegmovirus dna posttranslational glycosylation of corona-virus glyeoprotein e h inhibition by monensin requirements for transport of hsv- gtycoproteins to the cell surface membrane of human fibroblasts and vero cells charactcrisation of monoclonm antibodies and polyclonal immune sera directed against human cytomegalovirus virion proteins the effect of cytochalasin d and monensin on enveloped vaceinia virus release polymorphism of human cytomegalovirus glycoproteins characterized by monoclonal antibodies effect of herpes simplex virus type infection on the cellular dna polymerase activities of mouse cell cultures dna synthesis in chromatin prepa.rations from human fibrobia~sts infected by cyt~)megmovirus effect of -deoxy-d-glucose on cytomegalovirus-induced dna synthesis in human fibroblasts a) distinction ofvirm and host-derived glyeopolypeptides induced by "early" functions of human cytomegmovirus sodium butyrate seleetivly inhibits host celt gtycoprotein synthesis in human fibroblasts infected with cytomegalovirus unimpaired histon synthesis in human fibroblasts infected by human cytomegalovirus viral pol~eptides detected by a complement-dependent neutralizing routine monoclonai antibody to human cytomegalovirus action ofmonensin, a monovalent cationophore, on cultured human fibroblasts: evidence that it induces high cellular accumulation of glucosyl-and lactosylceramide (gluco-and lactecerebroside) inhibition of multiplication of enveloped viruses by glucose derivatives tartakoff am ( ) pertubation of vesicular traffic with the carboxylic ionophore monensin eleetrophoretic transfer of proteins from poiyacrylamide gels to nitrocellulose sheets: procedure and some applications effect of tunicamycin and monensin on biosynthesis, transport and maturation of bovine herpesvirus type glycoproteins induction of a host-specific chromatin-associated glycopolypeptide by human cytomegalovirus anti-complement immunofluorcscence establishes nuclear locatisation of human cytomegalovirus matrix protein characteldzation of herpes simplex virus induced deoxyribonucleic acid polymerase endo-[ -n-acetytglucosaminidase h sensitivity of precursor to herpes simplex virus type glycoproteins gb and gc this investigation was supported by the deutsche forschungsgemeinschaft (ra / - ). the authors are indebted to mrs. e. kotte and mr. b. becker for excellent technical assistance. key: cord- -paeosfiu authors: zhu, jinyan; xu, shuang; li, xueyan; wang, jue; jiang, yueqi; hu, weichen; ruan, wenke title: infectious bronchitis virus inhibits activation of the tlr pathway, but not the tlr pathway date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: paeosfiu various strains of infectious bronchitis virus (ibv) cause different forms of infectious bronchitis with different clinical signs. here, primary chicken embryo kidney (cek) cells and specific-pathogen-free (spf) chickens were infected with three pathogenic ibv strains, and it was observed that the tlr -myd pathway was inhibited but the tlr -tirf pathway was activated. after treatment with poly(i:c)-lmw, poly (i:c)-lmw/lyovec, and imiquimod, the replication of ibv was significantly suppressed after h. however, treatment with tlr pathway inhibitors such as pepinh-trif, celastrol, chloroquine, and bx resulted in increased replication of ibv after h. these results also showed that chloroquine and celastrol were most effective inhibitors of the antiviral response at hpi. infectious bronchitis is a contagious disease of poultry caused by infectious bronchitis virus (ibv), a member of the genus gammacoronavirus [ ] . different strains of ibv cause various pathologies [ , ] . avian toll-like receptor (tlr) and tlr recognize viral rna in endosomes and induce immune responses [ , ] . avian melanoma differentiation associated gene (mda ) is a rig-i-like receptor (rlr) that recognizes long viral rna in the cytoplasm [ ] . previous studies showed that tlr contributes to the host defense against severe acute respiratory syndrome coronavirus (sars-cov) and porcine epidemic diarrhea virus (pedv) [ , ] . stimulation of tlr hindered murine coronavirus infection, but stimulation of tlr did not affect murine coronavirus production [ ] . it was also shown that mda is critical for host defense during infection with murine coronavirus [ ] . ibv strains may different in their pathogenicity and induce different immune response. here, three ibv strains associated with three classic types of pathogenicity were chosen to explore potential differences in the innate immune responses they induce. the ah and tm strains of ibv were isolated from sick chickens with respiratory signs, kidney changes, and proventriculus changes from chicken farms. ibv strain h was obtained from qyh biotech company limited, beijing, china. chicken embryo kidney (cek) cells were prepared from the kidneys of -day-old specific-pathogen-free (spf) chicken embryos and were cultured in six-well plates ( × cells/well) at °c. for viral infection, cek cells were inoculated with ibv strain ah, tm, or h at a multiplicity of infection (moi) of for h at °c. mock-treated cek cells were incubated with sterile saline solution without virus and then cultured under the same conditions. the cells were collected at , , , or h post-inoculation (hpi), and total rna was extracted from the tissue samples and cells using trizol reagent (invitrogen, carlsbad, ca). all experiments were performed in triplicate. the extracted rna was treated with dnase i ( u/μg rna) for min and then reverse transcribed into cdna. the -μl reaction mixture contained μg of total rna, . μg of anchored oligo (dt ), and μl of easyscript rt/ri enzymemix in × es reaction mix (transgen biotech, beijing, china). the reaction mixture was incubated at °c for min and then at °c for min. the initial rna and dna concentrations were determined using a spectrophotometer (nanodrop technologies, wilmington, de). β-actin (actb) was selected as a control because it is a stably expressed and frequently used reference gene. the qpcr reaction mixture contained . μl ( μmol) of each primer, . these proteins were subjected to sds-page on a % acrylamide gel, and the separated protein bands were transferred to a pvdf membrane using a trans-blot (bio-rad, ca, usa). the membrane strips were tested for their reactivity with an anti-myd antibody, an anti-trif antibody, an anti-nf-κb antibody, or a β-actin antibody (cell signaling technology, ca, usa): for in vivo experiments, fifteen-day-old spf chickens were randomly divided into four groups (animal ethics approval no. - - ). they were challenged with . ml of a virus suspension containing eid of ibv strain ah, tm, or h or with . ml of sterile saline solution. the kidneys of these chickens were collected at days post-inoculation (dpi), since the virus titer in the kidney has been shown to reach a peak after - days [ , , ] . the tissues were stored in liquid nitrogen and used later for rna extraction and qpcr assays. to investigate the effect of innate immune molecules, agonists of tlr , tlr , and mda and inhibitors of key molecules of the tlr pathway were added to cek cells that had grown to - % as instructed by the reagent manufacturer (invivogen, ca, usa) as follows: μg of imiquimod (a tlr agonist) per ml for h, μg of low-molecular-weight (lmw) polyinosinic-polycytidylic acid (poly(i:c)) (a tlr agonist) per ml for h, . μg of poly (i:c)-lmw/lyovec (an mda agonist) per ml for h, μm pepinh-trif (a trif inhibitor) for h, μm celastrol (an nf-κb inhibitor) for h, μm chloroquine (an inhibitor of endosomal acidification) for . h, and μm bx (a tbk inhibitor) for h. the cells were then washed with pbs, and their viability was tested using trypan blue staining. the treated cells were incubated at an moi of with ibv strain ah, tm, or h for h at °c and washed again with pbs. control cek cells were infected with ibv without pretreatment and cultured under the same conditions. the cells were collected at , , , or hpi for qpcr using a pair of primers specific for the ibv n (fig. a-f) . the differences in mrna and protein expression suggested that transcription or translation of ifnβ could be regulated by the virus. expression of the myd , trif, and nf-κb proteins was detected by western blot. compared with the levels in uninfected cells, the expression of myd was significantly downregulated in all ibv-infected cells from to hpi. however, the expression of trif was significantly upregulated in all ibv-infected cells from to hpi. moreover, at hpi, the expression of both myd and nf-κb was significantly higher in cells infected with ibv strain h than in those infected with strain ah or tm (fig. g-i) . importantly, we found significant activation of the tlr -trif pathway and inhibition of the tlr -myd pathway at hpi by different ibv strains in cek cells. previous studies have shown the expression of innate immune molecules in chickens [ , , ] . we found that tlr and ifnβ mrna expression in the kidney was significantly downregulated in chickens infected with ah and tm ( fig. k and m) . however, there was no significant difference in mrna expression of tlr and mda in any the experimental groups ( fig. j and l) . the observed differences might be explained by the fact that more kinds of molecules are involved in the antiviral response in vivo than in vitro. the replication of strain ah was significantly inhibited compared to the control from to hpi, whereas the replication of strain h was significantly inhibited after hpi, and the replication of strain tm was significantly inhibited from to hpi (fig. ) . all of the inhibitors that were tested promoted ibv replication after hpi. after treatment with pepinh-trif, bx , or chloroquine, the rate of replication of ibv in ah-infected cek cells was significantly higher than in control cells at hpi, but the replication of ah in celastrol-treated cells was significantly higher at hpi. after treatment with pepinh-trif and bx , the replication rate of ibv strain tm was significantly higher at hpi. the rate of replication of tm was significantly higher at hpi after treatment with all four inhibitors. after treatment with chloroquine, the rate of replication of strain h was significantly higher at hpi, and the rate of replication of h in pepinh-trif-, celastrol-, and chloroquine-treated cells was significantly higher at hpi. at hpi, the most effective inhibitor of replication of ibv strain ah was chloroquine, but at hpi, it was celastrol. at hpi, the most effective inhibitor of tm was bx , but at hpi, it was chloroquine. from to hpi, the most effective inhibitor of h replication was chloroquine (fig. ) . previous research has suggested that ibv strains differ in their pathogenic mechanisms [ ] . tlr , tlr , and mda play critical roles in the activation of the innate immune response to ibv infection [ , ] . for example, tlr levels were significantly downregulated in the respiratory epithelial cells and lungs of -day-old chickens infected with the ibv connecticut strain [ ] . however, another report showed that tlr and tlr levels in the trachea and kidneys were upregulated from days to in -week-old spf chickens infected with ibv strain m [ ] . several studies have shown that the expression of tlr and tlr after ibv infection decreases after the passage of the infectious virus, which demonstrates the ability of ibv to adapt to the host environment [ , ] . mda has been shown to be upregulated in the trachea but downregulated in the kidneys [ ] . further research is required to clarify the complex regulatory mechanism in the kidneys after ibv infection [ , ] . ibv has a single-stranded rna genome, and tlr recognizes single-stranded viral rna, subsequently activating an immune response [ , ] . our results suggest that ibv inhibits the expression of tlr , but the mechanism by which this occurs is unclear. innate immune receptors are competitive determinants of cell fate [ ] . in the case of ibv, in addition to the structural proteins, more than nonstructural proteins and secretory proteins have important biological functions that still need to be elucidated. tlr activates an immune response after it recognizes viral double-stranded rna [ ] . we found that tlr was upregulated by ibv in cek cells. viral double-stranded rna is synthesized in cek cells late in ibv infection, so tlr probably plays an important role in the recognition of ibv during the late period of ibv infection. myd and trif were the adaptor molecules of tlr and tlr , respectively [ , ] . here, we found that myd protein expression was significantly suppressed, but trif was significantly upregulated. there is an evident correlation between activation and inhibition of immune recognition receptors and signal pathway molecules. nf-κb protein expression was upregulated at hpi in cells infected with ibv strain h , which is an efficient vaccine strain. this result might therefore identify another signaling pathway by which the immune response is activated after infection with h , such as the tlr pathway [ ] . in contrast to earlier reports, we found that ibv eid of ibv, and the kidneys of these chickens were collected at dpi. the expression of chicken tlr , tlr , mda , and ifnβ mrna was detected using qpcr. data were analyzed using unpaired t-tests or anova, followed by dunnett's multiple comparison test, using graphpad prism (graph-pad software, san diego, ca). the significance of the differences between the infected and mock-infected groups is indicated by the p-value strains with different pathogenicity induced different levels of mda mrna [ ] , indicating that tlr is probably the main pattern recognition receptor (prr) recognizing the ibv genome, rather than tlr or mda . imiquimod (also known as r ) is a ligand of tlr [ ] . poly(i:c) is recognized by tlr [ ] . poly(i:c)-lmw/lyovec is a preformed complex consisting of poly(i:c)-lmw and the transfection reagent lyovec. naked poly(i:c) is recognized by tlr , whereas transfected poly(i:c) is sensed by mda [ ] . both showed a strong effect on immune activation in this study. we suggest that the tlr pathway is the major immune pathway that inhibits early replication of ibv. trif and tbk are key molecules in tlr signaling pathway. pepinh-trif contains a -aa sequence that corresponds to the sequence of trif [ ] . bx inhibits the catalytic activity of tbk / ikkε by blocking its phosphorylation [ ] . celastrol is an effective inhibitor of nf-κb [ ] . chloroquine is an inhibitor of endosomal acidification [ ] . al of these compounds enhanced ibv replication after hpi in this study, consistent with the signal transmission by these downstream molecules. celastrol inhibited all of the ibv strains at fig. the effect of agonists and inhibitors on the replication of different ibv strains. agonists of tlr , tlr , mda and inhibitors of key molecules of the tlr pathway were added to the cek cells, which were infected with ibv strain ah, tm, or h at an moi of . the cells were collected at , , , or hpi, and ibv n and β-actin mrna expression was measured using qpcr. data were analyzed using unpaired t-tests or anova, followed by dunnett's multiple comparison test, using graphpad prism (graphpad software, san diego, ca). the significance of the differences between the treatment and control groups is indicated by the p-value hpi, since nf-κb is the downstream molecule of the immune pathway. in addition to celastrol, chloroquine also showed a strong ability to promote replication of ibv strains tm and h . this suggests that endosomal acidification and rna degradation are important for immune recognition of ibv. the three ibv strains tested showed different sensitivity to inhibitors. this study provides preliminary data on the immune responses induced by different ibv strains and their pathogenicity. future studies can use these data to analyze the mechanisms underlying the variations in ibv pathology and immune responses. these data also can be helpful for future ibv vaccine research. recognition of double-stranded rna and activation of nf-kappab by toll-like receptor porcine epidemic diarrhea virus infection induces nf-kappab activation through the tlr , tlr and tlr pathways in porcine intestinal epithelial cells differential innate immune responses induced by classical and variant infectious bronchitis viruses in specific pathogen free chicks use of the pharmacological inhibitor bx to study the regulation and physiological roles of tbk and ikappab kinase epsilon: a distinct upstream kinase mediates ser- phosphorylation and activation innate immune receptors as competitive determinants of cell fate essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production responses of the toll-like receptor and melanoma differentiation-associated protein signaling pathways to avian infectious bronchitis virus infection in chicks species-specific recognition of single-stranded rna via toll-like receptor and vaccination against infectious bronchitis virus: a continuous challenge induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens toll-like receptor and rig-i-like receptor signaling rig-i in rna virus recognition activation of the chicken type i interferon response by infectious bronchitis coronavirus molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor infectious bronchitis virus variants: molecular analysis and pathogenicity investigation recognition of singlestranded rna viruses by toll-like receptor protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles polymorphisms of chicken tlr and in different breeds celastrol, a novel triterpene, potentiates tnf-induced apoptosis and suppresses invasion of tumor cells by inhibiting nf-kappab-regulated gene products and tak -mediated nf-kappab activation differential involvement of bb loops of toll-il- resistance (tir) domain-containing adapter proteins in tlr -versus tlr -mediated signal transduction toll-like receptor signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection toll-like receptors and innate antiviral responses differential modulation of avian beta-defensin and tolllike receptor expression in chickens infected with infectious bronchitis virus role of adaptor trif in the myd -independent tolllike receptor signaling pathway induction of innate immune response following introduction of infectious bronchitis virus (ibv) in the trachea and renal tissues of chickens avian infectious bronchitis virus disrupts the melanoma differentiation associated gene (mda ) signaling pathway by cleavage of the adaptor protein mavs mda is critical to host defense during infection with murine coronavirus the establishment and characteristics of cell-adapted ibv strain h we would like to thank the national natural science foundation of china (# to wenke ruan) for financial support. conflict of interest the authors declare that they have no conflict of interest.ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. key: cord- -yc q fz authors: jie, tao; benqiang, li; jinghua, cheng; ying, shi; huili, liu title: preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: yc q fz porcine epidemic diarrhea virus (pedv) is prevalent in most parts of the world. owing to its antigenic variation, prevention of the diseases caused by this virus is difficult. in this study, two pedv isolates with similar growth kinetics were successfully propagated in vero cells. complete genome sequence analysis showed that they have a nt deletion in the orf gene and were classified into group , the same group that includes the classical cv strain. recombination analysis revealed that the event had occurred in the orf a gene, at – nt, among the two pedv isolates and the cv and dr strains. during their continuous propagation, nonsynonymous mutations occurred in the spike (s) gene of strain js- / between generations g and g , but there were no changes between g and g . we assumed that strain js- / might be attenuated by the th generation. piglets orally fed with js- / g showed no clinical symptoms, and no virus was detected in the feces and nasal fluid. in conclusion, js- / was successfully identified by screening, was attenuated after propagation in vero cells, and may serve as a candidate virus for vaccine preparations. porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea, an enteric disease characterized by acute watery diarrhea, dehydration, and vomiting and which affects pigs of all ages but has a high mortality rate among neonatal piglets [ ] . pedv was first described in england in , and until the end of the s, sporadic cases were reported throughout europe [ ] [ ] [ ] [ ] . since october , severe pedv outbreaks have occurred in the domestic pig population in china and caused enormous economic losses [ ] [ ] [ ] [ ] . pedv vaccines based on the classical pedv strain cv , namely the inactivated bivalent transmissible gastroenteritis virus (tgev) & pedv vaccine ( -present) and the attenuated bivalent tgev & pedv vaccine ( ) ( ) ( ) ( ) were widely used in china and played an important role in controlling the disease. nonetheless, an outbreak of diarrheal disease in and the high mortality rate among neonatal piglets indicated that the epidemic virus may have changed and escaped the immune response specific to the vaccine [ ] . it was subsequently confirmed that variants were responsible for the large-scale outbreak of the disease [ ] . the spike (s) protein of coronaviruses performs an important function: binding to cellular receptors and initiating the infection. it also induces neutralizing antibodies in vivo. mutations, including deletions and/or insertions, in the s protein may change the pathogenicity and tissue tropism of coronaviruses; and this mechanism may be the main reason for the virus escaping the immune response generated by vaccination [ ] . therefore, it is necessary to analyze the handling editor: sheela ramamoorthy. * liu huili huilil@ .com sequence and characteristics of the prevalent pedv strains for further exploration of these pathogenic mechanisms. in this study, we first determined the complete genome sequences and biological characteristics of pedv strains that have emerged in domestic pigs. subsequently, the genetic relationship between these and other pedv strains was analyzed. furthermore, we cultured the virus and screened for attenuated pedv strains by analyzing the genovariations that occurred during passaging -confirming the results in animal experiments. our results provide useful insights into the preparation of an effective and safe pedv vaccine. all animal experiments were conducted under the guidance of the institutional animal care and use committee at centers for disease control and prevention (cdc) and the laboratory animal care international accredited facility. strains jsls- / and js- / were isolated from the intestinal tissues of piglets with watery diarrhea and were sent to our laboratory for virus detection. sows were all inoculated with the bivalent killed or attenuated vaccines against tgev & pedv. as many as % of piglets died within days of birth. pigs from another farm died at ~ month of age following the development of watery diarrhea in the clinic. pedv-positive fecal samples were diluted -fold in pbs and then vortexed briefly, followed by centrifugation at , ×g for min. the supernatants were passed through . µm syringe filters and then inoculated onto confluent vero cells. after adsorption at °c for h, the cells were incubated in dulbecco's modified eagle's medium (dmem). the pedv strains were identified by rt-pcr and an indirect immunofluorescence assay (ifa). for the ifa, the vero cells were fixed with % ethanol and incubated with an anti-pedv monoclonal antibody for h, followed by incubation with a fluorescein isothiocyanate (fitc)-labeled goat anti-mouse igg antibody. finally, cell staining was examined under a fluorescence microscope. the pedv isolates were purified by a plaque method in vero cells. in brief, confluent cell monolayers in -well plates were infected with -fold dilutions of each virus isolate (to a total volume of . ml of pbs) for h at °c. the cells were washed twice with pbs and overlaid with modified eagle's medium containing . % of agar. the plates were then incubated at °c and % co . after days of incubation, the cells were stained with . % crystal violet [ ] . viral rna was extracted from a μl sample using the trizol reagent (takara, shiga, japan). the pedv cdna was synthesized by means of the primescript high fidelity rt-pcr kit (takara). full-length pedv genome amplification was performed with primers described previously [ ] . the ′ and ′ end sequences were determined with the ′ and ′ race kit (takara). for each amplicon, more than three independent clones were sequenced to determine the consensus sequence of a given genomic region. the clustalx (ver. . ) software was used to align the full-length genome sequences. phylogenetic analyses based on the s and open reading frame (orf ) genes or the entire genome were performed by the maximum-likelihood method with the general time-reversible nucleotide substitution model, where bootstrap replicates were implemented in the mega . software [ ] . growth kinetics of the pedv strains were measured in vero cells and compared. the viruses were added at a multiplicity of infection (moi) of to vero cells in a -well plate. after h, the cell medium was replaced with fresh dmem containing . µg/ml trypsin. culture samples collected at , , , , , and h postinfection were freeze-thawed twice and then centrifuged at , ×g for min at °c. the supernatants were collected and analyzed for the virus titer ( % tissue culture infectious dose [tcid ]/ml) following the reed-muench method [ ] . to evaluate the pathogenicity of a pedv strain, we analyzed and purchased pedv-negative pregnant sows. negativity for known enteric pathogens was confirmed by quantitative rt-pcr. pigs were orally inoculated with the js- / strain (different passages), -day-old neonatal piglets were given access to milk and water ad libitum (three piglets per group). the virus inoculum was tcid per animal. clinical symptoms, survival, and virus shedding (daily rectal and throat swabs) were then examined. virus shedding was quantified by rt-pcr with the following primers, pedv-f: ′-ttc ccg ttg atg agg tga t- ′, pedv-r: ′-aag cat tga ctg aac gac c- ′. serum and feces samples were collected from each piglet weekly and tested for igg and iga antibodies by elisa. we isolated six pedv strains in this study. due to their high identity, only two strains -jsls- / and js- / were chosen for genome analysis. they proliferated successfully in vero cells without trypsin (fig. a , b) and induced extensive cell death (lysis) without cell fusion (fig. c) . the plaque assay confirmed that they had a similar morphology (fig. d) . furthermore, their growth characteristics indicated that they had similar growth kinetics, with replication peaking h after infection (fig. e ). the genomes of the pedv isolates were sequenced after the th passage. the details for each gene are listed in table . complete sequence homology analysis of the pedv isolates and other pedv reference strains revealed that they shared high identity ( . %- . %) with domestic strains oh and fl . the phylogenetic tree based on the complete genomic sequences indicated that jsls- / and js- / isolates clustered in the same group together with classical strains cv and dr (fig. a) . next, analysis of the n-glycosylation sites within the s, membrane (m), and nucleocapsid (n) proteins indicated (table ). in the m protein, our two pedv strains and attenuated dr had three n-glycosylation sites, whereas cv had one more site. in the n protein, all the strains had six n-glycosylation sites, except for jsls- / which had one less. the full s sequences of our pedv isolates and of reference strains from korea, germany, the us, and china were compared. the phylogenetic tree, constructed by the neighbor-joining method, showed that the two pedv isolates from this study clustered within the same group as the classical strains, and genetically distant from the chinese pedv strains isolated during - (fig. b) . further comparison indicated that the s genes from our two pedv isolates were conserved, with amino acid identities of . - . %. nevertheless, they had obvious variation, relative to the domestic pedv strains isolated before , with amino acid identities of . - . %. analysis of the orf genes revealed . % homology in the encoded amino acid sequence between jsls- / and js- / isolates, whereas with other strains it reached % (e.g., ah/hf/ , heb/ / , and hlj/ qqhr/ ). this meant that there is a certain degree of variation between our two pedv isolates even though their identity is high. in addition, a nt sequence was found to be deleted in the orf gene in our two pedv isolates -a feature also found in the attenuated strains dr , hljby, ah-m, and js . this deletion is a typical characteristic of cell-adapted pedv strains [ ] . on the other hand, the orf gene of other domestic pedv strains isolated in recent years is intact in the same way as cv . it was therefore speculated that the pathogenicity of our pedv isolates was likely attenuated. the m protein is one of the important proteins of pedv for activating host immunity. amino acid comparison indicated that there were only three amino acid mutations between our pedv isolates, cv , and the attenuated dr : at amino acid positions , , and (data not shown). the sequence between amino acid residues and is a potential epitope, and the fact that most of the site mutations are located in this area implies antigenic diversity. by analyzing the amino acid sequences of the n protein, we found great differences among the pedv isolates, cv , and attenuated dr . compared with cv , strain js- / has amino acid mutations (data not shown). compared with attenuated dr , strain js- / has five amino acid mutations, whereas jsls- / has no changes. to determine the involvement of recombination events in the evolution of the isolates, we performed recombination analysis, using the rdp software, to compare our two isolates with representative chinese historical pedv strains from different clusters in the phylogenetic tree. the findings indicated recombination among the two pedv isolates and cv and dr had occurred in the orf a gene at site - (nt positions; fig. a) , with a higher recombination probability being detected for cv (fig. b) . the average p values for jsls- / and js- / were . × − and . × − , respectively (fig. c ). it has been reported that the pedv s gene demonstrates the biggest variability during evolution, prompting us to explore its key genovariation during cell propagation. these data will provide important information for researchers regarding cellular adaptation, virus attenuation, and pathogenicity. strain js- / was serially passaged in vero cells, and the th (g ), th (g ), th (g ), and th (g ) generations of the virus were selected and sequenced. comparison of the amino acid sequences revealed that there were missense mutations in the s gene (table ). three amino acid residues (at sites , , and ) were mutated by g , six (at sites , , , , , and ) more were mutated at g , and five more (at sites , , , , and ) were mutated by g . it is worth noting that the amino acid variations at passages g and g also participated in the dr attenuation process. nevertheless, the five other amino acid mutations were not seen in other pedv strains and may be specific to strain js- / . strains js- / g , js- / g , and js- / g at a titer of tcid were orally fed to pedv antibodynegative weaned piglets. after the virus challenge, the piglets fed with strain js- / g appeared asymptomatic, and no virus was detected in the feces and nasal fluid. by contrast, piglets infected with the js- / g strain developed watery diarrhea and died at days post-infection (dpi) (fig. a ). in addition, the piglets infected with the js- / g strain did not develop diarrhea but had to be detoxified for a period (fig. b) . we found that piglets inoculated with js- / g grew slowly before dpi, whereas the weight gain of piglets inoculated with js- / g was normal relative to that of control piglets (fig. c) . furthermore, infection with js- / g activated strong igg and iga antibody responses (fig. d) . these results confirmed that the js- / g strain was attenuated, and this change was attributed to its repeated propagation in tissue culture. porcine diarrheal disease has become epidemic in china in recent years, causing huge financial losses in the pig industry [ ] . pedv, tgev, and porcine rotavirus (porv) are the three main pathogens of this disease [ , ] . to trace the epidemiology of the recent outbreaks, porcine fecal samples were collected; our results revealing that pedv had a higher infection rate than both tgev and porv. this result is consistent with data from other reports [ , ] . to explore the evolution and pathogenicity of this virus, we next identified two pedv variants in porcine fecal samples from the shanghai and jiangsu provinces. of note, unlike the pedv variants isolated in china in recent years, our two isolates could propagate in vero cells successfully without trypsin. nevertheless, most of the pedv variants isolated in recent years can proliferate in mammalian cells successfully only in the presence of trypsin [ ] . it is unknown whether this phenomenon is related to the s gene, which is important for cell adaptation [ ] , or to the orf gene, which is a recognized marker for attenuated and virulent strains [ ] . our sequence analysis revealed that these two isolates clustered in the same group as classical strain cv , and all had a nt deletion in the orf gene. sun et al. [ ] reported that all newly isolated strains had intact orfs that could yield translation of a aa protein. these studies suggest that this area of the genome may be involved in determining cell tropism and pathogenicity of the virus [ ] . park et al. [ ] analyzed a cell culture-adapted pedv (passage ) strain with a smaller orf gene and found it to have lower virulence relative to the wild-type virus. this may mean that the pathogenicity of our two pedv isolates is different from that of the pedv variants isolated in recent years. the s protein of pedv has always been used as a marker of viral variation. under the pressure of herd immunity, the s gene of pedv mutates frequently, with some of the missense mutations altering viral antigenicity to aid in the virus's escape from preexisting immunity. thus, periodic vaccine updates may be required to ensure sufficient efficacy against emerging virus variants [ ] . in the present study, our data revealed that there is little variation in the s gene among the two pedv isolates, and that they are most similar to the attenuated dr strain. to further explore the role of the s gene in virulence, the js- / strain was successively cultured, and s gene variation during cultivation was analysed. of note, we found that there were three amino acid mutations between js- / g and js- / g , six between js- / g and js- / g , and five between js- / g and js- / g . it is worth noting that the amino acid variations between js- / g and js- / g were also identified in the dr attenuation process. this finding indicates that these missense mutations in the s gene may be related to viral pathogenicity. furthermore, animal experiments confirmed that piglets orally fed js- / g ( tcid ) had mild diarrhea and long-term excretion. on the other hand, piglets challenged with js- / g ( tcid ) appeared asymptomatic, with no virus detected in the feces and nasal fluid, indicating that g was attenuated and therefore is potentially safe for vaccine preparations. moreover, recombination analysis confirmed that this strain has a high probability of being recombined with cv and dr . most recently, the ch/hnqx- / strain was reported as a cv and dr strain recombinant with the highly virulent chn/ zmdzy/ strain. not surprisingly, this strain was detected on a swine farm that is located in the same province as the chn/zmdzy/ strain; both cv and dr pedv vaccines were used on this farm [ ] . therefore, it is possible that the virulence-enhanced cv and dr strains had been present in some areas, resulting in virus recombination. in conclusion, this study investigated the epidemiology behind recent outbreaks of porcine diarrheal disease in china and identified two pedv isolates from shanghai and jiangsu. furthermore, we successfully obtained an attenuated pedv strain by screening. the results should help our shanghai agriculture applied technology development program of china (no. t ), and the youth talent development plan of shanghai municipal agricultural system of china porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis new variant of porcine epidemic diarrhea virus, united states investigation into the role of potentially contaminated 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authors. key: cord- - bj eh d authors: pensaert, m. b.; debouck, p.; reynolds, d. j. title: an immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv , and several coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: bj eh d a possible antigenic relationship between the porcine enteropathogenic coronavirus-like agent (cvla) and known coronaviruses was examined by immunoelectron microscopy (iem) and by immunofluorescence (if). cvla did not show cross reactivity with infectious bronchitis virus, transmissible gastroenteritis virus (tgev), canine coronavirus (ccv) hemagglutinating encephalomyelitis virus (hev), neonatal calf diarrhea coronavirus (ncdcv) or feline infectious peritonitis virus (fipv). antigenic relationship was detected by iem between tgev and ccv, ncdcv and hev and by if between tgev and ccv, tgev and fipv, hev and ncdcv. in the third report of the international committee on taxonomy of viruses, the family coronaviridae is cited to contain definite members, probable members and possible members ( ) . the species of this monogenerie family are grouped mainly on physieo-chemieal characteristics. they are pleomorphie enveloped particles, averaging nm diameter, containing rna and essential lipid. they all bear unique definite projections. the eoronaviruses multiply in cytoplasm and mature by budding through endoplasmic retieulum ( ) . some eoronaviruses cause respiratory problems in birds and man, others are associated with enteritis in different species and some eoronaviruses affect multiple organs ( ) . antigenic relationships exist only between some members of this family (i ). in , a coronavirus-like agent (cvla) was isolated from an epizootic of diarrhea on belgian swine breeding farms ( ) . based on its morphologic characteristics, the cvla was suggested to be a tentative member of the family corona- - / t/ / /$ i. viridae ( ) . the cvla was shown by serologic cross protection studies to differ from the two knowm porcine coronaviruses, transmissible gastroenteritis virus (tgev) and hemagglntinating encephalomyelitis virus (hev) ( , ) . the purpose of the present report is to compare cvla antigenically with known coronaviruses. the demonstration of antigenic similarities to accepted coronaviruses would certainly contribute to definite classification of cvla within the family coronaviridae. this study was performed by means of immunoelectron microscopy (iem) and immunofluoreseence (if). both techniques have been used before to study serologic differences between virus species within, a virus family ( a, , ) . the coronaviruses selected for the present study were tgev, hev, neonatal calf diarrhea coronavirus (ncdcv), canine coronavirus (ccv), avian infectious bronchitis virus (ibv) and feline infectious peritonitis virus (fipv). origin of antigens the cvla was obtained by intestinal perfusion of a cesarean-derived colostrumdeprived (cdcd) piglet, previously inoculated orally with an intestinal homogenate containing the cv isolate ( ) . the cell culture-adapted purdue strain of tgev grown in sk- cells and the lth cell culture passage of the vw strain of t-iev ( ) grown in primary porcine kidney cells, were used as infected tissue culture fluid. the massachusetts- strain of ibv, in the form of allantoic fluid of inoculated embryonic eggs, was obtained from dr. s~'asoake, laboratory of avian pathology, state university of gent, belgium. ncdcv (norden laboratories vaccine strain) was grown in primary calf kidney ceils, then concentrated -fold by precipitation with per cent saturated ammonium sulphate. the american type culture collection strain - of ccv ( ) was grown in secondary dog kidney (dk/ ) cells. fipv was omitted from the antigens used for iem as starting material with satisfactory morphology" could not be obtained. preparation of specific antisera a monospeeific hyperimmune serum against the cv isolate of cvla was prepared in a cdcd pig. in the indirect fluorescent antibody technique using small intestinal cryostat sections of an experimentally inoculated pig, this antiserum bad a titer of : ( ). hyperimmune serum against a virulent belgian strain of tgev was raised in a conventionally reared pig. this hyperimmune serum had a virus neutralizing titer of : when tested against the cell culture adapted purdue strain of tgev. this serum had no detectable antibodies against the cvla and hev when tested by the indirect fluorescent antibody technique and by the seroneutralization test respectively. a monospecific hyperimmune serum against the vw strain of i-iev was prepared in a cdcd pig ( ). this serum had a virus neutralizing titer of l : t , in a microplate neutralization test with the cell culture adapted vw strain grown in pk-t cells ( ) . chicken ibv antiserum was provided by dr. si'a~oo~e, laboratory of avian pathology, state university of gent, belgium. this serum was raised in a spf chicken and had a hemagglutinationinhibition titer of : when tested against the massachusetts- strain. the ncdcv (british isolate) antiserum was obtained from a gnotobiotic calf and had a titer of : , in an indirect immunofluorescence test. ccv convalescent serum was obtained from a normal dog in the open population. this serum had a titer of : in a mierotiter neutralization test using tcid~ of ccv in dk/ cells. the iem test virus-containing fluids were clarified at ×g, at °c for minutes. the supernatant was sonified for seconds and clarified again at , × g, at ° c for minutes. antisera were inactivated at. °c for minutes. one hundred ~xl of antigen was mixed with ~ of an appropriate serum dilution which was determined in the homologous systems. the mixture was held at °c for hour and at °c overnight. one drop of the mixture was then placed on mesh formvar-eoated grids and stained with percent k-phosphotungstate, pit . i. grids were examined using a zciss em s- electron microscope. micrographs were taken at an instrumental magnification of , × az~d photographically enlarged to , ×. preparation of the antigens frozen sections from jejunum of a cdcd pig, experimentally infected with the cv isolate, were used us the source of cvla antigen ( ) . the preparation of the sections has been previously described (i ). frozen sections from the jejunum of a piglet, naturally infected with tgev, were used as the source of tgev antigen. these sections were shown to be free from cvla, hev and rotavirus. the vw strain of hev was cultivated in pk- cells grown on leighton coverslips. primary calf kidney celt monolayers were grown in mierotiter trays (stei~lin, england) and infected with ncdcv (norden laboratories). after hours the cell sheets were fixed with per cent acetone and used as antigen. coverslip cultures of dk/ cells, infected with ccv - were harvested after hours incubation. brain smears from fipv infected mice were obtained from dr. osteri~aus, national institute of public health, bilthoven, the netherlands. they were used as the source of fipv antigen. the propagation of fipv in mouse brain has been previously described ( ) . due to non-specific staining reactions, the ibv system could not be used for the present immunofluorescent studies. the globulin fraction of the hyperimmune scra against cvla, tgev and hev, mentioned above, was conjugated with fluoreseein isothioeyanate (fitc). dilutions of the conjugated antisera were tested for optimal fluorescence in their homologous system. the optimal dilution, usually : , was then used in the heterologous systems. the antisera against ncdcv and ccv, mentioned above, were used in an indirect if staining technique. in the homologous system, the ncdcv antiserum produeed bright fluorescence at a dilution of : and the ccv antiserum at a dilution of : . fitc conjugated anti-bovine and anti-dog globulins were produced in rabbits and obtained from nordic immunological laboratories, maidenhead, berks, u.k. the aseitie fluid from a eat naturmly infected with fipv was obtained from dr. pastoret, laboratory of virology, state university of ligge, belgium. this ascitie fluid had a virus-neutralizing titer of > when tested against the cell culture adapted purdue strain of tgev. the globulin fraction of this fluid was conjugated with fitc ~. the undiluted conjugate produced bright fluorescence in fipv-infected mouse brain smears. fluorescent antibody staining procedure indirect fluorescent antibody staining was carried out as follows. antigen-containing substrates were treated with the optimal dilution of antiserum. after an incubation period of minutes in a moistened chamber at ° c, the substrates were washed in changes of phosphat.e buffered saline solution (pbs) for minutes each. they were subsequently stained with the fitc eonjugatex] antiglobulins. after an incubation time of minutes at ° c in a moistened chamber, the substrates were washed as described above. finmly they were rinsed in distilled water for minute and dried in a warm air stream. all substrates were mounted with per cent glycerol in pbs except for the mierotiter trays which were read unmounted. direct fluorescent antibody staining was carried out by treating antigen-containing substr~ges with an optimum dilution of fitc-labelled antiviral serum as described above. the results of the i e m are shown in table . i m m u n e aggregates were observed in all homologous systems. t h e y were recognized by aggregation of widely spaced virus particles, surrounded by a fuzzy rim of antibodies. figure shows an example of a positive homologous and two negative heterologous reactions. cvla did not show detectable antigenic cross-reactivity with ibv, t g e v , ccv, t t e v or ncdcv. t g e v antiserum coated ccv antigen but not vice versa. an antigenic relationship was observed between ncdcv and h e v (fig. ) . a n t i s e r u m a g a i n s t a n t i g e n s c v l a the results of the immunofluoreseence tests are shown in table . conjugated antiserum to cvla reacted only with the homologous antigen, and cvla viral antigen was not detected by any of the other antisera. antigenic cross-reactivity was shown between tgev and ccv and between tgev and fipv. antiserum to tgev reacted with ccv, but did not show detectable antigenic cross-reactivity with fipv. on the other side, antiserum to ccv and to fipv both reacted with tgev. the antigenic relation between ccv and fipv was not studied. a "two= way" antigenic relationship between hev and ncdcv was also detected. the earlier results in which cvla was found to be antigenically unrelated to the known porcine coronavfi'uses, tgev and i-iev ( , t ) , are hereby confirmed. the present study did not reveal any evidence for a "one-way" or "two-way" cross-reactivity between the cvla and non-porcine eoronaviruses. since cvla cannot be cultivated in in vitro systems, only serologic tests such as iem and if could be used for examining cross-reactivity with coronaviruses. the results of the present study are expressed either as positive or negative because only one pai~icular serum dilution was used in each of the tests. the dilution of the serum was kept as low as possible in order to assure a maximal degree of sensitivity. even a low degree of cross-reactivity would, therefore, most likely have been detected. the present results suggests that cvla may represent a serologically distinct coronavirus species. such a feature is not unique within the family coronaviridae since the prototype species, ibv, does not appear to be related to other coronaviruses ( , ) . since the present study did not reveal further evidence for a more definite classification of the cvla, its morphological appearance remains the only feature for a tentative grouping within the coronavirus family. the existence of an antigenic relationship between hev and ncdcv has earlier been reported using virus neutralization ( ) , if ( ) and enzyme-linked immunosorbent assay (elisa) ( ). it is further corroborated by the present work using iem. the immunofluorescent cross reactivity of tgev antiserum with ccv, reported by pederse~ et al. ( ) , is confirmed by the present study. the relationship between these viruses can also be shown by iem. in contrast to the findings of pedersen et al. ( ) , ccv antiserum was found in the present experiments to cross-react in the indirect if with tgev. while the existence of different seretypes of ccv cannot be ruled out, other points should be taken in consideration in trying to explain these contradictory results. the antibody titer of the serum used may be important, particularly when the antigenic relationship between coronaviruses is examined. similar observations were made by i~eynolds and gagwes ( ) in examining the tgev-fipv relationship. furthermore, it cannot be excluded that the dog serum, used in the present study, contained homologous tge antibodies. the serum had been randomly collected. it is known that dogs can be naturally infected with tgev ( ). the present finding in which the dog serum, used in the if test, failed to aggregate tgev in the iem test is somewhat unexpected and is difficult to explain. in heterologous iem test, hyperimmune sera may be necessary to obtain positive reactions. the antibody concentration in the convalescent dog serum, used in the present study may have been too low to establish aggregation of tgev. the results of our if studies comparing fipv with tgev, are in agreement with the "one way" antigenic relationship existing between these viruses as earlier reported ( , t ). indeed, tgev antiserum failed to react with fipv. the "two way" antigenic cross reactivity between fipv and tgev, described by pederse~n et al. ( ) , could not be confirmed in the present studies. we gratefully acknowledge the financial support from the institute for encouragement of scientific research in industry and agriculture (iwonl), brussels, belgium. the technical assistance of miss chantal vanmaercke is gratefully appreciated. virus isolation and immunofluorescence in different organs of pign infected with hemagglutinating encephalomyelitis virus recovery" and characterisation of a coronavirus from military dogs with diarrhoea antigenic relationships amongst coronaviruses serological crossreactivity within the picoronaviruses as studied by electron microscopy. canad experimental infection of pigs with a new porcine enteric coronavirus, cv diagnosis of bovine coronavirus infections with hemadsorption-etution-hemagglutination assay (heha) and with enzyme-linked-immunosorbent assay (elisa) feline infectious peritonitis : a coronavirus disease of cats transmissible gastroenteritis in dogs: experimental intestinal infection with transmissible gastroenteritis virus classification and nomenclature of viruses feline infectious peritonitis {fip) virus. iv. propagation in suckling mouse brains antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species diagnosis of transmissible gastroenteritis in pigs by means of immunofluorescence characteristics of a corouavirus causing vomition and wasting in pigs tile corona, viruses: clinical and structural aspects with some practical implications a new coronavirus-like particle associated with diarrhea in swine a seroepizootiologie study of vomi$-ing and wasting disease virus in pigs virus isolation and serum antibody responses after infection of cats with transmissible gastroenteritis virus characterization of a calf diarrheal coronavirus [:h]tersuchungen fiber die antigenverwandtschaft dcr viren der felinen iufekti ser peritonitis und der transmissiblen gastroenteritis des schweines morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals received september , key: cord- -chgjpg v authors: takano, tomomi; azuma, natsuko; hashida, yoshikiyo; satoh, ryoichi; hohdatsu, tsutomu title: b-cell activation in cats with feline infectious peritonitis (fip) by fip-virus-induced b-cell differentiation/survival factors date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: chgjpg v it has been suggested that antibody overproduction plays a role in the pathogenesis of feline infectious peritonitis (fip). however, only a few studies on the b-cell activation mechanism after fip virus (fipv) infection have been reported. the present study shows that: ( ) the ratio of peripheral blood sig(+) cd (−) b-cells was higher in cats with fip than in spf cats, ( ) the albumin-to-globulin ratio has negative correlation with the ratio of peripheral blood sig(+) cd (−) b-cell, ( ) cells strongly expressing mrna of the plasma cell master gene, b-lymphocyte-induced maturation protein (blimp- ), were increased in peripheral blood in cats with fip, ( ) mrna expression of b-cell differentiation/survival factors, il- , cd ligand, and b-cell-activating factor belonging to the tumor necrosis factor family (baff), was enhanced in macrophages in cats with fip, and ( ) mrnas of these b-cell differentiation/survival factors were overexpressed in antibody-dependent enhancement (ade)-induced macrophages. these data suggest that virus-infected macrophages overproduce b-cell differentiation/survival factors, and these factors act on b-cells and promote b-cell differentiation into plasma cells in fipv-infected cats. feline coronavirus (fcov) belongs to group of the family coronaviridae. fcov is classified into two biotypes based on differences in pathogenicity: feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv). fecv infection is asymptomatic in cats. in contrast, fipv infection causes a fatal disease, feline infectious peritonitis (fip). the difference in pathogenicity between fecv and fipv in cats is considered to be associated with macrophage tropism of the viruses [ , ] . it has been proposed that fipv arises from fecv by mutation [ , , ] , but the exact mutation and inducing factors have not yet been clarified. when anti-fcov antibody-positive cats are inoculated with fipv, the onset time of fip is earlier than that in antibody-negative cats, and symptoms are severer [ ] . this phenomenon is known as antibody-dependent enhancement (ade) of fipv infection. a similar phenomenon has been observed with other virus infections, such as dengue and human immunodeficiency virus infections [ , , ] . ade of fipv infection has been reported to be induced by antibodies against fipv spike (s) protein [ , , ] . in ade-induced macrophages (ade macrophages), tnf-alpha production increases with viral replication, and this tnf-alpha acts on macrophages and promotes virus receptor (feline aminopeptidase n; fapn) expression [ ] . there are several events suggested to involve anti-fipv antibodies in fip development, other than ade. for example, antibodies produced due to fipv infection bind to the virus and form immune-complexes [ , ] . these complexes are deposited in micro blood vessels. complement binding to the deposit injures the vascular tissue and causes vasculitis (immune-complex-mediated vasculitis). hyperproteinemia also develops with an increase of gamma-globulin in fip cats [ ] . moreover, increased levels of interleukin (il)- , a cytokine involved in the survival of b-cells and their differentiation into plasma cells, in ascites t. takano Á n. azuma Á y. hashida Á r. satoh Á t. hohdatsu (&) laboratory of veterinary infectious disease, school of veterinary medicine, kitasato university, towada, aomori - , japan e-mail: hohdatsu@vmas.kitasato-u.ac.jp and culture supernatant of peritoneal exudative cells (pec) from fip cats have been reported [ ] . this suggests a close involvement of antibodies in fip pathogenesis. however, the mechanism leading to the alteration of b-cells into plasma cells has not been investigated. overproduction of the virus and tnf-alpha also occurs in ade macrophages in fipv infection [ ] , but it is not clear whether the production of b-cell differentiation/survival factors, such as il- , is involved in the induction of ade. in this study, we collected peripheral blood mononuclear cells (pbmcs) from fip cats and analyzed the b-cell surface antigens as well as measured the mrna expression level of the plasma cell master gene, b-lymphocyteinduced maturation protein (blimp- ). we also collected macrophages from fip cats and measured the expression levels of the viral rna and mrna of b-cell differentiation/survival factors: il- , cd ligand (cd l), and bcell-activating factor belonging to the tumor necrosis factor family (baff). furthermore, we investigated the relationship between fipv infection-induced ade activity of macrophages and il- , cd l, and baff mrna expression levels. type-ii fipv strain - ( tcid /ml) was administered orally to -to -month-old, specific-pathogen-free (spf) cats. thirteen cats that developed fip symptoms (fip cats), such as fever, weight loss, peritoneal or pleural effusion, dyspnea, ocular lesions, and neural symptoms, and thirteen -to -month-old spf cats as controls were used in this study. fip diagnosis was confirmed upon postmortem examination, revealing peritoneal and pleural effusions and pyogranuloma in major organs. all experiments were performed in accordance with the guidelines for animal experiments of kitasato university. felis catus whole fetus- (fcwf- ) cells was grown in eagles' minimum essential medium containing % l- medium, % fetal calf serum (fcs), u/ml penicillin and lg/ml streptomycin. feline alveolar macrophages and pbmcs were maintained in rpmi growth medium supplemented with % fcs, u/ml penicillin, lg/ml streptomycin, lm -mercaptoethanol, and lg/ml of polybrene. type-ii fipv strain - was grown in fcwf- cells at °c. fipv strain - was supplied by dr. m. c. horzinek of state university utrecht, the netherlands. phycoerythrin (pe)-conjugated anti-cd monoclonal antibody (mab) (anti-canine cd homologue of feline cd and human cd : mab cd . d ) and fluorescein isothiocyanate (fitc)-conjugated igg f(ab ) fragments of rabbit anti-feline igg(anti-feline sig ab) were used to stain feline b-cells. pe-conjugated mab cd . d was obtained from serotec ltd (uk). fitc-conjugate anti-feline sig ab was obtained from rockland inc. (usa). mab - - (igg a) used in the present study recognizes s protein of type-ii fipv, as demonstrated by immunoblotting [ ] . it has been reported that mab - - exhibits a neutralizing activity in fcwf- and crfk cells, but exhibits an enhancing activity in feline macrophages, depending on the reaction conditions [ ] . heparinized blood ( ml) was diluted twofold with phosphate-buffered saline (pbs) and subjected to ficoll-hypaque density gradient centrifugation at , rpm for min. the pbmc layer was collected, washed twice with pbs, and resuspended at cells/ml. a total of cells were incubated with pe-conjugated mab cd . d or fitc-conjugated anti-feline sig ab at °c for min. after the cells were washed three times with pbs containing . % nan , the number of stained cells was determined by counting , cells on a facs (becton dickinson, usa). albumin-to-globulin ratio for plasma samples, blood was collected from cats using a heparinized syringe and centrifuged at , rpm for min, and the supernatant was collected. the albumin and globulin levels were determined in plasma samples of spf cats and in fip cats, using an automatic analyzer. the albumin-to-globulin ratio was calculated by dividing the albumin levels by the globulin levels. feline alveolar macrophages were obtained from spf and fip cats by broncho-alveolar lavage with hank's balanced salt solution (hbss), as previously described by hohdatsu et al. [ ] . t. takano et al. rna isolation and cdna preparation rna isolation and cdna preparation were performed by the method of takano et al. [ , ] . determination of levels of feline gapdh mrna, il- mrna, cd l mrna, baff mrna, blimp- mrna and fcov n gene expression cdna was amplified by pcr using specific primers for feline gapdh mrna, il- mrna, cd l mrna, baff mrna, blimp- mrna, and the fcov n genes. the primer sequences are shown in table . pcr was performed by the method of takano et al. [ , ] . band density was quantified under appropriate uv exposure by video densitometry using scion image software (scion corporation, usa). il- mrna, cd l mrna, baff mrna, blimp- mrna, and the fcov n genes were quantitatively analyzed in terms of the relative density value to the mrna for the housekeeping gene gapdh. sequencing and analysis of blimp- and baff cdna pcr products ( ll) were electrophoresed with dna markers on a . % agarose gel. 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 sent to shimadzu corporation (japan) for sequencing, and the partial base sequences of blimp- cdna and baff cdna were determined. the sequences determined were then analyzed with the genetyx computer program (software development, japan). equal volumes a viral suspension (fipv strain - , tcid / . ml) and a mab - - solution were mixed and reacted at °c for h, and . ml of this reaction solution was used to inoculate feline alveolar macrophages ( cells) cultured in each well of well multi-plates. as controls, medium alone, virus suspension alone, or mab - - solution alone was added to feline alveolar macrophages. after virus adsorption at °c for h, the cells were washed with hbss and ml of growth medium. the cells and culture supernatant were collected every h thereafter. the cells were used for measurement of the fcov n genes, il- mrna, cd l mrna, and baff mrna, and the culture supernatant was used for measurement of the virus titer. confluent fcwf- cell monolayers in -well multi-plates were inoculated with ll of the sample dilutions. after virus adsorption at °c, the cells were washed with hbss, and ml of growth medium containing . % carboxymethyl cellulose was added to each well. the cultures were incubated at °c for days, fixed in % buffered formalin, and stained with % crystal violet. data were analyzed by student's t test. the data in fig. a and b were also analyzed by the mann-whitney test. p values . were considered to indicate a significant difference between compared groups. fig. b . the ratio of sig ? cd ? b-cells in pbmcs was not significantly different between spf and fip cats. in contrast, the ratio of sig ? cd -b-cells was significantly higher in fip than in spf cats. figure c shows (table ) . using these primers, rt-pcr was performed on pbmcs from fip cats, and a -bp band was specifically amplified. this amplified dna was sequenced, and the base sequence was partially identified (partial base sequence) (data not shown). the amino acid sequence was also deduced from the partial base sequence. homologies among the partial base sequences of feline, canine, bovine, mouse, and human blimp- mrna and the deduced amino acid sequences are shown in table . the blimp- mrna expression level in pbmcs was measured in spf and fip cats. rt-pcr was performed using feline blimp- -specific primers, and the blimp- mrna expression level in pbmcs was compared between fip and spf cats. the blimp- mrna expression level was significantly elevated in pbmc from fip cats, compared to that in spf cats (fig. ) . partial base sequence analysis of baff cdna, deduction of amino acid sequence, and comparison with other animal species il- , cd l, and baff are known as b-cell differentiation/survival factors. we investigated whether these factors in macrophages were increased in fip cats by measuring mrna expression. since the nucleotide sequence of feline baff mrna has not yet been determined, we partially sequenced the cdna of baff mrna. the sequences of mouse (genbank accession number: nm_ ), human (nm_ ), and bovine (xm_ ) baff mrna have been determined. primers corresponding to a highly conserved region were prepared for the detection of feline baff mrna (table ) . using these primers, rt-pcr of pbmcs from fip cats was performed, and a -bp band was specifically amplified. this amplified dna was partially sequenced (data not shown), and the amino acid sequence was deduced from this. homologies among the partial nucleotide sequences of feline, canine, bovine, mouse, and human baff mrna and their deduced amino acid sequences are shown in table . measurement of fcov n gene expression and il- , cd l, and baff mrna expression in alveolar macrophages of spf and fip cats the production of b-cell differentiation/survival factors in fip cats was investigated. macrophages, one of the target cells of fipv, were collected from fip cats. the il- , cd l, and baff mrna expression levels in these cells were measured by rt-pcr and compared to those in spf cats. fig. blimp- mrna expression levels in pbmcs of fip and spf cats. pbmcs ( cells) were collected from fip and spf cats, and the blimp- mrna was detected by rt-pcr. blimp- mrna was quantitatively analyzed in terms of their relative density compared to that of the mrna for the housekeeping gene gapdh virus replication in macrophages was investigated in fip cats by measuring fipv negative-strand rna expression. in all fip cats investigated, fipv negative-strand rna was expressed in alveolar macrophages (fig. a) . when the mrna expression levels of b-cell differentiation/survival factors were measured, expression of il- , cd l, and baff mrna was significantly enhanced in fip cats compared to spf cats (fig. b) . relationship between fipv replication and il- , cd l, and baff mrna expression levels in alveolar macrophages to investigate the relationship between fipv replication and b-cell differentiation/survival factor production in macrophages, alveolar macrophages from spf cats were inoculated with fipv alone or a mixture of fipv and anti-fipv s mab (mab - - ). the culture supernatant and cells were collected every h for days, and fipv replication and b-cell differentiation/survival factor production were measured. in the measurement of fipv replication, the virus titer in the macrophage culture supernatant was measured by the plaque method, and intracellular virus production was measured using fipv negative-strand rna expression as an index. to assess bcell differentiation/survival factor production, the il- , cd l, and baff mrna expression levels in the cells were measured. fipv negative-strand rna was strongly expressed in macrophages inoculated with a mixture of fipv and anti-fipv s mab compared to that in macrophages inoculated with the virus alone, and the virus titer in the culture supernatant was also significantly higher ( fig. a and b) . the mrna expression levels of all b-cell differentiation/survival factors, il- , cd l, and baff, were also significantly increased in macrophages inoculated with the mixture (fig. c) , suggesting that virus production increased in ade macrophages, in which b-cell differentiation/survival factor production also increased. in fip cats, the gamma-globulin level increases, and immune-complex-mediated vasculitis develops, suggesting that overproduced antibodies are closely involved in the pathogenesis of fip. however, only a few studies on b-cell characteristics and their activation mechanism after fipv infection in fip cats have been reported. here, we show that the ratio of sig-positive, cd negative cells increases in pbmcs of fip cats, and correlates with the albumin-to-globulin ratio in plasma samples. we also show that the blimp- mrna expression level is significantly elevated in pbmcs from fip cats. tedder et al. [ ] reported that cd molecules disappeared from the cell surface, when b-cells differentiated into antibodyproducing cells. shapiro-shelef and calame [ ] reported that blimp- is the master gene that causes the final differentiation of b-cells into plasma cells. thus, the number of plasma cells may increase in the peripheral blood of fip cats. plasma cells are mainly present in the bone marrow, spleen, and lymph nodes, but not in peripheral blood. an increase in plasma cells in peripheral blood may be evidence of enhanced b-cell activation. b-cells are stimulated by antigen-presenting cells (dendritic cells and macrophages) in lymphatic tissues, and start to differentiate into plasma cells. for b-cells to differentiate into plasma cells, various factors (cytokines, antigens, etc.) are necessary. we focused on il- , cd l, and baff as b-cell differentiation/survival factors. il- is involved in the differentiation of b-cells into plasma cells, and signal transmission via the il- receptor inhibits b-cell apoptosis [ , , ] . cd l inhibits the induction of bcell apoptosis by binding to cd expressed on the b-cell surface, activating b-cells [ , , , ] . baff induces an apoptosis-inhibitory gene, bcl- , by binding to the baff receptor expressed on b-cells, inhibiting b-cell apoptosis [ , ] . we clarified that il- , cd l, and baff mrna expression was increased in macrophages in fip cats. the il- , cd l, and baff mrna expression levels were also increased with virus replication in macrophages inoculated with fipv, suggesting that fipv replication a b fig. fcov n gene, il- , cd l and baff mrna expression levels in alveolar macrophages of fip and spf cats. a alveolar macrophages ( cells) were collected from fip and spf cats, and the fipv negative-strand rna was detected by rt-pcr. b alveolar macrophages ( cells) were collected from fip and spf cats, and il- , cd l and baff mrna was detected by rt-pcr. il- , cd l and baff mrna were quantitatively analyzed in terms of their relative density compared to that of the mrna for the housekeeping gene gapdh induced baff, il- , and cd l production in macrophages of fip cats, and these factors promoted b-cell differentiation into plasma cells. virus production and il- , cd l, and baff mrna expression were markedly enhanced in ade macrophages in vitro compared to macrophages inoculated with the virus alone. we reported previously that when the fipv antigen was detected by the fluorescent antibody method, - % of cells were positive when spf cat-derived alveolar macrophages were inoculated with fipv ? anti-fipv s mab, whereas - % of cells were positive when alveolar macrophages were inoculated with fipv [ , ] . it seems that the increased number of virus-infected macrophages leads to the over-expression of b-cell differentiation/survival factor mrna in macrophages. however, there is no evidence that this hypothesis is correct. a continued examination of the mechanism of ade activity in fipv infection would strengthen this hypothesis. we reported previously that ade activity in fipv infection of feline macrophages caused overproduction of tnf-alpha, which may lead to serious fip symptoms [ ] . the increased il- , cd l, and baff mrna expression levels in ade macrophages may also be closely involved in aggravation of pathogenesis, as well as tnf-alpha. that is, our data suggest that fipv re-infection induces ade and advances fip development in cats. however, addie et al. [ ] reported that fcov re-infection of anti-fcov antibody-positive domestic cats might not result in the development of ade. although the details are unclear, differences in the immunological condition of fcovinfected cats may be the reason why the phenomenon noted in ade does not occur in domestic cats. it is necessary to analyze mechanism of ade in detail. of the b-cell differentiation/survival factors measured, significant elevation of the ascitic and serum il- levels in fip cats has been reported [ ] . regarding cd l, tissue deposition of immune-complexes, similar to that in fip, is involved in the pathogenesis of systemic lupus erythematosus, rheumatoid arthritis, and sjogren's disease, in which a significantly increased serum cd l level has been reported [ ] . similarly, increased levels of immune-complexes in the blood of mice induced to overexpress baff have been reported [ ] . therefore, it is easily predicted that il- , cd l, and baff are involved in the development of immune-complex-mediated vasculitis. il- has also been reported to induce blimp- expression in b-cells, mainly by activating stat [ ] , suggesting that il- is involved in the enhanced blimp- mrna expression in pbmcs in fip cats. in this study, we showed that virus-infected macrophages overproduce b-cell differentiation/survival factors and that these factors act on b-cells and promote b-cell differentiation into plasma cells in fipv-infected cats. these findings may be important for the elucidation of the pathogenesis of fip. feline coronavirus infection risk of feline infectious peritonitis in cats naturally infected with feline coronavirus molecular and biological characterization of a murine ligand for cd gamma interferon/interleukin balance in tissue lymphocytes correlates with down modulation of mucosal feline immunodeficiency virus infection monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus macrophageand 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expression of type ii fipv receptor feline aminopeptidase n in feline macrophages 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 two receptors are required for antibody-dependent enhancement of human immunodeficiency virus type infection: cd and fc gamma r expression of c d receptors during human b cell differentiation: immunofluorescence analysis with the hb- monoclonal antibody antibody-dependent enhancement of virus infection and disease il- rescues the hyporesponsiveness of c-rel deficient b cells independent of bcl-xl, mcl- , and bcl- feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses key: cord- -rjl dpa authors: taguchi, f.; yamada, a.; fujiwara, k. title: asymptomatic infection of mouse hepatitis virus in the rat date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rjl dpa after intranasal inoculation of suckling rats mouse hepatitis virus multiplied mostly in the nasal epithelium; though there were no symptoms, antibodies were produced. antibodies were also demonstrated in adult rats. these findings suggest that the rat may be a natural host for the virus. , mhv-s multiplied in the "anterior part of the head", which includes the nasal bones as well as the nasal mucosa, and reached a maximum titer of about pfu/ . g on day . virus grew to much lower titres in the brain and low titres (less than pfu/ . g) were also detected in the lung, liver and spleen in some animals examined to days after virus inoculation. other tissues, such as salivary glands or blood, contained no infectious virus. limited to the nasal mueosa and brain (manuscript in preparation). two other strains of mhv, namely jhm and mhv-nu ( ), were also inoculated into -day-old rats and were found to multiply, but to a lesser extent than mhv-s. figs. a and b. necrotic changes (fig. a) and cytoplasmic immunofluorescenee (fig. b) in epithelial cells of the nasal mueosa. tissue from -day-old rats days after i.n. inoculation with pfu of mhv-s. bar represents . mm two-, -, -and t -week-old rats were also inoculated i.n. with t pfu of mhv-s and infectious viruses were assayed in the lung, brain, salivary glands and liver. in less than one-third of the total, were infectious viruses detected in the lung and salivary glands and the titers were less than t pfu/ . g. no infectious virus was demonstrable in the liver. sera were collected days after inoculation and examined for neutralizing antibody. table shows that almost all contained neutralizing antibody to a titre of > . the two rats inoculated at weeks and seronegative when tested at : were nevertheless positive at i : . so far only mice have been considered to be natural hosts of mhv and rat; has been excluded because highly virulent mhv inoculated by various routes did not cause any sy~lptomatic infection like that produced in the mouse ( , ) . however, this does not imply that mtiv does not infect the rat. in the present study, we clearly demonstrated that by the intranasal route mhv-s infects rats of various ages from the following criteria: . mhv replicated in the "anterior part of the head" and several other organs; . viral antigen was detected by immunoftuoreseenee in the epithelial cells of nasal mneosa where histopathological changes were also found ; . rats of all ages produced neutralizing antibodies after virus inoculation. i n some cases, neither infectious virus nor histopathologieal changes were demonstrable in infected rats, however, neutralizing antibodies were constantly detected in the sera of inoculated rats, revealing that mhv replication occurs in every cases, but m a y be below the detection level in some cases. two eoronaviruses, namely rat eoronavirus (rcv) ( ) and sialodaeryoadenitis virus (sdav) ( ) , are known to infect and cause disease in the rat. these viruses multiply first in the epithelial cells of the nasal cavity and thereafter, they manifest their organ tropism i.e., rcv goes to the respiratory system and woduees interstitial pneumonia ( , ) and sdav affects the salivary and lacrymal glands, and produces adenitis ( ) . the pathogenesis of nhv-s infection in rats resembles, in part, that of sdav or i~cv, in that these viruses cause the necrotic changes in the epithelial cells of the nasal mucosa at early stage of infection ( ) . however mhv is confined there, or affects slightly the central nervous system as it does in infections of mice (manuscript in preparation). b~tatt and his eoworkers showed that sdav multiplied in mice producing some histopathological changes in the respiratory system after i.n. inoculation ( ) and we demonstrated in the w e s e n t paper that {hv infected rats of any ages. these facts indicate that sdav and mitv are infectious for both mice and rats, so that both m a y play an important part in the survival of these viruses. a murine virus (jhm) causing disseminated encephalitis with extensive destruction of myelin. ii experimental infection of adult axenic rats with parker's coronavirus respiratory infection in mice with sialodacryoattenitis virus, a coronavirus of rats lethal enteritis in infant mice caused by mouse hepatitis virus a rnurine virus (jhm) causing disseminated encephalitis with extensive destruction of myelin. i. isolation and biological properties of the virus problems in checking inapparent infections in laboratory mouse colonies. an attempt at serological checking by anamnestic response mouse hepatitis virus and its pathogenic action antibodies to mouse hepatitis virus in human sera pathogenicity of mouse hepatitis virus for mice depending upon host age and route of infection isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis pathogenesis of sialodaeryoadenitis in gnotobiotic rats rat eoronavirus ( %cv): a prevalent, naturally occurring pneumotie virus of rats experimental viral hepatitis mouse hepatitis virus infection as a. highly contagious, prevment, enteric infection of mice difference in response to mouse hepatitis virus a, mong suseeptible mouse strmns age-dependent response of mice to a mouse hepatitis virus, mttv-s. japan we th~nk dr. t. sato (chugai pharmaceutical co., ltd) and dr. h. ikeda of the same institute for their many helpful discussions of the research.this work was supported partly by grant-in-aid for scientific research from ministry of education, science and culture, japan. key: cord- -xv vpji authors: xie, peng; li, yanling; li, yaling; liang, jianpeng; xiang, bin; lin, qiuyan; jin, jiaqi; ding, chan; xu, chenggang; ren, tao title: immune effect of a newcastle disease virus dna vaccine with il- as a molecular adjuvant delivered by electroporation date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: xv vpji newcastle disease (nd), caused by virulent newcastle disease virus (ndv) strains, has been one of the most problematic diseases affecting the poultry industry worldwide. conventional vaccines provide effective protection for birds to survive nd outbreaks, but they may not completely suppress ndv shedding. ndv strains circulate on farms for a long time after the initial infection and cause potential risks. a new vaccine with fast clearance ability and low viral shedding is needed. in this study, we used interleukin- (il- ) as an adjuvant and electroporation (ep) as an advanced delivery system to improve a dna vaccine candidate. the fusion (f) protein gene from an ndv strain of the prevalent genotype vii. . was cloned to prepare the vaccine. chickens immunized with the f gene dna vaccine co-delivered with an il- -expressing plasmid dna showed higher neutralizing antibody levels and stronger concanavalin-a-induced lymphocyte proliferation than those treated with the f gene dna vaccine alone. the co-delivered vaccine provided % protection, and less viral shedding and a shorter release time were observed in challenged chickens than when the f gene dna vaccine was administered alone. the use of f gene dna combined with il- delivered by electroporation is a promising approach for vaccination against nd. newcastle disease (nd) has been identified as an important poultry disease by the world livestock disease atlas, . it not only causes severe direct economic losses but also affects humans by decreasing food supplies. the causative agent of nd is avian orthoavulavirus , commonly named newcastle disease virus (ndv), a member of the family paramyxoviridae. vaccines remain important tools to control the disease in epidemic countries or regions, and co-application of attenuated and killed vaccines has effectively reduced the morbidity and mortality of the disease. however, the failure of vaccination to provide protection against the currently prevalent genotype vii nd has been reported [ ] . mutations occur during the viral replication process, and the natural evolution of wild-type ndv also poses a risk. as of , at least genotypes of ndv in class ii and one in class i have been identified, and this number has been increasing [ ] . epidemiology reports have suggested that emerging strains exhibiting antigenic variations may be responsible for recent nd outbreaks in africa and asia [ , ] . these facts suggest that the conventional vaccine is insufficient for long-term control of nd. incomplete inactivation of killed ndv vaccine may lead to infections in animals. studies have found some attenuated vaccines to be contaminated with exogenous viruses, including fowl adenovirus and chicken infectious anemia virus, [ , ] . in addition, traditional vaccines have the disadvantage of needing to be kept cold during shipping, which is difficult in some developing countries and remote rural areas. in , dna was first demonstrated to be effective for producing antigens in vivo [ ] . since then, dna vaccines have attracted increased interest because of their stability during transport and their safety [ ] . dna vaccines can mimic natural infections, can elicit a broad overall immune response and are stable at ambient temperatures. they can also be produced using polymerase chain reaction (pcr), which makes it easier to match the antigen to epidemic viral strains. in the last decade, a few dna vaccines have been licensed for use, including vaccines against west nile virus in horses [ ] , canine melanoma in dogs [ ] , and h n avian influenza virus in birds [ ] . due to the inefficiency of direct injection of naked plasmid dna, delivery methods including the use of electroporation (ep) [ ] , nanoparticles [ ] and biological carriers [ ] have been developed to increase the efficiency of uptake of dna vaccines. by using electrical pulses to generate transient pores in cell membranes, ep allows drugs, rna, dna, and proteins to be imported into cells more efficiently than traditional intramuscular (im) injection [ , ] . ep can also enhance vaccine efficacy by eliciting a cd + t cell response and inducing a local immune response [ , ] . another important strategy for improving dna vaccines is the use of molecular adjuvants. cytokines have been used for years as immune adjuvants to improve immune response of vaccines [ ] . one of them, il- , enhances the proliferation and cytolytic activity of t and nk cells, activates t helper cells, and induce the production of interferon-γ and other cytokines [ ] . il- adjuvant also increases the protective antibody response [ ] . notably, il- can induce strong mucosal immunity, making it especially advantageous in combating respiratory diseases, and it has shown remarkable efficacy in prevention of influenza and pneumococcal disease [ , ] . the genome of ndv encodes six structural proteins: nucleocapsid protein, matrix protein, phosphoprotein, fusion (f) protein, hemagglutinin-neuraminidase (hn) protein and large polymerase protein. the f and hn proteins are naturally expressed on the viral envelope and are recognized first by the immune system during infection. previous studies have shown that f-or hn-gene-based dna vaccines can induce the production of neutralizing antibodies (nabs) in birds that help them to survive ndv infections, and the efficacy of a dna vaccine based on the f gene has been shown to be superior to that of one based on the hn gene [ , ] . as the use of a vaccine strain homologous to the current circulating viruses is likely to reduce viral shedding [ ] , we chose the f gene of an ndv strain belonging to genotype vii, which is the prevalent genotype in china, to construct a dna vaccine. we evaluated the immunogenicity of the f gene dna vaccine combined with il- adjuvant in chickens and also compared the efficacy of im injection and ep. the aim of our work was to evaluate a new vaccine candidate for nd control and provide a reference for avian dna vaccine strategies. the ndv gm (chicken/china/gm/ ; genbank: dq . ) strain of genotype vii was used for challenge in the animal experiments, and the strain was classified as subgenotype vii. . according to the new classification system reported in [ ] . t cells used for transfection were maintained in our laboratory and grown in dmem (dulbecco's modified eagle's medium; gibco, thermo scientific, waltham, ma, usa) supplemented with % fetal bovine serum (fbs; gibco) at °c in % co . primary chicken embryo fibroblast (cef) cells for the neutralization test were prepared using -day-old spf chicken embryos provided by da huanong animal health products co., ltd. (guangdong, china) and were separated as described previously [ ] . the eukaryotic expression vector pcaggs containing a chicken β-actin promoter was obtained from the key laboratory of animal vaccine development in south china agricultural university. spf chickens were purchased from da huanong animal health products co., ltd., and housed in isolators. all animal experiments were approved by the south china agricultural university experimental animal welfare ethics committee. rna was extracted from the allantoic fluid of a chicken embryo inoculated with ndv strain gm and used as the template for f gene cloning. the rt-pcr products were inserted into the pcaggs vector to generate pcag-f. rna was extracted from peripheral blood mononuclear cells (pbmcs) of leghorn spf chickens, providing a template for cloning p and p subunits of chicken il- (chil- ). the full-length p and p genes were amplified separately by pcr. then, primer p -r-l was used with primer p -f to delete the tga stop codon and add a part of the linker nucleotide sequence, and the resulting amplicon was bp long. primer p -f-l was also used with primer p -r to delete the p atg and to add a part of the linker nucleotide sequence, and the resulting amplicon was bp long. finally, these amplified products were used as templates, and the primers il- -f and il- -r were used to obtain a fulllength il- gene with the linker sgggsggggsggggs between the p and p subunits. primers were designed based on the reference sequences obtained from the gen-bank database (ay . , ay . ). the pcr products were cloned into the restriction sites of the pcaggs vector to generate the construct pcag-il- . the primers used for pcr are listed in table . the plasmids pcaggs, pcag-f, and pcag-il- were introduced separately into t cells by transfection using lipofectamine™ (life technologies, usa) according to the manufacturer's instructions. protein expression was detected by indirect immunofluorescence assay (iifa) after hours. briefly, the cells were fixed with % paraformaldehyde, permeabilized with . % triton ×- , and blocked with pbs containing % bovine serum albumin. the cells were then incubated with a primary rabbit polyclonal antibody against ndv (diluted : in pbs) or that against il- (diluted : in pbs) at °c for h. ndv antibody was prepared by us and stored at the key laboratory of animal vaccine development in south china agricultural university (details not shown). the il- antibody was prepared as follows: briefly, the chicken il- gene was amplified and cloned into the vector pet- a (novagen; emd-millipore, billerica, ma), and the resulting plasmid construct was expressed in escherichia coli bl (de ) competent cells. the recombinant protein was purified and injected into rabbits to obtain polyclonal serum. fitc-labeled goat antirabbit igg (boster, wuhan, china; diluted : in pbs) was used as the secondary antibody. fluorescence signals were observed by fluorescence microscopy. forty -day-old white leghorn spf chickens were randomly divided into four equal groups (n = ) for vaccination experiments. for primary immunization, chickens in groups , , and were inoculated with μg of ddh o, -μg pcaggs, or μg of pcag-f, respectively, by im injection. chickens in group were inoculated with μg of pcag-f by ep. ep was performed using an in vivo gene delivery system (teresa, shanghai, china) as follows: the vaccine was delivered intramuscularly into the leg using a -ml syringe. the syringe was then pulled out, two silver needle electrodes were inserted into the muscles, and six electric pulses of v (each lasting for ms) were immediately delivered around the inoculation site at intervals of ms. after two weeks, the inoculation was repeated as a booster. three chickens were selected randomly to obtain pbmcs at and days after first immunization. serum samples were obtained from six randomly selected chickens in each group days after primary immunization. two weeks after the second immunization, chickens were challenged with ndv at a dose of eld in a -μl volume by oculonasal instillation. oropharyngeal and cloacal swabs were obtained , , , and days post-challenge (dpc) to detect viral shedding. briefly, swabs were suspended in ml of pbs with antibiotics (penicillin, u/ml; streptomycin, mg/ml) and inoculated into -day-old spf embryonated chicken eggs. the eggs were incubated at °c for h. the allantoic fluids were collected and tested for the presence of ndv using a standard hemagglutination (ha) inhibition (hi) test. symptoms and deaths were recorded for days. an additional -day-old spf chickens were randomly divided into four equal groups (n = ) to investigate the effect of chil- used as an adjuvant to the f gene dna vaccine. chickens in group were injected intramuscularly with ddh o; those in group , with μg of pcag-f; those in group , with μg of pcag-f and μg of pcag-il- ; and those in group , with μg of pcag-il- . all antigens were delivered at a final volume of µl by ep as described above. after two weeks, the chickens were administered an equal booster dose of plasmids by the same route. three chickens were selected randomly to obtain pbmcs at and days after the first immunization. serum samples were obtained days after the primary immunization from eight randomly selected chickens from each group for nab detection. two weeks after the booster, all chickens were challenged with ndv as in the first experiment. three randomly selected chickens were killed at dpc to measure virus titers in organs as reported previously [ ] . briefly, the collected tissues were homogenized in phosphate-buffered saline containing ampicillin and penicillin. virus titration was determined in tenfold serial dilutions of supernatant by inoculation into specificpathogen-free embryonated chicken eggs. virus titers were calculated based on the number of eggs that were positive by hemagglutination at each dilution. oropharyngeal and cloacal swabs of the remaining chickens in each group were obtained at , , , and dpc to detect viral shedding as described previously. all chickens were observed for days, and symptoms and deaths were recorded. various types of antibodies are produced in ndv infection, some of which neutralize the virus, such as antibodies induced by the f protein [ ] . in the first and second animal experiments, serum samples from six and eight randomly selected chickens, respectively, from each group were collected after immunization to evaluate the serum antibody response. the virus neutralization test was performed as follows: heat-inactivated sera were serially diluted twofold and incubated for h with tcid of ndv strain gm in dmem. serum-ndv mixtures were then applied to cef monolayers in a -well plate with four replicate wells per dilution. after days of incubation, virus was detected in the cell supernatant using a hemagglutination test. a positive ha result indicated that the virus had not been neutralized. the nab titer was defined as the highest serum dilution at which infectivity was inhibited in % of the wells. pbmcs were isolated from heparinized peripheral blood from three randomly selected chickens using chicken lymphocyte separation medium (haoyang, tianjing, china). lymphocyte proliferation was determined as reported previously [ ] . the isolated cells were washed twice with hanks' balanced salt solution, washed once in rpmi (gibco, usa), and then resuspended in rpmi containing % fbs, penicillin, and streptomycin at a final concentration of × cells/ml. the cells were then seeded in -well plates at a density of × cells per well. for the lymphocyte proliferation assay, the cultures were stimulated with μl of concanavalin a (cona, sigma, μg/ml) or not stimulated (negative control), both in triplicate, at °c in % co . after h, μl of mtt ( mg/ml) was added to each well, and the incubation was continued for an additional h. after incubation, the supernatant was discarded, and μl of dimethyl sulfoxide was added to dissolve the formazan crystals for min. the optical density (od) of each well was then determined at nm using a model microplate reader (bio-rad laboratories co., ltd., shanghai, china). the stimulation activity (od) was calculated using the following formula: od = (mean od of cona stimulated cells) − (mean od of unstimulated cells). statistical analysis was performed using graphpad prism software (graphpad software inc., san diego, ca, usa). all laboratory tests were performed in triplicate, and each value was measured three times. data are presented as mean values ± standard deviation (sd). mean values were analyzed using student's t-test. a -bp fragment of the f gene of ndv and a -bp fragment of the chil- gene were cloned and found to have no mutations. cells transfected with f and chil- plasmids showed stronger fluorescence than those transfected with the empty vector, indicating that both f protein and chil- were expressed (fig. ) . at dpc, some chickens in the ddh o group and pcaggs group started showing signs of depression, wryneck, and decreased feeding. a large amount of mucus was observed in the oral cavity when the swabs were collected. at dpc, the chickens started dying, and at dpc, all chickens in both groups were dead. in contrast, two out of ten chickens in the pcag-f/im group and one out of ten in the pcag-f/ ep group began to show minor symptoms at dpc, and only one of them (in the pcag-f/im group) died during the experiment (fig. ) . viral shedding was monitored by testing oropharyngeal and cloacal swabs obtained on , , and dpc (table ) . virus was detected in % of the oropharyngeal and cloacal swabs from chickens in ddh o group and the pcaggs group at dpc, and by dpc, no chickens in either group had survived. in the pcag-f/ im group, viral shedding was detected beginning at dpc, peaked at dpc, and ended at dpc. the pcag-f/ep group showed a lower shedding rate and shorter shedding duration than the pcag-f/im group. the protection and viral shedding rates differed between the pcag-f/ep and pcag-f/im groups, indicating that fig. expression of the f protein and chil- . t cells were transfected with pcaggs (a) or pcag-f (b) and incubated with polyclonal antibody against ndv or transfected with pcaggs (c) or pcag-il- (d) and incubated with polyclonal antibody against chil- . the photos were taken using a fluorescence microscope to detect fluorescein isothiocyanate signals from the secondary antibody fig. protection of chickens by the f gene dna vaccine delivered by im injection or ep. fourteen-day-old spf chickens (n = ) were inoculated with a dna vaccine preparation (ddh o/im, pcaggs/ im, pcag-f/im, or pcag-f/ep) and boosted after weeks. two weeks after booster vaccination, the birds were challenged with ndv and mortality for days was evaluated the two delivery methods might induce different immune responses in chickens. serum samples were collected days after the first immunization to measure the nab level. as shown in fig. a , as expected, no antibody was detected in the ddh o and pcaggs control groups. nabs were detected in five of the six chickens in pcag-f/ep group and four of the six chickens in the pcag-f/im group, demonstrating that the f gene dna vaccine could induce a humoral response. the mean nab level in the pcag-f/ep group was higher than that in the pcag-f/ im group, indicating that the ep method is superior to im injection for stimulating dna-plasmid-induced humoral immunity against the ndv f protein. a lymphocyte proliferation assay was performed to evaluate the ability of lymphocytes from immunized chickens to proliferate after nonspecific stimulation with a mitogen. in this assay, the mean od value for the pcag-f/im group was significantly higher than for the pcagss/im group (fig. b) . notably, the lymphocyte proliferation response for the pcag-f/ep group was significantly higher than that in the pcag-f/im group at or days after primary immunization, suggesting that ep might better sensitize lymphocytes to mitogen-induced proliferation. in the ddh o and pcag-il- /ep groups, clinical symptoms occurred at dpc, and members of both groups started dying at dpc (fig. a) . although the mortality in both groups was %, the death of chickens in the pcag-il- / ep group occurred later than in the ddh o group, suggesting that the il- construct alone has an antiviral effect that is, however, not sufficient to protect chickens against fatal ndv infection. all chickens in the pcag-f/ep and pcag-f+pcag-il- /ep groups survived with no clinical signs observed. in the ddh o and pcag-il- groups, virus was detected in respiratory organs and immune organs, while in the pcag-f/ep and pcag-f+pcag-il- /ep groups, no virus was detected (fig. b ). viral shedding lasted until dpc in the pcag-f/ep group but was only detected at dpc in % of the chickens in the pcag-f+pcag-il- / ep group (table ) . nabs were detected in all samples from the pcag-f/ep group except one collected days (fig. a ) after primary immunization. the average titer of nabs in the pcag-f+pcag-il- /ep group was higher than that in the pcag-f/ep group. this suggests that chil- helps the f gene dna vaccine to stimulate the host immune system, resulting in higher antibody levels. samples collected from the same group on days and showed no significant differences. the average od value in the lymphocyte proliferation test was significantly higher for the pcag-f+pcag-il- /ep group than for the pcag-f/ep group at days after the primary immunization (fig. b) . the average level of lymphocyte proliferation was higher for the pcag-il- / ep group than for the ddh o/ep group at and days after the primary immunization. dna vaccines have several advantages over conventional vaccines, including increased safety, minimal side effects, stability at ambient temperatures, and the ability to overcome interference by maternal antibodies. however, the low immunogenicity of dna vaccines are a major barrier to their application; antibody levels and other immune indicators tend to be low after dna immunization [ ] . ep can enhance the uptake of a dna vaccine by cells by promoting the formation of aqueous pores in cell membranes [ ] . ep has been used in several studies to enhance dna vaccines, and the results have been promising [ ] . oladele and colleagues constructed a dna vaccine based on the ha gene of h n avian influenza virus and found that ep delivery could enhance the antibody response, significantly reduce morbidity, and protect chickens from fatal ndv attack [ ] . in this study, we used ep to deliver a dna vaccine based on the f gene of ndv, and this provided % protection of chickens against virulent ndv challenge, which was better than conventional im injection ( %). the average neutralizing antibody level was also higher with ep than with im injection, indicating that ep administration makes the vaccine more effective. a lymphocyte proliferation assay is commonly performed to test the nonspecific response of the host after cona fig. protection conferred by pcag-f and pcag-il- alone or combined. fifty-two -day-old spf chickens (n = ) were inoculated with ddh o/ep, pcag-il- /ep, pcag-f/ep, or pcag-f+ pcag-il- /ep and boosted after weeks. two weeks after booster vaccination, the birds were challenged with gm. the mortality of ten chickens in each group in days was evaluated (a), and three other chickens were killed and viral titers in different organs were measured by inoculation of -day-old embryonated eggs (mean ± sd). viral titers in each organ were measured three times (b) / / / / / / / / pcag-f+pcag-il- /ep / / / / / / / / stimulation. as the nonspecific immune response plays an important role in defense against viral infections, lymphocyte proliferation assays are often performed to evaluate vaccine efficacy, generally together with other methods [ ] . at days after priming, the lymphocyte proliferation level of the pcag-f/ep group was higher than that of the pcag-f/ im group, suggesting that the use of ep for delivery resulted in enhanced lymphocyte proliferation. however, since there was no control group vaccinated with the empty plasmid by the ep route, the impact of f expression on lymphocyte proliferation in this study cannot be evaluated. the bird in the pcag-f/im group that did not survive the virus challenge had no detectable neutralizing antibodies. however, although all of the chickens in the pcag-f/ep group survived, they did not all have detectable neutralizing antibodies, indicating that antibodies may not be essential for protection and that cell mediated immunity might play an important role in plasmid dna-induced protection against ndv challenge. thus, our work demonstrates for the first time that ep can effectively improve the efficacy of ndv dna vaccines by enhancing the immune response. other delivery methods, including the use of liposomes, nano-chitosan or salmonella spp as a vector, have been used to improve ndv dna vaccines [ , ] . a dna prime-protein booster strategy can further increase the protection rate [ ] . a dna vaccine nano-encapsulated with hollow ag@ sio nanoparticles stimulates strong mucosal immunity when administered intranasally and has been shown to be protective against ndv. however, this is a high-cost and complicated procedure [ ] . viral shedding is another important indicator for vaccine evaluation. infected animals that shed virus can become new sources of infection, leading to rapid spreading. it has been reported that chickens inoculated with the commercial la sota vaccine continue to shed virus for days after challenge with genotype vii ndv, posing a great risk of virus transmission [ ] . both our previous experiments and those of others have shown that the use of a vaccine with higher homology to the challenge ndv strain result in less virus shedding than the use of a heterologous vaccine [ , ] . in this study, we chose an epidemiologically relevant ndv strain of genotype vii. . to provide the template for the vaccine and for challenge. although the f gene dna vaccine delivered by ep could protect chickens from lethal challenge with ndv, the virus was not quickly cleared. viral shedding still could be detected in oropharyngeal and cloacal swabs of % of the chickens at dpc. another strategy to improve the efficacy of dna vaccines is to add molecular adjuvants. il- is an important member of the interleukin family that is mainly secreted by dendritic cells and monocytes. it is an immunomodulator and a potential adjuvant, since it has been shown to enhance the immunogenicity of vaccines and to induce high-level cd + and cd + t cell responses [ , ] . to investigate whether chil- has potential as an adjuvant for the ndv f gene dna vaccine, we constructed an eukaryotic expression plasmid encoding chil- and combined it with the f gene dna vaccine. viral shedding was found to be reduced compared with the single dna vaccine group; only % of the oropharyngeal swabs from chickens were positive for the virus at dpc, and after that, all were negative by virus isolation. in this study, we verified that the co-administration of chil- and the f gene dna vaccine resulted in fast clearance of ndv, which would limit the transmission and circulation of the virus in the bird population. additionally, the mean level of nab in the pcag-f + pcag-il- /ep group was higher than that in the pcag-f group, indicating that chil- also enhances production of specific antibodies. mitogen-induced lymphocyte proliferation at d was also significantly higher for the pcag-f + pcag-il- / ep group than for the pcag-f/ep group, indicating that chil- might enhance lymphocyte proliferation when combined with the f gene dna vaccine. however, the enhanced lymphocyte proliferation might also have been due to the fig. immune responses induced by pcag-f and pcag-il- alone or combined. (a) serum samples were obtained days after primary immunization from eight randomly selected chickens from each group. all samples were tested three times on cef cells to obtain mean nab titers. (b) pbmcs were isolated from three chickens from each group at and days after the first immunization to measure proliferative activities. the error bars indicate the sd. *, p < . ; **, p < . ; ***, p < . fact that the total dose of plasmid dna was twice as high in the group receiving both plasmids. the nonspecific mitogen-induced lymphocyte proliferation observed might also have been at least partially induced by pathogen-associated molecular patterns (pamps) present in the plasmid dna preparation, such as lps or cpg motifs in the plasmid dna sequence. several attempts have been made to improve the efficacy of dna vaccines by adding molecular adjuvants. using complement degradation component as an adjuvant has been shown to increase the production of igg and iga induced by the f protein dna vaccine, accompanied by an increased protection rate [ ] . inoculation with il- and f protein together with salmonella typhimurium as a vector by the oral route has been shown to increase lymphocyte proliferation, but it does not promote antibody production or provide sufficient protection [ ] . viral shedding was not measured in either of the above-mentioned studies, and therefore, the impact of these approaches on shedding is not known. recently, another poxvirus-based dna vaccine targeting the hn protein and including il- as an adjuvant was evaluated [ ] . it was shown that il- can enhance a cell-mediated immune response. however, the vaccine did not provide adequate protection unless another adjuvant, mineral oil, was used simultaneously. since the amount of plasmid used in that study was much lower than the µg used here, and the minimal effective dose of our vaccine has not been determined, further studies are needed to investigate the synergistic effect between the hn or f protein and il- . furthermore, since only one strain of epidemic genotype vii was used as challenge virus for vaccine evaluation, the cross-protective effect of the vaccine against other genotypes still needs to be investigated. in summary, we have evaluated the effects of ep as a delivery method and il- as an adjuvant for ndv dna vaccines, and the results are promising. dna vaccines provide a level of safety and flexibility that is not achievable with conventional vaccines. the combination of ep administration and the use of il- as an adjuvant can effectively compensate for the low immunogenicity of the dna vaccine. further technological advancements will be needed to further reduce the cost of vaccines for large-scale application in the poultry industry. the current study can provide a reference for research on this and other avian dna vaccines. the f gene dna combined with chil- significantly enhanced the humoral immune response and lymphocyte proliferation in immunized chickens significantly reduced the rate of virus shedding. this vaccine is a promising new tool for nd control. author contributions peng xie and yanling li contributed equally to this work. px, yanling li, yaling li, jl, bx, jj and ql conducted the experiments. px and yanling li analyzed the data and wrote the paper. tr, cx and cd designed the experiments and revised the paper. all authors read and approved the final version of the manuscript. ethics approval and consent to participate animals were treated humanely and with regard for alleviation of suffering. all animal experiments were approved by the south china agricultural university experimental animal welfare ethics committee (permit number -v ). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the authors declare that they have no competing interests. tracing the origins of genotype viih newcastle disease in southern africa intranasal interleukin- is a powerful adjuvant for protective mucosal immunity immunologic responses to west nile virus in vaccinated and clinically affected horses electroporation enables plasmid vaccines to elicit cd + t cell responses in 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pathogenicity of contaminated exogenous virus generation and evaluation of a genetically attenuated newcastle disease virus rgm-viim as a genotype-matched vaccine advancements in dna vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity sulfated glucan can improve the immune efficacy of newcastle disease vaccine in chicken interleukin- gene adjuvant increases the immunogenicity of virus-like particles of human papillomavirus type regional variant strain direct gene transfer into mouse muscle in vivo preparation and efficacy of newcastle disease virus dna vaccine encapsulated in chitosan nanoparticles iga response and protection following nasal vaccination of chickens with newcastle disease virus dna vaccine nanoencapsulated with ag@sio hollow nanoparticles immune effect of newcastle disease virus dna vaccine with c d as a molecular adjuvant key: cord- -pghnl authors: wang, hai-ming; liu, tian-xin; wang, tong-yun; wang, gang; liu, yong-gang; liu, si-guo; tang, yan-dong; cai, xue-hui title: isobavachalcone inhibits post-entry stages of the porcine reproductive and respiratory syndrome virus life cycle date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: pghnl porcine reproductive and respiratory syndrome virus (prrsv) is a pathogen of great economic significance that impacts the swine industry globally. since the first report of a porcine reproductive and respiratory syndrome (prrs) outbreak, tremendous efforts to control this disease, including various national policies and plans incorporating the use of multiple modified live-virus vaccines, have been made. however, prrsv is still a significant threat to the swine industry, and new variants continually emerge as a result of prrsv evolution. several studies have shown that pandemic prrsv strains have enormous genetic diversity and that commercial vaccines can only provide partial protection against these strains. therefore, effective anti-prrsv drugs may be more suitable and reliable for prrsv control. in this study, we observed that isobavachalcone (ibc), which was first isolated from psoralea corylifolia, had potent anti-prrsv activity in vitro. although many biological activities of ibc have been reported, this is the first report describing the antiviral activity of ibc. furthermore, after a systematic investigation, we demonstrated that ibc inhibits prrsv replication at the post-entry stage of prrsv infection. thus, ibc may be a candidate for further evaluation as a therapeutic agent against prrsv infection of swine in vivo. porcine reproductive and respiratory syndrome virus (prrsv) is a positive-strand rna virus that is approximately kb in length and belongs to the family arteriviridae [ ] . prrsv is one of the most important viruses hindering the development of the swine industry globally, especially with the emergence of highly pathogenic prrsv (hp-prrsv) [ , ] . recently, new prrsv mutant strains have become pandemic in china [ , , , , , ] and are named nadc -like prrsv strains for their genomic similarity to the nadc strain isolated in the us in [ ] . nadc -like prrsv strains feature different recombinations and have produced many mosaic isolates, enhancing their genetic diversity. currently, the most effective methods to control viral diseases are vaccines and antiviral agents. however, vaccination against prrsv infection has achieved limited success, primarily due to the high genetic diversity of the virus [ , , , ] . in addition to genetic mutation, prrsv genetic diversity is also generated by recombination among different strains, including vaccine strains [ , , , , , ] . importantly, commercial vaccines cannot provide complete protection against heterologous prrsv infection [ , ] . the problems outlined above indicate that the use of vaccines may not be an ideal strategy for prrsv control; however, exploring effective anti-prrsv drugs may overcome these issues. isobavachalcone (ibc) is a prenylated chalcone of the flavonoid subclass that was first isolated from psoralea corylifolia in [ ] . ibc possesses a wide spectrum of biological activities, including antibacterial, antifungal, anticancer, anti-reverse-transcriptase, antitubercular and antioxidant functions [ ] . however, whether ibc has potential antiviral activity remains unclear. in the current study, for the first time, we observed that ibc has anti-prrsv activity. furthermore, after a systematic investigation, we demonstrated that ibc inhibits prrsv replication at the post-entry stage of prrsv infection. porcine alveolar macrophages (pams) were obtained from the lungs of -week-old specific-pathogen-free (spf) piglets, and monkey kidney cells (marc ) were cultured in dulbecco's modified eagle's medium supplemented with % fetal bovine serum (fbs). the highly pathogenic prrsv strain hun [ , ] and the nadc -like prrsv strain hen-l (isolated in our lab) were propagated and titrated in marc cells. ibc was purchased from shanghai tauto biotech company and was dissolved in ethanol and used at the concentrations indicated. the cytotoxicity of ibc was evaluated for pams and marc cells. briefly, twofold dilutions of ibc from a starting concentration of . μm were applied to % confluent cells in a -well plate. after incubation for h at °c, the cytotoxicity of ibc was evaluated using a cell counting kit- (cck , dojindo laboratories, japan) according to manufacturer's protocol. the cc was defined as the concentration of ibc that reduced the absorbance of treated cells by % relative to that of the cell control. a total of × marc cells were plated in a -well plate, and h later, the cells were infected with hun (multiplicity of infection [moi] = ) for h at °c. next, the culture medium was replaced with different concentrations of ibc diluted in dmem containing % fbs. cells were harvested at the indicated times for further western blot or immunofluorescence analysis. the total rna of all samples was extracted using an rneasy plus mini kit (qiagen, germany), and μg of total rna was reverse transcribed into cdna according to the manufacturer's protocol (primescript™ rt reagent kit with gdna eraser, takara). the real-time pcr procedure was performed using an agilent mx p real-time pcr system. the primers and probes were described in a previous report [ ] . cells were infected with hun for h and were then treated with various concentrations of ibc. after incubating for the indicated time, cells were fixed in % ethanol, permeabilized with . % saponin in pbs, and then blocked with % bovine serum albumin (bsa) for h at room temperature (rt). after blocking, the cells were incubated with a mouse anti-prrsv n protein monoclonal antibody (igg b, made in our lab) at a dilution of : for h at rt, and dsrna was stained with the mouse monoclonal antibody j (scicons, hungary) at a dilution of : for h at rt. after washing, an aliquot of : -diluted fitc-labeled antimouse igg (sigma) was used as the secondary antibody. for nuclear visualization, cells were treated with dapi (sigma). immunofluorescence was observed using a leica tcs sp confocal microscope. cells were washed twice with pbs and then prepared with ripa lysis buffer (solarbio). the cell lysates were centrifuged at , rpm for min at °c. the supernatants were collected, and protein concentrations were determined by bca assay. next, proteins from each sample were separated via % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), after which the protein bands were transferred onto a polypropylene fluoride (pvdf) membrane. the membrane was blocked with % nonfat milk for h at rt and then incubated with mouse anti-prrsv n protein monoclonal antibody ( : , made in our laboratory) or anti-β-actin antibody ( : , sigma) for . h at rt. after being washed three times with pbst ( . % tween in pbs), the membrane was incubated for h with hrp-conjugated anti-mouse igg antibody (sigma). protein bands were visualized with ecl chemiluminescence reagent (thermo scientific). western blot bands were quantified and analyzed using imagej. the relative intensity ratio of protein bands (n protein/β-actin) was calculated as an indicator of prrsv replication ability, with the value of the control group set as ; other ibc treated groups were normalized correspondingly. to further investigate the anti-prrsv activity of ibc, the drug concentration that could inhibit prrsv infections by approximately % ( % inhibitory concentration, ic ) was determined. marc cells were infected with prrsv (moi = ) for h at °c and then treated with twofold dilutions of ibc. total rna was isolated from cells at h postinfection (p.i.), and intracellular prrsv rna copy numbers were determined by rt-pcr. the percentage inhibition was calculated as [(rna copies in control cells -(rna copies in cells treated with ibc)] ÷ (rna copies of the control) × at the indicated ibc concentration, and the value for the control group was set as . the other ibc-treated groups were normalized correspondingly. a virus attachment and entry assay was performed by preincubating marc cells at °c for min, after which prrsv hun (moi = ) and μm ibc were added, and the cells were incubated for an additional h at °c to allow attachment of the virus to the cells. next, the cells were washed three times with cold pbs to remove unbound virus. the cells were then collected and either used to detect prrsv rna levels via real-time pcr (to assess viral attachment) or provided fresh medium containing μm ibc and incubated at °c for h. a temperature shift from to °c was performed to promote entry of the virus into the cells, and after a -h incubation, total rna was collected and analyzed by real-time pcr (to assess viral entry). marc cells were infected with prrsv hun (moi = ) at °c. after a -h incubation, cells were washed three times with pbs to remove unbound viruses and were replenished with % fbs. for the postinfection inhibition study, hun -infected cells were replenished with % fbs supplemented with or μm ibc at specific time points ( , , , or h). cells were harvested at h after ibc treatment, and the levels of prrsv replication were estimated by western blot analysis. origin . software was used for all graphical representations. the ic was calculated using non-linear regression. statistical significance was established using an independent t-test, and p-values less than . were considered significant. the structure of ibc is shown in figure a . to assess the anti-prrsv activity of ibc, we first evaluated the cytotoxicity of ibc on marc cells (fig. b) and pam cells (fig. c) , and the results indicated that cell viability was not significantly affected by ibc at concentrations up to . μm and that the cc of ibc for marc cells was . μm (data not show). the cytotoxic effect of the ibc solvent (alcohol) was also evaluated, the results of which indicated that the alcohol had no effect on marc cells (fig. b) and pam cells (fig. c) . next, we evaluated the anti-prrsv capabilities of ibc in marc cells that were infected with prrsv hun (moi = ) and then treated with different concentrations of ibc. we first determined progeny virus titers and found that ibc significantly inhibited prrsv replication in a dose-dependent manner (fig. d ). to confirm this finding, we also performed an indirect immunofluorescence assay by staining cells with a prrsv n protein antibody and then observed prrsv replication by fluorescence microscopy. as shown in figure e , prrsv replication was significantly blocked by ibc. western blot analysis revealed that prrsv n protein levels decreased markedly, indicating that increasing concentrations of ibc significantly inhibited prrsv replication (fig. f) . to determine whether this inhibition was strain specific, we additionally assayed a currently pandemic nadc -like strain (hen-l ) that was isolated in our lab and observed it also to be inhibited by ibc (fig. g) . thus, these data indicate that ibc inhibition of prrsv is not strain specific. furthermore, the antiviral activity of ibc against prrsv hun was both dose dependent and successful, with an ic of . μm (fig. ) and a selectivity index (si; the ratio of the cc to the ic ) of . . despite the evidence that ibc inhibits prrsv replication, the viral stage influenced by ibc remained unknown. to determine which step(s) in the viral life cycle are affected by ibc, we first performed time-of-drug-addition assays. the results indicated that ibc markedly inhibited prrsv when added early after infection and remained inhibitory when added up to h p.i., whereas prrsv inhibition was limited when ibc was added late, at h p.i. (fig. ) . these data suggest that ibc targets the early phase of the viral life cycle, as it was not able to prevent viral infection after the virus entered the post-replicative stages of infection. next, we tested whether ibc inhibited prrsv by disturbing prrsv attachment or entry. the results demonstrated that prrsv attachment was not influenced by ibc (fig. a) . interestingly, the prrsv entry assay results were the same as those of the attachment assay, demonstrating that neither prrsv attachment nor entry was influenced by ibc (fig. b ). the addition of ibc was inhibitory from to h p.i., whereas it had no effect on prrsv attachment or entry ( fig. a and b) . this inhibition at post-entry stages led us to speculate that ibc inhibits prrsv replication at the stage of viral rna synthesis. to test whether ibc blocked viral rna replication, we performed inhibition experiments using pam cells and stained for dsrnas, which are intermediates in viral replication, with the j antibody, which recognizes dsrna. the results indicated that dsrna levels were significantly decreased in ibctreated cells (fig. a and b) . these data clearly suggest currently, prrsv is a significant problem for the swine industry in china and was a veritable pandora's box for the chinese pig industry during the first outbreak [ ] . the current pandemic strains are highly diverse, and commercial vaccines can provide only partial protection against these strains [ , ] . thus, anti-prrsv drugs may be more suitable for prrsv control in the future. traditional chinese medicine (tcm) has been widely used as a source of novel drugs, and many crude tcm herbal extracts have been shown to inhibit prrsv replication [ , , ] . ibc is extracted from p. corylifolia, which itself is used in tcm [ ] . cheng et al. systematically analyzed seventeen compounds derived from tcms and tested their prrsv antiviral activity in vitro [ ] . two compounds, chlorogenic acid and scutellarin, were shown to have great anti-prrsv replication potential in their study, and the inhibition ratios of chlorogenic acid and scutellarin can reach . and . %, respectively, at the maximum noncytotoxic concentrations [ ] ; in contrast, the ibc inhibition ratio exceeded % at μm, an improvement over the maximum non-cytotoxic concentrations for the other tested compounds. this result indicated that ibc is more effective than chlorogenic acid and scutellarin. ibc has multiple biological activities; it potently abrogates akt and erk signaling pathways and exerts anti-proliferative effects on several human cancer cell lines, and it also induces apoptosis associated with the mitochondrial pathway [ , , ] . akt has been reported to play a positive role in prrsv randomly selected for statistical analysis. the mean rates of positive cells among the total cells in each group were calculated using imagej gene expression and entry, and prrsv replication may be dependent on the activation of pi k/akt [ , , , ] . the erk signaling pathway has been demonstrated to play an important role in the post-entry steps of the prrsv replication cycle and to enhance prrsv infection [ ] . the erk pathway may also contribute to hp-prrsv pathogenesis [ ] . apoptosis also influences prrsv replication, since the induction of apoptosis in marc cells increases virus replication [ ] . ibc inhibition of prrsv replication may occur through modulation of the above-mentioned pathway. ibc may be applicable as an efficacious and safe drug for the treatment of some cancers, such as neuroblastoma [ ] . ibc has been reported to inhibit sars-cov papain-like protease; however, whether ibc inhibits infectious virus was not tested in that study [ ] . this study is the first to report that ibc has potent antiviral activity, and we demonstrate here that it blocks prrsv replication at the initiation of viral rna replication in vitro. however, there are some unanswered questions that should be further explored in the future. first, whether ibc also shows anti-prrsv activity in vivo must be investigated. second, is the blockage of viral rna formation a direct or indirect effect? third, are there other viruses that are also inhibited by ibc? in conclusion, we evaluated whether ibc has antiviral potential as a prrsv infection treatment. the in vitro study established that ibc efficiently inhibited the replication of both hp-prrsv and the current chinese epidemic nadc -like strains without significant drug toxicity. thus, ibc may be a candidate for further evaluation as a therapeutic agent against prrsv infection of swine in vivo. commercial vaccines provide limited protection to nadc -like prrsv infection highly pathogenic porcine reproductive and respiratory syndrome virus induces prostaglandin e production through cyclooxygenase , which is dependent on the erk / -p-c/ebp-beta pathway genomic sequence and virulence comparison of four type porcine reproductive and respiratory syndrome virus strains in vitro screening for compounds derived 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epigallocatechin gallate analog-epigallocatechin gallate (egcg) palmitate against porcine reproductive and respiratory syndrome virus 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 in china nadc -like strain of porcine reproductive and respiratory syndrome virus efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus nadc -like acknowledgements this study was supported by the national key r&d program (grant number yfd ) and the national science foundation of heilongjiang (grant number zd ). key: cord- -sk xyd r authors: carossino, mariano; ip, hon s.; richt, juergen a.; shultz, kendra; harper, kimberly; loynachan, alan t.; del piero, fabio; balasuriya, udeni b. r. title: detection of sars-cov- by rnascope(®)in situ hybridization and immunohistochemistry techniques date: - - journal: arch virol doi: . /s - - -w sha: doc_id: cord_uid: sk xyd r in situ hybridization (ish) and immunohistochemistry (ihc) are essential tools to characterize sars-cov- infection and tropism in naturally and experimentally infected animals and also for diagnostic purposes. here, we describe three rnascope(®)-based ish assays targeting the orf ab, spike, and nucleocapsid genes and ihc assays targeting the spike and nucleocapsid proteins of sars-cov- . open reading frame ab the world is currently experiencing the devastating effects of the coronavirus disease (covid- ) pandemic caused by the newly emerged severe acute respiratory syndrome coronavirus (sars-cov- ). covid- has severely challenged the health care systems in most countries around the world, with increased demand for rapid diagnosis and treatment of seriously ill patients. it has been demonstrated that sars-cov- can infect domestic (i.e., cats and ferrets) and wild animals (e.g., tigers, lions, and mink), causing increasing concerns amongst animal owners [ ] . in situ hybridization (ish) and immunohistochemistry (ihc) techniques allow visualization of viral nucleic acid and protein antigens, respectively, within tissues and cells. these methods offer a semi-quantitative identification of target nucleic acids and proteins, respectively, while conserving topological information of expression within cells and tissues, with respect to specific cellular/tissue structures. this critical information is, in fact, lost with other detection methods, such as western blotting, qpcr/rt-qpcr, or single-cell rnaseq, for which cells and tissues must be dissociated. ish and ihc are well established and widely used in research and routine laboratory diagnostics [ , ] . since the s, rna ish has been a valuable tool for investigating molecular mechanisms of cellular and molecular pathology. currently, multiple approaches exist to carry out rna ish [ ] [ ] [ ] [ ] [ ] [ ] , and among them, the rnascope ® technology excels for robustness, specificity, and sensitivity [ , , [ ] [ ] [ ] [ ] . this technique takes advantage of a variation of the branched dna or "tree" amplification method. in contrast, ihc performance depends heavily on the existence of a specific antibody with high affinity for its antigen with minimum background staining and good performance in formalin-fixed tissues. handling editor: john ziebuhr. the development of suitable preclinical animal models is paramount for studying covid- pathogenesis and evaluating the efficacy of vaccines and therapeutics (i.e., antivirals). for this purpose, the development of sars-cov- -specific ish and ihc are both critical for the assessment of viral distribution, cell tropism, and cytopathology within tissues, complementing classical histopathology, various molecular tools, and serological assays. also, validation of these tools can be of significant utility for postmortem diagnosis of sars-cov- in animals (and humans) within the context of veterinary diagnostic laboratories using formalin-fixed tissues, which render the virus inactive and safer to test under bsl conditions. thus, the objective of this study was to develop rnascope ® ish and ihc methods for the detection of sars-cov- -specific antigen and rna in infected cells that can be utilized for both research (e.g., studies involving experimentally and naturally infected animals) and diagnostic purposes. for this study, confluent vero cells (ccl- ™, atcc, manassas, va, usa) were infected with the wa strain of sars-cov- (usa-wa / strain; bei resources, atcc, manassas, va, usa) at a multiplicity of infection (moi) of . twenty-four hours postinfection, mock-infected and sars-cov- -infected monolayers were fixed in % formalin, and cell pellets were embedded in paraffin. here, we briefly describe the sars-cov- -specific ish and ihc procedures. the list of reagents, including catalog numbers, as well as detailed protocols for these assay, can be obtained by contacting the authors. for rnascope ® ish, a total of three antisense probes targeting the nucleocapsid (n, nucleotide [nt] , - , ), spike (s, nt , - , ) and open reading frame ab (orf ab, nt - , ) of sars-cov- wa strain (gen-bank accession number mn . ) were designed and manufactured by a commercial company (advanced cell diagnostics [acd], newark, ca; table ). four micron sections of formalin-fixed paraffin-embedded mock-infected and sars-cov- -infected vero cells were mounted on positively charged superfrost ® plus slides (vwr, radnor, pa). the rnascope ® ish assay was performed using an rnascope . hd red detection kit (acd) as described previously [ , , ] . briefly, deparaffinized sections were subjected to target retrieval for min at - °c in x target retrieval solution, dehydration in % ethanol for min, and protease plus treatment for min at °c in a hybez™ oven (acd). slides were subsequently incubated with a ready-touse probe mixture for h at °c in the hybez™ oven, and the signal was amplified using a specific set of amplifiers (amp - ) as recommended by the manufacturer). the signal was detected using a fast red solution (red b: red a in a : ratio) for - minutes at room temperature. slides were counterstained with % gill hematoxylin i (sigma aldrich, st louis, mo) for min, and bluing was performed using . % ammonium hydroxide in water. slides were finally mounted with ecomount ® (biocare, concord, ca). probes specific for dihydrodipicolinate reductase b mrna of bacillus subtilis (dapb) and peptidylprolyl isomerase b (ppib) were used as negative and positive controls to assess the assay specificity and rna integrity, respectively. the antisense probes targeting the n, s, and orf ab genes generated equal and very strong intracytoplasmic and membranous signals in sars-cov- -infected vero cell pellets. in contrast, there was no staining in mock-infected vero cells or vero cells hybridized with the dapb probe (fig. ) . for ihc, μm sections of formalin-fixed paraffinembedded mock-infected and sars-cov- -infected vero cells were mounted on positively charged superfrost ® plus slides and subjected to ihc using three different antibodies directed to the nucleocapsid (n) and one antibody directed to the spike (s) protein (table ) . ihc was performed using the automated bond-max and a bond polymer refine red detection kit (leica biosystems, buffalo grove, il) as described previously [ ] . following automated deparaffinization, heat-induced epitope retrieval (hier) was performed using a ready-to-use citrate-based solution (ph . ; leica biosystems) at °c for min. sections were then incubated with each antibody ( table ) for min at room temperature, followed by a rabbit anti-mouse igg ( minutes) and/or a polymer-labeled goat anti-rabbit igg coupled with alkaline phosphatase ( minutes). fast red was used as the chromogen ( minutes), and counterstaining was performed with hematoxylin. slides were mounted using a permanent mounting medium (micromount ® , leica biosystems). infected and mock-infected sections were incubated without the primary antibodies as controls. the antibodies specific for the n and s proteins showed equivalent cytoplasmic labeling only in sars-cov- -infected vero cells (fig. ) . however, among the four antibodies used in this study, clone f , specific for the n protein, showed the most intense staining of the infected cells. here, we describe the development of three antisense probes for the detection of three gene targets of sars-cov- (namely the orf ab, s, and n genes) using the concurrently, sars-cov- -specific ihc assays were developed. of the four commercial antibodies used for ihc, the monoclonal antibody clone f specific for the n protein provided the best staining of sars-cov- -infected cells. the assays developed in this study are readily adaptable for the detection of sars-cov- in tissues from humans and animals, including those utilized as preclinical animal models of covid- for studying the efficacy of vaccines and therapeutics. furthermore, the development of ihc and ish tools is of utmost significance for understanding the pathogenesis of sars-cov- by characterizing the viral tissue distribution/cellular tropism in animal models and humans. most importantly, ish and ihc will complement other quantitative molecular methods in the assessment of vaccine and therapeutic efficacy studies by effectively analyzing the dynamics of viral distribution and clearance within a tissue/cellular context. the tools developed and reported here can be easily multiplexed using automated systems and substantially contribute to understanding sars-cov- pathogenesis. availability of data and material for further detail on protocols, please contact the authors directly. glickman l ( ) a critical needs assessment for research in companion animals and livestock following the pandemic of covid- in humans. vector borne zoonotic dis in situ hybridization and its diagnostic applications in pathology antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades in situ hybridization: methods and applications detection of viral infection and gene expression in clinical tissue specimens using branched dna (bdna) in situ hybridization rnascope: a novel in situ rna analysis platform for formalinfixed, paraffin-embedded tissues rnascope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma in situ detection of microrna expression with rnascope probes dramatically improved rna in situ hybridization signals using lna-modified probes detection of equine arteritis virus by two chromogenic rna in situ hybridization assays (conventional and rnascope(r)) and assessment of their performance in tissues from aborted equine fetuses defining hiv and siv reservoirs in lymphoid tissues rna in situ hybridization for epstein-barr virus and cytomegalovirus: comparison with in situ hybridization and immunohistochemistry characterization of inducible transcription and translation-competent hiv- using the rnascope ish technology at a single-cell resolution. front microbiol downregulation of microrna eca-mir- in seminal exosomes and enhanced expression of cxcl in the stallion reproductive tract are associated with long-term persistence of equine arteritis virus equine arteritis virus long-term persistence is orchestrated by cd + t lymphocyte transcription factors, inhibitory receptors, and the cxcl /cxcr axis key: cord- -bnf mvaf authors: desselberger, u. title: report on an ictv-sponsored symposium on virus evolution date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: bnf mvaf nan genes etc. however, for viruses it should be noted that not all genes encoding proteins of similar functions (e.g. polymerases) stem from one single root. viruses in the retroviridae seem to be of very ancient origin as retroelements have been found in eukarya, bacteria and now also in archaea; in the latter, there is evidence for at least four different lateral gene transfers [ ] . regarding the order of genes in a genome, patterns have often been maintained, but also often been rearranged. phylogenetic relationships have been found useful for virus identification, work on origin, speed and mechanisms of evolution, taxonomy, and the elucidation of transmission pathways (e.g. the transmission of hiv from a source to a victim [ ] ). a case is being made for the use of rankless taxonomy clades (these being monophyletic groups without grading) instead of hierarchical formal linnean taxa for classification (see phylocode; www.ohiou.edu/phylocode). such a tree-based, rankless system could be constructed independently of taxonomy. in the discussion, the positions of clades within accepted phylogenies and the role of the quasispecies concept in a phylogenetically based classification system were considered. alexander e gorbalenya (leiden university medical center) spoke about "using evolutionary models to learn about rna viruses". starting from the idea that evolutionary models lead to the generation of structure/function studies, which in turn may or may not verify the model, the concept was developed that biopolymer alignments represent evolutionary models. proof of concept was explored using sequence data for members of the flaviviridae, nidovirales and birnaviridae as examples. citing data from lindenbach and rice [ ] and the group of tautz (e.g. [ ] ), it was concluded that hepaciviruses (e.g. hepatitis c virus) and pestiviruses (e.g. bovine viral diarrhea virus) have more in common than was originally thought; however, the phylogenetic analysis of hepacivirus and pestivirus genomes is still a challenge to the taxonomy. the nidovirales were established as an order that comprises the coronaviridae, arteriviridae and roniviridae families. this conclusion was based on the finding that viruses in the nidovirales share the mechanism of discontinuous transcription [ ] and that they have motifs of their replicase enzymes in common [ , ] . however, the taxonomy of the coronaviridae is under further review [ ] . for instance, it has recently been found that the cysteine proteinases of an invertebrate nidovirus and of members of the potyviridae share unusual motifs [ ] . viruses in the birnaviridae (carrying dsrna genomes) and some (but not other) members of the tetraviridae (carrying ssrna genomes and infecting insects) share a unique arrangement of motifs in their replicases [ ] . a particular folding model of the replicase of infectious bursal disease virus, a member of the birnaviridae, has recently been tested and verified by the group of e mundt [ ] . in the discussion, the relationship between coronaviruses and influenza c viruses (sharing neuraminate-o-acetyl esterase motifs and functions) was noted. graham hatfull (university of pittsburgh, pennsylvania) spoke about "mycobacteriophage genomics and the origins of mosaicism". given that there are an estimated bacteriophages on earth (most of them in the sea), they represent an enormous genomic diversity and are also an excellent tool box with which to probe evolutionary theories. approximately tailed dsdna bacteriophages have been completely sequenced, and of those represent mycobacteriophages (of a genome size of approximately mbp). partial genomic sequences of of these phages (approximately mbp each) have been subjected to phylogenetic comparison and analyses [ , ] . their genes are closely packed and code for replication, integration, assembly and regulation functions. in the genomes, there is pervasive mosaicism, implying that horizontal exchange of genes has been an important component of their evolution. over % of the genes are only seen in mycobacteriophages but there are also some non-phage genes (which probably were picked up from host genomes). in terms of evolution and classification, each phage genome is considered to be a unique assembly of individual modules (a module either being an individual gene or a set of genes). in order report on ictv to arrive at its present resting place, each module has a different phylogenetic history. the models for the generation of mosaicism are targeted recombination and random illegitimate recombination, followed by selection ('recombination reassorts genetic modules'). in order to conceptualize evolutionary relationships, the model of a three-dimensional web-like (or 'sweb') reconstruction of events was proposed. this would allocate unique 'sequence space' to each phage without ranks or preconceptions. in the discussion, the issues of the stability of mosaic genomes, the speed of recombination during evolution, the lack of a species concept, and the integration and reactivation of mosaic genomes were considered. simon wain-hobson (institut pasteur, paris) described and analyzed "the enormous multiplicity of hiv infection in vivo and the end of clonality". in addition to a minimum point mutation rate of . /genome (increasing to /genome when nearing 'error catastrophe', see below), each hiv genome has undergone recombination events on average, i.e. recombination creates much more diversity than point mutations [ , , ] . in vitro, a single round of replication of hiv- in t lymphocytes generated on average recombination events per virus [ ] . hiv recombinants are frequently produced within individuals, and are even more frequently observed at epidemiological levels. siv recombinants are discovered within days of infection. a prerequisite of recombination is a multiply infected cell (either co-or superinfected); such cells have been found in hiv-infected patients [ ] . proviral sequences are randomly distributed on chromosomes; one chromosome can harbour several of them. there are also recombinant proviruses. there can be - proviral dna copies per cell, and the amount of dna in a cell can be increased threefold. one t cell can produce - hiv particles that are spread preferentially by cell-to-cell contact. within an individual, donor cells (mostly dendritic, i.e. antigen presenting cells) carry sequences different from those found in recipient cells (mostly t cells). upon multiple infections the recombination rate increases and can reach the level of self-destruction ('error catastrophe', see below). concomitantly, the ratio [number of virus particles (nvp)/pfu], already high for all members of the retroviridae, increases further. in these circumstances, an accurate phylogeny cannot be constructed. john coffin (tufts university school of medicine, boston ma, and national cancer institute, frederick md) spoke about "retrovirus evolution and drug resistance". for retroviruses, host-virus co-evolution has been known for some time. the formation of endogenous retroviruses (ervs) as integrated proviral sequences leads to indefinite vertical transmission in the host. ervs can be considered and analysed as representing fossil records of previous virus-host interactions [ ] . in human germlines, herv-k sequences are ubiquitous [ ] . the history of retrovirus evolution in humans is long. ervs represent - % of the human genome. strong parallels can be found in the phylogeny of erv of primates and that of primates themselves to the extent that the time points of evolutionary events in ervs and primates can be mutually determined. herv-k sequences entered human hosts approximately million years ago. every human individual carries - different ervs of which are considered as 'old' and as 'new'. the analysis of long terminal repeats (ltrs) of ervs has allowed distinct waves of infection to be identified. mutations have accumulated as the species evolved. however, the and ends of the ltrs have not always co-evolved (about / human proviruses have 'mismatched' ltrs). approximately % of sequence changes are consistent with evolution by point mutations; other changes are due to multiple recombination events. hervs are still active and can be reactivated. using examples of the development of resistance of hiv to the action of the antiviral drug -thiacytidine ( tc), mutations, selection, drift and linkage were recognized as genetic factors affecting the evolution of drug resistance. by using an ultrasensitive detection assay [ ] , direct sequencing of hiv rna from limiting dilutions and the application of mathematical methods, extensive recombination events and evidence for u. desselberger compensatory mutations were recognized as the main factors in the development of drug resistance. esteban domingo (centro de investigación en sanidad animal and universidad autónoma de madrid) spoke about "quasispecies dynamics and extinction of rna viruses". after an introduction in which basic genetic terms were defined (mutation and mutation rate, hypermutation, recombination, reassortment, segmentation etc), the quasispecies concept was presented according to which any sample of an rna virus represents a 'swarm' of closely related mutants. this composition allows the virus to adapt in a flexible way to changing environmental conditions. parameters of adaptability are: the number of mutations per genome ( - ), the population size (up to infectious particles/host organism), the genomic length ( . kb for hiv, - kb for other rna viruses, i.e. relatively small for all rna viruses), and the number of mutations needed to produce a phenotypic change (can be very small). mutant spectra matter for the quasispecies of many rna viruses (vesicular stomatitis virus, picornaviruses [poliovirus, foot-and-mouth disease virus (fmdv)], lymphocytic choriomeningitis virus (lcmv), bunyaviruses etc). hypermutated (pre-extinction) rna often interferes with the infectivity of clonal rnas. assignment of a quasispecies to a phenotype is indeterminate. quasispecies have both deterministic and stochastic features [ , ] . under bottleneck conditions (e.g. plaque-to-plaque passage in cell culture), the quasispecies spectrum will become narrower, and the fitness of the quasispecies to survive will decrease, due to the operation of muller's ratchet [ ] . the fidelity of the transcriptase/replicase will go in parallel with the viability of a quasispecies distribution; with decreasing fidelity of these enzymes, the viral sequences will transgress via an error threshold to become random sequences. for fmdv, a constant rate of . mutations/genome/plaque transfer has been found during plaque-to-plaque passage. in the presence of a mutagen, viral extinction was frequently observed in vitro [ ] . ribavirin, a licensed antiviral drug, was shown to be a mutagen as well. chronic infection of mice with lcmv was prevented (cured) by treatment of the animals with fluorouracil, a mutagen [ ] . in the discussion, the influence of the ratio [nvp/pfu] on viability was considered. marilyn roossinck (samuel roberts noble foundation, ardmore ok) asked "what determines the quasispecies population size? lessons from plant viruses". using examples from the tobamovirus genus and the bromoviridae family, it was shown that the mutation frequency depended on virus host interactions [ , ] . for brome mosaic virus, the control of diversity was located in rna segment , encoding the polymerase protein, and rna segment , encoding the cell-to-cell movement and coat proteins. bottleneck conditions limited diversity: of the (silent) mutants in a mixed inoculum, only were found in the th leaf and only in the th leaf from the site of inoculation. the transmission frequency differed for different mutants. viruses with large host ranges were found to have large quasispecies 'swarms' (or 'clouds' [ ] ). for further details see www.noble.org/virus evolution. ann palmenberg spoke about "rna structure and comparative picornavirology". for rna viruses, every viral base is to be regarded as a compromise forged by the totality of different selective pressures. those are mainly: protein recognition, mrna transcription, mrna translation, protein structure, and rna structure. different nucleic acids occur in different structural forms: in vivo, dna is usually in the b form, containing a wide major groove and rising by . a • /bp. duplex regions of rna occur in the a form, rising by . a • /bp and being more stable than dnas. the base stacking of rnas contributes hugely to their stability, and rna folding is largely driven by base stacking [ ] . evolutionary co-variance of nucleotides is sometimes observed; compensatory changes may stabilize a stem, and such observations may help to confirm an rna structure. the most stable forms of rna or dna contain a minimum of free energy [ , ] . using computer programmes developed by zuker's group, the optimal folding of rnas of relatively small size (picornaviruses) and large size (sars coronavirus) has been accomplished [ , ] . the question arose: how can one recognize if a calculated fold is real? after randomizing and refolding the rna sequence of encephalomyocarditis virus (in silico), a highly stable form (∆g of − kcal/mol) was obtained that was indistinguishable in stability from, and in the fold of, the real rna. thus, in reality 'the optimal fold' should be considered a myth; there is no single optimal structure. by mathematical derivation the number of alternative partners with which each base of an rna can interact (= p-num) can be obtained [ ] and plotted against the sequence; troughs of the curve indicate regions of few alternative partners. the p-num derivative is a 'quantitative measure of the propensity of that base to become involved with the same or alternative pairing partners in a collection of suboptimal folds' [ , ] ; it is thus a powerful parameter for locating wriggles in an rna structure. 'to maintain rna structure, evolution selects against better alternatives elsewhere in the genome'. the internal ribosome entry site (ires) of picornaviral rnas is a structural motif with a low p-num value. ires structures are similar to those of trnas in that they exhibit no significant sequence similarity, yet fold into virtually identical structures. the picornaviral cis-acting replication elements (cres), which display a cacaaa sequence to d polymerases, also have low p-num values, and again very different sequences adopt very similar -dimensional structures. vice versa, nucleotide sequence similarity does not always conserve rna structures. the aim of the talk was to show the significance of rna structural considerations for the evolution of viruses. at the conclusion of the symposium, andy ball thanked all speakers and discussants. the symposium was attended by approximately participants. comments and suggestions on drafts of this report by andrew ball, jean cohen, esteban domingo, mary estes, denis fargette, anne-lise haenni, mike mayo and ann palmenberg are gratefully acknowledged. classification of papillomaviruses quasispecies dynamics and rna virus extinction multiple molecular pathways for fitness recovery of an rna virus debilitated by operation of muller's ratchet improved free-energy parameters for predictions of rna duplex stability a comparative sequence analysis to revise the current taxonomy of the family coronaviridae the palm subdomain-based active site is internally permutated in viral rna dependent rna polymerases of an ancient lineage bacteriophages with tails: chasing their origins and evolution bacteriophage genomics a novel, endogenous retrovirus-related element in the human genome resembles a dna transposon: evidence for an evolutionary link human endogenous retrovirus k solo-ltr formation and insertional polymorphism: implications for human and viral evolution improved predictions of secondary structures of rna multiply infected spleen cells in hiv patients imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches dynamics of hiv- recombination in its natural target cells flaviviridae: the viruses and their replication molecular evidence of hiv- transmission in a criminal case the nonclonal and transitory nature of hiv in vivo don't forget about viruses topological organization of picornaviral genomes: statistical prediction of rna structural signals new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunodeficiency virus type rna in plasma reproducible nonlinear population dynamics and critical points during replicative competitions of rna virus quasispecies retroids in archaea: phylogeny and lateral origins the structure of a thermophilic archaeal virus shows a double stranded dna viral capsid that spans all domains of life generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure evolutionary history of cucumber mosaic virus deduced by phylogenetic analyses plant rna virus evolution synchronous loss of quasispecies memory in parallel viral lineages: a deterministic feature of viral quasispecies lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis virus (lcmv) new haven ct . sawicki sg, sawicki dl ( ) a new model for coronavirus transcription genetic diversity in rna virus quasispecies is controlled by host-virus interaction unique and conserved features of genome and proteome of sars-coronavirus, an early split off from the coronavirus group lineage of statistics and genomes vp of infectious bursal disease virus is an rna dependent rna polymerase the c-like proteinase of an invertebrate nidovirus links coronavirus and potyvirus homologs on finding all suboptimal foldings of an rna molecule prediction of rna secondary structure by energy minimization parc d'innovation, boulevard sébastian brandt, illkirch, france. -redaktion: sachsenplatz - key: cord- -nmkilkcx authors: reynolds, d. j.; garwes, d. j. title: virus isolation and serum antibody responses after infection of cats with transmissible gastroenteritis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: nmkilkcx transmissible gastroenteritis virus was administered orally to cats. no clinical disease resulted but infectious virus was isolated from faeces for up to days after infection and serum antibody was detected by neutralisation and immunofluorescence tests. transmissible gastroenteritis virus was administered orally to cats. no clinical disease resulted but infectious virus was isolated from faeces for up to days after infection and serum antibody was detected by neutralisation and immunofluorescence tests. . the spread of transmissible gastroenteritis virus (tgev) from infected pig herds and the recrudescence of infection after long periods have not been adequately explained. experiments on the host range of the virus have indicated that nonporcine hosts could have a role in the epidemiology of tge ( , , , , , t ) . indirect immunofluorescence (iif) and virus neutralisation (v n!) tests have been used recently to demonstrate antibody to tgev in cats. in holland and great britain samples from cats with clinical feline infectious peritonitis (fip) frequently have antibody to tgev in the i i f test ( ). however, neutralising antibody has not been found in sera from dutch cats with fip. american and dutch studies have recently demonstrated that sera from cats experimentally infected with f i p will cross-react, at low titre, with tgev-infected pig cells by immunofluoreseence but will not neutralise tgev in vitro ( , ) . this evidence has been used to suggest that an antigenic relationship exists between tgev and f i p virus. the potential of the cat to excrete tgev was studied by virus reisolation and the serological responses to tgev and f i p were monitored; thus the possible epidemiological role of the cat in tge could be assessed. in addition information was obtained on the possible origin and relationship of vn and i i f antibodies to tgev which occur naturally in cats, often at high titres ( , ). tgev strain kn , used as inoculum in this experiment, was prepared as a clarified per cent suspension of small intestine from an infected gnotobiotic piglet. i t had a titre of i. × pfu/ml. - / / / /$ . four twelve-week old kittens were obtained from an spf cat colony with no evidence of neutrmising antibody to tgev and no clinical history of fip. the kittens were housed separately in plastic isolators ( ). three animals (a, b and c) were given ml of virulent tgev orally, the fourth (d) was given ml phosphate buffered saline and retained as an uninfected control. animal a was sacrificed days after infection in an attempt to detect evidence of tgev replication in the tissues, whilst b and c were retained for days. animal d was given oral tgev days after the commencement of the experiment and days later (day ) it was hyperimmunized with . ml tgev viral antigen produced from cell cultured virus ( ), injected intraperitoneally. this dose was repeated days later (day ) and after another days (day ) the eat was killed and exsanguinated. control and infected animals were tested for tgev excretion by attempted isolation of virus from faeces for up to days after infection; occasionally rectal swabs were used to obtain fresh samples. cell growth medium was used to prepare per cent faecal suspensions and elute material from rectal swabs. both types of preparation were clarified by centrifugation at x g for minutes and . ml of the supernate was inoculated onto monolayer cultures of secondary adult pig thyroid (apt/ ) cells on glass eoverslips. the medium was changed after hours and days later any viral eytopathic effect was recorded, the medium was removed and passaged in fresh apt/ cultures and the eoverslips were stained to demonstrate virm antigen by indirect immunofluorescence, using paired sera from a gnotobiotic piglet before and after tgev infection, followed by fitcconjugated rabbit anti-swine globulin (nordic immunological laboratories, london). cultures with evidence of tgev antigen were passaged again in the presence of inhibitory levels of specific antiserum. recovery of tgev was judged successful if cells showed a characteristic cytopathic effect, produced cytoplasmic fluorescence with monospecific tgev antiserum alone and the infectivity could be neutralized by specific tgev antiserum. tgev was not isolated from the faeces of any animal before infection or from the uninfected control. uninfected eoverslip cultures, set up concurrently with infected samples, remained free of tgev. tgev was isolated from the faeces of animal b on days , , and after infection and from animal c on days , , and after infection. virus was not isolated from faeces of animal a in the days after infection nor from animal d either before or after oral administration of tgev. there were no signs of disease in the eats after infection. reisolation of tgev was attempted from animal a, killed days after infection and animals b and c killed days after infection. tissue suspensions were prepared and treated as described for faecal suspensions. tonsil, stomach, upper, middle and lower jejunum, ileum, caecum, colon, rectum, kidney, liver, spleen, lung, brain and mesenteric lymph node were the tissues used. pooled tissue suspensions from b and c were administered orally to -day old spf piglets and were also inoculated onto apt/ monolayers for antigen detection by the i i f test. the piglets showed no sig as of tge nor was there a detectable serological response. the coverslip cultures remained negative by immunofluoreseenee. the survival of infectious tgev in the inoculum was studied at ° and ° c to give an indication of the probable time course of viral inactivation in the host and in faeces. viral antigen was demonstrated by immunofluorcseence after infection of coverslip cultures by the method described for faecal virus reisolation. infectivity titres of tgev were determined by plaque assay on apt/ ceils in mm plastic petri dishes. infectivity was still present after but not days at ° c and was lost within hours at ° c. virus could be demonstrated by both plaque assay and immunofluorescence in infectious samples. blood was taken from the cephalic vein of animals b, c and d before and regularly after infection to monitor the serum for vn and i i f antibodies to tgev. a microtitre test ( ) was used to assay tgev neutralising antibody levels in serum and the responses of cats b, c and d are presented in figure as the toglo of the reciproem of the end-point dilution, calculated by kxrbeg's method ( ) . antibody could not be detected prior to infection and animal a remained negative for the three days prior to sacrifice. the uninfected control also remained negative. in animals b, c and d vn antibody was first detected approximately i week after exposure to tgev and the titres rose to maximum values of - -- .a. twenty-one days after first hyperimmunization cat d had developed a marked secondary response, with a vn antibody level of . which persisted until death. antibody to tgev was not detected in the i i f test before infection. following experimental infection with tgev serum antibody, detectable in the i i f test, developed and persisted similarly in the three cats tested (fig. ) . the i i f test titres were consistently lower than vn test titres. after hyperimmunization a marked increase in the i i f test titre was detected in d. this animal also developed antibody to apt/ cell antigens in the i i f test, although the titre was four-fold lower than that of specific antiviral antibody. antibody to f i p was assayed in an i i f test, by dr. n. c. pedersen~ university of california, davis, u.s.a. ( ) and in a suckling mouse brain-adapted f i p neutralisation test ( ) by drs. a. d. m. e. ost~r~avs and m. c. horzi~k, university of utrecht, the netherlands. no fip-specific antibody was demonstrable in the sera before or after oral infection with tgev. antibody was detected by the i i f test in cat d within days of the first hyperimmunization, however, reaching a titre of . which remained constant for the following days (fig. ) but this serum failed to neutralise f i p infectivity in the mouse brain system. this experiment demonstrates that cats exposed to tgev can excrete the virus for a period significantly longer than would be expected from passive transit of ingested virus. the eat could therefore constitute a practical epidemiological hazard to susceptible pigs. the ability of tgev excreted by the eat to infect pigs or other cats was not investigated but similar work ( ) demonstrated the ability of dogs and foxes to excrete tgev which could infect piglets. hence, contact between these carnivores and infected pigs should be prevented. a study of the replication site of tgev in the cat was not possible with the limited number of animals. virus isolation from only two of the animals tested may indicate some variation in host. susceptibility to infection although the serological responses were similar in all animals. f i p and tge, two antigenically-related viruses, are now known to infect eats experimentally but result in differing immune responses. following a single exposure to tgev, cats developed homologous antibodies, detectable by vn and i i f tests. antibody to f i p was detected only following hyperimmunization, which resulted in extremely high anti-tgev titres. experimental f i p results in no tgev neutralising antibody but antibody to both viruses is found using i i f tests with f i p titres exceeding tgev titres. these results provide serological evidence of a distant antigenic relationship between tgev and fip, perhaps due to an internal group antigen not involved in virus neutralisation. this situation can be compared to the closer relationship of the canine coronavirus to tge, agents which cross-react in vn and :[if tests ( , ) . hyperimmunization may be important in stimulating antibody to heterologous viruses. it is probable that infection with one or more tgev-related viruses could result in the complex serological picture in f i p cases previously described ( ) . f i p virus itself may have more than one antigenic type, which would also result in the differing responses found in f i p cases in the u.k., u.s.a. and the netherlands. we recovery and characterization of a coronavirus from military dogs with diarrhoea epizootiology of porcine transmissible gastroenteritis (tge) effect of precipitation with methanol on antigenic potency of tge virus epidemiological studies of transmissible gastroenteritis in swine feline infectious peritonitis (fip) virus. iii. studies on the multiplication of fip virus in the suckling mouse beitrag zur kollektiven behandlung pharmakologischer reihenversuche the effect of propagation of transmissfble gastroenteritis (tge) virus in pups and the lungs of baby pigs on the immunologic properties of the virus transmissible gastroenteritis in the neonatal dog infection of cats with tgev transmissible gastroen~eritis (tge) of swine. tile possible role of dogs in the epizootiology of tge seroepidemiology of feline infectious peritonitis virus infections using transmissible gastroenteritis virus as antigen serological studies of naturally occurring feline infectious peritonitis antigenic relationship of the feline infectious peritonitis virus to eoronaviruses of other species detection of transmissible gastroenteritis virus neutralising antibody in cats the production of gnotobiotic piglets and calves by hysterotomy under general anaesthesia micro-color test for assay of transmissible gastroenteritis virusneutralizing antibodies untersuchungen fiber die antigenverwandtschaft der viren der felinen infekti sen peritonitis (fip) und der transmissiblen gastroenteritis (tge) des sehweines authors' address" mrs. i). j. t~eynolds, institute for research on animal diseases, compton, newbury, berkshire :rg nn, england.i~eceived july t , key: cord- -hwki lk authors: abi, keha-mo; zhang, qi; zhang, bin; zhou, long; yue, hua; tang, cheng title: an emerging novel bovine coronavirus with a -amino-acid insertion in the receptor-binding domain of the hemagglutinin-esterase gene date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: hwki lk the hemagglutinin-esterase (he) protein of betacoronavirus lineage a is a secondary receptor in the infection process and is involved in the emergence of new betacoronavirus genotypes with altered host specificity and tissue tropism. we previously reported a novel recombinant bovine coronavirus (bcov) strain that was circulating in dairy cattle in china, but this virus was not successfully isolated, and the genetic characteristics of bcov are still largely unknown. in this study, diarrheic faecal samples were collected from a farm in liaoning province that had an outbreak of calf diarrhea (≤ months of age) in november , and all of the samples tested positive for bcov by rt-pcr. in addition, a bcov strain with a recombinant he (designated as swun/a / ) and another bcov strain with a recombinant he containing an insertion (designated as swun/a / ) were successfully isolated in cell culture (tcid : ( . )/ml and ( . )/ml, respectively). unexpectedly, we identified the emergence of a novel bcov variant characterized by a -nt bovine gene insertion in the receptor-binding domain in a natural recombinant he gene, suggesting a novel evolutionary pattern in bcov. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. betacoronavirus lineage a includes human coronavirus hku , human coronavirus oc (hcov-oc ), equine coronavirus, mouse hepatitis virus (mhv), canine respiratory coronavirus (crcov), porcine hemagglutinating encephalomyelitis virus, and bovine coronavirus (bcov). crcov and hcov-oc are closely genetically related to bcov [ , ] . members of this lineage have a unique gene encoding a surface hemagglutinin-esterase (he) protein that is not found in other coronaviruses the he protein serves as a secondary receptor in the infection process [ ] and is involved in the emergence of novel genotypes and in shifts in host specificity and tissue tropism [ ] . in addition, he protein can induce the production of neutralizing antibodies against the bcov [ ] . bcov causes gastrointestinal and respiratory diseases in cattle, leading to serious economic losses all over the word [ ] [ ] [ ] . no consistent genetic or antigenic markers have been identified for discriminating bcov in different clinical syndromes [ , ] , and the virus is thought to only has one genotype [ , ] . bcov can infect wild ruminant animals, including alpacas, sambar deer, and giraffes [ , , ] and probably can be transmitted to dogs [ , , ] . interestingly, a coronavirus isolated from human diarrheic samples has been shown to be related to bcov [ ] , indicating its public health significance. recently, we reported the wide circulation of a novel bcov strain with a recombinant he in dairy cattle in china [ ] . in this study, in order to monitor the epidemic of the recombinant strain, a total of diarrheic faecal samples were collected from a farm that had an outbreak of diarrhea in calves three months of age or younger in liaoning province in november, , and all of the samples tested positive for bcov by rt-pcr as described previously [ ] . full-length he genes ( nt) were successfully cloned from of the bcov-positive samples as described previously [ ] [ ] [ ] , and pcr products were purified and cloned into the pmd -t simple vector prior to sequencing. a handling editor: t. k. frey. the online version of this article (https ://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. [ ] clustered in a large independent branch (fig. ) , and three of the he gene sequences (genbank accession numbers: mn , mn , and mn ) clustered in an independent small branch. further analysis showed that all he genes identified in this study had undergone the same recombination event identified in our previous report [ ] . this suggests an increase in the prevalence of this recombinant, since it was found in only two out of five strains detected in the earlier study (samples collected in january [ ] ) but was present in of the isolates from the same farm characterized in this study (samples collected in november ). this indicates that the he recombinant bcov strain is still present in liaoning, china, and may be increasing in prevalence. the bcov he receptor-binding domain is composed of six surface loops, five of which are grafted onto the beta-sandwich core of the lectin domain, the r -loop, r -loop, r -loop, r -loop, and the rbs-hairpin, and the other is present on the esterase domain, the e-loop ( fig. a) . the isolates with a recombinant he were sequenced and found to contain the same amino acid (aa) variation (f v) in the r -loop that was described in our previous report [ ] . the r -loop in the lectin domain acts in ligand binding, and aa substitutions in this domain may affect receptor binding. for example, hcov-oc may have evolved during adaptation of bcov to a human host by gradually losing its lectin activity due to progressive accumulation of aa mutations in the r -loop of the lectin-binding domain in the he protein [ ] . unexpectedly, three isolates that clustered in an independent small branch all contained an identical insertion of nucleotides (aag gct act gtt ), resulting in four additional amino acids in the r -loop (fig. b ). this sequence was not present in any of the available bcov he sequences in the genbank database. interestingly, the inserted nucleotides were identified as originating from the bovine host, matching the sequence of a transcript variant of orf on bos taurus chromosome c (genbank accession no. xm ). it is worth noting that the r -loop is composed of aa (aa - ) in bcov he, and residues f , l , s , and n are essential for receptor-ligand interaction [ ] . a d model constructed based on the crystal structure of bovine coronavirus hemagglutinin-esterase (smtl id: cl . ) using the online software swissmodel (https ://www.swiss model .expas y.org/inter activ e) showed that the position of the -aa insertion in the r -loop between f and f in he in the recombinant bcov strains would alter the spatial conformation of the receptor-binding site relative to that in the he recombinant strains and prototype strains lacking this insertion ( fig. b and c) . mhv is also a member of betacoronavirus lineage a, and a variant has been reported with an aa insertion in the r -loop that causes a change in its receptor specificity to -o-ac-sia from -o-ac-sia [ ] . it is reasonable to speculate that the -aa insertion in the r -loop in he may affect he receptor binding in the bcov strain. the betacoronavirus he protein is involved in receptor binding, host spectrum and tissue tropism [ , , ] , and it would be valuable to further investigate the biological significance of this he variant. betacoronavirus he may have arisen from an influenza-c-like he fusion protein (hef), with transformation of the betacoronavirus he from a trimer into a dimer for increased plasticity of the receptor-binding site (rbs) compared to that of hef [ ] . the integration of a bovine gene into other viruses has been reported previously. for example, insertion of the bovine smt b gene into the genome of bovine viral diarrhea virus correlates with the cytopathogenicity of the virus [ ] . interestingly, the inserted sequence in he in this study may have originated from bovine chromosomal dna, which may provide an evolutionary advantage for bcov, and its identification should enhance our current understanding of the genetic evolution of bcov. the bcov strain with a recombinant he (designated as swun/a / ) and bcov strain with a recombinant he containing an insertion (designated as swun/a / ) successfully isolated in vero cell culture. a cytopathic effect (cpe) was observed after three blind passages, and typical cpe characterized by cell rounding, cell lysis, and detachment from the culture flask was observed after h at passage . after plaque purification, the tcid values of strains swun/a / and swun/a / were calculated as . /ml and . /ml, respectively, by the reed-muench method [ ] . to better understand the genetic evolution of swun/a / , genomic rna was successfully obtained from strains swun/a / and swun/ a / (genbank accession number: mn and mn ). the two complete genomes shared . % nt sequence identity with each other, and . %- . % nt sequence identity with all other bcov genomes with sequences in the genbank database. phylogenetic trees constructed by the neighbor-joining method based on the full-length genome sequence and the orf a, orf b and s genes showed that the two strains have a close genetic relationship (see appendix). thus, the bcov swun/a / strain might have evolved from the swun/a / strain. in summary, we report a novel bovine coronavirus variant with an insertion of nucleotides in the he gene that results in the addition of four amino acids in the receptorbinding portion of the he protein. this disruption could alter the receptor-binding site by changing its spatial conformation, and it may reflect an emerging pattern in the evolution of bcov. the betacoronavirus he gene is involved in the emergence of new betacoronavirus genotypes, host specificity, and tissue tropism [ , , ] . thus, further investigation of the biological and epidemiological characteristics of this variant bcov strain is warranted. isolation and sequence analysis of canine respiratory coronavirus betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase lectin activity structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution the murine coronavirus hemagglutinin-esterase receptorbinding site: a major shift in ligand specificity through modest changes in architecture monoclonal antibodies to bovine coronavirus: characteristics and topographical mapping of neutralizing epitopes on the e and e glycoproteins factors affecting calf mortality in iranian holstein dairy herds molecular and antigenic characterization of bovine coronavirus circulating in argentinean cattle during - market impacts of reducing the prevalence of bovine respiratory disease in united states beef cattle feedlots bovine respiratory coronavirus coronaviruses in cattle serological characterization of genotypically distinct enteric and respiratory bovine coronaviruses bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences detection and characterization of bovine-like coronaviruses from four species of zoo ruminants the infectivity and pathogenicity of a group bovine coronavirus in pups detection of a group coronavirus in dogs with canine infectious respiratory disease biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/esterase gene in dairy calves in china bovine coronaviruses associated with enteric and respiratory diseases in canadian dairy cattle display different reactivities to anti-he monoclonal antibodies and distinct amino acid changes in their he, s and ns protein molecular epidemiology of human coronavirus oc reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene comparative analysis of coronavirus hku genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku insertion of a bovine smt b gene in ns b and duplication of ns in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus a simple method of estimating fifty per cent endpoints acknowledgements this work was funded by the th five-year plan national science and technology support program (grant number key: cord- - e yu do authors: homberger, f. r. title: maternally-derived passive immunity to enterotropic mouse hepatitis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: e yu do maternally-derived antibody to enterotropic mouse hepatitis virus (mhv) strain y was transferred to pups by both intrauterine (igg) and lactogenic (iga and igg) routes. antibody present in the gastric whey of pups suckling immune dams dropped to undetectable levels by weaning age ( days post partum). mhv-specific igg was found in the serum of passively immune pups up to weeks of age. immune dams transferred equal levels of antibody to consecutive litters of pups, without evidence of decline. immunoblots showed that iga and igg in whey and serum were directed against nucleoprotein n and glycoprotein s. mhv-specific igm was not detected in any sample. coronaviruses are important pathogens in many species of birds and mammals, including man. they are usually associated with either respiratory or enteric disease. mouse hepatitis virus (mhv), family coronaviridae, genus coronavirus, is a common, highly contagious virus of laboratory mice. the mhv group contains numerous, closely related and highly mutable viruses with either respiratory or enteric tropism [ ] . mhv is important both as a natural pathogen of laboratory mice and as a model of coronaviral infection, particularly enteritis in the neonate. as with other enterotropic coronaviruses, outbreaks of enterotropic mhv in naive mouse populations cause enteritis with high mortality among neonates. older pups and adult mice show minimal or no apparent disease [ , ] . observations in enzootically infected mouse populations give reason to suspect that maternally-derived passive immunity is very significant in protecting newborn mice against apparent disease [ , , ] . the passive transfer of immunoglobulins from dam to young in mice differs from that of most other mammals. igg is absorbed in utero by means of specific receptors in the yolk sac wall, and is selectively absorbed postnatally for up to f.r. homberger days via igg receptors in the intestine [ ] . pilot studies with mhv-y, an enterotropic strain of mhv [t] , have shown that pups born to immune dams and transferred to naive dams at week of age, then challenged with mhv-y, all developed enteritis. in contrast pups born to naive dams and fosternursed by immune dams were protected against disease. this finding suggested that ingestion of immune milk is essential for passive immunity to enterotropic coronavirus infection and that locally active, intraluminal antibody such as iga, might play an important role in protecting pups from enteritis. the purpose of the present study was to evaluate the route of transfer and the role of the different immunoglobulins in passive immunity against enterotropic mhv and to determine which viral proteins are recognized by these immunoglobulins. mhv-y was isolated in nctc- cells during a natural outbreak of enteritis in infant mice [ ] , passaged in infant cd mice and used in the form of a clarified intestinal homogenate. mhv-s was obtained from the american type culture collection, bethesda, md and passaged twice in nctc- cells. nctc-l cell-adapted mhv- was obtained from the institut f/ir labortierkunde, universitfit ziirich, switzerland and passaged in nctc-l cells. outbread cd (crl-cd br) albino mice were obtained from charles river breeding laboratories, portage, mi in filtered containers. all mice were kept in autoclaved microisolator cages (lab products, maywood, nj) containing pine shavings. cages were changed aseptically in a laminar flow hood as previously described [ ] . all animals were seronegative to mhv. dams were orally inoculated with a single dose of mhv-y containing median neonatal enteritis doses of virus or with sterile tissue culture fluid (controls) and bred days later. pilot studies have shown that oral inoculation results in higher mhv antibody titers than parenteral inoculation and mimics more closely immunity in naturally infected animals. the day interval between immunization and breeding ensured that the dams were not shedding virus when the pups were born (approximately days after inoculation). adult mice were killed with carbon dioxide gas and neonates by decapitation. serial blood samples were taken with capillary tubes by periorbital puncture, under methoxyflurane (metofane, pitman-moore inc., washington crossing, n j) anesthesia. terminal blood samples were obtained from pups by decapitation and from dams by cardiac puncture. whey was collected by removing the stomach curds from pups, suspending them in phosphate buffered saline ( % w/v) and centrifuging the homogenate at , × g for min. the supernate was stored at - °c. serology sera and whey were tested for mhv-specific igg by an enzyme immunoassay (eia) using mhv-s-infected formalin-fixed nctc- cells as antigen [ ] . mhv-s is the prototype strain to which mhv-y, which grows poorly in cell culture, is most closely related eli. mhv-specific iga was best detected with an emzyme-linked immunosorbent assay (elisa) using purified mhv- as antigen. mhv- grown in nctc-l cells was pelleted at , x g for h. the resulting pellet was resuspended in ste buffer ( ram nac , passive immunity to enterotropic mhv mm tris, mm edta), layered on a continuous sucrose gradient ( %/ % w/v) and centrifuged for h at , x g. the visible band was collected, stored at - °c and diluted in bicarbonate buffer (ph . ) for coating wells of -well microtiter plates (dynatech laboratories, alexandria, va). a peroxidase-labeled goat-anti-mouse iga conjugate and abts substrate (both kirkegaard and perry laboratories, gaithersburg, md) were used in the elisa [ ] . sera of sentinel mice and of dams of replacement litters were screened for mhv antibody by indirect immunofluorescence assay [ ] . attempts to detect mhvspecific igm used all of the assays described above. immunoblots were performed on nitrocellulose paper using mhv-s as antigen. mhv-s was propagated in nctc- cells, and clarified culture fluid was treated overnight at °c with polyethylene glycol- ( g/ ml) in the presence of nac ( . g/ ml). the resulting precipitate was centrifuged at , x g for min. the pellet was resuspended in ste buffer, and layered on a discontinuous gradient ( %/ % sucrose in ste buffer) and centrifuged at , x g for rain. the visible band was collected and stored in aliquots at - °c. purified mhv-s was boiled for rain in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoretically separated on a . % acrylamide gel at ma for h. the proteins from the gel were then electrobtotted onto nitrocellulose paper at v for h [ ] . the nitrocellulose paper was stained with ponceau-s, cut into strips along the visible lanes and saturated in blocking buffer ( % fetal calf serum) overnight. nitrocellulose strips were twice washed for min in tris-buffered saline, covered with test serum or whey at different dilutions and incubated for h at °c. after two more wash cycles, phosphatase-labeled goat anti-mouse igg, iga, or igm conjugate was added and incubated for another h at °c followed by two rain washes. the strips were then stained with bcip/nbt ( -bromo- -chloro- -indolylphosphate/nitroblue tetrazolium) phosphatase substrate system (kirkegaard and perry laboratories) and air dried. the isotype and titer of mhv immtmoglobulins transferred in utero and in milk, as well as the duration of secretion during lactation were first examined. immune and naive adult females were bred and allowed to whelp. serum was collected from half of each litter immediately after birth before suckling to assess intrauterine transfer of passive immunity. the rest of the litter was sacrificed h later to collect serum and whey, then replaced by a litter from a naive dam to maintain the nursing stimulus. on days , , and post partum, the pups were removed for h and replaced by naive pups which had previously fasted for h. after feeding, serum and whey were collected from these pups. sera and whey were tested for mhv-specific iga, igg, and igm by eia or elisa. mhv-specific iga and igg but not igm could be found in the milk of immunized dams throughout the days of lactation (table ) . during this period, iga titers remained relatively constant, whereas igg titers peaked at to days and declined by days post partum. sera of pups sacrificed immediately after birth (day ) contained mhv-specific igg, indicating in utero transfer of antibody (table ) . virus-specific igm could not be detected at any time. in of the replacement litters used to collect whey on days , , and , igg antibody could be found in the serum (data not shown). this showed that igg can appear in the serum of naive pups after only h of suckling on an immune dam. toward the end of lactation, suckling activity declined and pups started to eat solid food. to find out how this change affected passive immunity, adult females were mhv-y-or sham-inoculated and bred days later. at , , , and weeks of age, two pups from each litter were sacrificed and serum and stomach contents were collected and tested. mhv-igg, but not iga or igm, could be found in the serum of pups suckling immune dams. mhv serum igg titers peaked at days (table ), when they reached levels higher than the ones found in their dams' sera collected immediately post partum. serum igg titers dropped markedly after weaning at days (table ) . mhv-specific igm could not be detected in sera from either pups or dams. stomach contents of pups suckling immune dams yielded constant igg titers and gradually declining iga titers up to the age of days, but neither isotype of mhv antibody was detected at or beyond days (table ). this coincided with the macroscopically obvious change of the stomach contents from milk curd to solid food mass. in a final experiment, the titer and duration of passively acquired mhv igg in serum were evaluated in first, second, and third litter pups weaned from immune vs. naive dams to determine the duration of detectable passive antibody and if the concentration of passively transferred antibody declined with sequential litters. pups from three consecutive litters born to immune and naive dams were exsanguated at , , , , and weeks of age and sera were tested for mhv-specific igg. naive males used for breeding also served as sentinels to verify that dams were not re-exposed to mhv, thereby causing booster infections. passively acquired mhv antibodies persisted in the sera of pups born and nursed by immunized females for to weeks. the mhv serum igg titers of two week old pups correlated closely with those of their corresponding dam, but dropped substantially during the next two weeks (table ). antibodies persisted for weeks in pups born to dams with low mhv igg serum titers and then declined to undetectable levels. in other animals, serum antibodies could be found through weeks. the duration of passive immunity did not decrease with the second and third litter of individual dams. to ensure that the persisting titers were not due to an inadvertant mhv infection, all sera were tested for iga, which could be found in the immunized dams later than five months after inoculation but not in any of the offspring (data not shown). selected whey and serum samples were also examined by western blot to determine the viral protein specificity of the immunoglobulin isotypes. serum of mice experimentally infected with other prototype strains of mhv crossreacted with mhv-s. mhv-jhm igg reacted with all three structural proteins of mhv-s, including glycoprotein s (fig. , lane ) . mhv-y serum igg reacted with the nucleoprotein n and glycoprotein s (fig. , lane ) . passively acquired mhv-y serum igg in pups (fig. , lane ) , as well as whey igg and iga (fig. , lanes and ) also reacted with n and s. reactivity to the m protein of mhv- -- --- i - ...... rdfitter nd e -- --- - - - - ....... "reciprocal mhv igg titer b reciprocal geometric mean of mhv igg titer, replicates c uninfected controls d<l: e dam died at weaning s was not detected in sera or whey of mhv-y-immune mice (fig. , lanes - and ). since igm against mhv could not be detected in any samples, sera from mice infected for , , , , or days with mhv-y were tested. all were igm-negative using elisa, eia, and western blot. the goat-anti-mouse igm conjugates that were used yielded positive results when tested by elisa and immunoblots with a different antigen (borrelia burgdorferi [ ] ). the mouse selectively transfers igg to fetuses in utero through yolk sac receptors and to pups for about two weeks via specific igg receptors on intestinal epi- this study demonstrated that igg to enterotropic mhv-y was transferred in utero as well as through the milk. the same results were found previously with dams immunized intraperitoneally with killed mhv strain dvim, another enterotropic strain of mhv [ ] . intrauterine antibody transfer could not be detected from dams inoculated oronasally with respiratory mhv-jhm [ ] . the present study also demonstrated lactogenic transfer of iga, but absorption from the intestine into the blood of pups did not occur. intraluminal iga and igg in the gastro-intestinal tracts of immune pups could be detected until the suckling activity declined and pups started to eat solid food. serum antibody in pups nursed by mhv-y-immune dams persisted much longer than expected. earlier studies with mice inoculated either oronasally or intraperitoneally with non-enterotropic mhv-jhm had shown that igg in the serum of passively immune pups persisted for about two weeks after the receptormediated absorption of igg in the intestine ceased at approximately two weeks of age [ , ] . in contrast, passively acquired igg to mhv-y persisted at low titer for more than weeks in some mice. this finding suggests that results of serologic monitoring should be interpreted cautiously among animals of this age group. the possibility must be considered that low positive results are due to maternally-derived passive immunity. the current study has also shown that an iga assay could be used to differentiate between passive and active immunity, since serum iga was only found in mice that were actively immune. compared with a similar study on respiratory strains of mhv [ ] , serum igg titers of dams inoculated with enterotropic mhv-y did not so obviously decline over the course of their first three litters. mhv igg titers passed on to the second or third litter were not substantially reduced. serum igg titers of pups dropped to the same level in all three consecutive litters after weaning and duration of passive immunity did not decrease in later litters. this was not due to repeated infections of the dams, since sentinel mice remained seronegative. mhv-specific igm was not detected in these experiments using three independent assays. literature reports of mhv igm could not be found, implying that no specific igm response is generated by mhv infection. in vitro studies showing that plasma cells in culture cease to produce igm when infected with mhv may support this contention [ ] . among the three major structural proteins of mhv, nucleoprotein n is moderately and membrane glycoprotein m highly conserved among mhv strains, whereas spike glycoprotein s is very variable. a fourth protein, hemagglutinin esterase he, appears in only a few mhv strains [ , , , ] . mhv-s virions possessed n, m, and s bands on immunoblots, but not he when stained with mhv-jhm antiserum. additional bands at kda are virusrelated proteins of cell origin antigenetically related to n protein and vary in expression dependent on culture conditions of the virus [ ] . in this study, mhv-y-specific igg and iga were directed against structural proteins n and s of mhv-s. that no specific reaction to the m protein was found is most likely due to insufficient antibody titer of the samples. 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lethal enteritis in infant mice mouse hepatitis virus: molecular biology and implications for pathogenesis etablierung des serologischen nachweises yon infektionen mit dem m/iusehepatitisvirus und dem pneumonievirus der maus mittels elisa. dissertation maternally derived immune resistance to fatal diarrhea in infant mice to mouse hepatitis virus experimental chronic lyme borreliosis in lewis rats ter meulen v ( ) influence of maternal immunity on the outcome of murine coronavirus jhm infection in suckling mice coronavirus jhm: tryptic peptide fingerprinting of virion proteins and intracellutar polypeptides an immunofluorescence test for the detection of serum antibody to rodent coronaviruses two enzyme immunoassays for the detection of antibody to rodent coronaviruses structural polypeptides of the murine coronavirus dvim western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus this work was supported by grant rr from the national center for research resources, national institutes of health, bethesda, md. the author was supported by a fellowship from the schweizerische nationalfond, bern, switzerland. i thank drs. stephen, w. barthold, and abigail l. smith for their invaluable help and debby beck and deborah winograd for their technical assistance. received march , key: cord- -kzaowysv authors: joshi, lok r.; okda, faten a.; singrey, aaron; maggioli, mayara f.; faccin, tatiane c.; fernandes, maureen h. v.; hain, kyle s.; dee, scott; bauermann, fernando v.; nelson, eric a.; diel, diego g. title: passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: kzaowysv passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (pedv). here, we investigated the immunogenicity of an orf virus (orfv) vector expressing the full-length spike (s) protein of pedv (orfv-pedv-s) in pregnant gilts and its ability to confer passive immunity and protection in piglets. three doses of orfv-pedv-s were given to two groups of pedv-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. one of the two groups immunized with the orfv-pedv-s recombinant virus was also exposed to live pedv orally on day post-immunization (pi). antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. the protective efficacy of orfv-pedv-s was evaluated after challenge of the piglets with pedv. pedv-specific igg, iga and neutralizing antibody (na) responses were detected in orfv-pedv-s-immunized and orfv-pedv-s-immunized/pedv-exposed gilts. pedv na, igg and iga were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after pedv challenge in comparison to control piglets. piglets born to gilts that received orfv-pedv-s and were exposed to live pedv showed stronger na responses and lower clinical scores when compared to piglets born to gilts immunized with orfv-pedv-s alone. these results demonstrate the potential of orfv as a vaccine delivery platform capable of eliciting passive immunity against pedv. porcine epidemic diarrhea virus (pedv), an alphacoronavirus in the family coronaviridae, is a single-stranded, positive-sense rna virus that causes porcine epidemic diarrhea (ped) in pigs [ ] . pedv infects pigs of all age groups resulting in an enteric disease with high morbidity. high mortality rates ( %- %) are observed in neonatal piglets; however, mortality is usually low in older animals [ ] . ped is characterized by vomiting, watery diarrhea and dehydration, which are usually followed by death in suckling piglets [ ] . after its introduction in the usa in , pedv caused the deaths of over million piglets, resulting in significant economic losses to the us swine industry [ ] . since then, significant investments and efforts to develop pedv vaccines have been made, with a few vaccines receiving a conditional license from the usda. the efficacy of these vaccines in protecting newborn piglets, however, is still unknown. piglets are born agammaglobulinemic due to the impermeable nature of the epitheliocorial swine placenta, and their immune system is immature, which makes them highly susceptible to pedv in the first weeks ( - ) of life. therefore, passive transfer of antibodies through colostrum and milk is critical for protection of neonatal piglets against pedv [ , ] . previous studies with transmissible gastroenteritits virus (tgev), a coronavirus that is closely related to pedv, have shown a high correlation between milk antibody levels and protection in piglets [ , ] . hence, a requirement for an effective pedv vaccine is the ability to induce high levels of antibodies in colostrum and milk with their subsequent transfer to piglets born to immunized sows. although several attenuated, killed, or subunit pedv vaccines have been developed, most of them fail to induce sufficient levels of lactogenic immunity and protection in newborn piglets [ , ] . thus, it is critical to develop improved alternatives to currently available pedv vaccines. recently, we demonstrated that a recombinant orf virus (orfv) expressing the full-length spike (s) protein of pedv (orfv-pedv-s) is capable of eliciting protective immune responses in immunized pigs [ ] . three-week-old piglets immunized intramuscularly (im) with the orfv-pedv-s recombinant virus were protected from clinical signs of ped after oral challenge with pedv and showed reduced virus shedding in feces [ ] . in the present study, we investigated the immunogenicity of orfv-pedv-s recombinant virus in pregnant gilts and its ability to induce passive immunity and protection in piglets born to immunized animals. the recombinant orfv-pedv-s was previously generated and characterized in our laboratory [ ] , propagated and titrated in primary ovine fetal turbinate cells (oftu, provided by d.l. rock, university of illinois) [ ] . pedv strain usa/co/ (co ) was obtained from the national veterinary services laboratory (nvsl) and propagated in vero- cells (atcc ® crl- ™) in the presence of . µg of tpck-treated trypsin per ml (sigma aldrich, st. louis, mo). six primiparous gilts were randomly assigned to three experimental groups as follows: group (g ), control (mem; n = ); group (g ), orfv-pedv-s-immunized (n = ); group (g ), orfv-pedv-s-immunized/live pedv-exposed (n = ) ( table ) . animals from g and g were immunized intramuscularly (im) with ml of the recombinant orfv-pedv-s containing . tissue culture infectious dose (tcid )/ml in mem. all animals received the first immunization on day and two booster immunizations on days and post-immunization (pi). the second booster was administered two weeks prior to the anticipated parturition date. in addition to the immunization regimen with orfv-pedv-s described above, gilts in g were exposed to live pedv orally ( × tcid in ml) on day pi. animals from g were sham-immunized with ml mem as described above. animals were housed in bsl- animal rooms, and two weeks prior to farrowing, each gilt was transferred to individual farrowing crates (two crates per room). all gilts received prostaglandin-f α ( mg) on day pi by im injection to induce parturition. animals from g and g farrowed on day pi, while animals from g farrowed on day pi. twelve piglets from each sow were randomly selected, kept with their mothers (n = per group) for days and allowed to suckle colostrum and milk ad libitum. excess piglets were euthanized to keep all sows with an equal number of piglets (n = ). all piglets (g , g and g ) were challenged orally on day post-birth with a virus suspension containing . × tcid of pedv strain co ( ml/piglet). animals were monitored daily for clinical signs and mortality, and the experiment was terminated on day pi or day post-challenge (pc). clinical scores were evaluated based in four criteria, which were modified from a scoring method described by lohse and collaborators [ ] as follows: a) well-being: normal = , slightly depressed = , depressed and lethargic = ; b) defecation: normal feces = , semi-solid and pasty = , watery feces = ; c) vomiting: no = , yes = ; d) body condition: normal = , × . × thin = , emaciated = . mean daily group scores and mortality rates were calculated and compared between different groups. piglets showing severe dehydration and emaciation were euthanized based on the independent evaluation of sdsu's veterinarian. serum was collected from sows on days , , , , , , , , , , and pi. additionally, colostrum and milk were collected from sows on days (day of farrowing) , , , , post-farrowing. serum and rectal swabs were collected from piglets on days (pre-colostrum), (pre-challenge), , , and post-birth. all animal studies were conducted at the sdsu animal resource wing (arw), following the guidelines and protocols approved by the sdsu institutional animal care and use committee (iacuc approval no. - a). indirect elisas using a truncated s protein (aa - ) were used to assess igg and iga antibody responses in animals immunized with the recombinant orfv-pedv-s virus as described previously [ ] . neutralizing antibody responses elicited by immunization with the recombinant orfv-pedv-s were assessed by fluorescent focus neutralization assay (ffn) as described previously [ ] . pedv s-specific igg and iga antibody responses were assessed in colostrum and milk by fmia. optimal assay conditions (amount of antigen, colostrum/milk and secondary antibody dilutions) were determined by a checkerboard titration [ ] . virus shedding was assessed in rectal swabs by rt-qpcr. viral rna was extracted from rectal swabs using a zymo viral rna extraction kit (zymo research, ca, usa) according to the manufacturer's instructions. primers and a probe targeting the pedv nucleocapsid protein were designed using primerquest tool (integrated dna technologies inc., usa). rt-qpcr was performed using a sen-sifast™ probe lo-rox one-step kit (bioline, ma, usa) following the manufacturer's instructions. genome copy numbers per ml were determined using a relative standard curve method. the amount of viral rna detected in feces was expressed as log genome copies/ml. gilts from both immunized groups (g and g ) developed pedv s-specific igg and iga responses ( fig. a and b). igg and iga antibodies were first detected on day pi in animals in g , and the highest level of antibodies was detected on day pi. similarly, in g , igg and iga antibodies were first detected on day pi, and their levels increased after the booster immunization on day . an anamnestic antibody response was observed in g gilts after they were exposed to live pedv on day pi (fig. a and b) . no spike-specific igg or iga antibodies were detected in the serum of control sows (g ). the ability of orfv-pedv-s to induce neutralizing antibody (na) responses against pedv was assessed using a ffn assay. na were first detected in serum a week after the first booster immunization (day pi; fig. c ). an increase in na levels was observed in both immunized groups (g and g ) after day pi (fig. c) . gilts in g , which were immunized only with orfv-pedv-s, had the highest neutralizing antibody titers on day pi, a week after the second booster immunization (day pi; fig. c ), whereas gilts in g , which were immunized with orfv-pedv-s and exposed to live pedv (day pi), had the highest antibody titers on day pi, with titers remaining constant thereafter until the end of the experiment (fig. c) . similar to the igg and iga responses, higher neutralizing antibody responses were observed in gilts in g when compared to g animals. no neutralizing antibodies against pedv were detected in control gilts (g ) in serum samples collected pre-farrowing/pre-challenge. animals in g seroconverted to pedv, presenting detectable levels of igg, iga and na a week after challenge of the piglets (day post-birth; fig. ). notably, a strong correlation between na and igg and iga levels was observed in sow serum (na vs. igg: r = . , p < . ; na vs. iga: r = . , p < . ; fig. d and e). these results confirmed our previous findings demonstrating the immunogenicity of orfv-pedv-s in pigs [ ] . high levels of s-specific igg antibodies were detected on days and post-farrowing, decreasing thereafter in animals from g and g ( fig. a) . colostrum collected from sows in g , exposed to live pedv, had higher levels of igg when compared to g on day post-farrowing ( fig. a) . high levels of s-specific iga antibodies were also detected in colostrum of animals from g and g on day post-farrowing (fig. b) . notably, the levels of s-specific iga antibodies remained elevated in milk up to day post-farrowing (fig. b) . iga antibody levels were higher in milk from animals in g when compared to animals in g after day post-farrowing (fig. b) . no spike-specific igg or iga antibodies were detected in colostrum/milk collected from control sows (g ) ( fig. a and b ). these results demonstrate the ability of orfv-pedv-s to induce s-specific igg and iga antibodies in colostrum and milk. passive transfer of antibodies from immunized sows to their offspring was assessed by elisa and ffn assays performed on serum samples collected from piglets pre-and post-ingestion of colostrum/milk. no s-specific antibodies were detected in serum samples collected on day one prior to ingestion of colostrum (fig. ) . notably, high levels of s-specific igg and iga antibodies were detected in the serum of piglets from g and g on day post-birth. piglets born to immunized sows had igg and iga antibodies in their serum until the end of the experiment on day post-birth (fig. ) , with higher levels of igg antibodies being detected until the end of the experiment. both igg and iga levels were higher in piglets born to sows in g when compared to piglets born to sows in g . no spike-specific igg or iga was detected in serum of piglets born to control sows (g ; fig. ). in addition to igg and iga, nas were detected in piglet serum on day post-birth. high levels of na were detected in piglets born to orfv-pedv-s-immunized sows until the end of the experiment on day pi (fig. c) . no neutralizing antibodies were detected in the piglets born to control sows (g , fig. c ). notably, a significant correlation between na titers in sow serum and piglet serum was observed (r = . , p = . ). additionally, a strong correlation between na and igg and iga antibody levels was observed in piglet serum (na vs. igg: r = . , p < . ; r = . ; fig. e and f). together, these results demonstrate passive transfer of pedv-specific igg, iga and nas from immunized sows to piglets through ingestion of colostrum and/or milk. all piglets born to immunized or control gilts were challenged orally with virulent pedv strain co on day post-birth. piglets were monitored daily for characteristic clinical signs of ped and mortality. daily average clinical scores were calculated for each group, and the mean daily scores are presented in fig. a . all piglets born to sows in g and g showed clinical signs of ped starting on day pc which lasted until day - pc. the highest clinical scores were observed in g piglets between days and pc. piglets in g started showing clinical signs on day pc, when moderate diarrhea was observed and continued until day pc. as shown in fig. a , more-severe clinical signs were observed in piglets from g (average daily scores ranging from to ), followed by g (average daily scores ranging from to ) and then g (average daily scores ranging from to . ). it is important to note that, while piglets in g and g experienced vomiting and diarrhea, none of the piglets in group experienced vomiting during experiment. virus shedding in feces was assessed by rt-qpcr using rectal swabs. piglets in g had a significantly lower level of viral rna in their feces (p < . ) in comparison to piglets in g and g on day pc (fig. b) . however, no significant differences in virus shedding in feces were observed between the three groups thereafter. the daily mortality recorded for piglets in the three groups is presented in fig. c . notably, twelve out of results are presented as group mean s/p ratios or na titers. the error bars represent +/-standard error of mean (sem). statistical significance was determined using two-way anova, and multiple comparisons were ran using tukey's test. a, b, and c represent statistical significance for g vs. g , g vs. g , and g vs. g , respectively. the significance level is < . . s/p, sample-to-positive ratio; the arrowhead represents the day of challenge with pedv. (d) correlation of piglet serum na levels with sow mean na levels. (e) correlation of group mean igg antibodies in piglet serum with piglet serum na levels. (f) correlation of group mean iga antibodies in piglet serum with piglet serum na levels. correlations were calculated using the spearman method with a % confidence interval using graphpad prism rt-qpcr in piglet feces expressed as log genome copy number per milliliter. data are presented as group means. error bars represent +/-sem. statistical significance was determined using two-way anova, and multiple comparisons were ran using tukey's test. a, b, and c represent statistical significance for g vs. g , g vs. g , and g vs. g , respectively. the significance level is < . . (c) survival curve demonstrating piglet mortality. the survival curve was gener-ated using the kaplan-meier method. statistical comparison between groups was performed using the log-rank test in graphpad prism . the p-value was adjusted using the bonferroni method. (d) correlation of piglet mortality with piglet group mean na levels. correlation of piglet mortality with group mean iga antibodies in sow colostrum/ milk. (f) correlation of piglet mortality with group mean igg antibodies in sow colostrum/milk. correlations were calculated using the spearman method with % confidence interval using graphpad prism ( / ; %) piglets in control g died by day pc, while only one piglet ( / ; %) in g died on day pc, and none of the piglets ( / ; %) in g died after the pedv challenge. interestingly, when the correlation between antibody levels in piglets and sows was compared to survival rates in piglets, moderate to strong correlations between na levels in piglet serum (r = . , p = . ) and iga and igg antibody levels in the sow colostrum/milk (r = . , p < . ; r = . , p = . , respectively) were observed (fig. d, e and f) . together, these results demonstrate decreased morbidity and mortality in piglets born to immunized gilts and a strong correlation between survival and antibody levels in colostrum and milk. in the present study, we assessed the ability of orfv-pedv-s to induce passive immunity against pedv following immunization of pregnant gilts. one of the goals of the study was to assess whether immunization with orfv-pedv-s alone would be sufficient or if live exposure to pedv would be required for protection of piglets born to immunized gilts. this underlies our experimental design, in which pregnant gilts were either immunized with orfv-pedv-s (g ) or with orfv-pedv-s followed by oral exposure to live pedv (g ). similar to our findings in -week-old weaned pigs [ ] , im immunization of gilts with orfv-pedv-s elicited pedv-specific igg, iga and nas. as expected, the booster provided by live pedv exposure in gilts from g led to higher antibody responses in serum, colostrum and milk of these animals. notably, passive transfer of antibodies from gilts to piglets was observed in both g and g , as pedv-specific igg, iga and nas were detected in serum of piglets born to immunized gilts following ingestion of colostrum and milk. na antibodies detected in piglet serum showed a strong correlation with igg and iga levels ( fig. e and f). although the antibody responses elicited by immunization with orfv-pedv-s alone were lower in comparison with those induced by immunization with orfv-pedv-s followed by live pedv exposure, the levels of antibodies induced by im immunization with orfv-pedv-s were sufficient to reduce neonatal mortality after oral challenge with pedv. antibodies present in sow colostrum and milk are derived either from serum or produced locally in the mammary tissue [ ] , and transfer of pathogen-specific antibodies from the sow to the piglet via colostrum and milk is critical for protection against the pathogens during the neonatal phase of the piglet's life. in this context, iga plays a key role in protection against enteric pathogens such as pedv, mainly because it is stable to proteolytic degradation in the intestine. by remaining in the gut lumen, pathogen-specific iga can effectively inhibit/ decrease virus infection/replication in the gut epithelium [ , [ ] [ ] [ ] . the results of this study show that increasing levels of iga in milk of immunized animals paralleled lower disease morbidity and mortality in piglets born to immunized gilts after challenge with pedv. while severe ped and high mortality ( %) were observed in piglets born to control gilts in g (no antibody responses detected), piglets born to gilts in g and g (high levels of iga in g and even higher in g ) showed less-severe clinical signs and reduced mortality rates ( and %, respectively). these results suggest that pedv-specific iga detected in piglets in g and g may have contributed to reducing disease severity and mortality in piglets. the possibility that other antibody isotypes or t cell-mediated immunity may have played a role in protection, however, cannot be formally excluded. one of the most important observations of our study was the reduced mortality in piglets from g ( % versus % in the control group, g ), which were born to gilts immunized im with orfv-pedv-s. notably, the lower piglet mortality in this group paralleled s-specific igg, iga and na responses detected in the gilts and passively transferred to piglets. together, these observations show that parenteral immunization with orfv-pedv-s is sufficient to induce protective levels of passive immunity against pedv. these results corroborate the findings of previous studies in which pedv specific-antibodies have been detected in serum and colostrum/milk and passively transferred to piglets after parenteral immunization with live, inactivated and/or subunit vaccine candidates [ ] . although the mechanism(s) underlying this phenomenon remain unknown, it is possible that systemic antibodies could be transferred from the sow's serum to colostrum and milk and then to the piglets. alternatively, the immunomodulatory properties of the orfv vector used here could potentially lead to migration of antibody-secreting plasma cells to the mammary gland, resulting in local antibody production. future studies assessing local immune cells in the mammary gland and antibody isotypes in the intestinal lumen of piglets born to immunized sows will be critical for dissecting the precise mechanism(s) underlying the protective immune responses elicited by im immunization with orfv-pedv-s observed here. in summary, this study demonstrates the ability of orfv-pedv-s to confer passive immunity against pedv following immunization of pregnant gilts. similar to our previous findings in -week-old weaned pigs [ ] , im immunization of gilts with orfv-pedv-s elicited pedv-specific igg, iga and nas responses. additionally, passive transfer of antibodies from gilts to piglets was observed, as pedv-specific igg, iga and nas were detected in serum of piglets born to immunized gilts following ingestion of colostrum and milk. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines porcine epidemic diarrhoea: new insights into an old disease. porcine health manag pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts virulence of current german pedv strains in suckling pigs and investigation of protective effects of maternally derived antibodies 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 classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus porcine epidemic diarrhea: a review of current epidemiology and available vaccines vaccines for porcine epidemic diarrhea virus and other swine coronaviruses immunogenicity of a recombinant parapoxvirus expressing the spike protein of porcine epidemic diarrhea virus experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain 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 the transfer of immunoglobulins igg, iga and igm from serum to colostrum and milk in the sow mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? strategies for design and application of enteric viral vaccines evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study we thank the staff of the sdsu animal resource wing (arw) for excellent care and handling of animals and for their invaluable help with the animal experiments. funding this study was funded by the national pork board grant no. - and in part by the national institute of food and agriculture hatch project no. sd - . the authors declare that there is a patent pending related to this work (us patent pending, p us ).ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. all animal experiments were reviewed and approved by the sdsu iacuc (approval no. - a). this article does not contain any studies with human participants performed by any of the authors. key: cord- -vnz x authors: chen, h. h.; stark, c. j.; atreya, c. d. title: the rubella virus nonstructural protease recognizes itself via an internal sequence present upstream of the cleavage site for trans-activity date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vnz x the substrate requirement for rubella virus protease trans-activity is unknown. here, we analyzed the cleavability of rv p -derived substrates varying in their n-terminal lengths ( – amino acids) from the cleavage site by the rv protease trans-activity. only substrates with at least amino acid residues n-terminal to the cleavage site were able to undergo cleavage. further, rubella sequence was found to be necessary in the n-terminal region of the substrate, whereas a heterologous sequence c-terminal to the cleavage site was tolerated. these results demonstrated a requirement for residues located between amino acids – of the rv p polyprotein, besides its cleavage site for rv protease trans-activity. this region overlaps with the starting site of the essential cis-protease activity of rv p polyprotein. this is a novel observation for a viral protease of the family togaviridae. a number of positive-stranded rna viruses employ viral nonstructural polyprotein processing as a strategy for genome expression by encoding their own (viral) proteases [ , , ] . thus, in order to achieve the expression of multiple proteins from a single message that are essential for regulation of viral replication and biogenesis, proteolysis of the viral polyprotein precursor is an essential event in most of the positive-strand rna viruses [ , ] . this is in contrast to the mrnas of their eukaryotic host cells, which mostly code for single proteins [ ] . in alphaviruses and rubella virus, members of the family togaviridae, two polyproteins are expressed: the nonstructural polyprotein, directly expressed from the genomic rna, and another polyprotein, expressed from a sub-genomic mrna synthesized during the viral infection [ ] . rubella virus is an enveloped virus of the genus rubivirus in the family togaviridae [ ] . the virion has a s ( nucleotide long) single-stranded, positive-sense, polyadenylated rna genome, which serves as the mrna following a series of events that lead to the uncoating of the viral particle upon entry into cells [ , , ] . in an infected cell, rv mrna first translates into a p nonstructural polypeptide [ ] . newly synthesized p often undergoes ciscleavage at nh -srgg / g-cooh into two mature nonstructural proteins, p and p , by a protease domain contained within the c-terminal region of p [ , , , ] . the viral p nonstructural polyprotein contains four conserved functional domains that are involved in viral rna synthesis and replication. they are sequentially located from the n terminus to the c terminus, as methyltransferase, protease, helicase, and rna-dependent rna polymerase domains based on bioinformatic analysis [ , ] . the first two motifs are located on the n and c termini of p , and the latter two are on the n and c termini of p [ , ] . rubella virus p -protease was identified to be a papain-like-cysteine protease [ ] . it utilizes divalent cations and also demonstrates trans-cleavage activity on homologous substrates in vitro [ ] as well as in vivo [ ] . further characterization of rv protease revealed that it exhibits zinc-binding activity as integral to its protease activity, thus suggesting that it is a novel viral metalloprotease [ , ] . within the c-terminal half of p , cys- and his- are the catalytic sites for this protease [ , ] . domains of rv protease required for cis-and trans-activity are mapped [ , ] . these studies have thus far focused on defining the catalytic sites, active domains, and cis-and trans-activity requirements of rv protease [ ] [ ] [ ] [ ] ] . experimental evidence suggests that p is actively involved in the synthesis of viral negative-strand rna, and its cleavage into p and p has been suggested to switch the complex to initiate positive rna synthesis [ ] . this leads to the logical hypothesis that, in the replication complex, those p molecules engaged in negative-strand rna synthesis must remain as p (i.e., lose their cis-protease activity) as long as they are required to participate in negativestrand rna synthesis. subsequently, the p -protease trans-activity cleaves these p molecules to signal the replication complex to switch to positivestrand rna synthesis mode. however, besides the presence of the cleavage site, what the internal sequence requirements of the p and its intermediaries for rv protease trans-cleavage activity are, and how the cis-activity of p is regulated to maintain optimal negative rna synthesis have not been identified. in this report, we analyzed the substrate features that are required of the rv protease trans-activity by using a truncated version of rv p (amino acid residues - ) that was previously shown to function as protease but had lost its cis-activity due to a substitution, g s, in the cleavage site, and a series of p -derived substrates that lack protease activity due to an amino acid substitution, c s, in the catalytic dyad [ ] . our analysis identified a region in the substrate corresponding to residues - of the p polyprotein that is required for the protease trans-activity, and this region overlaps with the starting site of the essential cis-protease activity of rv p polyprotein. potential implications of the requirement of the cis-activity region of p substrates for the rv protease trans-activity are discussed. plasmids capable of expressing functional rv protease and p -derived substrates in mammalian cells were constructed as follows: appropriate cdna segments representing various rv genomic regions were pcr-amplified and inserted into a mammalian expression plasmid, pcdna -his/max-c, driven by a cmv promoter. this plasmid also contains an in-frame xpress epitope tag for easy identification of the expressed protein by anti-xpress monoclonal antibody in immunobloting analysis (invitrogen inc, ca). two independent dna preparations for each plasmid were sequenced to ensure authenticity of the plasmids used in the study. rv trans-cleavage active protease-encoding cdna was amplified by pcr with a pair of primers (table ) , using a previously well-established infectious rv cdna template, pbrm -g s, which contains an active protease domain (catalytic site, c ) but lacks the cis-cleavage capability due to a substitution at residue g within the cleavage site (nh -srgg/g-cooh) to s (nh -srgs/g-cooh) [ ] . the amplified dna product was cloned at the bamhi and ecori sites in the expression plasmid described above. in this construct, in-frame with the protease sequence c-terminus, a green fluorescent protein (gfp) orf was also inserted, so that the expressed protease would have a higher molecular weight to distinguish it from its substrate in immunoblot analysis. this expression plasmid was designated as prvp (fig. a) . to evaluate the trans-cleavage activity of rv protease on p -derived substrates, a series of rv p -related polypeptide expression plasmids that express protein with different n-terminal lengths from the cleavage site ( , , , and residues) were constructed. this was achieved by using a previously well-established rv cdna template, pbrm -c s. the template contains rv cdna with the cleavage site (n -srgg/ g-c ) unmodified but lacks the protease activity due to a substitution at the catalytic activity residue (c s) of the enzyme [ ] . desired dna segments of pbrm -c s template with the same c-terminus (amino acid position ) but varying in length at the n-terminal end were amplified using appropriate sets of primers (table ) and cloned at the bamhi and ecori sites of the plasmid vector described above. the plasmids were designated, prvs- - , prvs- - , prvs- - , and prvs- - , producing proteins with -, -, -, and -residue n-terminal lengths from the cleavage site, respectively (fig. b) . to test whether replacement of p -related sequences c-terminal to the cleavage site by non-rubella sequences affect rv protease trans-activity, the plasmid prvs-gfp, which contains pcr-amplified rv protease substrate sequences representing polypeptide residues - (residues - represent the c-terminal side of the cleavage site), fused at its c-terminus to gfp orf in-frame, was created (fig. b) . similarly, to test whether a heterologous sequence on the n-terminal side of the cleavage site affects substrate recognition by the protease trans-activity, gfp orf was fused in-frame at the n-terminus of the rubella sequence in the plasmid prvs- - to create prvs-gfp- - (fig. b) . the template for the pcr in both cases was pbrm -c s. (a) shows pbrm -g s-derived p that was used as a pcr-template to clone the trans-active protease region. the p and p domains of p are indicated. the protease catalytic residue, cys at amino acid (c ) and a cleavage site substitution mutation (gly to ser) at p c-terminal residue, (g s, represented by 'x' in the p box and by a black circle on the top) are as shown. the mutation eliminates the cis-activity of the protease, leaving its trans-activity intact [ ] . numbers ( - ) on the side represent amino acid residues of p . the trans-active protease domain spans from amino acid position to [ ] . expression plasmid, prvp contains this protease domain in-frame flanked by an n-terminal xpress epitope tag (hatched box) and a c-terminal gfp orf. (b) shows pbrm -c s-derived p used as a pcr-template to produce a series of deletion substrates. the p and p domains on p were as shown. to serve as homologous substrates, the protease activity was eliminated due to a substitution at the catalytic residue, cys- to a ser [ ] (c s, represented by 'x' and by an open circle on the top) but with an intact cleavage site at the p c-terminal residue, (g ) as shown. expression plasmids containing rv p -related cdna prvs- - , prvs- - , prvs- - , and prvs- - (numbers denote position of amino acid residues within the p polyprotein representing the n and c termini of the substrate) used in the study were as illustrated. these plasmids express proteins with varying n-terminal lengths from the cleavage site ( , , and aa respectively as shown in parentheses under each construct box). cleavage in the substrate occurs at amino acid residue g . in prvs-gfp, the rv cdna insert was amplified from position - ( residues upstream of the cleavage site) and a gfp-orf was fused at its c-terminus, whereas in prvs-gfp- - , the gfp-orf was fused at the n-terminus of the amino acid residue-substrate. all plasmids shown here contain an in-frame n-terminal xpress epitope tag (hatched box) for immunoblot identification of the proteins rv-protease, and substrate plasmids were expressed in human embryonic kidney (hek) t cells (atcc, va) by transient transfection using lipofectamine- reagent as per supplier's protocol (invitrogen, ca). following a h incubation of cells at • c in a co ( %) incubator, cell lysates were prepared and subjected to polyacrylamide gel electrophoresis (either % or, - % gradient) with . % sodium dodecyl sulphate, followed by immunoblot analysis as described [ ] . substrates as well as the protease all had an xpress epitope tag at their n-terminus to detect the n-terminal side of the cleavage products. we evaluated the protease trans-cleavage activity on a series of rv p -derived polypeptides in which the protease catalytic site at amino acid position was mutated (c s) to serve as substrate (i.e. to lack protease activity, but retain the cleavage site). hek t cells were co-transfected with prvp (protease, fig. a) , and each of the substrate expression plasmids, prvs- - , prvs- - , prvs- - , and prvs- - . each substrate plasmid expressed rv p -derived polypeptides of different n-terminal lengths ( - residues) from the cleavage site (fig. b) . following transfection, cell lysates were prepared, and the proteins were subjected to immunoblot analysis. the substrates as well as the protease all had an xpress epitope tag at their n-terminus. our analysis of rv protease substrates revealed that homologous polypeptides of n-terminal -and -residue length from the cleavage site, expressed from prvs- - and prvs- - (fig. , lanes and , and kda, respectively), were able to undergo cleavage when co-expressed along with rv protease (fig. , lanes , , and , kda) with an expected size of the n-terminal cleaved products of -and -kda size (with additional xpress epitope residues), respectively (fig. , lanes and ) . however, sub- shown are longer exposures of the same blot to film. note that substrates generated from prvs- - and prvs- - were cleaved by the protease (">" identifies the cleaved products of and residues respectively in and ), whereas, -and -residue (from the cleavage site) representing substrates expressed from prvs- - and prvs- - were not cleaved by the protease ( and ). also note that the protease was able to cleave the rvs-gfp substrate with a gfp on the c-terminal side of the cleavage site to a lesser extent (< %), as a significant portion of the substrate remained uncleaved strates of n-terminal -and -residue length from the cleavage site (plus the xpress epitope residues), expressed from prvs- - and prvs- - , respectively (fig. , lanes, and , -and -kda), did not undergo cleavage when co-expressed along with the protease (fig. , lanes and ) as evidenced by the lack of xpress-tagged n-terminal cleavage products detected in the immunoblot analysis. (if the substrates were cleaved, the expected sizes of the n-terminal products would be kda and kda, respectively.) this clearly suggests that amino acid residues residing between residues - in the substrate are required in order for the substrate to be targeted by the trans-activity of the protease. we extended our analysis to identify any homologous sequence requirement of the substrate on the c-terminal side of the cleavage site. to address this, we utilized the plasmid prvs-gfp, which expresses an rv protease homologous substrate representing amino acids - (residues - represent the c-terminal side of the cleavage site), fused at its c-terminus to a gfp orf in-frame (fig. b) . when this plasmid was co-transfected along with rv protease plasmid (prvp) into hek t cells, we observed that the substrate (fig. , lane , -kda protein) was cleaved by the protease at the cleavage site, based on the expected molecular weight of the cleaved n-terminal product of kda (fig. , lane ) . this clearly suggests that the protease was able to cleave the rvs-gfp substrate with a gfp on the c-terminal side of the cleavage site, but to a lesser extent (< %), as a significant portion of the substrate was reproducibly observed to be the fact that -substrate was cleaved and -substrate was not suggested that trans-protease processing of the substrate either requires a minimum n-terminal length from the cleavage site in the substrate, or it recognizes an internal amino acid sequence within the -substrate that is lacking in the -substrate. if indeed an internal domain within the -substrate is being recognized by the protease, then this sequence should be present in the n-terminal region of -substrate. to verify the above hypothesis, we utilized plasmid prvs-gfp- - (fig. ) , which expresses a chimeric substrate in which the -substrate was extended at its n-terminus by gfp to compensate for the length (increased from to amino acids) along with the protease plasmid, prvp. immunoblot analysis (fig. ) demonstrated that when the protease (lane ) and a positive control substrate (lane ) were coexpressed, the substrate did undergo cleavage (lane ). although the n-terminal length was increased in the chimeric gfp- -substrate (lane ), it still failed to undergo cleavage when coexpressed with the protease (lane ), suggesting that it is not the n-terminal length from the cleavage site that is required of the substrate, but in fact it is the internal sequence present within the substrate that is recognized by the protease for trans-processing. the internal recognition domain identified in this report corresponds to amino acid [ ] . in the bottom panel, the protease substrate p is depicted with the location of the newly identified domain required for protease trans-activity (this report), which overlaps with the protease cis-activity starting site of p position - of rv p . this region overlaps with the n-terminal site of the essential cis-protease activity region on the p described by liang et al. [ ] . the location of the substrate recognition region (this report) relative to the essential cis-activity domain is illustrated in fig. . at this time we could not refine the recognition region further, as plasmids with deletions in this region of rv cdna could not be rescued in bacteria. in this report, we defined the minimal substrate (template) for proteolytic processing in trans by the rubella virus nonstructural protease using an epitopetagged protein expression system. results presented with rv protease (which has been functionally demonstrated to be similar to p activity [ ] ) demonstrated that the protease trans-activity recognizes a region n-terminal to the cleavage site in the p -derived substrates. thus, the trans-protease activity requires not only specific amino acids present at or proximal to the cleavage site, but also sequences upstream at a distance from the cleavage site. we also demonstrated that, on the c-terminal side of the cleavage site, heterologous residues unrelated to rv are somewhat tolerated by the protease, but we routinely observed (in three separate experiments) that the efficiency of the protease trans-activity on this type of substrate was less than % (illustrated in fig. , lane ) . this clearly suggests that the p domain residing on the c-terminal side of the cleavage site in p does influence the trans-activity of the protease. in another positivestranded rna virus, mouse hepatitis virus (mhv), one of the two virus-encoded proteases, plp- (pcp), demonstrates a homologous substrate length requirement on the c-terminal side of the cleavage site and the cleavage efficiency increases with increasing substrate and enzyme polypeptide length, although in this case, the protease recognition sequence on the substrate was not identified [ ] . the rv protease trans-activity-associated p internal sequence requirement, as identified here, is unique, and this is the first such report for the viruses that belong to the family togaviridae. in this study, we mapped the regions of the protease substrate required for trans-activity, which is reciprocal to the liang et al. study wherein they mapped the essential cis-and trans-activity regions of the protease itself and also demonstrated that the x domain (proline-rich, conserved in all m-group pcps) present in the rv protease is important for the protease trans-activity [ ] . they further speculated that this proline-rich x domain could serve as a protein-protein interaction domain that enhances the opportunity to meet its trans-cleavage substrate [ ] . however, in this report, we clearly demonstrate by deletion analysis that the x domain in the substrate is dispensable for recognition by rv protease trans-activity and the actual recognition region is located downstream of the x domain, overlapping with the n-terminal starting-site (a term coined by liang et al. [ ] ) of the essential cis-activity domain of the substrate. our results show that rv p -associated protease trans-activity requires a specific region within the p that represents p itself (illustrated in fig. ). taken together, our studies and those of liang et al. [ ] advance the field in enhancing our understanding of the molecular determinants that define the rubivirus protease trans-activity requirements that are essential for rv replication. in this report, we have shown that rv protease trans-activity demonstrates substrate specificity by requiring an internal sequence within the region that is n-terminal to the cleavage site. identification of a region in the p -related sequence-containing substrate (we utilized polypeptides from amino acid positions to of p or shorter) that is important for rv protease transactivity suggests that this region may offer a specific fold or a conformation to the p substrate to facilitate cleavage by the protease. since most protease-substrate interactions involve transient binding of the protease to its cognate substrate, it is conceivable that rv protease trans-activity on homologous substrates also involves transient binding of protease to the substrate, and such binding perhaps could occur within the sequence that is required on the substrate for protease transactivity. we attempted to perform coimmunoprecipitation experiments to establish the protease-substrate binding following cotransfection of cells with both plasmid constructs, but failed to obtain reproducible results, perhaps due to the transient nature of the interaction. however, if this binding truly occurs in the viral infection cycle, then, as rv protease is recognizing itself as substrate in the essential cis-activity region for the trans-activity (illustrated in fig. ) , it is tempting to speculate that the trans-activity may be regulating the cis-activity of p . this process could explain how p remains as p in the replication complex to initiate viral negative-strand rna synthesis. as discussed in the introduction, previous experimental evidence suggested that p initiates the synthesis of viral negative-strand rna, and its cleavage into p and p plays a critical role in switching the replication complex to the positive rna synthesis mode [ ] . in this context, our results leads us to speculate that perhaps the p -protease transactivity requirement of the p sequence within its essential cis-activity region (illustrated in fig. ) could transiently stall the p cis-protease activity, provided that p binds to p in this region, to allow the negative-strand rna synthesis to occur. this certainly is a verifiable future experimental direction to capture viral events that further enhance our understanding of the rubivirus proteases. role of calreticulin in rubella virus replication rubella virus characterization of the rubella virus nonstructural protease domain and its cleavage site molecular biology of rubella virus putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences viral proteinases rubella virus nonstructural protein protease domains involved in trans-and cis-cleavage activities the rubella virus nonstructural protease requires divalent cations for activity and functions in trans characterization of the zinc binding activity of the rubella virus nonstructural protease expression of the rubella virus nonstructural protein orf and demonstration of proteolytic processing the n-terminal conserved domain of rubella virus capsid interacts with the c-terminal region of cellular p and overexpression of p enhances the viral infectivity conservation of the putative methyltransferase domain: a hallmark of the 'sindbis-like' supergroup of positive-strand rna viruses virus-encoded proteinases of the togaviridae expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides site-specific protease activity of the carboxyl-terminal domain of semliki forest virus replicase protein nsp rescue of rubella virus replication-defective mutants using vaccinia virus recombinant expressing rubella virus nonstructural proteins proteolytic processing of rubella virus nonstructural proteins we thank dr. shirley gillam for providing m plasmid constructs pbrm -c s and pbrm -g s that were used in the study as pcr templates to generate the protease and substrate clones. we thank drs. kvk mohan and ye, cber for helpful critiques. hhc and cjs are supported by orise postdoctoral fellowships. key: cord- -cdvnqfjz authors: castilla, v.; mersich, s. e.; candurra, n. a.; damonte, e. b. title: the entry of junin virus into vero cells date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: cdvnqfjz the entry mechanism of junin virus (jv) into vero cells was studied analyzing the effect of lysosomotropic compounds and acid ph on jv infection. ammonium chloride, amantadine, chlorpheniramine and procaine inhibited jv production. the action of ammonium chloride was exerted at early times of infection. virus internalization was inhibited and viral protein expression was not detected. when the extracellular medium was buffered at low ph, the ammonium chloride induced block on jv infection was overcome. furthermore, jv was able to induce fusion of infected cells at ph . leading to polykaryoctye formation. taken together, these results demonstrate that jv entry occurs through an endocytic mechanism requiring a low ph dependent membrane fusion. junin virus (jv) is a member of the arenaviridae family of enveloped rna viruses. virions contain a genome composed of two segments of single-stranded rna and two major structural proteins, the nucleocapsid protein (np, mw - kda) and an external glycoprotein (gp , mw kda) [ ] . other minor proteins have also been described in purified virions or in infected cells [ , , ] . the main biological properties of jv are its ability to establish persistently infected cultures in-vitro and to produce chronic infections in the field mouse calomys musculinus [ ] . in humans, jv induces argentine haemorrhagic fever, an endemoepidemic disease with hematologic and neurologic signs, which mainly affects male rural workers [- ] . although some features of arenavirus replication have been described, the early events of the multiplication cycle remain poorly characterized. main interest has been focussed on the knowledge of viral rna transcription and replication, the characterization of the ambisense genome strategy and the study of protein structure and expression [- , ] . little is known about the process of virus attachment and entry into the cell. a brief report has recently suggested that the replication of the arenaviruses pichinde, mopeia and lassa was sensitive to some lysosomotropic compounds [ ] . two distinct pathways operate for the entry of enveloped viruses into animal cells: some viruses penetrate by direct fusion of the viral envelope with the plasma membrane while other viruses are taken up by an endocytic mechanism [ ] . the endocytosed virions travel to intracellular compartments where acidic conditions facilitate fusion between the viral envelope and the vesicle membrane releasing the nucleocapsid into the cytoplasm. thus, lysosomotropic agents such as weak bases and carboxylic ionophores that elevate the ph of acidic organelles, interfere with the virion uncoating and inhibit the replication [ , , ] . the uncharged form of any weak base has been proposed to enter the acidic intracellular compartments very efficiently, raising the ph as it becomes protonated and inhibiting hydrolytic enzymes [ ] . in this paper, we report studies on jv penetration into vero cells analyzing the effect of acidic ph and lysosomotropic bases on virus entry. monolayers of vero cells were grown in minimum essential medium (mem) supplemented with % calf serum and gentamycin. the iv strain of jv [ ] was used. stock solutions of amantadine (specia), ammonium chloride (fisher company), chlorpheniramine (schering-plough) and procaine (schering-plough) at concentrations of , , and mm, respectively, were prepared in culture medium and sterilized by filtration. ammonium chloride ( mm) was added to vero cells h before infection with jv or at , t, , or h post-infection (p.i.). in all cases supernatant cultures were harvested at h p.i. and extracellular virus yields were determined by plaque assay. vero cells were infected with jv (virus inoculum: × pfu) in the presence or in the absence of mm ammonium chloride. after adsorption for , , , , and rain at °c, infected cells were washed twice with cold phosphate buffer saline (pbs) and disrupted by freezing and thawing. the amount of infectious bound virus was then determined by plaque assay. cultures of vero cells were infected with jv at a moi of . after rain adsorption at °c two washes were done to remove excess inoculum and then cultures were warmed to °c for various times in the presence or absence of mm ammonium chloride. cultures were subsequently treated with proteinase k ( . mg/ml) in pbs for min at °c in order to remove external virus. protease treatment was stopped by the addition of mm phenyl methyl sulfonyl fluoride (pmsf), ~ bovine serum albumin (bsa) in pbs. cells were centrifuged min at g and the resultant pellet was washed and resuspended in mem. internalized virus was measured by an infectious center assay on vero cells. vero cells grown in coverslips were infected with jv (moi ) and mm ammonium chloride was added to the culture medium after adsorption. at h p.i. supernatants were removed. monolayers were washed with cold pbs, fixed in methanol and stained for cytoplasmic immunofluorescence with anti-jv purified immunoglobulins as previously described [ ] . veto cell monolayers in -wetl plates were adsorbed for h at °c with jv in mem containing . ~/o bsa, mm hepes, ph . . in a set of cultures cells were washed twice with pbs and then warmed to °c by the addition of pre-warmed medium buffered with mm hepes and mm - -piperazinediethene-sulfonic acid (pipes) at ph . , . , . , . , . and . , in the presence or in the absence of mm ammonium chloride. after h of incubation at °c, cells were washed twice with pbs and mem containing . ~ methylcellulose, ph . , was added. in other set of cultures, after adsorption cells were washed with pbs and incubated at °c for rain in mem buffered at different ph as indicated above. then, cells were washed with pbs and mem at ph . containing or not ram ammonium chloride was added. after h of incubation cultures were overlaid with methylcetlulose as above. in both set of cultures plaques were counted after days of incubation and percent of inhibition was calculated. vero cells were infected with jv at a moi of . at h after infection, medium was removed and cells were incubated in mem buffered at ph . or . as indicated above. then, monolayers were stained with giemsa and syncytia containing more than nuclei were counted. to determine the time at which lysosomotropic agents caused their inhibitory action, we next examined the effect of the time of addition of mm ammonium chloride on extracellular virus yields. a nearly complete inhibition of viral multiplication was observed when the compound was added one hour before or simultaneously with virus infection and maintained during all the period of incubation (fig. ) . when time of drug addition was delayed, virus production progressively increased, indicating that ammonium chloride inhibited an early step of the replicative cycle. in fact, no significant differences were found when the drug was added between and h p.i. since maximum inhibition in jv multiplication was obtained when the drug was present during the first hour after infection, we examined its effect on the early stages of replication. as shown in table , ammonium chloride had no effect on jv adsorption since the titers of virus adsorbed at °c in treated and untreated cells did not differ significantly. in both cases, virus adsorption occurred rapidly and the percentage of adsorbed virus was approximately %. to investigate whether ammonium chloride affects jv internalization, virus adsorbed cells at °c were warmed to ~c for various intervals in presence or absence of the compound and internalized virus was determined by infectious center assay. the amount of jv internalized virus in untreated cells sharply increased after min of incubation at °c and the uptake was apparently complete after . h (fig. ) . in the presence of ammonium chloride there was no virus internalization and a progressive decrease is observed in the number of infectious centers. the relatively small difference between the control and treated cells at the min time point might reflect the fact that part of the adsorbed virus was not removed by the proteinase k treatment. the action of weak bases on jv replication was confirmed by measuring viral protein production in infected cells by an immunofluorescence assay. the inhibition in the number of fluorescent cells in the presence of mm ammonium chloride was . % (fig. ) . the other lysosomotropic compounds also inhibited viral antigen expression (data not shown). in an effort to demonstrate that the entry of jv into vero cells is an acid ph-dependent process, we have finally studied the effect of different ph values on the internalization of jv particles that have been prebound to vero cells. in the absence of ammonium chloride, infectivity of jv was not affected at the ph range assayed ( . - . ) (data not shown). a high degree of inhibition was observed when cultures were incubated in medium containing ammonium chloride at ph . , . and . during h, while there was only . and . % inhibition at ph .t and . , respectively (fig. ) . at more acidic ph values the action of ammonium chloride was almost totally abolished. to assess that the acid ph is modifying the mechanism of viral entry and is not just neutralizing the ammonium chloride, jv bound cells were briefly treated with medium buffered at different ph for min and then incubated for h in neutral medium containing or not ammonium chloride. this treatment prevents endocytosis by inhibiting release into the cytoplasm of virus entered in endocytic vacuoles and allows virus entry only by fusion at the plasma membrane [ ] . under these experimental conditions, ammonium chloride inhibition was partially reversed at ph . (fig. ). to reinforce that the membrane fusion activity of junin virus is expressed at low ph, the formation of jv-induced syncytia in infected vero cells incubated in medium at ph . or . was quantitated. cell fusion was observed at h p.i. but only at ph . , there being more than nuclei in the syncytia whereas no polykaryon formation was seen at neutral ph (fig. ). these studies present evidence that junin virus entry occurs via a ph dependent endocytic mechanism mediated by a glycoprotein fusogenic activity. acidotropic bases are known to disrupt endosomal functions when their uncharged forms enter endosomes and lysosomes, become protonated, raise the ph and inhibit the hydrolytic enzymes [ ] . we have demonstrated here (figs. , ) . the main action of a m m o n i u m chloride on jv replication was exerted at an early step in the multiplication cycle (fig. ) . however, virus attachment which occurred at °c was not inhibited as it is shown in table . virions b o u n d to cells at °c are compelled to internalize when the temperature of incubation is raised to °c. when jv internalization, the next stage of the viral cycle, was studied a significant inhibition was observed during the first v. castilla et al. hour after adsorption. in the presence of ammonium chloride an increasing reduction in the number of proteinase k-resistant infectious centers was detected (fig. .) it might be due to the lysosomal degradation of endocytosed virions unable to fuse with endosomal membranes, as seen for other enveloped viruses. these data were indicative of a process of low ph-dependent endosomal fusion for jv uptake. in fact, a bypass of the ammonium chloride block of jv infection was achieved when the extracellular medium was at a ph below . (fig. ) , suggesting that the acidic conditions would probably trigger direct fusion of virus envelope with the cell membrane. the location of penetration is determined by the ph threshold of fusion activity [ ] . for some viruses such as semliki forest virus, fusion occurs at a ph . in early endosomes, whereas influenza virus x- requires ph . and fuses in the late endosomes [ , , ] . for jv entry, fusion seems to occur in endosomes where the ph is . or lower. the acid environment may be responsible for structural changes in jv external proteins allowing membrane fusion and facilitating viral uncoating as described for influenza virus [ ], rubella virus [ ] , west nile virus [ ] and tick-borne encephalitis virus [ ] . it is possible to induce fusion in some model systems by mimicking the low ph intracellular conditions. in particular, mann et al. [ - have shown that sindbis virus infected cells express a fusion function after treatment at acid ph. we demonstrated that junin virus can mediate cell fusion at ph . producing polykaryocytes in which over % of the cells in the monolayer participate (fig. ) . thus, this result offers indirect support for the conclusion that the route of jv entry is ph-dependent in vero cells. major expression of gp , the main external envelope glycoprotein [ ] , was observed in the surface of jv infected cells at h p.i., by immunofluorescence assay (data not shown). thus, gp might be responsible for jv-induced membrane fusion in the endosome or in the cell surface. this proposal is also supported by results obtained with c , a host-range mutant of jv, with an alteration in gp detected by peptide mapping and a blockade in adsorption-penetration pathway [ , ] . in conclusion, our data demonstrate for the first time that jv enters the cell by a receptor mediated endocytic pathway and that low ph is neccesary for viral internalization through a fusing activity. further experiments are in progress to determine the precise role of gp in virus entry and the nature of the conformational changes at low ph leading to membrane fusion. the entry of african swine fever virus into vero cells arenavirus gene structure and organization protein structure and expression among arenaviruses antigenic relationships among attenuated and pathogenic strains of junin virus coto ce (t ) a comparison of junin virus strains growth characteristics, cytopathogenicity and viral polypeptides polypeptide synthesis in junin virus infected bhk- cells membrane fusion activity of the influenza virus hemagglutinin early events in arenavirus replication are sensitive to lysosomotropic compounds the uncoating and infection of the flavivirus west nile on interaction with cells: effects of ph and ammonium chloride junin virus structure epitope model of tick-borne encephalitis virus envelope glycoprotein e: analysis of structural properties, role of carbohydrate side chain and conformational changes occurring at acidic ph kinetics of endosome acidification detected by mutant and wild type semliki forest virus association between the ph-dependent conformational change of west-nile flavivirus e protein and virus-mediated membrane fusion attenuation of murine coronavirus infection by ammonium chloride virus entry into animal cells polykaryocyte formation mediated by sindbis virus glycoproteins ph-dependent solubility shift of rubella virus capsid proteins weak bases and ionophores rapidly and reversibly raise the ph of endocytic vesicles in cultured mouse fibroblast lectin affinity of junin virus glycoproteins genetic organization of junin virus, the etiological agent of argentine hemorrhagic fever reduced virulence ofa junin virus mutant is associated with restricted multiplication in murine cells damonte eb (t ) a mouse attenuated mutant of junin virus with an altered glycoprotein the entry of junin virus into vero cells the effects of oligosaccharide trimming inhibitors on glycoprotein expression and infectivity of junin virus helenius a (t ) protein mediated membrane fusion fusion of influenza virions in an intracellular acidic compartment measured by fluorescence dequenching membrane fusion process of semliki forest virus: low ph-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells argentine hemorrhagic fever we thank s. coronato for her technical assistance. this work was supported by grants from the consejo nacional de investigaciones cientificas y t cnicas (conicet) and universidad de buenos aires. e.b. damonte is member of the research career from conicet. key: cord- -kkkrkgg authors: belsy, acosta; odalys, valdés; alexander, piñón; clara, savón; angel, goyenechea; grehete, gonzalez; guelsys, gonzalez; luis, sarmiento; pedro, más; guadalupe, guzmán maría; alina, llop; pilar, perez breña ma; inmaculada, casas title: molecular characterization of adenoviral infections in cuba: report of an unusual association of species d adenoviruses with different clinical syndromes date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: kkkrkgg adenoviruses are common pathogens that are responsible for a wide variety of infectious syndromes. the objectives of this study were to identify and characterize members of different adenovirus species at the molecular level and to describe the correlation between viruses and clinical syndromes during a period of years. between and , of respiratory specimens ( %) from patients with acute respiratory tract infection tested positive for adenovirus. four adenovirus isolates from samples sent for enterovirus isolation were also analyzed. this research identified confirmed cases of human adenovirus infection by pcr and/or viral culture. the most common diagnosis was upper respiratory infection ( %). human adenovirus d was the major species found ( %), followed by human adenovirus c ( %) and human adenovirus b ( %). human adenovirus was the major serotype found producing bronchiolitis, followed by human adenovirus . in patients with upper respiratory infection, the major serotype found was human adenovirus . viruses of the species human adenovirus d were identified in seven ( %) cases of acute febrile syndrome. four isolates from clinical materials obtained from patients with encephalitis, acute flaccid paralysis and meningoencephalitis were identified as belonging to the species human adenovirus d. our data demonstrate a surprising result about the identification of an unusual association of viruses of the species human adenovirus d with different clinical syndromes. this observation could be evaluated as a possible indicator of the emergence of a novel strain but further studies are required. human adenoviruses (hadvs) are members of the family adenoviridae, whose members infect hosts across a broad spectrum of vertebrates. there are serotypes of hadv in the genus mastadenovirus, distributed in six species, a-f (formerly subgroups or subgenera) on the basis of their physicochemical, biological and genetic properties. a new serotype isolated recently ( ) has been proposed to represent a new species g [ ] . human adenoviruses are associated with sporadic infection, and community and institutional outbreaks. these viruses cause a variety of clinical manifestations, such as conjunctivitis, pneumonia, gastroenteritis, and hemorrhagic cystitis. some serotypes can occasionally infect tissues of the central nervous system (cns) and cause aseptic meningitis, meningoencephalitis, and encephalitis. they can cause especially severe disease in infants, young children, immunocompromised persons, and transplant recipients [ , , , , , , ] . serosurveys suggest that virtually all people are exposed to hadv during childhood [ , ] . they can remain in an asymptomatic carrier state until at least young adulthood [ ] , and virus may be actively shed long after symptomatic infection [ ] . over the past decades, neutralization tests, elisa, and virus isolation had been used for the detection and identification of adenovirus serotypes. however, these methods are relatively complicated, labour-intensive, and timeconsuming, and they have low sensitivity. [ , , ] . these disadvantages have limited their use. amplification of the viral genome by pcr has been introduced as a convenient and powerful alternative for molecular diagnosis. additionally, genome amplification allows further characterization of the adenovirus serotype by sequence analysis [ , , ] . the objectives of this study were the identification and molecular characterization of different hadv isolates and to describe the correlation between viruses and clinical syndromes during a period of years. between october and september , respiratory specimens (nasopharyngeal swabs and pharyngeal washes) from patients with acute respiratory tract infection (arti) were sent specifically to the national reference laboratory of respiratory viruses for testing respiratory viruses, including influenza virus a, b and c, human respiratory syncitial virus (hrsv), human parainfluenza virus - , hadv, human coronavirus, human rhinovirus and human metapneumovirus (hmpv). routine virological testing for respiratory pathogens was performed using a combination of direct immunofluorescence, isolation in cell culture and pcr assays [ , , ] . in addition, four samples (three stools and one cerebrospinal fluid) from the enterovirus (ev) laboratory with previous viral isolation without identification were analyzed. nasopharyngeal swabs and pharyngeal washes were collected in ml of virus transport medium (mem, gibco-brl, life technologies, paisley, scotland; penicillin u/ml, and streptomycin lg/ml, biowhittaker, ma; mycostatin u/ml, sigma; bovine serum albumin . %, merck, darmstadt, germany) the specimens were frozen and stored at - °c until the analysis was carried out. a human embryonic fibroblast cell line was used for primary isolation of ev. tubes with % confluent monolayers were inoculated with . ml of homogenized samples. cells were fed with ml of % fetal calf serum in basal medium eagle and visualized for cytopathic effect (cpe) twice a week. when a cpe that was not typical of ev was observed, the monolayer was scraped and tested for adenovirus antigen by immunofluorescence with a specific monoclonal antibody (chemicon, temecula, ca). total viral rna/dna from -ll aliquots from clinical samples or supernatant of infected cell culture was extracted using the guanidinium thiocyanate method as described previously by casas et al. [ ] . the lysis buffer included copies of the cloned, amplified product of the internal control described by coiras et al. [ ] . it was used for checking the extraction process, the amplification efficiency, and the presence of inhibitors in the clinical specimens. after processing, the dried pellet was resuspended in ll of rnase-free sterile water. negative controls consisting of rnase-free sterile water (sigma) were treated following the same procedure. for each assay, known positive controls, derived from infected viral cells, were added. in all cases, nucleic acid extracts ( ll) were examined for influenza virus a, b and c, hadv, hrsv a and b [ ] , human parainfluenza virus - , coronaviruses, rhinoviruses, and ev [ ] , using nrt-pcr. in addition, a multiplex npcr assay for human herpesvirus was used in the case of a patient with encephalitis [ ] . the protocols employed have been published previously and used for clinical diagnosis. three different biosafety cabinets were used for master mix preparation, sample handling and primary reaction product handling. different laboratory wear and coats were used for each cabinet. amplicon detection was done in a different room. specimens that were positive for hadv were processed by two independent nested reactions with pmol of adhex f, nt to ; ( cccittyaaccacc accg ) and adhex r, nt to ; ( katg gggtaragcatgtt ) or pmol of adhex f, nt to ; ( caacacctaygastacatgaa ) and adhex r, nt to ; ( acatccttbc kgaagttcca ) degenerate primers as described previously [ ] . these nested primer pairs were designed to bind inside the hexon protein coding region of the adenovirus genome [ ] . the pcr products were purified with qiaquick pcr purification kit (qiagen) according to the manufacturers's protocol and were sequenced in both directions using the primer pairs described above. sequences were obtained using an automatic dna sequencer (abi prism ; applied biosystems) and a big dye terminator cycle sequencing kit version . (applied biosystems). the fragment size sequenced corresponded to amino acids, from amino acid position - of the hexon protein. this fragment is located at the external surface l of the hexon protein monomer but does not overlap with the hrv domain [ ] . the nucleotide sequences obtained were first analysed using the chromas software (version . ). the forward and reverse sequences were combined using bioedit and were compared and aligned with the corresponding previously published sequences of prototype viruses available from genbank, using the clustal x (version . ) program. specimens that yielded identity scores c %, were considered to be good genotype matches. phylogenetic trees were inferred using programs from the mega package (version ) and reconstructed using the neighbourjoining method. the evolutionary distances were estimated using kimura's two-parameter method [ ] . the statistical significance of a particular tree topology was evaluated by , replicates of boostrap re-sampling. the criteria for serotype assignation were the shortest distance from a prototype strain, as deduced from the phylogenetic tree ( fig. ) , and the highest similarity value obtained after sequence comparisons. the genbank accession numbers of the nucleotide sequences presented in this study are: eu -eu , eu , eu -eu and eu -eu . in the present report, the nested pcr method used was able to detect different hadvs in clinical samples and supernatant culture with a sensitive internal control system to assure the quality of reaction conditions in each individual tube. in this investigation, confirmed cases of hadv infection were identified by pcr and/or viral culture ( table ) . the monthly collection of clinical samples is shown in table . human adenovirus was detected throughout the year; however, samples ( %) were collected between june and september. two significant outbreaks of hadv infection were identified, one in july of and the other in september of . more than half of the positive samples ( %) were from children younger than years of age, and % of them were under months of age. thirty-two percent were from patients between and years, and % were from adults between and years of age. no difference in gender distribution was observed. the most common diagnosis was upper respiratory infection ( %). medical records documented the following symptoms at initial presentation: nasal congestion ( %), sore throat ( %) and cough ( %). all patients were febrile. fourteen ( %) patients had bronchiolitis. acute febrile syndrome ( %) was defined as the concurrence of fever, cold symptoms, headache, weakness, vomiting and diarrhoea, a common but non-specific symptom. in these cases, the results of diagnostic assays for the detection of other potentially pathogenic enteric microorganisms were negative. an interesting finding was the detection of hadv in a patient years of age who was admitted with a diagnosis of encephalitis. no human herpesvirus, influenza virus, ev or flavivirus genomes were found in the cerebrospinal fluid of this patient. human adenovirus dna was detected in the supernatant of a cell culture infected with viruses obtained from fecal specimens taken from a patient with acute flaccid paralysis (afp), as well as in two cases of meningoencephalitis. overall, % of hadv positive patients were hospitalized. partial hexon nucleotide sequences for different hadv types were obtained. a blast search in the genbank database for all of the amplicon sequences determined in the present study was done. a % agreement with existing genbank sequences for serotypes , , , and was obtained. an agreement ranging from to % was obtained for prototype strains of human adenovirus d. an alignment of the hexon gene sequences was computed by using sequences from representative serotypes of species b (hadv ), c (hadvs , , and ), and d (hadvs , , , , , , , , , , , , - , , , and - ) . in order to evaluate the phylogenetic relationships between individual prototype viruses and positive cases of hadv infection by pcr and/or viral culture, phylogenetic trees were constructed using a phylogeny reconstruction algorithm (the neighbour-joining method) and a nucleotide substitution method . phylogenetic and sequence similarity analysis permitted accurate species classification and serotype identification except for species d hadv, for which serotypes could not be clearly defined. the resulting phylogenetic tree showed three different clusters, represented by the species b, c and d. the bootstrap values ( fig. ) were, in general, quite high, and especially at nodes between species. however, these values were less good for some nodes within species d. the assignment of a serotype to a clinical isolate was done by calculating the shortest distance between the isolate and a prototype strain. a total of ( %) hadvs were serotyped, and typing results yielded some interesting observations. the major species found was hadv-d, with clinical cases ( %), followed by hadv-c, with cases ( %), and hadv-b, with two cases ( %). in bronchiolitis cases, hadv- ( %) was the major serotype, followed by hadv- ( %) and single cases of hadv- and hadv-d. in the group of patients with upper respiratory infection, the major serotype found was hadv- ( %). hadv- ( %) was the second-most common serotype, and five specimens were typed as hadv-d; one of them recovered in and four in . lastly, two specimens recovered in were hadv- . hadv-d was the species identified in nine ( %) cases detected during an atypical outbreak of acute febrile syndrome in july of . four isolates from clinical materials obtained from patients with severe disease such as encephalitis, afp and meningoencephalitis were identified as hadv-d. hadvs are categorized by species (hadv-a, hadv-b , hadv-c, hadv-d, hadv-e and hadv-f), and further by serotype (ad -ad ), and cause a wide spectrum of illnesses in both children and adults, including those involving the cns. a new recently isolated serotype ( ) has been proposed to belong to a new species g [ , , ] . more than half of the positive samples ( %) were from children younger than years of age, and % of them were under months of age. the highest incidence of adenoviral infection occurs in children from months to years of age, although outbreaks have been noted when susceptible hosts, such as military recruits, adolescents, visitors to summer camps, and sometimes nursing home residents, congregate together. explanations for the relative lack of illness in the youngest or in older individuals include the presence of transplacentally acquired maternal antibody in young infants and the development of neutralizing antibody to the most common adenoviral strains in the majority of children older than years old. the age distribution of hadv in developing countries appears to be similar to that in developed countries [ ] . the results in this study showed that adenovirus infections were more common in children younger than years of age, and % of them were under months of age. most patients were hospitalized ( %); nevertheless, none of these had severe respiratory infections. in cuba, infants are considered special patients by pediatricians. this report also describes the seasonal variation of adenovirus infections in cuba. respiratory viral agents usually have characteristic seasonal patterns in temperate and tropical climates. in temperate climates, the majority of isolations of respiratory viruses are in the winter. in central america, there are few studies published about the epidemiological characteristics of adenoviruses. the cuban island is located in the caribbean sea at the entrance of the gulf of mexico. the climate of cuba is semitropical, and two seasons are generally recognized: a rainy season from may to october and dry season from november to april. cuba is often hit by hurricanes from june to november, resulting in great economic loss and temporarily interrupted sanitary conditions. the average minimum temperature is °c ( °f), and the average maximum is °c ( °f). the warmest month is july with °c ( °f). in this study, adenovirus was detected throughout the year; however, two significant outbreaks of hadv infection were identified, the first in july of , when hurricane dennis hit severely the western part of cuba, and the second in september of . we believe that the effect of temperature and rainfall appears to be a determinant of the timing of those outbreaks. figure displays a phylogenetic tree built from an alignment of the nucleotide sequences of the amplicons obtained from clinical samples and each serotype prototype. as shown in fig. , each serotype is clearly distinct. serotypes belonging to the same species cluster together. in cuba, human adenovirus circulates in the pediatric population throughout the year. furthermore, members of adenovirus species c of are commonly involved in respiratory diseases in the pediatric population. two previous studies have been reported on the associations between specific clinical syndromes and hadv serotypes. the analysis of the occurrence of hadv-c revealed that hadv- and hadv- were the predominant serotypes in children with acute respiratory diseases, and hadv- was the most prevalent one associated with conjunctivitis [ , ] . human adenovirus respiratory infections have usually been associated with species b, c, and e [ ] . hadv-c includes hadv- , hadv- , hadv- , and hadv- . these serotypes are commonly associated with febrile respiratory illness in children and are noted to be endemic in certain regions or in epidemics [ ] . the significant proportion of infection with hadv- , hadv- , hadv- and hadv- among patients with respiratory infection is consistent with previous reports [ , , , ] . however, we detected hadv-d in a child with bronchiolitis, and in cases of upper respiratory infection. hadv-d are rarely isolated in respiratory illness surveys, and strong causal correlations are generally lacking. nevertheless, this association was previously described by casas et al. [ ] . on the other hand, hadv-d was identified in a group of patients with acute febrile syndrome and in four isolates from clinical materials obtained from patients with encephalitis, afp and meningoencephalitis. sporadic cases or small outbreaks of neurological disease following adenovirus infection are well documented [ , , , ] . the high prevalence of infection with hadv-d in previously healthy patients is not consistent with previous reports. this association was unexpected, and a detailed analysis of this sample set will be published in a separate report. human adenovirus is unique among the common respiratory viruses in that it can spread to organs other than the respiratory system, resulting in conjunctivitis, gastroenteritis, acute hemorrhagic cystitis, and meningoencephalitis. the liver, spleen, pancreas, kidney, or heart may also be involved in disseminated infections, both in previously healthy people and in immunocompromised patients (e.g., infants, patients with aids, transplant recipients) [ ] . hadv-d has been frequently associated with severe adenovirus infection in these patients. in our study, we reviewed the medical records and did not find that the patients suffered from aids or another type of immunodeficiency. however, we thought that is important to take into account that the highest percentage of hadv-d was identified in children. the identification of such a large number of hadv-d infections is extremely interesting and a major and unique finding. we recognize that the limited sequence data analyzed ( nt in a relatively conserved region of the hexon) for typing did not generate enough results. the sequencing of the hypervariable regions of the hexon gene (hvr alone or hvr - ) and fiber gene of select strains will certainly provide a solid basis for confirming the species/serotype identification [ , , ] . we used the method previously published by casas et al. [ ] , to facilitate sequencing and typing of hadvs using the hexon protein coding region of the hadv genome. this method was extensively validated using clinical samples and prototype strains, and we also noted unusual associations between specific serotypes and clinical presentation. the primers and pcr conditions have also been extensively validated. some years ago, adenovirus infection was considered to have little consequence. however, much has changed since these early epidemiological studies were conducted. in contrast to the modest number of hadvs recognized years ago, unique serotypes are now recognized, and a new serotype isolated recently ( ) has been proposed to belong to a new species g. different serotypes have been found to have different tissue tropisms that correlate with different clinical manifestations of infection. partial epidemiological investigations have revealed that, among some specific serotypes, multiple genetic variants exist that often have quite different geographical distributions and associated virulence [ , , , ] . our study expands the range of hadv serotypes that have been reported elsewhere in association with major clinical syndromes. further independent testing is needed to verify these associations. however, future studies of hadv should not exclude these rare serotypes, and they should be kept in mind. unfortunately, because adenoviruses can be asymptomatically shed for prolonged periods of time, recovery of hadv from the upper respiratory tract or stool samples by culture does not confirm it as the cause of a specific disease. accordingly, recovery of hadv should always prompt an effort to identify any additional or alternate potential explanations for symptoms present at the time of a positive viral culture [ , ] . additional research is needed for the development of better rapid methods to detect and to quantify hadv in various body fluids (blood, stool, and throat). finally, our findings, combined with the existence of several reports concerning the association of hadv with different syndromes confirm that members of the family adenoviridae, mainly the serotypes belonging to hadv-c are common causal agents of respiratory infection. in contrast, serotypes of hadv-d could be involved in the aetiology of acute respiratory infection and neurological disorders in previously healthy people. however, this observation needs to be confirmed in a larger study. our identification of hadv-d associated with different syndromes may signify the emergence of new genomic variants that have the potential to spread globally. further studies of such typing with other molecular typing methods are necessary. these reports show that surveillance, serotyping and molecular characterization methods need to be improved in order to identify emerging adenovirus variants. authors wish to thank all of the physicians who provided samples and clinical data. we also acknowledge all technicians and researchers of the virology deparment of the instituto de medicina tropical pedro kourí, la habana, cuba, who collaborated with this investigation. thanks to dr enrique tabarés, from universidad autónoma de madrid, spain, for his help. rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction outcome and clinical course of patients with adenovirus infection following bone marrow transplantation family adenoviridae infections in , infants and children in a controlled study of respiratory tract disease. ii. variation in adenovirus infections by year and season intravenous cidofovir therapy for disseminated adenovirus in a pediatric liver transplant recipient new method for the extraction of viral rna anddna from cerebrospinal fluid for use in the polymerase chain reaction molecular identification of adenoviruses in clinical samples by analysing a partial hexon genomic region molecular and clinical characteristics of adenoviral infections in taiwanese children in - 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rbrzv o authors: choudhary, manohar lal; anand, siddharth p.; sonawane, nupoor s.; chadha, mandeep s. title: development of real-time rt-pcr for detection of human metapneumovirus and genetic analysis of circulating strains ( - ) in pune, india date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: rbrzv o human metapneumovirus (hmpv) is an important respiratory virus implicated in respiratory infections. the purpose of this study was to develop a one-step real-time rt-pcr assay that can detect all four lineages of hmpv and to identify the hmpv lineages circulating in pune, india. conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. a total of clinical samples that were positive for different respiratory viruses (including samples that were positive for hmpv) were tested using the real time rt-pcr assay, and the specificity of the assay was observed to be %. using in vitro-synthesized rna, the sensitivity of the assay was ascertained to be copies of the target gene per reaction. phylogenetic analysis of the nucleoprotein (n) and attachment glycoprotein (g) genes confirmed that this assay detected all lineages of hmpv. a , b and b strains were observed during the study period. our assay is highly sensitive and specific for all known lineages of hmpv, making it a valuable tool for rapid detection of the virus. a and b were the predominant subtypes circulating in pune, western india. human metapneumovirus (hmpv), first isolated from children with acute lower-respiratory-tract infections (alrti) in the netherlands in , is an enveloped, non-segmented rna virus that belongs to the family paramyxoviridae and the genus metapneumovirus [ ] . subsequently, it has been reported globally [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it frequently causes both upper and lower acute respiratory tract infections (artis) in people of all age groups [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in immunocompromised patients [ , ] . several groups have described reverse transcription polymerase chain reaction (rt-pcr)-based assays to detect hmpv targeting the nucleoprotein, fusion or polymerase genes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mpv has been classified into two broad groups: group a, with subgroups a and a , and group b, with subgroups b and b [ ] [ ] [ ] . more recently, the a group has been split into two clades (a a and a b) [ ] . however, there is limited information on the prevalence and genetic diversity of human metapneumovirus (hmpv) strains circulating in india [ ] [ ] [ ] . it is important to establish diagnostic hmpv assays which detect all subtypes of the virus. this study describes the development of real time rt-pcr assay for the detection of all four hmpv lineages (a , a , b , b ) in respiratory specimens and genotyping of circulating strains from july to august in pune, western india. clinical samples (nasal/throat swab) referred to the national institute of virology (niv), pune, for diagnosis of h n pdm from july to august were retrospectively used in this study with the approval of niv's human ethics committee [ ( )/ec-i/ ] as per indian council of medical research (icmr) guidelines. this included outpatient and admitted cases in pune, maharashtra, india. a total of clinical specimens were tested to measure the sensitivity and specificity of the assay. rna was extracted using a magmax- viral rna isolation kit according to the manufacturer's instructions. fifty microlitres of clinical specimen was used as starting material, and the rna was finally eluted in ll of elution buffer. primer and probe design primers and probe were designed using the hmpv sequences available in the ncbi database. representative sequences of nucleoprotein genes of prototype sequences of a , a , b and b subtypes [ , ] were aligned, and primers were designed from conserved regions of the consensus sequence to ensure that all lineages would be amplified and detected using this assay. primers were checked for cross-reactivity, self-dimerization, hairpin formation and secondary structure formation using ncbi blast, oligocalc software (http:// www.basic.northwestern.edu/biotools/oligocalc.html) and oligoanalyzer. primer and probe sequences are given in table . sequencing primers were designed for the nucleoprotein (n) and attachment glycoprotein (g) genes as shown in table . the entire nucleoprotein gene was amplified using t hmpvnfp and hmpvnrp primers ( table ). the forward primer was tagged with a universal t promoter sequence at its ' end. pcr amplification was performed on a geneamp pcr system using an invitrogen superscript iii one step rt-pcr kit. the master mix for rt-pcr consisted of ll x buffer, ll enzyme mix, lm forward primer, lm reverse primer, and ll of rna template to make a -ll reaction. thermal cycling conditions were °c for min for reverse transcription, initial denaturation at °c for min, cycles of three steps - s at °c, s at °c, s at °c -and final extension at °c for min. in vitro-synthesized rna was made using a t riboprobe kit (promega, leiden, the netherlands) as per manufacturer's instructions. in vitro-synthesized rna was quantified using a nanophotometer (implen, germany), and the rna copy number was calculated. the in vitro-transcribed rna was then serially diluted tenfold and used to determine the analytical sensitivity of the real-time rt-pcr. real-time rt-pcr assay was performed on an applied biosystems abi machine using an invitrogen superscript iii one-step qrt-pcr kit (invitrogen, california, usa). different parameters of the assay, such as annealing temperature and time and primer and probe concentrations, were optimized. a standard curve was plotted using tenfold serially diluted in vitro-synthesized rna, testing each concentration in duplicate. a typical -ll pcr reaction consisted of lm forward and lm reverse primer, . lm of taqman probe, . ll buffer, ll superscript iii enzyme, and ll rna template. real-time rt-pcr thermal cycling conditions were as follows: °c for min for reverse transcription, initial denaturation at °c for min and cycles of s at °c and s at °c. sequencing primers were designed for the nucleoprotein (n) and attachment glycoprotein (g) genes. the glycoprotein sequence is highly variable, and hence, subgroupspecific primers were designed as shown in table . the nucleoprotein gene was amplified as described earlier, and the pcr product was purified using a qiaquick pcr purification kit according to the manufacturer's instructions. sublineages were determined using blast analysis of n gene sequences. after determining the subgroup, the glycoprotein gene was amplified using a subgroup-specific forward primer (hmpvgafp for a and a ) (hmpvgbfp for b and b ) and a reverse primer hmpvlrp (table ) . pcr reaction setup and conditions for the g gene were the same as those used for the n genes. internal primers ( studies for assessing the detection limit of the assay were performed using in vitro-transcribed rna from template of lineage a and b viruses. the standard curve was plotted using tenfold serial dilutions of the in vitro-synthesized rna in duplicate from copies per reaction to . copies per reaction to check the pcr efficiency of the assay, and it was found to be approximately % (fig. ) . the slope of the standard curve was found to be - . (acceptable value = - . to - . ). the analytical sensitivity of the assay was copies of in vitro-synthesized rna per reaction for both lineages. the sensitivity and specificity were determined by testing hmpv-positive samples (n= ), known positive samples of respiratory viruses other than hmpv (n= ), and samples that were negative for any of the respiratory viruses (n= ). clinical specimens that were confirmed previously as positive by multiplex rt-pcr [ ] were used in this study. a total of samples were tested, as shown in table . no cross-reactivity was observed with other respiratory viruses, and all samples that were positive for hmpv by multiplex rt-pcr were positive in the realtime rt-pcr assay. one known rsv-positive sample also tested positive for hmpv in the assay. both rsv and hmpv were found to be present in the sample, as shown by sequencing, confirming a dual infection. phylogenetic analysis of the nucleoprotein gene sequences showed homology with hmpv reference sequences, as shown in fig. for the nucleoprotein gene, % sequence homology was observed between the samples and reference sequence (can - ) at the nucleotide level, and - % at the amino acid level, for the a genotype. also, the sample sequences showed % amino acid sequence identity to indian strain ind/ - / op. for the b lineage, sequence identities between the sample sequences and reference strains (nl/ / ) were between and % at the nucleotide level and - % at the amino acid level. the amino acid sequence identity between the sample sequences and indian strain ind/ - / op was between and %. for the b lineage, sequence identity between the sample sequences and reference strains (can - ) was % at the nucleotide level and between and % at the amino acid level. as expected, the nucleoprotein sequences were highly conserved, both at the nucleotide and amino acid level, showing - % identity between the sample sequences and reference sequences. twenty-seven randomly selected hmpv-positive samples were sequenced for the glycoprotein gene. of the sequences, sequences aligned with the a subtype, with the b subtype, and with the b subtype (fig. ) . of the sequences, were from , were from , and one was from . from the two phylogenetic trees it was observed that the b subtype was observed only in . however in , the a and b subtypes were also found in co-circulation with the b subtype, as shown in fig. . glycoprotein gene sequence alignments of the a subtype showed % sequence identity between sample and reference sequences (can - , nl/ / , kol/ / ) at the nucleotide level and % with can - and kol/ / , and % with ind/ - at the amino acid level. the b subtype sequences for the g gene showed % sequence identity with nl / at the nucleotide level and % at the amino acid level, but only % at the nucleotide level and . % at the amino acid level with a strain from eastern india (kol/ / ). the sequence homologies are similar to those reported by agrawal et al. [ ] . the b subtype g gene sequences showed % identity to the reference sequence hmpv - at the nucleotide level and % to can - at the amino acid level. again, homology to an indian strain (ind/ - ) was lower, showing % sequence identity at the amino acid level. the deduced g proteins of different hmpv strains had different lengths. changes in the stop codon were observed among strains of different lineages. those from subgroup a a were aa in length with the stop codon uag (nt ), a b had aa and utilized stop codon uaa at nt position , whereas those from subgroup b were aa in length and terminated at nt position using the stop codon uga. strains from the b subgroup had two different stop codons, resulting in a g protein with aa using a uag stop codon at nt position and one with aa using a uaa stop codon at nt position . the attachment glycoprotein of the b subtype has potential o-glycosylation sites on average. the region spanning amino acids to (extracellular domain) was checked for potential glycosylation sites. g-values of c . were considered to indicate potential o-glycosylation sites. the extracellular domain (amino acids - ) of the a subtype showed an average of o-glycosylation sites. also, a subtype sequences exhibited a higher ratio of serine to threonine than those belonging to the b subtypes. there are four distinct genetic lineages of hmpv, a , a , b and b , which exhibit substantial diversity. kuypers et al. [ ] developed an assay in which detection of all fig. phylogenetic analysis of the attachment glycoprotein (g) gene. a phylogenetic tree was constructed with mega . software using the maximum-likelihood method. bootstrap probabilities greater than % are shown at the branch nodes. reference sequences for each genotype were obtained from genbank b known subtypes of hmpv was achieved by using two primer/probe sets for the fusion gene in a one-step realtime rt-pcr reaction. each set was designed to amplify isolates belonging to subtype a or b but to be used together in one reaction mix. pabbaraju et al. [ ] developed a twostep real-time rt-pcr using a fusion gene with a lineagespecific primer-probe and a universal probe for detection of all hmpv lineages. maertzdorf et al. [ ] designed two sets of primers and probes within the nucleoprotein gene to detect all known genetic lineages of hmpv in a one-step real-time rt-pcr. the original nl-n assay developed by maertzdorf et al. [ ] performed poorly with subgroup b viruses, and it was further modified by klemenc et al. [ ] to improve its sensitivity for detecting all four lineages of hmpv. we have designed a primer-probe set using conserved regions of the nucleoprotein gene to detect all known genetic lineages of hmpv in a one-step real-time rt-pcr. our assay detected copies of in vitro-synthesized rna per reaction for both lineages. a panel of clinical samples that had previously been found to be positive for hmpv by multiplex pcr [ ] was confirmed to be positive by our real-time rt-pcr assay. this assay was also concordant with an additional panel of samples that were negative for hmpv (table ). these results suggest that our one-step real-time rt-pcr assay is sensitive for detecting all genetic lineages and specific, as it did not exhibit cross-reactivity with other respiratory viruses. phylogenetic analysis of the nucleoprotein and glycoprotein genes in the present study showed that both group a and groups b hmpv were co-circulating in pune, western india, during the study period. phylogenetic analysis reveals that in both and , a , b and b strains were seen in circulation, with predominance of the a and b subtypes. during the same period, the a lineage was predominantly detected in southwest china [ ] , okinawa, japan [ ] and new york, usa [ ] . a and b strains were found to be co-circulating in our study population in and , but the b subtype was seen only in in our study. of all the samples sequenced, only one sequence belonging to a subtype was seen. agrawal et al. [ ] reported a higher prevalence of subgroup a ( %) than subgroup b ( [ ] . in central and south america, subtypes a and b were the predominant strains circulating in - [ ] . in italy, b was the predominant subtype, followed by a and b in - , and in - , lineage a was the predominantly circulating lineage, followed by a [ ] . the hmpv lineage a has been reported to cause hmpv epidemics globally [ ] . this is the first study from india where the complete n gene from all circulating subtypes has been analysed. sequence analysis of the g gene revealed homology to reference strains similar to that described previously [ ] . the predicted g proteins of different hmpv strains had different lengths, which may be attributed to amino acid substitutions and insertions, deletions, and/or changes in the stop codon. five different lengths of g proteins were observed in this study, with or amino acids in subgroup a , or amino acids in the b subgroup, and in b . this was the first report showing an hmpv strain with amino acids circulating in pune, india. we need to further monitor hmpv cases from pune and other parts of india to gain insights into the circulating patterns of this particular strain of hmpv. this will help us to better understand the mechanism of emergence of new strains of the virus. in conclusion, we have developed a one-step real-time rt-pcr assay for the detection of hmpv from all four genetic lineages. our assay is sensitive and highly specific and can be used for future implementation in a diagnostic setting. phylogenetic analysis shows that the a and b lineages are predominantly circulating in pune. a newly discovered human pneumovirus isolated from young children with respiratory tract disease children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus infection in japanese children first detection of human metapneumovirus in children with acute respiratory infection in india: a preliminary report human 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the role of human metapneumovirus in upper respiratory tract infections in children: a -year experience study of human metapneumovirus-associated lower respiratory tract infections in egyptian adults an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility for the elderly in oregon rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults severe metapneumovirus infections among immunocompetent and immunocompromised patients admitted to hospital with respiratory infection humane metapneumovirus (hmpv) associated pulmonary infections in immunocompromised adults-initial ct findings, disease course and comparison to respiratory-syncytialvirus (rsv) induced pulmonary infections comparative evaluation of realtime pcr assays for detection of the human metapneumovirus molecular assays for detection of human metapneumovirus real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages detection of human metapneumovirus rna sequences in nasopharyngeal aspirates of young french children with acute bronchiolitis by real-time reverse transcriptase pcr and phylogenetic analysis detection and quantification of human metapneumovirus in pediatric specimens by real-time rt-pcr diagnosis and epidemiological studies of human metapneumovirus using real-time pcr detection and characterisation of human metapneumovirus from children with acute respiratory symptoms in northwest england real-time reverse transcriptase pcr assay for improved detection of human metapneumovirus analysis of the genomic sequence of a human metapneumovirus antigenic and genetic variability of human metapneumoviruses global genetic diversity of human metapneumovirus fusion gene novel human metapneumovirus sublineage genetic variability of attachment (g) and fusion (f) protein genes of human metapneumovirus strains circulating during detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in india genomic analysis of four human metap-neumovirus prototypes development of a multiplex one step rt-pcr that detects eighteen respiratory viruses in clinical specimens and comparison with real time rt-pcr molecular epidemiology of human metapneumovirus from phylogenetic analysis of human metapneumovirus from new york state patients during february through genotype variability and clinical features of human metapneumovirus isolated from korean children human metapneumovirus strains circulating in latin america human metapneumovirus-associated hospital admissions over five consecutive epidemic seasons: evidence for alternating circulation of different genotypes evolutionary dynamics analysis of human metapneumovirus subtype a : genetic evidence for its dominant epidemic real-time rt-pcr for human metapneumovirus acknowledgments the authors would like to thank the director for support, and the indian council of medical research (icmr) for funding the study. we would like to acknowledge mr. santosh kumar jadhav from the bioinformatics group and the technical team of the influenza group for their help. key: cord- -ab pnct authors: sugiyama, k.; amano, y. title: hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ab pnct the hemagglutination (ha) and receptor destroying enzyme (rde) activities of a newly isolated mouse enteric coronavirus (designated as dvim) are described. dvim agglutinates mouse or rat red blood cells (rbc) at ° c. at ° c the agglutination was rapidly reversed. the optimal ph for ha and for rde activities using mouse red cells were shown to be . and . respectively. hemagglutination by dvim was not inhibited by pretreatment of rbcs withvibrio cholerae filtrate or by pretreatment with influenza-a neuraminidase. therefore, the dvim receptors on rbcs differ from the receptors of influenza-a, and the rde activity of dvim acts specifically on this receptor. in addition, an analysis of the dvim polypeptides showed that the virions contain five major, vp (m.w. , ), vp ( , ), vp ( , ), vp ( , ), vp ( , ) and two minor, vp a ( , ), vp b ( , ) polypeptides. vp and vp b were digested by bromelain, suggesting that they constitute the surface glycoproteins. coronaviridae are a family of lipid-containing rna viruses which exhibit a unique morphology. the virions have widely-spaced surface projections which form a radiating "corona" around the particles as shown by electron microscopy ( , ) . the diarrhea virus of infant mice (dvim), a newly-isolated mouse enteric coronavirus, is antigenically related to other coronaviruses by complement fixation assay but clearly distinguishable by neutralization assay ( ) . the existence of a receptor destroying enzyme (rde)-like activity has not been reported for any of the coronaviruses. we report here a rde activity associated with dvim virions which differs from that of ortho-or paramyxo- * - / / / /$ . viruses. t h e s t r u c t u r a l p o l y p e p t i d e s of d v i m were a n a l y s e d b y p o l y a e r y l a m i d e gel eleetrophoresis. the mouse enteric coronavirus (dvim) was kindly provided by dr. kozaburo sate (central laboratory of shionogi pharm. co., osaka, japan). d v i m was passaged in balb/c- t cell cultures in eagle's minimal essential medium containing percent fetal calf serum. virus inoculation was carried out with tcid~ per l~oux bottle. a t hours post-infection virus was harvested by three freeze.thaw cycles and stored at -- ° c. influenza virus strain a/ri/ , a/victoria/ / , a / n j / / and hemagglutinating virus of j a p a n ( h v j or sendal virus) strain z were grown in the allantoic cavity of -day-old embryonated ehieken eggs as previously described ( ) . the same purification processes were employed for each virus at ° c. alter clarification of culture fluid b y centrifugation at , × g for minutes, virions were concentrated by eentrifugation at , × g for minutes, resuspended in . m n t e buffer ( . ~ tris-c , .i ~c nac , . m edta), p i t . , layered onto a discontinuous gradient of sucrose ( : : percent w/w), and centrifuged at , × g for hours. virions were collected from the interphase between and percent sucrose solutions and were dialysed against n t e buffer. for polypeptide analysis, virions were repurified by linear sucrose gradient ( to pereent w/w) eentrifugation at , × g for minutes. routine h a tests were carried out as previously described ( ), using . percent mouse rbcs in dulbeeeo's pbs, containing . percent bovine serum albumin. alternatively, . percent avian rbcs, or . percent other m a m m a l i a n rbcs were used. the titer was recorded as the reciprocal of the highest virus dilution causing a detectable ha. hemadsorption tests were done on dvim-infected b a l b / c - t cell monolayers grown on glass cover slips. a . percent suspension of mouse rbcs were adsorbed for hours at ° c. cover slips were washed and prepared for analysis by light microscopy. the experiment for elution kinetics was performed on identical eell-monolayers at various incubation periods. to test for r d e -l i k e activity, cells were incubated at indicated temperatures, prepared for microscopy and photographed. the elution ratio was expressed as the percentage of remaining rbcs enumerated per photographic field. sodium dodecyl sulfate (sds)-page was performed on . percent polyacrylamide gels by the method of maize~ et al. ( ) , using m m diameter tubular gels m m long. purified virus was solubilized in percent sds, percent -mereaptoethanol, percent glycerol, and . percent bromphenol blue, for t. minutes at t ° c. after preeleetrophoresis for one hour at ma/gel, samples were applied and eleetrophoresis was carried out at ma/gel in .i ~ sodium phosphate buffer, p h . , for hours, at room temperature. gels were fixed overnight with percent sulfosalieylie acid and stained with . percent coomassie brilliant blue in percent methanol. the gels were destained with several changes of percent methanol in percent acetic acid. gels were stained for carbohydrate and lipid with schiff's reagent and oilred-o, respectively, following the methods of hietgholzep~ et al. ( ) . stained gels were scanned with a densitometer (joko gel scanner) at n m for peptides, n m for carbohydrates, and n m for lipids, respeetively. the approximate molecular weights of the polypeptides were determined by the m e t h o d of st~apiao et al. ( ) . percentage composition of each strueturm polypeptide was determined from the photometric scans using a joko scanning planimeter. r e d blood cells of seven different species were tested for agglutinability with dvim, and the results are summarized in table . only mouse and r a t i~bcs were a g g l u t i n a t e d at ° c, indicating a restricted receptor range for the h a activity. mouse i~bcs agglutinated by d v i m at ° c, could be liberated from agglutination by incubation a t ° c ( table ). the d v i m sample had a titer of , h a u at ° c, which decreased to h a u after minutes of incubation at ° c. this indicates t h a t in addition to its h a activity, ;[)vim virions appear to possess a l~de-like a c t i v i t y which differs from the i~de a c t i v i t y shown for p a r a m y x oand myxoviruses, which is n o t active at ° c. avian red cells were prepared as . percent suspensioss in pbs (ph . ), mammalian red cells were prepared as . percent in the same pbs hemagglutination test were carried out at ° c as usual, then the microplate was incubated at ° c, for minutes the h a titer was stable at ° c. however, h a appeared to be reversable only under some p h conditions, when incubated at ° c, as shown in table . the optimal p h for h a a c t i v i t y is relatively wide, whereas r d e -l i k e a c t i v i t y appeared optimal at a p h of about . and exhibited no a c t i v i t y at p h . , at ° c. tile h a a c t i v i t y was stable at ° c, up to hours, b u t was i n a c t i v a t e d at ° c, within minutes (data n o t shown). k. sugi¥~a and y. ai,~ano: table . bromelain, a proteolytic enzyme which removes t h e club-shaped projections from the surface of other coronaviruses ( % , ), affected both h a a c t i v i t y and infectivity of dvim. trypsin did not affect h a a c t i v i t y or infectivity, while pepsin affected only h a activity. the h a titer was reduced slightly by np- t r e a t m e n t , and infectivity was destroyed. e t h y l ether destroyed both virus h a a c t i v i t y and infectivity. ha-titrations were carried out with . percent mouse red blood cells in indicated ph, at ° c, thereafter the microplate was incubated at ° c for minutes the d a t a in tables and the infected cells exhibit virus-specific enzyme aeti~dties associated with these syncytia. fig. shows the elution kinetics of red blood cells from the syncytia, induced by dvim hours after infection, at ° and °c respectively. approximately to per cent of rbcs were liberated within minutes at ° c while the liberation at °c progressed slowly. therefore, the cell surfaces of dvim-induced syncytia exhibit the same h a and rde-like activities associated with virions. if liberated i~bcs are reexposed to dvim-infeeted monolayers, no hemadsorption is observed. the same syneytia could again hemadsorb freshly prepared rbcs (fig. ) . this implies that hemadsorption of dvim does not correspond to "pseudo-hemadsorption" described by bucknall et al. ( ) . furthermore, the elution of red blood cells from syneytia appears to be a result of enzymatic digestion of rbc receptors by a gde-like activity situated on the syneytia surface. this activity was dependent upon incubation temperature, similar to rde-like activity of dvim virions. to confirm the receptor destroying activity of dvim, the effect of the purified virus on red blood cells was examined. mouse rbcs treated with dvim were reagglutinated b y influenza-a viruses or hvj, but could not be reagglutinated by ])vim virions (table ) . i n contrast, r d e treated mouse rbcs could still be fully agglutinated by dvim (table ). influenza-a hemagglutination of i~bcs was reduced in proportion to the concentration of rde. i k. sugiyama a n d y. amano: ~fouse r e d blood cells were t r e a t e d w i t h r d e a t indicated concentration, dissolved in . m ca-borate buffer (ph . ), at ° c for minutes, after which t h e cells were w a s h e d a n d resuspended as a . p e r c e n t suspensions i~ p b s a n d used for h a titration. i i d e was o b t a i n e d from vibrio eholerae v a r i a n t of s t r a i n filtrate (takeda p h a r m . co. osaka, j a p a n ) . control t~bcs were n o t t r e a t e d with t h e r d e fig. . r e a d s o r p t i o n of freshly p r e p a r e d i~bc to pre~dously exposed syncytia. h e madsorbed s y n e y t i u m which were i n c u b a t e d at ° c to release i~bcs for minutes, were exposed to freshly p r e p a r e d rbcs a n d p h o t o g r a p h e d above results strongly suggest that dvim possesses an enzymatic activity which reacts with surface components of rbcs. however, this enzymatic action appears to differ from the bacterial neuraminidase and that of influenza-a virions. since dvim is a newly isolated coronavirus, its structural polypeptides have not been characterized. therefore we analysed the virion polypeptides by sds-page. electrophorescd polypeptides, when stained with coomassie brilliant blue, revealed a mimmum of seven bands. a densitometer scan of stained polypeptides of dvim and hvj are shown in figs. a and b, respectively. the five major polypeptides of dvim were designated as vp to vp in decreasing order of electrophoretic mobility, as shown in fig. a . two additional minor bands v p l a and v p l b were observed between vp and vp . the approximate molecular weight and molar ratios of the structural polypeptidcs, are presented in table . preliminary analyses indicated that three polypeptides, vp , vp b and vp are glycoproteins, while vp reacted with oil-red- , suggesting it may be a lipid-containing protein. a densitometer scan of a stained polyacrylamide gel of the polypeptides of virus particles treated with bromelain ( . mg/ml at °c for minutes) is shown in fig. . as can be seen from this figure, vp and vp i b were digested by the bromelain treatment, suggesting that these polypeptides are found on the surface of the virion. polypeptides v p l a and vp were also reduced, and only trace amounts of them were still observed, suggesting these polypeptides may also be surface polypeptides. vp was unaffected by bromelain treatment, suggesting that it represents an "inner core" protein. direct ha (i. e. without pretreatment of the virus) and hemadsorption have been described for several eoronaviruses, c- ( , ) , hev ( , ) , ibv ( ), and bovine coronavirus ( ) . the hemagglutinating ability of c- required several passages in suckling mouse brain ( ) , and massachusetts strain of ibv ( ) required both sucrose gradient purification and digestion with phospholipase c for activation. the ha of dvim, reported here, was detectable directly in the culture fluid. the ratio of infectivity to ha varied between . and . × tcid / . ml/ha unit. dvim hemagglutinated the rbcs of only two species tested, while other coronaviruses, c- , hev and ibv, were less specific. accordingly, ha of dvim was expressed only using two types of red blood cell species, mouse and rat, and was also detectable at low temperatures in a stable pattern. the formation of "prozone" described by bing~am et al. ( ) was not observed for dvim. the instability of ha and hemadsorption at ° c suggests that a viral enzyme may be responsible for uncoupling the virus from the red blood cell receptor. the possible enzymatic activity of dvim was demonstrated on homologous virus receptors of mouse rbcs without any effect on receptors for heterologous viruses, influenza-a and hvj. furthermore, it was also shown that bacterial neuraminidase (rde) had no effect at all on the receptor for dvim; nevertheless, the receptors for influenza-a viruses were destroyed by the same treatment. additionally, even after purification through a sucrose density gradient, dvim retains its receptor-destroying activity, thereby showing that this activity is viral-specific and associated with virions. our preliminary results strongly suggest the existence of some type of receptor-destroying enzyme such as that of influenza c virus ( , ). the optimal ph of dvim ha activity was distinct from that of the receptordestroying activity. it has not been determined whether these activities may reside on a single polypeptide or on two different polypeptides. because the surface projections of dvim are somewhat different morphologically compared to other coronaviruses, it will be of interest to study the exact nature of the hemagglutinating and receptor-destroying activity of dvim. the receptor-destroying activity of dvim appears to be unique, when compared to that of ortho-and para-myxoviruses. purified dvim contains seven species of polypeptides, five major and possibly two minor polypeptides. these results compare favorably to those obtained for human coronavirus oc ( ), ibv ( ) , bovine coronavirus ly- ( ) and other murine eoronaviruses ?¢ii-iv- ( ) and j h m ( ) . the classification of the polypeptides of d v i m as glyco-or glycolipoprotein m u s t be considered as tentative at this point since the chemical nature of these complexes cannot be precisely defined by differential staining techniques. the number and size of gtyeoproteins of eoronaviruses which have been reported previously b y m a n y investigators, were shown to vary. i t appears t h a t our v p i (mw , ) corresponds to a large polypeptide (mw , -- , ) reported for other coronaviruses ( , , , , , , , , ) , because it is glyeosylated and appears to be situated on the surface of the virion. other d v i m virion polypeptides also appear to be surface glyeoproteins. furthermore, some of the investigators agree t h a t coronaviruses contain a polypeptide having an a p p r o x i m a t e molecular weight of , , corresponding to our vp , which is not glyeosylated and tends to comprise a large proportion of the virus protein ( , , , ) . polypeptide vp represents percent of virus protein and is not affected b y bromelain digestion at all. this suggests t h a t v p is also located in the "inner core" ( , , ) of the virion. although a considerable a m o u n t of v p was not observed in fig. , the possibilityt h a t the v p could 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polyacrylamide gels characterization of a coronavirus. . glycoproteins of the viral envelope; tryptie peptide analysis structural polypeptides of the marine eoronavirus jhm received february , key: cord- -u wd rn authors: stohlman, s. a.; weiner, leslie p. title: stability of neurotropic mouse hepatitis virus (jhm strain) during chronic infection of neuroblastoma cells date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: u wd rn a line of mouse neuroblastoma cells which was chronically infected with the neurotropic strain (jhm) of mhv, a member of the coronavirus group, was established. these cells, designated n(j), exhibited typical mhv cytopathic effects (cpe) at all passage levels along with the continual production of infectious virus. most cells were positive for viral antigen by immunofluorescence. viral particles consistent with the morphology of mhv were found by electron microscopy. the uninfected neuroblastoma cell line did not contain a detectable population of cells resistant to jhm, and persistence did not elicit the production of interferon. no plaque morphology or temperature sensitive mutants were selected for in the n(j) culture, and we were unable to detect the presence of either a defective or defective interfering virus population. the addition of low concentration antiviral antibody modulated the infection to a carrier culture with viral antigen in the cytoplasm of the cells, but no infectious virus was produced, and the cells lacked both surface viral antigen and cpe. possible mechanisms of viral persistencein vitro are discussed. mouse hepatitis virus, a member of the coronavirus group, is endogenous to mice and has been shown to occur predominantly as a natural, latent infection in. vivo ( ) and also to be able to establish a latent infection in vitro ( ) . the jhm strain has almost complete neurotropism characterized by encephalomyelitis with both acute and chronic demye]ination and an inability to produce apparent hepatitis in its natural host ( , , ) . chronic demyelination following intracerebral inoculation was found in only per cent of surviving balb/c mice, indicating that a subpopulation of jhm may he responsible for chronic infection. mechanisms of persistence in vitro have been previously analyzed in attempts to - / / / /$ . s.a. stohl~a~ and leslie p. weinei~: be~ter understand chronic and inapparent infections in vivo ( , ; ) . to approach the study of possible mechanisms of chronic infection in vivo, persistence of jhm virus in vitro was established and studied. mouse hepatitis virus (mhv), strain jhm ( , ) , used in these experiments was derived from the eighth consecutive suckling mouse brain passage which was prepared as previously described ( ) . the mouse neuroblastoma c clone n a cells of a strain mice ( ) were kindly suppiied by dr. m. oldstone of scripps clinic and research foundation, and the dbt cell line ( ) was kindly supplied by dr. a. hirano of the university of tokyo. both cell lines were maintained in dulbecco's modification of eagle's mi~mm essential medium (dmem) supplemented with t per cent fetal bovine serum (fbs). nctc cells (american type culture collection) were mainrained in dmem supplemented with per cent fbs and per cent horse serum. all incubations were per cent co~ at ° c unless otherwise specified. supernatant virus was assayed following serial dilution in dmem plus per cent fbs. monolayers of either dbt or nctc eetis in mm plastic plates were inoculated with . ml of virus suspension. following adsorption at room temperature for minutes, plates were overlaid with dmem plus per cent fbs containing . per cent agarose. plaques were visualized in the dbt monolayers at hours post infection following a second agarose overlay containing / , neutral red. plaques were visualized in nctc cell monolayers at hours post infection with . per cent. crystal violet following fixation with per cent formalin for minutes. for the determination of cell-associated virus, the infected monolayers were washed one time with dmnm and removed with a rubber policeman. the cells were suspended in dmem plus per cent fbs and sonieated at ° c for t to t minutes in a bra:nsonie ultrasonic cleaner. the resulting suspension eontai~fing less than per cent of the original cells intact was then assayed without further treatment as described for supernagant virus. infectious centers were determined on monolayers of nctc or dbt cells. monolayers of cells for assay were removed with . per cent trypsin, washed two times with deem, and counted in a hemoeytometer. following incubation at room temperature for minutes in the presence of anti-jhm hyperimmune aseitie fluid ( per cent plaque reduction neutralization titer = / ~ ), serial dilutions were plated on the indicator monolayers and fixed with . ml of dmem plus per cent fbs containing . per cent agarose. after solidification of the primary overlay, the remaining agarose overlay ( . ml) was added to the plates. plaques were visualized as described above. cell culture supernatants for interferon assay were prepared by eentrifugation at × g for minutes and , × g for hours. the ph was adjusted to . , and following incubation at ° c for hours, the ph was returned to . . after dialysis against phosphate buffered saline (pbs), ph . , at °c for hours and a final centrifugation at , × g for hours, the preparations were filter sterilized and stored at -- ° c until use. in addition to positive control mouse interferon prepared in l cells (american type culture collection) using uv-inaetivated newcastle disease virus (ndv) ( ) , n.i.h. positive control reference mouse if (g - - ) was used. interferon preparations were assayed by the per cen~ plaque reduction method using vesicular stomatitis virus (vsv) as the challenge virus. following serial two-fold dilution of the interferon preparations in eagle's minimal essential medium containing per cent newborn calf serum, ml of each dilution was added to confluent l cell monolayers in mm plates. after hours incubation at ° c, the fluid overlay-was aspirated, and the monolayers were challenged with approximately plaque forming units (pfu) of vsv in . ml. for immunofluorescence studies, cells were seeded on mm glass coverslips. the covers]ips were washed times with pbs, air dried for minutes, fixed at room temperature in acetone for minutes, and stored at -- ° c until use. prior to staining, coverslips were washed once in pbs and drained. mouse anti-jhm ser~rn prepared as previously described ( ) or mouse anti-jhm hyperimmune ascitie fluid prepared in adult male dub ici~ mice ( ) were used as a source of antiviral antibody. following incubation with antiserum for minutes at ° c, the coverslips were washed times with pbs, and fluorescein isothiocynate conjugated goat anti-mouse igg (grand island biological company, grand island, new york) containing . per cent evan blue was added. after an additional minutes at ° c the coverslips were washed times in pbs and mounted on per cent glycerol in pbs and examined under a leitz ortholux microscope. fresh, unfixed specimens for surface antigen were stained following the above procedure. the :nj cell line was established by infecting the mouse neuroblastoma n a cells ( ) with jhm virus at a multiplicity of infection (moi) of . . following the acute phase of jhm growth in n a cells, characterized by cell fusion and eytolysis, a small number of ceils remained which rcpopulated the culture vessel in to days. during the first passages, the cells grew at approximately per cent of the rate of uninfected n a cells, but, by passage and subsequently, the nj cells grew at the same rate as the n a cells. as the nj cells approach confluency, the culture begins to exhibit increasing s)~cytia formation. the nj cells have shown evidence of viral cpe at all passage levels, and intraeellular viral antigen ( fig. ) was detected by immunofluoreseent staining in to i per cent of the cells. viral antigen detected by immunofluorescence did not vary with passage level. viral antigen in both single cells and syncytia was diffuse throughout the cytoplasm, while only syncytia were found to express viral antigen at the cell surface. infectious virus was assayed at -- days after subcnlturing when the cells became confluent (fig. ) . initially there was a decline in infectious virus during the first passages. during this phase, the nj culture was not resistant to superinfection by jhm virus. following superinfeetion, an abbreviated, acute infection took place with increased syncytia formation followed by cellular degeneration. there was little change in the virus titer. by passage , infectious virus reached a level (approximately pfu/ml) which has remained constant for the remaining passages analyzed. at, passage levels , , and , the nz cells were refractory to superinfeetion with jhm. following superinfection, there was no increase in either cpe or amount of supernatant or cell-associated infectious virus. infectious center assays showed a decrease in the n u m b e r of cells producing infectious virus with continued passage, although no decrease in the percentage of fluorescent, cells was found. cells producing detectable infectious virus decreased from per cent at passage to per cent at passage , there was no change in either the levels of infectious virus produced or in the c p e evident following passage . all single cell clones isolated h'om the n a cell line were found to be susceptible to jitm virus, and mthough there were differences in the viral induced cpe at hours post infection, all clones progressed to massive cpe by hours post infection and yielded essentially identical peak levels of supernatant virus (approximately × pfu/ml). interferon activity was not detected in either undiluted or supernatants concentrated times from passage level , , or of the nj culture. although interferon was not detectable, the nj cultures were tested for resistance to superinfection by heterologous viruses at passages and ( , ) . no decrease in the yield of either vesicular stomatitis virus (vsv) or encephalomyocarditis virus (emc) was detectable when nj cells were infected at an moi of or . however, at an moi of t, the deld of progeny from the nj cells was significantly reduced compared to that obtained from control n a cells (table ) . . expressed as logic pfu/ml b multiplicity of infection the possible evolution of viral variants in this in vitro system was studied by using a suckling mouse brain (smb) virus pool heterogenous with respect to plaque morphology to initiate the infection. this pool of virus elicits acute encephalomyelitis and demye]ination with chronic and recurrent demyelination in mice ( , ) . the nj virus population was assayed at alternate passage levels ( fig. ) for virus and the relative numbers of plaque variants. neither during establishment nor at any passage level up to an including the th passage was there a selection for plaque morphology variants. the cell-associated nj virus, like the cloned and uncloned parental jhm, shows no differential ability to form plaques at °, °, or ° c. no differences were found in the ability of the infected cells to form plaques at either °, °, or ° c, excluding the possibility that a temperature sensitive (ts) defect might prevent maturation of infectious virus at ° c. in addition, over nj jhm virus clones were prepared from passage levels and and tested for ts characteristics. none of the clones tested showed a temperature defect. transmission electron microscopy indicated that most of the cells had intraeellutar vacuoles containing eoronavirns-like particles typicm of those seen in n a cells acutely infected with jhm and in other eoronavirus infected cells ( ) . all mnltinucleate cells contained virus particles while only -- per cent of the uninucleate cells had vacuoles containing viral particles. intracellular particles which appeared to be devoid of central staining material were found, and negatively stained virions from the nj culture also appeared to be differentially strained with phosphotungstic acid. studies were carried out to detect the presence of defective interfering (i)i) or defective particles in the nj culture. to determine if nj supernatants contained di particles, monolayers of )bt cells were infected with concentrated nj supernatants at a m i of and challenged with jhm at a moi of . and . . no difference in superrtatant virus yield was found. similar experiments using the non-neuro-adapted a strain of mhv, which grows to t -fold higher titer than jhsi as challenge virus, also lailed to show any differenees ill virus yield. attempts were made using both dbt ( ) and cl ( ) cells as hosts to see if di particles could be generated by undiluted serial passage. no decrease in virus yield or cpe was evident following serial passages in either cell type. preparations of radiolabeled jhm virus from acutely infected n a cells alld h~ jhm were compared in csci equilibrium density gradients to detect the presence of defective particles. a single peak of radioactivity (density -- . ) was found in both gradients, and there was no differenee in the rna: protein ratios of the two preparations. although these experiments do not completely rule out the production of di particles by the nj cells none are detectable by these techniques. n] jhm virus retained the ability to replicate and to produce cpe in cell lines susceptible to the parental jhm. intracerebral inoculation of mice with nj jtt~ from nj passage levels , , and produced encephalomyelitis with demyelinating lesions like those seen following infection with the parental virus ( , ) . nj the effects of antibody addition to the nj culture were studied using heatinactivated antiviral antibody at a final dilution of / (plaque reduction neutralization titer of / ). antibody was added to the culture either immediately after the cells were suspended in fresh medium for passage or after -- hours when the majority of the cells had attached. following to days incubation, the cultures were examined for virus, cpe, and both surface and cytoplasmic antigen. figure shows that following the initial passage in the presence of antibody, there was an increase in both the supernatant and cell associated virus titer, which rapidly declined until after serial passages no infectious virus was detectable. surface antigen and cpe were lost following passages. intracellular viral antigen was retained for passages in the presertce of antibody and subsequent passages in the absence of antibody. no infectious virus could be detected after passage in the absence of antibody. infectious virus was not released from these cells following incubation for passages at high ( ° c) or low ( ° c) temperatures nor following co-cultivation with cell lines susceptible to jhm virus at either °, °, or ° c. the mechanism of in vitro persistence by the jhm strain of mhv, in contrast to most other reported persistent infections by i~na viruses remains unknown. since jhm virus represents one of the animal models of virus induced demyetination with an ongoing chronic myelin breakdown ( , , , ) , the mechanisms by which this virus can estabhsh a chronic neurological process is of great interest. the studies described in this paper were carried out to determine if in vitro persistence could select for a minor pre-existing or new mutant virus population capable of chronic infection in vivo. the nj culture was resistant to both homologous virus challenge at passage and beyond and to challenge with heterologous virus infection at low moi and thus differs from cultures producing di particle ( ) or the agent described by te~ mevl~ and manti~ ( ) . interferon, which produces both heterologous and homologous virus resistance was not detectable in nj culture supernatants. most cells in the nj culture showed viral antigen which supports the possibility that resistance of the nj culture may be the result of intrinsic interference ( , ) ; however, no mechanism for homologous virus resistance has been determined. the selection of temperature sensitive and plaque morphology mutants ( ) have also been suggested as possible mechanisms responsible for the establishment or maintenance of persistence. the nj virus population showed no selection of ts or plaque morphology mutants through the th passage. in addition to the parameters already discussed, nj jhm was tested for another biological property unique to jhm, namely its ability to cause demyelination following i.c. inoculation into susceptible mice. the nj virus retained this property and produced lesions identical to those caused by the parental virus. antiviral antibody addition to the nj culture at passage increased yields of both cell-associated and supernatant virus. enhancement of murray valley encephalitis virus and rabbit pox virus plaque formation has been described in cells treated mth virm specific antisera ( , ) . furthermore, the addition of non-neutralizing antiviral antibody to leucocytes infected ~dth dengue virus enhances virus growth ( ) perhaps by increasing virus adsorption to phagocytic cells, thereby increasing the apparent moi by facilitating entrance of the infections virus into the cell cytoplasm while escaping the defense mechanisms of the phagocyte. in n a cells no active phagocy~osis of colloidal carbon particles was found. in addition, the nj culture is refractory to superinfection with jhm virus at passage and beyond. therefore, we must postulate that the effect of the antibody must not be enhanced viral uptake but a modulation of the cell surface that increases viral synthesis. the syncytia are the most likely cells affected since they are the only cell type with detectable celt surface antigen. subculturing in the presence of antibody cured the culture of cpe and surface fluorescence and of infectious virus. the cells did not, however, lose internal viral antigen and remained resistant to superinfection by ,jhm. they did not revert to viral woduction and were still resistant to jhs~ infection following passages in the absence of antibody following the "cure". antibody can, therefore, modulate a jti vi infection having infectious virus and cpe to a persistent infection with no infectious virus and no viral antigen at the cell surface. thus, it is possible that immune modulation, similar to that seen in vitro occurs in vivo and contributes to establishing chronic infection. a similar process has been postulated as the mechanism for the maintenance of herpes virus in the nervous system ( ). these studies described in this report show that in vitro persistence of jhm virus in the n a cell line is not mediated by a detectable change in the in vitro properties of the virus and in this respect is similar to in vitro persistence by other rna viruses implicated in chronic neuropathology ( , ) . the nj jitm virus also retained its special neurotropism in rive. the in vitro properties of nj jhm are similar to a jhm virus isolated from the brain of a chronically infected animal which also showed no deteetame changes in its in vitro or in vivo properties (st~)hlman, unpublished data). the in vitro antibody studies indicate that the host response may be the critical element in the establishment and maintenance of persistence in rive. we wish to acknowledge debra dipaolo, alan ttiti, and arthur i-ienry for excellent technical assistance. this investigation received financial support from public t~[ealth service grants i f ns - and t~ ns - (national institute of neurologieai and communicable disorders and stroke) and from the i~roe foundation. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological properties of the virus latent virus as exemplified by mouse hepatitis virus (mhv) nasoencephalopathy of mice infected intranasaity with a mouse hepatitis virus, st, rasn jttm antibody-enhanced dengue virus infection in primary leukoeytes the enhancement of virus infectivity by antibody mouse hepatitis virus-induced recurrent demyelination mouse hepatitis virus (miiv- ) plaque assay and propagation in mouse cell line dbt cells persistent noneytoeidal vesicular stomatitis virus infections mediated by defective t particles that suppress virion transcriptase mechanism of sindbis virus-induced intrinsic interference with vesicular stomatitis virus replication influence of host cell on residual infectivity of neutralized vesicular stomatitis virus genesis and maintenance of a persistent infection by canine distemper virus a carrier cell line of measles virus in lu cells ultrastrueture of animal viruses and bacteriophages: an atlas temperature-sensitive mutant viruses and the etiology of chronic and inapparent infections persistent infections of tissue culture cells by rna viruses isolation of a latent murine hepatitis virus from cultured meuse liver cells in vitro differentation of a mouse neuroblastoma maintenance of latent herpetic infection : an apparent rote for anti-viral igg dengue virus-induced modifications of host cell membranes enhanced grm~h of a routine eoronavirus in transformed mouse cells persistent geovirus infection of cho cells resulting in virus resistance pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) persistent parainfluenza type t ( / ) infection of brain cells in tissue culture interferon production by inactivated newcastle disease virus in celt cultures and in mice key: cord- -b kg qpo authors: saeng-chuto, kepalee; stott, christopher j.; wegner, matthew; senasuthum, raweewan; tantituvanont, angkana; nilubol, dachrit title: retrospective investigation and evolutionary analysis of a novel porcine deltacoronavirus strain detected in thailand from to date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: b kg qpo porcine deltacoronavirus (pdcov) in thailand was first detected in . we performed a retrospective investigation of the presence of pdcov in intestinal samples collected from piglets with diarrhea in thailand from to using rt-pcr. pdcov was found to be present as early as february . phylogenetic analysis demonstrated that all pdcov variants from thailand differ from those from other countries and belong to a novel group of pdcov that is separate from the us and chinese pdcov variants. evolutionary analysis suggested that the thai pdcov isolates probably diverged from a different ancestor from that of the chinese and us pdcov isolates and that this separation occurred after . electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. porcine deltacoronavirus (pdcov) causes an enteric disease in pigs that is characterized by diarrhea and is clinically similar to porcine epidemic diarrhea (ped) and transmissible gastroenteritis (tge) [ ] . pdcov, a recently emerged rna virus of the family coronaviridae, genus deltacoronavirus, was first reported in hong kong in during an investigation to identify novel coronaviruses [ ] . in february , pdcov was first detected in pigs with clinical diarrheal disease on farms in ohio, usa. a subsequent retrospective investigation reported revealed that pdcov had been present in the us as early as [ ] . pdcov has since been identified in canada, china, south korea and laos [ , , ] . in thailand, pdcov was first reported in [ ] . however, it is unknown whether pdcov was present in thailand prior to . we therefore conducted a retrospective study to detect the presence of pdcov intestinal samples from thailand that had been submitted for the identification of enteric diseases. positive samples were subjected to full-length genome characterization followed by genetic analyses to compare thai strains of pdcov to those from other countries and to investigate the genetic similarity and the evolutionary relationships among them. a total of intestinal samples were assayed for the presence of pdcov using rt-pcr. intestinal samples were from -to -day-old piglets with clinical diarrheal disease collected from january to december and stored at - °c. the presence of other viruses causing enteric disease, including porcine epidemic diarrhea virus, and transmissible gastroenteritis virus was detected using rt-pcr. in brief, viral rna was extracted from the samples using nucleospin rnavirus (macherey-nagel inc., pa, usa) in accordance with the manufacturer's instructions. complementary dna (cdna) was synthesized from the extracted rna using m-mulv reverse transcriptase (new england biolabs inc., ipswich, ma, usa). pcr amplification was performed on cdna using specific primers for the n gene of pdcov, the spike gene of pedv, and the n gene of tgev as described previously [ , , ] . samples positive for pdcov were then subjected to fulllength genome sequence characterization. twenty-six pairs of primers were used to amplify the different regions of pdcov [ ] . pcr amplification was performed using platinum Ò taq dna polymerase high fidelity (invitrogen, ca, usa) in accordance with the manufacturer's protocol. the pcr products were purified using a nucleospin plasmid kit (macherey-nagel inc., bethlehem, pa, usa). the -and -terminal regions were determined by using a kit for rapid amplification of and cdna ends ( and -race, clontech, japan). sequencing was performed at first base laboratory sdnbhd (selangor, malaysia) using an abi prism xl dna sequencer. the full-length pedv genome sequence was assembled, and nucleotide and deduced amino acid sequences were aligned and analyzed using the clustalw program [ ] . the percentages of homology between the sequences at the nucleotide and amino acid levels were calculated. to investigate its evolutionary relationships to foreign pdcov strains and estimate the divergence time of pdcov in thailand, phylogenetic analysis of the full-length nucleotide sequence of the pdcov isolate collected in this study, together with other pdcov isolate sequences (supplementary table ), was performed using the bayesian markov chain monte carlo (bmcmc) method implemented in the program beast v . . [ , ] . a beast run was performed based on the gtr?g?i model with a coalescent bayesian skyline tree prior and a clock model for each analysis using million generations with sampling of every , generations and the first % discarded as burn-in. tracer v . was used to confirm that post-burn-in trees yielded an effective sample size (ess) of [ for all parameters. the resulting tree was viewed and generated in figtree v . . . the results of testing for pdcov, pedv, and tgev in intestinal samples are shown in table . of intestinal lopburi and chonburi. these four provinces contain three major swine-producing areas of thailand. these results suggest that pdcov is widespread throughout thailand. it is noteworthy that samples ( . %) were positive for pedv and that samples that were positive for pdcov were also positive for pedv. the results suggest the presence of mixed infections with pedv and pdcov in pigs. the production records of the herd in which pdcov was first detected showed that there had been three diarrhea outbreaks during and , and both pedv and pdcov were detected. in three diarrhea outbreaks, the pre-weaning mortality rate was . , . and . %, respectively. however, we were unable to define the relationship between pedv and pdcov regarding to the primary or secondary causes of the diarrhea outbreaks. whether coinfection with these viruses would necessitate more-complex control methods or would result in more-frequent, repetitive outbreaks requires further investigation. the pairwise nucleotide and amino acid sequence identity values of the full-length genome sequences of the four thai pdcov isolates are shown in table . these sequences were . . % identical at the nucleotide and . - . % identical at the amino acid level and were more closely related to each other and to variants from china and the united states than to thai strains isolated in , suggesting that the thai pdcov population is continuously changing. phylogenetic analysis based on the full-length genome sequence demonstrated that pdcov isolates are divided into two groups, the chinese-and us-like-groups, as was reported previously [ ] . all four thai pdcov isolates were grouped in a novel cluster separate from variants from the united states and china but were more closely related to the chinese isolates than to the us pdcov isolates (fig. ) . the currently circulating isolates of pdcov share a common ancestor that was present around , with % hpd values covering years from to (fig. (fig. ) . the analysis of amino acid changes also suggests that they are from a different lineage. in conclusion, we retrospectively investigated the presence of pdcov in thailand and found that pdcov has been present in thailand since at least february and that it is present in mixed infections with pedv. interestingly, the pdcov variants in thailand are different from variants in other countries and belong to a novel group of pdcov strains. evolutionary analysis suggested that the thai pdcov isolates likely diverged from a different ancestor from that of the chinese and us pdcov isolates, and the separation occurred after . this study provides interesting results associated with the emergence and the evolution of pdcov. further investigation, including more retrospective studies of available samples, will provide a better understanding of this pathogen. porcine deltacoronavirus in mainland china beast: bayesian evolutionary analysis by sampling trees bayesian phylogenetics with beauti and the beast . pathogenicity of porcine deltacoronavirus strains in gnotobiotic pigs detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex rt-pcr complete genome characterization of korean porcine deltacoronavirus strain kor/knu - full-length genome sequence of porcine deltacoronavirus strain usa/ia/ / the genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in thai the first detection and full-length genome sequence of porcine deltacoronavirus isolated in lao pdr different lineage of porcine deltacoronavirus in thailand, vietnam and lao pdr in pcrbased retrospective evaluation of diagnostic samples for porcine deltacoronavirus in thailand emergence of porcine deltacoronavirus in us swine genetic diversity of orf and spike genes of porcine epidemic diarrhea virus in thailand clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice 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 acknowledgements this work was supported by the national research council of thailand (grant number ) and the agricultural research development agency (public organization). we gratefully acknowledge the graduate scholarship program of chulalongkorn university and the funding provided by the special task force for activating research (star), swine viral evolution and vaccine research (svevr), chulalongkorn university. the study was funded by the thailand research fund (grant number mrg ) and the national research council of thailand (grant number ). conflict of interest the authors declare that they have no conflicts of interest related to this work.ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. key: cord- -g vcy ea authors: carstens, e. b.; ball, l. a. title: ratification vote on taxonomic proposals to the international committee on taxonomy of viruses ( ) date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: g vcy ea in accordance with the statutes of the international committee of taxonomy of viruses (ictv), the final stage in the process of making changes to the universal scheme of virus classification is the ratification of taxonomic proposals by ictv members. this can occur either at a plenary meeting of ictv, held during an international congress of virology meeting, or by circulation of proposals by mail followed by a ballot. therefore, a list of proposals that had been subjected to the full, multi-stage review process was prepared and presented on the ictvonline web pages in march . this review process involved input from the ictv study groups and subcommittees, other interested virologists, and the ictv executive committee. for the first time, the ratification process was performed entirely by email. the proposals were sent electronically via email on march to ictv life members ( ), ictv subcommittee members ( ), and ictv national representatives ( ). in accordance with the statutes of the international committee on taxonomy of viruses (ictv), the final stage in the process of making changes to the universal scheme of virus classification is the ratification of taxonomic proposals by ictv members. this can occur either at a plenary meeting of ictv, held during an international congress of virology meeting, or by circulation of proposals by mail, followed by a ballot. therefore, a list of proposals that had been subjected to the full, multi-stage review process was prepared and presented on the ict-vonline web pages in march . this review process involved input from the ictv study groups and subcommittees, other interested virologists, and the ictv executive committee. for the first time, the ratification process was performed entirely by email. the proposals were sent electronically via email on march , to ictv life members ( ), ictv subcommittee members ( ), and ictv national representatives ( ). members were then requested to vote on whether to ratify the taxonomic proposals, within a -month deadline. international committee on taxonomy of viruses attempts to keep an up-to-date list of all members, including national representatives, but this is a daunting task. as a means of promoting contact between ictv and its national members and to ensure that lists of members are up to date, member societies of iums are obliged to either renew ictv national members or replace them every three years. in effect, national members have a -year tenure that can be renewed indefinitely. national societies, which are members of the international union of microbiological societies, are encouraged to contact ictv directly through the ictv secretary with the names of their national representatives. complete contact information can be obtained from the ictv web site: http://ictvonline.org. the following are the taxonomic proposals that were ratified by ictv members in april . (this is not strictly necessary as a taxonomic proposal because it concerns entities below the species level, but it is left in to clarify this reorganization of the herpesviridae. these viruses are reassigned to new taxa below). ( . - v are not strictly necessary as taxonomic proposals because they concern entities below the species level, but they are left in to clarify this reorganization of the herpesviridae). . v. to rename callitrichine herpesvirus , an unassigned species in the subfamily gammaherpesvirinae in the family herpesviridae: saguinine herpesvirus . . v. to assign the following viruses to the species human enterovirus c in the existing genus enterovirus in the family picornaviridae: human poliovirus , human poliovirus , human poliovirus . (this is not strictly necessary as a taxonomic proposal because it concerns entities below the species level, but it is left in to clarify this reorganization of the picornaviridae p- p. to create a new genus and to name it elaviroid. . p. to assign the species eggplant latent viroid as the type species of the new genus prokaryote virus subcommittee ampullaviruses . b- b. to create a new genus and to name it ampullavirus. . b. to designate the species acidianus bottle-shaped virus as the type species of the new genus created in . b. . b. to create the following as a species of the new genus created in . b: acidianus bottle-shaped virus. . b- b. to create a new family and to name it ampullaviridae. . b. to designate the following genus as part of the new family created in . b: ampullavirus. globuloviruses . - b. to create a new unassigned genus named globulovirus. . b. to designate the species pyrobaculum spherical virus as the type species of the new genus created in . b. . b. to create the following as a species of the new genus created in . b: pyrobaculum spherical virus. . b- b. to create a new family and to name it globuloviridae. . b. to designate the following genera as belonging to the new family created in . b: globulovirus. . b. to create the following as species in the genus globulovirus in the family globuloviridae: thermoprotheus tenax spherical virus . lipothrixviruses . b. - b. to create a new genus in the family lipothrixviridae and to name virus taxonomy: eighth report of the international committee on taxonomy of viruses . b. to create the following as a species in the genus rudivirus in the family rudiviridae: acidianus rodshaped virus . . g. to spell out greek letters in all taxon names, including the names of virus species, in full roman typography: e.g. alpha, beta, gamma, delta,…lambda, mu, etc. (note: while this proposal may not be popular in some quarters, it is forced on the ictv by computer search capabilities which are beyond our control). the ictv executive committee unanimously nominates the following eminent virologists and long-term secretaries of the ictv to life membership of the ictv: as a result of the vote, all of the above taxonomic proposals were approved by the membership. they are now a part of the official ictv taxonomy. a list of the approved taxa can be found on the ictv online web site. the most recent report of ictv was published in [ ] . ictv is currently updating this information and expects to publish its th report in .for further information, please visit the ictv web pages at http://www.ictvonline.org and http://talk.ictvonline.org/. key: cord- - yvs a authors: han, tae-hee; chung, ju-young; hwang, eung-soo; koo, ja-wook title: detection of human rhinovirus c in children with acute lower respiratory tract infections in south korea date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: yvs a recently, hrv-c was identified as a new species of hrv, but its spectrum of clinical disease is still not clear. the purpose of this study was to investigate the molecular epidemiology of hrvs in children with acute lower respiratory tract infections (lrtis). a total of hrv-positive samples that were negative for other respiratory viruses were sequenced. hrv-a was detected in , hrv-b in , and hrv-c in of these samples. all hrv-c-positive patients showed favorable clinical outcomes. we confirmed the presence of hrv-c in children with lrtis, but its association with clinical severity is not clear. human rhinoviruses (hrvs) are the most frequent cause of acute respiratory illness worldwide [ ] [ ] [ ] [ ] [ ] . although hrvs are most commonly associated with mild upper respiratory tract disease, infection of lower airways does occur [ , ] . lower respiratory tract infections (lrtis), especially in infants, the elderly, and immunocompromised patients are increasingly being reported [ ] [ ] [ ] . hrvs are currently classified into two species, hrv-a and hrv-b, in the genus rhinovirus of the family picornaviridae [ ] . phylogenetic analysis of the vp /vp and vp coding regions indicated the presence of serotypes in genetic group a and serotypes in genetic group b [ , ] . in recent studies, a member of a newly identified species, hrv-c, has been suggested as an etiologic agent in children with acute respiratory disease such as bronchiolitis, pneumonia, and asthma exacerbation [ ] [ ] [ ] [ ] [ ] . the purpose of this study was to investigate the molecular epidemiology of hrvs in children hospitalized with acute lrtis in south korea. from january to december , a total of nasopharyngeal aspirates were collected from hospitalized children (male/female, / ; median age, months; range of age, - months) with acute lrtis at sanggye-paik hospital, seoul, south korea. all specimens were tested for the presence of human respiratory syncytial virus (hrsv), influenza virus a, influenza virus b, parainfluenzavirus, adenovirus, human metapneumovirus (hmpv), human bocavirus (hbov), and human coronaviruses (- e, -oc , -hku- , and -nl ) by rt-pcr, as described in our previous study [ ] . from the hrv-positive samples, a total of samples that were negative for other respiratory viruses were included in this study for subsequent sequence analysis. viral rna was extracted from each sample using a qiaamp viral mini kit (qiagen gmbh, hilden, germany) according to the manufacturer's instructions. rna was quantitated using nanodrop (thermo scientific, wilmington, de, usa). reverse transcription was performed on . lg of . after incubation at °c for h, the samples were heated for min at °c to stop the reaction. semi-nested pcr for amplification of * bp of the noncoding region of hrvs from clinical specimens was performed. primer p - (caagcac ttctgtywcccc nt - , reference strain l ) was used as the forward primer, and multiple primers were used as reverse primers: p - (acggacacccaaagtag, nt - ), p - (ttagccacattcaggggc, nt - ), p - (ttagccacattcaggagcc, nt - ) and p - (ttagccgcattcagggg, nt - ), as described previously [ ] . a first round of pcr was performed on the samples using p - and p - , followed by a second round of pcr using p - , p - , p - , and p - . pcr was done using the following reaction conditions: initial denaturation at °c for min; cycles of °c for s, °c for s, and °c for min; and final extension at °c for min. in the pcr reaction, a forward primer ( accractactttgggtgtccgtgt , position - in hrv-c ) and a reverse primer ( tciggiadyttccaicaccaicc , position - in hrv-c ) were used to generate a * -bp pcr product encompassing a portion of the untranslated region, the full viral capsid protein (vp) gene, and a portion of the vp gene of the hrv genome. the pcr was done using the following reaction conditions: initial denaturation at °c, min; cycles of °c for s, °c for s, and °c for min; and final extension at °c for min. the amplicon was purified using a qiaquick kit (qiagen gmbh, hilden, germany) and sequenced in both directions using a bigdye terminator v . cycle sequencing kit (applied biosystems, foster city, ca, usa). sequencing products were resolved using an abi xl autoanalyzer (applied biosystems, foster city, ca, usa). nucleotides sequences were aligned using bioedit v . and presented in a topology tree, prepared in mega . [ ] . partial ncr sequences (* bp, nt - of genbank accession no. l ) for the hrv strain were submitted to genbank (fj - , fj - ) . a total of single hrv-positive specimens from children hospitalized with acute lrtis were sequenced after performing rt-pcr based on the ncr region. phylogenetic analysis based on -ncr gene analysis showed that of the hrv strains were hrv-a, were hrv-b, were hrv-c, and the species was undetermined for ( fig. ) . rt-pcr assays based on the vp /vp region were performed to determine the species, and phylogenetic analysis was possible with specimens, which showed that were hrv-a, were hrv-b, and were hrv-c. nine strains for which the species was not determined from the noncoding region belonged to hrv-a in cases and hrv-b in (fig. ) . hrv-c was detected in patients ( boys and girls, months to months of age (mean age months, median age months)) and the diagnoses were asthma exacerbation in patients, bronchiolitis in , and pneumonia in . none of the hrv-c-positive patients required admission to the intensive care unit, and their clinical outcomes were favorable. hrv-c was detected mostly in the spring, while hrv-a showed a peak in september . co-circulation of hrv-a and hrv-c was noted in spring and autumn. to our knowledge, this is the first study to confirm the presence of hrv c infection in children with acute lrtis in korea. recently, novel hrv species were identified and their members were reported to be associated with acute respiratory tract infections with febrile wheeze, asthmatic exacerbation, influenza-like illness, pneumonia and rhinitis [ , , , ] . although several novel hrv species have been identified due to the development of molecular methodology, it is difficult to compare these novel hrvs because different regions of the genome have been used for analysis. in recent studies [ , ] , molecular typing of rhinovirus using the -ncr region has been suggested to be a simple and reliable method for classifying hrv serotypes, because analysis of the vp- or vp -vp region requires multiple primer pairs for rt-pcr. an association of members of novel hrv species with severe respiratory tract infections [ , ] and a global distribution of members of novel species in respiratory specimens have fig. phylogenetic tree of clinical viral isolates (n = ) based on analysis of * bp from the noncoding region (nt - of hrv l ). the phylogenetic tree was built using the neighborjoining method with the kimura two-parameter estimation. bootstrap values from , replicates are shown next to the branches. hrvc strains include strains from korea (kr), strains from wisconsin (eu and eu ), strain qpm from australia (ef ), strain - from hong kong (ef - ), strain nat from ucsf (ef ), and strain nat from ucsf (ef ). group b strains include strains from korea, hrv (ef ), and hrv (ef ). group a strains include strains from korea. nine strains from korea did not belong to groups a-c. been reported based on analysis of vp- and - genomes [ ] . lee et al. [ ] and kiang et al. [ ] reported that a genuine hrv-c, distinct from hrv-a and hrv-b, could be identified by pcr analysis based on the ncr region, and some strains that appeared to represent novel species, including the qpm strain described by mcerlean et al. [ ] , the strains by lamson et al. [ ] and hong kong strains [ ] , may be hrv-a variants rather than hrv-c. in the present study, phylogenetic analysis of the ncr region showed that qpm, hrv-c strain and hrv x were grouped into the hrv-c species, but strains could not be identified. in subsequent analysis of the vp /vp based on analysis of * bp from the vp /vp region. the phylogenetic tree was built using the neighbor-joining method with the kimura twoparameter estimation. bootstrap values from , replicates are shown next to the branches. nine strains whose groups were not determined from noncoding region belonged to hrv-a and hrv-b. the scale bar indicates the estimated number of substitutions per bases region, all of the strains that were not identified by ncr region analysis were identified as hrv-a (in cases) or hrv-b (in cases). these results indicate that the ncr may be useful for classifying novel species of hrv, but identification of serotype based on comparison of nucleotide sequences from the ncr should be used with caution. in this study, hrv-c infection did not require admission to the intensive care unit and prognosis was good, which is different from what has been found in previous studies [ , ] . in conclusion, hrv-c and hrv-a were co-circulating in children hospitalized with lrtis in korea in , implying a possible role of hrv-c in lrtis. however, further studies are needed to standardize diagnostic methods for detection of hrv-c infection and to determine its association with a severe clinical course. rhinoviruses. in: fields virology rhinoviruses infect the lower airways virological and serological analysis of rhinovirus infections during the first two years of life in a cohort of children asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection role of rhinovirus in hospitalized infants with respiratory tract infections in spain viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation virological diagnosis in community-acquired pneumonia in immunocompromised patients picornaviridae phylogenetic analysis of rhinovirus isolated collected during successive epidemic seasons clinical features and complete genome characterization of a distinct rhinovirus genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children a diverse group of previously unrecognized human rhinoviruses are common cause of respiratory illness in infants characterization of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis mass taq polymerasechain reaction detection of respiratory pathogens, including a new rhinovirus genotype, that causes influenza-like illness in new york state during novel human rhinoviruses and exacerbation of asthma in children detection of viruses identified recently in children with acute wheezing mega : molecular evolutionary genetics analysis (mega) software version . global distribution of novel rhinovirus genotype an assay for -noncoding region analysis of all human rhinoviruses prototype strains a recently identified rhinovirus genotype is associated with severe respiratory tract infection in children in germany molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality acknowledgments this study was partly supported by research grant ( ) by inje university. key: cord- -foaf vyl authors: weiss, marianne; horzinek, m. c. title: the proposed family toroviridae: agents of enteric infections date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: foaf vyl nan in and two new viruses detected in fecal material from cattle and horse, respectively, were described ( , ), which could not be assigned to any known virus family. results gained since then from morphological, biochemical and serological studies demonstrated the unique t~atures of these viruses and justified the proposal of a new virus family, provisionally named "toroviridae" (from latin torus= a doughnut shaped ring) ( ) . berne virus, the best studied representative was isolated from a rectal swab of a diarrheic horse during routine diagnostic work in berne (switzerland) in ( ) . in woode et at. ( ) described the isolation of a virus during an acute epizootie of neonatal calf diarrhea in breda, iowa, u.s.a. (breda virus ). antigeniemly related viruses were later found in feces from a colostrum-deprived calf in iowa (breda virus ) ( ) and from to months old diarrheic calves in ohio, u.s.a. ( ) . a morphologically similar virus (lyon virus) detected in cattle in lyon, france ( , ) , was shown later to possess an antigenic relatedness to the berne (bev) and breda (brv) viruses. beards et al. ( ) reported in particles resembling bev and brv in stool specimens of children and adults with diarrhea, which reacted with antibodies against bev and brv in immunoelectron microscopy. similar particles were seen by schaap in feces of children with gastroenteritis in rotterdam ( ) . consequently, members of the torovirus family presently recognized are enteric viruses from three different, species. from serological studies, however, the presence of toroviruses in other animals became evident. this review summarizes the current knowledge of the properties of this new group of viruses. in negatively stained preparations toroviruses are pleomorphic and measure to nm in their largest diameter. they were described as spherical, oval, elongated or kidney-shaped particles (fig. l) consisting of" a peplomer-bearing envelope and a sausage-like internal structure with transverse striation (estimated periodicity about . nm) ( , ) . bev projections measure nm in length. they have a drumstick shape and consist of a thin stalk carrying a distal sphcrule ( ) . brv were described to possess . - . nm pcplomers; few particles showed irregularly arranged processes of - nm thought by woode et al. to represent tissue debris ( ). the longer peplomers were more frequently seen in brv than brv preparations. particles from human feces were surrounded by a halo of - nm projections; occasionally a second ring of small peplomers was noticed, partly superimposed upon the first. longer projections were only occasionally observed ( ) . in thin sections through bev infected cells (horse kidney, embryonic mule skin, equine dermal cells) densely staining spherical, elliptiem and elongated particles were detected ( , ) . a clear distinction between an inner structure of high electron density, apparently corresponding to the nucleocapsid, and a less dense outer region can be made. spherical and elliptical particles enclosing a crescent-shaped core are prevalent in the extracellular space. enveloped twin circular structures with a light centre are interpreted as cross-sections through virions containing a hollow tubular nucleocapsid bent into an open torus (fig. ) . bacilliform viruses with a rodlike core are on the right electron mierographs of bev particles, on the left schematic interpretations of the viral structures seen in the corresponding photographs are shown, a virion with a toroidal core within a circular particle out, line. the indicated section plane leeds to a biconcave structure with twin circular cross-sections of the core, b. section plane cuts the nueleocapsid only once, c. d elliptical virion with little resolution of the interior, e rod-shaped particle, f circular structure with an electron-lucent center corresponding to ~ cross-section through a rod-shaped particle, g virion with a c-shaped nucleocapsid; in contrast to a the envelope follows the smaller curvature of the torus, h cross-section through g cutting the nucleocapsid twice encountered in cytoplasmic vacuoles. cross-sections through these rod-like particles revealed three concentric circles of high electron density. the outer circles measured and nm, respectively, in diameter. the innermost circle of highest electron density (diameter nm) is thought to represent a transversal section through the nueleoeapsid. its electron lucent centre is indicative for the tubular structure of the core. thin sections through brv infected intestinal cells of calves ( , , ) showed elongated viral particles with rounded ends measuring × . nm. in cross-sections a core of high electron density with an electron lucent, central channel could be discerned from a fuzzy outer membrane of medium electron density. for both viruses the core was reported to measure - nm in diameter ( , , ) . the length of the nueleocapsid can only be approximated; it depends on the orientation in space of the virion and the plane of section. in bev a mean length of nm (_+ nm, n = ) was calculated, but cores exceeding nm in length have been encountered ( , ) . it was concluded that, toroviruses are enveloped, peplomer-bearing particles "containing an elongated tubular nueleocapsid of presumably helical symmetry. the eapsid may be bent into an open toms, conferring a disk-or kidney-shaped morphology to the virion (largest diameter - nm) or straight, resulting in a rod-shaped particle (dimensions × nm)" ( ). proteins: in polyacrylamide gel electrophoresis (page) bev and brv proteins showed quite similar patterns of molecular weights (mol. wts). metabolically labelled bev preparations revealed structural proteins in the range of - , , , and kd ( , ) . in radioiodinated purified intact bev the and kd proteins were labelled; when the preparation was treated with triton x- , the kd polypeptide was labelled in addition ( fig. ) . radioiodinated brv polypeptides were encountered with apparent mol. wts of , , k and and in the k range ( ) . the and kd proteins of bev are both phosphorylated. the kd protein is the :most prevalent protein in bev, accounting tbr about per cent of the total protein mass. it has i~na binding properties and was found in an intraeellular substructure of higher density than tlhe virion (d = . g/ ml in csc ). it is therefore suggested to represent the main capsid protein ( ) . in radioimmune precipitation (rip) it, was recognized preferentially by heterologous sera (cattle), another indication that the kd protein corresponds to an internal, evolutionary conserved, and broadly crossreactive protein. second in abundance (about per cent of the virion protein mass) is the kd protein. it is neither phosphorylated nor glycosylated. after treatment, of virions with triton x- the kd polypeptide was found in slowly sedimenting material, an observation indicative of its membrane association. the kd protein is therefore a likely candidate for an envelope protein ( ) , (horzinek et al., unpublished results). the low molecular weight polypeptide of b r v does not comigrate in page with the kd nucleocapsid protein of bev and seems to be larger. its migration behaviour was affected by ether extraction suggesting a membrane association and a correspondance to the kd protein of bev ( ) . no function could be attributed to the phosphorylated kd protein so far; it is assumed to serve as a matrix protein. the high molecular weight virion protein in the range of - kd of bev is glycosylated, probably by n-linked oligosaccharides since tunicamycin, an antibiotic known to inhibit n-linked oligosaccharide synthesis, prevented the formation of infectious virus as well as appearance of the - kd band in page of infected cells ( ) . in rip the - kd protein of bev was preferably recognized by a horse antiserum with high neutralization activity ( ) . radioiodinated b r v preparations contained genome: bev replication is not inhibited by dna nueleotide analogous which indicates the presence of an rna genome ( ). one type of single stranded rna molecule with a mol. wt of about . × was isolated from virus particles. the virion rna was shown to be infectious and a positive polarity is suggested. polyadenylation of genomie rna was apparent from oligo dt affinity chromatography and ti l~nase fingerprints. (horzlnet~ et al., unpublished results). in a linear sucrose gradient a virion buoyant density of . to . g/ml was determined for bev ( ) . under the same conditions brv banded at . g/ml. for bev and bt~v sedimentation coefficients of and s, respectively, were estimated ( ) . in addition to the main infectivity ( s) peak a second virus specific peak of slower sedimentation ( to s) was detected in isokinetie sucrose gradients of bev; these particles contained smaller virus specific rna molecules and the kd protein. they were non-infectious and are probably non-interfering. also in bi%v preparations a second peak ( s) with a hemagglutinating activity was encountered. nature and significance of these subviral particles require further study. t~esistanee bev is readily inactivated by heat but well preserved if stored at temperatures below - ° c ( ) . desiccation and freeze-drying resulted in insignificant losses of infectivity. bev is very sensitive to lvv-ix'raditation. a high stability to extreme hydrogen ion concentrations was noted; infectivity titers remained unchanged in a ph range between . to . . pronase and b. subtilis proteinase reduced bev virus infectivity whereas treatment with trypsin and chymotrypsin remained without effect. neither phospholipase c, %nase nor doc ( . per cent) affected the titer of purified bev preparations. triton x- in contrast, lead to rapid inactivation with a constant level of residual infectivity. organic solvents and formalin destroyed the viral infectivity completely. in vivo: brv in calves was shown by if and em to replicate in epithelial cells of the colon, the caudal part of the jejunum and the ileum. no viral antigen was discovered in the epithelium of the anterior part of the jejunum nor in subepithelial tissue. cells of both the crypts and villi in mid to lower jejunum and ileum are infected as are most cells of the large intestine ( , , , , ) . in vitro: bev, originally isolated in secondary horse kidney cells, can be propagated in cell cultures of equine origin (horse kidney, embryonic horse lung, embryonic mule skin and equine dermal cells) ( ) . attempts to grow bev in cells originating from man, monkey, cattle, pig, rabbit, mouse, hamster, were unsuccessful (weiss, unpublished results). neither brv nor the human torovirus particles could be adapted to gro~h in culture so far ( , ) . hemagglutination brv and bev possess hemagglutinating activity whereas no such property could be demonstrated for the human torovirus particles up to now. hemagglutination of bt~v was only obtained with mouse and rat erythrocytes (ecs). all attempts with ecs from other species (human group o, bovine, hamster, guinea pig, chicken, turkey, goose) remained without success ( ). in contrast, bev agglutinates (in decreasing order) ecs from human (group ), rabbit and guinea pig but not from rat and mouse or other ( ) . by electron microscopy bev particles were shown to bind with the peplomers to the ec surface (fig. ) . evidence was obtained that, the human erythroe ~e receptors for bev are glycoproteins or glyeolipids. the strain p / of bev is the only equine torovirus strain isolated so far ( ) . in contrast several brv isolates were obtained from cattle. three of them were compared antigenically and were found to be related ( ) . no distinction between them was noted in immunofluorescence (if) tests. on the basis of results obtained in hemagglutination inhibition (hi), enzymelinked immunosorbent assay (elisa) and immunoelectron microscopy (iem) however, they were divided into serotypes: serotype (= breda ) is represented by the iowa isolate , serotype (= breda ) by the ohio isolate and the iowa isola.te . bev and brv share antigens as evidenced by cross reactions in different, serological tests. bev preparations reacted in seroneutrmization, elisa and rip with bovine sera ( , ) , and positive reactions were noted between brv antigens and bev antibodies in if and elisa but not in a hi test (woode, personal communication). a mouse serum raised against breda and known to recognize not. only the homologous proteins but also the and kd proteins of the heterologous brv in rip, inhibited hemagglutination of the heterologous serotype to a low but significant degree and neutralized the infectivity of bev ( ) . monoclonal antibodies produced against bev and recognizing the - kd protein in rip showed neutralizing and hemagglutination inhibition properties ( ) (fig. ) . from these data and the observation that a horse field serum with a high neutralizing activity against bev preferentially recognized the - kd protein ( ) it is concluded that the erossreaeting antigens are represented by the high mol. wt glyeoproteins. their apparent involvement in neutrmization and tti makes them candidate for peplomer proteins. the preferential recognition of the kd nueleocapsid protein of bev by heterologous cattle sera mentioned above may indicate a broadly reacting group specitic antigen. a positive if was noted with lyon virus and bev antibodies in horse sera, and antisera t?om cattle positive for lyon reacted in seroneutralization, elisa and rip with bev ( , ) . evidence of a reaction of the particles in human feces with sera containing antibodies against brv and bev, respectively, were obtained in iem ( ), (flewett personal communication). further tests, however, are needed to confirm the antigenie relatedness of the putative human torovirus with bt~v and bev. for bev a purification procedure was developed ( ) . supernatants from infected tissue cultures were mixed with ammonium sulphate (end concentration to per cent) and the resuspended and clarified precipitate was layered on top of a linear to per cent (w/w) sucrose gradient,. after centrifugation to equilibrium and fractionation, the samples were monitored for the presence of virus antigen by indirect elisa. for some experiments, e.g. preparation of elisa antigen ibr serology, a lower degree of purity was sufficient. in this case the precipitated and resuspended material was sedimented through a per cent sucrose layer onto a per cent sucrose cushion. brv was purified from diarrheic feces ( , ) . fecal material was diluted : to : and clarified by low speed centrifugation. depending on the degree of purity desired, the supernatant was either used directly, pelleted at , to , × g; or further purified by the methods described for bev. in bev infected e. derm cells an increase in extracellular infectivity was noted between the th and th hour after infection, and a plateau was reached at about hours. a pronounced cytepathic effect (cpe) was evident about hours post infection. in the presence of actinomycin d and a-amanitin, bev replication was drastically decreased when the drugs were added during the first hours after infection. uw preirradiation of the cells also interfered with bev multiplication. from these results it appears that bev replication depends on some nuclear function of the host cell ( ) . (fig. ) . in infected e. derm cells bev is assembled by budding of a preformed rigid nucleocapsid through intracytoptasmic membranes ( ) . tubular structures, representing nucleocapsids, are formed in places distant from the budding site. they are often seen in immediate proximity of cytoplasmic accumulations of an electron dense granular substance supposed to consist of ~iral material. tubules were not only encountered in the cytoplasm but budding of virus particles occurs predominantly into the golgi system but was also observed through membranes of the rough endoplasmic reticulum and into the perinuclear space. the following sequence of events was reconstructed: the intracytoplasmic nucleoeapsid becomes attached to the membrane with one of its rounded ends and subsequently sideways. during budding the eapsid apparently acquires its definite diameter and electron density. as a result of the budding process an enveloped bacilliform virus particle is found free in the lumen of the cytoplasmic cisternae (fig. ) . virus containing vesicles merge with the peripheral plasma membrane and release their contents. during transition from the intravesieular to the extracellular state the morphology of virus particles changes from the rod-like form to the characteristic torus form (fig. ) . b r v morphogenesis cannot be followed sequentially since virus propagation has so far not been achieved in tissue culture. the available data, however ( ) , indicate that the brv morphopoiesis is similar to that of bev. tubular structures were encountered in the cytoplasm and nucleus and enveloped viral particles were seen predominantly in vesicles of the golgi system. virus containing vesicles appear to move to the cell surface and to release their contents by fusion with the plasma membrane. all brv particles encountered in ultrathin sections were elongated and bacilliform. berne virus was isolated from a rectal swab of a diarrheic horse ( ); whether it had caused this disease could not be proven. in a few experimental and naturally oecuring bev infections clinical signs were not noted ( ) . consequently, a disease picture cannot be attributed to bev so far. brv in contrast, was first isolated during an acute epizootic of calf diarrhea ( ) , and was later shown to cause diarrhea of varying severity, both in colostrum-deprived and gnotobiotic calves ( , ) . in man the torovirus-like particles were also found in association with diarrhea ( ) . pathological and histopathological data are only available from brv infected calves ( , , i ). lesions were seen in the intestinal mueosa of the middle to caudal part of the jejunum, of the ileum, cecum, spiral colon and descending colon. they consisted of villus atrophy and necrosis of epithelial cells covering villi and crypts. in addition, an acute inflammatory response with cellular infiltration and subtle changes in capillaries were noted in the altered regions. the infected cells showed a distension of the cytocavitary network, a dilation of the golgi complex, the appearance of autophagolysosomes, shortened mierovilli and degenerated mitochondria. viral antigen and particles could be demonstrated in the affected parts of the intestine by indirect if and em, respectively. toroviruses have been demonstrated in the horse, in cattle and man, and they were occasionally seen in pigs (saif, unpublished results). these are not the only host species of toroviruses. from serological examinations evidence was obtained that bev-related viruses are prevalent in other ungulates (cattle, goat, sheep, pig), in tbral mice and in laboratory rabbits ( ) . in per cent of cattle, per cent of goat, per cent of sheep, and per cent of pig sera tested high antibody tigers cross-reacting with bev in seroneutralization were detected. low neutralization titers were found in sera of laboratory rabbits and in two species of wild mice (apodemus sylvaticus, ciethrionomys glareolus); they may indicate the presence of more distant serotypes. the high tigers encountered in ungulates, however, are thought to be due to viruses serologically more closely related to bev. neutralization tests are known to be very sensitive and specific. assays for group specific antigens should elucidate the actual prevalence of toroviruses in different animal species and probably explain erratic inhibitions obtained with feline and human sera in the seroneutralization tests. it is not known whether toroviruses are restricted to their respective hosts or interspecies transmission occurs. toroviruses are widespread in horse and cattle populations as evidenced by the presence of antibody titers. in switzerland, per cent of randomly collected sera (n= ) from adult horses contained antibodies reacting with bev in a seroneutralization test. antibodies against bev were also encountered in small numbers of randomly collected equine sera from germany, france, italy and the u.s.a. ( ) . in cattle sera from the u.s.a. (n = ) . per cent were reported positive in an elisa using brv as an antigen (t ). the way of spread of torovirus infections is not known. brv was experimentally transmitted by oral administration ( , ) , and an oral-fecal route of transmission is likely to occur in nature. only few data on morbidity and mortality of torovirus infections are available so far. the infection rate apparently can be high. in a beef herd in iowa from which brv was originally isolated, out of newborn calves developed diarrhea and animals died ( ) . a sudden seroconversion indicating an infection with bev was noted in all animals of a herd of foals in switzerland ( ) ; no clinical signs had been observed in this case, in the same herd, followed serologically to the age of one year, maternal antibodies against bev were detected. three to months after birth their titers had dropped below detection level. in calves brv antibodies as measured by elisa were encountered f~om the th month of age onwards; young calves became seronegative about weeks after birth (va:z de boom et al., unpublished results). several intestinal viruses have been recognized during the last fifteen years (e.g. astro-, calici-, parvo-, corona-viruses). enteric infections are also caused by the toroviruses, a group of animal viruses clearly separated from other families by their distinctive properties. three members are known so far, but others will certainly be discovered. some features determined for bev, e.g. stability to low ph, resistance to sodium deoxycholate, trypsin and chymotrypsin, prove them as well adapted to an intestinal environment. little, however, is known at, present of their pathogenic potency. bev has to be considered as a virus hi search of disease and the significance of torovirus particles seen in connection with diarrhea in humans needs further study. brv were shown to cause severe disease in newborn calves deprived of maternal antibodies. it is unknown, however, whether they are able to induce symptoms in normally reared calves alone or only in combination with other agents. apart from their eventual clinical and epidemiological significance, toroviruses will attract special interest due to their novel virion architecture. particles with such a polymorphic appearance in negatively stained preparations most certainly have been seen by many electron microscopists and dismissed as non viral. characterization of this family of viruses will studies with an unclassified virus isolated from diarrheic calves purification and partial characterization of a new enveloped i~na virus (berne virus) toroviridae: a taxonomic proposal po~ilenz jf { ) diagnostic methods for the newly discovered "breda" group of calf enteritis inducing viruses studies of an enteric "breda" virus in calves. nd ann meet conf res workers in anita dis nouveaux virus intervenant dans l' tio-logie des ent~rites n onatales des bovins nouvelle morphologie virale assoei e £ une infection bovine. note pr iminaire an enveloped virus in stools of children and adults with gastroenteritis that, resembles the breda virus of calves morphogenesis of berne virus (proposed f~mily toroviridae) cellular lesions in intestinal mucosa of gnotobiotic calves experimentally infected with a new unclassified bovine virus (breda virus) astrovirus and breda virus infections of dome cell epithelium of bovine ileum a morphologic study of the replication of breda virus (proposed family toroviridae) in bovine intestinal cells berne virus is not "coronavirus-like the nucleocapsid of berne virus the surface proteins of breda virus resistance of berne virus to physical and chemical treatment comparative studies on three isolates of breda virus of calves antibodies to berne virus in horses and other animals an elisa for serologic studies on breda virus the peplomers of berne virus the haemagglutinating activity of berne virus provide new insights at the molecular level, especially concerning virus replication. key: cord- -mgzmzeyr authors: olofsson, s.; norrild, bodil; andersen, Åse b.; pereira, leonore; jeansson, s.; lycke, e. title: populations of herpes simplex virus glycoprotein gc with and without affinity for the n-acetyl-galactosamine specific lectin ofhelix pomatia date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: mgzmzeyr two fractions of herpes simplex virus glycoprotein gc were isolated and characterized by means of immunosorbent-purification with monoclonal antibodies against gc and helix pomatia lectin (hpa) affinity chromatography. about per cent of the glycoprotein gc population demonstrated affinity for the lectin, compatible with presence of n-acetylgalactosamine as terminal sugar of the oligosaccharide. the hpa-binding populations of gc appeared as two electrophoretic bands with lower molecular weights than the non-binding gc. the gc subfraction without affinity for the hpa was subjected to treatments aiming to desialylize the carbohydrate moiety. only per cent of the initially non-reactive fraction of gc became reactive to hpa after the treatments, suggesting that masking of penultimate n-acetylgalactosamine by sialic acid was not a main reason for lack of hpa affinity. results of treatment with alkaline na bh( ) demonstrated presence of oligosaccharide-peptide linkages sensitive to β-elimination suggesting o-glycosidic type of linkage. the subfraction of gc demonstrating affinity for hpa as well as gc devoid of hpa binding capacity both revealed affinity for con a. therefore n-glycosidically as well as o-glycosidically linked oligosaccharides seemed to be present on the one and same glycoprotein. on the basis of the results presented we assume that the glycosylation of hsv glycoprotein gc may lead to, at least, two populations of the glycoprotein gc, one with terminal n-acetylgalactosamine residues of oligosaccharides -glycosidically linked to the polypeptide and the other without affinity for hpa. however, both populations of gc contain similar proportions of oligosaccharides of the high mannose or complex types with n-glycosidic carbohydrate-peptide linkages as indicated by their affinity for con a. all known glycoproteins of enveloped viruses contain high mannose or complex type ohgosaccharides linked to the polypeptide via an n-glycosidic linkage between asparagine and n-aeetylglueosamine (glcnac) ( ) . under conditions of tunicamyein (tm) inhibition of gtycosylation in virus-infected cells, aberrant polypeptides will be formed resulting in loss of viral infectivity ( , , , , , ) . according to prevailing concepts, the main function of n-glycosidic oligosaecharides is to stabilize the polypeptide conformation and the loss of infectivity is caused only indirectly by the absence of oligosaccharides due to such secondary events as increased sensitivity to intracellutar proteolytic degradation or unspecific aggregation of underglycosylated viral glycoproteins ( ) . we have found that the type-specific glycoprotein gc of herpes simplex virus type (hsv- ) contains oligosaccharides with affinity for the n-acetylgalactosamine-binding helix pomatia lectin (i-ipa). most likely the oligosaceharide constituent of tt~e hpa-binding hsv glyeoprotein is linked to the peptide via an -glycosidic bond between n-aeetylgalaetosamine (galnac) and a serine or threonine of the peptide ( , ) . such o-glycosidic linkages of hsv ( ) , vaecinia ( ) and coronavirus ( , ) glycoproteins have been described recently. it is tempting to compare the hpa-binding i-isv gtycoproteins with several biologically highly active glyeoeonjugates such as blood group substances and lymphocyte differentiation antigens which also demonstrate hpa-binding activities ( , i) . for these types of antigens it is likely that the carbohydrate itself mediates the biological activity, a phenomenon which contrasts to the less specific biological significance of n-glycosidic oligosaceharides. recently, the function of so called stage-specific differentiation antigens have been blocked by addition of competing carbohydrate haptens ( ) . thus, the hpa-binding oligosaccharides of hsv glyeoproteins might constitute a carbohydrate structure with a more defined biological significance, probably as a recognition structure. in the present study the distribution of i-ipa-binding activity among i.isv glyeoproteins was investigated, and it was found that only a subfraetion of gc contains hpa-binding oligosaccharides while the other fra.etion contains -glycosidic oligosaeeharide without affinity for itpa. the prototype virus i-isv- (f) was propagated in vei~o cells maintained as monolayer cultures in minimal essential medium (mem) supplemented with per cent foetm calf serum (fcs). the i-isv- (hfem) ts b (i ) was a generous gift from dr. a. buehan, university of birmingham, england. this mutang, which was defective in gb at non-permissive temperature was used as a marker for ga and gb ( ) . monoclonal antibodies ttybridoma cells were produced as previously described ( ) and aseites preparations containing monoelonm antibodies against glycoproteins gc (i-ic ), ga/gb (h ) or gd (tii) ) were used. v e r o m o n o l a y e r cells grown in cm e tissue culture flasks ( × cells) were infected w i t h itsv- using a m u l t i p l i c i t y of infection of p f u / c e l l a n d a v o l u m e of ml m e m c o n t a i n i n g per cent fcs. after h o u r of a d s o r p t i o n at ° c t h e virus was aspirated a n d t h e cells overlayered from to hours postinfeetion b y t h e a d d i t i o n of ml m e m w i t h per cent fcs a n d t. y, ci/ml m e d i u m of d-( - -c)-glucosamine (specific a c t i v i t y . mci/mmol, a m e r s h a m , e n g l a n d ) . a t t h e e n d of t h e labelling period t h e cells were scraped off, washed once in p b s a n d collected as a cell pellet after e e n t r i f u g a t i o n for m i n u t e s at × g. the cell pellet from . × l s glueosamine labelled h s v infected vei~o ceils was p r e p a r e d as previously described ( ) . briefly, p a c k e d cells were m i x e d w i t h . ~ tris-hydrochloride, p i t . , a n d homogenized in a t i g h t fitting dounce homogenizer. after e e n t r i f u g a t i o n at × g t h e m e m b r a n e s r e m a i n i n g in t h e s u p e r n a t a n t were pelleted at , × g for hour. the pelleted m e m b r a n e s were suspended in . g l y e i n e -n a o h buffer, p h . , a n d centrifuged a t , × g for hour. finally, t h e w a s h e d m e m b r a n e s were suspended in s, g l y e i n e -n a o h buffer c o n t a i n i n g per cent t r i t o n x -t . t h e suspension was homogenized a n d centrifuged a t , × g for hour. the s u p e r n a t a n t was used in t t p a -a f f i n i t y c h r o m a t o g r a p h y . the t r i t o n x -i solubilized m a t e r i a l was applied on columns (diameter m m ) w i t h m l of h p a coupled to sepharose mb (pharmaeia, sweden). t h e gel was equilibrated w i t h . ~ tris buffered saline a n d . per cent t r i t o n x -f a n d was developed a t m l per h o u r w i t h t h e same buffer. proteins specifically b o u n d were eluted w i t h . ~ galnae. hsv- proteins w i t h or w i t h o u t affinity for h p a were p r e c i p i t a t e d w i t h tca a n d w a s h e d in order to c o n c e n t r a t e eluted protein. after wash t h e proteins were dried a n d s. olofsson et al.: solubilized in buffer and reacted with specific monoclonal antibodies added in optimal amounts. the antigen-antibody complexes were bound to fixed staphylococci (strain cowan-i) ( ) . the immunopreeipitates were washed extensively in p b s supplemented with . ~ nac , . per cent (w/v) sds and . , per cent (v/v) tritonx- . after further washing in the same buffer, supplemented with . ~ nac , the proteins were dissolved in disruption mixture for sds-polyacrylamide geleleetrophoresis. sds-polyacrylamide geleleetrophoresis was done according to morse et al. ( ) . the separation gel was . percent (w/v) acrylamide cross-linked with . percent (w/v) diatyltartardiamide. the gels were stained and autoradiography was done on k o d a k x r p - x -c r o a t film. desialylation to r e m o v e sialic acid h s v glycoprotein gc without affinity for h p a was treated in amounts of less t h a n m g with of neuraminidase ( unit) overnight at ° c in . acetate buffer, pi-i . . alternatively, aliquots of gc without affinity for i-ipa were subjected to t r e a t m e n t with . ~ i~eso at, ° c for hour. the solution was neutralized with solid na co~ and desalted by sephadex g- gel. filtration. the alkaline borohydride t r e a t m e n t was carried out as previously described ( ) . after fractionation by h p a chromatography t:he material was desalted on sephadex g- and dissolved in . ~ n a o h and . ~ nabtt . this mixture was incubated for hours at room t e m p e r a t u r e ( -- ° c) under nitrogen in tefloncapped tubes. the reaction was stopped by adding glacial acetic acid and the results of the ~-elimination reaction were assayed by sephadex g gel filtration. preparations of membrane proteins of hsv- infected vero cells were subjected to h p a affinity chromatography (fig. ) . two fractions one with (bound) and the other without affinity (unbound) for the leetin were collected. the hpa binding material was eluted with . m galnac. approximately per cent of the hsv glycoprotein preparation demonstrated affinity for hpa. in order to identify the glycoproteins present both fractions were immunoprecipitated with monoclona] antibodies directed against ga/gb, gc or gd. precipitated proteins were subsequently characterized in sds-polyacrylamide gels. the glycoprotein with affinity for h p a demonstrated gc specificity (fig. , slot ), but with a higher electrophoretic mobility than the nonbinding gc (fig. , slot ). the precursor to gc was also identified. presence of glycoprotein ga/gb was not observed (fig. , slot ) . the glycoproteins without affinity for h p a contained ga/gb, gc and gd (fig. , slots and ) . the data on gd is not shown. purification of gc was made possible by binding extracts of c-glucosamine labelled infected cells on sepharose immunosorbent columns containing monoclonal antibodies reactive with gc (fig. ) . material passing through the column and designated pool i was identified as a mixture of ga/gb, gd and their precursors as based on the eleetrophoretic mobilities observed. pool i i i.e. the specifically bound protein represented to : per cent of the radioactive material loaded on the column, was identified as gc and precursors gc. two additional potypeptide bands were detected corresponding to proteins with molecular weights of k and k (fig. , slot ) . the lectin affinity chromatography with immunosorbent-purified gc demonstrated that about per cent of the isolated gc was biospecifically bound to hpa (fig. ) . both unbound and hpa-bound immunosorbent purified gc were subjected to sds-page. two polypeptide bands with affinity for itpa were identified and both proteins had electrophoretic mobilities that were significantly higher than that of the major band identified without affinity for the leetin (fig. , slots and ) . these data together with those presented in fig. therefore indicate that among the hsv glyeoproteins ga, gb, gc and gd only two subspecies of gc have affinity for hpa. if the hpa-binding subfraction comprises an underglycosylated precursor to gc, one would expect that the non-binding fraction may consist of sialylized g~teoproteins with masked hpa-binding oligosaccharides. for removal of such masking structures tt~e gc fraction without hpa affinity was treated with neuraminidase or dilute it s before hpa chromatography (fig. ) . however, only about per cent of the originally hpa negative gc fraction became hpa positive after treatment with neuraminidase or h s o . therefore the fraction of gc without affinity for the hpa seemed not to contain sialylized glyeoprotein gc with galnae as penultimate sugars. . the autoradiographie image of the immunosorbent purified, c glucosamine labelled gc with affinity for ttpa. identified on sds polyaerylamide gel. slot , total extracts from hsv- (hefm) t s b infected cells grown at ° c. slot , gc isolated from pool w of an t t p a column without a purification on immunosorbent. the protein was precipitated with monoeloanl antibodies to gc (analogous to fig. , slot ) . slot , the fraction of immunosorbent purified gc which could bind to hpa. slot , immunosorbent purified gc (analogous to fig. , slot ). notiee the difference in the electrophoretie mobility between g c isolated by immunosorbent chromatography (slot ) and the sub-population of gc which had h p a affinity (not , marked with arrows) binding of gc to hpa suggests presence of terminal galnae and also presence of o-linked oligosaccharides. the finding that only a subfraetion of gc contained hpa binding activity even after desialylation raised the question whether or not the subfraction which primarily did not bind to hpa contained ohgosaccharides o-glycosidically linked to the peptide. immunosorbent purified gc was therefore fractionated by hpa affinity chromatography and the subfraction not binding to hpa subsequently subjected to treatment with alkaline nabh . before and after treatment with nabtt the fractions of c-glucosamine labelled gc was studied on sephadex g , untreated or mocktreated material was found in the void volume exclusively (panel a, fig. ). after treatment with alkaline nabh release of labelled material was demonstrated. the molecular weights of these structures corresponded to or more. the results were interpreted as ~elimination of o-linkages between oligosaccharides and peptides of gc. experiment, s with pronase-digested underglycosylated hsv glycoprotein produced in the presence of tunieamycin indicate existence of at least two classes of relatively large tunicamycin-resistent oligosaccharides; with and without affinity for hpa (onofsso~ et al., submitted for publication). these data support the conclusion that more than one type of o-linked sugar may be attached to hsv glycoproteins. oligosaceharides with n-glycosidical linkages of the high mannose type or moderately branched complex type exhibit affinity to con a ( ) . testing of the hpa binding and non-binding fractions of gc with con a affinity chromatography revealed approximately the same abundance of biospecific binding of both the gc fractions to con a (fig. ) . these data indicate that n-glycosidieally linked the viral glycoproteins (ga/gb, gc and gd) synthesized in hsv- infected cells are glycosylated in several discrete steps ( , , ) . we have found that the type-specific glyeoprotein, gc, of hsv- contains oligosaccharides binding to hpa, a lectin with specific affinity for galnac ( ) . this sugar is genera~ily absent in normal n-glycosidic oligosaccharides but is present in oligosaccharides linked o-glycosidically to a serine or a threonine residue of the poiypeptide. recent findings that glycoprotein gc is sensitive to weak alkaline borohydride treatment ( ) and contains tm resistent oligosaccharides (olo~sso~ et al., submitted for publication) further supports that gc, in addition to the n-glycosidic carbohydrate chains, contains o-glycosidically linked oligosaccharides. in the present study the immunochemical specificity of the hsv glycoproteins without affinity for hpa and those with affinity for the lectin were identified by immunoprecipitation with monoclonal antibodies against hsv specified glyeoproteins. the glycoprotcins without affinity for hpa contained ga/gb, gd and the majority of gc, whereas only glycoproteins with the immunological specificity of gc were found in the fraction binding to hpa. the data thus emphasized that of the hsv-glycoproteins only gc contains oligosaccharides with affinity for hpa. using immunosorbent-purified glycoprotein it could be clearly demonstrated that glyeoproteins with hpa-binding oligosaccharides constituted a subfraction comprising about per cent of the glycosylatcd gc population (fig. ) . however, our data also show that gc without affinity for hpa contains o-linked carbohydrates, and that o-glycosidic as well ~s n-glycosidic linkages exist on the one and same gc molecule (figs. and ) . the eleetrophoretic analysis of the hpa-binding gc subfraction revealed two polypeptides, one with a molecular weight of k which corresponds to the partially glycosylated precursor of gc (pgc) and one with a molecular weight of t k which is intermediate between that of the fully glycosylated gc and that of pgc. the low molecular weights of hpa-binding gc molecules relative to the bulk of glyeoproteins with gc specificity suggest that gc with affinity for hpa constitute partially glycosylated gc intermediates. if we assume that, the hpabinding oligosaecharides represent such precursors to larger o-glycosidic ohgosaccha.rides lacking hpa-affinity it might be suspected that hpa-binding sites were blocked by terminal sialic acids as observed for hpa-binding oligosaccharides of t lymphoeytes ( , ) . however, our experiments with two different procedures for elimination of sialic acids only marginally increased the hpa-binding activity of gc, suggesting that sialylation of hpa-binding sites was not a main reason for absence of hpa binding propert:ies. the hpa-binding glycoprotein might therefore represent a fully glycosylated fraction of gc, that is glycosylated differently than the bulk of gc. one possible explanation would be that the biosynthesis of the gc-associated o-glycosidic oligosaecharides at some point is branched and follows different * pathways, one leading to hpa-binding oligosaccharides and the other to oligosaceharides with terminal structural properties without affinity for hpa. this diversity m a y be caused either solely by host cell glyeosyl transferases or by one or more virus specified or virus modified enzymes acting in concert with the cell specified glycosyl transferases. we have previously pointed out the changes of kinetic properties of glyeosyl transferases after hsv infection of cells ( ) . presence of competing glycosyl transferases leading to biosynthetic branching of a core structure into different types of otigosaccharides with and without blood group a activity has been demonstrated with - inked carbohydrates of the sheep submaxillary mucin ( ) . in presence of tm only trace amounts of glycoprotcins ga/gb and gc were demonstrable on the surface of the infected cells as shown by iodination of the intact cells ( ) and by indirect immunofluorescence with monoclonal ab hc- of fixed cells (unpublished data). in fact gc was the only glycoprotein transported to and inserted into the plasmamembrane in significant amounts in presence of tm. the gc exposed on the cell surface lacked reactivity in the antibodydependent cell-mediated c)~otoxicity test a finding which suggests that presence of n-glycosidic oligosaccharides were essential for the adcc reaction ( ) . on the other hand the presence of - inked carbohydrates seems sufficient for transport of glycoprotcins to the plasmamembrane and different functions of the two classes of oligosaccharidcs on the itsv glycoprotcin gc might thus be discerneable. helix pomatia agglutinin: selectivity of binding to lymphocyte surface glycoproteins on t cells and certain b cells membrane proteins specified by herpes simplex viruses. v. identification of an fe-binding glycoprotein synthesis and processing of glycoproteins gd and gc or herpes simplex virus type a role of oligosaccharides in glyeoprotein biosynthesis stage-specific embryonic antigen involves u ( -> ) fueosylated type blood group chains synthesis and maturation of g]ycoproteins of enveloped animal viruses tunieamycin resistent glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein a new surface marker on t lymphocytes of human peripheral blood idukso~, ).: antiviral activity of tunicamyein on herpes simplex virus tunieamycin inhibits glycosylation and multiplication of sindbis and vesicular stomatitis virus lectins. their chemistry and application to immunologsz cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins separation of the intergral membrane glycoproteins e i and e of semliki forest virus by affinity chromatography on eoncanavalin a-sepharose anatomy of herpes simplex virus (tisv) dna. x. mapping of viral genes by analysis of polypeptides and functions specified by ttsv × t i s v recombinants coronavirus glyeoprotein e , a new type of viral glyeoprotein the effect of tunieamycin on the synthesis of herpes simplex virus type glyeoproteins and their expression of the cell surface effects of glueosamine -deoxyglueose and tunicamycin on glyeosylation, sulfation and assembly of influenza virai proteins effect of tunicamycin on the morphogenesis of semlild-forest virus and i~ous sarcoma virus o-glycosidic carbohydrate-peptide linkages of herpes simplex virus glycoproteins unusual lectin binding properties of a herpes simplex virus type -specific glycoprotein altered kinetic properties of sialyl and galactosyl transferases associated with herpes simplex virus infection of gmk and b t l k cells ). : serological analysis of herpes simplex virus types and with monoclonal antibodies. inf. i m m u n differential immunological reactivity and processing of glyeoproteins biology and chemistry of euearyotic cell surfaces. miami ~ri:nter symposia supression of glyeoprotein formation of semliki forest, influenza, and avian sarcoma virus b~-tunicamyein biogenesis of vaccinia: carbohydrate of the hemagglutinin molecule membrane proteins specified by herpes simplex virus. i. identification of four glycoprotein precursors and their products in type -infected cells we thank dr. richard emmons for support of the work done at the california department of health services and dale dondero for excellent technical assistance.the work done at university of gsteborg was sponsored by the swedish medical research council (grant no ), the national swedish board development (project kb - / - ) and the medical faculty of g teborg.the work done at the university of copenhagen was sponsored by the carlsberg foundation, the novo foundation and king christian the tenth's foundation. received october , key: cord- - ufgwxxg authors: lai, m. m. c.; fleming, j. o.; stohlman, st. a.; fujiwara, k. title: genetic heterogeneity of murine coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ufgwxxg several mouse hepatitis viruses (mhv) with different pathogenicity were studied by oligonucleotide fingerprinting. two strains, mhv-k and mhv-d, which were isolated in japan and, which cause anaplasia and necrosis of bone marrow and diarrhea, respectively, were found to be closely related to mhv-a , the prototype mhv. two other mhv strains, isolated from nude mice, were found to have diverged extensively from the known mhv strains. the mhvs isolated from separate cloned neuroblastoma cell lines persistently infected with jhm strain were also found to have diverged more markedly than the corresponding virus maintained under the conditions of lytic infection. genetic divergence during persistent infection may be one of the mechanisms by which the mhv diverges. mouse hepatitis virus (mhv), a member of the coronaviridae, is an p~na virus containing a single-stranded rna genome of . × a molecular weight ( ) . it has been isolated from mice in many parts of the world and has been associated mainly with hepatitis and encephalitis ( , ) . it repiieates in a variety of tissue culture cells of rodent origin, including fibroblasts and neuronal cells ( ) . mhv infection in vitro generally results in the lysis of infected cells, but can also lead to persistent infection ( , , ) . in nature, the virus survives by establishing latent infections in the gastrointestinal tract of mice ( ) . viruses of this group have also been shown to cause enteric infections and wasting diseases in nude mice ( ) . thus, the various strains of mouse hepatitis virus exhibit a wide range of pathogenic potentials. we have previously studied several mhv strains by oligonucleotide fingerprinting, and found that they were widely divergent (t ). however, some strains are much more closely related, e.g. mhv-a and mhv- , suggesting a close genetic origins (t ). this approach is useful in determining the evohltionary relationship among various mhv strains. in this study, we have further examined several mouse hepatitis virus strains isolated in japan. two of these viruses revealed extensive genetic homology with the prototype mitv-a strain. thus, these strains appear to have remained genetically stable in nature. we have also compared the jhm strain of mhv (mhv-jhm) passaged under the conditions of lyric infection with those maintained as persistent infections in vitro. the viruses maintained under the latter conditions exhibit much more variability, suggesting that persistent infections may be a mechanism contributing to the genetic heterogeneity of the current mhv strains. the mhv-d strain was isolated from an infant mouse with fatal diarrhea, and has been shown to cause hepatitis and diarrhea in suckling mice ( , , ) . mi-iv-k was isolated from all asymptomatie adult nude mouse (nu/nu, balb/c). this strain causes bone marrow anaplasia and necrosis as well as hepatosplenic hemopoiesis ( ). mttv-i~tuu was isolated from a nude mouse with wasting syndrome and hepatitis ( ) , and causes fatal wasting disease in nude mice but is not pathogenic in euthymic mice ( ) . these three strains were all isolated in japan. mhv-i~ was isolated from a nude mouse and was a generous gift of dr. michael collins, microbiological associates, bethesda, maryland. the two mhv-a strains studied were originally obtained from dr. c. bond of the university of california at san diego and from dr. k. ]~lolmes of the uniformed services university, bethesda, maryland. all viruses were grown in dbt cells as previously described ( ) . the in vitro persistent infection was established by growing mhv-jhm in a mouse neuroblastolna cell line ( ) . single cell clones were obtained in the presence or absence of anti-jhm hyperimmune aseitic fluid. two of the viruses were rescued from the nonprodueer cell clones by fusing with dbt cells in the presence of polyethylene glycol ( ) . these two viruses, designated cs i and cs , were found to be cold-sensitive mutants ( ) . two other virus strains, a and a , were obtained from the virusproducing persistently infected cell clones. the same procedures as described by lai and sto~il~a~-( ) were followed. the ~ p-]abeled virus was extracted with phenol in the presence of per cent si)s, and the rna was prepared by separation on -- per cent sucrose gradients in an sw rotor at , rpm for hour. the s l%l~a peak was collected and used for oligonucleotide fingerprinting. the analysis of ~ °-labeled s rna by two-dimensional polyacrylamide gel eleotrophoresis followed essentially the same procedures as described previously (]i, ). to study the genetic relationship of different mhv strains causing various diseases in mice, we first examined the oligonucleotide fingerprints of two of the ikhvs isolated in japan. one, mhv-d, was isolated from a suckling mouse with fatal diarrhea ( ) and could cause a similar disease in experimentally infected. infant mice ( ) . the other, mhv-k, causes anaplastie and necrotic lesions in bone marrow of mice (t ). the ~ p-labeled s rnas of these two viruses were digested with rnase tt, and separated by two-dimensional polyaerylamide gel electrophoresis. as shown in fig. , these two viruses have very similar oligonueleotide fingerprints, suggesting a close genetic origin. n{ost surprisingly, the fingerprints of mhv-d (fig. b) is indistinguishable from that of mhv-a (fig. c) , the prototype mhv, which we have studied previously ( ) . these data suggest that the mhv-d might have been derived from the mhv-a strain quite recently. the oligonucleotide fingerprint of mhv-k (fig. t a) was also similar to that of mhv-a , but, contained six additional oligonueleotides which were not present in mhv-d or nhv-a . the very high extent of homology between mhv-k and mhv-a suggests that mhv-k is also a recent derivative of mitv-a . the finding that ~hv-k contained six more large tl-o]igonucleotides than mhv-a suggests either that mtiv-k has greater genetic complexity than mhv-a or that mitv-k is heterogeneous. to distinguish between these two possibilities, several subelones of mttv-k were plaquepurified and analyzed by oligonuclcotide fingerprinting. such an analysis showed that all of the mhv subclones are similar but not identical, suggesting that the virus is indeed heterogeneous; however, all of them are very similar to mhv-a (data not shown). the nihv-a strain used here for comparison is the same strain we have examined previously ( ) . it has relatively weak pathogenicity ( ) . no clinically apparent symptoms were induced by the intracranial injection of plaqueforming units of mhv-a into mice (data not shown). however, the nhv-a strains maintained in other laboratories have much higher hepatotropic properties, inducing very severe hepatitis (k. holmes, personal communication). we therefore compared the mhv-a strains obtained from different laboratories. we found that their oligonueleotide fingerprints are indistinguishable (data not shown). therefore, these two mhv-a strains are essentially identical, or have only very minor differences that could not be detected by this technique. several mhvs have been isolated from latently infected nude mice which had developed wasting syndrome. these virus strains are of relatively low virulence in euthymic mice but produced fatal wasting diseases in nude mice ( ) . in order to assess the genetic origin and the pathogenic potentims of these viruses peculiar to nude mice, we examined the oligonucleotide fingerprints of two of these isolates : one, designated nnu, was isolated in japan ( ) comparison with other known [hv isolates ( ) revealed that they were also very distinct from any other mhv strains studied so far. thus, these viruses represent quite unique mhv isolates. to assess the genetic stability and variability of mitv and to study the possible mechanism of viral divergence observed as above, we examined the heterogeneity of viral genomic sequences in viruses isolated from persistently and latently infected cells. the persistent infection was established by infection of a mouse neuroblastoma cell line with the j h m strain of mhv ( ) . the viruses were rescued from separate cell lines derived from single cell clones by polyethylene glycol-mediated fusion with dbt or cl cells ( ) . two of these rescued viruses, cs and cs , have been shown to be cold-sensitive ( ) . as shown in fig. : the oligonucleotide fingerprints of both cs t (fig. b) and cs (fig. c) are very similar to that of the small-plaque strain of the parental j h m (jhiw-ds) (fig. a) ( ) . however, they both contain some oligonucleotides which are unique for each strain. in addition, cs does not contain three oligonucleotides present in the jh /lds. this result suggests that these two viruses represent the variants of jhm-ds, which were selected during persistent infection. two other viruses, a (fig. e ) and as (:pig. f), were produced spontaneously from two different persistently infected neuroblastoma cell clones. these two viruses have oligonucleotide fingerprints similar to that of a large-plaque variant of jht { (jhm-dl), i.e. they contain a unique spot z, but lack the spot. x, the latter of which is characteristic of jhm-ds ( fig. a; ) . however, they also contain at least one unique oligonueleotide spot not seen in the oligonueleotide fingerprints of either jhm-dl or jhm-ds, suggesting that these two viruses are derivatives of jhm-dl. the isolation of jhm-ds and jhm-dl variants h'om the persistently infected cells was in sharp contrast with the viruses isolated under the conditions of lytic infection. as we have shown previously, mhv-jtim produced plaques of varying size on i)bt cells ( ) . we have randomly picked at least plaques on dbt cells and analyzed them by oligonucleotide fingerprinting. only two fingerprint patterns were observed, one with the ds-type fingerprint and the other with the dl-type ( ) . the jhm-ds was subsequently serially passaged for at least passages on dbt cells via lytic infection. the cloned viruses were studied at passages and . the oligonucleotide fingerprints of the viruses passaged times were exactly identical to that of jhm-ds (data not shown). no viruses of other genotypes were isolated, suggesting that mhv viruses are relatively stable during lyric infection, whereas persistent or latent infection in the-~euroblastoma cell line tends to select for various types of variants. we have previously studied six strains of mouse hepatitis virus isolated at different geographical locations and found them to be genetically heterogeneous ( ) . in this report, we have analyzed additional mhv strains, some of which were isolated in japan. one surp ~sing finding is the genetic similarity of those strains isolated in japan to the mhv-a strain, the miiv prototype. these strains might, therefore, represent, very recent derivatives of mhv-a or vice versa. this finding also suggests that these strains have remained relatively stable during passages through animals. it is interesting to note that these genetically highly related viruses have different pathogenicity: the mtiv-a used in our laboratory is weakly pathogenic ( ) , n[hv-d causes diarrhea ( ), mhv-k causes bone marrow diseases ( ), while another closely related strain mhv- causes severe hepatitis ( ) . some of the genetic differences observed among these strains might be associated with the pathogenicity of the respective viruses. however, it is equally possible that these differences are not related to viral pathogenicity. in this regard, it is worth noting that several strains of mhv-a have different pathogenieities and yet they could not be distinguished by oligonueleotide fingerprinting. it is possible that there are minor genetic differences among these viruses or that the different pathogenicity was contributed by host factors. this issue requires complete nueleotide sequence analysis of these and additional miiv strains. the finding that the viruses rescued from the persistently in~ected cells were more heterogeneous than those passaged under the conditions of lyric infection is similar to the observation with vesicular stomatitis virus ( ) . in that case, the vsv underwent progressive genetic changes as the cells were passaged in vitro ( ) . similar long-term culturing of cells infected with other mhv strains has not been carried out; nevertheless, it is remarkable that the viruses rescued by cell fusion or produced spontaneously from these persistently infected cells are different from the parental viruses, while the randomly selected viruses maintained under the conditions of lyric infection are genetically stable. it is unclear why these cell lines would select for these unusual virus variants. it is noteworthy, however, that these variants produce lytic infection upon reinfection of the neuroblastoma cell line, and preliminary data indicated that. these viruses do not produce diseases different from those induced by the parental viruses (s. a. s~ohlma~¢. unpublished results). whether the selection of such variants in vivo contributes to the pathogenicity of the viruses is not clear at the present time. the propensity of the persistently infected cells to select for viral mutants might be one of the mechanisms by which the mhv diverges. it is also noteworthy that the two viruses rescued by fusion of ]atently infected cell clones with the permissive cells were the derivatives of jhm-ds, while the viruses spontaneously produced by the persistently :infected cells were jhm-dl derivatives. these two viruses, i.e. ds and dl, differ in their ability to cause c:hronic demyelination ( ) . there might be a correlation between the dl ~nd the ds viruses and the ability to yield virus progeny under these conditions. such a possibility is being studied in our laboratories. mouse hepatitis, reo- and theiler viruses latent virus as exemplified by mouse hepatitis virus (mhv) persistent infection with mouse hepatitis virus jhm strain in dbt ceil culture isolation of low-virulent mouse hepatitis virus from feces in infected mouse breeding colony isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis evolution of multiple genome mutations during long-term persistent infection by vesicular stomatitis virus evolution of a coronavirus during persistent infection in vitro pathology of diarrhea due to mouse hepatitis virus in the infant mouse isolation of mouse hepatitis virus from infant mice with fatal diarrhea hepatosplenic myelosis in naturally occurring mouse hepatitis virus infection in the nude mouse mouse hepatitis virus a : mrna structure and genetic localization of the sequence divergence from hepatotropic strai~ ?¢ii-tv- the i%na of mouse hepatitis virus comparative analysis of rna genomes of mouse hepatitis viruses pathogenic murine coronaviruses. i, characterization of the biological behavior in vitro and virus-specific intraeellular rna of strongly neurotropic jhmv and weakly neurotropic a v viruses coronaviridae. in: comprehensive virology murine eoronaviruses: isolation and characterization of two plaque morphology variants of the jh v~ neurotropic strain characterization of the coldsensitive murine hepatitis virus mutants rescued from latently infected cells by fusion rescue of a positive-stranded i%na virus form antigen-negative neuroblastoma cells stability of neurotropic mouse hepatitis virus (jhm strain) during chronic infection of neuroblastoma cells persistent infection with mouse hepatitis virus of low virulenee in nude mice the biology and pathogenesis of coronaviruses we thank the excellent technical a~sistance of todd lasman, todd kennell, and chris jatton. we also wish to thank claudia troesch, alisa young, and raymond mitche]i for excellent editorial assistance. this work was supported by a research grant -a from the national multiple sclerosis society. key: cord- -b ncmkjg authors: song, jie; hu, yajie; li, weiyu; li, hui; zheng, huiwen; chen, yanli; dong, shaozhong; liu, longding title: transcriptome analysis following enterovirus and coxsackievirus a infection in respiratory epithelial cells date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: b ncmkjg enterovirus (ev-a ) and coxsackievirus a (cv-a ) are the major pathogens responsible for hand, foot and mouth disease (hfmd), but the mechanism by which these viruses cause disease remains unclear. in this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with ev-a and cv-a in human bronchial epithelial ( hbe) cells. using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of hfmd. as a result, a total of common differentially expressed genes were present in both ev-a - and cv-a -infected cells. a trend analysis of these genes showed that of them displayed the same trend in ev-a and cv-a infection, including upregulated genes and downregulated genes. these genes were further used to conduct go, pathway, and coexpression network analysis. it was discovered that enriched go terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (gpi)-anchor biosynthesis and dna replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as tbck and gpc) might be involved in the progression of hfmd. finally, we randomly selected differentially expressed genes for qrt-pcr to validate the transcriptome sequencing data. the experimental qrt-pcr results were roughly in agreement with the results of transcriptome sequencing. collectively, our results provide clues to the mechanism of pathogenesis of hfmd induced by ev-a and cv-a . electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. enterovirus (ev-a ) and coxsackievirus a (cv-a ) have been identified as the most common etiological agents causing hand, foot and mouth disease (hfmd), which is usually seen in the asia-pacific region in infants and young children. both of these two viruses belong to the species enterovirus a, family picornaviridae. they have a positivesense single-stranded rna genome with an approximate length of nucleotides, consisting of four structural viral proteins (vp to vp ) and seven nonstructural proteins ( a to c and a to d) [ ] . clinically, most hfmd cases are mild and self-limiting, but some cases progress rapidly and are accompanied by severe neurological complications, such as aseptic meningitis, encephalomyelitis, and acute flaccid paralysis, and can further deteriorate into fatal myocarditis and pneumonia [ ] . in recent years, the number of these serious cases and deaths has been increasing and has caused global concern. fortunately, an inactivated ev-a vaccine with completely independent intellectual property rights has been successfully developed by three vaccine organizations, including beijing vigoo biological co., ltd. (vigoo), sinovac biotech co., ltd. (sinovac), and the institute of medical biology, chinese academy of medical science (cams). this vaccine is currently available on the market and has been approved by the china food and drug administration (cfda) [ ] . this vaccine shows high efficacy and a satisfactory safety profile to provide protection against clinical ev-a -associated hfmd, but it does not provide cross-strain protection against cv-a -induced hfmd. furthermore, it has no therapeutic effect on patients who have already been infected with ev-a [ ] . in previous studies, it was demonstrated that hfmd patients with ev-a and cv-a infections presented with significantly different clinical manifestations. ev-a infections frequently lead to severe central nervous system (cns) complications and even death, while cv-a infections often result in milder symptoms that resolve within a few weeks [ ] . nevertheless, over the past years, accumulating evidence has revealed that hfmd in patients infected with cv-a can also develop into a serious stage with neurological complications, and the overall condition of these patients and their clinical symptoms are generally consistent with those of severe hfmd patients infected with ev-a [ ] . data from the china national center for disease control and prevention shows that there were . million cases of hfmd in china in , including deaths. therefore, it is urgently necessary to strengthen basic research on the replication and pathogenesis of ev-a and cv-a , which could provide a theoretical basis for the development of hfmd-specific therapeutic drugs. rna sequencing (rna-seq) can be used to investigate differences in gene expression at a genome-wide level. rna-seq has the advantages of more accurate quantification, higher repeatability, a wider detection range, and more reliable analysis than other methods [ ] . in addition to analyzing gene expression levels, rna-seq can also be used to discover new transcripts, single nucleotide polymorphisms (snps), and splice variants to provide information about allele-specific gene expression [ ] . indeed, a large number of studies have been conducted to analyze the transcriptome expression profiles of ev-a and cv-a infections. for example, lui et al. analyzed the transcriptome profiles of ev-a -infected colorectal cells and found that ev-a activated a signaling pathway that might participate in inhibiting viral replication [ ] . yao et al. carried out a transcriptome analysis of ev-a -infected human rhabdomyosarcoma (rd) cells and found that ev-a - a protein could be considered a key inducer that triggered cellular apoptosis and death in rd cells mediated by thioredoxin-interacting protein (txnip) [ ] . xu et al. reported that differentially regulated mrnas were associated with the host cellular pathways that directed cell cycle/proliferation, apoptosis, and cytokine/chemokine and immune responses in sh-sy y human neuroblastoma cells infected with ev-a [ ] . this finding suggests that the changed mrnas might be involved in the pathophysiological mechanisms of ev-a infection in human neural cells. jin et al. performed transcriptome sequencing in cv-a -infected hek t cells and found that cv-a can upregulate the expression of scarb and ecm receptor [ ] . song et al. also studied transcriptome changes in cv-a -infected rhesus monkey peripheral blood mononuclear cells and discovered that inflammatory cytokines, such as il- and il- , were notably increased after cv-a infection [ ] . however, no studies have analyzed and compared the pathologic attributes in ev-a and cv-a infections. previous studies have demonstrated that the respiratory tract is an important route for hfmd transmission; therefore, the interaction between ev-a /cv-a and airway epithelial cells should be investigated [ ] . in the current study, we aimed to discover significant differentially expressed genes in ev-a -and cv-a -infected respiratory epithelial cells through transcriptome sequencing. we then investigated the potential pathogenesis of hfmd induced by ev-a and cv-a via systematically analyzing these differentially expressed genes by bioinformatics analysis, which might provide clues about the mechanisms of hfmd and possible molecular targets for hfmd treatment. monolayers of human bronchial epithelial ( hbe) cells were purchased from jennino biological technology (guangzhou, china). the cells were seeded at a density of × cells per well in -well sterile plastic culture plates and grown in a base medium of roswell park memorial institute (rpmi)- medium containing % (vol/vol) fetal bovine serum (fbs), units of penicillin per ml, μg of streptomycin per ml and mm l-glutamine at °c in a % air and % carbon dioxide (co ) incubator. for transcriptome study, hbe cells were infected at a multiplicity of infection (moi) of . with enterovirus (ev-a ; subgenotype c , genbank no. eu . ) or coxsackievirus a (cv-a ; subgenotype b, genbank no. jn . ), which were isolated from an epidemic in fuyang, china, in and from an hfmd patient in guangxi, china in , respectively. both viruses were incubated with cells for one hour to attain virus attachment, and rpmi- medium containing % fbs was then added. the cells were incubated for a further h, h and h for virus propagation. it was observed that the proportion of cells infected with each of these viruses at the hour time point had reached about % (fig. s ). at each time point, cells were collected using rpmi- medium and centrifuged twice in phosphate-buffered saline (pbs) at °c for min at rpm. finally, the harvested cell pellets were snapfrozen in liquid nitrogen and stored until rna extraction. for the control sample, the same process was carried out with the exception that ml of sterile pbs was used in place of the virus. total rna was extracted using an rneasy ® mini kit (qia-gen, usa) as recommended by the manufacturer. the extracted rna was cleared of contaminating genomic dna by treatment with rnase-free dnase i (takara bio, japan). the concentration of rna was measured using a nanodrop spectrophotometer (thermo scientific, usa), and the rna quality was determined using an ultrospec pro uv/visible spectrophotometer (ge healthcare, uk). samples with an absorbance ratio (a /a ) of . to . were subjected to rna integrity analysis using an agilent ® bioanalyzer. samples with an rna integrity number (rin) greater than were used for rna-seq library construction. briefly, mrna was enriched using oligo (dt) beads, and poly(a)-containing mrna was then purified using dynabeads (life technologies, usa) and further fragmented into smaller pieces with fragmentation buffer using rnase iii and an ion adaptor. next, the rna fragments were reverse transcribed and amplified to make first-strand complementary dna (cdna) with random primers, followed by second-strand cdna synthesis. the second-strand cdna was further purified, adenylated at the ' ends, and ligated with sequencing adaptor. these fragments ( - bp in size) were subjected to pcr amplification using phusion high-fidelity dna polymerase (neb, beijing, china), and the products were sequenced on an illumina hiseq™ platform (illumina, usa). the raw rna-seq paired-end reads were filtered to remove the "dirty" reads, i.e., those containing sequencing adapters, reads with % > q < % bases, and reads of low quality (reads with ambiguous bases "n") using the fast qc package (http://www.bioin forma tics.babra ham.ac.uk/proje cts/ fastq c/). the obtained "clean" reads were mapped against the human reference genome grch using hisat software. the fragments per kilobase of transcript sequence per million base pair (fpkm) values of each gene were determined by the length of the gene and read count mapped to the gene with cufflinks (version . . ) [ ] . pca was employed for quality control, to identify problems with the experimental design, to find mislabeled samples, and to visualize variations between the expression analysis samples by using the data from clean reads. for gene expression analysis, the expression level of each gene was calculated by determining the number of reads and was further normalized by a variation of the fpkm method. to identify genes that were differentially expressed between the two samples, cufflinks was applied to calculate the t-statistic and the p-values for each gene. we calculated the expression ratios of h/ h, h/ h and h/ h as fold changes. all p-values were adjusted by the benjamini and hochberg approach to control the false discovery rate (fdr). genes with a two-fold change and an adjusted p-value < . were regarded to be differentially expressed. to find commonalities between ev-a -and cv-a -infected hbe cells, we constructed a venn diagram using the common differentially expressed genes in each group. the common differentially expressed genes were subject to unsupervised hierarchical clustering using euclidean distance and average linkage to measure the cluster similarity/ dissimilarity. a trend analysis was further utilized to identify commonalities between ev-a and cv-a infection. the common differentially expressed genes in the ev-a and cv-a infections that had the same trend were identified as genes with similar changes. the database for annotation, visualization and integrated discovery (http://david .abcc.ncifc rf.gov/), which utilizes gene ontology (go) to identify the biological process, molecular function, and cellular components of common differentially expressed genes, was applied in the current study. in addition, the biocarta and reactome database (http:// www.genom e.jp/kegg/), which uses the kyoto encyclopedia of genes and genomes (kegg) database, was applied to identify pathways of common differentially expressed genes in this study. the fdr-corrected p-value threshold was set at . , which denotes the significance of go term enrichment and pathway correlation. coexpression network construction was based on the gen-mania algorithm by using the common differentially expressed genes. the construction of coexpression networks is conducive to finding potential mechanisms associated with differentially expressed genes. to validate the transcriptome results, we designed pairs of primers using primer premier . to perform qrt-pcr analysis targeting seven upregulated (blm, gbp , hdac , nnmt, pnisr, rnf and taf ) and three downregulated genes (ano , irx and pcdh ). briefly, reverse transcription was first performed using total rna ( μg) isolated as described above with a prime script ® rt reagent kit (takara, japan) according to the manufacturer's protocol. next, qrt-pcr was carried out using μl of cdna template, . μl of gene-specific primers (supplementary table s ), μl of sybr ® premix ex taq™ ( ×), . μl of rox reference dye ii ( ×), and water up to μl. the qrt-pcr reaction program was as follows: predenaturation at °c for s, followed by cycles of denaturation at °c for s, annealing at °c for s, and extension at °c for s on a fast real-time pcr system (applied biosystems, usa). relative expression levels of the chosen genes were determined by normalizing the data for the target transcripts against the glyceraldehyde- -phosphate dehydrogenase (gapdh) transcript data by the -ΔΔct method. three independent biological replicates for each sample and three technical replicates for each biological replicate were analyzed. using the illumina hiseq™ platform, sequencing of the seven cellular samples generated approximately . million raw reads, from which . million clean reads were obtained, with an average of . million clean reads per sample. through rrna trimming and alignment to the human reference genome sequence grch , . million reads ( . %) were mapped to the genome. the detailed data on raw reads, clean reads, rrna-trimmed reads and mapping reads in each group are shown in table . the mapping reads were used for pca to assess the discrete degree analysis of each group. the results showed a clear separation of groups between ev-a and cv-a infection (fig. a) . the virus-infected groups were significantly shifted from the control group. thus, these data showed differences between the infection groups. to investigate the transcriptome responses to ev-a and cv-a infection in hbe cells, the differentially expressed transcripts based on the criterion of a twofold change and an adjusted p-value < . were screened. the distribution of gene expression levels is shown in fig. s using the data of log (fpkm). a total of , differentially expressed genes were found to be significantly differentially expressed, with , upregulated and , downregulated differentially expressed genes. the number of upregulated and downregulated genes in each group is listed in fig. b . a venn diagram was constructed to find the common differentially expressed genes in all groups, and it showed that differentially expressed genes cooccurred in each group (fig. c) . these common differentially expressed genes were further applied to unsupervised hierarchical clustering analysis. as illustrated in fig. d , by clustering the differentially expressed genes detected in all infected samples, the ev-a and cv-a infections could be perfectly separated. to further explore commonalities between the ev-a and cv-a -infected hbe cells, trend analysis of the common differentially expressed genes was carried out to reveal differentially expressed genes that had the same change trends. the results showed that upregulated and downregulated genes showed a similar trend in both the ev-a -and cv-a -infected groups (fig. ) . to investigate the biological processes that contribute to changes in the transcripts during the development of ev-a and cv-a infection, these upregulated and downregulated genes were used separately to analyze the go, related pathways, and coexpression network. regarding the go bp terms, the upregulated genes were enriched in nine terms (fig. ) , and the downregulated genes were enriched in five terms (fig. ) . the upregulated genes were markedly enriched in five mf terms (fig. ) , and the downregulated genes were markedly enriched in three mf terms (fig. ) . regarding the cc terms, five terms were enriched in the upregulated genes (fig. ) , and five cc terms were enriched in the downregulated genes (fig. ) . additionally, kegg pathway enrichment analysis for the significantly differentially expressed genes was used to identify the pathways related to the genes. our data showed that only two pathways were associated with the upregulated genes (fig. a) and one was related to the downregulated genes (fig. b) . ultimately, the construction of a coexpression network was implemented to identify the molecular interactions (fig. ). ten differentially expressed genes from the rna-seq data were randomly chosen for validation by qrt-pcr (fig. ) . the results demonstrated that the expression of blm, gbp , hdac , nnmt, pnisr, rnf and taf was upregulated, whereas the expression of ano , irx and pcdh was downregulated. these findings were consistent with the rna-seq expression profiles. over the last years, ev-a and cv-a have become important emerging viruses that pose a threat to global public health, especially in children under five years old. they both cause hfmd outbreaks with many cns-complicated cases and deaths in different parts of the world, particularly in the asia-pacific region [ ] . vaccination is considered to be one of the most effective ways to protect against ev-a and cv-a infections. therefore, researchers have been working on vaccine development in recent decades, and there are currently three vaccine organizations in china that have developed an inactivated whole ev-a vaccine that is safe and has good efficacy for protecting against ev-a -associated hfmd in children [ ] . however, there are no vaccines against hfmd caused by other enteroviruses, including cv-a . hence, it is urgent to explore the pathogenesis of hfmd triggered by ev-a and cv-a infection. transcriptome sequencing technology is able to identify all transcripts, even when detailed genetic information or a reference genome is lacking [ ] . there have been many reports on transcriptome analysis of the effect of ev-a and cv-a infection on different sensitive cells. for example, transcriptome analysis revealed differentially expressed genes, including scarb , mir- - p, and enriched ecm-receptor interaction and circadian rhythm pathways involved in the pathogenesis of hfmd in cv-a -infected hek t cells [ ] . characterization of critical functions of lncrnas and mrnas in rd cells and mouse skeletal muscle infected by ev-a using rna-seq demonstrated that lncrnas may participate in ev-a infection-induced pathogenesis by regulating immune responses, protein binding, cellular component biogenesis, and metabolism [ ] . data from transcriptomics profiling in ev-a -infected human colorectal cells showed altered expression of human genes involved in critical pathways, including the immune response and the stress response, which provided valuable insights into the host-pathogen interaction between human colorectal cells and ev-a [ ] . these studies indicated that the changes in the transcriptome of cells infected with ev-a and cv-a may be different when different cells are examined, which is of great significance for exploring potential pathogenic mechanisms. however, at present, there are no relevant studies on transcriptome comparison between ev-a and cv-a infections in hbe cells. hence, in this study, we adopted this technology to obtain information about the pathogenic mechanisms of hfmd induced by ev-a and cv-a infection. it was discovered that there was a significant difference between infections caused by ev-a and cv-a , because the pca data clearly showed that the groups for ev-a infection and cv-a infection were individually gathered together. applying a venn diagram enabled us to find common genes whose expression changed after ev-a and cv-a infection. these common differentially expressed genes were used to perform hierarchical cluster analysis, and the heatmap result showed that these genes were mainly clustered into two categories-either significantly upregulated or significantly downregulated, suggesting that ev-a and cv-a infections result in largely similar transcriptome-level changes. thus, these common genes might be involved in the pathogenesis of hfmd. next, we analyzed the expression trends of the differentially expressed genes and found that they were all classified into expression trends, including five upregulated trends after ev-a and cv-a infection, four downregulated trends after ev-a and cv-a infection, and one opposite trend after ev-a and cv-a infection. the differentially expressed genes that showed the same trends were the genes that we chiefly focused on and were applied in the subsequent go, pathway, and co-expression network analysis. the upregulated genes were enriched in nine go-bps and mainly included positive regulation of transcription, dna-templated transcription, initiation, histone acetylation, regulation of endocytosis, gpi anchor biosynthetic process, protein polyubiquitination, preassembly of gpi anchor in er membrane, cellular response to dna damage stimulus, and dna replication. the downregulated genes were enriched in five go-bps, mainly including cellular response to heat, positive regulation of phosphorylation, branching involved in labyrinthine layer morphogenesis, positive regulation of epithelial cell proliferation involved in lung morphogenesis, and specification of loop of henle identity. previous studies have shown that the above go-bps are intimately associated with viral infections. there is growing evidence that histone deacetylase (hdac ) plays a very important role in natural immunity [ ] . for example, in rna-virus-infected hosts, hdac is able to bind to rig-i and catalyze rig-i deacetylation, which is essential for rig-i to recognize double-stranded rna. in addition, hdac can also promote ifn production by catalyzing the deacetylation of β-catenin, which further hinders viral replication [ ] . hence, these studies imply that the enriched "histone acetylation" might participate in the infection and replication process of ev-a and cv-a . moreover, the enriched go-bp "positive regulation of phosphorylation" was also observed to be involved in viral infections. for instance, epstein-barr virus (ebv)-encoded bglf kinase could directly downregulate the nf-κb signaling pathway by phosphorylating coactivator uxt [ ] . furthermore, ebv nuclear antigen (ebna ) can stimulate its own nuclear entry by phosphorylating s in the nuclear localization signal [ ] . thus, these studies indirectly indicate that the enriched "positive regulation of phosphorylation" might be a potential mechanism through which hfmd is induced by ev-a and cv-a infection. kegg pathway enrichment analysis for these dysregulated genes is useful for identifying related pathways and molecular interactions among genes. the upregulated genes were markedly enriched in two pathways, namely, glycosylphosphatidylinositol (gpi)-anchor biosynthesis and dna replication, while the downregulated genes were only markedly enriched in one pathway, namely, biosynthesis of antibiotics. among these pathways, the gpi-anchor is considered to play an important role in the infection and pathogenesis of many viruses. for example, enk et al. found that the herpes simplex virus (hsv)- -encoded mir h could target the gpi anchoring pathway, which further reduced the expression levels of several immune-modulating proteins and finally enhanced viral spread and enabled evasion of elimination by natural killer cells [ ] . amet et al. demonstrated that a deficiency of gpi-anchor could inhibit the production of infectious hiv- and render virions sensitive to complement attack [ ] . therefore, changes in the "gpi-anchor" pathway might promote the spread of ev-a and cv-a . in addition, it has been reported that ev-a can affect dna replication in host cells through its nonstructural protein d and then block the cells in s phase [ ] . moreover, another enterovirus (cv-a ) can also block host cells at g /g through its nonstructural proteins c and d by affecting dna replication [ ] . thus, it is clear that the enriched "dna replication" pathway might contribute to the development (c) venn diagram displaying the number of ev-a -specific, cv-a -specific, and ev-a / cv-a -common genes at different time points after ev-a and cv-a infection. (d) hierarchical heatmap showing the overlapping differentially expressed genes in each group. red indicates genes that were expressed at higher levels, and green indicates genes that were expressed at lower levels. each column represents one group, and each horizontal line refers to one gene. the cutoff values of log fold change > or < - and a false discovery rate of < . were applied ◂ fig. trend analysis of the differentially expressed genes in hbe in response to ev-a and cv-a infection at different time points postinfection. the genes that were upregulated in both the ev-a and cv-a infections are indicated in red. the genes that were downregulated in both the ev-a and cv-a infections are indicated in blue. the genes that showed opposite changes in the ev-a and cv-a infections are indicated in black of hfmd caused by ev-a and cv-a . regarding the "biosynthesis of antibiotics" pathway, no reports have shown it to be associated with viral infection. however, since this pathway appeared in ev-a and cv-a infection, more research on this pathway might be warranted. coexpression network analysis for these dysregulated genes was carried out to identify key genes that regulate the pathogenesis of hfmd during ev-a and cv-a infection. in the coexpression network of upregulated genes, tbck is located at a key node position. a previous study confirmed that mutations in the tbck gene could lead to neurological diseases such as neuronal cerebello-lipidosis and neurodegeneration [ ] . in addition, tbck might play an important role in cell proliferation, cell growth, and actin organization by modulating the mtor pathway [ ] . as mtor is a pivotal pathway of autophagy activation, we speculate that tbck may be involved in the induction of autophagy induced by ev-a and cv-a infection. in the coexpression network of downregulated genes, gpc / is located at a key node position. however, gpc family proteins are mainly involved [ , ] . there was no indication that gpc had any function in immunity or virus infection. in conclusion, transcriptome sequencing technology and bioinformatics approaches were employed to identify the differentially expressed genes in hbe cells in response to ev-a and cv-a infection. go and pathway enrichment analysis, combined with the construction of coexpression networks can greatly contribute to a better understanding of the genes that are involved in ev-a and cv-a infection. however, the present study had several limitations. this study involved in vitro experiments, which might not conflict of interest the authors declare that they have no conflicts of interest. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. glycosylphosphatidylinositol anchor deficiency attenuates the production of infectious hiv- and renders virions sensitive to complement attack homozygous tbc domain-containing kinase (tbck) mutation causes a novel lysosomal storage disease-a new type of neuronal ceroid lipofuscinosis (cln )? glypican- binds to frizzled and plays a direct role in the stimulation of canonical wnt signaling epstein-barr virus bglf kinase downregulates nf-kappab transactivation through phosphorylation of coactivator uxt hdac regulates cellular viral rna sensing by deacetylation of rig-i hsv microrna modulation of gpi anchoring and downstream immune evasion the role of glypicans in hedgehog signaling analysis of rna-seq data using tophat and cufflinks transcriptome analysis reveals dynamic changes in coxsackievirus a infected hek t cells innate immunity evasion by enteroviruses linked to epidemic hand-foot-mouth disease enterovirus seroepidemiology in taiwan in and comparison of those rates in characterization of critical functions of long non-coding rnas and mrnas in rhabdomyosarcoma cells and mouse skeletal muscle infected by enterovirus using rna-seq ev vaccine, an invaluable gift for children elucidating the host-pathogen interaction between human colorectal cells and invading enterovirus using transcriptomics profiling ev infection induces neurodegeneration via activating tlr signaling and il- production coxsackievirus a : epidemiology, diagnosis, and vaccine differential gene expression confirmed by qrt-pcr. the rna-seq data and qrt-pcr validation results were compared ev vaccine, a new tool to control outbreaks of hand, foot and mouth disease (hfmd) rna-seq perspectives to improve clinical diagnosis next-generation sequencing and the impact on prenatal diagnosis homozygous boricua tbck mutation causes neurodegeneration and aberrant autophagy global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with ca infectioninduced hfmd regulates bbb permeability and promotes cns lesions following ca infections by directly targeting mmp rna sequencing: the teenage years identification of a phosphorylation site within the p protein important for mrna transcription and growth of parainfluenza virus coxsackievirus a induces cell cycle arrest in g /g phase for viral production global transcriptomic analysis of human neuroblastoma cells in response to enterovirus type infection transcriptomic analysis of cells in response to ev infection and a(pro) as a trigger for apoptosis via txnip gene enterovirus infection and vaccines enterovirus mediates cell cycle arrest in s phase through non-structural protein d dynamic interaction of enterovirus and dendritic cells in infected neonatal rhesus macaques pkc alpha regulates sendai virus-mediated interferon induction through hdac and betacatenin key: cord- - yl ren authors: traavik, t. title: development of a modified immunoelectroosmophoresis method for uukuniemi and runde virus serology date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: yl ren in search for a suitable method for sero-ecological screenings for arboviruses in norway, efforts were undertaken to make the immunoelectroosmophoresis technique more sensitive than here to fare in detection of antibodies. the aim was to make it comparable to haemagglutination inhibition test in sensitivity, retaining the advantages in specificity, simplicity and capacity. this has been achieved by: . using concentrated virus as antigen. . performing electrophoresis in a gel consisting of an agar-agarose mixture in optimal concentrations. . “sandwiching” the first specific electrophoretic run with an anti-species antiserum to the tested sample. the isolation of uukuniemi (uuk) group viruses and a new coronavirus-like agent, runde virus, from norwegian ixodes ticks ( , , , ) , were intended to be followed up promptly by sero-ecological surveys by a standard haemagglutination inhibition test (hai). it was soon decided, however, that certain disadvantages of hai, with regard to our specific needs, would make it worthwhile to search for another screening method. after having solved the problem of making potent ha antigens for our weakly haemagglutinating viruses ( ) , the remaining disadvantages of hai were linked to the question of unspecific serum inhibitors, and to its broad group reactivity. we intended to perform sero-eeological screenings comprising human beings, small mammals, passerine birds, sea birds, wild ruminants, domestic animals and t. tt~aavi~: : reptiles. however, little is known about the qualitative and q u a n t i t a t i v e contents of serum inhibitors in m a n y of the groups above. therefore it was desirable to use a serological screening method which eliminated the inhibitor problem b u t was comparable to h a i in sensitivity. the use of the immunoelectroosmophoresis (ieop) technique has attracted increasing interest in virus serology due to its speed, simplicity and modest requirements for reagents. b u t its use has been hampered b y the lack of sensitivity, which is partly due to the anodal m o v e m e n t of most immunoglobulins under the conditions previously employed ( ) . this paper reports how the adoption of relatively simple measures makes i e o p equal to h a i in sensitivity for detection of antibodies to norwegian arbovirus strains. since it eliminates the serum inhibitor problem and also has various praetiem advantages over hai, it has become our standard screening method. the original isolations of the five virus strains used in these experiments are described elsewhere ( , ) . they were isolated from suspensions made from lxodes ticks by intraeerebral inoculations of --~ days old baby mice [born: nmri (spf)]. sf e and by e are uukuniemi (uuk) group viruses from i. ricinus ticks. ru e is an u u k group virus from i. uriae, while ru e and t~u e are coronavirus-like agents isolated from i. uriae. the two latter strains seem identical and we have tentatively termed them runde virus. ru e differs antigenieally from the two other u u k viruses. tahyna virus (strain ) was kindly provided by dr. v. danielova, institute of parasitology, czechoslovak academy of sciences, prague as a per cent lyophilized suckling mouse brain suspension from the rd passage. tribee and tickborne encephalitis (tbe) virus were kindly placed at our disposition by dr. m. gresikova, institute of virology, slovak academy of sciences, bratislava. ten per cent lyophilized suckling mouse brain suspensions from the th and the rd passage, respectively, were used. the following kinds of antigenic preparations have been utilized in these experiments : crude suckling mouse brains (smb antigen) are per cent suspensions of infected suckling mouse brains in pbs ph . with . per cent bovine mbumine (apbs). suerose-aceton extracted suckling mouse brains (sa antigen) were prepared according to clarke and casals ( ), but the finm lyophilization step was omitted. cell culture antigens (bhk antigens) were prepared by growing the viruses in bhk /c cells in roux bottles. the infectious culture fluids are concentrated to times by precipitation with per cent polyethylene glycol / . per cent nac at ph . and ° c ( ) overnight, followed by lowspeed centrifugation and resolution of precipitates in apbs. finally sonication is performed. for tbe, hela bristol cells were used instead of bhk. it was found beneficial to include lipoprotein-absorption by the colloidal silieagel aerosil (degussa, frankfurt a. main) for m antigens ( , ) . this was done in order to avoid lipoprotein deposition around the electrophoresis wells. this may in some instances interfere with the interpretation of specific precipitation lines. uninfected mouse brains and bhk-cultures were treated in parmlel and used as control antigens. each mouse initially received between and bmlds (baby mouse lethal doses) in . ml brain suspension mixed with an equal volume freund's complete a d j u v a n t (difco) intraperitoneally. thereafter weekly injections without a d j u v a n t were given. after another week, an injection identiem to the first one was performed. seven to days later paracentesis and bleeding from the retroorbitm sinus were done. reference mouse immune aseitic fluids to u u k (strain s ), t b e (rsse) and tribec had been obtained from the yale arbovirus research unit. rabbits received injections of virus grown on bi-ik cells, concentrated times by peg/nac and sonieated. they first received ml intraperitoneally, and then ml intravenously and : weeks later. the rabbits were bled by cardiac puncture before the immunization started, and weeks after the last injection. the antisera were adsorbed with packed b h k cells. all sera were inactivated at o c and stored at -- ° c. the antispeeies sera used in these experiments were r a b b i t antimouse ig (ram/ig) and r a b b i t anti-human ig (rahu/ig) from nordic immunol. lab., tilburg, the netherlands. human sera the h u m a n sera were selected from the files of the virus laboratory, h a u k e l a n d hospital, bergen. the sera had been remitted for all kinds of suspected virological diseases and controls. our only criterion for inclusion was that the patient should live in or near by areas infested by . ricinus. the technique is a modification of the m e t h o d of anter. et al. ( ) , employing tile commercial i-iepascreen eleetrophoresis apparatus (spectra biologicals, oxnard, california). for the electrode trays as well as for the gel a barbital buffer with an ionic strength of . and a p h of . (spectra biologieals) is employed. gels are made on precoated lantern slides, × cm. twelve ml moulten per cent agarose are poured on to each slide, giving an average gel thickness of . ram. three double-rows of wells are cut in the gel. the well diameters and interdistances are ram. two slides are run in the apparatus at the same time. r u n n i n g time is ~ minutes. the reagents are applicated by the aid of a ~ oxford automatic pipette. i n screenings, this technique permits the simultaneous testing of unknown samples. the following paramters have been tested: a) the optimal antigen preparations and concentrations. b) the composition of gels. mixtures of baeto agar (difco) and agarose (l'industrie biologique frangaise ) from . -- . to . -- . per cent have been used. c) the effects of antispeeies antiserum. after the termination of the first electrophoresis run, the antiserum well was filled with antispeeies (anti-mouse or anti-human ig) and the electrophoresis was continued for ~ minutes. this sandwiching should theoretically trace immuneeomplexes which are not big enough to be read directly as precipitates. d) the effects of varying well-diameters and -interdistances. when screening unknown samples, the specificity of i e o p precipitation lines was secured b y the sensitive gel precipitation method (chi) (closed hexagon immunodiffusion) ( ) . antigens were allowed to diffuse for hours before filling of antisermn wells. r e p a t e d fillings of wells were necessary in some instances. w h e n needed, sera were concentrated by polyaerylamide gel (lyphogel, gelman instr. co.) ( ) . i n all i e o p runs, positive and negative controls for antigens as well as antisera were included. ( ), using microtitration equipment (cooke engineering co., alexandria, va). for u u k viruses optimum conditions were pit . at ° c, for t~unde virus pill . at ° c ( , ) . cft was performed by standard procedures ( ) using units of complement in the final test,. the optimum antigen dilutions used for antibody detection was found by checker-board titrations. i n per cent agarosc gel, crosstitrations showed that all types of antigens had to be diluted to obtmn maximal sensitivity in antibody-detection. the celt culture antigens had the highest titers, and also effected a -- -fold increase in the titers of the antisera. sandwiching the reactions with rabbit anti-mouse serum gave only a two-fold rise in antibody titers. these results are illustrated in table . i n contrast, if an mlbalanced system is encountered, i.e. the antigen is too strong to attain optimal sensitivity, the effect of antispecies serum is much more striking, on occasions it has increased the titer of antiserum b y a factor of . a remarkable higher sensitivity was demonstrated by mixing increasing amounts of ~gar into the electrophoresis gel. the concentration of antigen needed for optimm conditions had to be elevated in parmlei to the relative agar-concentration. b y using a gel composed of . per cent agar and . per cent agarose, and concentrated b h k antigens, results comparable to the h a i titers were obtained for the reference sera with all the viruses tested. this is demonstrated in table . the reasons for the improvement was clearly seen b y immnnoelectrophoresis as demonstrated in figure . with increasing agar concentration in the gel, more of the antibodies move cathodically, but at the same time the anodal movement of the antigens are hampered, so that more concentrated antigens are needed. under balanced conditions, i.e. near to equivalency, the effect of "sandwiching" is not very pronounced, raising end-point titers b y -- dilution steps a antibody titers are given as reciprocal titers b ag--antigen, ab--antibody rabbit antimouse ig (tables , ) . however, when unbalanced systems are met with, "sandwiehing" is improving the results considerably (table ) . the ability to detect antibodies and antigen increased to some e x t e n t with dilution of antigen and a n t i b o d y as d e m o n s t r a t e d b y checker-board titrations. (figs. l b and d) . in figures i a and b mouse hyperimmune sara were applieated in the wells, and eleetrophoresis was performed for minutes. the corresponding viruses, sa antigens, were applieated in the throughs, and diffusion took place overnight. in figures e and d antigens were eteetrophoresed and the corresponding antisera applieated in the throughs. the viruses used ware from above: by e , i{u e and i{u e a marked prozone was seen for all viruses. the prozone effect could be restricted to some degree b y "sandwiching", b u t the effectiveness of this varied from one experiment to another, and could no~ be relied upon. consequently, the optimal dilution of antigen is considered the one giving precipitation lines for all antibodydilutions, discriminating the question of absolute end-point titer. these features are illustrated in figure . increasing the well diameter to m m a n d varying the interdistances between a n d m m gave no benefit in a n t i b o d y detection. increased interdistanccs effected the optimal r u n n i n g time adversely. some sera which had been found sero-positive to u u k virus in h a i were titrated in parallel in h a i , the original i e o p a~nd i e o p with . per cent agar to . per cent agarose. the results are shown in table . there was no correlation antibody titers given as reciprocal values b concentration of agar mentioned first e rabbit antihuman ig in a second electrophoresis run table antibody preparation b) the precipitating antibodies of the antisera and mouse aseitic fluids could be adsorbed by using homologous virus-antigens, but not by using any hegerologous antigen. e) when anti-human antiserum was used to sandwich reactions with mouse antisera or immune ascitic fluids, and anti-mouse antiserum was used to sandwich human sera, no increase in tigers was seen. neither anti-human nor anti-mouse angisera could improve the results obtained by rabbit sera. d) the uuk group viruses by e and ru e had earlier displayed a oneway serological relationship in chi ( ) . antiserum to by e reacted with both viruses, while antiserum to i{u e reacted with the homologous virus phenomenon was seen also in ieop. during the past few years, the ieop method has found its main application in the detection of hepatitis b surface antigen (hbsag) (t, , , ) . it also has been used to some extent in the diagnosis of certain bacterial ( ) and fungal infections ( ) . in virology, the ieop method has been employed in studies of myxoviruses ( ), poxvirus ( ), herpes simplex ( ) and some plant viruses ( ) . recently, tsotsos ( ) examined the possibilities of using ieop for typing and subtyping of various viruses belonging to major groups. however, very few researchers have investigated the possibility of using ieop as a method for detection of viral antibodies. the main advantages of ieop over other serological methods would be its simplicity, speed, the possibility of testing simultaneously many specimens, and its modest requirements for reagents and equipment. obviously, however, the assumption has been that this could not compensate for the insensitivity of the method in detection of. antibodies. even so, ieop modifications have been elaborated for screening of antibodies to influenza virus ( ), california encephalitis virus ( ), rubella virus ( ) and measles virus ( ) . bug in all these cases, the sensitivity in antibody detection seemed inferior to the methods routinely used for these viruses. even in the future, sero-eeological screenings wili be an integrated part of efforts to evaluate the extent and significance of arboviruses. the most commonly used technique for this purpose has been hal in addition go the unreliability connected with unspeeifle inhibitors, hai has certain other major disadvantages (apart from the fact that haemagglutinafion is not common to all arboviruses). the most serious objection to hai as a sero-ecological tool is its very broad reactivity. cross reactions occur between viruses which are not even transmitted by the same vector, but which might nevertheless circulate within the same areas. we have shown, for the norwegian arbovirus strains, as has already been demonstrated for other virus groups ( ) , that ieop is virus type and even subtype specific, and thus offers advantages over the hai test in this regard for sereecological screenings. the low consumption of serum is another very important factor. in many instances very limited amounts of blood can be drawn from each individual of animal species that are to be investigated, and one often wishes to perform tests against more than one antigen. the modifications which have been described in this paper to achieve a very sensitive ieop test for antibody detection, are all simple and inexpensive. some of the same effects have been obtained for hepatitis b diagnostics by employing discontinuous buffersystems ( ) . as pointed out by kelkai~ and nipi~adt~ar (t ) it is, however, a much simpler and cheaper solution to establish the optimal agaragarose mixture for a given antigen/antibody system. high-grade concentration of sera is a laborious and timeconsuming task, and often is prohibited by the sm~]l volumes of blood available. provided the opportunity to concentrate the antigen, the modifications used in this work to obtain a sensitive i e o p for antibody detection might prove valuable also with other viruses. the relative failure experienced with tbe virus in these investigations m a y illustrate the problem of concentration. tbe has a considerably smaller molecular mass than the other viruses used, and according to tsetses ( ) , this needs to be compensated for by the use of a more concentrated antigen. counterelectrophoresis for detection of hepatitis associated antigen : methodology and comparison with gel diffusion and complement fixation hepatitis-associated antigen: improved sensitivity in detection california arbovirus (la crosse) infections. ii. preeipitin antibody 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of measles virus-specific nueleoeapsid antibodies by means of counterimmunoeleetrophoresis immunoeleetrophoretischer nachweis yon "hepatitis-associated antigen immunoosmophoresis, a rapid and sensitive method for evaluating viruses a sensitive modification of the ouchterlony technique. detection of hepatitis associated antigen in a dosed hexagonal system the isolation of an agent related to uukuniemi virns from norwegian ixodes ricin.us ticks. acta path. microbioh stand a method for production of antisera to hepatitis b antigen subtypes, and the distribution of subtypes d and y among norwegian hepatitis b patients. acta path. microbioh scand tick-borne viruses in norway uukuniemi group viruses isolated in norway runde" vinls, a coronavirus-iike agent associated with seabirds and ticks improvement of arbovirus ha antigens by treatment with a colloidal silica gel and sonication identification and typing of viruses and detection of antiviral antibodies by counteriminunoeleetrophoresis enhanced detection of australia antigen in serum hepatitis by discontinuous eounterimmunoelectrophoresis key: cord- -l w jhsw authors: lasecka, lidia; baron, michael d. title: the molecular biology of nairoviruses, an emerging group of tick-borne arboviruses date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: l w jhsw the nairoviruses are a rapidly emerging group of tick-borne bunyaviruses that includes pathogens of humans (crimean-congo hemorrhagic fever virus [cchfv]) and livestock (nairobi sheep disease virus [nsdv], also known as ganjam virus), as well as a large number of viruses for which the normal vertebrate host has not been established. studies on this group of viruses have been fairly limited, not least because cchfv is a bsl human pathogen, restricting the number of labs able to study the live virus, while nsdv, although highly pathogenic in naive animals, is not seen as a threat in developed countries, making it a low priority. nevertheless, recent years have seen significant progress in our understanding of the biology of these viruses, particularly that of cchfv, and this article seeks to draw together our existing knowledge to generate an overall picture of their molecular biology, underlining areas of particular ignorance for future studies. new viral diseases appear with increasing frequency. just in the last two years we have seen the appearance of a new livestock virus (schmallenberg) and a new human pathogen (mers coronavirus). in some cases these viruses appear to be completely new, in others they have 'emerged' into our awareness as human use of different habitats changes, leading to increased contact with carriers of disease, whether those carriers are ''bush meat'' or the many insect and tick species that can act as vectors of disease. one such group of viruses that is rapidly becoming more important is the genus nairovirus in the family bunyaviridae. this genus includes a number of human and livestock pathogens, as well as a collection of other viruses about which little is known, not even the host in which they naturally circulate. the nairoviruses show a number of unique features, and the purpose of this article is to summarise our current knowledge of the molecular biology of these viruses and highlight areas where it is to be expected that research will soon bear fruit. a primary characteristic of the viruses of the genus nairovirus, distinguishing them from most of the other members of the family bunyaviridae, is that they are all transmitted by ticks [ ] . based on antibody cross-reactivity, nairoviruses are classified into seven serogroups (table ) [ , , ] , of which the most important are the crimean-congo haemorrhagic fever (cchf) group, which includes the human pathogen crimean-congo haemorrhagic fever virus (cchfv), and the nairobi sheep disease (nsd) group, to which belong nairobi sheep disease virus (nsdv) dugbe virus (dugv) and kupe virus (kupv). kupv [ ] and finch creek virus [ ] are the most recently discovered viruses of this genus. cchfv is arguably the most important from a human perspective, causing haemorrhagic fever in humans, with mortality up to % (reviewed in refs. [ ] and [ ] ). after dengue virus, this is the second most widespread of the arboviruses that are pathogenic to humans; cases of cchf have been reported in sub-saharan africa, the former soviet union, bulgaria, turkey, china, pakistan, india, the arabian peninsula, northern greece, iraq and iran [ , - , , - , ] . although cchfv can infect several mammalian species, it appears to cause disease only in man [ , ] . because of its importance as a human pathogen, most of the available data on nairoviruses come from studies on this virus, though studies on other members of the group have also contributed, notably on nsdv, a virus first identified nearly a hundred years ago as a tick-borne virus that causes severe haemorrhagic gastroenteritis in sheep and goats, with mortality rates of up to % in susceptible populations [ , , ] . interestingly, an asian virus causing a similar disease, ganjam virus (gv), was recently identified, based on genetic and serological studies, as being the same virus as that causing nsd in east haemorrhagic gastroenteritis in sheep and goats [ , ] . antibodies to nsdv/gv have been reported in humans, but otherwise only laboratoryacquired infections have been seen [ , , ] dugbe virus (dugv) frequently isolated from ticks infesting livestock in which dugv appears to be apathogenic [ , , ] . while one case of human infection has been described [ ] , the link to dugv was circumstantial kupe virus (kupv) crimean-congo haemorrhagic fever group crimean-congo haemorrhagic fever virus (cchfv) haemorrhagic fever in man [ , , ] hazara virus (hazv) africa [ , , ] . it is not possible to say yet whether gv is a variant of nsdv or vice versa, but it is clear that this virus also has a wide distribution. nairoviruses, like the members of the other genera of the family bunyaviridae, are enveloped viruses that appear spherical in the electron microscope, with a diameter of approximately nm [ , , ] . as with all bunyaviruses, the genome consists of three segments of negative-sense rna [ ] (reviewed in refs. [ ] and [ ] ). these are termed the small (s) segment, encoding the nucleocapsid (n) protein, which forms a complex with each rna segment [ , ] ; the medium (m) segment, which encodes a polyprotein that is processed into two mature glycoproteins (gn and gc) as well as one or more non-structural proteins [ , , , , ] ; and the large (l) segment, which encodes the viral rna-dependent rna polymerase (rdrp) [ , ] . viral replication takes place in the cytoplasm, and viral budding occurs in the golgi [ , ] (fig. ) . during viral replication, the genome segments are used as the template for synthesis of both mrna (transcription) and complementary rna (crna) (replication), where crnas are then used as the template for the synthesis of progeny genomic viral rna (vrna) [ ] . the ' and ' untranslated regions (utrs) of the s, m and l segments contain the minimum cis-acting elements necessary for transcription, replication and packaging [ , ] . the terminal ' and ' non-coding regions of each segment are complementary to each other and highly conserved among different nairoviruses [ ] . the first nine nucleotides are usually the same in different segments, which suggests that this sequence is recognised by the viral polymerase to initiate viral transcription/ replication [ ] . it is not clear whether the complementarity between ' and ' ends reflects an interaction between the ends of segments or the requirement for the same ' sequence in genome and anti-genome rnas to act as the attachment site for the viral polymerase [ , ] (reviewed in refs. [ ] and [ ] ). as with other bunyaviruses, the nairoviruses utilise capped primers snatched from host mrna for initiation of their mrna transcription [ ] . the initiation of crna and vrna synthesis occurs by a different mechanism, which has not yet been fully worked out. the fact that polymerase slippage occurs during mrna synthesis [ ] , coupled with the observation that the rna segments of cchfv, dugv and nsdv contain a '-terminal pyrimidine [ , , ] , while viral rna polymerases can initiate rna synthesis only by attaching purines [ ] , suggests that initiation of rna replication occurs in a prime-and-realign manner. the two membrane glycoproteins (gn and gc) are believed to determine cell tropism and the ability of the viruses to infect susceptible cells via recognition and binding of one or more cellular receptors. the specific cellular receptor(s) used by nairoviruses are currently unknown, but the gc protein is thought to be involved in virus attachment to the target cell receptor, as antibodies against gc, but not gn, appear to protect cells from cchfv infection in plaque reduction neutralisation assays [ , ] . the ectodomain of cchfv gc contains an epitope that is highly conserved among strains [ ] . this fragment is probably exposed in the virus, as antibodies against this epitope are neutralising for different strains of cchfv and protect mice from challenge with cchfv [ ] . similarly, the gc of dugv was also demonstrated to be targeted by neutralising antibodies [ ] . antibodies to the gn protein were found to be non-neutralising in vitro, though could still be protective in vivo [ ] . based on the finding that an independently folding section of this gc protein bound to cchfv-susceptible cells, and using this part of gc as a probe [ ] , nucleolin has been suggested as a putative cchfv receptor. nucleolin is a cellular protein that is abundant in the nucleus but can also be expressed on the cell surface of several cell types [ , , , , ] , including mononuclear phagocytes, endothelial cells and hepatocytes which are known to be targets for cchfv [ , ] . although nucleolin has also been suggested to act as a receptor for several other viruses, such as parainfluenza virus type [ ] , human immunodeficiency virus (hiv) [ ] , coxsackie b virus [ ] and respiratory syncytial virus (rsv) [ ] , further studies are required to determine whether nucleolin acts as the primary receptor for cchfv; in particular, infection studies with wild-type (not cell-culture-adapted) viruses are required to confirm the biological relevance of any potential receptor candidates. after binding to their receptor, viruses fuse with the plasma membrane or internalise through one of several endocytosis pathways to gain entry into the intracellular environment (reviewed in refs. [ ] and [ ] ). cchfv entry is dependent on the low ph of the endosomal compartment, which appears to be required in the first steps of the virus entry post-internalisation [ , ] , and the virus has been shown to use clathrin-dependent endocytosis, while caveolin- is dispensable for its entry [ , ] . studies using dominant negative rab and rab indicate that cchfv fuses with early, but not late, endosomes to gain access to the cytoplasm [ ] . cholesterol also appears to be important for cchfv replication, especially at early stages after binding and internalisation; it is possible that depletion of cholesterol from cells traps viral particles in endosomes [ ] or interferes with clathrin-mediated endocytosis [ ] . the internalisation of cchfv virions was shown to be dependent on intact microtubules [ ] . further investigation of cchfv infection showed that disruption of the cell microtubules inhibited viral rna replication [ ] , and both intact actin and microtubules are essential for correct distribution of the n protein to the perinuclear area [ , ] . as nairoviruses enter via endocytosis and bud in the golgi compartment, the observation of a redistribution of viral proteins and suppression of cchfv assembly/egress by microtubule modification (depolarisation and stabilisation) [ ] is not surprising, and this might be caused by interference with the endogenous secretory pathway, which also occurs along microtubules [ ] (reviewed in ref. [ ] ). disruption of actin filaments in cchfv-infected cells also drastically reduced replication of the virus [ ] . while the n protein is often located in close proximity to the golgi apparatus, where generation of new viral particles occurs [ ] , n does not interact directly with golgi membranes [ , ] . the n protein appears to interact with the l protein, even in the absence of viral rna, as expressed n protein seems to redistribute most of the l protein into the perinuclear area containing n in transfected cells [ ] . the n protein of nairoviruses, at approximately kda, is almost twice of size of the n proteins of other bunyaviruses, with the exception of those of hantaviruses, which, at approx. kda, are similar in size [ , ] . the n protein is the most abundant protein in the virion and encapsidates newly synthesised vrna and crna; this process is necessary for completion of the replication cycle and packaging of the genome into virions [ , , ] . viruses of different genera in the family bunyaviridae appear to adopt different mechanisms of rna encapsidation, e.g. some bunyavirus n proteins recognise generic ssrna, while others recognise specific structures in the vrna [ , , , , ] . there is a need to understand the mechanism by which nairoviruses encapsidate genomic and antigenomic rna; inhibition of vrna encapsidation could be a target for potential antiviral drugs, e.g., using an rna decoy to bind the viral n protein, as has been suggested as an antiviral treatment for hepatitis b virus (hbv) [ ] , or selecting rna-binding proteins that target specific packaging sequences in the viral genome [ ] . the recently determined crystal structure of the n protein of cchfv shows that it is built from two major domains: a globular head and an extended stalk [ , , ] . the globular domain is the larger and is formed from both n-terminal and c-terminal helices, while the stalk domain is formed by a group of internal helices (the exact numbering of the helices varies between the several papers publishing the structure of this protein). three potential rna-binding regions were identified in the n protein structure. two positively charged grooves are located in the globular domain: the smaller one is under the stalk domain, and the larger one is in the opposite site of the globular domain. the third positive groove is located in the stalk domain. the positively charged residues forming the rna-binding site appear to be well conserved across the genus nairovirus, and several residues were identified as crucial for rna binding and virus replication, e.g., k and q in the smaller groove and k and h in the larger groove of the head domain [ , ] . the n protein of cchfv was also crystallised as a linear oligomer, where three monomeric n subunits were organised in head-to-tail manner, with the stalk domain of one monomer interacting with an oligomeric groove located at the base of the head domain of an adjacent molecule [ ] . in addition, the linear oligomers of n were predicted to interact in a dimeric manner to form an antiparallel double superhelix [ , ] . further predictions suggest that that there are nine n molecules per turn of the superhelix, which is Å in diameter [ ] . additionally, a positively charged crevice located on the outside of the double superhelix is predicted to serve as an additional rna binding cleft [ ] . studies performed by guo and collaborators [ ] suggest that the cchfv n protein, when expressed without rna, predominantly exists as a monomer, and the nairovirus n protein is only a weak binder of nonspecific rna in this monomeric state [ , , , ] . this suggests that nairovirus n binds rna only in the oligomeric state and/or that the n recognises specific structures of viral rna, as was shown for viruses of the genus hantavirus [ , ] . however the fact that the nairovirus n protein does not form oligomers without rna suggests that its oligomerisation is rna-stabilised, where monomeric n protein requires binding to rna to form an oligomer, a mechanism that was previously suggested for influenza a virus [ , ] . in a similar fashion, the n protein of rift valley fever virus (rvfv) can form only a short oligomeric form in the absence of rna [ ] . superimposing the structures obtained for the cchfv n protein by two different groups, using two cchfv isolates, revealed the head and stalk with very similar folds, but with a transposition of the stalk domain when comparing these two molecules; results which suggest a flexibility of the stalk domain and the possibility of different conformations of n for different functions or different states of the n-rna complexes (e.g., transcription vs replication) [ ] . additionally, comparison of the crystal structure of the n protein in the monomeric and oligomeric forms suggests that the stalk domain changes its conformation upon oligomerisation, probably by binding to the oligomerisation groove on the head domain of the adjacent molecule [ ] . such flexibility of the stalk domain has also been shown for the n proteins of rvfv and lasv, which, during binding to an oligomerisation groove on an adjacent n molecule, undergo conformational changes exposing an rna-binding groove [ , ] . these structural data have led to suggestions of possible models for the initiation of transcription and replication. transcription of viral mrnas by nairoviruses utilises short capped rna fragments ( - nt in length) derived from host mrnas as primers [ ] . incubation of the cchfv n protein with primer-length ssrna resulted in a conformational change of the stalk domain, which resulted in disruption of the oligomeric interactions and release of the monomeric n protein from the antiparallel double superhelix [ ] . as discussed by wang et al., presentation of the capped primers to the ribonucleoprotein (rnp) may initiate conformational changes in the stalk domain, leading to the release of monomeric n and exposing vrna to the primer and the viral polymerase. given that head-to-stalk interactions between two adjacent n molecules have also been observed for rvfv, lasv, and bunyamwera virus (bunv) [ , , ] , it seems likely that the model proposed for cchfv may be biologically valid. the n protein of cchfv has also been shown to have nuclease activity specific for dsdna and ssdna (but not for rna); the importance of this dnase activity and its function in the virus life cycle are unknown [ ] . the residues involved in the nuclease activity are located in the globular head domain of the n protein and are conserved in all nairoviruses [ ] . in contrast, the n protein of lasv, the head domain of which exhibits high structural homology with that of cchfv, has rna-specific nuclease activity [ , , ] . the n protein of nairoviruses also contains a conserved sequence signature specific for catalytic motif ii (cmii) of n- adenine-specific dna methylases (lasecka and baron, unpublished) (fig. ) , where the conserved motif nppw could be involved in substrate binding or in catalytic activity [ , ] . the motif is located on an exposed loop of the stalk domain, and the function and potential importance of this motif still need to be determined. the methylation of dna is used for regulation of gene expression, but so far there is no indication that the n protein of nairoviruses travels to the nucleus; the protein does not contain a classical nuclear localisation signal (nls), nor does it accumulate in the nucleus of infected or transfected cells. n methylation is also used as a posttranscriptional modification of mrna, and rna methyltransferases appear to contain motifs very similar to the cmi and cmii motifs identified in dna methyltransferases (reviewed in ref. [ ] ); further studies are required to determine if the nairovirus n protein can modify its own or host cell rnas in this way. like those of members of the other genera of the family bunyaviridae, the m segment of nairoviruses contains a single open reading frame (orf) encoding a polyprotein that is co-and post-translationally cleaved into the mature viral glycoproteins [ ] . the glycoproteins of most nairoviruses are still poorly characterised, and most of the available data come from studies on cchfv, largely through studies on proteins expressed from plasmids. the processing of the cchfv m polyprotein to generate the mature glycoproteins appears to be more complex than that of other bunyaviruses, as it involves first the generation of glycoprotein precursors through the action of the signal protease in the endoplasmic reticulum (er) followed by further cleavages to give rise to the full set of mature glycoproteins, a process that employs other cellular proteases [ , , , ] (fig. ) . the virions of most of the nairoviruses contain two mature glycoproteins, gn and gc [ , , , , , , ] ; however, two nairoviruses, hazara virus and clo mor virus, have been shown to contain three structural glycoproteins [ , ] . the full nairovirus m-encoded polyprotein appears to have six hydrophobic regions (tm -tm ), which could function as transmembrane helices [ , ] and act either as classic secretory signal peptides (tm ), membrane anchors (tm , and ), or a combination of both (tm , ) (fig. ) . signal cleavage motifs are found after tm (releasing the amino terminus of the gn precursor, pregn) and tm (releasing the gc precursor pregc). sequence inspection revealed a signal cleavage signal immediately after tm , suggesting that there might be a separate protein released, consisting of the sequence between the distal ends of tm and tm ; such a protein has been shown to be produced by cchfv and has been termed ns m [ ] . further non-structural glycoproteins have been identified in cchfv-infected cells, referred to as the mucin-like domain (a highly o-glycosylated peptide), gp , gp and gp , all of which are released from infected cells; gp and gp contain both the gp protein and the mucin-like domain [ ] . a model for the generation of the cchfv glycoproteins is summarised in figure based on a number of studies [ , , , ] . co-translational cleavage in the er by cellular signalase generates the -kda pregn (containing the mucin-like domain, gp , and gn), the predominantly cytoplasmically oriented ns m , and the -kda pregc. the pregn is further cleaved, either in the er or the golgi compartment, to separate mucin-like domain/gp from [ , , ] gn. this cleavage occurs at a conserved rrll; motif and is effected by the host's subtilisin kexin isozyme- /site- protease (ski- /s p) [ , ] . this cleavage has been shown to be critical for virus replication and subsequent infectivity, and lack of the cleavage prevents incorporation of the glycoproteins into viral particles [ ] . a similar tetrapeptide (rkpl) is found amino acids downstream of the signalase cleavage site in pregc, which, although it is not processed also by ski- /s p, is predicted to be utilised by a related subtilisin-like protease in the er/cis-golgi to generate the mature gc ( kda) [ , ] . the mature gn interacts (directly or indirectly) with the gc, and both are translocated to the virus-assembly sites in the golgi [ , , ] . the interaction of mature gn with gc is essential for the gc to travel from er to golgi, as gn, but not gc, contains a golgi localisation signal [ ] . the ectodomains of gn and gc appear to be sufficient for heterodimer formation and transport to the golgi, indicating that at least partial golgi-targeting information is located in the gn ectodomain [ ] . the final processing of the glycoproteins takes place in the trans-golgi, where the mucin-like domain and gp are separated from each other by a furin-like protein convertase at the rskr; cleavage site [ , ] . this cleavage is not required for cchfv gn maturation [ ] , and it appears that not the entire pool of mucin-like domain/gp polyprotein is being cleaved, as the nonstructural proteins termed gp and gp contain both the mucin-like domain and gp and are resistant to resolution into smaller proteins by denaturation in sds and urea [ ] . resistance to denaturation also suggests that gp is probably not a dimer of gp [ ] . interestingly, the mucin-like domain/gp and gp can fold independently of the rest of gn and are secreted even when expressed on their own in transfected cells [ ] . no specific biological function has been assigned to the mucinlike domain, gp , gp or gp . the mucin-like domain of the ebola virus glycoprotein has been shown to play a major role in pathogenesis, including involvement in the observed increase of endothelium permeability [ , ] , and it will be important to see if this is also true of cchfv, especially given the changes visible in the endothelial cells of cchfv patients. the ns m protein, when expressed in transfected cells on its own, is transported to the golgi [ ] . while the function(s) of the nairovirus ns m have still to be determined, the fact that gn requires ns m for maturation may mean that ns m is necessary for virus replication [ ] . glycosylation is an important post-translational modification of secreted and membrane proteins that can influence protein folding, transport and function. n-linked glycosylation in particular is known to regulate protein folding, association with chaperones [ ] , transport, cellular localisation [ , ] , and even virus infectivity (reviewed in ref. [ ] ). the n-terminal part of the nairovirus m polyprotein, which contains the mucin-like domain, is heavily o-glycosylated, while the adjacent gp domain appears to have few o-glycosylation sites [ , ] . both domains are also n-glycosylated, the mucin-like domain containing five potential sites and gp two [ ] . of two predicted n-glycosylation sites in each of the mature gn and gc proteins, only one is functional in gn (n ), while both sites in gc (n and n ) are glycosylated [ ] . however, only the glycosylation of the gn is essential for gn maturation, correct localisation, and transport of itself and other cchfv glycoproteins [ ] . given that the gn glycosylation sites are conserved among cchfv strains [ ] , it is likely that correct glycosylation of the cchfv gn is critical for virus viability. recently, nmr has been used to determine a solution structure for the c-terminal (cytoplasmic) tail of cchfv gn, showing that this region contains a dual zinc-finger domain, the sequence of which is highly conserved among nairoviruses [ , ] . classical bba-zinc finger domains have been shown to take part in protein-protein interactions (reviewed in ref. [ ] ), and it is possible that the dual zincfinger domain in the c-terminus of the nairovirus gn protein is involved in interaction with rnps, which would help drive assembly/budding of the virus. analysis of the sequences of m segments of other nairoviruses suggests that the general model proposed for processing of the m polyprotein described above for cchfv holds true for the other nairoviruses; m polyproteins show similar membrane topology, with six transmembrane regions and conserved signalase cleavage sites after the transmembrane domains tm , tm and tm (fig. ) . glycoprotein maturation from precursor proteins has also been observed for dugv, where pregn is around kda and pregc around kda [ ] . an exception to this general similarity is erve virus (ervev), which does not contain tm and tm , its m polyprotein lacking the entire amino acid sequence between tm and the gc ectodomain. this suggests that ervev lacks an ns m protein. most of the nairoviruses for which we have sequence data appear to have a pregn domain that is about amino acids shorter than that seen in cchfv, due to a much shorter mucin-like domain, as was previously described for dugv [ ] . another difference is that other nairoviruses do not contain a furin-cleavage site (rskr) following the o-glycosylated mucin-like domain. the ski- /s p-like protease cleavage tetrapeptides in many nairoviruses appear to be different to those proposed for cchfv; however, all appear to fit the consensus subtilisin cleavage sequence (r/k)x(hydrophobic)z; (where x is any amino acid and z is preferably f, k, l or t but not v, p, e, d, c) [ ] . this may reflect differences in adaptation to specific tick or mammalian hosts, and further studies on the importance of these various stages in the maturation of the glycoproteins for different hosts are clearly required. the l segment of nairoviruses, which contains a single open reading frame (orf) of approximately kb, encoding a protein of approximately kda, is almost twice as long as the l proteins of most other bunyaviruses, with the exception of the tospoviruses, in which the l segment is approximately kb in length [ , , , ] (fig. ) . despite this difference in length and sequence, nairovirus l proteins still show the four conserved functional regions previously described for other bunyavirus l proteins [ , , , , ] . the bunyavirus l proteins contain the rna-dependent rna polymerase (rdrp), and the most conserved region of the l segment among nairoviruses is the region corresponding to the coding sequence for the core catalytic domains of the rdrp [ , , ] . within this polymerase module (also called region ) can be distinguished six conserved motifs [ , ] (fig. ) . motifs a through d are conserved among all rna-dependent polymerases [ , ] ; motif pre-a, upstream of motif a, is present in all rna-dependent rna polymerases and reverse transcriptases, and motif e, which is downstream of motif d, is conserved in segmented negative-strand rna viruses [ , , ] . motifs a, c and d are predicted to bind nucleoside triphosphates (ntps) and are therefore likely to be involved in the catalytic functions of the polymerase [ , ] , while motifs b and e are predicted to take part in template and/or primer positioning [ ] . motif pre-a is also predicted to be involved in template positioning [ , ] . the fact that the inter-motif distances are more or less constant suggests that the polymerase module functions in a structurally dependent manner [ ] . upstream of the polymerase module are regions and (fig. ) which are conserved in bunyaviruses and arenaviruses [ , ] , while downstream of the polymerase module is region (fig. ) , which, although originally suggested to be specific for bunyaviruses [ ] , appears to be conserved in other segmented negative-strand rna viruses [ ] . protein sequence analysis shows that the distances between regions appear to be conserved among bunyaviruses, with the exception of the interval between regions and , where nairoviruses appear to have much longer amino acid sequences than other bunyaviruses (fig. ) . region , based on sequence similarity with other viruses, appears to be responsible for capsnatching endonuclease activity [ , , ] ; however, this needs to be confirmed experimentally. in the case of nairoviruses, the rdrp accounts for only one-third of the entire l protein (fig. ) , with regions of unidentified function in both the amino (n) and carboxy (c) termini [ ] . all bunyaviruses have a significant c-terminal section after the rdrp, although the function of this region is so far unknown in any of the viruses of this family, and there appear to be no cross-genera-conserved motifs [ ] . n-terminal to the rdrp motifs, the l proteins of nairoviruses contain several additional domains that are unique to this genus, of which the ovarian-tumour (otu)like protease domain is the most studied [ , , , , ] . this domain belongs to a larger papain-like cysteine protease family also found in other viruses (e.g., blueberry scorch virus (blscv) of the genus carlavirus, and equine arteritis virus (eav) and porcine respiratory and reproductive syndrome virus (prrsv) of the genus arterivirus), in saccharomyces cerevisiae, in drosophila melanogaster and in mammalian cells [ , ] . curiously, the region containing the otu domain also contains a sequence resembling a topoisomerase-like domain, located at amino acids , and therefore lying between amino acids that form part of the of the otu catalytic site -cysteine and histidine [ , ] . the consensus topoisomerase motif the approximate size, expressed as the number of amino acids (aa), is indicated for each protein, and the diagram is drawn approximately to scale. see text for details of the various motifs. adapted from ref. [ ] (skxxy) is not conserved across nairoviruses, being mostly slxxy in cchfv and nsdv/gv, but the activesite tyrosine is conserved across all nairoviruses so far sequenced. given the structural similarities between topoisomerases and strand-specific recombinases [ ] , this motif may indicate a role for this region of l in rna strand manipulation as well as its function as a protease. alternatively, nairovirus l proteins may include their own topoisomerase activity, rather than having to recruit the host-cell topoisomerase i, as has been shown for at least one non-segmented rna virus [ ] . downstream of the otu domain, the l protein of nairoviruses contains a c h -type zinc finger domain and a leucine zipper motif [ , ] , both of which are highly conserved among nairoviruses, but the function of which in the viral replication cycle is still to be determined. interestingly, the zinc-finger domain and leucine zipper motif are located in region and region , respectively, of the nairoviral l proteins but do not appear to be present in these regions in the l proteins of other bunyaviruses. the otu-domain protease activity is dispensable for virus genome replication [ ] , and most recent studies have focussed on the effects of this enzymatic activity on host proteins. mammalian otu-domain proteins are primarily deubiquitinating enzymes (dubs), responsible for cleaving the modified peptide bond that links ubiquitin (ub) to hostcell proteins or to other ub molecules. most mammalian dubs are only able to deubiquitinate and usually have a limited set of targets that they de-conjugate in this way (reviewed in ref. [ ] ). the otu domains of nairoviruses, in contrast, are capable of de-conjugating not only ub but also other ubiquitin-like peptides, notably the interferonstimulated gene protein (isg ), removing these peptides from a variety of protein targets [ , , ] . it has been shown that amino acids to of the l proteins of cchfv, nsdv or dugv are sufficient for enzymatic activity, and conserved active site residues that are critical for its catalytic activity have been identified [ , , ] . in the last few years, the crystal structures of the otu domain with and without a ubiquitin molecule have been determined [ , , ] . the cchfv otu showed an overall similar structure to yeast otu , but with an additional domain formed by two antiparallel b-strands that allow the viral otus to bind both ub and isg [ , ] . specific cleavage targets, other than host ubiquitinated or isgylated proteins, for the viral otu-like protease have not yet been described. as several potential cysteine-protease-like cleavage sites have been identified in the l protein sequence of nairoviruses [ ] and some viral proteins containing an otu-like protease domain have also been shown to undergo autoproteolytic cleavage to generate multiple mature proteins, e.g., the replicase of blscv [ ] , it has been suggested that the l proteins of nairoviruses may also be autoproteolytically cleaved into an active rna polymerase and protein(s) with additional function [ ] . we have raised specific antibodies to the nand c-termini of the nsdv l protein and shown that such cleavage does not occur in infected cells (lasecka and baron, unpublished). early studies on nairoviruses showed that, unlike other bunyaviruses, they do not shut off host protein synthesis [ , ] , so the viruses must have a different way of controlling the immediate host cell responses to infection, such as the innate immune response and apoptosis. cchfv infection in cultured cells induced apoptosis, albeit at late stages of infection [ , ] . one of the ways in which nairoviruses might induce apoptosis has been suggested to be via the induction of er stress [ ] . certainly cchfv and dugv have both been shown to induce er stress [ ] , and we have observed the same in cells infected with nsdv (lasecka, unpublished data). during replication, nairoviruses synthesise large amounts of their glycoproteins, which mature in the er and golgi [ , , ] and are likely to overload the normal er protein synthesis machinery. as long as the apoptosis occurs after the virus has completed the assembly and release of progeny virus, apoptosis is not a major problem, but it is unclear as yet whether nairoviruses take active steps to inhibit apoptotic pathways. there is the possibility that apoptosis may be delayed or inhibited in cchfv infections by the presence of a highly conserved caspase- cleavage site (devd) in the n protein [ , ] , which may act as a decoy substrate for caspase- . although this motif has been suggested to be involved in control of apoptosis [ , ] , the cleavage of n protein by caspase- is not required for replication/transcription of a cchfv minigenome [ ] . in addition, this devd motif appears to be inaccessible to caspase- in the oligomeric form of cchfv n, suggesting that only n monomers are caspase- sensitive [ ] . cchfv also appears to be the only nairovirus that contains the devd motif [ ] ; other nairoviruses, such as nsdv/gv, do not have the motif, yet are still highly pathogenic. the innate immune response to viral infection has been described in a number of recent reviews [ , , , ] . cells detect viral pathogen-associated molecular patterns (pamps) (e.g., double-stranded rna (dsrna) or dna with unmethylated cpg motifs) using pattern recognition receptors (prrs), of which the most well-known are the toll-like receptors (tlrs), which scan extra-cytoplasmic spaces including the interior of endosomes and lysosomes, and the cytosolic proteins melanoma-associated differentiation gene- protein (mda- ) and retinoic acidinducible gene-i protein (rig-i), which detect virus-associated pamps in the cytoplasm. recognition of a pamp leads to activation of the prr, followed by an intracellular signalling cascade leading to the transcription of interferon b (ifnb) mrna. secreted ifnb works in an autocrine and paracrine manner by binding to the ifna/b receptor of both infected and uninfected cells. this in turn activates a signalling pathway, which up-regulates interferon-stimulated genes (isgs), including ifna, which increases the stimulus to other isgs. finally, the products of isgs (directly or indirectly) lead to the cell entering the so-called antiviral state, in which many proteins are present that can inhibit different viruses. interferon and its actions have strong inhibitory effects on the replication of nairoviruses. for instance, treatment of human endothelial cells with ifna significantly reduced the yield of cchfv [ ] , while the replication of cchfv and dugv is impaired in vero cells stably expressing the human mx (mxa) protein; mxa is the product of an isg and appears to act through sequestration of the n protein of these viruses in a perinuclear region [ , ] . however, as with other viruses, nairoviruses have developed mechanisms to evade this innate antiviral response. some of these mechanisms appear to be unique to one virus; others appear to be common to all the viruses in the genus that have been studied. for example, cchfv can delay ifna/b production in infected cells; andersson et al. showed that, in cchfv-infected cells, an increase in ifnb mrna could only be detected h after infection, which leaves the virus a replication window with no antiviral state [ ] . this delay is related to a delay in translocation of the transcription factor interferon regulatory factor- (irf- ) to the nucleus [ ] and hence a delay in the induction of isg , one of the genes whose transcription is regulated by irf- . the isg protein is a cytoplasmic protein (p ) that is involved in the global inhibition of translation in response to infection (reviewed in ref. [ ] ). the delay in isg expression correlates with earlier findings that nairoviruses do not appear to shut off cellular protein synthesis [ , ] . in contrast to the findings with cchfv, ifnb induction was seen in nsdv/gv-infected cells after about hours; however, during those hours, the virus actively blocked induction of ifnb [ ] . nsdv/ gv infection also blocked the signalling pathways activated by external ifna or ifnc, blocking the activation of transcription from promoters normally activated by one or the other of these cytokines by directly inhibiting the phosphorylation of the transcription factors stat and stat [ ] . of the cytoplasmic prrs, rig-i is activated by rnas with a ' triphosphate group, such as viral vrnas and crnas, while mda- is activated by binding dsrnas [ , , , ] . like other negative-sense rna viruses, nairoviruses do not produce detectable amounts of dsrna during replication [ ] and hence avoid activation of a number of dsrna-sensitive prrs (e.g., mda- , tlr ) and dsrna-dependent enzymes (e.g., dsrna-activated protein kinase r [pkr] and ' '-oligoadenylate synthetase [ ' ' oas] ). the newly synthesised vrna and crna of nairoviruses, as for other bunyaviruses, is cotranscriptionally encapsidated by the n protein, minimising the exposure of vrnas and preventing formation of dsrna intermediates [ , , ] . it is thought that the major sensor for bunyavirus infection is therefore rig-i. 'triphosphate groups are present on rna molecules generated during viral replication but are absent on cellular rnas, as cellular mrna contains a cap structure at the ' end, and the transcription products of other cellular rna polymerases (rna polymerases i and iii) contain a monophosphate at the ' end. by utilising capped, short nucleotide sequences snatched from host mrna to initiate viral mrna transcription [ ] , nairoviruses prevent recognition of viral mrna by prrs. cchfv further avoids activation of rig-i as its vrna and crna have a monophosphate group rather than the triphosphate group found at the ' end of most viral rnas [ ] , though the mechanism of ' monophosphate generation during replication of cchfv remains unknown. strategies to generate 'monophosphates on viral rna have been shown for other viruses: htnv utilises a prime-and-realign mechanism to generate ' and ' complementary ends, where a viral endonuclease (which is probably also involved in cap-snatching) is proposed to remove '-terminal extensions, leading to a ' monophosphate on the final rna [ ] . interestingly, this mechanism is not common to all bunyaviruses, as rvfv was shown to contain triphosphate groups at the ' ends of its vrnas, which can activate ifnb via the rig-i pathway [ ] . it will be interesting to see if other nairoviruses adopt the rna processing seen for cchfv. it is possible that bunyaviruses that have developed methods of blocking the rig-i-activated pathway (e.g., by the activities of a nonstructural protein such as the phlebovirus nss protein) can have triphosphates at the ' end of their vrnas while viruses that do not have an nss activity must generate monophosphates to avoid activating the rig-i pathway in the first place [ , , , ] . ubiquitin and ubiquitin-like molecules play important roles in the initiation and maintenance of immune response. for example, they are essential for the action of cytokines such as ifna/b and tumour necrosis factor alpha (tnfa) (reviewed in ref. [ ] ). ubiquitination allows for the activation of nuclear factor kappa b (nf-jb) by targeting the inhibitor of nf-jb (i-jb) for degradation [ ] . k -linked ubiquitination activates several molecules of the ifnb induction pathway, including rig-i, mitochondrial antiviral signalling protein (mavs), tank-binding kinase- (tbk ), ijb kinase-e (ikk-e), tumour necrosis factor receptor-associated factor (traf ) and traf [ , , , ] . in addition to modulation of the innate immune signalling, ubiquitination also plays an important role in antigen presentation by major histocompatibility complex (mhc) class i and ii proteins [ ] . isg is an ifninduced -kda ubiquitin-like molecule that is composed of two-ubiquitin-like domains [ , ] . although the precise role of isg in modulation of protein function is unknown, conjugation by isg is also known to modify hundreds of cellular proteins, including several of those involved in the antiviral response, e.g., pkr, mxa, stat , rig-i, janus kinase (jak ), and irf- [ , , , ] . both ub and isg are synthesised as precursors, which are cleaved in order to expose the conjugation sequence (lrlrgg) by which they are attached to other proteins. the conjugation is mediated by a sequence involving activating enzymes (e ), conjugating enzymes (e ) and protein ligases (e ) [ , , ] (reviewed in ref. [ ] ). removal of these conjugated proteins is carried out by cellular deubiquitinating enzymes and deisgylating enzymes, which have roles, as expected, in negative feedback regulation of ifn induction and action [ , ] . as described in the discussion of the nairovirus l protein, the n-terminus of these proteins contains an otu proteaselike domain [ ] similar to that often found in mammalian dubs, and experimental evidence showed that this protease domain in the viral protein indeed deconjugated ub and isg . global ubiquitination and isgylation levels were greatly reduced in nsdv/gv-infected cells [ ] , and overexpression of the amino-terminal end of the l proteins of cchfv, nsdv or dugv in cell culture resulted in a similar reduction in ubiquitin-and isg -conjugated proteins [ , , , ] . the l protein otu domains of several nairoviruses have been shown to block ifnb induction and the actions of type i and type ii ifns [ , , ] . interestingly, at high enough concentrations, even catalytically inactive otus are capable of blocking ifnainduced transcription [ , ] . this suggests that catalytically inactive otu domain proteins are still capable of sequestering specific ubiquitinated or isg ylated targets by binding to them. the nsp protein of eav and prrsv also contain otulike domains [ ] . recently, it was shown that both the nsp of eav and the otu domain of cchfv l protein are able to deubiquitinate rig-i and hence block rig-i-mediated activation of ifnb [ ] . interestingly, the rna of neither cchfv nor eav appears to activate rig-i [ , ] , so the fact that these viruses have evolved specific mechanisms to inhibit the rig-i-mediated induction of ifnb suggests that rig-i still has a function in the antiviral response to these viruses. however, unlike cchfv, the vrna of nsdv/gv does activate transcription from the ifnb promoter (lasecka and baron, unpublished observations). the ability to directly block the rig-i pathway would therefore be particularly important for this virus. comparison of the otu domains of different nairoviruses revealed some differences between their affinity for different types of poly-ub and isg [ ] . for instance, cchfv shows a higher affinity for k -poly-ub than k -poly-ub, and the cchfv otu was more active in the deubiquitination of host proteins than the otus of either nsdv or dugv [ , , ] . on the other hand, while the ervev otu appears to bind any poly-ub weakly (when compared to those of cchfv and dugv), it had higher affinity for isg [ ] . this may indicate that different nairoviruses have adopted slightly different ways of utilising their core deubiquitinating and deisgylating activities, which might reflect the wide range of pathogenicity caused by these viruses, or differences in the requirements imposed on these viruses by their arthropod hosts, about which we know very little. isg is not as strongly conserved as ubiquitin among different species, even mammals, so there can be real effects of species preference in isg binding/cleavage, e.g., the cchfv otu appears to show a preference for isg of human origin over that of mouse origin, while ervev appears to recognise both human and mouse isg s equally [ ] . the better binding of the murine isg by the ervev otu may be associated with the homology between the isg of mouse and the white-toothed shrew from which ervev is commonly isolated [ , ] . nairoviruses share many of their features with other bunyaviruses, e.g., replication in the cytoplasm, budding in the golgi, and their coding and rna replication strategy. from phylogenetic studies, the members of the genus nairovirus appear to be most closely related to those of the genus phlebovirus of all bunyaviruses [ , , , ] . however, nairoviruses possess many features not found in other bunyaviruses. nairoviruses appear to have complex processing of their glycoproteins, which involves the actions of cellular proteases such as ski- /s p-like proteases and furin. the proteins of nairoviruses also contain domains that have not been observed in other bunyaviruses, such as the l protein otu domain. nairoviruses express a secreted mucin-like domain, which may play an essential role in the pathogenicity of the virus [ , ] . structural similarity between the nairovirus n protein globular domain or the rdrp region of its l protein and equivalent proteins of arenaviruses has been taken to suggest that the nairoviruses are more closely related to arenaviruses than to members other genera of the family bunyaviridae [ , ] , with some authors even suggesting that the current classification of the nairoviridae might need re-evaluation in the future [ ] . several areas for future study stand out. given that the nairoviruses are, in general, tick-borne, while most other bunyaviruses are insect-borne, it is to be expected that the interaction of these viruses with their arthropod hosts will be specific to the virus genus and need specific study. fortunately, expertise with handling ixodid ticks and tick cell lines is rapidly increasing, and it is to be hoped that our understanding of the replication of these viruses in their tick hosts will catch up with our knowledge of what is happening in mammals. the nairoviruses have been more resistant to the development of successful reverse genetics than e.g. the orthobunyaviruses or the phleboviruses, and development of such a system for nairoviruses will be invaluable in helping us understand the roles of various nairovirus-specific domains such as the topoisomerase-like domain, c h -zinc finger domain, leucine zipper motif, and otu domain of the l protein, or the ns m and mucinlike domain from the m segment, both in mammalian and arthropod hosts. the ability to create targeted mutations will also enable us to more rapidly develop stably attenuated viruses that could act as vaccines. presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of crimean-congo hemorrhagic fever virus molecular basis for ubiquitin and isg cross-reactivity in viral ovarian tumor domains identification of a novel c-terminal cleavage of crimean-congo hemorrhagic fever virus pregn that leads to generation of an nsm protein human mxa protein inhibits the replication of crimean-congo hemorrhagic fever virus role of actin filaments in targeting of crimean congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells type i interferon inhibits crimean-congo hemorrhagic fever virus in human target cells crimean-congo hemorrhagic fever virus delays activation of the innate immune response analysis of oropouche virus l protein amino acid sequence showed the presence of an additional conserved region that could harbour an important role for the polymerase activity nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and rna polymerization dugbe virus ovarian tumour domain interferes with 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the protein flexibility in rna binding identification of nucleolin as a cellular receptor for human respiratory syncytial virus nfkappab pathway: a good signaling paradigm and therapeutic target sequence motifs characteristic for dna [cytosine-n ] and dna [adenine-n ] methyltransferases. classification of all dna methyltransferases thunderclap headache caused by erve virus different modes of ubiquitination of the adaptor traf selectively activate the expression of type i interferons and proinflammatory cytokines general review of tick-borne diseases of sheep and goats world-wide arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling uncovering ubiquitin and ubiquitin-like signaling networks sequence analysis of l rna of lassa virus virus glycosylation: role in virulence and immune interactions crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski the inner structure of uukuniemi and two bunyamwera supergroup arboviruses structure of crimean-congo hemorrhagic fever virus nucleoprotein: superhelical homooligomers and the role of caspase- cleavage the proteins and rnas specified by clo mor virus, a scottish nairovirus synthesis of bunyavirus-specific proteins in a continuous cell line (xtc- ) derived from xenopus laevis tensaw virus genome sequence and its relation to other bunyaviridae double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses interferon and cytokine responses to crimean congo hemorrhagic fever virus; an emerging and neglected viral zonoosis ubiquitin and ubiquitin-like proteins as multifunctional signals nairobi sheep disease. in: th (ed) foreign animal disease crimean-congo hemorrhagic fever crimean-congo haemorrhagic fever: a seroepidemiological and tick survey in the sultanate of oman the erve virus: possible mode of transmission and reservoir identification of a putative crimean-congo hemorrhagic fever virus entry factor origin and evolution of retroelements based upon their reverse transcriptase sequences genomic analysis reveals nairobi sheep disease virus to be highly diverse and present in both africa, and in india in the form of the ganjam virus variant identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site electron microscopic and antigenic studies of uncharacterized viruses. ii. evidence suggesting the placement of viruses in the family bunyaviridae the ubch ubiquitin e enzyme is also the e enzyme for isg , an ifnalpha/beta-induced ubiquitin-like protein human isg conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways rna-binding proteins that inhibit rna virus infection acknowledgments the laboratory of mdb receives funding from the uk biotechnology and biological science research council (bbsrc). ll was the recipient of a bbsrc phd studentship. key: cord- -vgf lz authors: sheppard, m.; werner, w.; tsatas, e.; mccoy, r.; prowse, s.; johnson, m. title: fowl adenovirus recombinant expressing vp of infectious bursal disease virus induces protective immunity against bursal disease date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: vgf lz the right hand end nde i fragment ( . – map units) of the fowl adenovirus serotype (fav- ) was characterised so as to allow the location of an insertion site for recombinant vector construction. infectious bursal disease virus (ibdv) vp gene from the australian classical strain / , under the control of the fav- major late promoter/leader sequence (mlp/ls) was inserted into a unique not i site that was generated at . map units. this recombinant virus was produced without deletion of any portion of the fav- genome. when administered to specific pathogen free (spf) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the fav- /vp recombinant induced a serum vp antibody response and protected chickens against challenge with ibdv v , an intermediate virulent classical strain. birds were not protected when the recombinant was delivered via the conjunctival sac. infectious bursal disease virus (ibdv) induces an immunosupressive disease of chickens. the primary effect of this disease is the destruction of b-lymphocytes [ ] and consequently the severe impairing of the chicken to develop antibodies to other avian pathogens or vaccines [ ] . vaccination of breeding hens sensitised by natural exposure, by live vaccine, or inactivated oil-emulsion vaccine, produces a long lasting high serum antibody response [ , ] . maternal antibodies are then transferred to the progeny chickens via the yolk sac providing protection for the first few critical weeks after hatching [ ] . in recent times an emerging problem of bursal disease in the poultry industry has been with antigenic variants in the usa [ , , ] and the very virulent strains in europe [ ] . these new antigenic strains of ibdv are able to escape from maternally derived antibodies induced by the classical strains. the emergence of these new strains has resulted in changes to vaccination regimes, with broilers being vaccinated in ovo or at - weeks of age when maternal antibody has declined, with more virulent vaccines. more recently there has been the successful use of an antibody-virus complex vaccine [ ] . ibdv is a member of the family birnaviridae, and has two segments of double-stranded rna [ ] . a precursor polyprotein vp -vp -vp , encoded on the larger genomic a segment is processed by autoproteolysis to produce viral proteins vp , vp and vp [ , , , ] . the amino acid sequence is highly conserved between the classical strains and antigenic variations are confined to the central region of the protein between residues to [ , , ] , the conformational discontinuous host protective epitope [ ] . virus neutralizing (vn) mouse monoclonal antibodies are primarily directed against vp [ , , ] . this protein has been the focus of attempts to produce new vaccines by recombinant dna technology. vp expressed in e. coli reacted with a range of vn monoclonal antibodies, although the protein had been injected in milligram quantity [ ] . yeast expressed and purified vp was incorporated into an oil emulsion vaccine that was able to induce both virus neutralizing and elisa antibodies in spf chickens [ ] . these antibodies were passed on to the progeny and gave protection when challenged with virulent ibdv. vp was also expressed in a fowlpox virus recombinant [ ] . significant levels of protection were provided by vaccination with this recombinant, although the level of protection was lower than that provided by an inactivated oil emulsion vaccine containing whole ibdv [ ] . finally, a herpesvirus of turkeys (hvt) recombinant expressing vp induced protection against virulent ibdv challenge [ ] . adenoviruses have a number of advantages as vectors including, stable dna that is readily manipulated, the virus is relatively easily propagated, many of the serotypes have low pathogenicity in man or animals and the virus can induce cellular, humoral or mucosal immune responses. human adenovirus (hav) vectors have been successfully used to express a variety of viral and cellular genes. the first immunogenic protein expressed by a hav vector was the hepatitis b surface antigen [ ] . since that time, hav vectors have been constructed that express a wide variety of foreign immunogens including the herpes simplex virus glycoprotein b [ , , , ] , respiratory syncytial virus f and g genes [ ] , human immunodeficiency virus gp [ , ] , rabies virus glycoprotein gene [ , , ] pseudorabies virus genes [ ] , epstein-barr virus genes [ , ] , porcine respiratory coronavirus spike antigen [ ] , and expression of luciferase in an bovine adenovirus vetor [ ] . fowl adenoviruses (fav) have a number of features that make them attractive to the poultry industry as a vector for vaccine delivery. included in these features are (i) the ease of propagation, (ii) the high titres achievable ( - pfu/ml), (iii) stability of its genome, (iv) the ease of administration (water, aerosol or injection), (v) a large range of serotypes that vary in virulence [ ] [ ] [ ] and (vi) a genome that is relatively easy to manipulate and can tolerate insertion and expression of foreign dna. in this report we describe the construction of a recombinant fav serotype containing the vp gene from ibdv and demonstrate in vaccination via various routes that this recombinant can protect spf chickens from ibdv infection and bursal damage. fav serotype (fav- ) and was grown in chicken kidney (ck) cells derived from week old specific pathogen free chickens (spf unit, maribyrnong, victoria). the ibdv challenge virus was v , an intermediate virulent classical strain, was supplied by arthur websters (castle hill, australia). an expression cassette was constructed for insertion into the fav- genome. the fav- major late promoter and splice leader sequences (mlp/ls) ( [ ] ; genbank accession number af ) were inserted into puc along with a multiple cloning site (mcs) and an sv polya recognition sequence (fig. a) . the expression cassette was flanked by not i sites to allow subsequent insertion into the not i site engineered into the fav- nde i fragment . the right hand end nde i fragment of fav- was cloned (fig. ) and sequenced (genbank accession number af ). a unique not i site was inserted into the bam hi site ( . map units). ck cells were transfected with the nde i fragment containing the not i site together with the spe i fragment which contained nearly the entire fav- genome except for the right hand end . kb and overlapped with the nde i fragment by bp. homologous recombination between these two dna fragments resulted in a complete and viable fav- but with a unique not i site in the genome (fig. ) . the ibdv vp gene from australian strain / [ ] was inserted into the mcs of the expression cassette (fig. b ) restriction enzyme digestion and dna sequencing confirmed the construct. the expression cassette was isolated as a not i fragment and cloned into the not i site of the cloned fav- ndei fragment . the plasmid containing the vp gene was linearized and transfected with the fav- spe i fragment into ck cells. the resultant recombinant virus designated as rfav- /vp (fig. b) , was plaque purifed three times. genomic dna was prepared and checked by restriction enzyme analysis and southern blotting to confirm the presence of the expression cassette and vp gene within the not i site of fav- nde i fragment (data not shown). the immunogenicity of the expressed vp was confirmed in an antigen elisa using the neutralising monoclonal antibody - [ ] . three week old spf birds were divided into two groups. one group was vaccinated with rfav- /vp intravenously (i.v.) at a dose of pfu in approximately l of inoculum. a second group was vaccinated i.v. with fav- at a dose of pfu [ ] . birds were bled two weeks post-vaccination (p.v.) and then every week until six weeks p.v. antibodies to fav and vp were assessed by elisas. three week old spf birds were divided into five groups. one group was vaccinated with rfav/vp i.v. at a dose of pfu in approximately l of inoculum. a second group was vaccinated i.v. with fav- at a dose of pfu. for comparative purposes, a commercially available inactivated oil emulsion ibdv / vaccine (bursavac k, websters) was included in the trial. this vaccine was administered in a . ml volume by the subcutaneous (s.c.) route, on the inside of the thigh muscle. a fourth group was unvaccinated. birds in groups - were challenged with ibdv strain v instilled via the conjunctival sac in l of inoculum. group , unvaccinated, were not challenged. all birds were bled weekly for three weeks prior to challenge. at days post-challenge, all birds were euthanised, the bursa removed and divided, half used for immunological testing (antigen elisa) and other half for immunohistology. in order to further explore possible vaccination strategies, one day old spf chickens vaccinated either intraperitoneally (i.p.), intramuscularly (i.m.), s.c. or instilled via the conjunctival sac with either fav- or rfav- /vp . eight groups of day old spf chicks, per group, were vaccinated with rfav/vp via conjunctiva pfu (group ), i.p. pfu (group ), i.p. pfu (group ), i.p. pfu (group ), i.m. pfu (group ), s.c. pfu (group ) or with fav- pfu via conjunctiva (group ) or unvaccinated (group ). birds in groups - were challenged with ibdv v instilled via the conjunctival sac in l of inoculum. all birds were bled weekly for three weeks prior to challenge with ibdv v . at days post-challenge, all birds were euthanised and bursa removed for immunological testing in an antigen elisa and immunohistology. antibody and antigen-capture elisa for ibdv has been described previously [ ] . briefly, for the detection of antibodies to ibdv the microtitre plates were coated with a standardised amount of purified virus, followed by serial dilutions of the chicken sera. affinity purified goat anti-chicken igg (h + l) peroxidase labelled antibody (kpl, australia) was added, followed by the substrate , -azino-bis( -ethylbenzthiazoline) sulfonic acid (abts). serum antibody titres were expressed as the reciprocal of dilutions giving an od nm of . . the ibdv antigen capture elisa was performed by preparing a % (w/v) bursal homogenate and applying this undiluted or serial dilutions to microtitre wells coated with monoclonal antibody - [ ] followed by conjugated rabbit anti-chicken igg hrp. bursae were considered negative for infection when no antigen was detectable in the undiluted % (w/v) bursal homogenate. for the detection of fav- antibodies, microtitre plates were coated with a standardised amount of purified fav- , followed by serial dilutions of chicken sera. affinity purified goat anti-chicken igg (h + l) peroxidase labelled antibody was added, followed by substrate abts. plates were read at od nm . pooled negative sera determined the cut off at . od nm . immunoperoxidase staining of thin sections of bursa were performed. briefly, paraffin embedded tissue sections were prepared on glass slides. sections were treated with trypsin, blocked with % horse serum and then incubated with monoclonal antibody - [ ] . a biotinylated horse anti-mouse antibody was applied followed by vectastain (immunodiagnostics, australia), then peroxidase substrate (dab) and counterstained with haematoxylin. the nde i fragment of . kb at the very right hand end of the fav- genome was cloned (fig. a) the plasmid pgem zf(+) mcs was modified to contain hind iii and bam hi sites (pms ab). the left portion of nde i fragment was cloned as a nde i-bam hi fragment into pms ab. the terminal bam hi fragment was generated by polymerase chain reaction, with an apa i site of the itr, and cloned into pms ab already containing the nde -bam hi fragment resulting in plasmid pms . a not i site was inserted into the bam hi site of pms , resulting in plasmid pms . the recombinant rfav- /not i was generated by digesting pms with nde i-apa i and mixing with fav- spe i fragment using a calcium chloride transfection procedure to transfect ck cells. resulting plaques were plaque purified and tested for the presence of a unique not i site aataaa sequence downstrem from this restriction site. it is not known whether this region contains an actual coding or non-coding sequence. if this small putative orf is not transcribed, then a relatively large region of up to bp (not including the itr) could possibly be deleted, allowing extra room for insertion of foreign dna. in the first construction, the bam hi site in the fav- nde i fragment was replaced with a not i site and the resulting plasmid transfected with spe i fragment to produce a recombinant fav- containing a unique not i site (rfav- /noti) (fig. ) . the recombinant was plaque purified three times and genomic dna analysed by restriction digestion and southern blot comparison to parental fav- dna (data not shown). in order to confirm that the insertion of a not i site had not interrupted any functional transcription unit, day old spf chickens were vaccinated via the conjunctival sac with pfu of rfav- /not i. at day and b the recombinant fav- /vp was generated as follows: the plasmid pms (nde i fragment /not i) was digested with not i and the not i fragment containing the mlp/sl-vp -poly a expression cassette from pms ligated in, resulting in plasmid pms . the plasmid pms was digested with nde i-apa i and mixed with fav- spe i fragment and transfected into ck cells using a calcium chloride procedure. virus was plaque purified and screened for the presence of the vp gene day post vaccination, kidneys were removed, homogenised and plated onto ck cell monolayers. cultures were passaged - times until cytopathic effect (c.p.e.) was observed. genomic dna was prepared, restriction digested with not i, southern blotted and probed with fav- nde i fragment . the results (not shown) confirmed that rfav- /not i replicated in vivo and that no functional transcription unit was interrupted. we then constructed a second recombinant, not deleting any of the fav- genome but took advantage of the % genome packaging availability of the virus capsid [ , ] . the vp gene from the australian strain / of ibdv [ ] was inserted into the major late promoter expression cassette (fig. b) . the vp expression cassette was then cloned into the not i site of nde i fragment in plasmid pms . the resulting recombination expression cassette plasmid pms was transfected fav- spe i fragment , a recombinant virus, rfav- /vp isolated and plaque purified three times (fig. b) . the recombinant was analysed by restriction digestion and southern blots, probed with ibdv vp dna or fav- nde i fragment (data not shown). the immunogenicity of the vp expressed by the recombinant in cell cultures was further analysed in an antigen capture elisa. the recombinant expressed vp was shown to react strongly with the neutralising monoclonal antibody - [ ] . in order to confirm the expression of the ibdv vp by the recombinant in vivo, three-week-old spf chickens were inoculated with either pfu of fav- or pfu of rfav- /vp i.v. all chickens were pre-bled, then at two weeks p.v., and then weekly until week six p.v. the sera was tested by elisa for antibody to ibdv (vp ). table shows that detectable antibodies to vp were present days p.v. in birds vaccinated with rfav- /vp . generally, vp antibodies in order to assess the efficacy of the rfav- /vp , three-week-old spf chickens were inoculated with pfu of rfav- /vp i.v. (group ), pfu of fav- i.v. (group ), or with a killed commercial ibdv vaccine k s.c. (group ). two other groups were unvaccinated (groups and ). all chickens were prebled, then bled weekly for three weeks post vaccination. the sera were tested by elisa for antibody to ibdv. table shows the results of experiment . as was shown in experiment , detectable antibodies to vp were present days post vaccination with rfav- /vp (not shown) and increased by days post inoculation. high vp antibody levels correlated with high fav- antibodies in the recombinant vaccinated group . to examine the efficacy of the recombinant vaccine, groups - were challenged with ibdv strain v . group birds (unvaccinated) were not challenged. chickens vaccinated at weeks i.v. with rfav- /vp were protected from challenge with ibdv v . in contrast, birds vaccinated with fav- or unvaccinated birds were not protected from challenge as shown in table . all chickens vaccinated with either bursavac k or rfav- /vp had detectable levels of vp antibodies and unvaccinated chickens or chickens vaccinated with fav- only, had no detectable vp antibodies (not shown). at days post challenge, the bursa from all chickens were extracted and tested for the presence of ibdv antigen by elisa. the presence of ibdv antigen in the bursa indicates the failure to protect against ibdv challenge. the results a bird # m -m are group , fav- vaccinated; bird # r -r are group , rfav- /vp vaccinated. group birds (bursavac k vaccinated-challenged) were negative for vp antigen (ag), negative for fav- ag and positive for vp antibodies (ab) (results not shown). group (unvaccinated-challenged) were negative for vp ab, negative fav- ab at day pre-challenge (results not shown). group birds (unvaccinated -no challenge) were all negative for vp ag and ab elisas (results not shown) b vp antibody titres at day pre-challenge c fav- antibody titres at day pre-challenge d -; undetectable in undiluted bursa homogenates e ±; negative using titrated bursa homogenates, weakly positive using undiluted bursa homogenates f positive result using undiluted bursa homogenate presented in table show birds vaccinated with fav- produced detectable levels of ibdv (vp ) antigen present in the bursa. all these birds were negative for vp antibody in pre-challenge sera. all recombinant vaccinated birds had high levels of vp antibodies in pre-challenge serum and either showed no detectable ibdv antigen in the bursa or in three cases, only detectable in undiluted bursa homogenates. this indicates that in these three cases some ibdv was getting into the bursa. birds that were vaccinated with the recombinant fav- /vp and tested negative for ibdv antigen by elisa were also negative for ibdv antigen by immunohistology. to further investigate the efficacy of the rfav- /vp , groups of day old spf birds were vaccinated via the conjunctival sac, i.p., i.m. or s.c. table shows the results of the experiment. birds vaccinated by the i.p. route developed antibodies to fav- and vp in a dose dependent manner, as shown by groups , and . all routes, except via conjunctiva were successful in establishing an immune response sufficient to protect birds from challenge with ibdv v . unvaccinated birds were not protected. all birds protected showed no evidence by immunohistology of ibdv antigen in the bursae days post challenge, further indicating that the rfav- /vp was successful in eliciting a protective immune response. a geometric mean vp antibody titres at day pre-challenge b geometric mean fav- antibody titres at day pre-challenge c rec, rfav- /vp ; all birds were bled before vaccination and tested in vp and fav- antibody elisa. none contained detectable antibodies and the results are not shown. birds were bled at days post vaccination and antibody titres to vp and fav- determined. groups to were challenged at day with live ibdv (bursavac s). protection assessed by the number of birds with vp antigen titres of less than , negative for immunoperoxidase staining and showing no bursal depletion in histopathology d protection assessed by negative antigen elisa and no antigen detected by immunohistology since the early s adenoviruses, herpesviruses and poxviruses have been investigated as candidates for use as live vectors for the delivery of vaccines. generally, when constructing an adenovirus vector, either the e region (replication competent) or the e region (replication defective) is deleted and a foreign gene is inserted [ , , , , , , , , - , , ] . we chose to avoid both of these regions and instead inserted the ibdv vp gene into a non-coding region that was identified in the right hand end of the genome just upstream of the inverted terminal repeat. this site was chosen as it did not appear to disrupt any fav gene expression. the fav major late promoter and leader sequences [ ] were chosen to express the ibdv vp gene as opposed to a heterologous promoter. packaging ability and stability of recombinant human adenoviruses have been shown to be limited to approximately % of the original genome [ , ] . the fav- genome is some kb larger than the hav genome. based on these criteria, it is possible that a foreign insert of up to . kb could be integrated into the fav- genome without requiring deletion of any part of genome. the vp expression cassette consisting of fav- mlp/ls vp coding region and sv polya region totaled . kb and it was decided to insert this expression cassette without deleting any of the fav- genome. the nde i fragment ( . to map units) was chosen as a potential region for the possible insertion of the expression cassetle. sequencing of this fragment revealed a number of unidentified open reading frames and a bam hi restriction site bp from the right hand end of the genome. sequence analysis suggested that this site was in a transcriptionally active region. a not i site was inserted into the bam hi site as not i does not cleave the fav- genome at any other site or the expression cassette and a recombinant virus was generated which contained a unique not i site. inoculation of spf birds, re-isolation in ck monolayers and analysis by restriction digesion demonstrated that this recombinant was stable and viable in vitro and in vivo. it was concluded that this region of fav- was not transcriptionally active and therefore ideal for the insertion of an expression cassette. the mlp/sl-vp -polya expression cassette was inserted into the not i site of nde i fragment , and the resulting fragment was used to generate a recombinant fav- that contained the ibdv vp gene (rfav- /vp ) by homologous recombination. the vp antigen expressed by the recombinant was immunogenic as tested by its reactivity with the conformational dependent neutralising monoclonal antibody - . vaccination of three week old spf birds i.v. with pfu of the recombinant produced an antibody response to vp of ibdv. this experiment clearly demonstrated that a recombinant fav expressing vp was capable of inducing an immune response in spf chickens to vp . the next experiment examined whether the induction of this response to vp was sufficient to protect birds from challenge with ibdv. vaccination with rfav- /vp protected birds from challenge with live ibdv v as no ibdv was detected in bursal homogenates by antigen elisa and immunoperoxidase staining of tissue section. thus, the recombinant stimulated an immune response that was able to prevent ibdv from replicating in the bursa. all birds that were naive, that is unvaccinated or fav- vaccinated, were not protected from challenge with ibdv v . ibdv antigen was detected by the antigen elisa on bursa homogenates and by immunohistology for the presence of ibdv in tissue sections. therefore, in these naive birds, there were deficient antibody levels to provide protection against ibdv challenge. fahey et al. [ ] demonstrated that there was a direct correlation between maternal serum antibody levels and protection with titres of or greater providing complete protection against ibdv. further, it is now known that serum ibdv antibody levels correlate well with serum neutralising antibody levels, implying that the majority of the serum neutralising antibodies are directed against vp (ignjatovic and sapats, pers. comm.). the fact that the rfav- /vp reported in this study induced pre-challenge antibody titres averaging greater than directed specifically against vp (the major protective antigen) demonstrated the use of this recombinant virus as an effective vaccine vector, not only against ibdv, but potentially against other poultry pathogens. vaccination with the recombinant via a number of routes was examined. although the systemic routes used in this report are not what are commercially acceptable for delivery of vaccines, the data demonstrated that a recombinant adenovirus vector could potentially be developed for commercial application. bird s vaccinated systemically (i.v., i.p., i.m., s.c.) developed antibodies to fav- and vp . birds vaccinated by instillation of the conjunctival sac, developed antibodies to fav- later than that seen for vaccination by systemic routes, but failed to produce any detectable antibodies to vp . vaccination of chickens via the conjunctival sac is essentially oral administration, as fluid delivered drains into the naso-pharyngeal cavity and is swallowed by the bird. virus re-isolation experiments showed that recombinant virus could be found in the caecal tonsils but was less easily isolated from kidney, liver or spleen ( - passages in ck cells) compared with systemically vaccinated birds ( passage in ck cells). therefore, fav- replication in natural infection is mainly in the gut and has limited systemic spread. the inefficiency of fav- in crossing the gut is probably due to differences in the cell receptor binding regions present on the fiber. it has been shown for some human adenoviruses that a few amino acid changes in the fiber can determine tissue tropism [ ] . in hav type , a region has been identified which binds to the mhci alpha domain found on the surface of epithelial and ␤-lymphoblastoid cells [ ] . no receptor binding regions have been identified for any fav fiber. it has been demonstrated with fav serotype [ ] , that virulence is function of the short fiber, and that swapping the short fiber from a hypervirulent to a mildly virulent strain resulted in the corresponding phenotypic change. unlike serotype , which has a short fiber [ ] , with the long fiber not yet located, serotype has both a short [ ] and long fiber (unpubl.) in similar positions on the genome relative to that of serotype (celo) [ ] . further, it appears that the long fiber of fav- is of similar amino acid sequence length to the short fiber (unpubl.) it is likely that the lack of virulence of fav- , presumably determined by the short fiber, and correlated with tissue tropism, explains why vaccination via conjunctiva faild to induce a serum vp antibody response sufficient to protect birds from challenge. however, when delivered by systemic inoculation, fav- has the ability to bind and infect cells of other organs and subsequently replicate. this report represents the first construction of a fowl adenovirus viral vector, and demonstrated that it could be used to express an antigen (ibdv vp ) that could produce a protective immune response in a challenge model. the sequence of the vp gene from / is identical to that of the challenge strain v (ignjatovic and sapats, personal communication). v is an intermediate virulent classical strain, which is being used as a vaccine against the very virulent strains of europe and asia. it will be of great interest to further test this recombinant and examine whether it can protect commercial broilers against all very virulent ibdv strains and the antigenic variant ibdv strains of 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and mice replication-defective recombinant adenovirus expressing the epstein-barr virus (ebv) envelope glycoprotein gp / induces protective immunity against ebv-induced lymphomas in the cottontop tamarin isolation and characterization of variant infectious bursal disease viruses. abstracts rd american veterinary medical association the major late promoter and bipartite leader sequence of fowl adenovirus dna sequence of the fowl adenovirus serotype short fiber gene differentiation of infectious bursal disease viruses directly from infected tissues with neutralizing monoclonal antibodies: evidence of a major antigenic shift in recent field isolates naturally occurring neutralizing monoclonal escape variants define the epidemiology of infectious bursal virus in the united states acute infectious bursal disease in poultry: isolation and characterization of a highly virulent strain determination of optimum formulation of a novel infectious bursal disease virus (ibdv) vaccine constructed by mixing bursal disease antibody with ibdv comparison of the efficacy of four inactivated infectious bursal disease emulsion vaccines authors' address: dr. m. johnson, csiro, australian animal health laboratory, private bag nr. , geelong , australia.received august , key: cord- - movyeb authors: garcía-luque, i.; ferrero, m. l.; rodríguez, j. m.; alonso, e.; de la cruz, a.; sanz, a. i.; vaquero, c.; serra, m. t.; díaz-ruíz, j. r. title: the nucleotide sequence of the coat protein genes and ′ non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: movyeb the nucleotide sequence of the coat protein genes and ′ non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. the deduced coat protein of an italian isolate of pepper mild mottle virus (pmmv-i) consists of amino acids and its ′ non-coding region is nucleotides long. they have been found to be very similar in sequence and structure to those previously reported for a spanish isolate (pmmv-s). in contrast, a dutch isolate termed p codes for a coat protein of amino acids and its ′ non-coding region is nucleotides long, which may have arisen by duplication. the nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. the term paprika mild mottle virus (pammv) is proposed. different tobamoviruses such as tobacco mosaic virus (tmv), tomato mosaic virus (tomv), or tobacco mild green mosaic virus (tmgmv) have been described infecting pepper crops [ , , , ] . in addition, the introduction of commercial pepper cultivars and hybrids with incorporated resistance genes to common strains of tmv and tomv has led to descriptions of several tobamoviruses that were able to infect these resistant pepper cuttivars and were given different names [ , , , , , , , , , , [ ] [ ] [ ] [ ] . these resistance-breaking tobamoviruses of pepper have been characterized to different extents by their biological and serological properties and most of them share similar properties such as the ability to induce mottling diseases in pepper, the inability to infect tomato plants, and the induction of necrotic local lesions in some hosts which are much smaller than the ones produced by tmv or tomv in the same plants [ , , , -i. these viruses have been differentiated by breeders as pathotypes p , p , , and pi , based upon their ability to overcome the resistance conferred by an allelic series of capsicum spp. genes known as l , l , and l , respectively [ ' , ] . in every case, the hypersensitive resistance is manifested through the induction of necrotic local lesions. in a previous work, a tobamovirus infecting tmv-resistant pepper crops grown under plastic in the southeast region of spain [ ] was identified as a p , pathotype [ ] and based upon its biological, biochemical and serological properties, it was termed pepper mild mottle virus strain s (pmmv-s) to differentiate it from an italian isolate [ ] , here referred as pmmv-i which was identified as a p , , pathotype [ ] . in order to define the regions of the viral genome involved in the induction of the hypersensitive resistance genes against the tobamoviruses in capsicum spp., we have previously determined the complete genome sequence of pmmv-s. in this work, we present the sequences of the coat protein cistrons and ' non-coding regions of pmmv-i and of a dutch virus isolate termed p , identified as a p pathotype [ ] . the data obtained made it possible to clarify the status of the tobamoviruses involved in resistance breakage of the different capsicum spp. genes, which is important for resistance breeding. in addition, the nucleotide and amino acid sequence homology values found, indicate that the dutch isolate p should be considered as a new virus within the tobamovirus group, different from pmmv-s and pmmv-i which seem to be strains of the same virus. the origin of pmmv-s has been reported [ ] . pmmv-i is an italian isolate of pmmv [ ] kindly supplied by dr. m. conti (italy). the dutch p isolate from pepper [ ] was a kind gift from dr. a. th. b. rast (netherlands). viruses were purified from systemically infected nicotiana clevelandii gray plants and virion rnas were prepared from purified virus particles by sds-phenol extraction as previously described [ , ] . the resistance-breaking characteristics of the purified viruses were confirmed by inoculation to the appropriate capsicum spp. plants [ . the synthesis and cloning of cdna was done essentially as described [ ] . an oligonucleotide ( ' gggggattcgaaccc), complementary to nucleotides (nt) to and to from the ' end of pmmv-i and p isolate rnas, respectively, was used for priming first strand synthesis. double-stranded (ds) cdna was size-fractionated in % polyacrylamide slab gels, ligated to hin dii-digested puc plasmid and used to transform e. coli strain dh c~. due to specific reorganization in most of the cdna clones which contained the coat protein coding region of the p virus, another set of clones was prepared by using an oligonucleotide ( ' tgggccccatacccgggg), complementary to the last ' end nt two pepper resistance-breaking tobamoviruses of this rna as primer for first strand cdna synthesis. the ds cdna was digested with hin diii, fractionated by electrophoresis in a % agarose gel, and the nt long fragment was eluted and cloned into the hin diii-hin dii sites of puc . clones were analyzed for the presence of coat protein-related sequences by colony hybridization, using as radioactive probe a cdna fragment which contained the last nt of pmmv-s rna labelled by nick-translation [ ] . the ' end nucleotide sequences of both pmmv-i and p rnas were determined by the direct cleavage method [ ] on fragments obtained by partial rnase t digestion of the ' end labelled rnas as described [ ] . dna sequences were determined by the chemical degradation procedure [ ] and the dideoxy chain termination method [ ] . for sequencing, subclones were obtained by restriction enzyme or bal digestions [ ] . no sequence variation was found among the different clones analyzed. sequences were analyzed with the dnastar computer programs (dnastar, inc., u.k.). folding of the non-coding region was performed with the rna/star computer program developed at the university of leiden [ ] . multiple sequence alignments were obtained using the clustal v program of higgins and sharp [ ] and higgins et at. [ ] . trees were generated by the neighbor-joining method [ ] . the nucleotide sequence data will appear in the embl, genbank, and ddbj nucleotide sequences databases and accession numbers x and x . pmmv-i r n a and c d n a clones ( fig. a) were used to determine the nucleotide sequence of the ' terminal bases. from the nucleotide sequence analysis (fig. a) , it is predicted that the coding region for the coat protein (cp) is nt long, followed by a non-coding region of bases. the cp is composed of amino acids with a calculated mr . the length of pmmv-i cp is, therefore, two amino acids shorter than the one previously reported for it ( amino acids) [ ] . the alignment of the cp open reading frame (orf) and the ' non-coding region of pmmv-i and pmmv-s (fig. ) shows that both viruses have much sequence identity at the nucleotide level. there are a total of nt changes in the cp coding region and nt changes plus one deletion in the non-coding region. most of the changes ( ) take place at the third base of the codons. only three amino acid exchanges are scattered along the protein (fig. ). of these base changes, are pyrimidine transitions, the most abundant substitution. these exchanges lead to a greater amount of t/a vs. c/g content in this particular region of pmmv-i respect to pmmv-s. it is also noticeable that the variability of the non-coding region ( . %) is less than that in the cp o r f ( . %), indicating that its nucleotide sequence may be under greater functional constraints. of the three amino acid exchanges detected in the cp of pmmv-i, two of them are of the conservative type (ser to thr and ala to vat at positions table shows a comparison of the amino acid composition of the cps of pmmv-s and pmmv-i deduced from the nucleotide sequence and those determined for pmmv-i [ and capsicum mosaic virus (camv), a tobamovirus isolated from pepper in australia [ ] . the differences observed in the amino acid contents may be ascribed to the methods employed for their determination, rather than to differences in the cps, as different isolates of tmgmv [ , ] have been found to have strictly conserved cps. as the molar content of methionines in both the pmmv-s and camv cps also coincide and are greater than that of pmmv-i, it can be inferred that camv is closer to pmmv-s than to pmmv-i. the last nt ofpmmv-i rna may be folded in the same possible structure as that previously proposed for other tobamoviruses [ , , , ] which consist of three pseudoknots preceeding the trna-like structure (fig. a) . the ' non-coding regions of both pmmv-i and pmmv-s can adopt the same overall structure because the nucleotide changes (the a to g transition at position and the deletion at position ) (fig. ) take place in loops of the structure (fig. a) . the only structural difference will be caused by the a to t transversion at position as this will modify the stem of the anticodon arm in the small bulging loop (fig. a) [ ] . as this is a variable region within the trna-like structure [ ] , it is unlikely that biological differences between the viruses would be caused by this change. two pepper resistance-breaking tobamoviruses gaaaacccggcaaatcccaacacagctgaaatagcatctgctactcagcgtgttgat~atgccacggttagtattagggcltgtattaataatcttatgaacgagct tgcgcgtggtacg ggtatgttaaatacggtctccttcgagactatttctaacttgacctggactaccgcagctactacataagtagtttaataagtaaa ' gactatatatagtcagcggtgtatgcttgatac acagtgtttatccctccacttaaatcgaagggcggttgtggtcatcactacatttatgtagtgcaacttg~agaagatgaggtggtacataccaaaatgtacagtggttttccctccact tgaat cgaagggttagttgttggagttttcacgtgagacgtt ggtgcaacgtaactgcgtgtacaactgtaaaagtaaaaaggggttcgaatcccccctttaccccgggtatggggccca~h i the nucleotide sequence of the ' terminal region of the p viral r n a determined from its r n a and from c d n a clones (figs. b and b) showed that f ia id vs i n i lf v r i sdgeng r st p it ns i i t s sl ps f f $ i l v ns i e pf qs gdv--y y p it i i duplicated sequences in b with altered secondary structure are marked by an asterisk. the computer-predicted structure was slightly modified in order to maintain the folding previously determined for other tobamoviruses [ , ] its cp o r f is bases long, followed by a non-coding region of nt. the predicted cp is composed of amino acids with a calculated mr . comparisons of the amino acid and nucleotide sequence of its cp coding region with those from other tobamoviruses ( table ) shows a greater degree of amino acid sequence identity with those of pmmv-s ( . %), t m g m v ( . %), and pmmv-i ( , %) than with other tobamoviruses, and its nucleotide sequence identity being greatest with that of t m g m v ( table ). in contrast, the greatest nucleotide sequence identity in the non-coding region was with those of tomv and odontoglossum ringspot virus (orsv) ( table ). the alignment of the p t cp amino acid sequence with those from the most closely related tobamoviruses (fig. ) shows that, in addition to the rnabinding domains [ ] , there is a well-conserved domain located at residues to , with only one amino acid substitution (asn to gln), which is unique for this virus among the closest tobamoviral cps. the sequence is more variable in the central part of the cp coding region which includes a nt insertion that gives a longer protein. this region has been previously shown to be of very variable length [ ] . the p cp have , and non-conservative amino acid substitutions compared with those from t m g m v , tmv and tomv, respectively (fig. ) . they are concentrated at position (val to ash), in the region located between residues to , and in the one-third carboxy part of the protein. the number of non-conservative amino acid substitutions with respect to pmmv-s and pmmv-i cps are and , respectively (fig. ) . they correspond to amino acid residues (ile to gln), (ser to val), (ala to arg), (leu to ala), (tyr to leu), (leu to ser), and (met to asn). this last exchange table . nucleotide (n) and amino acid (a) sequence identities of the p and pmmv-i coat protein (cp) genes and the ' non-coding regions (nc) and those of other tobamoviruses [ ] coincides with the only non-conservative substitution present in the pmmv-s and pmmv-i cps (fig. ) . the " non-coding region of the p viral rna (fig. b) is unusually long and contains bases, with a duplicated sequence domain (positions to and to ) (fig. b) , which has also been recently found to be repeated in the ' non-coding region of orsv [ ] . similar duplication features have also been reported in the ' non-coding regions of viruses belonging to the tobravirus group [ ] as well as in viroids [ ] . the ' terminal nt can be folded in a similar structure to that of other tobamoviruses [ , , ] (fig. b) . in the trna-like structure, the anticodon arm (ac arm) consists of a hairpin with a five nt bulging loop. as the g u g anticodon in the loop is an anticodon for histidine, this rna may also be charged with histidine like most other tobamoviruses [ , ] . however, in the stalk structure the double helical segments v and vi are not present (fig. b) . the double helical segment iv is preceded at its ' end by a bases long fragment which may also adopt a similar configuration with six double helical segments, i'-vi' (fig. b) , able to form three pseudoknots and a second truncated trna-like structure at its ' end, in which the aminoacylacceptor arm (aa arm) is absent, and the ac arm is much shorter than the one found downstream. the tobamovirus phylogenetic tree obtained (fig. ) shows that both pmmv-i and pmmv-s are located in the same branch as tmv and tomv, the two most closely related tobamoviruses so far described. this clustering corresponds with those previously proposed [ , ] , even though different types of data were used for constructing the dendrograms; namely amino acid compositions of the cps, the peptide patterns of the k proteins, and the amino acid sequences of the cps deduced from their nucleotide sequences. the p isolate is linked with tmgmv in a different branch indicating that it has evolved independently from both pmmv-s and pmmv-i. in this sense, and based upon previous serological findings [ , ; our unpubl, data] , the hungarian tomv-ob [ ] should map in the same branch as p since the grouping of tobamoviruses correlates well with their serological relationships. the comparison of the nucleotide and amino acid sequence here presented for pmmv-i with that of pmmv-s [ ] shows that they are strains of the same virus even though their differential ability to overcome the l and l resistance genes of capsicum spp., respectively [ , ] . therefore, we think that other resistance-breaking tobamoviruses of pepper which have been previously shown to be similar in biological and serological properties to these viruses [ , , , , , ] should also be referred to as strains of pmmv. the degree of dissimilarity found in the nucleotide sequence and amino acid content of the coat protein of the dutch p isolate, another tobamovirus of pepper that can break the resistance conferred by the capsicum l gene, with respect to either pmmv-i or pmmv-s and the rest of the known tobamoviruses, indicates that this virus should be considered as a distinct virus within the tobamovirus group. we propose the term of paprika mild mottle virus (pammv) to designate it because the mottling symptoms induced by this virus in pepper cultivars are hardly distinguishable form the ones induced by pmmv-s and pmmv-i. it is possible that the amino acid exchanges observed among the cps of these tobamoviruses could be involved in their differential ability to overcome the resistance conferred by the different hypersensitive resistance genes in capsicum spp. plants as it has been previously shown for similar structural changes in the cp of tmv and tomv [ , which are responsible for the induction of the hypersensitive reaction in n. sylvestrisplants. however, it is also possible that other regions of the viral genome could be implicated in this characteristic as is found with some strains of tomv which are able to break the tm i and tm resistance genes in tomato plants [ , , ] . it is possible that the unique non-conservative met to asn substitution at position in the cp could be responsible for pmmv-i being able to overcome the resistance conferred by the capsicum l s gene. similarly, it has been previously reported that a single amino acid substitution at position (ser to phe) in the cp of tmv confers the ability to induce the hypersensitive response in n. sylvestris plants which carry the n' resistance gene [ ] . it is noticeable that pmmv-s is located closer to the branch point than pmmv-i in the phylogenetic tree (fig. ) . thus, pmmv-i seems to have evolved further from its common ancestor pmmv-s. this might have occurred because pmmv-i has been positively selected since the introduction of the l resistance gene in pepper crops. as mentioned above, part of the ' non-coding region of the p virus seems to have arisen by duplication. in the trna-like structure of brome mosaic virus (bmv), it has been found that certain regions such as the anticodon nucleotide sequence [ ] or the sequences which correspond to the internal transcription promoter of eukaryotic trnas, often referred as b-box, are necessary for the efficient replication of the virus [ , , and references herein]. the sequences equivalent to the b-box (ggtcgnnc) are located in the aa arm of the trnalike structure of the tobamoviruses. interestingly, none of the equivalent regions are repeated in the p viral rna. takamatsu et al. [ ] have recently demonstrated that the double helical segments v and vi in the ' non-coding region of tmv are not necessary for its replication, while the maintenance of stem region i and the u a a a u sequence in segment ii are essential for its efficient replication. in contrast with the trna-like structure, double-helical segments i and ii correspond to the duplicated sequences of p and orsv. the a to g substitution in the segment ii of the p virus (ugaau) is the only difference found in the duplicated structural elements. the functional significance, if any, of the conservation of these motifs is unknown at present. it is possible that this region may be recognized by host and/or viral factors involved in the viral replication mechanism and also help maintain the structure of the encoded motif. recombination phenomena have been described in many animal and plant r n a viruses [ , , , , , ] , and it is thought that it occurs as a copychoice mechanism, when the replicase is synthesizing one of both strands involved in secondary structures [- , ] . the phenomenon described here, indeed, seems similar. therefore, the nucleotide differences between the upstream and downstream sequence s in the ' non-coding regions of p and orsv may result from recombination with another tobamovirus coinfecting the same cell as has been described for polioviruses [ ] , although they also could be caused by a greater evolutionary rate in the duplicated sequences. prediction of rna secondary structure, including pseudoknotting, by computer simulation sequence of cowpea chlorotic mottle virus rnas and and evidence of a recombination event during bromovirus evolution a tobamovirus causing heavy losses in protected pepper crops in spain nucleotide sequence of the genomic rna of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper correlation of co-ordinated amino i acid substitutions with function in viruses related to tobacco mosaic virus nucleotide sequences of ' and ' non-coding regions of pepper mild mottle virus strain s rna evolutionary changes in tmv pepper strains as a result of repeated host passages strains of tmv and genes for resistance in capsicum genetic recombination between rna components of a multipartite plant virus modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of bmv rnas containing deletions within the multifunctional ' end nucleotide sequence analysis of the movement genes of resistance breaking strains of tomato mosaic virus recombination between satellite rnas of turnip crinkle virus the amino acid composition of the coat protein of a tobamovirus from an australian capsicum crop a new pepper strain of tomato mosaic virus point mutations in the coat protein gene of tobacco mosaic virus induce hypersensitivity in nicotiana sylvestris mutant viral rnas synthesized in vitro show altered aminoacylation and replicase template activities an unusual strain of tobacco mosaic virus from pepper a classification of the tobamoviruses based on comparisons among their k proteins sequence and structure at the genome 'end of the u -strain of tobacco mosaic virus, a histidine-accepting tobamovirus characterization of a spanish strain of pepper mild mottle virus (pmmv-s) and its relationship to other tobamoviruses tobamovirus classification nucleotide sequence of tobacco mosaic virus rna some viruses affecting tomatoes and peppers in argentina resistance to tobacco mosaic virus in capsicum, with reference to the sansum latent strain a simple and very efficient method for generating cdna libraries the complete nucleotide sequence of tobacco rattle virus rna- fast and sensitive multiple sequence alignments on a microcomputer two pepper resistance-breaking tobamoviruses clustal v: improved software for multiple sequence alignments molecular cloning, sequencing and expression in escherichia coli of the odontoglossum ringspot virus coat protein gene domains in viroids: evidence of intermolecular rna rearrangements and their contribution to viroid evolution the mechanism of rna recombination in poliovirus a point mutation in the tobacco mosaic virus capsid protein gene induces hypersensitivity in nicotiana sylvestris high-frequency rna recombination of murine coronaviruses molecular cloning: a laboratory manual similarities among plant viruses (+) and ( -) rna termini imply a common ancestry with promoters of eukaryotic trnas occurrence of pepper mild mottle virus in pepper cuttivars from italy and spain the present status of tomato and pepper viruses sequencing end-labeled dna with base-specific chemical cleavages two strains of tobacco mosaic virus, one of which is seed-borne in an etch immune pungent pepper nucleotide sequence of a cloned cdna copy of tmv (cowpea strain) rna, including the assembly origin, the coat protein cistron, and the ' non-coding region nucleotide sequence of the coat protein cistron and the ' noncoding region of cucumber green mottle mosaic virus (watermelon strain) rna two concomitant base substitutions in the putative replicase genes of tobacco mosaic virus confer the ability to overcome the effects of a tomato resistance gene, tin- mutations in the tobacco mosaic virus -kd protein gene overcome tm- resistance in tomato transfer of the movement protein gene between two tobamoviruses: influence on local lesion development nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome a tobamovirus infecting capsicum in australia serological comparison of an australian isolate of capsicum mosaic virus with capsicum tobamovirus two pepper resistance-breaking tobamoviruses direct chemical method for sequencing rna introductory remarks on strains of tmv infecting peppers in the netherlands the three-dimensional folding of the trna-iike structure of tobacco mosaic virus rna. a new building principle applied twice two anomalous tobravirus isolates: evidence for rna recombination in nature mutational analysis of the coat protein gene of tobacco mosaic virus in relation to hypersensitive response in tobacco plants with the n' gene the neighbor-joining method: a new method for reconstructing phylogenic trees dna sequencing with chain termination inhibitors le virus de la mosaique du tabac chez le piment. i. apparition en france du pathotype pi the complete nucleotide sequence of the genomic rna of the tobamovirus tobacco mild green mosaic virus new strain of tobacco mosaic virus (tmv) in pepper mutational analysis of the pseudoknot region in the ' noncoding region of tobacco mosaic virus rna tobamoviruses of pepper, eggplant and tobacco: comparative host reactions and serological relationships five pseudoknots are present at the nucleotides long ' noncoding region of tobacco mosaic virus rna serologicalidentification of four tobamovirusesinfecting pepper pepper mild mottle virus. aab descriptions of plant viruses pepper mild mottle virus, a tobamovirus infecting pepper cultivars in sicily t ) bell pepper mottle virus, a distinct tobamovirus infecting pepper crossover regions in foot-and-mouth disease virus (fmdv) recombinants correspond to regions of high local secondary structure the authors thank m. v. lafita for typing the manuscript. m. l. f. and e. a. were supported by fellowships from fundaci n ram n areces. the work was supported by grants from cicyt (agr - and agr - ) and fundacidn ramdn areces. authors' address: j. r. diaz-ruiz, centro de investigaciones biol gicas, c.s.i.c., vel/tzquez , e- madrid, spain.received july , key: cord- -he vekrt authors: lambeth, l. s.; yu, m.; anderson, d. e.; crameri, g.; eaton, b. t.; wang, l.-f. title: complete genome sequence of nariva virus, a rodent paramyxovirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: he vekrt nariva virus (narpv) was isolated from forest rodents (zygodontomys b. brevicauda) in eastern trinidad in the early s. initial classification within the family paramyxoviridae was based mainly on morphological observations including the structure of nucleocapsids and virion surface projections. here, we report the characterization of the complete genome sequence of narpv. the genome is , nucleotides in length, conforming to the rule-of-six, and has a genome organization typical of most members of the family, with six transcriptional units in the order ′-n–p-m-f–h-l- ′. the gene junctions contain highly conserved gene start and stop signals and a tri-nucleotide intergenic sequence present in most members of the subfamily paramyxovirinae. sequence comparison studies indicate that narpv is most closely related to mossman virus, which was isolated from wild rats (rattus leucopus) in queensland, australia, in . this study confirmed the classification of narpv as a member of the subfamily paramyxovirinae and established the close genome organization and sequence relationship between the two rodent paramyxoviruses isolated almost a decade apart and from two locations separated by more than , km. members of the family paramyxoviridae are pleomorphic enveloped viruses possessing a nonsegmented negativestrand (nns) rna genome [ ] . they are divided into two subfamilies, paramyxovirinae and pneumovirinae. the subfamily paramyxovirinae is currently classified into five genera: respirovirus, morbillivirus, rubulavirus, avulavirus and henipavirus. the subfamily pneumovirinae consists of two genera: pneumovirus and metapneumovirus [ ] . paramyxoviruses are well-known pathogens of humans (measles virus, mumps virus, etc.) and livestock animals (newcastle disease virus, rinderpest virus, etc.). the emergence of henipaviruses (hendra and nipah viruses) further highlighted the broad host range and the severity of the diseases that can be caused by novel paramyxoviruses [ , ] . bats are increasingly recognized as an important reservoir of novel viruses, including paramyxoviruses, coronaviruses and, potentially, filoviruses [ ] . at least five bat paramyxoviruses have been identified, which include hendra virus, nipah virus, tioman virus, menangle virus and mapuera virus. there is anecdotal evidence suggesting that porcine rubulavirus may also originate from bats [ , ] , and indian bats may carry a paramyxovirus antigenically related to simian virus and parainfluenza virus [ ] . while it is not clear why bats seem to be an ideal reservoir host for many different viruses, one suggested possibility is the direct result of their great species diversity and population abundance. if these are the main drivers for virus distribution and evolution, one would expect to find even more viruses in rodents, since they are the most diverse mammalian animals on earth [ ] . sendai virus (sev), although first isolated from a human specimen, is believed to have rodents as its natural reservoir [ ] and represents the first and best characterized paramyxovirus of rodent origin. since then, at least four other paramyxoviruses, mossman virus (mospv), j virus (jpv), beilong virus (beipv) and nariva virus (narpv) have been isolated from rodents. the complete genome sequences of the first three rodent viruses were determined previously by our group [ , , ] . here, we report the full-length genome sequence of narpv to complete the molecular analysis of all known rodent paramyxoviruses. narpv was isolated from forest rodents, zygodontomys b. brevicauda, trapped in the nariva swamp in eastern trinidad in the early s [ , ] . the virus grew in suckling mouse brain and formed syncytia in vero and bhk cells. narpv was identified as a member of the family paramyxoviridae, mainly based the structure of its nucleocapsids (approximately nm in diameter and mean length . lm) and its virion morphology, being enveloped, spherical and pleomorphic with surface projections [ ] . the virus displayed no serological cross-reactivity with a range of known paramyxoviruses at the time of isolation, including parainfluenza virus types - , mumps virus, newcastle disease virus and measles virus [ ] . narpv resembles members of the genera respirovirus and rubulavirus in that it generates only cytoplasmic inclusion bodies in virus-infected cells [ ] . in this respect, it differs from members of the genus morbillivirus that produce both cytoplasmic and nuclear inclusions in infected cells. however, haemagglutination and cell-binding studies performed on guinea pig and monkey red blood cells suggested that narpv, like measles virus, does not use sialic acid receptors on red blood cells as do respiroviruses and rubulaviruses [ ] . the data presented in this study confirmed the classification of narpv as a member of the subfamily paramyxovirinae. although narpv could not be classified into any of the existing genera, it is clear that narpv is most closely related to mospv, confirming its rodent origin. analysis of the narpv h protein also revealed the lack of the consensus nrkscs sequence motif known to be important for sialic acid binding for the hn proteins of respiroviruses and rubulaviruses [ ] . virus culture and rna isolation vero cells were infected with narpv or salem virus (salpv) [ ] , which was used as a driver in the cdna subtraction experiment described below, and incubated at °c until the appearance of syncytia. upon reaching approximately % cpe, the medium was replaced with pbs, and cells were collected in pbs. cells were pelleted by centrifugation at g for min, and virus in the supernatant was then harvested by ultracentrifugation at , g for min. rna extraction was performed using the rneasy mini kit (qiagen) according to the manufacturer's instructions. after elution from the column in rnase-free water, rna concentration was determined using a gene quantii (pharmacia). the rna concentration was adjusted to approximately lg/ll for subsequent applications. genome characterization by cdna subtraction and gap-filling pcr a clontech pcr-select cdna subtraction kit (clontech, usa) was used to select virus-specific fragments as previously described by our group [ , , ] . double-stranded cdna was made using random hexamer oligonucleotide primers and lg each of total rna as prepared above from pelleted narpv (tester cdna) and salpv (driver cdna). digestion, adaptor ligation, hybridization and pcr reactions were then carried out as described in the instructions provided with the kit. nested pcr products from both subtractions were size-purified on a % agarose gel in three fractions ( . - . , . - . and . - . kb) using the qia-quick pcr gel extraction kit (qiagen). the three purified fractions were cloned, and plasmids with insert sizes bp or greater were randomly selected and sequenced. for filling the gaps that were not covered by fragments obtained from the cdna subtraction above, random cdna was synthesized using the omniscript rt kit (qiagen) with random hexamer primers. virus-specific primers were designed using either cdna subtraction-derived sequences or consensus sequences of published paramyxovirus genomes and made by a commercial provider (geneworks, australia). the platinum pcr supermix kit (invitrogen, usa) was used to perform pcr on the cdna template synthesized as described above. pcr products were visualized with ethidium bromide on - % agarose gels and purified, using either the qiaquick pcr purification kit (qiagen) or the qiaquick gel purification kit (qiagen), prior to dna sequencing. the sequences of the genome and anti-genome termini were determined using a modified procedure from a previously published method [ ] . virus growth and rna extraction were conducted as described above. total rna (containing both genome and anti-genome rna) was used for cdna synthesis using the thermoscript rt-pcr system kit (life technologies, usa) and virus-specific primers located within approximately - nt of the genome termini. reverse transcriptase reactions were incubated at °c for min followed by rt inactivation at °c for min and treatment with rnase h. the firststrand cdna was purified using the qiaquick pcr purification kit (qiagen) prior to ligation with the anchor oligonucleotide ( -gaagagaaggtggaaatggcgt tttgg, -phosphorylated and -blocked) using t rna ligase (new england biolabs, usa). the ligated product was amplified by pcr using a virus-specific primer, nested with respect to the first primer used for cdna synthesis, and a -nt adaptor primer complementary in sequence to the adaptor. when required, a hemi-nested pcr, using the same adaptor primer and an additional (third) nested virusspecific primer, was also performed. the pcr products obtained were gel purified as described earlier and either sequenced directly or cloned before sequencing, in which case at least six individual clones were sequenced to ensure a reliable consensus sequence. purified pcr products or plasmid dna were sequenced using the bigdye Ò terminator v . kit (applied biosystems, usa) and an abi prism dna sequencer (applied biosystems). every nucleotide in the genome was sequenced with a minimum of threefold redundancy, at least once in each sense and at least once directly from pcr products without cloning. the clone manager and align plus programs in the sci ed central software package (scientific and educational software, usa) were used for routine sequence data management and analysis. sequence similarity searches were conducted using the blast service at the national center for biotechnology information (ncbi). phylogenetic trees were constructed using the neighbour-joining algorithm with bootstrap values determined by , replicates in the mega software package [ ] . the full-length genome sequence of narpv has been deposited into genbank under the accession number fj . accession numbers for other sequences used in this study are listed below. for viruses where full-length genome sequence was not available, individual protein sequences were used and are indicated by the abbreviated gene letter in parentheses following the accession number. the new naming convention for paramyxovirus abbreviations, as proposed in the th ictv report [ ] , was used in this paper for those viruses which have not been formally classified. atlantic salmon paramyxovirus (asapv) eu ; avian paramyxovirus type (apmv ) characterization of the narpv genome similar to strategies used for characterization of paramyxovirus genomes used in our group, a cdna subtraction approach was used initially to obtain narpv-specific sequences. this was then followed by pcr to fill in the ''gaps'' using specific primers designed from narpv nariva virus genome sequences obtained from the cdna subtraction or degenerate primers designed using highly conserved consensus sequences of known paramyxoviruses in the subfamily paramyxovirinae. finally, the genome terminal sequences were obtained using a modified race technique as described in ''materials and methods''. as shown in fig. a , the narpv genome organisation is similar to those of most members of the subfamily paramyxovirinae, containing six transcriptional units in the order -n-p/v/c-m-f-h-l- . at , nt, the narpv genome is at the lower end of the genome size spectrum known for members of the subfamily paramyxovirinae, varying from , nt for porcine rubulavirus [ ] to , nt for beilong virus [ ] . the genome length of narpv conforms to the rule-ofsix [ ] , a characteristic known to be true for all sequenced members of the paramyxovirinae. as with all other members of the paramyxovirinae, narpv has a leader of nt. the trailer of narpv is nt long. the first and last - nt of paramyxovirus genomes are highly conserved and complementary in nature and are critical elements of the genome and antigenome viral rna polymerase promoter [ ] . this is also true for narpv (fig. a) . when the leader sequence was aligned with those of other subfamily members (fig. b) , it was evident that narpv's genome end sequence is most similar to that of mospv and least similar to those of rubulaviruses or avulaviruses. it is worth noting that the narpv genome has a c residue at the fifth position from both ends, which is unique among known members of the paramyxovirinae. this region contains the promoter for replication and transcription and has been shown by reverse genetics to be important for pathogenicity and attenuation of certain paramyxoviruses [ ] . overall comparison of deduced sizes and amino acid sequences of all proteins indicated that narpv is most closely related to mospv (table ) genome is significantly larger ( , nt) than that of narpv due to the longer untranslated regions (utrs) located at the end of most genes ( fig. b and table ). the narpv genome has a coding capacity of approximately %, which is higher than the average of % for members of the subfamily paramyxovirinae [ ] , whereas the presence of longer utrs has resulted in a lower coding capacity of % for the mospv genome. transcription of individual genes of paramyxovirinae members is carried out by a stop-and-reinitiation mechanism controlled by conserved sequences at the gene borders [ ] . members of genera respirovirus, morbillivirus and henipavirus have a conserved trinucleotide intergenic region (igr), whereas viruses in the genera rubulavirus and avulavirus have igr of variable length and sequence. the gene start, stop and igr sequences of narpv are listed in table (shown as cdna sequence in the antigenome ? sense). the sequence comparison suggests that the transcriptional regulatory elements of narpv are most similar to those of the mospv genome. the coding strategy of the multi-cistronic p gene of paramyxovirinae members is an important genomic feature used for classification. similar to most subfamily members, the non-edited mrna of the narpv p gene codes for the p protein, whereas the mrna with a single g-insertion at the rna editing site codes for the v protein. both mrna species have the coding capacity for a c protein, which is coded in an alternative reading frame. however, insertion of two g's results in an mrna containing a stop codon immediately after the editing site, suggesting that narpv does not code for a w-like protein present in certain subfamily members such as henipaviruses [ ] . the narpv p gene has the editing site sequence -actaaaaggg gca- , which is identical to that in the mospv genome [ ] . a for the p/v/c gene of narpv, there are two different types of mrna made via rna editing, each encoding a different protein product, i.e., p (non-edited mrna) and v (? g) b the putative c protein can be made from either of the two possible mrna species molecular features of deduced narpv structural proteins the n proteins of narpv and mospv share a % sequence identity. as for other paramyxovirus n proteins, the most variable region is located in the c-terminus. the sequence identity increases to % when the n-terminal -aa regions are compared. the region containing the highly conserved paramyxovirinae n-protein motif f-x -y-x -u-s-u-a-m-g (x = any aa; u = aromatic aa) has identical sequence between narpv and mospv, both having the sequence fapgnypllwsyamg. the p and v proteins of narpv are slightly acidic in nature, with pi values of . and . , respectively. the sequence identities between these two proteins of narpv and mospv are much lower than that observed for their n proteins (table ). it is interesting to note that the difference in size of the two p proteins is due to a -aa deletion in the narpv p protein (or a -aa insertion in the mospv p protein), present in the region upstream of the p/v editing site (fig. a) . since the ''deletion'' area was located close to the stop codon of the narpv c orf, the conservation of these proteins was largely unchanged as this resulted in only a -aa truncation in comparison to the mospv c protein (fig. b) . it is also interesting to note that, in overlapping coding regions, there are significantly higher amino acid sequence identities between the c proteins and v proteins of the two viruses than the p proteins. for the c and p common region, the percentage sequence identify is % for c and % for p. for the v and p common region, it is % for v and % for p. the m protein of narpv is aa in length. the m proteins of narpv and mospv have the highest sequence identity among all the deduced proteins and a very similar pi, indicating the basic nature of the proteins (table ) . m is also the only protein that has an identical size between the two viruses. the f protein of narpv, like the f proteins of other paramyxoviruses, is predicted to be a type i integral membrane protein. the predicted cleavage site of the a alignment of the full-length p protein sequences with identical residues shaded. the overlapping coding regions for the c proteins and v-specific polypeptide are indicated by the light grey letters c and v above. b alignment of the full-length c protein sequences. c alignment of the v-specific protein sequences narpv f protein, sgrnk, is dibasic and does not conform to the consensus sequence motif for cleavage by furin, r-x-k/r-r [ ] . it is interesting to note that all of the rodent paramyxovirus f proteins characterized so far have the mono-or dibasic cleavage site instead of the more common multi-basic furin cleavage site observed in most other paramyxoviruses. their cleavage sites resemble those of the bat-derived henipaviruses and the fish-derived asapv (see fig. ). the predicted cleavage site of the narpv f protein is immediately followed by a highly conserved -aa putative hydrophobic fusion peptide as for all known paramyxovirus f proteins. the mprpv f protein contains potential sites for n-linked glycosylation. unusually, are located in the f region, and of the are also conserved in the mospv f subunit. in contrast, only are located within the f subunit, and none of them were conserved in the mospv f protein. like other members of the paramyxovirinae, the narpv h protein is predicted to be a type ii integral membrane protein with a hydrophobic domain located at the n-terminal region (aa - ) functioning both as the signal sequence and the transmembrane anchor. for certain paramyxoviruses, the attachment protein can be made in a soluble form by using an alternative in-frame atg codon within the signal/anchor sequence [ ] . for narpv h protein, an in-frame atg codon was present at aa , which has the potential to produce a soluble h protein if translation initiates from this atg codon. the sequence surrounding this atg codon (tgcatgt) does not have a strong conservation to the kozak consensus sequence (accatgg). this is also true for the first atg codon of the predicted h orf, which has the sequence aaaatgg. it is therefore possible that both atg codons are used in vivo, albeit probably with different efficiencies. the -aa motif (nrkscs) present in the predicted neuraminidase active site found in respirovirus and rubulavirus hn proteins [ ] is absent in the narpv h protein. it had the sequence aydgca at the corresponding site, with only the c residue conserved. the mospv g protein has the sequence vfdgcs in this region. there are three potential n-linked glycosylation sites predicted for the narpv h protein at n , n and n . although mospv g protein also has three predicted n-linked glycosylation sites at n , n and n , only one site at n for narpv and n for mospv is located at the same location in the protein. for narpv h protein, the n site is located very close to the transmembrane domain and is therefore unlikely to be glycosylated in vivo. the l proteins of narpv and mospv share more than % sequence identity. the six strongly conserved linear domains identified within the l proteins of nonsegmented negative-strand (nns) rna viruses by poch et al. [ ] can also be identified within these two l proteins. it is interesting to note that in the most conserved domain iii, both l proteins contained the gdne sequence motif, which has only been found in hev, niv and tuppv, and is different from the highly conserved gdnq motif found in all other known viruses in the order mononegavirales [ ] . phylogenetic trees were generated by neighbour-joining methods from sequence alignments of deduced aa sequences of the six major proteins of all known members of the subfamily paramyxovirinae. a representative tree based on the n protein aa sequences is shown in fig. , which clearly shows the close relationship between narpv and mospv and indicates that these two viruses, together with jpv, beipv, tuppv and salpv, cannot be placed in any of the five existing genera in the subfamily. phylogenetic trees based on other proteins give similar results (data not shown). the discovery of hev about years ago opened a new chapter in the genetic diversity of paramyxoviruses. it not only challenged the notion that paramyxoviruses have relatively uniform genome sizes, it also revealed several genetic features not observed in previously known paramyxoviruses, such as the lack of both haemagglutination and neuraminidase activities in the attachment protein, the lack of a multi-basic cleavage site in the f protein and the replacement of the highly conserved gdnq sequence by gdne in the l protein. interestingly, many of the newly discovered paramyxoviruses seemed to contain genome features that keep pushing the ''boundaries'' of known paramyxoviruses in terms of genome size, genome organization and gene sequences. this is best demonstrated by genomes of the jpv and beipv. they are much larger than most other paramyxovirus genomes, contain several novel genes and have an exceptionally large gene for the attachment protein [ , ] . another interesting example is fdlpv [ ] . at , nt, the fdlpv genome is relatively small among the known members of the subfamily paramyxovirinae but contains a seventh gene, u (for unknown), positioned between the n and p/v genes. although rodents represent the most diverse mammals on earth, sev remained the only known paramyxovirus of rodent origin until the recent characterization of mospv, jpv and beipv by our group [ , , ] . in this context, we decided to determine the genome structure and sequence of narpv, another unknown rodent paramyxovirus. it is obvious that sev and the rest of rodent viruses characterized to date do not share a common ancestor virus. in contrast, jpv and beipv are closely related both in genome organization and size and in gene sequences. in this paper, we have shown that narpv and mospv also share many genetic features, not only confirming their rodent origin, but also suggesting that these two viruses evolved from a common progenitor virus. it is worth noting that although jpv/beipv and narpv/ mospv represent two quite different groups of rodent paramyxoviruses in terms of genome size and organization, their common proteins do share more sequence identity than any other known paramyxoviruses, as shown by the phylogenetic tree in fig. . the only common feature we could identify among all known rodent paramyxoviruses was the lack of the multibasic cleavage site present in most other paramyxoviruses. the only other paramyxoviruses with a monobasic cleavage site are henipaviruses of bat origin and the only known paramyxovirus of fish origin, asapv (fig. ) . the biological significance of this observation is not known at the present time. in conclusion, the characterization of the narpv complete genome sequence in this study further highlights the great genetic diversity present among the paramyxoviruses of wildlife origin, ranging through bats, rodents, reptiles and fish. further studies are required to determine whether these rodent viruses have the potential to infect and cause diseases in humans and livestock animals. the taxonomic status of these viruses is yet to be determined. due to their major differences in genome size, genome organization and deduced amino acid sequences, it is most likely that these viruses will be classified into new genera within the subfamily paramyxovirinae. molecular phylogeny and divergence time estimates for major rodent groups: evidence from multiple genes bats: important reservoir hosts of emerging viruses tioman virus, a novel paramyxovirus isolated from fruit bats in malaysia hendra and nipah viruses: different and dangerous sendai virus, the mouse parainfluenza type : a longstanding pathogen that remains up-to-date arg-arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway the complete genome sequence of j-virus reveals a unique genome structure in the family paramyxoviridae nariva virus: further studies, with particular reference to its hemadsorption and hemagglutinating properties complete genome sequence of fer-de-lance virus reveals a novel gene in reptilian paramyxoviruses family paramyxoviridae paramyxoviridae: the viruses and their replication beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded rna viruses full-length genome sequence of mossman virus, a novel paramyxovirus isolated from rodents in australia site-directed mutagenesis of a conserved hexapeptide in the paramyxovirus hemagglutininneuraminidase glycoprotein: effects on antigenic structure and function isolation of a new parainfluenza virus from a frugivorous bat, rousettus leschenaulti, collected at poona, india sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains identification and phylogenetic comparison of salem virus, a novel paramyxovirus of horses the membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein g are synthesized from alternative initiation codons prevalence of rabies and lpm paramyxovirus antibody in nonhematophagous bats captured in the central pacific coast of mexico mega : molecular evolutionary genetics analysis (mega) software version . nariva virus, a hitherto undescribed agent isolated from the trinidadian rat, zygodontomys b. brevicauda (j. a. allen & chapman) optimized rapid amplification of cdna ends (race) for mapping bacterial mrna transcripts electron microscopic evidence of nariva virus structure emerging paramyxoviruses genome diversity of emerging paramyxoviruses full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the americans nariva virus genome acknowledgments we thank dr. n. karabatsos for supplying the nariva virus stock, kaylene selleck and eric hansson for technical assistance, tony pye for providing the automated sequencing service, and drs. glenn marsh and jackie pallister for critical review of the manuscript. key: cord- -zdhg axx authors: shirato, kazuya; maejima, madoka; hirai, asuka; ami, yasushi; takeyama, natsumi; tsuchiya, kotaro; kusanagi, kouich; nunoya, tetsuo; taguchi, fumihiro title: enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: zdhg axx porcine epidemic diarrhea virus (pedv) is the major causative agent of fatal diarrhea in piglets. to study the pathogenic features of pedv using a mouse model, pedv with virulence in mice is required. in pursuit of this, we adapted a tissue-culture-passed pedv mk strain to suckling mouse brains. pedv obtained after ten passages through the brains (mk-p ) had increased virulence for mice, and its fusion activity in cultured cells exceeded that of the original strain. however, the replication kinetics of mk and mk-p did not differ from each other in the brain and in cultured cells. the spike (s) protein of mk-p had four amino acid substitutions relative to the original strain. one of these (an h-to-r substitution at residue , ) was first detected in pedv isolated after eight passages, and both this virus (mk-p ) and mk-p showed enhanced syncytium formation relative to the original mk strain and viruses isolated after two, four, and six passages, suggesting the possibility that the h-to-r mutation was responsible for this activity. this mutation could be also involved in the increased virulence of pedv observed for mk-p . the group i coronavirus porcine epidemic diarrhea virus (pedv) is an enveloped rna virus with a single, positive-stranded genome about kb in length [ ] . pedv is the causative agent of pig diarrhea and induces a loss of appetite and weight in adult pigs, whereas it is lethal in piglets [ ] . however, our understanding of the pathogenesis of diseases caused by pedv is limited due to a lack of appropriate small-animal models. coronaviruses cause various diseases in a species-specific manner, and they grow in cultured cells established from susceptible host species; human, feline, and porcine coronaviruses grow only in cells isolated from the respective species [ ] . one of the major factors determining species specificity is the cellular receptor, which interacts with a given virus, but not with other viruses, when the virus initially encounters cells [ ] . some of the group i coronaviruses, such as porcine transmissible gastroenteritis virus (tgev) and human coronavirus e (hcov- e), utilize aminopeptidase n (apn) [ , , , ] . however, the receptor of pedv has not yet been identified, although there was a controversial report on a pedv receptor protein [ ] . expression of the receptor protein for mouse hepatitis virus (mhv) and severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) in cultured cells confers susceptibility to each of these viruses [ , , ] . expression of the tgev receptor in mice from a transgene rendered otherwise resistant mice susceptible to tgev [ ] . however, this approach is not available for pedv. an alternative approach is to isolate viruses that have adapted for growth within the animals and which exhibit virulence. such adaptations have been reported for two other coronaviruses, ibv and oc [ ] . in the present study, we isolated a strain of pedv that was more virulent than the original tissue-culture-adapted virus after passage through mouse brain cells. the adapted virus showed enhanced cytopathology compared with the original virus. vero cells were obtained from the american type culture collection (atcc; manassass, va, usa). the cells were maintained in dulbecco's modified eagle's medium (dmem; sigma, st. louis, mo, usa) containing % fetal calf serum (fcs). the pedv strain used in this study was an mk strain isolated from the jejunum of a pig that had exhibited diarrhea in in japan, and which had been passed nine times in vero cell cultures. pedv was maintained in vero cells using a slightly modified version of a reported method [ ] . briefly, vero cells were infected with the virus. after h, the cells were washed with phosphate-buffered saline (pbs) and cultured in dmem containing % tryptose phosphate broth (tpb) and . lg/ml trypsin (sigma) for h. next, the cells and supernatant were pooled, ultrasonicated, and stored at - °c until use. the pedv infectious titer was determined by a plaque assay using vero cells. briefly, vero cell monolayers grown in -well plates coated with type i collagen (agc techno glass, chiba, japan) were inoculated with serially diluted virus samples. after h to allow for adsorption of the virus, the inoculum was removed, the cells were washed with pbs, and dmem containing % tpb and . lg/ml trypsin was added. for titration of the virus, half of the concentration of trypsin was used to drive cell fusion in a procedure that is gentle compared with viral propagation. after a -h incubation, the cells were fixed and stained with pbs containing . % crystal violet and % formalin under ultraviolet light. after staining for h, the cells were washed with water and dried, and the plaques were counted under a light microscope as described for the infectious titration of human coronavirus e [ ] . the viral titer in the brains of the mice is given as plaqueforming units (pfu)/ ll of % homogenate, whereas the titer in the supernatant is given as pfu/ml of supernatant; the cell titer is given as pfu/well of a -well plate. isolation of a murine-adapted variant of pedv specific-pathogen-free (spf) pregnant icr mice were obtained from japan slc (shizuoka, japan). all animal experiments were approved by the committee on experimental animals, national institute of infectious diseases, japan, and all experimental animals were handled according to the guidelines of the same committee. all spf mice were confirmed by seromonitoring to be free from infection with mhv, lactate dehydrogenase virus, and other pathogenic microorganisms. newborn (within h of birth; -day-old) mice were used in this study. pedv strain mk ( , pfu) was inoculated intracerebrally (i.c.) into the brains of -day-old mice (n = - ). five days post-infection (p.i.), the mice were sacrificed and their brains collected, pooled, and homogenized in pbs to yield a % suspension, which was centrifuged at , rpm for min at °c. next, ll of the supernatant from the % brain homogenate was inoculated i.c. into suckling mouse brains, and the brains were collected as described above. this cycle was repeated ten times, and the virus obtained was designated mk-p . mk-p was amplified once by vero cell culture to match its condition to that of the original virus, then stored at - °c until use. to investigate viral virulence and replication in mouse brains, -day-old mice were infected i.c. with , pfu of pedv and monitored daily for clinical features; they were also weighed daily. pbs was used in a mock infection. to examine viral replication in the brains, the mice were euthanized on the indicated day p.i., their brains were collected, and % homogenates were prepared as described above. to determine the replication kinetics in cultured cells, vero cells were infected with pedv at a multiplicity of infection (m.o.i.) of . . after h, the cells were washed with pbs and cultured in dmem containing % tpb and . lg/ml trypsin. at the indicated hour p.i., the supernatant and cells were collected separately, the cells were ultrasonicated, and the viral titers were determined using a plaque assay as described above. sequence analysis viral rna was extracted with trizol ls reagent (invitrogen, carlsbad, ca, usa) following the manufacturer's protocol. first-strand cdna was synthesized using m-mlv reverse transcriptase (takara-bio, shiga, japan) and oligod(t) to determine the statistical significance of the survival curves, log-rank and generalized wilcoxon tests were performed. significance was determined in all other cases using unpaired t tests. p \ . was considered to be statistically significant. to obtain a murine-adapted variant of pedv, passage through suckling mice was performed. zero-day-old mice were inoculated i.c. with the mk strain of pedv, and the brains of the infected mice were collected four or days later. the brains were pooled and used for the next round of mouse inoculation. surprisingly, mice infected with the non-adapted, original strain (mk) showed clinical signs and mortality; these observations differ from those reported for hcov oc and ibv [ ] . however, in subsequent passages, clinical signs in the infected mice, such as trembling, tremors, and growth retardation, became more obvious. the process was stopped after ten passages, at which point the variant was designated mk-p . the original mk strain and the mouse-adapted variant mk-p were inoculated onto vero cells in the presence of trypsin and cultured for h. as shown in fig. , more syncytium formation was seen in the cells infected with mk-p than in those infected with the original strain. the mean diameter of the mk-p syncytia was significantly greater than that of the mk syncytia ( ± vs. ± lm; n = , p \ . ). the neurovirulence of mk-p in suckling mice was examined. zero-day-old mice were infected i.c. with , pfu of virus and monitored daily for clinical symptoms and mortality. all mice infected with mk and mk-p succumbed to infection; however, the mice infected with mk-p died more quickly than the mk-infected mice ( fig. a ; n = , p \ . ). the mice were also weighed daily ( fig. b; n = ). the weights of the mice infected with mk or mk-p increased for - days after infection. subsequently, the weight of the mice decreased beginning days p.i., whereas the mice infected with mk began to lose weight days p.i.- days later than the mk-p infected mice. the body weight of the mice infected with mk-p was determined days p.i. because most of the mice had succumbed by - days p.i. the mock-infected mice did not exhibit weight loss or clinical symptoms. as described above, a mouse-adapted variant of pedv was successfully isolated. the growth kinetics of the original strain and mk-p were examined in suckling mouse brains. mk and mk-p exhibited similar growth kinetics in suckling mouse brain, peaking on day p.i. (fig. a) . their growth kinetics in cultured vero cells was also determined. some coronaviruses tend to remain intracellular, and viral stocks are prepared by ultrasonication or repeated freeze-thawing cycles. to compare the amounts of budded and intracellular virus, the titers in the supernatant (fig. b) and cells (fig. c) were determined separately. similar to the growth kinetics in the brains of the mice, the growth pattern in vero cells did not differ significantly between mk and mk-p (fig. b, c) . these results suggest that the increased virulence in suckling mice and syncytium formation in cultured cells is not due to increased growth in the brain or cultured cells. because the s proteins of coronaviruses are reported to be associated with viral pathogenicity [ ] , table ) . among these, the amino acid substitutions at positions and , resulted in a polarity change (hydrophobic to hydrophilic). to determine at which brain passage the amino acid changes occurred, we determined the primary sequences of s protein from the viruses isolated after two, four, six, and eight passages in mouse brain. as shown in table , the amino acid at position differed at the second passage, while those at positions and changed simultaneously at the fourth passage. the amino acid change at position , occurred at the eighth passage. as described above, the plaque size of mk-p was larger than that of the parental strain (fig. ) . this larger plaque morphology was also detected in pedv after eight passages, whereas the viruses isolated after four and six passages showed syncytium formation similar to that in mk (fig. ) . these results suggest that the amino acid change at position , (h to r) first detected at the eighth passage, which is located in the cytoplasmic tail (ct) region, contributed to the larger plaques of mk-p , though other genes must be considered. two different coronaviruses, oc and ibv, have successfully adapted to grow in mice and show virulence after brain-to-brain passage in suckling mice [ , ] . these viruses initially showed low virulence (i.e., clinical symptoms such as tremor and rigidity and lethargy in suckling mice), but the virulence increased with repeated passage in mouse brains. in this study, the passage of pedv in suckling mouse brains clearly strengthened its virulence, similar to the adaptation seen in oc and ibv. unexpectedly, the original mouse non-adapted pedv showed significant virulence for suckling mice. the primary sequences of s protein from the viruses isolated after repeated passages were determined. pedv passed in mouse brains showed mutations in the s protein, which had accumulated after ten passages. the h-to-r mutation at residue , , which was first detected after eight passages, is likely responsible for the enhanced cell-cell fusion activity, as only those viruses with this mutation showed significantly larger syncytia compared with the original pedv; the s protein is a major determinant of the fusion activity of coronaviruses [ , ] . it would be interesting to find out whether this substitution in the s protein is responsible for the enhanced fusion activity by expressing s protein alone. those mutations found in the mouseadapted viral s proteins could be responsible for the increased virulence; however, other genes have been reported to be involved in the virulence of such coronaviruses as mhv and sars-cov [ , , , ] . to identify the genes responsible for the increase in virulence, we would need the entire genomic sequences of the original and mouse-adapted pdevs; additionally, reverse-genetic analyses would be essential. pedv differs from most coronaviruses in that it grows in cultures of cells derived from animal species not susceptible to pedv; the virus grows efficiently in vero cells derived from the kidneys of green monkey, which is not a natural host of the virus [ ] . few cases are known in which coronaviruses multiply in cells derived from non-susceptible host animals. sars-cov can grow in a variety of cells established from several animal species [ , , , ] , and it is somewhat capable of growing in the same animals; however, efficient infection is restricted to cells from animals that support sars-cov infection [ ] . the growth potential of sars-cov in a variety of animal cells correlated with the utilization of its receptor molecule, ace [ ] . most known coronaviruses become infective in cells when their functional receptor molecule is expressed by transfection with a plasmid encoding the receptor molecule [ ] . this suggests that vero cells susceptible to pedv infection express a functional receptor. in our study, both mouse-adapted and non-adapted mk strains of pedv could infect and grow in suckling mouse brains, suggesting that a receptor molecule utilized by both viruses exists in suckling mouse brain. to determine which cell types in the brain express the receptor, we are attempting to identify the cell types that are infected by pedv in suckling mouse brain. this will provide valuable information related to the receptor molecule utilized by pedv. in summary, a highly neurovirulent variant of pedv was successfully isolated by passage following the i.c. infection of -day-old suckling mice. the s protein of the variant had four amino acid substitutions, which might be responsible for its enhanced cell-cell fusion activity. of these substitutions, the h-to-r substitution at position , in the ct region of the s protein is likely to be the determinant of the high fusion activity. to address this possibility, the expression of wild-type and mutant s proteins in cultured cells and an analysis of their fusion activity are necessary. reverse-genetic analyses of the entire pedv genome are also required to assess the effect of s protein mutations on the virulence and pathogenesis of pedv. p p p p p fig. plaque morphology of pedv isolates after mouse brain passage pedv passed in mouse brains two (p ), four (p ), six (p ), eight (p ), or ten (p ) times as well as the original mk (p ) was inoculated onto vero cells. cell fusion assays were performed as described in the legend to fig. aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv growth of infectious bronchitis virus in suckling mice brain susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme and infection can be blocked by soluble receptor coronavirus receptors. in: siddell sg (ed) the coronaviridae proteasemediated entry via the endosome of human coronavirus e severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate the molecular biology of coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus angiotensin-converting enzyme is a functional receptor for the sars coronavirus growth in suckling-mouse brain of ''ibv-like'' viruses from patients with upper respiratory tract disease pathology and virus dispersion in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus via different inoculation routes mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice inactivation of expression of gene of mouse hepatitis virus strain jhm does not affect virulence in the murine cns a new coronavirus-like particle associated with diarrhea in swine reverse genetic characterization of the natural genomic deletion in sars-coronavirus strain frankfurt- open reading frame b reveals an attenuating function of the b protein in vitro and in vivo severe acute respiratory syndrome coronavirus infection of golden syrian hamsters coronavirus e susceptibility in man-mouse hybrids is located on human chromosome coronaviruses: structure and genome expression single-amino-acid substitutions in open reading frame (orf) b-nsp and orf a proteins of the coronavirus mouse hepatitis virus are attenuating in mice feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i feline aminopeptidase n is a receptor for all group i coronaviruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins acknowledgments we thank shutoku matsuyama, miyuki kawase, and makoto ujike for their valuable comments and encouragement. this work was supported by a grant-in-aid for scientific research (b; no. ) from the ministry of education, culture, sports, science, and technology of japan. passage number i i t t t t t t m m m m a d d d d d , h h h h r r fig. the replication kinetics of mk (squares) and mk-p (circles). a viral replication in suckling mouse brain. zero-day-old mice were infected i.c. with , pfu of pedv. at the indicated days p.i., the brains were collected and % homogenates were prepared. the viral titer in the brains was determined using a plaque assay with vero cells and is expressed as pfu/ ll of % homogenates (n = ). b, c viral replication in cultured cells. vero cells were infected with pedv at an m.o.i. of . . viral titers in the supernatants (b) and cells (c) were determined separately using plaque assays (n = ). the viral titer in the supernatant is given as pfu/ml of supernatant, while that in the cells is given as pfu per well of a -well plate key: cord- - ie v wy authors: ramírez-salinas, g. lizbeth; garcía-machorro, jazmín; rojas-hernández, saúl; campos-rodríguez, rafael; de oca, arturo contis-montes; gomez, miguel medina; luciano, rocío; zimic, mirko; correa-basurto, josé title: bioinformatics design and experimental validation of influenza a virus multi-epitopes that induce neutralizing antibodies date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ie v wy pandemics caused by influenza a virus (iav) are responsible for the deaths of millions of humans around the world. one of these pandemics occurred in mexico in . despite the impact of iav on human health, there is no effective vaccine. gene mutations and translocation of genome segments of different iav subtypes infecting a single host cell make the development of a universal vaccine difficult. the design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. in this work, we used the predicted amino acid sequences of the neuraminidase (na) and hemagglutinin (ha) proteins of different iav subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant iav strains. these peptides were tested using immunological assays to test their ability to induce production of antibodies against iav. we immunized rabbits and mice and measured the levels of igg and iga antibodies in serum samples and nasal washes. rabbit antibodies against the peptides p and p (both of which are hybrids of na and ha) recognized ha from both group (h , h , and h ) and group (h and h ) iav and also recognized the purified na protein from the viral stock (influenza a puerto rico/ / ). igg antibodies from rabbits immunized with p and p were capable of recognizing viral particles and inhibited virus hemagglutination. additionally, intranasal immunization of mice with p and p induced specific igg and iga antibodies in serum and nasal mucosa, respectively. interestingly, the igg antibodies were found to have neutralizing capability. in conclusion, the peptides designed through in silico studies were validated in experimental assays. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. influenza a virus (iav) is a lipid-enveloped, singlestranded, negative-sense rna virus belonging to the family orthomyxoviridae. the viral envelope contains three transmembrane proteins (na [neuraminidase], ha [hemagglutinin] and m [proton channel]) on the viral surface and one protein (m [matrix protein]) below the membrane. the viral core contains the nucleoprotein (np), viral rna, and three polymerase proteins (pb , pb , and pa) [ ] . iav is classified into subtypes based on two major antigens: the surface spike glycoproteins na and ha [ ] . all iav subtypes are known to cause infections in birds, which are their natural reservoir [ ] . humans are infected principally by the iav subtypes h n , h n , h n , h n , and h n [ ] . influenza pandemics have become serious socioeconomic and public-health problems worldwide. moreover, seasonal flu causes approximately , to , deaths per year [ , ] . iav epidemics and pandemics are attributed to mutations in the viral rna genome. mutations involving surface proteins (na and ha) result in structural protein changes that cause a loss of antibody recognition against the virus. this is one reason why new flu vaccines need to be designed for each seasonal influenza or pandemic influenza strain. the development of vaccines is the major method used to prevent iav infection and represents one of the most important contributions by the immunology field to public health [ ] . an important strategy is to identify conserved epitopes that may be used to design new vaccines that are capable of conferring broad protection. currently, the primary goal is to develop vaccines that protect by eliciting antibody responses against multiple subtypes and strains of influenza viruses [ ] [ ] [ ] . these broadly neutralizing antibodies (bnabs) generally target conserved and functional regions or epitopes on the major surface glycoproteins: hemagglutinin (head and stem), neuraminidase, and m e [ ] [ ] [ ] . the hemagglutinin (ha) is the main surface glycoprotein of influenza virus, which mediates the adsorption and penetration of the virus into host cells [ ] . each molecule of ha comprises a membrane distal globular head composed of ha , which contains the receptorbinding site (rbs), and a stem region, which encompasses the fusion machinery [ ] . most bnabs are directed against the ha protein. the receptor-binding site is a functionally conserved region on the ha globular head domain that is a target for bnabs that inhibit viral entry by preventing ha binding to its host receptor [ , ] . since the stem region contains the most conserved epitopes for antibody recognition, antibodies produce against this region have a higher neutralization breadth than rbs-targeted bnabs. these stem-binding bnabs inhibit virus replication by blocking attachment and preventing conformational changes that are essential for membrane fusion [ ] [ ] [ ] . na is the second most abundant glycoprotein on the surface of influenza a and b viruses, and conserved domains or epitopes in na induce bnabs that protect against viruses of a single subtype [ ] . thus, na epitopes may use in universal influenza vaccines [ , [ ] [ ] [ ] [ ] . although na-specific antibodies can control infection by several mechanisms, the main mechanism is the inhibition of enzyme activity [ , , ] . thus, "universal vaccines consisting of conserved domains or epitopes from ha and na could be more broadly protective than single proteins. the rational design of peptide-based vaccines is based on computational procedures that employ knowledge of the antigen recognition of different protein targets, such as the major histocompatibility complex (mhc), t-cell receptor, and/or b-cell receptor. these procedures are performed at the molecular level [ ] using information from the protein data bank. this strategy offers many advantages, such as the production of short immunogenic peptides without the risk of infection and safe handling of biological samples under room-temperature conditions. additionally, peptides can be synthesized in vitro to decrease production costs and can be handled without strict conditions due to their stability [ ] . peptide vaccines are classified according to the immunological cell type involved in the immune response (i.e., b-cell or t-cell epitopes) and are used to induce specific immune responses. t cells recognize peptides coupled to mhc from antigen-presenting cells, and the mhc is then coupled to the t-cell receptor (tcr). following the formation of the peptide-mhc-tcr complex, t cells receive several biological signals that are capable of activating the immune system to act against microorganisms or cancer cells [ ] . in addition, activation of b cells by epitopes can increase the protection spectrum of vaccines, making them more protective than those with limited immune system activation capacity [ ] . in this study, we selected a set of peptides (p) from ha and na (including hybrids) that were capable of generating antibodies against iav. first, a protein sequence bioinformatics study was performed using the ha and na proteins to obtain multi-epitope peptides characterized by conserved residues and promiscuous properties. the epitope selection process included criteria related to their protein surface localization. then, three-dimensional ( d) models of the target epitopes were constructed and subjected to molecular dynamics (md) simulations to explore their thermodynamic properties. finally, the epitopes were used in experimental assays to explore their immunogenic properties in animal models. we retrieved the full primary sequences of the ha and na proteins reported between and from the ncbi influenza virus resource [ ] . we aligned the ha and na sequences for each subtype using the muscle/ebi server [ ] and the clustal x . . program [ ] . we constructed a three-dimensional ( d) structure model based on the na consensus sequence (yp_ . ) of the h n virus by employing the swiss model server [ ] [ ] [ ] [ ] . the na monomer a (pdb id: ti ) was used as a template. for the h n ha consensus sequence (table ) , we used the modeller . program to build d models of multi-chain ha by employing a multi-trimer template ha (pdb id: ruy). the stereochemical quality of the models was evaluated using ramachandran plots and pdbsum [ ] (data not show) and errat servers [ ] (data not shown). we submitted the na (yp_ . ) and ha consensus sequences (table ) we selected epitopes based on promiscuity criteria among the iav subtypes, the degree of conservation, and their surface localization on the quaternary structure models of the proteins that were built (fig. s ), selecting six peptides (p , p , p , p , p and p ) without conjugation to keyhole limpet hemocyanin (klh). p is located in the middle of ha, whereas p -p and p are located in globular heads of ha. p is located in in globular head of na (fig. s) . we determined the degree of exposure of the regions by visual inspection, using the vmd program [ ] . once the most promising epitopes were identified, we added linkers of different length and structure ( - glycine residues) to control the distance between two selected epitopes and to maintain multi-epitope properties, discarding hybrids in which the added glycines or the neighboring peptides were predicted to be immunogenic. additionally, epitopes previously identified by our research group were selected as candidates for the construction of hybrid peptides to evaluate their capacity to increase immunological responses [ , ] . we predicted proteasome and immunoproteasome cleavage using the proteasome cleavage prediction server, pcps (http://imed.med.ucm.es/tools /pcps/index .html), to verify epitope structural stability. the pcps analysis identified fragments of peptides ranging from to residues in length. these peptide sequences were submitted to the netmhc . [ , ] , netmhcii . [ , ] and pred balb/c (http://antig en.i r.a-star.edu.sg/predb albc/.) servers to determine whether they maintained their immunogenic properties. we built d models of the selected multi-epitopes using the pepstrmod server (http://osddl inux.osdd.net/ragha va/ pepst rmod/, accessed on march ). we subjected these multi-epitopes to md simulations using the namd . program [ ] with charmm as the force field [ ] . the constant number-of-particles, pressure, and temperature (npt) ensemble and the periodic boundary conditions were used. a constant temperature ( k) was maintained using a langevin thermostat set at a constant pressure ( atm) maintained by an isotropic langevin barostat [ ] . these pressure and temperature values represented standard physiological conditions. energy minimization was performed with steps in the conjugate-gradient algorithm with restraints on the peptide backbone, followed by steps without restraints. the system was heated for ps and equilibrated for ps with restraints to the α-carbon and then finished with no restraints and k. these md simulations were run for ns. we calculated the root mean square deviation (rmsd), which measures whole-protein motions while generally considering the α-carbon coordinates in relation to md simulation times, root mean square fluctuations (rmsf), which measure the α-carbon coordinates in relation to md simulation time per residue, and radius of gyration (rg), which measures the protein compactness from the center of the protein to the periphery according to the d coordinates in the structural analyses of multi-epitope md simulations from the carma program [ ] . the secondary structure was determined using the visual molecular dynamics (vmd) program [ ] . nine peptides were designed, but eight peptides (including p in p ) were selected (table ) to be synthesized (peptide . inc.) according to theoretical studies for experimental testing as described in the following experimental procedures ( table ). the conjugation of peptides with the klh molecule was performed using succinimidyl -(n-maleimidomethyl)cyclohexane- -carboxylate (smcc), which is an heterobifunctional crosslinker that contains n-hydroxysuccinimide (nhs) ester and maleimide groups that allow covalent conjugation of amine molecules in klh with the sulfhydryl groups of cysteine residues located in the middle of each hybrid peptide. we immunized eight rabbits (from harlan mexico) with peptides (p , p , p , p and p ) without conjugation to klh and hybrid peptides (p , p and p ) conjugated to klh at three time points with during seven-day intervals. for the primary immunization, µg of peptide plus complete freund's adjuvant (sigma chemical co.) was administered by the subcutaneous route. the second immunization ( days later) included µg of peptide plus incomplete freund's adjuvant administered by the subcutaneous route. the third immunization was administered by the intramuscular route days after the primary immunization and contained µg of peptide in ml of saline solution. seven days after the last immunization, we anesthetized the rabbits with pentobarbital via the intraperitoneal route. serum samples were obtained from blood extracted by cardiac puncture and stored at - °c. screening was carried out to select the peptides that induced the highest antibody titer in rabbit serum. for this purpose, five groups of peptides were used. six balb/c mice for each peptide were injected three times at seven-day intervals via the intranasal route with µg of the peptide (p , p , p , p and p ) plus µg of cholera toxin (ct) mucosal adjuvant. seven days after the last immunization, we anesthetized the mice with pentobarbital. samples were obtained from blood extracted by cardiac puncture and washes of the nasal cavity with pbs, respectively. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals, in accordance with mexican federal regulations for animal use and care (nom- -zoo- ) and was authorized by the comité interno del cuidado y uso de los animales de laboratorio (cicual) at esm-ipn. the protocol was approved by the committee on ethics of the escuela nacional de medicina y homeopatia of instituto politecnico naciocal, ipn (protocol number enmh-cb- - ). mice were anesthetized with ether for intranasal immunization, and for biological assays, mice and rabbits were killed using pentobarbital. all efforts were made to minimize suffering and to use biological samples immediately. we determined anti-peptide antibody levels in serum samples from immunized rabbits and serum samples or nasal washes of mice immunized with peptides, using an indirect enzymelinked immunosorbent assay (elisa). for biological assays, the mice or rabbits were killed using pentobarbital, and their biological samples were used immediately. each sample was tested in duplicate. briefly, -well plates were coated with µl of different peptides ( µg of peptide per ml in carbonate-bicarbonate buffer [ mm na co and mm nahco , ph . ]). the plates were incubated overnight at °c, followed by three washes with phosphate-buffered saline containing . % tween- (pbst). blocking was performed using pbst plus % fat-free milk for h, and the samples were then washed with pbst. serial dilutions of serum samples from nine immunized rabbits ( : to : ), serum from immunized mice (from : to : ) and nasal washes (from : to : ) were prepared and added to the plates, which were incubated overnight at °c and washed with pbst, after which μl of a : dilution of goat anti-rabbit igg peroxidase (thermo scientific) for rabbit serum and rabbit anti-mouse igg peroxidase ( : ) or rabbit anti-mouse iga ( : ) peroxidase (thermo scientific) for mouse samples was added to each well. the plates were incubated for h at room temperature and washed with pbst. the enzymatic reactions the endpoint titer was defined as the reciprocal of the highest analyzed dilution that gave an absorbance value above . . influenza a virus strain puerto rico/ / was grown in madin-darby canine kidney (mdck) cells. the cells were cultured in dulbecco's modified eagle medium nutrient mixture f- (dmem/f- ) (caisson) supplemented with % fetal bovine serum (sfb) (mediatech) at °c and % co . the viral stock was prepared in serum-free medium and frozen at - °c for later use as described previously [ ] . viral particles that had been isolated from infected mdck cells and purified using serum-free medium were trapped in wells coated with concanavalin a (con a), which immobilizes the detergent-solubilized viral glycoproteins of the viral envelope. briefly, -well plates (costar) were coated with μl of con a (sigma-aldrich) per well at a concentration of μg/ml in pbs, ph . , for h. the wells were washed three times with pbs containing . % triton x- (pbs-tx) and incubated with solubilized and inactivated influenza a virus strain puerto rico/ / (serum-free virus stock) in pbs-tx for h. after the wells were washed with pbs-tx, the unreacted cona binding sites were blocked with rpmi medium containing % fbs (rpmi- % sfb) for h. dilutions of the heat-inactivated rabbit serum samples were made in rpmi- % sfb and incubated for h at room temperature. the positive control was a : dilution of serum from a patient who was positive for influenza virus [ ] . rabbit pre-immune serum samples were used as negative controls. the wells were washed again and incubated with μl of the appropriate peroxidase-conjugated anti-igg (h+l) (santa cruz) as the secondary antibody. the wells were washed again and then incubated for h with μl of , '-azino-bis ( -ethylbenzothiazoline- -sulfonic acid) (abts) (sigma-aldrich), and h o was added as a substrate. absorbance values were determined at nm [ ] . levels of antibody against ha and na were determined using recombinant ha (rha) proteins purchased from serum samples from immunized rabbits were inactivated by heating at ± °c in a water bath for min, and the total igg were purified by affinity chromatography using immobilized protein a sepharose cl- b (sigma-aldrich) as described by contis-montes de oca et al. [ ] . dilutions were made in pbs and added to a final volume of μl in -well round-bottom plates using pre-immune serum as a control. the virus concentration was then adjusted to hemagglutination units (hau) per μl in pbs and added to samples, except for the negative controls, which consisted of pbs without virus. the plates were incubated for h at °c while human red blood cells (hrbcs) of type "o" were washed twice with x pbs, ph . , and centrifuged at × g for min at - °c. the cells were then resuspended in a final concentration of . % in x pbs, and μl of this suspension was added to each well. the plates were incubated again for h at °c, and the effects were observed [ ] . the hi titer was the highest dilution that caused total inhibition of agglutination. hrbcs with pbs were used as a negative control, and hrbcs plus virus at hau were used as a positive control. sera were tested at dilutions from : to : . neutralizing antibodies were titrated as described previously [ , ] briefly, serial dilutions of the heat-inactivated test sera in duplicate starting from : were mixed with pfu of influenza a virus strain puerto rico/ / for h at °c and added to mdck cells at a density of , cells per well in -well plates. positive and negative control sera and virus back titration to confirm the viral inoculum were included. at h after infection, serum-free dmem/f- medium with µg of tpck (l- -tosylamide- -phenylethyl chloromethyl ketone)-treated trypsin was added to each well, and the plates were incubated for h at °c. then, µl of overlay medium was added to each well, and the plates were incubated for days under the same conditions. the supernatant was discarded, and plaques were visualized by staining with naphthol blue-black dye. the result is reported as the reciprocal of the dilution that protects % of the cells from a cytopathic effect due to infection. three independent assays were conducted in duplicate, and the results are presented as the mean and standard deviation of one representative assay. the statistical significance of differences among groups was determined by anova, followed by tukey's test. differences with p values less than . were considered significant. the analysis was performed using the prism program (graphpad, san diego, ca.). the half-maximal effective concentration (ec ) was determined using the statistical program sigmaplot for windows version (systat software inc., san jose, ca, usa). (fig. a-d) of the influenza a h n glycoproteins na and ha (fig. e) were obtained from multiple alignments. these consensus protein sequences were used to identify the immunogenic regions in ha and na (fig. ) . sequences that were conserved during evolution (data not shown), present in different influenza virus subtypes, or located on the surface (exposed to solvent, see fig. s ) were predicted to be easily accessible to antibodies with neutralizing potential [ ] , as demonstrated for hiv [ ] . six peptides (from h n as reference), including four from ha (p , p , p , p and p ), one from na (p ), and one from na reported previously by us [ ] were selected as described above. multiple sequence alignments of ha peptides from different subtypes of influenza a virus (h n , h n , h n , h n and h n ) showed a degree of conservation for each of the selected peptides when h n (a/california/ / ) was used as a reference ( % identity, fig. ). p was conserved to different degrees with respect to subtypes h n (a/puerto rico/ / ), h n and h n (table ) . the most similarity was observed with h n (a/puerto rico/ / ) and the lowest with h n (see fig. a ), suggesting that p could induce immunogenic responses against h n , h n , h n , h n and h n as a multi-vaccine. p and p were most similar to the corresponding sequences of pandemic h n (a/puerto rico/ / ), see table ), suggesting that they were potential candidates for specific vaccines and immunodiagnostics, whereas p was most similar to the corresponding sequence of h n (a/puerto rico/ / ) and had a low degree of similarity to h n sequences. however, multiple alignment of na sequences showed that p was more similar to the corresponding sequences of the h n (a/brisbane/ / [h n ]) and h n subtypes than to those of other influenza virus subtypes (see table ). furthermore, p , p and p could work as a multi-vaccine epitope to provide broad protection against unspecified influenza a virus infections. one additional parameter that the epitopes needed to achieve for good recognition by mhc and tcr was low structural mobility [ ] . this was investigated using md simulations [ ] measuring structural motions dependent on structural flexibility of hybrid peptides with long sequences (fig. ) . it is known that lower flexibility of peptides is associated with a better immune response [ ] . as shown in fig. , p yielded larger rmsd values in md simulations than compared to p and p (approximately - ns and higher fluctuation close to ns) ( fig. a) . from ns to ns, p showed less motion with minimal oscillations compared to p and p , suggesting that it might be more immunogenic. based on rmsf values, p exhibited more mobility than p and p . additionally, p had higher rg values (approximately ns) compared to p and p . these results agree with some of the biological results described in the following paragraphs. the peptides that bind to hla-dr, hla-dq, hla-dp, human mhc class ii alleles and mouse i-e d and i-a d molecules are shown in table . all of the peptides contain identical or very similar sequences (epitopes) that are pre- dicted to bind to both human and mouse mhc-ii alleles. for example, p is an epitope that binds to human hla-drb * , hla-drb * , hla-drb * alleles and mouse i-a d allele. in summary, the selected peptide contains epitopes that are capable of interacting with human and mouse (balb/c) mhc-ii alleles. these results show that the predicted peptides contain helper t-cell epitopes, which could activate t-cell-dependent responses, such as igg and iga antibody responses. the peptides p , p and p contain three epitopes, whereas p only contains one epitope. the hybrid peptides p and p contain four epitopes, and p contains only one that is likely to induce an immune response against ha and na proteins [ , ] . table . these peptides are predicted to contain several t-cell epitopes, some of which are predicted to bind to both human and mouse mhc-i alleles. for example, p contains the sequence fqnihpiti, which binds to human hla-b* and mouse h -d d alleles. the peptides were found to contain different number of epitopes that are recognizable by cytotoxic t cells. p , p and p have four epitopes, and p and p have three. the epitopes that bind to mhc-i molecules could activate cytotoxic t cells that contribute to protection against invasion by influenza virus [ ] [ ] [ ] . using the b-cell epitope prediction program abcpred, we identified several linear b-cell epitopes (table ). p , p and p were predicted to contain two b-cell epitopes, whereas the hybrid peptides were predicted to contain - epitopes. therefore, hybrid peptides could induce stronger mucosal and systemic humoral immune responses [ ] . to confirm the immunogenicity of these peptides in vivo, we analyzed sera obtained from rabbits, and the peptides that induced the highest antibody titers in rabbits were used to immunize mice intranasally. igg in sera and iga in nasal washes, as well as neutralizing antibodies, were then detected. the unconjugated p induced the strongest igg immune response in rabbits (fig. a) . this result means that p could be immunogenic because it induced igg antibodies in the absence of klh [ ] . according to epitope predictions, p contains t-cell (mhc ii) and b-cell epitopes and is located in the middle of protein with º of exposure. p was included in peptides p and p . p did not induce an immunogenic response when it was hybridized to p to yield p ; however, it induced a stronger igg immune response when it was hybridized to p (na) to yield p . as expected, the peptides p and p induced the strongest immune response when conjugated to klh. however, p induced a poor immune response (fig. ). p hybridized with p to yield p resulted in an immune response that could be attributed to p . although p is present in p , it is located far from klh. the position of the peptide in the hybrid could therefore explain the experimental results. p in the p -klh complex could be located at the klh protein surface, and this location makes proteasome processing for presentation by mhc more difficult than the p -klh complex. the better immunogenic response obtained with p alone or p hybridized with p to yield p might have been due to the fact that p is very rich in basic residues, which have been shown to be important for immunogenicity [ ] . p was capable of inducing an immune response despite having a lower molecular weight than p and p . similar results have been reported previously for another peptide [ ] . as reported previously, the immunogenicity of p could be due to its na epitope [ ] . as mentioned above, we tested the immunogenicity of peptides conjugated to klh to investigate the effect of klh on the response to p . surprisingly, p still induced a weaker immune response than p despite the presence of p (fig. , panel b) . these results confirmed that the peptide sequence was of key importance for immunogenicity. the carriers could play an important role due to their ability to affect the recognition pattern on mhc regardless of the presence of basic residues that are capable of recognizing important sites, as reported by carrillo-vazquez et al. [ ] . the igg immune response is a t-cell-dependent response because the class switch of the igg isotype requires the interaction of peptides with mhc ii molecules and t helper (cd t) cells [ , ] . thus, our results indirectly demonstrate that immunogenic peptides induce t helper cells restricted to mhc-ii. using elisa, the immunogenicity of peptides in the mucosal compartment was examined in mice that were immunized intranasally with peptides mixed with ct (fig. ) . the igg and iga antibody responses to the peptides were similar in the serum samples. intranasal immunization induced higher titers of igg and iga antibodies to p , lower titers to p , and very low titers to the unconjugated peptides p , p , and p (fig. , panels a and b) . these results suggest that p requires other structural changes for proteasome protection or immunological transport in the p hybrid. to investigate the antibody responses in the mucosal compartment, the levels of igg and iga antibodies against the peptides were measured in nasal washes (fig. , panels c and d). we observed that p , p and p induced higher titers of igg antibody, whereas the iga antibody response was highest against p and p , followed by p , p and p . surprisingly, all peptides (both conjugated [p and p ] and unconjugated [p , p , and p ] peptides) induced were added to microplates that had been coated with different peptides. igg antibodies were detected using a secondary ab specific for rabbit igg ( : ). sera from rabbits immunized with either the unconjugated peptide or with hybrid peptides p and p conjugated with klh showed the highest titer ( : ), whereas sera against the remaining peptides had a lower titer (a < . ; : ). samples of serum from a pre-immune rabbit were used as controls. no peptide-specific igg responses were found in pre-immune sera. individual samples were run in duplicate, and the data are shown as the mean ± sd high iga antibody titers in nasal washes, but peptide yielded the highest titers. the production of iga antibodies in nasal mucosa requires that dendritic cells process and present peptides on class ii mhc molecules (pmhc) to cd + t cells. in turn, these helper cells promote iga class-switching recombination and affinity maturation of iga-committed b-cells [ ] . thus, our results indicate that the peptides induced cd + t helper cells that recognize peptides in the form of an mhc-ii-peptide complex [ , , ] . in summary, intranasal immunization of mice with p and p along with ct induced specific igg and iga antibodies in serum and nasal mucosa. these antibodies could protect the host against virus invasion. according to the current concept, iga is the dominant antibody involved in protection against infection in the nasal compartment (upper respiratory tract), whereas serum igg, which diffuses into the lower respiratory tract, is predominantly involved in the protection of the lungs [ ] [ ] [ ] . finally, to test for substantial heterosubtypic binding activity, sera against p and p were tested for reactivity with purified hemagglutinin proteins from different subtypes and strains. we focused on testing p and p due to the promising results obtained with these peptides. peptide-specific igg or iga antibody titers in sera and nasal washes of mice immunized with different peptides. serial dilutions of sera (panels a-b) or nasal washes (panels c-d) from mice inoculated with different peptides were added to microplates that had been coated with specific peptides. antibodies were detected using a sec-ondary ab specific for mouse igg ( : ) or iga ( : ). samples of serum from pre-immune mice were used as controls, and no peptide-specific igg or iga responses were found in pre-immune sera or nasal washes, respectively. individual samples were run in triplicate, and the data are shown as the mean ± sd as shown in fig. a -g, the response patterns of the sera anti-p and anti-p against the hemagglutinins was similar for most hemagglutinins, with the exception of a/brisbane/ / (h n ) and a/puerto rico/ / (h n ), both of which are h n strains (fig. f and g) . both sera reacted similarly against the h n subtype. the responses of p and p against the h n subtype were not clear from the bioinformatics studies (see fig. ). the linear epitopes could not be evaluated due to their low similarity to the h n protein sequence, suggesting that a conformational epitope was involved. conformational epitopes are capable of inducing immune responses despite being discontinuous epitopes and are recognized by antibodies as linear epitopes in a tertiary structure [ ] . antibodies produced in response to the a/california/ / pandemic virus cross-reacted with several influenza virus strains, including a/brisbane/ / (h n ), a/puerto rico/ / (h n ), a/aichi/ / (h n ), and a/anhui/ / (h n ) [ ] [ ] [ ] [ ] . the origin of these crossreactive antibodies may be cross-reactive t and b cells specific for conserved epitopes in the hybrid peptides p and p . various studies have demonstrated that cross-protection against influenza virus strains may be due to cross-reactive t cells [ ] [ ] [ ] . a previous in silico analysis provided a list of potential cross-reactive t-cell epitopes, including the sequences cpkyvkstk and hagaksfyknl, which were present in p , p and p [ ] . also, as shown in fig. , p and p induced antibodies capable of . serial dilutions of sera from rabbits inoculated with peptides con-jugated to klh were added to microplates that had been coated with purified recombinant hemagglutinins ( . μg). antibodies against p and p had similar binding activity to the majority of the rhas, except for a/brisbane/ / and a/puerto rico/ / , both of which are h n subtypes. as controls, serum samples from preimmunized rabbits were tested against the specific peptide recognizing purified na protein from a viral stock in elisa studies measuring igg antibodies. according to cona envelisa studies, p and p are capable of inducing antibodies in rabbits that recognize wild-type viral antigens (immobilized with cona) from an influenza virus h n strain at dilutions of : and : ( fig. ) with significant statistical differences (one-way anova, p < . ) compared to pre-immune sera at the same dilutions. these absorbance values are equal to values others have reported for patients who were previously infected with influenza virus [ ] . in the hemagglutination inhibition assay, it was found that sera from rabbits that had been inoculated with p and p inhibited hemagglutination at a dilution of : and : , respectively (table ) . there are reports in which viral antigens (surface glycoproteins) can be immobilized to be recognized by antibodies in an elisa, as reported elsewhere for influenza virus and dengue virus [ , ] . in this study, antibodies from rabbits immunized with p and p recognized viral particles of strain (a puerto rico/ / ) immobilized with cona (fig. ) , and they also inhibited hemagglutination with titers higher than (fig. ) , which decreases the risk of infection to % [ ] . the above coincides with the neutralizing capacity of antibodies induced in mice immunized with peptides and (reciprocal of prnt titer of and , respectively; table ). these results indicate that in silico studies can be used to identify potential protective epitopes. in summary, we compared amino acid sequences ( fig. ) to provide a plausible explanation for the observed cross-protection between h n and other influenza viruses [ ] . only p alone produced a response in vivo. the observed responses to p and p could have been due to coupling to klh. additionally, p contained p and p contained a recently reported peptide [ ] . however, the responses against h n were apparently not due to the presence of linear epitopes, suggesting that the peptides were capable of adopting d structures that formed a conformational epitope [ ] . rabbit antibodies to p and p recognized ha proteins from both group (h , h , and h ) and group (h and h ) influenza a viruses belonging to three different clades: the h clade (h , h , and h ), the h clade (h ), and the h clade (h ). additionally, the antibodies also recognized purified neuraminidase from the viral stock and viral particles immobilized in the trapping elisa, and they were able to inhibit hemagglutination and neutralize the virus. na-specific igg antibody titers in sera from rabbits immunized with different peptides. serial dilutions of sera from rabbits inoculated with peptide (p ), and peptide (p ) were added to microplates previously coated with neuraminidase protein ( . µg). igg antibodies were detected using a secondary ab specific for rabbit igg ( : ). individual samples were run in duplicate, and the data are shown as the mean ± sd fig. trapping elisa. antibodies from rabbits immunized with p or p recognized influenza virus immobilized with cona. preimmune serum samples were used as negative controls, and a human serum was used as a positive control (p < . , pre-immune vs. immunized rabbit) the humoral immune responses against ha and na are the primary means of resistance to influenza virus infection [ ] ; therefore, hybrid peptides composed of ha and na peptides can be used as potent synthetic vaccines. because antibodies against the hybrid peptides p and p had broad heterosubtypic activity against the antigenically diverse h , h , h , h , and h influenza subtypes, these peptides might be used as broad-spectrum agents for prophylaxis (to induce neutralizing antibodies) and also for treatment of human or avian influenza infections, since the antibodies recognize the neuraminidase protein and could potentially prevent the release of new viral progeny during infection. the use of bioinformatics tools enabled the identification of potential epitopes for vaccine purposes. some of these epitopes could act as immunogenic agents alone, offering several advantages, such as low cost and ease of handling without special treatment. introduction to molecular biology of influenza a viruses the influenza virus: antigenic composition and immune response host adaptation and transmission of influenza a viruses in mammals seasonal, avian, and novel h n influenza: prevention and treatment modalities seasonal influenza vaccine dose distribution in countries evolution and ecology of influenza a viruses development of high-yield influenza a virus vaccine viruses prospects of ha-based universal influenza vaccine advances in universal influenza virus vaccine design and antibody mediated therapies based on conserved regions of the hemagglutinin broadly neutralizing antibodies against influenza viruses vaccine options for influenza: thinking small neuraminidase as an influenza vaccine antigen: a low hanging fruit, ready for picking to improve vaccine effectiveness structural basis of preexisting immunity to the h n pandemic influenza virus structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution a perspective on the structural and functional constraints for immune evasion: insights from influenza virus structural insights into the design of novel anti-influenza therapies naction! how can neuraminidase-based immunity contribute to better influenza virus vaccines? mbio influenza neuraminidase as a vaccine antigen theoretical analysis of the neuraminidase epitope of the mexican a h n influenza strain, and experimental studies on its interaction with rabbit and human hosts contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines influenza a: biología, vacunas, y origen del virus pandémico a/h n design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach peptides for immunological purposes: design, strategies and applications the molecular bases of delta/alphabeta t cellmediated antigen recognition identification of b cell and t cell epitopes using synthetic peptide combinatorial libraries the influenza virus resource at the national center for biotechnology information muscle: multiple sequence alignment with high accuracy and high throughput clustal w and clustal x version . swiss-model: modelling protein tertiary and quaternary structure using evolutionary information the swiss-model workspace: a web-based environment for protein structure homology modelling the swiss-model repository and associated resources automated comparative protein structure modeling with swiss-model and swiss-pdbviewer: a historical perspective pdbsum additions knowledgebased validation of protein-structure coordinates derived by x-ray crystallography and nmr-spectroscopy vmd: visual molecular dynamics a continuous peptide epitope reacting with pandemic influenza ah n predicted by bioinformatic approaches mhc class ii epitope predictive algorithms reliable prediction of t-cell epitopes using neural networks with novel sequence representations nn-align an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction scalable molecular dynamics with namd all-atom empirical potential for molecular modeling and dynamics studies of proteins constant-pressure molecular-dynamics algorithms software news and updates. carma: a molecular dynamics analysis program neutrophils extracellular traps damage naegleria fowleri trophozoites opsonized with human igg influenza viruses: an introduction simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in bhk- cells: comparison of the bhk suspension test with standard plaque reduction neutralization differences in antibody responses of individuals with natural infection and those vaccinated against pandemic h n influenza immuno-fluorescence assay of leptospiral surface-exposed proteins neutralization of virus infectivity by antibodies: old problems in new perspectives structural and thermodynamic approach to peptide immunogenicity virus-specific t cells as correlate of (cross-)protective immunity against influenza the effector t cell response to influenza infection antiviral cd + t cell effector activities in situ are regulated by target cell type b cell responses to influenza infection and vaccination t cell activation exploring the immunome: a brave new world for human vaccine development bronchus-and nasal-associated lymphoid tissues mechanism and regulation of class switch recombination protection against influenza virus infection in polymeric ig receptor knockout mice immunized intranasally with adjuvant-combined vaccines a nasally administered trivalent inactivated influenza vaccine is well tolerated, stimulates both mucosal and systemic immunity, and potentially protects against influenza illness mucosal iga responses in influenza virus infections; thoughts for vaccine design how a single amino acid change may alter the immunological information of a peptide heterosubtypic neutralizing monoclonal antibodies cross-protective against h n and h n recovered from human igm+ memory b cells immunity to pre- h n influenza viruses confers cross-protection against the pandemic swineorigin a (h n ) influenza virus vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections preexisting human antibodies neutralize recently emerged h n influenza strains pre-existing immunity against swine-origin h n influenza viruses in the general human population assessment of seasonal influenza a virus-specific cd pandemic h n swine-origin influenza a virus seasonal influenza can poise hosts for cd t-cell immunity to h n avian influenza immunoinformatic comparison of t-cell epitopes contained in novel swine-origin influenza a (h n ) virus with epitopes in - conventional influenza vaccine denv- subunit proteins fused to cr receptorbinding domain (p )-induces specific and neutralizing antibodies to the dengue virus in mice hemagglutination inhibiting antibodies and protection against seasonal and pandemic influenza infection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations rafael campos-rodríguez citlicampos@gmail.com key: cord- -i a igc authors: nagata, s.; okamoto, y.; inoue, t.; ueno, y.; kurata, t.; chiba, j. title: identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: i a igc thirteen monoclonal antibodies (mabs) to the glycoprotein (g) of vesicular stomatitis virus (vsv) serotype indiana were prepared and examined for their effects on various biological activities of vsv, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. competitive binding assays with these mabs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the g protein. in some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. the mabs to all the epitopes but one (epitopes – ) reacted with the denatured g protein in a western immunoblot analysis. four of the epitopes (epitopes , , , and ) were involved in neutralization and two (epitopes and ) in hemagglutination inhibition. none of the mabs inhibited the adsorption of radiolabeled vsv to bhk- cells; the mabs to epitope slightly enhanced the virus adsorption. all neutralization epitopes except epitope (epitopes , , and ) were associated with inhibition of vsv-mediated cell fusion. these results show a direct spatial relationship between the epitopes recognized by the mabs and functional sites on g protein and further insights into the structure and function of g protein. many enveloped viruses including vesicular stomatitis virus (vsv), family rhabdoviridae, genus vesiculovirus, transfer their nucleocapsids to the cytoplasm of host cells by the adsorption and receptor-mediated endocytosis, followed by fusion with the endosomal membrane [ , ] . the glycoprotein (g) of vsv is the sole protein anchored in the viral envelope and plays a critical role in this early stage of virus infection. many biological properties of g protein are associated with the virus entry [ ] , which include adsorption to host cells [ , ] , hemagglutination (ha) [ , ] , and mediation of in vitro cell-cell fusion [ , ] . the cell-cell fusion occurs only at low ph, which mimics the acidic environment of the endosomal lumen [ , ] . as expected from its central role in infection, the g protein also gives rise to and reacts with neutralizing antibodies [ ] . in recent years, much effort has been made to reveal the structurefunction relationships of the g protein, especially regarding its role in fusion [ , , , , , ] , but the underlying molecular mechanisms are still poorly understood. one approach to the structure-function relationship of surface glycoproteins of viruses is to analyze for the sites and effects of the monoclonal antibody (mab) binding [ , , , ] . production of mabs against g proteins of two major serotypes of vsv (indiana and new jersey) has been reported by two research groups [ , , , ] . these mabs have mainly been used to map neutralization and non-neutralization epitopes on g protein and to analyze the mutation leading to antigenic variations of g protein [ , , [ ] [ ] [ ] ] . the effects of the mabs specifically reacting with g protein on biological functions other than neutralization have not been reported. in the present study, we prepared thirteen mabs specific for seven distinct epitopes on g protein of vsv-indiana and examined for their effects on various biological activities of vsv including in vitro infection, ha, adsorption to the cells, and mediation of cell-cell fusion. our findings defined the spatial relationship between the epitopes recognized by the mabs and the functions of g protein. the san juan strain of vsv-indiana originally provided by dr. r. r. wagner, university of virginia, was obtained from dr. k. yamamoto, national institute of health, tokyo. the virus stock was prepared by infecting bhk- cells (japanese cancer research resources bank) at a multiplicity of . pfu/cell. virus harvested at h postinfection was concentrated by ultrafiltration and ultracentrifugation [ , ] , and purified by sucrose density gradient centrifugation [ ] . this preparation containing . mg/ml of viral protein ( . x pfu/ml) was stored at - °c. the protein content of the preparation was determined with bca protein assay reagent (pierce chemical co., rockford, il, u.s.a.) with bovine serum albumin (bsa) as a standard. virus infectivity was determined by plaquing on monolayer cultures of bhk- cells [ ] . the g protein was extracted from the purified virus with mm octyl- -d-glucopyranoside (sigma chemical co., st. louis, mo, u.s.a.) as described by petri and wagner [ ] . after removal of the nucleocapsids by ultracentrifugation at , x g, the supernatant containing g protein was dialyzed against mm hepes (ph . ) containing . m naci. g protein thus obtained was free from any other virus protein in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) stained with coomassie brilliant blue. for immunization, female balb/c mice were subcutaneously injected twice each with gg of the purified g protein emulsified in the same quantity of freund's complete and freund's incomplete adjuvants, respectively. then, additional two intraperitoneal injections with gg of g protein were given. three days before fusion for hybridoma production, the final gg of g protein was injected intravenously. the spleen cells of the immunized mice and sp /o-ag , balb/c mouse non-secretory plasmacytoma cells, were fused with polyethylene glycol according to oi and herzenberg [ ] or by the novel vsv-mediated cell fusion method described previously [ , ] . media preparation and hat selection of hybridomas were described previously [ ] . in weeks, the hybridomas were screened for production of anti-g protein antibody by enzyme-linked immunosorbent assay (elisa) with purified g protein as the antigen (see below). the positive cultures were cloned several times by the limiting dilution method. the isotypes of the specific antibodies were determined with a mouse monoclonal antibody isotyping kit (amersham international, buckinghamshire, england). thirteen hybridomas were established and each was over-grown in about of a serumfree medium (iscove's modified dulbecco's medium, sigma) containing mg/ml of bsa, mm sodium pyruvate (gibco, grand island, ny, u.s.a.), ~tg/ml of bovine insulin (sigma), gg/ml of iron-saturated human transferrin (miles scientific, naperville, il, u.s.a.), ~tm -mercaptoethanol, gm ethanolamine, . ~tg/mt of linoleic acid, . ~tg/ ml of oleic acid, . gg/ml of palmitic acid, and gg/ml of gentamicin. mab in the culture supernatant was precipitated with ammonium sulfate at % saturation and the precipitate was further purified by high performance liquid chromatography on hydroxyapatite beads [ ] or by affinity chromatography on protein g-sepharose (pharmacia, uppsala, sweden). the eluate was concentrated to ml by membrane ultrafiltration ( , tool. wt. cut-off centriprep; amicon, danvers, ma, u.s.a.), and dialyzed against phosphate-buffered saline (pbs). the antibody concentration was determined from absorbance at nm with an extinction coefficient of . per mg of protein. for use as negative controls in various assays, two mabs prepared at national institute of health were purified by the same procedure. one of them was ig g specific for the core antigen of feline immunodeficiency virus (unpubl.) and the other was ig g a specific for sheep red blood cells (not crossreactive with goose erythrocytes) [ ] . production of anti-g protein antibody in hybridoma culture supernatants was examined by elisa. wells of microtiter plates (costar ; costar, cambridge, ma, u.s.a.) were coated with the purified g protein ( gg/ml) in mm sodium carbonate buffer (ph . ) for h at room temperature. the wells were washed with pbs containing . % (v/v) tween (pbs-tween) and blocked overnight at °c with . % (w/v) gelatin in pbs. after washing, each culture supernatant was added to the wells and the plates were incubated for t h at room temperature. the antibody bound was detected by incubation for h at room temperature with alkaline phosphatase-conjugated goat anti-mouse igg + m (tago ; tago inc., burlingame, ca, u.s.a.) diluted , -fold in pbs-tween. the enzyme reaction was started by adding mg/ml of p-nitrophenylphosphate (wako pure cemical ind., osaka, japan) in % (v/v) diethanolamine (ph . ) containing . mm mgc . the s. nagata et al. absorbance at nm was measured with an eia autoreader (sanko junyaku co., tokyo, japan). as a positive control, a , -fold dilution of mouse immune serum was used. this control usually showed an absorbance of approximately . after incubation for rain at room temperature. the well with absorbance higher than . was regarded as positive. purified mabs were titrated by the same elisa to compare the relative reactivities, in which the wells of the plates were coated with vsv virions ( gg/ml) instead of g protein. the relative reactivity was defined as the concentration of mab needed to attain % of the absorbance value of the positive control. purified vsv was separated by sds-page in % polyacrylamide gel under reduced conditions. the proteins separated were then blotted onto immobilone membrane (millipore corp., bedford, ma, u.s.a.) according to towbin et al. [ ] . the blots were allowed to react with culture supernatants of established hybridomas, and specific bands were visualized with alkaline phosphatase-conjugated goat anti-mouse igg + m (tago ) and the bcip/ nbt phosphatase substrate system (kirkegaard & perry lab. inc., gaithersburg, md, u.s.a.). a -gg portion of each purified mab was mixed with gg of biotinyt n-hydroxysuccinimide ester (nhs-lc-biotin, pierce) in . ml of . m sodium bicarbonate (ph . ). after incubation for h at room temperature, the mixtures were dialyzed extensively against pbs at °c; bsa was then added to a final concentration of mg/ml. for competitive binding assay, the wells of plates were coated with purified vsv ( ~tg/ ml) and blocked with % (w/v) unfatted bovine milk in pbs. serial -fold dilutions of unlabeled competitor mab ( x - to x - mg/ml) were added to the wells ( ~tl/ well) and the plates were incubated for h at room temperature. subsequently, gl of biotinylated mab was mixed with competitor mab. the concentrations of biotinylated mabs were gg/ml for b and b , . gg/ml for v b , and . gg/ml for the other mabs. these concentrations were about half-maximal in their titration curve and were within the range where the binding was linear. after incubation overnight at °c, biotinylated mab bound was detected with a , -fold dilution of alkaline phosphataseconjugated streptavidin (bethesda research lab., gaithersburg, md, u.s.a.). dilution was made in pbs-tween containing % (w/v) unfatted bovine milk. the enzyme reaction and absorbance determination were carried out as described above. all assays were performed in duplicate and the results were expressed as the percentage of binding calculated with the formula: average ofabsorbance in the presence of competitor binding(%) = x average ofabsorbance in the absence of competitor for the neutralization assay, the stock of vsv was diluted to a final concentration of approximately , pfu/ml with bicarbonate-free eagle's minimum essential medium (mem, nissui pharmachemical co., tokyo, japan) containing . mg/ml of bsa and mm hepes (ph . ). the virus was mixed with an equal volume of each of serial twofold dilutions of each purified mab (from gg/ml) in the same medium. the mixtures were incubated for h at °c and then plated in duplicate on monolayers of bhk- cells in -well culture plates for plaque assay ( gl/well). the neutralization antibody titer was defined as the reciprocal of the highest dilution reducing more than % of the plaques of the control without mab. hemagglutination inhibition (hi) was assayed with or hemagglutinating units of vsv in v-bottom microtiter plates as described by halonen et al. [ ] , except that goose erythrocytes were used after the treatment with trypsin (sigma) at l~g/ml for rain at °c. this pretreatment of the erythrocytes enhanced ha, thus increasing the sensitivity of hi [ ; unpubt, data] . the reciprocal of the highest dilution of purified mab causing complete inhibition of hemagglutination was taken as the hi titer. for adsorption assays, s-labeled vsv was prepared by the addition of ~ ci/ml of l-[ slmethionine (amersham) to the infection medium as described by bailey et al. [ ] . the radiolabeled virions were concentrated and purified as described for the unlabeled virus. the final preparation was free of contaminating labeled materials as judged by sds-page and autoradiography. the final preparation contained . x l° pfu/ml ( . mg/ml viral protein) with a specific activity of . x cpm/~tg. the radiolabeled virus absorbed to cells was quantified essentially as described by matlin etal. [ ] . bhk- cells grown to confluency in -well culture plates (about cells/well) were washed twice with the binding medium, bicarbonate-free mem buffered with mm hepes (ph . ) containing mg/ml of bsa, and cooled for rain on ice. the radiolabeled purified virus ( , cpm) was mixed with each purified mab at various concentrations in the binding medium. the mixtures were incubated for h at °c, chilled, and then plated in duplicate on the bhk- cell ( gl/well). after incubation for h on ice, unbound virus was removed. the cells were washed four times with the binding medium. the cells bound with the virus were solubilized in . ml of solubable (nen research product, boston, ma, u.s.a.), and its radioactivity was measured with a liquid scintillation counter. the average radioactivities bound to the cells in the presence of mab were expressed as the percentage of the radioactivity bound in the absence of mab. nonspecific interaction of the virus with the ceils and the surface of the plates was minimized by adding bsa to the binding medium. the addition of bsa reduced the nonspecific binding of labeled vsv to the surface of the plates from . % to less than . % of the input radioactivity. the amount of virus used was within the range where the radioactivity bound to cells increased proportionally with the amount of the input virus. the effect of mabs on vsv-mediated cell fusion was assayed by inhibition of polykaryon formation of bhk- cells [ , ] . the cells in -well culture plates (about . x celts/well) for - h were washed twice with the ice-cold binding medium (bicarbonatefree mem buffered with mm hepes, ph . , containing mg/ml of bsa). the purified virus ( gg) in gl of the cold binding medium was applied onto the cells. after incubation for h on ice to allow viral adsorption, free virus was removed, and the cells were treated on ice for min with ~g/ml of each mab in ~. of the binding medium. after removing the mab solutions, . ml of prewarmed ( °c) acidic medium, bicarbonate-free mem buffered with mm mes (ph . ), was added for triggering fusion and the plates were incubated for min at °c. the medium was replaced with . ml of the prewarmed binding medium and the cells were incubated for an additional hour at °c. after fixation with % formalin in pbs and staining with hematoxylin, inhibition of polykaryon for-marion was examined under a phase-contrast microscopy. the amount of the virus used in this assay was enough to induce fusion in about % of the cells. two fusion experiments yielded stable hybridomas secreting m a b s specifically reacting with g protein of vsv. their characteristics are listed in table . their relative reactivities varied over a -fold range, but were still within a relatively high range c o m p a r e d with those of other m a b s to different antigens determined by us. all the m a b s except for p f reacted with g protein in western blotting analysis. competitive binding e l i s a was carried out a m o n g these m a b s to classify the epitopes of g protein recognized by them. typical results o f the competitive binding assay are shown in fig. , in which the binding o f biotinylated a m a b to g protein was challenged by several unlabeled mabs. we observed four types of competition. in addition to homologous m a b ( a ), p f completely inhibited the binding. c partially inhibited the binding. f , p e or p f a all mabs had ~: light chain b relative reactivity was defined in elisa as the mab concentration needed to attain an absorbance of . when a positive control (immunized mouse serum diluted , -fold) had an absorbance value of . c reactivity to g protein in western blotting analysis d epitopes (antigenic determinants) were identified by competitive binding assay among mabs as described in table table ). the mabs were assigned to seven distinct epitopes on g protein based on the complete inhibition. when an unlabeled mab inhibited the binding of a biotinylated mab at gg/ml (at least -fold excess of biotinylated mabs) to less than % and when this inhibition was observed in pair-wise assays, both mabs were considered to share the same (or a closely adjacent) epitope. these seven epitopes were designated as ep to ep . the mabs to the same epitope showed similar patterns of partial inhibition or enhancement of binding against mabs to different epitopes (table ) . only mabs to ep were subgrouped into two based on the pattern; mabs to ep a had no effect on the binding of mabs to ep , whereas mab to ep b enhanced the binding of mabs to ep . the mabs were assayed for the neutralizing activity by the plaque reduction test ( table ). the mabs assigned to four (ep , ep , ep and ep ) of the seven epitopes had neutralizing activities. the neutralization titers of the mabs to ep and ep were about -times higher than the others. inhibition of hemagglutination by the mabs was also examined ( table ). the mabs assigned to two epitopes (ep and ep ) had higher hi activity than the others. goose erythrocytes used in this experiment were pretreated with trypsin to enhance the sensitivity of hi. untreated erythrocytes gave similar results, although higher mab concentrations were required (data not shown). titers for neutralization represent the reciprocal of the highest twofold dilution of purified mab ( gg/ml initial) causing more than % reduction in the plaque number b titers for hi represent the reciprocal of the highest twofold dilution of purified mab ( gg/ml initial) inhibiting ha caused by or hau of vsv. control ig g and ig g a mabs and the serum-free medium for growing hybridomas showed no hi activity (< ) we examined mabs for the influence on the adsorption of radiolabeled vsv to bhk- cells. mabs to ep and ep had very little, if any, effects on the virus adsorption (fig. a and c) similar to the control mabs (fig. h) . mabs to ep , ep , ep and ep slightly reduced the virus binding (fig. d, e, f and g). even at the highest concentration ( gg/ml), they exerted partial inhibition ( - % of the binding of control). on the other hand, mabs to ep slightly enhanced the vsv adsorption only at certain concentrations (fig. b) . in another experiment with a different preparation of radiolabeled vsv with a higher specific activity, similar results were obtained: no mab completely inhibited the virus binding and mabs to ep enhanced the virus binding (data not shown). we examined mabs for the effects on vsv-mediated polykaryon formation of bhk- cells to test whether the mabs inhibit the fusion induced by vsv. extensive cell fusion was induced by acid treatment of the virus-bound cells (fig. o) but not by the same treatment of unbound cells (fig. p) . mabs reacting with ep (fig. g and h) , ep ( fig. i) , and ep (fig. ) completely inhibited polykaryocyte formation. the other mabs (fig. a-f, j, and k) p f + + --+ a + +, +, and -neutralization titers of > , , >f and > , respectively for % reduction of plaques, shown in the third column of table b + and -hi titers against h a u of >t and ~< , respectively, given in the th column of table c _ no inhibition or slight inhibition of adsorption ( - %); e enhancement of adsorption (~> %) in the virus binding assay shown in fig. d + inhibition of fusion; -no inhibition e not tested to the cell fusion activity. w e reported here for the first time identification of the g protein epitopes associated with h a and fusion activities. in the competitive binding assay, the m u t u a l complete competition o f paired m a b s revealed the presence of at least seven distinct epitopes on g protein of vsv-indiana. in addition, some topographical relationships a m o n g some o f these epitopes were suggested by partial inhibition or enhancement o f the binding o f m a b s ( table ). in particular, association a m o n g ep , ep , and e p w o u l d be quite possible since the m u t u a l binding enhancement o f the respective m a b s was observed. such enhancement is p r o b a b l y due to an a d v a n t a g e o u s allosteric alteration of g protein after binding with the first m a b , thereby resulting in increased binding o f the second m a b . similar competitive binding assays with anti-g protein m a b s were reported by volk etal. [ ] and le-francois and lyles [ , ] , w h o d e m o n s t r a t e d and epitopes on g protein of vsv-indiana, respectively. the enhancement of binding was f o u n d also by lefrancois and lyles [ , ] . o f the seven epitopes identified on g protein, all b u t one (ep - ) reacted with respective m a b s even in western blot analysis. these are p r e s u m a b l y linear epitopes not dependent on the secondary structure. the m a b s assigned to four epitopes (ep , ep , ep , and ep ) had vsv-neutralizing activity, although of varying efficiency. previous reports also demonstrated the same number of neutralizing epitopes on g protein of vsv-indiana [ , ] . mabs to ep and ep showed hi activity. the proximity between these hi epitopes were suggested by the competitive binding assay and it is likely that these epitopes concurrently form the functional domain for the ha activity. these hi epitopes were not always neutralization epitopes and were different from the fusion-inhibition epitopes. this indicates that the sites involved in ha activity are different from those involved on the other functions. in general, viral ha is equivalent to the viral attachment to cells. however, the hi mabs did not inhibit the vsv binding to bhk- cells. the little correlation between these two activities is likely ascribed to the difference of the target cells and/or conditions in these assays. "l;he result of another experiment showed that the hi mabs markedly inhibit the binding of radiolabeled vsv to goose erythrocytes, in which the binding is measured under the same condition as the hi assay (data not shown). attempts to identify the cell-binding domain of g protein were unsuccessful in this study. in the binding assay, no mab completely inhibited the vsv adsorption (fig. ) . mabs to ep , ep , ep , and ep at high concentrations slightly inhibited vsv adsorption, but such low inhibitory effects were not related to the efficient neutralization or hi. the lack of the complete inhibition suggests that all neutralizing mabs prepared in this study block the virus infection at a step subsequent to adsorption. on the other hand, a certain concentration of mabs to ep , one of the neutralization epitopes, rather enhanced vsv adsorption. although it is difficult to explain the biological significance of this enhancing effect, a similar enhancement of the vsv adsorption by immune serum was reported by schlegel and wade [ ] . they suggested that the vsv-antibody complex binds to a different or an additional cell binding site, thus altering the adsorption efficiency. the same explanation may be given to the enhancing effect of mabs to ep . in the fusion inhibition assay, mabs reacting with three epitopes (ep , ep , and ep ) inhibited the vsv-mediated cell fusion. these epitopes are probably located on or close to the fusogenic domain of g protein. although hydrophobic domains involved in fusion have been identified in several viral fusion proteins [ , ] , such a domain has not been identified in g protein of vsv [ ] . a most recent finding that introduction of a glycosylation site into residue of g protein resulted in fusion-defective mutant suggests that the residues to are involved in the fusion activity [ ] . some of our fusion-inhibiting mabs may recognize these residues and the location of the fusion-inhibition epitopes will be required. we found the presence of multiple epitopes related to the fusion activity. this suggests that these different regions of g protein contribute to the fusion activity in partnership and might explain the lack of highly hydrophobic fusion sequence in the g protein of vsv. all fusion-inhibiting mabs had the neutralizing activity. in the vsv infection process, after endocytosis of the b o u n d virion, the fusion of viral envelope with the endosomal m e m b r a n e is necessary for the entry of the nucleocapsids into the cytoplasm [ , ] . fusion-inhibiting mabs neutralize vsv probably by blocking the fusion stage of the infection process. to analyze further the structure-function relationship of g protein, our studies are currently aimed at locating these epitopes on the g protein by testing the reactivity of the mabs to g protein fragments expressed in escherichia coli. effects of deae-dextran on infection and hemolysis by vsv. evidence that nonspecific electrostatic interactions mediate effective binding of vsv to cells glycosylation is not required for the fusion activity of the g protein of vesicular stomatitis virus in insect cells restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components identification of distinct antigenic determinants on semliki forest virus by using monoclonal antibodies with different antiviral activities monoclonal antibodies to the glycoprotein of vesicular stomatitis virus (new jersey serotype): a method for preliminary mapping of epitopes surface structure of vesicular stomatitis virus a cell line expressing vesicular stomatitis virus glycoprotein fuses at low ph effect of glycosylation on the conformational epitopes on the glycoprotein of vesicular stomatitis virus (new jersey serotype) hemagglutinin of rabies and some other bullet-shaped viruses membrane fusion of envelope viruses: especially a matter ofproteins epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector the glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody biological activities of monoclonal antibodies reactive with antigenic sites mapped on the g glycoprotein of la crosse virus the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus i. analysis of neutralizing epitopes with monoclonal antibodies the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus ii. monoclonal antibodies to nonneutralizing and cross-reactive epitope of indiana and new jersey serotypes antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants point mutations in glycoprotein gene of vesicular stomatitis virus (new jersey serotype) selected by resistance to neutralization by epitope-specific monoclonal antibodies spontaneous mutations leading to antigenic variations in the glycoproteins of vesicular stomatitis virus field isolates ph-dependent hemolysis and cell fusion of rhabdoviruses virus entry into animal cells pathway of vesicular stomatitis virus entry leading to infection transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of madin-darby canine kidney cells. i. morphological evidence biological properties of vsv glycoprotein. ii. effects of the host cell and of the glycoprotein carbohydrate composition on hemagglutination vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different ph values production of monoclonal antibodies by the use of ph-dependent vesicular stomatitis virus-mediated cell fusion preferential generation ofmonoclonal igg-producing hybridomas by use of vesicular stomatitis virus-mediated cell fusion membrane fusion in fertilization, cellular transport, and viral infection immunoglobulin-producing hybrid cell lines vesicular stomatitis virus membrane proteins and their interactions with lipid bilayers functional epitopes on glycoprotein of vsv reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis analysis ofmurine coronavirus surface glycoprotein functions by using monoclonal antibodies neutralized vesicular stomatitis virus binds to host cells by a different "receptor topographical mapping of epitopes on the glycoproteins of murine hepatitis virus- (strain jhm): correlation with biological activities electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications sequences of the major antibody binding epitopes of the indiana serotype of vesicular stomatitis virus monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity rhabdovirus biology and infection: an overview cell fusion by semliki forest, influenza, and vesicular stomatitis viruses a fusion-defective mutant of the vesicular stomatitis virus glycoprotein fatty acid acylation is not required for membrane fusion activity or glycoprotein assembly into vsv virions growth, purification and titration of rhabdoviruses. in: mahy bwj (ed) virology: a practical approach high performance liquid chromatography of mouse monoclonal antibodies on spherical hydroxyapatite beads we thank dr. kiichi yamamoto for the gift of vsv-indiana and drs. shudo yamazaki and robert r. wagner for their useful information on this strain. we thank also dr. yoshio yamakawa for his help with hplc and dr. akiko taniguchi for her assistance in some assays. several helpful discussions with dr. michiyuki matsuda are gratefully acknowledged. we thank also mr. john meissner and mr. russell nash for their critical reading of the manuscript. we thank also the members of department of pathology, national institute of health, for their encouragement. key: cord- -gvom f authors: traavik, t. title: improvement of arbovirus ha antigens by treatment with a colloidal silica gel and sonication date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: gvom f a remarkable increase in ha titers for weakly haemagglutinating norwegian arbovirus strains, uukuniemi and runde viruses, was achieved by including treatment with the colloidal silica gel aerosil in the antigen preparation scheme. by combining this procedure with sonication, the titers of sucrose-aceton extracted, infected suckling mouse brains could be increased several hundred times. good antigens also were obtained from virus grown in bhk /c cell cultures and concentrated by polyethylene glycol /nacl. rubella virus ha antigen and hb(s)ag were adsorbed to the gel, and excluded from a preparation by treatment with aerosil. this indicates a limitation to the universal use of the method, presumably related to the particle size. due to speed, sensitivity, economy and simplicity the haemagglutination inhibition test (hai) has been a standard test in arbovirus serology. this applies to both identification of virus isolates and serological surveys and diagnostics. for production of arbovirus haemagglutinating (ha) antigens, the classical sucrose-aeeton extraction method (sa) ( ) with infected suckling mouse brains has been almost undisputed, although methods based on infected cell culture fluids ( , ) and infected mouse aseites fluids ( ) have also been reported. the arbovirus strains isolated in norway up to date ( , , , ) , have all presented modest yields of ha antigens by the conventional sa method. tweenether treatment ( ) has not increased the tigers appreciably. the low titers of suckling mouse brain preparations necessitate a high consumption of mouse litters and frequent performance of the rather cumbersome and laborious sa method. for unknown reasons, variations in the quality of the antigen from batch to batch are adding to the difficulties. consequently, there is a marked need for a reliable and simple method to prepare high-titered ha antigens from infected tissues or cell cultures. since the haemagglutination inhibitors in virus preparations appear to be of lipoprotein n a t u r e ( , , ), a procedure for i-ia antigen production might be based on a compound which removes lipoprot, eins without interference with the q u a n t i t y or configuartion/composition of the genuine ha antigens. the colloidal silica gel "aerosil" has demonsbrated capability to adsorb serum lipoproteins a n d also the hepatitis b antigen (hbsag) ineluding the dane particle ( , t ) . i t seemed worthwhile to investigate whether this compound would adsorb inhibitors to ha, leaving the viruses/ha antigens in solution. since a beneficial effect of sonieation on arbovirus h a titers has been reported ( ), it was decided to include ultra sound t r e a t m e n t into the investigations. the strain sf e was isolated from engorged ixodes ricinus ticks (nymphs) collected from migrating passerine birds captured at store fcerder (an island in the oslofjord) in may . by hai it was demonstrated to be serologically related to the $ prototype uukuniemi virus and that it also had the same morphology (i , ). the strain i~u e was isolated from unengorged ixodes uriae (females) collected in the seabird colony at runde in september . in the electron microscope it presents a morphology which closely resembles that of the corona virus group (t , ). a tween-ether extracted l~ubella ha antigen from behringwerke has also been used in these investigations. antigen preparations crude suckling mouse brain preparations (smb antigens) consist of approximately per cent suspensions of infected brain material in borate saline ph . or in pbs ph . . sf e was used at the fourth mouse brain passage, titering . logs bmlds /ml (baby mouse lethal doses). the fifth smb passage of i~u e which titered . logs bmld~ /ml eonstitut, ed the antigenic source for this virus. to get rid of tough debris, %he brain suspensions were centrifuged once at × g for minutes. sucrose-aceton extracted suckling mouse brains (sa antigens) were prepared according to clarke and casals ( ), but the final lyophilization step was omitted. virus precipitates from cell culture fluids (bhk antigens) were prepared as follows: polyethylene glycol (peg) (maerogolum , norsk medicinaldepot), g/ ml, and nac , . g/ ml, were dissolved in culture fluids harvested from virus infected b t i k /c cultures ( ). the phi was adjusted to . , and precipitation took place at ° c overnight. on the following morning the culture fluids were centrifuged at × g for minutes. the supernates were discarded, and the precipitates redissolved in pbs with . per cent bovine serum albumine or borate saline with . per cent albumine (babs). the antigens used in these experiments were made by redissolving precipitates in / of the originm volume. the strains employed in these experiments had the following passage history: sf e had passed times in mice, and thereafter undergone to passages in cell culture. ru e was used after mouse brain passages and -- passages in b h k / c cells. the rubella i-ia antigen was used as such. aerosil (degussa, frankfurt a. m~in), according to the manufacturer is a colloidal silica consisting of aggregated nm particles with a surface area of about me/gram. aerosil adsorption this was performed as follows : the antigen solutions were mixed with , , , , and mg aerosil per ml. the mixtures were treated with a swirlcr (cenco instrumenten, breda, the netherlands) to ensure a homogenous suspension to be effected. the tubes were placed in a waterbath with automatical shaldng (type , j. kottermann, k.g. tianigsen, germany) for hours at ° c and centrifuged for minutes at × g. the antigen-containing supernate was pipetted off and kept, while the sedimented silica gel was discarded. ha and h a l tests were performed essentially as described by cla~xe and casals ( ), modified for microtitration equipment (cooke eng. co., alexandria, virginia) and using chicken erythroeytes instead of the recommended goose cells. the ha activity was tested with the following antigen preparations of each virus parallelly: a) untreated ; b) aerosil-treated; e) sonieated ; d) aerosil-treated and then sonicated ; e) sonieated and then aerosil-treated. uninfected suckling mouse brains, sa extracted suckling mouse brains and culture fluids from uninfected bhk /c cultures had been treated as the antigens and were included in every test as controls. ha activity was investigated in the p i t range of . -- . at -{ ° c, room temperature and ° c. i n order to confirm the virus specificity of observed ha reactions, specific mouse antisera, and a serum pool from uninfected mice were utilized in hai. six units of antigen were employed in these tests. infected brain suspensions and peg treated cell culture concentrates were titrated by intraeerebral inoculation in suckling mice after aerosil absorption, incubation with shaking in waterbath at ° c and untreated. preeipitin activity was titrated by immunoelectroosmophoresis in per cent agarose gel as described elsewhere ( , ) . b y employing mg aerosil per ml, the h a titers were increased from -to -fold, depending on the kind of antigenic preparation, for sf e a n d i~u e . by using mg silica gel per m], a further -to -fold increase was obtained. no beneficial effect was recognized b y increasing the concentration above this level. accordingly a n aerosil concentration of mg/ml was chosen throughout the experiments. the rubella h a antigen initially titered -- . this was reduced to to after t r e a t m e n t with mg aerosil per ml and to nil when mg/ml was used. at mg/ml some h a activity appeared. this m a y be due to inefficient mixing. these results are summarized in table . dependence on ph and temperature sf e t virus was earlier shown to have a marked p h dependence in the h a reaction, with m a x i m u m activity at p h . -- . for sa preparations. the p h effect on r u e was not as manifest, nearly constant h a titers being obtained at p h . -- . . after aerosil t r e a t m e n t the p h spectrum for h a was broadened, although s f e still possessed a m a x i m u m at p i t . -- . (table ) . the i n c u b a t i o n temperature had earlier been shown to affect the h a of sf e severely. the highest titers were obtained at ° c, while nearly no i-l~_ activity was recorded at ° c. the influence of varying temperatures was less for r u e , b u t somewhat higher titers and clearer end-points were obtained at ° c. after absorption with silica gel, sf e i haemagglutinated at ° c, although titers were lower t h a n at ° c. for ~u e titers were essentially the same at ° c a n d ° c, b u t sedimentation patterns were better at the lower temperature (table ) . no differences were recorded between the two sonication procedures applied. i n some instances a -to -fold titer increase was registered, b u t at other times h a t, iters were unaffected b y sonication alone (table @ the e//ect o/aerosii on in]ectivity and precipitins i t was shown, as demonstrated in table , t h a t ~he drop in infectivity titer seen for lgu es was m a i n l y due to the i n c u b a t i o n at ° c. sf e was unaffected b y aerosil treatment. preeipitin titers were not affected for a n y of the viruses. t. tr-~kawk : the results are summarized in table ~ there was no obvious difference between performing sonication before or after aerosil treatment, but the combination of these two procedures proved valuable. as can be seen the highest titers were obtained with sa extracted mouse brain preparations. but fairly acceptable and useful ha antigens can be produced from cell culture fluids and crude suckling mouse brains. the ha reactions observed are virus specific as demonstrated by homologous serum inhibitions and negative reactions for control preparations. ha titers in reciprocal values b untreated the experiments described have demonstrated the ability of the colloidal silica gel aerosil to absorb supposed ha inhibitory factors from virus antigen preparations. the two viruses investigated are large, -- nm in diameter ( , , , ) . the results obtained with tween-ether extracted rubella virus and with hbsag ( ), when aerosil actually absorbs the virus specific antigens, points to a limitation to the method, presumably presented by the size of the virion. the exact limit is not known at present, but the data available indicate that the critical diameter is somewhere between and nm. it seems unlikely that the aerosil absorption method can be applicated to the smaller alpha and flaviviruses. methods used in the past to improve the capability of low-titered ha antigens or to bring non-haemagglutinating viruses to react, have been based on detergents ( , ) or enzymes ( , ) . reaction conditions must be carefully controlled with regard to concentration and time in order to obtain satisfactory results. since the effect of aerosil seems to be due to a simple absorption effect without any interference with chemical composition or configuration, the problems mentioned are eliminated. the results obtained with virusinfected cell culture fluids after precipitation with peg /nag and aerosil absorption imply that antigens of this kind represent a useful alternative to the conventional suckling mouse brain antigens. the inferiority in ha titers will be more than compensated by the gain in resources and economy and the simplicity of production. the preparation of arbovirus hemagglutinins by sonication and trypsin treatment production of hemagglutinin by dengue virus in hela cells techniques for haemagglutination inhibition with arthropod-borne viruses polyphosphoinositides as receptor substances for certain groups of arboviruses arbovirus haemagglutinins. differential susceptibility to trypsin nonspecific inhibitors of arbovirus hemagglutinin concentration and purification of vesicular stomatitis virus by polyethylene glycol "precipitation use of sarcoma to prepare haemagglutinating and complement-fixing antigens for viruses in adult mice studies on the structure of a haemagglutinating component of a group a arbovirus (sindbis) simple method for preparation of haemagglutinating arbo-a virus antigens from brains of suckling mice haelnaggtutination with arthropode-borne viruses and its inhibition by certain phosphotipids a method for production of arthropodborne viral haemagglutinins in tissue culture on the adsorption of australia antigen to a colloidal silica., and some possible applications hepatitis-free and stable human serum for intravenous therapy the isolation of an agent relatedto uukuniemi virus from norwegian ixodes ricinus ticks tick-borne viruses in norway uukuniemi group viruses isolated in norway runde virus", a coronaviruslike agent associated with seabirds, and ticks key: cord- -lzvtgwox authors: hasoksuz, m.; lathrop, s.; al-dubaib, m. a.; lewis, p.; saif, l. j. title: antigenic variation among bovine enteric coronaviruses (becv) and bovine respiratory coronaviruses (brcv) detected using monoclonal antibodies date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: lzvtgwox bovine coronavirus (bcv) causes neonatal calf diarrhea (cd) and is associated with winter dysentery (wd) in adult dairy cattle. it can also be isolated from the respiratory tracts of cattle entering feedlots. monoclonal antibodies (mabs) specific for the hemagglutinin esterase (he) and spike (s) surface proteins of bovine enteric coronavirus (becv) strains and two bovine respiratory coronavirus (brcv) strains were tested against becv strains and recently isolated brcv strains, in order to characterize the antigenicity of bcv strains with varied tissue tropisms. all mabs had high immunofluorescence (if) titers against becv and brcv strains, indicative of conserved cross-reactive epitopes. in hemagglutination inhibition (hi) tests, the s-mabs were more broadly reactive than he-mabs. the brcv and cd mabs were more broadly reactive in hi than the wd mabs. the ha activity of the mebus vaccine cd strain was not inhibited by any of the mabs tested. the hi activity of brcv strain r was unique among the brcv isolates. in virus neutralization assays, mabs to the brcv strain r neutralized all becv strains tested. antigenic variation exists among both becv and brcv strains, but it cannot be attributed soley to the clinical origin of the strain. bovine coronavirus (bcv) causes calf diarrhea (cd), is associated with winter dysentery (wd) in adult dairy cattle, and may contribute to respiratory disease in feedlot cattle [ , ] . there are several studies of the antigenicity of becv isolates using polyclonal or monoclonal antibody [ , , , ] , but there are no studies of brcv isolates using mabs. although some biologic and antigenic differences between becv strains have been detected, it is unclear if these differences are distinctive between becv (cd, wd) and brcv strains, and if such differences can be used to differentiate and identify the sources of individual strains. in this investigation, mabs to brcv strain r , mabs to the cd strain db , and mabs to the wd strain dba were tested by if, hi and vn to determine the antigenic relatedness of becv and brcv strains. the becv strains (tables , ) were plaque isolated or cloned by limiting dilution as described previously [ , ] . the respiratory strains (tables , ) were isolated in hrt- cells from cattle in ohio feedlots from - and were cloned by limiting dilution [ , ] . monoclonal antibodies to cd strain db , wd strain dba, and brcv strain were produced as described previously [ , , ] . briefly, female balb/c mice were given an intraperitoneal (ip) injection of . ml of inactivated and purified bcv strains ( g protein/mouse). mice with if titers to bcv ranging from , - , and hi titers of ≥ were used for mab production. the hybridoma supernatants were screened by if, then by fluorescent focus neutralizaiton (ffn) and hi tests against homologous virus strains. the if and ffn tests were performed using hrt- cell cultures grown in well microplates as described previously [ ] . the cd, wd and brcv hybridomas which were positive for ffn and hi antibodies to bcv were used to produce ascites by injection of the cloned ( - times) hybridoma cells into pristane-primed mice [ , , ] . the mice were injected with . ml of hybridoma cells ( × cells/ml) and - days later ascites were collected. the isotypes and subisotypes of the mabs were determined by immunodiffusion assay of hybridoma supernatant fluids with monospecific anti-mouse immunoglobulin sera (icn biomedicals, aurora, oh) and are summarized in tables and . the hemagglutination inhibition (hi) test was performed using -well ubottom plates [ ] . monoclonal antibodies to brcv and becv were serially diluted -fold with veronal buffered saline (vbs) and mixed with the same volume of ha units of purified bcv [ ] and incubated at • c for h. after incubation, a % mouse erythrocyte suspension was added and incubated for h at • c. hi titers were expressed as the reciprocal of the highest dilution of mab which completely inhibited ha (pellet formation). hi titers of ≥ are indicated by a plus (+) in table , while a lack of hi (titer of < ) is indicated by a dash (−). two of the cd strains of becv, including the mebus vaccine strain, failed to react with any of the mabs tested by hi ( table ). the third cd strain, db , was the most broadly reactive of the becv strains tested, as its hemagglutination was inhibited by of the mabs tested. among the wd strains, each strain reacted with only one classification of mab (cd, wd or brcv). among the dba mabs, wd- , directed against the s protein was more reactive than either of the mabs directed against the he protein. brcv strains r and r shared similar hi patterns when tested with all of the mabs. brcv strains r and r reacted similarly with of the mabs, but differed when tested against of the brcv mabs. all of the mabs, from both enteric and respiratory strains, failed to inhibit the hemagglutination caused by brcv strain r . the hi results showed that for the dba mabs, the s-mabs were more broadly reactive in hi than the he-mabs (table ) . because the s protein is more efficient than the he protein in hemagglutination [ ] , it follows that the s-mabs would be more efficient in inhibiting hemagglutinaiton than the he-mabs. the brcv and cd mabs were more broadly reactive in hi than the wd mabs. a cpe reduction virus neutralization (vn) test was performed using hrt- cell cultures grown in -well microplates as described previously [ ] . briefly, serial -fold dilutions of mabs were mixed with the same volume of virus suspensions containing tcid / . ml and then incubated at • c for min. four wells of hrt- cells were inoculated with . ml of each virus-serum mixture and incubated for - days at • c. neutralizing antibody titers of mabs were expressed as the reciprocal of the highest dilution that inhibited % of the cpe. vn titers of ≥ are indicated by a plus (+) in table , while a lack of vn titer (< ) is indicated by a dash (−). the mabs tested were more broadly reactive in vn tests ( table ) than in hi tests (table ) . whereas the mebus vaccine and sdc calf diarrhea strains failed to react with any of the mabs in hi testing, both were neutralized by mabs directed against both db cd and r brcv strains. additionally, the mebus cd strain was also neutralized by mabs directed against the dba wd becv strain. the vn reactivity patterns of the mebus cd strain and the dba wd strain were identical to one another. the cd strain db had a very similar pattern, varying only in regard to its lack of neutralizaiton by the wd- mab. the brcv mabs tested in vn successfully neutralized all bcv strains, both enteric and respiratory. five of the brcv strains were neutralized by mabs directed against the db cd strain, and all cd strains were neutralized by both brcv mabs. brcv strain r is unique, as it was in hi testing, failing to react with any of the cd or wd mabs. based on hi and vn testing with mabs, we found that antigenic and biologic variation exists among bcv strains, but that this variation was not always related to the clinical source of the isolates. this is in agreement with previous studies using polyclonal antisera and becv isolates [ , , , , , ] . tsunemitsu et al. [ ] tested bcv strains isolated from nasal swabs and feces of calves and found them to be indistinguishable when tested by iem, ha, if, and vn, using hyperimmune serum. tsunemitsu et al. [ ] determined the hi and vn titers of polyclonal antibodies against various becv strains and found that the reactivity patterns for these strains could not be grouped according to their clinical origin (cd or wd). using the same isolates, but testing them with the mabs produced in this study, rather than polyclonal hyperimmune antiserum [ ] , similar hi patterns were found, which could not be predicted based on clinical origin. reynolds et al. [ ] found that hyperimmune antiserum to three different bcv isolates ( respiratory, enteric) showed vn with the homologous and heterologous isolates and significant cross-reactions with eight other isolates, which were of intestinal and respiratory origin. using polyclonal antiserum and mabs directed against the s protein of the mebus cd strain, michaud and dea [ ] found that wd isolates from quebec were all closely related, using polyclonal antibodies, but could be classified into distinct antigenic subgroups based on their reactivity with the s-mabs of the mebus strain in vn and hi tests. this finding is in agreement with the results of the present and our previous study [ ] : becv isolates were closely related using polyclonal antiserum, but could be classified into to antigenic subgroup based on vn and hi test results. with mabs, milane et al. [ ] indicated that variations (unrelated to clinical source) affecting the antigenic determinant of the he protein occurred among the becv strains isolated in quebec and that these strains were grouped differently by their mab hi titers from the prototype mebus strain. similarly, in the present study, the ha activity of the mebus strain was not inhibited by any of the mabs tested. the brcv isolate r was unique among the respiratory isolates when tested against the becv mabs, a finding consistent between hi and vn tests and in testing with both mabs and hyperimmune sera [ ] . however, using the brcv-mabs, this strain was antigenically related to the other brcv strains when tested by hi and vn. differences in vn and hi test results are to be expected, as there was no correlation between these activities [ ] . disparate vn and hi results have been reported for mabs to the s and he proteins for the quebec bcv isolates [ ] , mabs to the s protein for the mebus strain [ ] and mabs to the he protein for the wd strain (bcq. ) [ ] . moreover, mabs were reactive with separate epitopes on the bcv he or s protein. the he protein contains three antigenic regions, a, b, and c. domain a is further subdivided into two epitopes, a and a , which react with neutralizing antibodies [ ] . the s protein has three antigenic regions, two (a, b) or (a, b, c) or which react with neutralizing antibodies [ , ] . in the present study, the db mabs, cd- and cd- reacted with a hemagglutinating epitope, and a third db mab (data not shown) reacted with a non-hemagglutinating epitope, although both epitopes were separately involved in vn activity. this was also true for most of the mabs, as they demonstrated vn activity with strains for which they had failed to inhibit hemagglutination. this finding is in agreement with e -ghorr, et al. [ ] that their mabs against the he protein were both neutralizing, but only one had hi activity. in addition, the r mab brcv- inhibited ha activity, but it had no neutralizing activity. this is in agreement with michaud and dea [ ] who reported that he-mabs to the mebus strain had hi activity, but no neutralizing activity. the results of this study indicate antigenic and biologic variation, as detected by mabs, exists among bcv isolates, but that this variability is not always related to the clinical source of the isolate. one brcv strain, r , and to some extent the mebus vaccine strain, represent unique bcv isolates, distinct from the other enteric and respiratory strains, based at least on one-way hi and vn reactivity patterns using mabs. cell culture propagation of coronavirus isolated from cows with winter dysentery monoclonal antibodies to bovine coronavirus: characteristics and topographical mapping of neutralizing epitopes on e and e glycoproteins monoclonal antibodies to bovine coronavirus glycoprotein e and e : demonstration of in vivo virus neutralizing activity a serological comparison of bovine coronavirus strains isolation of bovine respiratory coronavirus from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronavirus epidemiologic factors and isotypespecific antibody responses in serum and mucosal secretions of dairy calves with bovine coronavirus respiratory tract and enteric tract infection production and characterization of monoclonal antibodies against an avian group a rotavirus bovine coronavirus respiratory infections in feedlot cattle coronavirus infection of the bovine respiratory tract characterization of monoclonal antibodies to bovine enteric coronavirus and antigenic variation among the quebec isolates characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates studies on the relationship between coronavirus from the intestinal and respiratory tracts of calves winter dysentery in dairy herds: electron microscopic and serological evidence of an association with coronavirus infection enteropathogenic coronaviruses the s protein of bovine coronavirus is a hemagglutinin recognizing - -acetylated sialic acid as a receptor determinant antigenic variation among transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the s protein of tgev antigenic and biological comparisons of bovine coronaviruses derived from neonatal calf diarrhea and winter dysentry of adult cattle isolation of coronavirus from feces and nasal swabs of calves with diarrhea monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus the hemagglutinin/esterase glycoprotein of bovine virulent and avirulent coronavirus: sequence and functional comparison between strains we thank j, mccormick, k. gadfield and p. nielsen for technical assistance, and dr. d. hodgins and k. sestak for helpful discussions. we thank the american association of bovine practitioners for partial support of this research. received february , key: cord- - hmrt h authors: di martino, barbara; di profio, federica; melegari, irene; robetto, serena; di felice, elisabetta; orusa, riccardo; marsilio, fulvio title: molecular evidence of kobuviruses in free-ranging red foxes (vulpes vulpes) date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: hmrt h red foxes (vulpes vulpes) are susceptible to viral diseases of domestic carnivores. in this study, by screening rectal swabs collected from red foxes in italy, we identified kobuvirus rna in five samples. based on analysis of partial rdrp and full-length vp genes, all of the strains shared the highest identity with canine kobuviruses (cakvs) recently detected in the us, the uk and italy. these findings provide the first evidence of the circulation of these novel viruses in foxes. demonstrating that non-domestic carnivores are susceptible to kobuvirus infections. here we report the detection of kobu-like viruses in faecal samples obtained from italian red foxes (vulpes vulpes) that are genetically very similar to cakvs. between september and may , individual rectal swabs were collected from red foxes in northern italy (valle d'aosta and piemonte regions) submitted to the national reference center for wild animal diseases (cermas). the samples were obtained from animals of both genders ( females and males) found dead or shot during the regular hunting season. twelve foxes were under year of age, and were adults. fresh faecal samples were placed into sterile containers and stored at - °c until tested. rna was extracted from ll of % (wt/vol) faecal suspension using trizol ls (invitrogen, ltd, paisley, uk) procedure. the final rna pellet was resuspended in ll of rnase-free water and used directly in rt-pcr assays or stored at - °c. viral dna was extracted from the supernatants of faecal homogenates by boiling for min and chilling on ice as described previously [ ] . kobuvirus rna was detected 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 kobuviruses, following reaction conditions described previously [ ] . the samples were also tested by rt-pcr for canine coronaviruses (ccovs) [ ] and canine noroviruses (novs) [ , ] , and by pcr for canine parvovirus (cpv- ) [ ] . novel primers (vp rf-f, -gcgggcgaatcctt caac- , and vp rf-r, gcgacctttcggagcgcc- ) to amplify the complete vp gene were designed by visual inspection of an alignment containing the kobu-like sequences obtained in this study and the corresponding conserved regions of the cakvs and aivs available on the ncbi website. all of the amplicons were purified using a qiaquick gel extraction kit (qiagen gmbh, hilden, germany) and sequenced directly using bigdye terminator cycle chemistry and a dna analyzer (applied biosystems, foster city, ca). rt-pcr products for which no sequences were obtained by direct sequencing were cloned into the pcr . vector (invitrogen, ltd, paisley, uk). plasmid dna was purified using a miniprep kit (qiagen gmbh, hilden, germany); three clones per product were sequenced. basic local alignment search tool (blast; http://www.ncbi.nlm.nih.gov) and fasta (http://www. ebi.ac.uk/tools/sss/fasta/) with default values were used to find homologous hits. sequence editing and multiple alignments were performed with the bioedit software package, version . [ ] . a phylogenetic tree (neighbor joining and p-distance model) with bootstrap analysis ( , replicates) was constructed using the mega software package, version . [ ] . out of faecal specimens, five ( . %) were found to be positive for kobuvirus rna, and two ( . %) for ccov rna. canine novs and cpv- were not detected. kobuvirus was the single identified enteric virus in four specimens, while a mixed infection with both pathogens (ccov ? kobuvirus, n = ) was found in one sample. all of the samples were obtained from adult animals. none of the foxes that were positive for ccov or kobuvirus rna showed pathological lesions indicative of enteritis. by fasta and blast analysis of the partial d region amplified with the primers univ-kobu-f/univ-kobu-r, the five fox strains (genbank accession no. kf -kf ) shared . - . % nt sequence identity with each other and displayed the closest relatedness ( . - . % nt) with the cakvs previously found in the us, the uk and italy [ , , , ] . identity to feline kobuviruses [ ] ranged from . % to . %, while nt sequence identity to human aivs was . - . %. the full-length sequence of the vp gene was determined for each of the five kobuvirus strains (genbank accession no. kf -kf ). sequence analysis indicated that the fox kobuviruses were highly related to each other ( . - . % nt and . - % aa identities) and to the cakv strain uk/ (accession no. kc ) ( . - . % nt and - % aa identities) recently identified in a healthy dog in the uk [ ] . neighbor-joining phylogenetic analysis based on the vp sequences was carried out with a selection of strains representative of the genus kobuvirus, including murine kobuvirus [ ] , human aivs, wild-boar kobuvirus [ ] , the ferret kobuvirus strain mpkov [ ] , and the prototype strains porcine kobuvirus s- [ ] and bovine kobuvirus u [ ] . based on inspection of the tree (fig. ) , all of the fox kobuvirus sequences formed a tight cluster with the cakvs recently identified in the uk and in the us [ , , ] , sharing a common root with murine kobuvirus and human aivs. historical evidence shows that foxes are susceptible to viral diseases of domestic carnivores, including infections with canine distemper virus [ , ] , cpv- [ ] , canine adenoviruses [ , ] , ccov [ ] , and canine herpesvirus [ ] . in the present study, we detected kobuviruses in freeranging red foxes with a prevalence rate of . % ( / ). investigations based on the molecular analysis of the fulllength vp gene revealed that the five fox strains were most closely related to the cakvs ( . - % aa) recently identified in dogs [ , , ] . therefore, the fox kobuviruses detected in this study may be considered members of genotype cakv- within the species aichivirus a. in addition, this is the first study demonstrating the presence of ccovs in free-ranging red foxes in italy. although cakvs were found in both asymptomatic and diarrhoeic dogs, the pathogenic potential of these novel kobuviruses in carnivores remains to be elucidated. in this preliminary study, all of the cakv-like viruses detected were identified in adult foxes found dead or shot during the regular hunting season without apparent pathological enteric lesions. accordingly, it is unclear if these viruses may have a role in the aetiology of diarrhoea in foxes. future large-scale virologic investigations by screening faecal samples collected from both diarrhoeic and healthy animals are needed to assess the distribution and pathogenic potential of cakv-like viruses in foxes. in conclusion, this study provides direct evidence of the circulation of cakv-like viruses in red foxes, reinforcing the previous observations that kobuvirus infections are probably as frequent in wild animals as they are in domestic animals [ , ] . additional studies are warranted in order to determine the significance of these findings and to evaluate the epidemiological and clinical importance of these novel viruses in wild carnivores. furthermore, as fox kobuviruses are newly recognized viruses and information on their diversity is still limited, a definitive characterization should rely on sequence analysis of the complete genome. fig. neighbor-joining phylogenetic tree based on the vp gene ( bp) of the kobuvirus strains detected in red foxes. the tree was generated using the neighbor-joining method and p-distance correction, supplying a statistical support with bootstrapping of replicates. the scale bar indicates nucleotide substitutions per site. the markers indicate the sequences detected in this study ratification vote on taxonomic proposals to the international committee on taxonomy of viruses prevalence and genetic diversity of aichi virus strains in stool samples from community and hospitalized patients molecular epidemiology of canine adenovirus type and type in free-ranging red foxes (vulpes vulpes) in italy antigenic analysis of canine parvovirus strains isolated in italy phylogeny and prevalence of kobuviruses in dogs and cats in the uk detection and genetic characterization of feline kobuviruses a real-time pcr assay for rapid detection and quantitation of canine parvovirus type in the feces of dogs canine kobuviruses in diarrhoeic dogs in italy bioedit: a user-friendly biological sequence alignment and analysis program for windows / /nt 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 mega : integrated software for molecular evolutionary genetics analysis and sequence alignment viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses canine distemper epizootic among red foxes novel norovirus in dogs with diarrhea the fecal viral flora of wild rodents development of a nested pcr assay for the detection of canine coronavirus 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 porcine kobuvirus in wild boars (sus scrofa) metagenomic analysis of the ferret fecal viral flora survey on viral pathogens in wild red foxes (vulpes vulpes) in germany with special emphasis on parvoviruses and analysis of a dna sequence from a red fox parvovirus rational optimization of generic primers used for norwalk-like virus detection by reverse transcriptase polymerase chain reaction 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 detection of canine coronaviruses genotype i and ii in raised canidae animals in china acknowledgments this work was financed by grants from the university of teramo, italy, and from the italian ministry of university and research. conflict of interest 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- -wyzb v a authors: forsyth, m.; al-nakib, w.; chadwick, p.; stanway, g.; hughes, p. j.; leckie, g.; almond, j. w.; tyrrell, d. a. j. title: rhinovirus detection using probes from the ′ and ′ end of the genome date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: wyzb v a this study investigated the abilities of cdna probes from the ′ and ′ ends of the genome of human rhinoviruses (hrv-) , , and b to detect rna from rhinovirus serotypes. the results show that probes from the ′ end of the genomes of hrv- , , and b detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. in contrast, all the ′ end probes were specific for the homologous virus. however, along hrv- probe detected a large number of serotypes. it was concluded that such cdna probes would not detect all serotypes with equal efficiency. synthetic oligonucleotides corresponding to short but highly conserved regions in the ′ non coding region may overcome this problem. rhinoviruses are the major causative agents of the common cold [ ] . in the majority of healthy individuals, the infection results in a short illness of some - days duration characterized by rhinorrhoea, nasal obstruction, sore throat and pharyngitis [ ] . however, in immunocompromised individuals particularly children and in patients with obstructive airways disease, rhinovirus infection may result in more serious lower respiratory tract involvement [ , ] . furthermore, recent community studies in michigan, u.s.a., suggested that rhinoviruses can be isolated from up to percent of adults (over years of age) with lower respiratory tract involvement [ ] . in these individuals the median duration of illness was as long as weeks [ ] . we have recently shown that a new synthetic anti-rhinovirus agent, r , can sucessfully suppress illness in volunteers challenged with a rhinovirus [ ] . it is anticipated that with further progress in the field it may be possible to treat these infections. however, rapid virus identification prior to treatment would be essential since these antivirals are specific for rhinoviruses. until recently, rhinoviruses could only be identified by growth in a sensitive cell culture. such procedures are time consuming, labour intensive and require considerable expertise. although, it is now possible to detect rhinovirus antigens directly in nasal washings using immunologically based methods such as elisa [ ] , the diversity of serotypes, recently estimated to be around [ ] makes efficient detection of all serotypes difficult. we have therefore, attempted to overcome this problem by developing procedures based on r n a detection. a previous study has shown that a c d n a probe from the ' non-coding region of hrv- detects . % of the rhinoviruses investigated, but the sensitivity of detection was variable and presumably depended on the degree of genomic homology of particular serotypes with hrv- [ ] . in this study we therefore increased the number of probes used to include the ' ends of hrv- , b and and the ' ends of h r v - b [ ] and [leckie et al., in prep.] . the aim was to find a probe that would hybridise with r n a from various serotypes with equal efficiency and we therefore studied the reactions of the new probes with r n a from rhinovirus serotypes. stocks of rhinoviruses including animal rhinoviruses such as the calf rhinovirus sdi and bovine rhinovirus ec and other control viruses, namely influenza a and b (flu a and b), coronavirus e and coxsackie a (coxa ) were prepared as previously described [ ] . each stock was titrated in microtitre plates and titres expressed as tcids /ml. table shows the final titres of the virus stocks as used in this study. viral rna was extracted using the method of rotbart et al. [ , ] . briefly, . ml of each virus preparation was mixed with an equal volume of a : mixture of x ssc-- % formaldehyde. the mixture was then spotted onto nitrocellulose filters that had been pre-soaked in x ssc as described earlier [ ] . details of the methods used to prepare and label probes used in this study have been described previously [ , ] . briefly, probes were produced from m templates containing rhinovirus cdna cloned in the appropriate orientation. the hrv- ' construct contained nucleotides - and was produced by cutting a recombinant plasmid with psti and bglii and ligating into the m mpl psti and bamhi sites. the corresponding ' probe comprised positions - contained within an hpai fragment ligated into m cut with sinai. the hrv- b probes represented positions - ( ') located within a psti hindiii fragment which was ligated into these sites of m and - ( ') located in a psti fragment. the hrv- ' end probe was prepared from a . kb cdna clone of hrv- designated prg . a base pair fragment representing nucleotides - of the hrv- sequence (unpublished) was subcloned into m mp in the positive sense orientation. both of the ' end probes used in this study were prepared from a . kb hrv- cdna clone pr covering the ' region of the genome. the short ' end probe was prepared from a base pair fragment representing nucleotides - . the total length of the hrv- genome is excluding poly a tail. the long ' end probe was prepared from a base pair fragment from pr covering nucleotides - . in both cases the fragments were subcloned into the phage vector m mp in the positive sense orientation. the templates were used to produce radioactive cdna probes complementary to viral sense rna by extension of an m universal primer in a reaction performed by the klenow fragment of dna polymerase . annealing of the primer/template was achieved by boiling together ( min) in a mixture ( lal) comprising primer ( ng), template ( lag), mm tris-hc , ph . , and . mm mgci . the solution was allowed to cool to room temperature and to it was added dgtp, dctp dttp (to a final concentration of . ram), p_ datp ( laci) klenow fragment ( units) and water to give a final volume of ~tl. the reaction took place at room temperature for minutes after which the radioactive dna was separated from unincorporated nucleotides by passage through a sephadex g column. the probes were hybridized with viral rna as described previously [ , ] . the strength of the hybridization signals was assessed visually by two independent observes and the signal was classified as very strong (+ + + +), strong (+ + + ), good (+ +), positive (+), weak ( -), or no signal ( ). comparison of the hybridization results with the titres of the virus stocks (table ) shows that generally there is no direct relationship between the titres and the strength of the hybridization reaction. for example, the hrv- probe hybridised very strongly (+ + + +) with hrv- even though the titre was low (< tcids /ml) but only weakly with hrv- a ( + ) which had one of the highest titres of the viruses tested (> tcids /ml) ( table ) . it therefore appears that the efficiency of detection is more directly related to other factors, the most important of which is probably the degree of rna homology between the rna of the different rhinoviruses. table shows the strength of the signal observed using the various viruses and seven probes. it can be seen that the reactions varied greatly in intensity. thus, the hrv- , ' end probe gave a very strong signal (+ + + +) when hybridized with rna from hrv- , , and and a strong signal (+ + +) when reacted with rna from hrv- , , , , , , and . similarly, a ' end probe from hrv- gave a very strong signal (+ + + +) with rna from hrv- , , and . the ' end probe from hrv- b hybridized very strongly (signal + + + + ) with rna from hrv- and and strongly (+ + + ) with rna from hrv- a, , and . in contrast to the ' end probes, those from the ' end ( , , and nucleofides in length for hrv- , b, and , respectively) detected only the homologous virus in the conditions of the assay. however, the longer probe ( nucleofides in length) from hrv- , detected many more viruses (table ) . indeed, this probe hybridized very strongly (+ + + + ) with rna from hrv- , , , , , , and and reacted strongly (signal + + +) with rna from hrv- , , , , and suggesting that these viruses are closely related to hrv- in the ' end region of the genome and perhaps reflects the conservation of the polymerase sequences among these viruses. both ' and ' end probes from hrv- , - , and - b reacted with their respective viruses very strongly (+ + + + ). table shows the percentage of rhinovirus serotypes detected by each probe according to the strength o f the hybridization signal. thus . , , and . % of viruses investigated were detected (signal > + ) by hrv- , , and b, ' end probe, respectively, while % were detected by the long ' end hrv- probe. similarly, . , . , and . % of rhinoviruses gave a good hybridization signal ( > + + ) with ' end probes from hrv- , , and b, respectively, while . % gave a similar signal with the long hrv- , ' end probe. as can be seen from table , none of the control respiratory viruses such as influenza a, b, and coronavirus e, gave positive hybridization signals in any experiments during this study. coxsackie a , gave a positive signal with hrv- ' end probe suggesting some genomic homology with hrv- . these hybridization tests were repeated three or more times and the results were shown to be reproducible. the data suggest that c d n a hybridization with different probes show a different relationship between rhinovirus serotypes from that based an other properties. for example, hrv- which shares the same cellular receptor as hrv- (both are included in the major receptor group) [ , ] reacted more strongly (signal + + + ) with the ' end probe from hrv- b, a serotype in the minor receptor group than with hrv- (signal +). similarly, hrv- which shares the same cellular receptor as hrv- b (both are included in the minor receptor group) [ , ] reacted more strongly (signal + + + ) with the ' end probe from hrv- , a serotype from the major receptor group, than with hrv- b (signal +). moreover, this relationship is also different from that based antigenic crossreactivity [ ] . it is interesting to note that r n a from hrv- hybridized extremely well (signal + + + +) with both hrv- and b ' end probes suggesting a strong genomic homology between hrv- and these two viruses. similarly, r n a from hrv- reacted very well with the ' end probe from both hrv- and b indicating a close genomic relationship between hrv- and these two serotypes in the ' end. furthermore, rna from hrv- and hybridized extremely well with both ' and ' end probes from hrv- implying that these viruses have strong genomic homology with hrv- in both ends of the genome. in contrast, rna from hrv- , t, , and (signal < +) and and (signal + to -) did not hybridize well with any of the probes investigated probably indicating that these viruses are more divergent from hrv- , , and lb. the findings of this study are that probes from the ' end of the genome of rhinoviruses detect a large number of rhinoviruses, although the detection rate is variable and apparently depends on the strength of genornic homology among the different serotypes. in contrast, probes from the ' end of the genome (of some nucleotides) of hrv- and b detected only the homologous virus under the hybridization conditions of the assay. however, a similar size probe from the ' end of hrv- detected many more serotypes. in contrast, a shorter probe ( nucleotides in length) also from the ' end of hrv- , detected only the homologous virus, thus indicating that the detection rate is highly influenced by probe length. both ' and ' end probes detected the homologous viruses with equal efficiency. it was interesting to note that the ' end hrv- probe was more efficient than the other probes in detecting a larger number of rhinoviruses. this is somewhat surprising since comparative sequence analysis indicates that hrv- is relatively diverse from the majority of rhinoviruses studied. it might therefore be thought that probes from hrv- b and hrv- , which are more typical rhinoviruses, would prove to give a higher detection rate. the data presented in this paper are interesting in that they show that cdna probes are unlikely to be useful in detecting all rhinoviruses with equal efficiency despite an earlier prediction that the ' end non-coding region was likely to be relatively highly conserved throughout the rhinovirus genus [ ] . these results are therefore in agreement with our earlier findings with the hrv- probe [ ] and show that although there is considerable homology in the ' end noncoding region of many rhinoviruses it is still not sufficient for a probe prepared from this region to detect all the different rhinovirus serotypes with equal efficiency. furthermore, in tests on clinical material, both the identity of the infecting serotype and its concentration in nasal secretion would vary widely. in this study there were great variations in the signal given by the different serotypes even though the titres of virus used were usually greater than tcids /ml which is much higher than that normally found in nasal washing (often < l tcids /ml). recent work with synthetic oligonucleotides corresponding to short but highly conserved regions in the ' end non-coding region of the rhinovirus genome [ ] suggests that such probes will detect all rhinovirus serotypes with equal efficiency [ ] . further studies are in progress. many rhinovirus serotypes share the same cellular receptor detection of human rhinoviruses and their molecular relationship using cdna probes rhinovirus detection by cdna: rna hybridization common cold viruses--rhinoviruses suppression of colds in human volunteers challenged with rhinoviruses by a new synthetic drug (r ) synthetic oligonucleotides as diagnostic probes for rhinoviruses isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses antigenic grouping ofg rhinovirus serotypes the common cold: control? direct detection of rhinoviruses by an enzyme-linked immunosorbent assay provocation of airflow limitation by viral infection: implication for treatment a collaborative report: rhinovirus---extension of the numbering system from to the nucleotide sequence of human rhinovirus b: molecular relationships within the rhinovirus genus the association of rhinoviruses with lower respiratory tract disease in hospitalized patients rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes use of subgenomic poliovirus dna hybridization probes to detect the major subgroups of enteroviruses factors affecting the detection of enteroviruses in cerebrospinal fluid with coxsackievirus b and poliovirus i cdna probes rhinovirus detection using cdna probes the complete nucleotide sequence of common cold virus: human rhinovirus dr. glyn stanway is supported by a grant from the medical research council number g ca. key: cord- - tok mqk authors: nanda, s. k.; johnson, r. f.; liu, q.; leibowitz, j. l. title: mitochondrial hsp , hsp , and hsp bind to the ′ untranslated region of the murine hepatitis virus genome date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: tok mqk we have previously shown that mitochondrial-aconitase binds specifically to the ′ terminal nucleotides of the murine hepatitis virus (mhv) rna along with three additional proteins of , and kda to form a stable rna-protein complex. supershift and western blot assays have identified these three proteins as mitochondrial hsp (mthsp ), hsp , and hsp . a series of co-immunoprecipitation assays have established that these four mhv rna binding proteins are associated, even in the absence of mhv rna. however, the presence of a synthetic rna containing the sequence bound by these four proteins does increase the amount of co-precipitated protein, in particular the amount of hsp which is brought down with antibodies directed against hsp and mthsp . we have provided evidence for the interaction of these four proteins with the ′ end region of mhv rna in infected cells by a series of immunoprecipitation rt-pcr assays. we believe it is likely that mhv rna interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mthsp , hsp , and hsp . murine hepatitis virus (mhv) is a prototypic member of the family coronaviridae, and contains a single-stranded, positive-sense rna genome approximately kb in length [ ] . viral proteins are translated from six to seven subgenomic mrnas as well as from the genome. the virus-specific subgenomic and genomic rnas make up a -coterminal nested-set [ , ] and contain a leader sequence of approximately nucleotides (nt) at the end [ , ] . coronaviruses perform their entire replication program in the cytoplasm of infected cells. following uncoating, coronaviruses express the largest known replicase polyproteins, which in turn are proteolytically processed to yield a large number of mature proteins including rna-dependent rna polymerase (rdrp) [ , ] . the rdrp, perhaps in association with host proteins, directs the synthesis of negative-sense rnas from the end of the viral genome. analysis of the structure of defective interfering (di) rnas indicated that approximately nucleotides (nt) at the terminus, and nt at the terminus are required for di rna replication in mhv-infected cells [ , ] . the cis-acting signals for the synthesis of negative-strand rna are contained within the last nucleotides plus the poly (a) tail at the end of the mhv genome [ ] . specific binding of host cellular proteins to two distinct sites within the -utr of mhv-jhm genomic rna has been reported [ ] . one site, the (+) protein binding element [ (+) ] , was mapped within the most nt of the genomic rna [ ] , the other site was mapped to nucleotides - upstream from the end of the viral genome [ ] . cytosolic protein extracts from murine cl- cells assayed using a probe containing the (+) element formed three rna-protein complexes, with the slowest migrating and presumably largest complex, complex , being the most abundant [ , ] . recently, we established that mitochondrial aconitase (m-apo-aconitase) binds specifically to the last nucleotides of the utr of mhv rna [ ] . however, the interaction of purified m-apo-aconitase by itself was not stable under electrophoretic conditions. four different proteins with molecular weights of kda (m-apo-aconitase), kda, kda and kda were required to form a complex with the last nucleotides of the mhv utr which was stable enough to survive electrophoresis. in this work we have identified the kda protein bound to the mhv protein binding element as mitochondrial hsp (mthsp ), the kda protein as hsp , and the kda protein as hsp . murine cl- cells were cultured in dulbecco's minimal essential medium supplemented with % fetal bovine serum at • c in a % co atmosphere [ ] . the origin and growth of the jhm strain of mhv (mhv-jhm) virus used in this study have been described previously [ ] . infected cell extracts were prepared from confluent cl- monolayers infected with mhv-jhm at a multiplicity of infection (m.o.i.) of pfu/cell. theatpase activity of the post-mitochondrial lysate was measured by determining the amount of free radioactive p i liberated by [γ- p]atp hydrolysis. the assays were done essentially as previously described [ ] except that the partially purified post-mitochondrial lysates (approximately µg total protein) were incubated at • c with purified m-apo-aconitase ( - µg, graciously supplied by m.c. kennedy, gannon university, erie, pa) and mhv-jhm utr rna in atpase buffer ( mm hepes, ph . , mm kcl, mm mgcl , . % np- , mm dtt, mm atp containing µci [γ- p]atp plus cocktails of protease and phosphatase inhibitors). at the times indicated, the reaction was stopped by the addition of µl of acid washed charcoal (sigma) ( . % in mm hcl and mm h po ) to bind free nucleotides. the samples were centrifuged at , rpm for min, and the radioactivity mthsp , hsp and hsp bind to the mhv -utr contained in the supernatant was determined by liquid scintillation counting. control reactions were carried out in the absence of purified m-apo-aconitase or by replacing the enzyme with bsa. all assays were performed in triplicate and mean values and standard deviations calculated. mouse monoclonal antibodies directed against mthsp , hsp and hsp were obtained from stressgen biotechnologies corp (victoria, b.c., canada) and affinity bioreagents inc. (golden, colorado). a rabbit antiserum against m-aconitase was provided by dr. richard s. eisenstein, university of wisconsin, madison [ ] . the monoclonal antibody directed against the mhv nucleocapsid gene, antibody - - , has been described [ ] . monoclonal antibodies directed against a t -tag and anti-sv t ag (ab- ) were purchased from novagen (madison, wi) and oncogene science (cambridge, ma) respectively. a mouse monoclonal antibody directed against phosphotyrosine (py ) was purchased from bd transduction laboratories (lexington, ky). in vitro transcription reactions were carried out with [α- p]utp to generate radiolabeled probes [ ] . after transcription, the radiolabeled rnas were purified through sep-pak light c cartridges (waters, milford, ma). the concentration of radiolabeled rna was measured spectrophotometrically. crude cytoplasmic lysates and post-mitochondrial lysates were prepared from cl- cells as previously described [ ] . rna-protein binding reactions were performed in a volume of µl as described previously [ ] . briefly, extracts containing - µg of total protein, to ng of one of the p-labeled mhv (+) rna probes, µg heparin (sigma), ng of poly(i)-poly(c) (sigma) and % glycerol were incubated at • c for min in binding buffer ( mm tris ph . , mm mgcl , mm dtt, mm kcl) and then digested at • c for min with rnase t (calbiochem). rna-protein complexes were subjected to nondenaturing polyacrylamide gel electrophoresis, dried, and autoradiographed as described [ ] . for supershift assays approximately µg of protein in the post-mitochondrial lysate was incubated either with . or µg of monoclonal antibody, or with buffer ( mm tris/hcl, ph . , and . % np- ) alone in a final volume of µl at • c for h. after the incubation the mixture was used for the standard rna binding assay. a biotinylated synthetic rna ( -aguaaaugaa ugaaguugaucauggccaauugg aaga- ) corresponding to nucleotides - at the end of the mhv genome (counting the first nucleotide upstream from the poly(a) tail as position ) was purchased from dharmacon research (boulder, co). aliquots of biotinylated rna were cleaved and deprotected as per the manufacturer's guidelines and bound to magnetic strepavidin beads (perseptive biosystems, farmingham, ma) as described previously [ ] . the beads were washed free of unbound rna with buffer b ( mm tris, ph . , mm mgcl , mm edta, mm dtt, % glycerol, mm pmsf, µg/ml leupeptin, µg/ml aprotinin . µg/ml pepstatin) and a preparation of mhv (+) rna binding proteins, partially purified by sequential ion exchange and heparin agarose binding steps [ ] , was added to the beads in the presence of heparin (sigma), poly(i)-poly(c) (sigma), and trna, at concentrations of µg/µl, ng/µl, and µg/µl, respectively. the binding reaction was incubated for h at • c and washed four times with buffer b. the bound proteins were eluted by boiling the beads in × sds loading buffer. s. k. nanda et al. samples (either immunoprecipitates or rna affinity purified proteins) were added to sdslysis buffer preheated to • c and boiled for min. proteins were separated on either % standard or - % gradient (w/v) sds-polyacrylamide gels and transferred to nitrocellulose membranes (biorad) using a semi-dry blotting procedure [ ] . membranes were blocked overnight in tbst ( mm tris-hcl, ph , mm nacl, . % (v/v) tween ), % (w/v) dried milk powder, incubated for h with primary antibodies in tbst, % (w/v) dried milk powder, washed in tbst, and incubated with a second antibody conjugated to horseradish peroxidase for h. membranes were developed using an ecl (enhanced chemiluminescence) detection kit (amersham pharmacia biotech, piscataway, nj) and visualized by exposure to biomax light film (kodak, rochester, ny). uv cross-linking was performed as described previously [ ] with the modification that after uv crosslinking and digestion with rnase a samples were immunoprecipitated and the labeled proteins resolved by sds-page. uninfected and infected cl- cells were lysed in ml of ice-cold lysis buffer containing mm tris-cl ph . , mm nacl, mm edta, mm sodium pyrophosphate, mm orthovandate, % np- , mm naf, µg/ml leupeptin, µg/ml pepstatin and mm phenylmethylsulphonyl fluoride. lysates were clarified by centrifugation at , × g for min. immunoprecipitation was carried out using dynabeads protein a (dynal biotech, lake success, ny) as per the manufacturer's recommendations. antibodies were bound to protein a immobilized on the magnetic beads and cross-linked using dmp (dimethyl pimelimidate) in . m triethanolamine ph . . antibodies cross-linked to the protein a magnetic beads were used to capture the antigens from the cl- cell lysates. immunoprecipitates were washed several times with pbs, extracted in × sds-buffer and then separated by sds-polyacrylamide gel electrophoresis and analyzed by western blotting. fifty microliters of dynabeads-protein a suspension per reaction were washed twice with . ml of rnase-free . m na-phosphate (pbs, ph . ) and resuspended in µl of pbs. approximately µg of each monoclonal antibody tested [ - - , directed against jhm strain n protein ( ) , anti-mitochondrial hsp , anti-hsp , anti-hsp , µl of anti-t -tag, anti-sv t ag] was added to magnetic beads, and rotated slowly for min at room temperature. the test tubes were then placed in a magnetight separation stand (novagen) for min and supernatants were removed. the beads were washed twice with . ml of rnasefree . m na-phosphate (ph . ) containing . % bsa. five hundred microliters of binding buffer [ mm tris (ph . ), mm kcl, mm mgcl , and mm dtt] used for rnase protection/gel mobility shift assays was used for the last wash and subsequently discarded. two hundred micrograms of mock-infected and mhv-jhm infected post-mitochondrial fractions were resuspended in binding buffer and added to the beads.antigen-antibody binding reactions were rotated for h at room temperature. the immunocomplexes bound to the beads were washed three times with binding buffer. proteins and protein-rna complexes binding to the beads were eluted in µl of mm tris (ph . ), % sds, and mm edta at • c for min. the eluted material was treated with µg of proteinase k ( mg/ml) at • c for min, extracted twice by phenol-chloroform and nucleic acids precipitated by ethanol in the presence of µg mthsp , hsp and hsp bind to the mhv -utr glycogen carrier. rna was pelleted by centrifugation and used as template for reverse transcription (rt). rt was performed using a mhv-jhm specific primer, spanning nt to at the end of jhm genome ( gtagtgccagatgggtta ), and superscript ii (invitrogen) at • c for h. synthesized cdna was used as a template for pcr. the positive sense primer for pcr was the same primer as used in rt reactions. the negative sense primer corresponds to the complementary sequence from nt to at the end of jhm genome ( gtgattcttccaattggc ). amplification was performed at • c for sec, • c for sec, • c for sec for cycles; at • c for sec, • c for sec, • c for sec for the next cycles, at • c for sec, • c for sec for the last cycles; followed by a min extension at • c. ten nanograms of the plasmid de was used as a positive control in pcr reactions. rt-pcr was also carried out to detect the abundant cellular gapdh mrna. the sequence of the rt primer for the synthesis of gapdh cdna is: -gccaaaagggtcatcatctc- . the rt primer also serves as the positive sense primer for pcr. the sequence of the negative sense primer for gapdh is: -gtagaggcagggatgatgttc- (primers were provided by dr. george davis, tamus-hsc). pcr reactions underwent cycles of amplification (each cycle consists of at • c for sec, • c for sec), followed by a min extension at • c. pcr products were resolved by agarose gel electrophoresis. we have recently demonstrated that m-apo-aconitase is one of the four proteins which bind to the last nucleotides upstream of the poly(a) tail of the mhv genome [ (+) protein binding element] as an rnp complex using gel shift/rnase protection and uv-cross-linking assays. in these assays the addition of a rnase t digestion step prior to electrophoresis increases both the resolution and sensitivity of gel shift assays by digesting unbound rna and rna nonspecifically bound to protein, and by decreasing the size and apparent heterogeneity of the rna-protein complexes [ ] . the digested rna runs as a smear with the dye front at the bottom of the gel, with the majority of the digested probe running out of the gel. three rna-protein complexes are detected by this assay. the largest complex, complex , is typically the most abundant complex detected, with the two faster migrating complexes being produced in variable amounts [ , ] . in the course of experiments investigating the binding of m-apo-aconitase to the (+) protein binding element, we determined that the addition of atp and purified m-apoaconitase to cytoplasmic lysates increased rna binding activity (fig. a , compare lanes and ), in gel shift/rnase protection assays. the formation of the largest complex, complex , was most dramatically affected (black arrow, fig. a ). it should be noted that the lysates used in these experiments contain a large amount of m-aconitase, only a small fraction of which is the apo-enzyme which binds mhv rna [ ] . the addition of atp to the binding reactions in the absence of added m-apo-aconitase produced only a small increase in rna binding activity (fig. a , lane ). when atp was omitted from the binding reaction (fig. a , lane ) no increase in rna binding activity was detected in the presence of m-apo-aconitase. these effects were consistently observed in multiple experiments. substituting bsa for m-apo-aconitase in these binding reactions resulted in no increase in rna binding activity (data not shown). these finding led us to generate a working hypothesis that the lysate might possess anatpase activity and thatatp hydrolysis generated energy for a conformational change in m-apo-aconitase which resulted in more efficient rna binding. we therefore analyzed rna binding activity in the presence of atp-γs, a non-cleavable analogue of atp. the substitution of atp-γs for atp did not stimulate rna binding activity (fig. a, to further investigate the role of atp in the rna binding reaction, we measured the ability of rna binding reactions containing m-apo-aconitase, atp, and cytoplasmic lysate to hydrolyze [γ- p]atp, as described in materials and methods. as shown in fig. b , atp was hydrolyzed in the complete binding reaction. only minor amounts of atpase activity were observed when purified m-apo-aconitase was omitted from the reaction or when it was replaced with bsa. similar results were obtained in multiple experiments. thus stimulation of rna binding activity and atpase activity were both dependent on the presence of cytoplasmic lysate, m-apo-aconitase, and atp. atpase activity was strictly dependent on the presence of mgcl in the reaction buffer, since no activity was detected in the absence of mgcl or in the presence of mm edta (not shown). thus cytoplasmic lysate exhibits a cation dependent atpase activity characteristic of members of the hsp family of chaperones [ ] . we have previously shown by northwestern blotting and affinity chromatography that in addition to m-apo-aconitase, three other proteins with molecular masses of approximately , , and kda bind to rna corresponding to the last nucleotides of the mhv genome [ ] . the presence of an atpase activity in cytoplasmic lysates which was stimulated by the addition of m-apo-aconitase and rna containing the (+) protein binding element, plus the association of hsp family members with proteins destined to be imported into mitochondria [ ] , suggested to us that the kda protein might be a member of the hsp family. to investigate this possibility we performed a "supershift" assay with antibodies to mthsp and hsp/hsc . these two monoclonal antibodies to related hsp family members do not cross-react. the antibody specific for mthsp produced a supershifted rnp complex ( fig. a, lane , gray arrow) . although the amount of the supershifted rna-protein complex was relatively small, it was quite specific; antibody to the closely related hsp/hsc protein failed to produce a supershifted band ( fig. a, lane ) as did cytoplasmic lysate in the absence of antibody ( fig. a, lane ) , although both contained rnaprotein complex (indicated by the black arrow). the relatively small fraction of rna-protein complexes which were supershifted suggests that an excess of mthsp was present in the binding reaction over that which bound to mhv rna in complex . alternatively, complex might be formed by a heterogeneous population of proteins and only those containing mthsp are supershifted, or the antibody used had a relatively low affinity for mthsp . labeled rna incubated with antibody to mthsp in the absence of cytoplasmic lysate did not produce any rna-protein complexes, only small rnase t digestion products were detected (fig. a, lane ) . similar experiments were attempted with an antisera to mitochondrial aconitase (kindly provided by dr. richard s. eisenstein, university of wisconsin, madison) but were unsuccessful due to large amounts of ribonuclease present in this rabbit polyclonal antisera (data not shown). identical experiments using a labeled rna containing an nucleotide mutation known to interfere with formation of the rna-protein complex [ ] , as expected, failed to contain either the shifted or supershifted complexes (not shown). mthsp undergoes tyrosine phosphorylation under stress conditions [ ] . it has been reported previously that infection with mhv- induces the tyrosine phosphorylation of cellular proteins [ ] ; thus it seemed logical to investigate the phosphorylation status of the mthsp proteins in the rnp complex. preincubation of the cytoplasmic lysate with anti-phosphotyrosine antibodies led to the presence of two supershifted rnp complexes with a corresponding decrease in the intensity of the unshifted rnp complex (fig. b, lane ) . these data suggest that at least one of the proteins binding to the mhv (+) protein binding element is tyrosine phosphorylated, possibly mthsp . hsp potentiates the function of the hsp family of protein both in vitro and in vivo [ ] . this suggested to us the possibility that the kda mhv (+) binding protein could be hsp . to investigate this hypothesis we performed a "supershift" assay with an antibody to hsp . as shown in fig. c , preincubation of crude post-mitochondrial lysate with anti-hsp antibody prior to our standard rnase protection/gel mobility shift assay resulted in the presence of two additional slowly migrating protected rnp complexes (lane ) compared to our standard assay (lane ). the result of the supershift assay suggested that the kda protein we had observed upon uv-crosslinking was hsp . the finding that two of three rna binding proteins which we had identified were hsp family members suggested to us that the kda protein might be hsp . a supershift assay with anti-hsp produced a supershifted band, as shown in fig. c , lane , confirming our suspicion that the kda protein was hsp . the finding that antibodies to hsp and hsp only supershifted a relatively small portion of rna-protein complex suggests that for these two chaperones, only a small fraction of these molecules participated in the formation of mhv rna-protein complexes. alternatively, complex might be formed by a heterogeneous population of proteins and only those containing hsp or hsp are supershifted, or the antibodies used had relatively low affinities. to confirm the results of our gel shift analyses, we employed two additional sets of immunologic assays. previously we showed that four proteins with molecular weights of kda (m-apo-aconitase), kda, kda and kda bound to an rna affinity reagent corresponding to the mhv (+) protein binding element and were eluted with sds-page sample buffer [ ] . mhv (+) rna binding proteins were affinity purified in the presence of trna, heparin and poly(i)-poly(c) to block non-specific binding to the affinity matrix as described in materials and methods, resolved by sds-page and electrophoretically transferred to membranes. immunoblot analysis of the material eluted from the specific rna affinity matrix with anti-mthsp (fig. a, lane ) or anti-hsp (lane ) antibodies demonstrated immunoreactive species with molecular masses of approximately kda and kda, respectively. these experiments confirmed the identity of the kda protein as mthsp and the kda protein as hsp . the second immunologic assay we employed was an immunoprecipitation assay after transferring label from a p-labeled rna probe containing the mhv (+) protein binding element to its cognate protein binding partners by uvcrosslinking. immunoprecipitation with anti-hsp pulled down four proteins protein binding element were partially purified and then subjected to affinity purification with a biotinylated rna immobilized on magnetic beads as described in materials and methods. bound proteins were eluted by boiling the beads in × sds-page loading buffer, resolved by sds-page ( - % gradient gel), and transferred to a nitrocellulose membrane. the membrane was probed with either anti-mthsp antibody ( ) or with anti-hsp antibody ( ) . the positions of molecular weight markers are indicated. b post-mitochondrial lysates were incubated with ng of p-labeled mhv (+) rna for min at • c and digested with limiting amounts of rnase t as described previously [ ] . the rna-protein complexes formed were irradiated with uv light for min. the samples were digested with rnase a ( µg/µl) and directly immunoprecipitated with anti-mthsp ( ) or irrelevant antibody ( ) (fig. b, lane ) . a parallel immunoprecipitation reaction with anti-mthsp antibody produced the identical result (fig. b, lane ). an irrelevant control polyclonal rabbit antibody (fig. b, lane ) failed to precipitate any labeled proteins.a control immunoprecipitation reaction with a monoclonal antibody to the hsp/hsc mthsp , hsp and hsp bind to the mhv -utr (cytoplasmic) protein also failed to precipitate any labeled proteins (not shown). immunoprecipitation of uv-cross linked rnp complexes with anti-hsp also pulled down all four proteins, although the signals were weak even after days of exposure (fig. b, lane ) as opposed to a one day exposure for the other immunoprecipitation experiments. the immunoprecipitation reactions were all performed after treating the rna-protein complex with rnase. the finding that each of these antibodies pulled down the same four proteins after extensive rnase a digestion suggests that these four proteins bound to the mhv (+) containing rna as a complex rather than as four proteins binding to different regions on this rna. this data is consistent with our previous glutaraldehyde crosslinking experiments which suggested that there were protein:protein interactions amongst all four proteins ( , , and kda) which made up rnp complex [ ] . a multi-protein rnp complex can form either by binding of a pre-formed multiprotein complex to an rna, or multiple proteins may recognize the rna separately, and only establish protein:protein contacts after they have bound to the rna. in order to clarify the situation we immunoprecipitated rnp complexes formed in uv-crosslinking reactions using unlabeled rna containing the mhv (+) protein binding element. these immunoprecipitates were compared to immunoprecipitates prepared in parallel from partially purified cytoplasmic lysate in the absence of rna. both sets of immunoprecipitates were subjected to immunoblot analysis. when we used anti-mthsp or anti-hsp to pull down the rnp complex or lysate in the absence of added rna, and blotted the immunoprecipitated material separated by sds-page with anti-hsp , hsp could be detected in both the cases, whereas samples immunoprecipitated with non-specific antibody did not contain hsp (fig. a) . however, the signal for hsp in immunoprecipitates with anti-hsp was considerably augmented by the addition of rna. when the immunoprecipitation experiments were carried out with anti-mthsp , anti-hsp , or anti-hsp , and the immunoprecipitated materials were blotted and probed with anti-m-aconitase, m-aconitase was detected irrespective of the presence of rna, but was not detected in immunoprecipitates with non-specific antibody (fig. b) . however, the signals for m-aconitase were greatly increased by the presence of rna in the immunoprecipitation reactions with anti-hsp and anti-hsp . thus the association of hsp with hsp and with m-aconitase is stimulated by the presence of mhv (+) containing rna. these data suggest that these proteins are recruited to an rna-protein complex. to search for the presence of specific mhv rna-protein complexes in cells rather than formed after the in vitro addition of synthetic rna probes we performed a series of immunoprecipitation-rt-pcr assays. mhv rna-protein complexes ( , , ) or with primers for the abundant mrna for gapdh ( , , ) . pcr products were resolved by electrophoresis in a . % agarose gel mthsp , hsp and hsp bind to the mhv -utr were immunoprecipitated from post-mitochondrial lysates prepared from mock infected and mhv-jhm infected cl- cells with various monoclonal antibodies and amplified by rt-pcr with primers directed against the untranslated region of the mhv genome. a monoclonal antibody to the mhv nucleocapsid (n) protein, a protein which is known to bind to mhv rnas, was used as a positive control. antibodies to mthsp , hsp , and hsp all co-precipitated mhv rna which could be amplified by rt-pcr (fig. a, lanes - ) . the assay appeared to be specific in that monoclonal antibodies to irrelevant antigens (lanes and ) did not bring down complexes containing mhv rna. to further investigate the specificity of this assay we performed a second set of immunoprecipitation reactions and attempted to detect mrna for the highly expressed house keeping gene gapdh as well as for mhv specific rnas (fig. b ). antibodies to mthsp , hsp , and hsp captured mhv rna (lanes , , ) , but failed to precipitate gapdh mrna (lanes , , ) . the relative amount of mhv rna captured by the three anti-hsp antibodies in this experiment correlates well with the immunoprecipitation uv-crosslinking experiment shown in fig. b . thus we have now demonstrated that mthsp , hsp , and hsp bind to mhv rnas present in infected cells. the untranslated regions of most positive stranded rna viruses interact with host factors during their replication. we have previously shown that m-aconitase binds specifically to the last nucleotides of the utr of mhv rna along with three other proteins with molecular weights of approximately kda, kda and kda to form an rnp complex [ ] . in this study, we have used supershift assays and western blot assays of rna affinity purified protein to identify the three additional components of this rna-protein complex as mthsp , hsp , and hsp . we have also demonstrated this four protein -rna complex in postmitochondrial extracts of mhv infected murine cells by immunoprecipitation of rna-protein complexes followed by rt-pcr assays. both m-aconitase and mthsp are nuclear encoded, posses mitochondrial targeting sequences, and are thought to translocate into mitochondria shortly after synthesis. mthsp plays an important role in importation of proteins into the mitochondrial matrix [ ] . however mthsp has been documented to be present in extra-mitochondrial locations by biochemical fractionation, immunofluorescent labeling and confocal microscopy, and immunoelectron microscopy [ , , ] . this protein is also known as a glucose response protein (grp ); a senescencerelated gene product, mortalin; and as a peptide binding protein, pbp . this compendium of names reflects the different intracellular compartments and functions which have been described for this protein in addition to its role in refolding and import of mitochondrial proteins. mammalian hsp s have a predominantly cytoplasmic distribution in the cell [ ] . although the functional hsp complex is predominantly localized to mitochondria in eukaryotes [ ] , detailed immunoelectron microscopy studies in a wide variety of cells and tissues show that - % of hsp immunoreactivity is present at discrete extra-mitochondrial sites, including unidentified cytoplasmic vesicles [ , , ] and secretory granules [ ] . mhv replicates in the cytoplasm, making it unlikely that mhv interacts with these proteins inside mitochondria. we believe that it is most likely that mhv rna interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mthsp , hsp , and hsp . the alternative scenario which would allow this interaction to occur, requires export of at least m-aconitase from mitochondria. hsp , hsp , and mthsp are all molecular chaperones. the folding of most newly synthesized proteins in the cell requires the interaction of a variety mthsp , hsp and hsp bind to the mhv -utr of protein cofactors known as molecular chaperones. these molecules have been identified in most cellular compartments of different organisms, and recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding [reviewed in [ ] ]. there are several families of chaperones; those most involved in protein folding are the -kda heat shock protein (hsp ; dnaj), -kda heat shock protein (hsp ; groel), and -kda heat shock protein (hsp ; dnak) families. hsp family members are regulated by hsp (dnaj or its homologs). these essential and ubiquitous partner proteins make up the dnaj family, with hsp (hdj- ) [ ] and hsdj (hdj- ) [ ] being the best studied human homologs. they function to increase atp turnover, facilitate chaperone action, and promote a more stable interaction with protein substrates [ , ] . the atpase activity we detected in partially purified lysates during rna binding reactions is consistent with the known properties of hsp 's atpase activity. it also suggests the possibility that mthsp is interacting with a dnaj domain containing partner, such as hsp during rna binding reactions. gel supershift assays with anti-phosphotyrosine antibodies (fig. b ) demonstrate that at least one of the proteins in the complex formed with mhv (+) containing rnas and m-aconitase, mthsp , hsp , and hsp is tyrosine phosphorylated. although m-aconitase, hsp , and mthsp contain predicted potential tyrosine phosphorylation sites, only mthsp has been experimentally demonstrated to undergo tyrosine phosphorylation [ , ] . thus mthsp is the protein most likely to be responsible for the supershift we observed with antiphosphotyrosine antibody. immunoprecipitation/western blot assays with anti-mthsp and anti-phosphotyrosine antibodies indicate that a portion of the mthsp is tyrosine phosphorylated, at least in mouse fibroblasts which contain mhv (+) rna binding activity (nanda and leibowitz, unpublished) . the phosphorylation/dephosphorylation status of many rna binding proteins plays a major role in terms of their interaction with rna. enzymatic treatment of crude lysates with alkaline phosphatases greatly inhibits their rna binding activity (nanda and leibowitz, unpublished) . this marked decrease in rna binding activity after dephosphorylation of the lysate argues in favor of an important role for mthsp tyrosine phosphorylation in the formation of the rnp complex. previously published northwestern blot data [ ] suggest that all four of the proteins are individually able to interact with mhv (+) containing rna. a series of co-immunoprecipitation assays (fig. ) have established that these four proteins are associated, even in the absence of rna. however, the data clearly indicated that the presence of rna enhanced the association of hsp with hsp and with m-aconitase. the addition of rna also enhanced the association of m-aconitase with hsp , but did not affect the association of mthsp with hsp or m-aconitase. one possible interpretation of these results is that those associations which we observed to be rna independent are due to the known associations of these proteins with each other (mthsp with m-aconitase, hsp s with hsp ), whereas the remainder are dependent on rna and the formation of an rna-protein complex. our data do not distinguish between two possibilities, formation of this four protein rnp complex from the separate proteins as opposed to rna binding to a pre-existing four protein complex. in either case our results clearly indicate that these four proteins form a complex with mhv-jhm (+) containing rna. we have established here that m-aconitase, mthsp , hsp and hsp interact with each other as well as with mhv rna. the nature of the interaction between hsp and hsp is complex. the interaction between the j domain and the hsp atpase domain is well established. an interaction between hsp and the peptide-binding domain of hsp also occurs in vitro. other hsp family members have been shown to bind to rna substrates and may guide the appropriate folding of these rnas and affect subsequent regulatory processes such as mrna stability and/or translation [ , , ] . thus it is quite possible that hsp potentiates the activity of mthsp and hsp , and thus stabilizes the interaction of m-aconitase with the rna. it is quite interesting that tomita and colleagues have recently reported that a yeast hsp family member, ydj p, is involved in forming brome mosaic virus replication complexes active in negative-strand rna synthesis, and suggested that a chaperone system involving ydj p participates in viral replicase folding or assembly into the active replication complex [ ] . it has recently been demonstrated that groel, the e. coli hsp homolog, can associate with lipid membranes while remaining functional as a protein folding chaperone [ ] . replicating mhv rna has been localized to early endosomal vesicles [ ] . it is possible that the association of hsp with the viral rna may contribute to its intracellular localization. this could occur if the interaction of hsp (likely in association with the other three proteins which make up the rna-protein complex we have identified) with the rna assists the intracellular translocation of rna-protein complexes [ ] . groel has also been suggested to be part of a protein complex that protects bacterial transcripts from rnase e-mediated degradation [ , ] . molecular chaperones rarely function alone; rather they function together in complex pathways [ ] . in mammalian cells, components of the mitochondria, namely grp (mthsp ) and hsp , a homolog of bacterial groel, were found to associate with each other and transiently interact with newly synthesized mitochondrial proteins [ ] . only a few partnerships between various hsp s and hsp s have been elucidated. the most well-studied chaperone partnership is one found in e. coli between dnak, a chaperone of the hsp class, and dnaj, an hsp . both genetic and biochemical evidence indicates a functional partnership between these two chaperones [ , , ] . however, a chaperone complex containing mthsp , hsp and hsp along with m-aconitase has not been reported previously. we have provided evidence for the existence of such a complex and for its interaction with the end region of mhv rna. the precise functional significance of this interaction for mhv replication is still unclear, although our previous work is consistent with its having an enhancing effect on viral rna stability [ ] . it is clear that a single hsp protein may interact with more than one hsp family member [ ] . our knowledge of the interactions between the various chaperones themselves, as well as with newly synthesized mthsp , hsp and hsp bind to the mhv -utr proteins or other chaperone target proteins is incomplete. it will be interesting to see how modulating the expression of these hsps affects mhv replication. the hsp and hsp chaperone machines dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver regulation of hsp function by a eukaryotic dnaj homolog intracellular processing of the n-terminal orf a proteins of the coronavirus mhv-a requires multiple proteolytic events identification of groel as a constituent of an mrna-protection complex in escherichia coli ) p , a member of the heat shock protein family, undergoes tyrosine phosphorylation in response to oxidative stress mammalian hsp and hsp proteins bind to rna motifs involved in mrna stability mammalian -kda stress protein (chaperonin homolog). identification, biochemical properties, and localization protein folding in vivo: unraveling complex pathways requirement for hsp in the mitochondrial matrix for translocation and folding of precursor proteins analysis of cis-acting sequences essential for coronavirus defective interfering rna replication the molecular biology of coronaviruses further characterization of mranas of mouse hepatitis virus: presence of common -end nucleotides cytoplasmic protein binds in vitro to a highly conserved sequence in the untranslated region of ferritin heavy-and light-subunit mrnas increased hepatotropism of mutants of mhv, strain jhm, selected with monoclonal antibodies the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm escherichia coli dnaj and grpe heat shock proteins jointly stimulate atpase activity of dnak identification of the cis-acting signal for minus-strand rna synthesis of a murine coronavirus: implications for the role of minus-strand rna in rna replication and transcription deletion mapping of a mouse hepatitis virus defective interfering rna reveals the requirement of an internal and discontinous sequence for replication mitochondrial hsp ssc : role in protein folding a specific host cellular protein binding element near the end of mouse hepatitis virus genomic rna the bovine papilloma virus e protein has atpase activity essential to viral dna replication and efficient transformation in cells murine hepatitis virus strain induces the macrophage prothrombinase fgl- through p mitogen-activated protein kinase activation subcellular localization of the hsp -homolog encoded by beet yellows closterovirus regulation of the heat-shock protein reaction cycle by the mammalian dnaj homolog, hsp cell-cycle dependent tyrosine phosphorylation on mortalin regulates its interaction with fibroblast growth factor- the two mammalian mitochondrial stress proteins, grp and hsp , transiently interact with newly synthesized mitochondrial proteins mitochondrial aconitase binds to the -untranslated region of the mouse hepatitis virus genome human cdna encoding dnaj protein homologue cloning of a cdna for heat-shock protein hsp , a human homologue of bacterial dnaj mammalian hsp /dnaj homologs: cloning of novel cdnas and a proposal for their classification and nomenclature extramitochondrial localization of mortalin/mthsp /pbp /grp interaction of erythropoietin rna binding protein with erythropoietin rna requires an association with heat shock protein mthsp , hsp and hsp bind to the mhv maturation of the polymerase polyprotein of the coronavirus mhv strain jhm involves a cascade of proteolytic processing events cloning and some novel characteristics of mitochondrial hsp from chinese hamster cells functional interaction of heat shock protein groel with an rnase e-like activity in escherichia coli immunoelectron microscopic localization of the -kda heat shock chaperonin protein (hsp ) in mammalian cells mitochondrial-matrix proteins at unexpected locations: are they exported? coronavirus mrna synthesis involves fusion of non-contiguous sequences sequence relationships between the genome and the intracellular rna species , , , and of mouse hepatitis virus strain a mutation of host dnaj homolog inhibits brome mosaic virus negative-strand rna synthesis evidence for a lipochaperonin: association of active protein-folding groesl oligomers with lipids can stabilize membranes under heat shock conditions electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication molecular chaperones in pancreatic tissue: the presence of cpn , cpn and hsp in distinct compartments along the secretory pathway of the acinar cells the nh -terminal amino acids of the escherichia coli dnaj protein stimulate the atpase activity of dnak and are sufficient for lambda replication a conserved motif at the end of mouse hepatits virus genomic rna required for host protein binding and viral rna replication specific binding of host cellular proteins to multiple sites within the end of mouse hepatitis virus genomic rna analysis of sequence-specific binding of rna to hsp and its various homologs indicates the involvement of n-and c-terminal interactions this work was supported by in part by national multiple sclerosis society grant rg -b- and a generous gift from the stearman family. we gratefully acknowledge dr. claire kennedy for providing us with purified m-aconitase and richard eisenstein for providing us with antim-aconitase antibody. the authors would like to thank dr. lori bernstein and dr. van wilson for their helpful comments and reading of the manuscript. we also thank elena belyavskaya for her help and encouragement. key: cord- -shwxhak authors: zhang, dan; mao, haiyan; lou, xiuyu; pan, junhang; yan, hao; tang, hongfeng; shu, yan; zhao, yun; cheng, xiaoli; tao, hong; zhang, yanjun; ma, xuejun title: clinical evaluation of a panel of multiplex quantitative real-time reverse transcription polymerase chain reaction assays for the detection of respiratory viruses associated with community-acquired pneumonia date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: shwxhak we developed a panel of multiplex quantitative real-time reverse transcription polymerase chain reaction (mqrt-pcr) assay consisting of seven internally controlled qrt-pcr assays to detect different respiratory viruses. we compared the new mqrt-pcr with a previously reported two-tube mrt-pcr assay using clinical sputum specimens. the mqrt-pcr assay performed comparably with the two-tube assay for most viruses, offering the advantages of quantitative analysis, easier performance, lower susceptibility to contamination, and shorter turnaround time in laboratories equipped with conventional real-time pcr instrumentation, and it could therefore be a valuable tool for routine surveillance of respiratory virus infections in china. wider application. first, an automated capillary electrophoresis device is needed, and many laboratories in the centers for disease control and prevention (cdc) in china do not have this specialized laboratory equipment. second, the size differences of some amplicons are not large enough for reliable judging of results by untrained staff. third, there is a need to open tubes after pcr amplification to analyze the pcr products by qiaxcel automatic electrophoresis, which significantly increases the risk of cross-contamination. given that the laboratories from all of the provincial and most municipal cdc laboratories in china have access to conventional real-time pcr instrumentation, in this study, we aimed to develop a multiplex quantitative real-time rt-pcr (mqrt-pcr) consisting of a panel of seven internally controlled qrt-pcr assays to detect different respiratory viruses: human coronavirus (cov) e, cov nl , cov oc , covhku , parainfluenza virus (piv) , piv , piv , piv , influenza virus (iv) types a and b, human respiratory syncytial virus (rsv) types a and b, human rhinovirus (hrv), human metapneumovirus (hmpv), human adenovirus (adv), and human bocavirus (hbov). clinical evaluation of the mqrt-pcr assay panel was conducted and compared with the two-tube assay in parallel. the mqrt-pcr assay panel consisted of seven separate assays with primer/probe sets covering human respiratory viruses: assay , iva and ivb; assay , cov oc and cov e; assay , cov nl and covhku ; assay , piv , piv , piv , and piv ; assay , hmpv and hbov; assay , hrv and adv; assay , rsva and rsvb. primers and probes were designed based on conserved target regions of the viral genomes, using primer premier software version . [ ] . for all primer and probe sequences, blast analysis was performed in silico to ensure specificity, and no cross-reactivity was observed. subsequently, in the initial development of the mqrt-pcr assay panel, the sensitivity of each assay for each virus type/subtype was thoroughly evaluated individually using serial tenfold dilutions ranging from to copies of in vitro rna transcripts (for rna viruses) or cloned plasmids (for dna viruses). the specificity of each assay was also extensively examined using in vitro rna transcripts and cloned plasmids as well as archived samples. no obvious cross-reaction was observed in the multiplex assay panel. the limit of detection was copies per reaction for piv , piv , rsva, hbov and adv, and copies per reaction for the other virus type/ subtypes. the individual reactions within each assay were distinguished using probes with different fluorophores. the sequences of the primers and probes and the target genes are listed in table . each assay also contained an endogenous rnase p (human genome ribonuclease p) gene as an internal control. each mqrt-pcr assay was carried out individually in a -μl reaction volume. rt-pcr buffer mix ( . µl) was combined with . µl of each primer/probe set and µl of × enzyme mix (agpath-idtm one-step rt-pcr kit; applied biosystems, waltham, massachusetts, usa). nucleic acid extracts ( µl) were added to seven wells of each primer/probe set. the following cycling conditions were used on a real-time pcr instrument (applied biosystems): min at °c, min at °c, cycles of s at °c and s at °c. threshold cycle (ct) values were determined by manually adjusting the fluorescence baseline to fall within the exponential phase of the amplification curves, and above any background signal. a test result was considered positive if a well-defined curve was obtained that crossed the threshold cycle within cycles. a mixture of plasmids was included accordingly as external positive controls in all runs to monitor assay performance. for comparison, the two-tube assay was performed using a one-step rt-pcr kit (qiagen, hilden, germany) in a -μl volume according to previously described protocols, and the products were analyzed using qiaxcel automatic electrophoresis equipment with a qiaxcel dna high resolution kit. if results were discordant between the mqrt-pcr assay panel and the two-tube assay, both tests were repeated concurrently to evaluate any problems relating to sample degradation or potential hands-on error. assignment of samples as having concordant or discordant results was based on the results of duplicate testing by both methods. if the results were still discordant, rt-pcr was then performed, followed by sequencing using a pair of primers from other literature (data not shown) [ , ] . a total of sputum specimens were obtained from patients with cap who were admitted to the children's hospital, zhejiang university school of medicine, china, between february and july . ages ranged from month to years, and ( . %) were under years old. trained staff collected sputum by adding ml of transport medium and pipeted the samples into separate aliquots, which were then stored at − °c. total rna/dna was extracted from µl of clinical sample using a qiaamp viral rna mini kit (qiagen). the extracts were eluted into µl of dnase-and rnase-free water and stored at − °c. all of the extracted nucleic acids were tested using both the mqrt-pcr assay panel and the two-tube assay. of samples tested, ( . %) and ( . %) were positive for one or more viruses by the mqrt-pcr assay panel and two-tube assay, respectively. piv , e and hku were not detected by either of methods. the overall agreement in detection of each pathogen between the two-tube assay and mqrt-pcr assay panel is shown in table . all the positive specimens with discordant results that were confirmed by mono-pcr and sequencing to be true positives are shown in table . we compared the performance of the mqrt-pcr assay panel with that of the two-tube assay and found that viruses in samples were detected only by the two-tube matrix iva-f gac cra tcc tgt cac ctc tga c iva-r ggg cat tyt gga caaakcgt cta cg iva-p fam-tgc agt cct cgc tca ctg ggc acg -bhq ivb nucleoprotein ivb-f ccc acc rag caa caa acg ivb-r cct tcc gac atc agc ttc hrv nd hrv assay and confirmed as true positives by sequencing [ , , ] . these viruses included hrv (n = ), piv (n = ), oc (n = ) and hmpv (n = ). the two-tube assay appeared to have superior sensitivity to the mqrt-pcr assay panel for detecting these four viruses. we further tested the performance of the mqrt-pcr panel using archived clinical samples that were positive for four viruses ( for hrv, and five each for piv , oc and hmpv). complete agreement with the two-tube assay results was achieved in nearly half of the samples (five for hrv, three for piv and oc , and two for hmpv). these discrepancies are most likely due to primer design, since we selected different conserved regions of the same genes or different gene targets for these two methods. for example, two primers (hmpv- and hmpv- ) were designed to amplify the l and n genes of hmpv in the two-tube assay, while the mqrt-pcr assay panel was designed for amplifying the f gene. the hrv-positive specimens that were positive in the two-tube assay but negative in the mqrt-pcr assay panel were from cases of hev infection. hrv and hev are closely related viruses. using this method, it is difficult to distinguish the ' untranslated regions of these groups of viruses, resulting in possible cross-reactivity. the primers of the mqrt-pcr assay panel were designed for the detection of the hn gene of piv , while the two-tube assay was designed for amplifying the ha gene. this discrepancy might lead to higher sensitivity of the two-tube assay for the detection of piv . in this study, the samples were not collected in winter (february to july). the prevalence of viruses can vary by geography and season. piv infections were most commonly detected in the spring and summer, although infections do occur year round. only four specimens tested positive for piv , and lower respiratory tract infection with piv and piv occurred less frequently than with piv and piv in previous studies [ ] [ ] [ ] [ ] . in the united states, e, oc and hku have been shown to follow different seasonal patterns, with outbreaks of e occurring in winter, oc in spring and autumn, and hku in summer [ , ] . oc and hku were detected in this study following the same seasonal patterns. although piv , e and nl were not detected in this study, we detected a few stock clinical samples previously identified as piv , e and nl using both the mqrt-pcr panel and two-tube assays. this demonstrated that both assays worked well for detection of these three viruses (data not shown). the two-tube assay requires that the tubes are opened after pcr amplification for qiaxcel automatic electrophoresis, whereas the mqrt-pcr assay panel is a closed system that effectively avoids contamination. by splitting a panel into seven multiplex reaction assays, individual mixes can be modified easily if needed without affecting the other mixes, which can allow for more-efficiently targeted testing based on epidemiological findings. another advantage of the mqrt-pcr assay panel is the saving of hands-on time. the assay panel requires - h to complete samples, including min to prepare the pcr mixture, while the two-tube assay usually takes - h. also, the mqrt-pcr assay panel is easily integrated into the workflow of laboratories using conventional real-time pcr platforms and can be performed after little training. in addition, the human rnasep gene as an internal control is readily detected to validate the rna extraction procedure and to prevent sampling and rt-pcr errors. notably, the mqrt-pcr assay panel allows for quantitative analysis, which should become increasingly helpful for epidemiological studies, for assessing clinical outcome according to virus type or viral load, and for evaluating antiviral agents [ ] . the mqrt-pcr assay had some limitations. first, the mqrt-pcr assay panel required seven parallel assays with only moderate throughput in each assay. it should be optimized to develop a single-tube mqrt-pcr to detect as many viruses as possible in one assay. second, for the detection of hrv, piv , oc and hmpv, the sensitivity of the mqrt-pcr assay panel was lower than that of the two-tube assay. competition for reagents might have decreased sensitivity for detection of these viruses in a multiplex format. third, our study was limited by the failure to collect samples throughout the entire year. increasing the sample size and year-round detection would be important improvements. in summary, this study demonstrates the clinical performance of an mqrt-pcr assay panel for the detection of viral respiratory pathogens. the mqrt-pcr assay panel is easy to use, and brings us closer to the mainstream adoption of molecular diagnostic testing. the further optimization, evaluation and widespread use of the mqrt-pcr assay will provide a valuable tool for routine surveillance of respiratory virus infection in china. of their right to keep information confidential. written informed consent was obtained from parents or caregivers. estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in countries: a systematic analysis for the global burden of disease study global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global burden of childhood pneumonia and diarrhoea pneumonia mortality among children under in china from to : an analysis from national surveillance system epidemiology of viral pneumonia causes of death in children younger than five years in china in : an updated analysis community-acquired pneumonia requiring hospitalization among u.s. children 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intergenic non-coding rnas expressed in the main developmental stages in drosophila melanogaster added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection comparison of automated microarray detection with real-time pcr assays for detection of respiratory viruses in specimens obtained from children comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses parainfluenza virus types , , and in pediatric patients with acute respiratory infections in beijing during epidemiology and clinical presentation of the four human parainfluenza virus types seasonal trends of human parainfluenza viral infections: united states outbreak of pneumonia in a long-term care facility: antecedent human parainfluenza virus infection may predispose to bacterial pneumonia epidemiology and clinical characteristics of human coronaviruses oc , e, nl , and hku : a study of hospitalized children with acute respiratory tract infection in guangzhou, china epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method we acknowledge the children's hospital, zhejiang university school of medicine, and the center for disease control and prevention of zhejiang province for providing sputum. key: cord- -u npu ng authors: attoui, h.; mohd jaafar, f.; biagini, p.; cantaloube, j.-f.; de micco, p.; murphy, f. a.; de lamballerie, x. title: genus coltivirus (family reoviridae): genomic and morphologic characterization of old world and new world viruses date: journal: arch virol doi: . /s sha: doc_id: cord_uid: u npu ng we report a genomic and morphologic study of the european eyach (eya) virus (genus coltivirus, family reoviridae) and a comparative analysis with the american colorado tick fever (ctf) virus (the type species of the genus). the previously established, but distant, antigenic relationship between these viruses was strengthened by genetic findings (presence of cognate genes, amino acid identity between and %, similar conserved terminal motifs, suspected read-through phenomenon in segment of both viruses) and by indistinguishable ultramicroscopic morphologies. moreover, putative constitutive modifying enzyme activities were suspected to be carried out by homologous viral proteins (rna-dependent rna polymerase, methyl/guanylyl transferase, ntpase). these findings, together with the comparative analysis to genomes of south-east asian isolates, support the recent classification of arboviruses with segments of dsrna within two distinct genera (genus coltivirus and genus seadornavirus) and raise interesting questions about the evolutionary origins of coltiviruses. the previously proposed hypothesis that eya virus was derived from an ancestral virus introduced in europe with the migration of lagomorphs from north-america, would imply a divergence date between american and european isolates of over million years ago (mya). this analysis allows for the first time to propose an evolutionary rate for virus dsrna genomes which was found to be in the order of (− ) to (− ) mutations/nt/year, a rate similar to that of dsdna genomes. for many years after the discovery of yellow fever virus [ ] and the confirmation of carlos finlay's earlier finding that the virus is transmitted by mosquitoes, arthropod-borne viruses (arboviruses) were considered to be a singular unique group. it was even supposed that the arboviruses formed a natural taxonomic group [ ] . however, in the s and s it became evident that the various arboviruses fall into several taxonomic groups [ ] . among the last of the arboviruses to be characterized and classified were those that are now members of the family reoviridae, that is the member viruses of the genera orbivirus, coltivirus and seadornavirus. this was despite the very early discovery of some member viruses: e.g., the first african horse sickness virus was discovered by m'fadyean in [ ] and colorado tick fever virus was discovered by florio in [ ] . it was not until - that the first biophysical, biochemical and morphological characterization of these viruses permitted their taxonomic placement [ , ] . members of the family reoviridae have dsrna genomes divided into to segments [ ] . the member viruses of the genus orbivirus (type species bluetongue virus) have segments, the members of the genus coltivirus (type species colorado tick fever virus) have segments, and the members of the recently identified genus seadornavirus (type species banna virus; previously coltivirus subgroup b) have segments [ , ] . among the member viruses of these taxa, the coltiviruses stand out because of their role as human pathogens. the new world virus, colorado tick fever virus, the etiologic agent of colorado tick fever, is widespread in the rocky mountain region of north america. its distribution matches that of its vector ticks: dermacentor andersoni [ ] , other dermacentor species, ixodes species, haemaphylis leporispalustris and otobius lagophilus [ ] . the antigenically related old world virus, eyach virus, was isolated in europe in [ , ] and indirectly incriminated in human neurological disease shortly thereafter [ ] . its distribution matches that of its vector ticks: ixodes ricinus and ixodes ventalloi. other members of the genus are the california hare coltivirus s - - from california and the salmon river virus from idaho. the first basis for distinguishing the coltiviruses from other viruses and relating them to each other was serologic cross-reactivity. the viruses exhibit no cross-reactivity with other viruses, but colorado tick fever virus and eyach virus cross-react in complement-fixation tests. the next basis for distinguishing the coltiviruses was virion morphology and morphogenesis. at the time of the first electron microscopic study of colorado tick fever virus in , only a few other arbovirus had been observed to have similar morphology, namely african horse sickness virus and bluetongue virus, two of the founding members of the genus orbivirus which was formalized in [ , , , ] . after eyach virus was isolated in , its morphology was studied cursorily, but until now little has been formally reported. since , the use of serology and electron microscopy for taxonomic purposes has declined as knowledge of the molecular biology and genetics of the viruses has advanced; at first this advance followed upon analysis of the nucleotide sequence of the smallest viral genome segments, then upon that of the entire genome [ ] . we present here the complete genomic sequence of the prototype strain of eyach virus and a comparative analysis of the genomic sequences of colorado tick fever virus, additional strains of eyach virus, and california hare coltivirus s - - . we also present here a morphologic and morphogenetic comparison of colorado tick fever virus and eyach virus as a way of extending molecular comparisons to at least on set of phenotypic characters. these analyses have allowed us to consider the close phylogenetic relationships among the member viruses of the genus coltivirus and the more distant relationships among the various genera of the family reoviridae. eyach virus (eya virus) strain fr and eya virus strain fr were kindly provided by c. chastel, as lyophilized infected mouse brain. eya virus strain gr and california hare coltivirus s - - (ctfv-s - - ) were kindly provided by n. karabatsos as frozen mouse brain. the colorado tick fever virus (ctf virus) strain florio was purchased at the american type culture collection. ctf virus strain florio, eya virus strain fr and eya virus strain fr were propagated by intracerebral inoculation of suckling mice. the mice were killed on day post-infection and brains were homogenized in pbs by vortexing with glass beads or prepared for electron microscopy (below). eya virus strain gr, obtained as frozen infected mouse brain, was used for genome extraction without further propagation. california hare coltivirus s - - was adapted to cell culture by passages in bhk- cells. the virus was then propagated in bhk- cells at • c under % co , using eagle's minimum essential medium supplemented with % fetal bovine serum and penicillin g, kanamycin, streptomycin, at iu/ml, g/ml and g/ml, respectively. dsrna extraction and preparation ctf virus strain florio and california hare coltivirus s - - : dsrnas were extracted from virus infected cell cultures using a guanidinium isothiocyanate procedure (rna now, biogentex), as described previously [ , ] . eya virus strain fr : dsrna was extracted from infected mouse brains after homogenization in phosphate buffered saline using the same rna now method. eya virus strains gr and fr : dsrna was extracted from individual mouse brains using the same method. because eya virus cannot be grown in cell culture, the sequence of its dsrna genome segments was obtained by a strategy based on specific stepwise elimination of dsrna segments (designated the "sequential segment subtraction method", " sm") [ ] . this involves rnase h hydrolysis of specific rna template segments, after their hybridization the extracted viral dsrnas from california hare coltivirus s - - , eya virus strain fr and eya virus strain gr were copied into cdna in the presence of random hexanucleotides and mumlv superscript reverse transcriptase (gibco brl) as described previously [ ] . pcr primers were designed from the sequences of the first, sixth, seventh and twelfth genome segments of ctf virus and eya virus (primer sequences are shown in table ). the protocols used for pcr amplification, cloning and sequencing were as described previously [ ] . the methods used for the cloning and sequencing of ctf virus strain florio has been described previously [ ] . the vp sequences of ctf virus strain florio and eya virus strain fr were compared with the sequences of putative rna-dependent rna polymerases of representative strains of viruses representing eight genera of the family reoviridae. genbank accession numbers are provided in table . all sequence alignments were generated by the clustal w program [ ] and the blast program contained in the dnatools program package (version . . , s.w. rasmussen). phylogenetic analyses were performed with the mega program [ ] using the p-distance determination algorithm. sequence relatedness was reported as percentage identity or percentage genetic distance. tree drawing was performed with the treeview program [ ] . comparison of sequences obtained in this study with those in databases was performed using the ncbi blast program (http://www .ncbi.nlm.gov/blast). the pfam program (http://www.sanger.ac.uk/pfam/search.shtml) was used to search for previously described protein family-domains. the motif program (http://www.motif.genome.ad.jp) was used to analyse theoretical protein sequences for the presence of known functional amino acid motifs. thin section electron microscopy brains of suckling mice infected with ctf virus strain florio were randomly cut into mm blocks and processed for sectioning. tissue was fixed for min in . % glutaraldehyde, postfixed for min in % phosphate-buffered osmium tetroxide, dehydrated in a graded ethanol series, and embedded in an araldite-epon mixture [ ] . scraped cell monolayers were pelleted by centrifugation at g for min and thereafter processed as brain tissue except for a reduction of glutaraldehyde fixation time to min. sections were cut with glass knives and stained with lead citrate [ ] . calibration of magnifications was made from photographs of a , line per inch diffraction grating replica (ladd research industries, inc., burlington, vermont, usa) taken at each magnification step used. negative contrast electron microscopy carbon-coated grids prepared according to the technique of simpson and hauser [ ] were rendered hydrophilic by exposure to ultraviolet irradiation. very small quantities of infected mouse brain tissue were teased apart in large drops of % sodium silicotungstate stain, ph . [ ] . grids were floated on the drops and dried by touching to filter paper. cell cultures were differentially centrifuged prior to negative staining. low speed pellets ( g, min) and ultracentrifuge pellets ( , g, h) were dispersed in drops of stain and treated in the same way as brain tissue. magnification calibration was also performed at the kilovoltage and magnification steps used for photographing negative contrast specimens. spraying and pseudoreplication techniques were employed but did not yield further resolution of virus structure. the sequence of the segments of the genome of ctf virus strain florio has been determined: genbank accession numbers are af , af -af , af , af , u and u , respectively for segments to . california hare coltivirus s - - genome sequence partial sequences of segments , , , and of california hare coltivirus s - - , as determined in this study, has been deposited in genbank: accession numbers are af ( bp), af ( bp), af ( bp) and af ( bp). full-length sequences of segments - of eya virus have been determined: genbank accession numbers are af to af . the full-length sequence of segment of eya virus strain fr was found here to be exactly the same as the previously published partial sequence of the same segment [ ] . partial sequences of segments , , , and of eya virus strain fr and eya virus strain gr, as determined in this study, have been deposited in genbank: accession numbers are af ( bp) and af ( bp), af ( bp) and af ( bp), af ( bp) and af ( bp), af ( bp) and af ( bp). the size of the various segments of ctf and eya viruses are shown in table . the largest open reading frame (orf) was determined for each segment. this permitted us to deduce the sizes of the -and -non-coding regions (ncr), and those of the putative proteins encoded by each segment (see table ). the proteins encoded by the various dsrna segments were designated vpx, where x refers to the number given to the rna segment, based on its size. comparison of the genomic sequence of ctf virus, three strains of eya virus, california hare coltivirus s - - and the seadornaviruses genomic sequences of ctf virus strain florio, california hare coltivirus s - - , eya virus strain gr, eya virus strain fr and eya virus strain fr were compared with those of viruses which are now recognized as members of the genus seadornavirus. the latter included banna virus strain bav-in strain bav-in strain bav-in and strain bav-ch, and kadipiro virus strain kdv-ja . these comparisons indicated a maximum of % amino acid identity between member viruses of the two genera. this low level of similarity is comparable to values found when comparing viruses in different genera of the family reoviridae. as a frame of reference, the level of amino acid identity between homologous proteins of different viruses within various genera across the family reoviridae is between and % [ ] . analysis of the ncrs of the various genomic dsrna segments permitted the identification of conserved motifs located at each terminus. in the positive strand of each genome segment of ctf virus strain florio, the motif [ -sacuuuugy- , where y = c or u, s = g or c] was found in the -ncr. the motif [ -wugcagus- , where w = a or u] was found in the -ncr. the -and -terminal tri-nucleotides of all segments were found to be inverted complements. in all segments except segment the -ncr tri-nucleotide was gac; in segment it was cac. in all segments except segment the -ncr tri-nucleotide was guc; in segment it was gug. in the positive strand of each genome segment of eya virus strain fr , the motif [ -gacawuuk- , where k represents g or u] was found in the -ncr. the motif was found in the -ncr. the -and -terminal tri-nucleotides of all segments were found to be inverted complements. in all segments the -ncr tri-nucleotide was gac. in all segments the -ncr tri-nucleotide was guc that is the same as in ctf virus. analysis showed the terminal motifs of the two viruses to be quite similar. the consensus sequence was found in the -ncr and in the -ncr. the conserved terminal sequences of ctf virus and eya virus although similar, differ in the nucleotide at the fifth position of -ncr of the positive strand (u in case of ctf virus and a or u in the case of eya virus). a comparison of these sequences to those of member-viruses of different genera of family reoviridae is presented in table . this comparison showed that the last nucleotide at the termini is a cytosine residue for viruses belonging to the different genera of family reoviridae except phytoreoviruses which have a uracil residue. the first nucleotide at the termini is a guanosine residue for viruses belonging to the different genera of family reoviridae except fijiviruses and cypoviruses which have an adenine residue. comparisons of nucleotide and amino acid sequences showed that all of the genome segments of eya virus have cognate segments in the ctf virus genome (fig. ) . according to the standard designation of segments in which numbers are assigned in a decreasing order of size (s being the largest and s the smallest), there is one change in homologous eya virus and ctf virus segments: segment of eya virus strain fr is homologous to segment of ctf virus strain florio and vice versa. identification of rna-dependent rna polymerase motifs motifs found in the polymerase sequences of all members of the family reoviridae, were identified in the vp of eya virus and ctf virus. these are motifs sg (positions - ) and gdd (positions - for ctf virus and - for eya virus). the search for sequence homologies using the local blast program revealed matches between vp of ctf virus and eya virus (amino acid to ) and the rna-dependent rna polymerases of nilaparvata lugens reovirus (accession number d ; identity: %, similarity: %) and rice ragged stunt virus (accession number u ; identity: %, similarity: %). these findings are consistent with the hypothesis that the first genome segments of ctf virus and eya virus encode the viral rna-dependent rna polymerase (vp (pol)). analyses of amino acid sequences using ncbi's blast program, showed that amino acid - of the vp of ctf virus and amino acid - of vp of eya virus matched (identity: %, similarity: %) a sequence of the dna adenine methyltransferase of chlorella virus sc- a (family phycodnaviridae, accession number u ). the analysis of this region of the chlorella virus dna methyltransferase showed that it is conserved among a number of dna methyltransferases. analyses of vp showed the presence of a potential phosphamide linkage site (amino acid - of ctf virus: lnydky and for eya virus: lnyikh) comparable to that found in the protein encoded by segment of members of the genus orthoreovirus (position - : anpdkf, ). this protein sequence is thought to be involved in the fixation of guanosine forming the cap structure found on the positive sense strand of orthoreovirus dsrna. the ncbi's blast program revealed that the region between amino acid and of vp of ctf virus resembled a number of nucleotidebinding proteins, such as myosin and kinesin and two distinct purine nucleotide phosphohydrolases (ntpases). these ntpases are the archaeoglobus fulgidus purine ntpase ( % similarity, amino acid - , accession number ae ) and the sulfolobus acidocaldarius purine ntpase ( % similarity, amino acid - , accession number y ). in the homologous eya virus protein (vp ), the sequence between amino acid and also resembled nucleotide-binding proteins, such as myosin, kinesin and atpases. the vp sequence of ctf virus and eya virus contains the pattern [hhhhgx gksxnhhhhdd], where h indicates a bulky hydrophobic residue (amino acid to ) corresponding to a nucleotide-binding site in a number of protein modification enzymes [ , ] . the local blast analysis showed that the region of the protein between amino acid and is homologous to proteins encoded by segment of rice ragged stunt virus (genus oryzavirus, family reoviridae) and nilaparvarta lugens reovirus (genus fijivirus, family reoviridae). in these latter viruses, segment respectively encodes the non-structural protein ns , and a core protein which has nucleotide-binding activity. moreover, in the same region (amino acid to ), the pfam analysis also revealed the presence of a kinase domain. the rgd sequence (arginine-glycine-aspartic acid) is characteristic of a number of cell binding proteins and in the case of viruses this motif is thought to mediate the binding of viral structures to cellular integrins [ , ] . three rgd sequences were identified in the vp (amino acid - ), vp (amino acid - ) and vp (amino acid - ) of ctf virus. all of them are conserved in the homologous eya virus proteins. rgd motifs in vp and vp are present at the peaks of a single hydrophilic domain, as shown using a kyte and doolittle hydropathy plot [ ] . these rgd sequences may therefore be functional. an additional rgd motif is present in a hydrophilic region of ctf virus vp (amino acid - ) but this site is not conserved in eya virus vp . other significant sequence features -read-through phenomenon analysis of the nucleotide sequence of segment of both eya virus and ctf virus, revealed the presence of an opal stop codon belonging to the class of so-called "leaky" stop codons. this stop codon is flanked at its -end by a cytosine residue, a configuration that has been shown in retroviruses [ ] and alphaviruses [ ] to allow "read-through." this phenomenon results from the incorporation of arginine, cysteine or tryptophan in the usual stop codon [ ] . accordingly, two proteins might be synthesized, the first encoded by the orf between nucleotides and (orf encoding the protein vp ), the second encoded by the orf between nucleotides and (orf encoding vp ). comparison of the nucleotide sequences of segment of ctf virus and eya virus shows that the sequence variation is mainly a function of the third position of the codon. the orf variations at the third position of the codons were calculated using the number of differences estimation method and found to represent . % of the overall variation in this orf, while the remaining . % were shared by positions and . an identical magnitude of variation was found in the part of orf beyond the leaky stop codon. clearly, this nucleotide variation was not randomly distributed ( . % at the third position versus . % at positions . this result indicates that this region of segment is submitted to the same kind of genetic selective pressure as regions encoding viral proteins. it is therefore very likely that this region of orf is translated during viral replication. in addition, using the kyte and doolittle hydropathy plotting system (with an amino acid sliding window) on the deduced proteins (beyond the suspected leaky stop codon) showed that the putative proteins of ctf and eya viruses are very similar (fig. ) . this indicates that the function of the encoded proteins of the two viruses is highly conserved. the amino acid identity between homologous proteins of ctf and eya viruses ranged from to %, with similarity ranging between and % (fig. ) . the most variable proteins are those encoded by segments , and with amino acid identities of , and %, respectively. when sequences of segments , and of california hare coltivirus s - - , eya virus strain fr and eya virus strain gr were included in analyses, the same magnitude of variability was observed, as reported in table . in the case of the protein encoded by segment (the viral rna-dependent rna polymerase) high identity values were found ( - %), as seen in table . analyses of the amino acid sequences of the viral polymerase proteins (pol) of member viruses of particular genera in the family reoviridae indicated relationships that could be used to develop a sense of the overall phylogenetic relationships within the family. there was - % amino acid identity between the various rotaviruses, - % between the various phytoreoviruses, - % between the various orbiviruses, and - % between the various orthoreoviruses. however, fig. . thin section electron microscopy the same progression of events in the maturation of ctf virus and eya virus was observed in mouse brain and in cell culture. the initial discernible change in cells was the appearance in the cytoplasm of granular areas (identified as viral inclusion bodies, vib) of moderate electron density. these vibs had definite yet unbounded margins and some involved large portions of the cytoplasm. electron dense particles, nm in diameter, were found scattered throughout these granular matrices and especially at their edges; these particles were indistinguishable from cores of progeny virus particles (figs. , ) . the observation of vibs reveals possible internal structures that could correspond to early stages of morphogenesis, reinforcing the hypothesis that these granular areas correspond to the site of virus assembly. as infection progressed increasing numbers of virions appeared at the periphery of the granular matrices. virions consisted of a homogeneous dense core, nm in diameter, surrounded by an electron lucid layer, considered to be the capsid itself. the total diameter of virions was - nm (figs. , ). an additional envelope layer was found surrounding some virions located within cisternae of the endoplasmic reticulum. this envelopment appeared to be a consequence of passage of virions through endoplasmic reticulum membranes (figs. , ) . enveloped particles had a diameter of - nm. no difference in any morphologic or morphogenetic character was discernable between ctf virus and eya virus. with both viruses large numbers of multiple filaments appeared in complex arrays in association with virions and granular matrices (fig. ) .arrays of these filaments had cross-striations. these filaments might be equivalent to the "tubules" consisting of the ns protein [ ] , observed in orbivirus infected cells. within fig. . cytoplasm of a neuron of a suckling mouse infected days earlier with eya virus. individual virions were found within envelopes derived from cytoplasmic membranes. some viruses, notably coronaviruses, arteriviruses and flaviviruses, migrate in this way to the cell surface and are released by exocytosis -however, we were not able to prove this mechanism of shedding here. thin section stained with lead citrate. magnification × , . eya virions are indicated by arrows neurons in the brains of suckling mice infected with ctf virus arrays of these filaments occurred within nuclei as well as in cytoplasm. with both viruses, in both suckling mice and cell cultures, relatively few virions were found free in extracellular spaces, even late in infection. instead, virions accumulated within cells at various stages of degeneration. virus release appeared to occur via cell lysis. negative contrast electron microscopy ctf virus and eya virions were observed in negatively stained preparations; they were identical. virions were round or polygonal in outline with regularly spaced polygonal surface features, which seemed to lie flat on a rather smooth virion surface. virion mean diameter was nm (range - nm) (fig. a) . quite prominent in all preparations were delicate round particles nm in diameter (fig. b) . these were made up of regular subunits. from observations of partially degraded virus particles and virus particles variably penetrated by stain, and by analogy with the morphological details of orbiviruses and reoviruses, it was concluded that the nm particles represent the virion inner capsid and the nm particles complete virions. as has been observed in the past, the detailed surface structure of ctf virus and eya virus capsids seemed quite discernable, seemed quite available for analysis of symmetry. however, so few particles have been photographed in a way suitable for such analysis that this has not yet been done. colorado tick fever virus was identified more than fifty years ago by florio and colleagues as the etiologic agent of colorado tick fever, one of the most important tick-borne viral diseases in north america [ ] . the virus is the type species of genus coltivirus. in its natural habitat the virus is transmitted between small mammals and the tick dermacentor andersoni and other ixodid ticks [ , , ] . antigenic variants of the virus have been described; these have been isolated from ticks and vertebrates [ , ] . two related but distinct viruses have been identified in north america: one is california hare coltivirus s - - , isolated as the name implies in california from a black-tailed jackrabbit lepus californicus [ ] ; the other is salmon river virus, isolated in idaho from a human patient with a ctflike illness [ ] . a virus related to ctf virus, identified as eyach virus, has been isolated on several occasions in europe [ , ] . this virus exhibits a one-way cross-reaction with ctf virus in complement-fixation tests, but each virus has a distinct neutralization profile. more recently, comparison of the sequence of genome segment of ctf virus and eya virus confirmed this serological relationship [ ] . for some years the genus coltivirus was divided into two subgroups, a and b. subgroup a included ctf, eya and the other north american and european viruses. subgroup b comprised viruses isolated from mosquitoes and humans in southeast asia. however, recently dsrna hybridisation analyses, serological analyses and comparison of the genomic sequences of the various viruses led to dropping the a and b subgroup designation and construction of a distinct genus, the genus seadornavirus, for the asian mosquito-borne viruses [ , ] . genetic characterization of the coltiviruses was for some years limited to hybridisation analyses of genomes segments of field isolates of ctf virus; this work only confirmed the narrow range of ctf virus genetic diversity and suggested possible reassortment in nature of segments and [ ] . recent progress in viral genomics, such as the analyses of the nucleotide sequence of full genomes of the two major coltiviruses, as reported here, have permitted a more systematic approach and many new insights into the viruses and their evolutionary relationship. these sequence analyses have shown that the organization of the genomes of ctf and eya viruses is similar: (i) the genomes of the viruses are of comparable size (∼ , bp); (ii) each of the segments of genomic dsrna of the viruses are of comparable size; (iii) each genome segment is flanked by similar -and conserved non-coding sequences; and (iv) there is strong evidence that in segment of each virus there is a read-through phenomenon. these findings are reinforced by the identification, in the cognate genes of the viruses, of sequences encoding similar motifs (functional domains) in nonstructural viral proteins as found in certain other viruses and organisms. however, the paucity of available data on coltivirus proteins did not permit clear resolution of many structure-function relationships. an important exception is the unambiguous identification of the viral rna-dependent rna polymerase encoded in segment of the viruses. the specific sg and gdd motifs found match polymerase motifs found in the genomes of other member viruses of the family reoviridae. motifs for protein modifying enzymes including nucleotide phosphohydrolases (ntpases), guanylyltransferases and methyltransferases i and ii were also identified. such constitutive enzymes are known to be involved in capping positive strands of virion rna segments in other member viruses of the family reoviridae. classification criteria within the family reoviridae, based on the quantification of genetic relatedness, have been formally accepted by the international committee on taxonomy of viruses in its seventh report [ ] . in particular, the degree of relatedness of vp (pol) amino acid sequences has been accepted as a means of delineating genera and assigning viruses to particular genera. a vp (pol) amino acid identity of less than % has been taken as a criterion for placing viruses in different genera [ ] . accordingly, the comparison of the ctf virus vp (pol) sequence with that of eya virus (amino acid identity %) unambiguously places the two viruses in the same genus (genus coltivirus). moreover, on the same basis this genus may be considered distinct from all other genera -the amino acid identity between coltivirus vp (pol) and all other member viruses of the family reoviridae is less than %. in particular, thevp (pol) amino acid identity between the coltiviruses and the member viruses of the genus seadornavirus (which are also arthropod-borne and also have segment dsrna genomes) is less than % [ ] . a number of other molecular and biological characters support the separation of the coltiviruses and seadornaviruses into distinct genera: (i) the genomes of ctf virus and eya virus are significantly larger than those of banna virus and kadipiro virus (∼ , bp vs. ∼ , bp); (ii) the g + c content of ctf and eya virus genomes is %, which is significantly higher than the % calculated from the full-length genome sequences of genetic analyses have indicated that the most important differences between the member viruses of the genus coltivirus are found in the genes encoded in segments , and (these segment designations are the same for ctf and eya viruses). amino acid identities here are %, % and %, respectively, lower than those seen for other segments of the two viruses. together with their geographic and ecological isolation and their distant serological relationship, there seems to be a consistent basis for defining the two viruses as distinct species within the genus. the same basis is likely to be useful in the future for the demarcation of other species within the genus. of course, two phenotypic characters representing the expression of a large part of the genome of ctf virus and eya virus are the structure of the virion itself and the complex morphogenetic steps leading to the construction of the virion. in this regard, the authors enjoyed reviewing studies of ctf virus done more than years ago and comparing those observations with unpublished studies of eya virus. the viruses proved indistinguishable, but there is still more to be done here: the elegant structural studies that have been done in recent years on reoviruses, rotaviruses and orbiviruses (bluetongue virus) using computer analysis of cryoelectron microscopy images and x-ray crystallography should be done on the coltiviruses. the overall similarity in virion strucural design of the member viruses of the family reoviridae belies the differences that have evolved as the member viruses of the various genera have occupied such different niches. from the need to withstand a wide ph range and digestive enzymes in the vertebrate intestinal tract and transmission by the fecal-oral route (reoviruses, rotaviruses) to the need to withstand the systemic environment in diverse arthropod vectors and vertebrate hosts and transmission by arthropod bite (orbiviruses), to the need to withstand the unique physiology of plants and plant cells (fijiviruses, phytoreoviruses), very different characters, especially of virion surface structure, are called for. aspects of the functional qualities of these structures should be addressed by comparative structural studies. the identification of genetic, serologic and morphologic/morphogenetic relationships between ctf virus and eya virus raises interesting questions about their evolutionary origin. several hypotheses have been proposed: as a frame of reference, the molecular evolutionary rate of the polymerase gene of tick-borne flaviviruses (single stranded rna viruses) has been estimated as × − mutations/nt/year [ ] . this rate is based on host divergence of the various viruses between , and , years ago. even though there is little information on the molecular evolutionary rate of dsrna viruses, it seems extremely unlikely that the coltiviruses could be evolving at a rate consistent with that in the first hypothesis. on the other hand, several lines of thought favor the second hypothesis: (i) it seems reasonable to expect that the stabilizing effect of double-strandedness, as seen with dsdna genomes, should apply to dsrna genomes and therefore the molecular evolutionary rate of viruses with such genomes might be expected to approach that of dsdna genomes (perhaps less the extra stabilizing effect of dna proofreading enzymes); (ii) it has been proposed that the alternating life cycles of arboviruses in arthropods and vertebrates provides a stabilizing effect -mutations, especially in polymerase genes, that might provide a survival advantage in one host might represent a disadvantage in the other host -so the replication and transmission of the parental type might be favoured overall; (iii) tick-borne viruses may evolve slower than viruses transmitted by other arthropod vectors or directly from vertebrate host to host -tick vectors remain dormant for long periods during which virus replication cycles must be reduced, and tick vectors remain localized thereby limiting virus radiation and dispersal; (iv) the powerful evolutionary effect of reassortment, so common with dsrna viruses, may be abrogated by the econiche isolation and habits of the tick vectors of the coltiviruses -that is, the level of reassortment seen with the other genus of dsrna viruses, the orbiviruses, might reflect habits and habitats of their culicoides spp. and mosquito vectors rather than the viruses themselves; (v) we know nothing about other coltiviruses or similar viruses, viruses that might "fill in the blanks" between ctf virus and eya virus and provide support for one or another evolutionary hypothesis. for example, it is not inconceivable that another virus might be discovered that would indicate that ctf virus was derived from an eya-like virus rather than the opposite. perhaps the most intriguing aspects of this examination of the various hypotheses for the molecular evolution of the coltiviruses pertain to: (i) the overall molecular evolutionary rate of viral dsrna genomes in general, and (ii) the more ancient evolutionary history of the divergence of the ancestors of the member viruses of all the genera of the family reoviridae and the other dsrna viruses. another intriguing aspect of this examination of the genomes of the coltiviruses has been the discovery that part of the eya virus vp is clearly distinct from the homologous region of ctf virus vp (amino acid identity < %), whereas the rest of the proteins are quite similar (amino acid identity > %). the distinct region of eya virus vp is similar (∼ % similarity) to a sarcolemmalassociated protein of the host rabbit in europe, oryctolagus cuniculus. this raises the question of a possible recombination event between the genome of an ancestor of eya virus and an mrna of its lagomorph host. recombination in dsrna viruses (distinct from reassortment) has been reported (e.g., in rotaviruses), but only between viral genomes. recombination between viral and cellular rnas has been seen in member viruses of the genus pestivirus (family flaviviridae; positive-stranded ssrna viruses) [ ] . clearly, the significance of the similarity between the eya virus protein and that of its host warrants further investigation. the genetic analyses reported here have allowed us to resolve several questions pertaining to the taxonomy of the coltiviruses, but they have raised even more questions. extending these genetic analyses to a better understanding of the natural history of the coltiviruses will take much more work. one key will be developing a better understanding of the nature and extent of coltivirus variation in nature and a better understanding of the evolution of the various viruses in their complex and geographically and ecologically isolated niches. in this regard, we can hope that more and more coltiviruses will be discovered in nature and characterized in reference laboratories. another key will be developing a better understanding of the variation in structure-function relationships of the various viral proteins, the architectural organization of the virions, and the nature of variances in viral phenotypes, and pathotypes. taken together, the morphologic, serologic, biologic, and genetic characteristics that we have studied so far begin to paint the picture of a coherent set of viruses that have solved survival demands in unique fashion. the viruses are entrenched in their econiches and represent in some instances important threats to human health. the viruses are interesting adversaries, worthy of further investigation. fifty years with viruses complete sequence determination and genetic analysis of banna virus and kadipiro virus: proposal for assignment to a new genus (seadornavirus) within the family reoviridae sequence determination and analysis of the full-length genome of colorado tick fever virus, 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"eukaryotic" protein kinase superfamily antibodies against some arboviruses in persons with various neuropathies african horsesickness reoviridae molecular characterization of pestiviruses plastic embedding mixtures for use in electron microscopy orbiviruses and coltiviruses physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus -a new taxonomic group. ii. electron microscopic studies colorado tick fever virus: an electron microscopic study treeview: an application to display phylogenetic trees on personal computers electron microscopy of neurotropic african horse sickness virus surgeons us army ( ) the etiology of yellow fever: a preliminary note eyach, an arthropod-borne virus related to colorado tick fever virus in the federal republic of germany the use of lead citrate at high ph as an electron opaque stain in electron microscopy rgddependent entry of coxsackievirus a into host cells and its bypass after cleavage of vp protein by intestinal proteases complete nucleotide sequence of reovirus l gene and deduced amino acid sequence of viral mrna guanylyltransferase structural components of vesicular stomatitis virus the alphaviruses: gene expression, replication, and evolution bluetongue virus structure clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice ) virus taxonomy. seventh report of the international committee on taxonomy of viruses population dynamics of flaviviruses revealed by molecular phylogenies the authors wish to thank c. chastel and n. karabatsos for providing eya virus strain fr , eya virus strain fr , eya virus strain gr and the california hare coltivirus s - - . we also thank dr c. goldsmith for preparation of some of the photomicrographs. this study was supported by eu grant 'reo id' number qlk - - . the "unité des virus emergents" is an associated research unit of the institut de recherche pour le développement (ird). this study was supported in part by the ird. key: cord- - sniu j authors: fennestad, k. l.; mansa, b.; larsen, s. title: pleural effusion disease in rabbits: observations on viraemia, immunity and transmissibility date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: sniu j baby rabbits surviving infection with pleural effusion disease virus (pedv) developed viraemia persisting for at least six months. only the infectious serum samples collected during the first months of disease could transfer the typical ped. six months after neonatal infection, virus concentration in serum was ( ) to ( ) rabbit-infectious doses per ml, the level of igg appeared elevated, and serum rendered non-infectious by ether-treatment had a protective effect in passive immunisation experiments. no evidence of glomerulonephritis or deposits of immunoglobulins could be demonstrated in the kidneys. during the nursing period pedv was transmitted from infected baby rabbits to two out of four dams, but not to control litter-mates. after the nursing period control rabbits, caged together with the viraemic rabbits for to days, remained free from pedv infection. the demonstration of pleural effusion disease (ped) virus as passenger of rabbit testicular suspensions of treponema pallidum in several laboratories shows that this virus can be transmitted from rabbit to rabbit by testicular fluids at intervals of -- days, i.e. the customary time between intratesticular inoculation and harvest of treponemes from the testes ( , , ) . ped is a little known rabbit disease first described in from the scandinavian countries as intercurrent death among rabbits inoculated with t. pallidum ( , ) . the viral nature of the aetiological agent was reported in ( ) , but as yet pedv has not been demonstrated convincingly by culture, electron microscopy, - / / / / $ . or by a specific serological method ( , , , , ) . recently, it has been suggested that the virus may be antigenically related to human coronavirus strain e ( ) . in experimental studies ped virus (pedv) freed from treponemes showed a remarkable persistence in rabbits. by serial passages of blood or pteural fluid pedv could readily be maintained in rabbits by serial passage at intervals of -- , -- , and days. this increase in passage intervals reduced morbidity from fatal disease to almost subclinical infection. rabbits from the -day passages failed to develop clinical signs of ped on re-inoculation with virus ( ) . the present experiments were carried out to study the persistence of pedv in blood of neonatally infected rabbits and the transmissibility of the infection by contact. the experimental design also permitted observations on the nature of immunity to ped. all animals were obtained from the closed colony belonging to statens seruminstitut. (sse:cph); they were fed a pelleted diet and water was given in bottles. cardboard trays with wood shavings were used as bedding material and changed once a week. male albino rabbits, aged -- months, were used for demonstration of the pedv, for serial passages, titration and protection experiments. before use, these rabbits had been employed once for pyrogen testing of protein fractions of human blood. each of four dams with offspring was placed in a metal cage with a floor area of approximately . m . two to four baby rabbits in each litter were infected with pedv, while the remaining animals in the cage served as uninfected controls (of. table ). the dam was removed from the cage days after infection of her offspring and, after an additional period of -- days in a separate cage, challenged with the pedv. after removal of the dam, an age-matched control baby rabbit was introduced into each of two cages (nos. and ) without uninfected cage mates. infected and uninfected rabbits remained together in the same cage for a period of -- days, and their blood was examined for pedv at -day intervals from time of inoculation. the origin of the highly virulent copenhagen strain of pedv and the stock pool of infectious serum used for inoculation have been described previously ( ) . each baby rabbit received subcutaneously . ml virus pool diluted : in pbs (ph . ) corresponding to approximately . × rabbit-infectious doses of pedv ( ) . for the subcutaneous challenge we used t ml of i : diluted pleural fluid, corresponding to approximately a rabbit-infectious doses. this fluid came from two virus stock pools of pleura] fluid kept at minus ° c. each pool originated from ~ rabbits succumbing -- days after inoculation during serial passages of the copenhagen strain of pedv ( ). rabbit test ]or ped v the rabbit test described previously was used for the demonstration of pedv ( ). briefly, the inoeulum to be examined was given subcutaneously to a -- month-old rabbit. fever together with iridocyclitis or death with necropsy findings characteristic of ped or both were considered as evidence of the presence of :pedv in the inoculum. challenge days after inoculation served to demonstrate presence or absence of immunity to pedv. baby rabbits and other rabbits that died were examined as described previously ( ) . for the demonstration of pedv in dead baby rabbits . -- ml blood, pleural, or peritoneal fluid was used as inoculum in testing for pedv. at -day intervals blood samples were obtained from all infected and control animals. an amount of . ml blood from each sample, mixed with . ml pbs (ph . ), was used as inoculum. from post inoculation (p.i.) day and at the subsequent -day intervals . ml serum from each of the infected rabbits was also examined in the same way. the infected rabbits were sacrificed and examined days after infection. the control animals were placed in individual cages after -- days of contact with infected cage-mates and challenged with pedv days later. serial passages in rabbits by subcutaneous inoculation of infectious serum from p.i. days , , and days were carried out at -day intervals using one rabbit per passage. inoculmn for the passages was . ml serum mixed with . ml pbs. each rabbit used for passage was challenged with pedv days after inoculation to study immunity to the original virus. serum samples were obtained h'om the infected rabbits days after infection. each sample was divided into two portions and one portion was treated with ether to destroy infectivity of pedv ( ). this was done by shaking undiluted serum with an equal volume of diethyl ether at room temperature for about minutes. the ether phase was removed and the aqueous phase was re-extracted similarly with ether a total of four times. the residual ether was removed by aeration with nitrogen. ethertreated serum and untreated serum from each rabbit were diluted tenfold in pbs, and to ml of serum dilution -s were given intravenously to series of rabbits. rabbits receiving ether-treated serum were challenged subcutaneously hours later with rabbitdnfectious doses of pedv. rabbits receiving untreated serum were challenged in the same manner days after serum injection. after challenge the animals were observed for clinical signs of ped for up to days. serum from infected and control rabbits was examined days after infection or after -- days of contact. protein concentration was measured by refractometry (american optical hand refraetometer). the igg concentration was determined by the single radial immunodiffusion method ( ) . the agarose plates contained . izl swine anti-rabbit immunoglobulins (dako, denmark) per cm . a purified igg preparation ( ~ per cent igg) from normal rabbit serum was used as reference preparation. the serum samples were diluted with pbs (ph . ) to a concentration of -- g protein per and sedimentation analysis was carried out at , rpm and at ° c in the beckman model e ultracentrifuge with schlieren optics. the photographic plates from the eentrifugation were enlarged for drawings, which were used for calculation of the sedimentation rates and for the planimetric determination of the relative concentrations of the s, s and s components. the kidneys from four infected rabbits were removed at necropsy on p.i. day . tissue blocks, fixed in lillies neutral buffered formalin, were embedded in paraplast ® for light microscopy. sections were cut at and lira and stained with h & e, pas ~ h, and silver methenamine -~ i-i & e. tissue for immunofluoreseence microscopy (ifm) was frozen in a dry ice-alcohol mixture and stored at minus ° c. blocks were embedded in tissue teek ®-gelatine (ames lab.) ai~d cut into fm sections at minus °c on a leitz histoeryotome. the details of preparation of specimens and the ifm procedures have been given previously ( ) . fluorescein isothiocynate conjugated antisera (nordic hnmunol. lab.) from goat, specific for rabbit immunoglobulins (g , g , iga, igm, fe ~, fab) and from swine, specific for rabbit igg (fc + fab), were used in diiution : , during the nursing period, i.e. from inoculation of the baby rabbits until post inoculation (p.i.) day , seven of the inoculated baby rabbits died. one died within hours after inoculation, another was devoured by the dam, and a third was killed on p.i. day because of enterocolitis. two died with pleural and peritoneal effusions on p.i. days and , respectively, but in the latter a haemorrhagie intussusception of the colon was also present. no cause of death could be established in the remaining two rabbits dying on p.i. days and . table shows the results of examination of the baby rabbits and their dams during nursing period. pedv was demonstrated in two dead baby rabbits examined on p.i. days and , but not in two others dying on p.i. days and . on p.i. day pedv was demonstrable in blood of all six surviving infected . and ) . two of the four dams (nos. and ) were found to be protected when challenged with pedv, indicating that these two dams had become infected during the nursing period. after the nursing period two baby rabbits died on p.i. days and with pleural and peritoneal effusions. the remaining four infected baby rabbits, the five litter-mates, and two additional controls (introduced in cage nos. and ) appeared clinically normal until sacrifice or challenge. no gross lesions were observed at necropsy of the infected rabbits. this indicates that viraemia was present for six months in all four infected rabbits and that transmission of pedv infection from these rabbits to their agematched cage-mates did not take place. all rabbits inoculated with blood specimens obtained days after infection responded with clinical disease typical of ped. this response was also seen in two of the four rabbits inoculated with blood from p.i. day . inoculation with blood or serum specimens from p.i. day or later never resulted in clinical disease typical of ped; at most a transient fever was observed. to observe if the virus present in blood would retain inability to provoke clinical disease, serial rabbit passages of infectious serum from p.i. days , t , and were carried out at -day intervals. table shows the number of rabbit passages required before infectious serum regained a capacity to produce disease corresponding to that of t/he early isolates and the miginm virus. evidently, with continued viraemia ~n increasing number of passages was needed, and with two late isolates from the same rabbit clinical disease was not produced in spite of many passages. to determine virus concentration in serum obtained days after infection, decreasing amounts of serum were given intravenously to series of rabbits, which were then challenged days later, i.e. at a time when homologous antibody in the inoculum no longer could be expected to exert a protective effect. as seen from table , rabbits receiving from ml serum to ml of serum dilutions - to - failed to develop clinical disease typical of ped on challenge. this indicates a virus concentration per ml of serum of ~ to rabbit-infectious doses. the protective effect of the same sera, after infectivity had been destroyed by ether-treatment, was studied by challenge one day after serum injection. table shows that two of four rabbits, receiving the largest dose of serum, failed to develop clinical disease typical of ped. the table also shows the day of death from ped. using mortality rather than the full clinical picture as evidence of ped, it would appear that rabbits receiving ml or more of ether-treated serum had a lower mortality ( out of ) than rabbits receiving .t ml or less ( out of ). this suggests a protective effect of all four ether-treated sera. table shows the protein and igg concentra$ions in serum from the neonatally infected rabbits and their control cage-mates days after infection. the concentrations of the s, s and s components are also listed. the higher contents of igg and the s component in serum fl'om infected rabbits as compared with the controls suggest that the infected rabbits developed an igg antibody response during the pedv infection. no pathological changes were found in the kidneys of the four rabbits with persisting viraemia. examination for deposits of immunoglobulins in the kidney tissues also gave negative results. the presence of pleural and peritoneal effusions in four baby rabbits, dying on p.i. days , , and , respectively, provides strong evidence of death from ped. accepting only these four deaths as caused by pedv, the time of death was considerably delayed in baby rabbits as compared with older animals. k.l. fens;estad, b. mansa, and s. larse~: in -- month-old rabbits of the same stock, infected by the same route and with the same highly virulent pedv strain, most deaths occm~red within the first week after infection and pleural effusion was the characteristic finding. the remaining deaths occurred in ttle nd or rd week with characteristic pleurat and peritoneal effusions ( ) . the delay in occurrence of death from ped among the baby rabbits probably reflects a low reactivity between virus and host rather than resistance to infection, since pedv was demonstrated in all baby rabbits surviving infection. the persisting viraemia after neonatal infection is perhaps less surprising. in young adult rabbits pedv could regularly be transmitted from rabbit to rabbit. at intervals of days, using only one animal per passage ( ) . the reported failure of one attempt to extend this interval to and days, respectively, does not exclude the possibility that longer persisting viraemia may also exist in the adult rabbit. the few failures to demonstrate chronic viraemia in the present experiments may be explained by variation in virus concentration or perhaps differences in susceptibility to infection of the animals used in the rabbit test. the lack of clinical response in rabbits inoculated with infectious serum from p.i. day or later may indicate a simultaneous presence of virus and virus antibody in the inoculum. however, when serial rabbit passages were carried out at -day intervals clinical disease was produced only in a single instance after the first passage. it may also be argued that the lack of response was due to a low virus concentration in serum. the virus concentration on p.i. day was measured to to rabbit-infectious doses. the same concentrations of the original virus regularly produce clinical disease (unpubl. observations). this may suggest a change in virulence of the virus, production of defective interfering particles etc. further investigations of the virus-host interactions are required. the increased level of igg in infectious serum from p.i. day is considered a sign of formation of humoral antibodies to pedv. apparently, these antibodies were unable to neutralize circulating virus. nevertheless, the antibodies had a protective effect when pedv in serum was destroyed by ether. the question whether virus was attached to immunoglobulins, forming circulating infectious virus-antibody complexes, was not examined. in other viral infections associated with circulating infectious virus-antibody complexes, glomerutonephritis and deposits of host immunoglobulins have been demonstrated ( ) . the failure to demonstrate such kidney lesions in the present expei~ments may be due to the age of the animals at time of infection as observed in experiments with aleutian disease virus (i ). cross-infection between infected and uninfected rabbits in the same cage occurred from offspring to dam in two of four cases during the early stage of infection. this transmission is explained by the intimate contact between dam and offspring during the acute stage of infection. the lack of cross-infection by contact between baby rabbits before and after weaning indicates that direct transmission of ped is probably a rare occurrence. this assumption is in accordance with observations on ped among adult rabbits inoculated with treponemes contaminated with pedv. ped was present as an intercurrent disease at statens seruminstitut from to . during this period evidence indicating cross-infection to adjacent or other cages was never found and ped remained strictly confined to animals inoculated with contaminated treponemal suspensions. pleural effusion disease in rabbits. clinical and post mortem observations. acta path. microbiol, seand pleural effusion disease in rabbits. interferon in body fluids and tissues after experimental infection. acta path. microbiol, scand pleural effusion disease agent as passenger of treponema pallidura suspensions from rabbits spontaneous deaths among rabbits inoculated with treponema paltidu.m less than weeks before fever after inoculation of rabbits with treponema pallidum demonstration of a virus-like agent contaminating material containing the stockholm substraln of the nichols pathogenic treponema patlidum screening out a virus-like agent from the testicular suspension of the nichols pathogenic t. pallidum. with observations on certain characteristics of the agent immunofluorescent microscopy findings in minimal or no change disease and slight generalized mesangioproliferative glomerulonephritis. acta path. microbiol, scand intcreurrent death of rabbits after inoculation with treponema pallidum in the netherlands. th iclas syrup immunochemical quantitation of antigens by single radial immunodiffusion immune complex disease associated with viral infections reduced severity of lesions in minks infected transplacentally with aleutian disease virus rabbit cardiomyopathy associated with a virus antigenically related to human coronavirus strain e key: cord- -d hnqkh authors: choi, c. s.; joo, h. s.; molitor, t. w. title: temperature dependent replication of porcine parvovirus isolates date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: d hnqkh the replication of four porcine parvovirus isolates, nadl- , nadl- , kbsh, and kresse, in swine testes cells were compared at temperatures of , , and °c. replication of the kresse isolate was restricted at and °c as evidenced by progeny virus, virus polypeptide and viral dna synthesis, but not at °c. in contrast, replication of kbsh was restricted at °c, but not at or °c. findings from this study support the contention that replication of kbsh and kresse isolates are temperature dependent. temperature dependent replication has been reported for various families of viruses including members of the orthomyxoviridae [ ] , herpesviridae [ , ] , picornaviridae [ , ] , paramyxoviridae [ ] , and coronavirus [ ] . temperature-sensitive (ts) mutants have also been described for parvovirus, h [ ] , kilham rat virus [ ] , and porcine parvovirus [ , ] . based on previous studies demonstrating temperature-dependent replication of parvoviruses as well as other families of viruses, we proposed to evaluate putative temperaturedependent replication of selected ppv isolates. since the mean body temperature of a pig is . °c rather than °c, a difference in temperature-dependent replication in vitro may help to explain the differences observed in the replication of these isolates in swine. established swine testes (st) cells were used throughout this study for the propagation of ppv in vitro as previously described [ ] . ppv isolates employed included nadl- and nadl- , obtained from dr. w. mengeling (nadc, ames, ia), kbsh from dr. p. tattersall (yale university, new haven, ct), and kresse from dr. j. kresse (nvsl, ames, ia). nadl- was isolated from a dead fetus and has been propagated solely in swine fetuses. nadl- had been passaged eight times in porcine embryo kidney cells before we obtained the isolate, and has been passaged an additional three times in st cells at °c in our laboratory before use in this study. kbsh, which had been previously propagated approximately times in kb cells at °c [ , had been passaged twice at °c in st cells in our laboratory before use in this study. kresse was originally isolated from skin lesions of young pigs [ ] , was propagated twice in st cells and once in swine fetuses. using these ppv isolates, stock virus preparations was prepared by inoculating a multiplicity of infection (m.o.i.) of . fluorescent focus forming unit (ffu)/cell onto st cells at °c. virus was extracted from cells by cycles of freezing and thawing in te buffer ( mm tris, mm edta, ph . ), as previously described [ , and titrated by indirect fluorescent antibody (ifa) staining [ ] . virus titers were expressed in ffu. stock virus preparations were stored at - °c. the preparation of stock viruses of all four isolates was prior to the studies on temperature-dependent replication. studies examining putative temperature-dependent replication were initiated by absorbing m,o.i, of each of the virus isolates onto to % confluent monolayers of st cells at either , , or °c for h. the virus inoculum was removed, medium was replenished and cell incubation was continued at the same temperatures. at h intervals, for days post-inoculation, aliquots of cell-free supernatants or cell extracts were tested directly for virus antigen by hemagglutination [ ] or indirectly for infectious virus by ifa via the inoculation of -fold dilutions onto fresh st cells [- ] . to evaluate viral protein synthesis, [ s]-methionine radiolabelled polypeptides were immunoprecipitated from infected cell lysates [ ] . st cells were infected at an m.o.i, of for h, pulsed with ~tci/ml of [ s]-methionine for h and immunoprecipitated with rabbit anti-ppv sera. the immunoprecipitated polypeptides were electrophoresed in sds-containing . % polyacrylamide gels, and were exposed to x-ray film (dupont, boston, ma). to evaluate ppv dna synthesis, slot blot hybridization was performed on viral dna extracted from cultured cells by a modified hirt procedure [ ] . extracted dna was blotted onto nylon membranes (hybond-n, amersham) in the minifold ii apparatus (schleicher and schuell, keene, dh). hybridization was performed by incorporating a p radiolabelled rna probe following procedures previously described [ ] . the rna probe was synthesized using sp rna polymerase from a sp plasmid containing a , bp fragment of the ppv genome [ ] . the specific activity of the probe was approximately . x vcpm/lag. nadl- and nadl- isolates showed similar replication profiles for the production of infectious virus in cell-free supernatants from the temperatures tested (fig. a, b) . the concentration of infectious virus was lowest in ceils (fig. c, (fig. ) . similar patterns of cell associated virus were detected from both nadl- and nadl- infected ceils at the temperatures (fig. a, b) , whereas marked differences were again observed for kresse and kbsh-infected cells (fig. c, d) . the pattern of cell associated virus from kresse and kbsh-infected cells was similar to that described above for . after radio-labelling, cell iysates were immunoprecipitated with rabbit anti-ppv sera, and subjected to sds-page extracellular virus with the exception that higher titers were observed than those of cell free virus. in addition, cell-associated ha antigen was detected from kbsh-infected cells after day pi (data not shown), at which time no antigen was detected in cell free supernatant. since marked differences in replication of kresse and kbsh isolates were observed at and °c evidenced by both intracellular and extracellular infectious virus and ha antigen, further characterization of viral polypeptide production was attempted. synthesis of viral polypeptides at , , and °c was examined by immunoprecipitation of radiolabelled polypeptides. temperature dependent differences in the number and quantity of viral polypeptides were observed in cells infected with kresse and kbsh and propagated at either , or °c (fig. a) . viral polypeptides (vp and vp ) were observed in st cells infected with the kresse isolate at °c (fig. b, lane ) , but were present in lower concentrations from infected st cells propagated at °c and completely absent at °c. viral polypeptides (vp and vp ) were detected from kbsh-infected cells propagated at both and at °c, but at a higher concentration at °c. no viral polypeptides were detected from uninfected control cells cultured at these temperatures. to further examine the mechanism of temperature-dependent replication of ppv isolates, the synthesis of viral d n a was evaluated by nucleic acid hybridization of cell extracts (fig. ) . nadl- d n a was detected from cells incubated at both and °c in equivalent amounts (fig. a ). viral d n a was detected from cells infected with kresse isolate and propagated at °c ( fig. b, lane ) , but little or no viral dna was detected from infected cells propagated at °c (lane ) or °c (lane ). viral d n a was detected from st cells infected with kbsh and propagated at and °c (fig. c, lanes and ) , but little or no d n a was detected at °c (fig. c, lane ) . viral d n a was not detected from uninfected cells cultured at these temperatures. in mouse coronavirus, wild type virus replicates rapidly in the brain and spreads to the liver causing lethal hepatitis. in contrast the ts mutant replicates less extensively within the brain and could not be detected in the blood or liver [ . similarly, gauntt et at. [ ] have indicated that the difference in biological properties of ts mutants of coxsackie virus at temperatures above °c may ultimately offer insight into differences in pathogenicity observed in neonate mice. these three mutants showed differences in viral rna and polypeptide synthesis as well as virus assembly at and °c. fujisaki et al. [ ] and izumida et al. [ ] have established attenuated ppv strains by serial passages at low temperatures, to °c. no clinical signs, viremia or virus shedding were observed from sows inoculated with these viruses, but the pregnant sows immunized with these viruses and challenged with virulent virus gave birth to normal piglets. present studies showed that nadl- and nadl- virus isolates, which are pathogenic to mid-term gestation swine fetuses, exhibited similar in vitro replication patterns at all temperatures. in contrast, replication of kbsh, which is non-pathogenic to swine fetuses, was restricted at °c but not at °c. the replication of kresse virus isolate, which shows a broad range of pathogenicities, causing fetal death and skin lesions in young pigs, was more favorable at °c than at or °c. the mechanisms for restricted replication at non-permissive temperature appeared to be different between kbsh and kresse isolates. although both isolates showed comparable production of infectious virus at non-permissible temperatures, differences were noted at early stages of replication. at h pi, infectious virus, albeit at very low titers, were detected from kbsh-infected cells incubated at °c. in contrast, no virus was detectable until days pi for kresse virus infected cells incubated at °c. when viral potypeptides were evaluated at h pi, lower concentration of polypeptides was detected from kbsh-infected cells at °c, but were not detected at all from kresse-infected cells incubated at °c. in addition, lower concentrations of viral dna were detected from kbsh-infected cells at °c but there was no detectable viral dna from kresse-infected cells at °c when tested at h pi. these findings indicate that replicative restriction at non-permissible temperature may be due to a defect in virus assembly for kbsh, but replication of kresse appeared to be compromised at the initial stage of replication. differences between production of ha antigen and infectious virus were also noted. intracellular ha antigens, but not extracellular antigen, from kbsh-infected cells was detectable only after days pi at °c (data not shown). similar findings was found in kresse-infected cells at °c. while infectious virus was detectable after days pi, no ha antigen was detecable up to days pi. these findings indicate that greater than ffu are required for ha unit, coincident with a previous study [ ] . infectious ppv particles contain structural capsid polypeptides (vp , vp , and vp ), while empty, non-infectious virus particles consist of only capsid polypeptides (vp and vp ) [ ] . since viral polypeptides were evaluated at h pi following a h pulse, insufficient infectious virus was produced to demonstrate vp capsid polypeptides. in the early stages of ppv replication, empty particles are predominantly produced and afterwards, vp is post-translationally cleaved from vp [ ] . collectively, these results indicate that the ability of an isolate to replicate at temperatures near or above the mean physiological temperature of swine, . °c, may be essential for virulence. the mechanism for restriction of ppv isolates at non-permissive temperatures and whether adaptation of ppv isolates at non-permissive temperatures can be accomplished awaits further investigation. a temperature-sensitive mutant ofpoliovirus type altered in capsid assembly partial characterization of temperature-sensitive mutants of pseudorabies virus virus isolation is associated with herd infertility, abortion and stillbirth in pigs pathogenicity of a skin isolate of porcine parvovirus in swine fetuses inhibition of porcine parvovirus replication by empty particles nuclear accumulation of measles virus nucleoprotein associated with a temperature-sensitive mutant isolation and characterization of a temperature-sensitive uncoating mutant of pseudorabies virus establishment of an attenuated strain of porcine parvovirus by serial passage at low temperature preliminary characterization of coxsackie virus b temperature-sensitive mutants parvovirus as contaminants of permanent cell lines. isolations from - porcine parvovirus: replication in and inhibition of selected cellular functions of swine alveolar macrophages and peripheral blood lymphocytes establishment of the attenuated strain of porcine parvovirus for the live vaccine and its biological-immunological characteristics a search for parvoviridae (picornaviridae) a) observation of the pathogenicities of porcine parvovirus infection b) rapid diagnostic techniques for detection of parvovirus infection in mummified fetuses induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a is associated with restriction of viral replication in the brain parvovirus infection in pigs with necrotic and vesicle-like lesions pathogenesis of in utero infection: experimental infection of five-week-old fetuses with porcine parvovirus reproductive disease experimentally induced by exposing pregnant gilts to porcine parvovirus porcine parvovirus. properties and prevalence of a strain isolated in the united states porcine parvovirus: virus purification and structural and antigenic properties of virus polypeptides porcine parvovirus dna: characterization of the genomic and replicative form dna of two virus isolates kbsh parvovirus: comparison to porcine parvovirus temperature-sensitive mutants of fowl plaque virus defective in the intracellutar transport of hemagglutinin oronasal and intramuscular vaccination of swine with a modified live porcine parvovirus vaccine: multiplication and transmission of the vaccine virus covalent association of protein with replicative form dna of parvovirus h- replication process of the parvovirus h- . v. isolation and characterization of temperature-sensitive h- mutants defective in progeny dna synthesis isolation and characterization of thermosensitive mutants from kilham rat virus, a rodent parvovirus the authors thank dr. t. blackwell for his valuable discussions. we also acknowledge l. alexander and r. cronje for assistance in preparation of this manuscript. this work was supported in part of grants crsr- - and crsr- - from the u.s. department of agriculture. received march , key: cord- - rhdxzj authors: sato, k.; inaba, y.; matumoto, m. title: serological relation between calf diarrhea coronavirus and hemagglutinating encephalomyelitis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rhdxzj neutralizing (nt) and hemagglutination-inhibiting (hi) antibodies to calf diarrhea coronavirus (cdcv) and hemagglutinating encephalomyelitis virus of swine (hev) (strain n) were detected in high proportions of normal adult cattle and pigs in japan. since comparison of nt and hi titers in the serum samples suggested an antigenic difference between the viruses, cross nt and hi tests of these viruses were carried out with antisera raised in rabbits. the homologous nt titers were markedly higher than the heterologous titers. in hi tests essentially the same results were obtained. these findings indicate the presence of a marked difference in antigenic make-up between cdcv and hev. nt and hi tests can clearly differentiate the viruses, although there is some cross reaction. neutralizing (nt) and hcmagglutination-inhibiting (hi) antibodies to calf diarrhea, coronavirus (cdcv) and hemagglutinating encephalomyelitis virus of swine (hev) (strain n) were detected in high proportions of normal adult cattle and pigs in japan. since comparison of nt and h i titers in the serum samples suggested an antigenic difference between the viruses, cross nt and h i tests of these viruses were carried out with autisera raised in rabbits. the homologous nt titers were markedly higher than the heterologous titers. in h i tests essentially the same results were obtained. these findings indicate the presence of a marked difference in antigenic make-up between cdcv and hev. nt and h i tests can clearly differentiate the viruses, mthough there is some cross reaction. $ coronaviruses have been reported to be divided into two serological groups by the aid of the fluorescent antibody technique ( ). calf diarrhea coronavirus (cdcv) and hemagglutinating encephalomyelitis virus (hev) n of swine were classified in one group, together with mouse hepatitis virus type and human coronavirus c . the other group comprized feline infections peritonitis virus, transmissible gastroenteritis virus of swine, canine coronavirus and human coronavirus e. viruses in each group were antigenically related to each other to varying degrees, but were antigenieally unrelated to eoronaviruses of the second group. during the studies on cdcv and h e v we happened to test some normal bovine and swine sera for neutralizing (nt) and hcmagglutination-inhibiting (hi) antibodies to these viruses and obtained data suggestive of a marked antigenic difference between the viruses. accordingly we conducted a direct comparison between the viruses by nt and h i tests using antisera raised in rabbits. cdcv passaged in bovine embryonic kidney cells ( ) was kindly supplied b y dr. c. a. mebus, university of nebraska, u.s.a. i n our l a b o r a t o r y the virus was shown to readily replicate and induce cytopathie effect in cultures of a continuous cell line, bek- , derived from bovine embryonic kidney, thus providing a sensitive, practical assay m e t h o d and a satisfactory source of the virus ( ). i n the present s t u d y virus grown in bek- cells was used. strain n of h e v ( ) was kindly supplied b y dr. k. hirai, gifu university, j a p a n , and was grown in p r i m a r y swine k i d n e y cultures. nt tests were carried out b y the serum dilution m e t h o d using tube cultures of b e k -i cells with cdcv and those of p r i m a r y swine kidney with h e v . virusserum mixtures were incubated at ° c for hour before inoculation into tube cultures per serum dilution. the virus dose per tube was tcid . the antibody titer was expressed as the reciprocal of the highest serum dilution which shouted neutralization in at least one of the tubes. an a n t i b o d y titer of or higher was t a k e n as positive. h i tests were carried out b y the microtiter m e t h o d ( , ) . infectious culture fluid was used as h a antigen. f o r h i tests the serum was inactivated at ° c for minutes and t r e a t e d with kaolin to remove non-specific inhibitors and with packed chicken erythrocytes to remove antibodies to the erythrocytes. the antigenserum mixtures were incubated at room t e m p e r a t u r e for hour, mixed with chicken e r y t h r o e y t e suspension and incubated at room t e m p e r a t u r e for hour. the h i a n t i b o d y titer was expressed as the reciprocal of the highest serum dilution showing complete h i and titers of or higher were taken as positive. serum samples from normal adult cattle and pigs were tested for nt and h i antibodies to cdcv and hev. many of the bovine and porcine serum samples contained n t and h i antibodies to these viruses, indicating dissemination of the viruses or antigenically related viruses in these animals. f u r t h e r m o r e , the bovine sera had m a r k e d l y higher n t titers to cdcv t h a n h e v , while the porcine sera showed similar n t titers against b o t h viruses; nt titer ratio, ttev/cdcv, ranged from : to : with bovine sera and from : / to : with porcine sera. representative results are shown in table . h i titers of bovine sera were table t t i l also higher to cdcv than hev, although they were low; hi titer ratio, hev/ cdcv, ranged from : to : . hi titers of porcine sera were very low to both viruses. hi titers of representative sera are also shown in table i . these results suggested that there are antigenic differences between these two viruses. therefore, we conducted a direct comparison between cdcv and hev by cross nt and i-ii tests. the antisera used were prepared in specific pathogen free rabbits with virus grown in cultured cells and concentrated by ultracentrifugation (i). the animals received an intravenous dose of the virus suspension, followed at and weeks intervals, by two intramuscular doses of the virus suspension mixed with freund's complete adjuvant, and sera were obtained weeks after the last dose. the results are summarized in table . the homologous nt titers were much higher than the heterologous titers. in hi tests similar results were also obtained. all the serum samples obtained before the immunization were invariably negative in these tests. these findings show the presence of a marked difference in the antigenic makeup between these two viruses. nt and i-ii tests can clearly differentiate the viruses, although there is some cross reaction. replication of bovine eoronavirus in cell line b e k - culture neonatal calf diarrhea : propagation, attenuation and characteristics of a coronavirus-like agent characteristics of a eoronavirus (strain n) of pigs antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species characteristics of a coronavirus causing vomition and wasting in pigs hemagglutination by calf diarrhea coronavirus key: cord- -zfbu authors: teo, jeanette; pietro, patrizia di; biagio, floriana san; capozzoli, monica; deng, yi-mo; barr, ian; caldwell, natalie; ong, kian-leong; sato, mitsuharu; tan, rosemary; lin, raymond title: vereflu™: an integrated multiplex rt-pcr and microarray assay for rapid detection and identification of human influenza a and b viruses using lab-on-chip technology date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: zfbu threatening sporadic outbreaks of avian influenza and the h n pandemic of highlight the need for rapid and accurate detection and typing of influenza viruses. in this paper, we describe the validation of the vereflu™ lab-on-chip influenza assay, which is based on the integration of two technologies: multiplex reverse transcription (rt)-pcr followed by microarray amplicon detection. this assay simultaneously detects five influenza virus subtypes, including the pandemic influenza a (h n ), seasonal h n , h n , h n and influenza b virus. the vereflu™ assay was clinically validated in singapore and compared against reference methods of real-time pcr, virus detection by immunofluorescence of cell cultures and sequencing. a sensitivity and specificity of . % and . %, respectively, was demonstrated for pandemic h n ; . % and %, respectively, for seasonal h n ; . % and . %, respectively, for seasonal h n ; . % and %, respectively, for influenza b. additional evaluations carried out at the world health organization (who) collaborating centre, melbourne, australia, confirmed that the test was able to reliably detect h n . this portable, fast time-to-answer ( hours) device is particularly suited for diagnostic applications of detection, differentiation and identification of human influenza virus subtypes. hemagglutinin (ha) and neuraminidase (na) subtypes [ ] . influenza b viruses are not subtyped. historically, only three ha subtypes, h , h and h , have been responsible for pandemic outbreaks. this includes the outbreak caused by a novel influenza a (h n ) virus of swine origin [ ] . in contrast, all virus subtypes and combinatorial possibilities of ha and na have been detected in wild birds and poultry [ ] . since , highly pathogenic avian influenza (hpai) viruses, in particular the h n subtype, have jumped the species barrier and caused ongoing sporadic fatal outbreaks in humans [ ] . therefore, the looming threat of pandemics from influenza viruses cannot be overlooked, and assays that enable rapid, accurate identification and subtyping of influenza viruses is pertinent for surveillance and outbreak management. the increasing popularity of chip-based dna microarrays reflects their tremendous diagnostic potential and utility for rapid detection and subtyping of influenza viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] . of late, a handful of respiratory virus microarrays have been produced commercially, such as the infiniti Ò flu a-sh n quad assay (autogenomics, inc., carlsbad, ca), the fluchip (indevr, boulder, co) and the fda-cleared vrnat assay (verigene Ò , northbrook, il). the last of these is designed to detect influenza a and b viruses and respiratory syncytial viruses a and b using a glass slide array. the infiniti Ò flu a-sh n quad assay and the fluchip assays are, however, still for research use only. in our study, we describe the development and validation of the vereflu tm assay, which, to our knowledge, is the first commercially available integrated lab-on-chip (loc) device that combines reverse transcription (rt)-pcr amplification followed by amplicon hybridization to a dna microarray for the specific detection of clinically important influenza virus subtypes, including the pandemic influenza (h n ) and h n viruses. vereflu tm assay -system overview the hardware that is required to run the chips comprises a computer, a thermal control system (tcs), which enables pcr thermal cycling, and a microarray optical reader (lean s.r.l., medolla, italy). the accompanying software controls the instrumentation and also generates the analysis report. the workflow of the system and the processing time of each step is listed below. . extraction of viral rna (vrna) from the sample. this can be done either manually using spin-columns or using automated extraction systems (* min). . the sequences were aligned using the bioedit sequence alignment editor version . . . , and highly conserved regions resulting from the alignment were used for probe and primer design. as shown in table , ha-specific primers were designed for pandemic and seasonal h n , h n , h n and influenza b viruses. additional primers and probes were designed against the na segments of pandemic h n and h n to facilitate subtyping. reverse primers were synthesized with the addition of a -terminal cy label (sigma proligo, singapore). the capture probes were designed using the variable regions of the genes ( table ). the assay comprised influenza-virus-specific pcr primers and a set of forward and reverse psii primers for the amplification of a pcr-positive control of plant origin (table ) . together, these primers constituted the vereflu tm primer mix a. there were influenza targeted capture probes, rt-pcr control probes, and positive and negative hybridization control probes (table ) . singapore a total of respiratory samples (throat and nasal swabs) were collected from patients presenting influenza-like illness with fever c °c, cough, sore throat, headache and muscle ache. the samples were collected from may to august by sentinel physicians participating in a local influenza surveillance programme. the specimens were transported from their site of collection to the national public health laboratory (nphl), singapore, where sample processing was carried out. all specimens were subjected to influenza virus detection by real-time rt-pcr and shellvial culture followed by immunofluorescence (if) assay. swabs taken from patients using flocked swabs (copan diagnostics, murrieta, ca) were transported in ml of copan universal transport medium (utm). utm specimens were divided into two aliquots. one aliquot was used for viral culture using standard shell vial technique and the other was subject to nucleic acid extraction using an ez biorobot (qiagen, hilden, germany). extracted rna was eluted in ll of buffer, and the nucleic acid samples were stored at - °c until required for pcr analysis. real-time rt-pcr detection of influenza a virus has been described by spackman and colleagues [ ] . samples that were positive for influenza a virus were further subtyped into h or h using the protocol described by the public health laboratory services branch, centre for health protection (chp), department of health, hong kong sar, china (http://www.chp.gov.hk/files/pdf/ chp_protocols_for_the_detection_of_human_swine_ influenza.pdf). the detection of pandemic influenza a (h n ) was based on the nucleoprotein (np) gene, using primers and probes designed by the nphl. the detection of influenza b virus has been described by krafft et al. [ ] . all rt-pcrs reactions were set up using quantitect probe pcr kits (qiagen) with the amplification performed on a lightcycler . real-time pcr system (roche, mannheim, germany). pcr protocols, primer and probe sequences are available upon request from the authors. for the isolation of influenza virus, the aliquoted specimen in transport medium was passed through a . -lm acrodisc syringe filter (pall corp., ann arbor, mi) and inoculated into a shell vial (diagnostic hybrids, inc., columbus, oh) containing a monolayer of madin-darby canine kidney (mdck) cells. after two days of incubation at °c, the direct if test using fluorescein-isothiocyanateconjugated (fitc) monoclonal antibodies (light diagnostics, concord road, billerica, ma) specific for influenza a and b viruses was used to detect and differentiate between the viruses. a zeiss inverted fluorescent microscope was used to examine the samples for positively staining cells. influenza-virus-positive cultures were stored at - °c for future use. virus isolates in mdck cell culture and clinical specimens of influenza a virus, including pandemic influenza a (h n ) virus, were submitted to the who collaborating centre for reference and research on influenza at victorian infectious diseases reference laboratory (vidrl) from who national influenza centres, regional laboratories and hospitals from australia, new zealand, and the asia/pacific region. viruses were grown in mdck cells and/or embryonated chicken eggs [ ] . virus growth was monitored by observing cytopathic effect, and the presence of haemagglutination activity was observed using turkey red blood cells as described previously [ ] . the isolates were tested using a standard haemagglutination inhibition assay (hai) against a panel of reference viruses and their homologous ferret antisera [ ] . vrna was extracted from either clinical specimens or from viruses grown in mdck cell cultures as described previously [ ] . real-time rt-pcr was also performed to determine the type and subtype of viruses from clinical specimens as described previously [ ] . vereflu tm assay pcr amplification the multiplex rt-pcr reaction was performed using a quantitect virus?rox vial kit (qiagen, ontario, canada) in a total volume of ll. the rt-pcr master mix was prepared by adding the following components: ll of x qt virus nr master mix, ll of vereflu tm primer mix a, ll of rnase-free water, . ll of rnase inhibitor ( u/ll), . ll of qt virus rt mix, ll of the rna template and ll of psii amplification control. psii is in vitrotranscribed rna derived from the chloroplast-encoded gene psba from a mungbean plant (genbank: gq ). the rt-pcr mix was pipetted into chip inlets leading to the pcr micro-reactor where amplification takes place. the chip was sealed and placed into the tcs. the cycling parameters used were as follows: min at °c; min at °c; cycles of sec at °c, sec at °c and sec at °c. upon completion of rt-pcr, the chip was extracted from the tcs and unsealed. for hybridization of amplicons to the microarray, . ll of hybridization mix was pipetted into the chip inlets such that the final buffer composition was mm phosphate buffer, . mm kcl, mm nacl, x denhardt's solution, . % tween and . nm each of the positive hybridization controls at , at , at ( table ). the chip was re-sealed and placed into the tcs. heat denaturation of amplicons was performed at min at °c, followed by a -min hybridization at °c. after the hybridization, the chips were unsealed and washed for min at room temperature in falcon tubes filled with ml of . x ssc/ . % sodium dodecyl sulfate and dried by centrifugation at rpm for min in a -ml falcon tube. the dried chip was scanned in an optical reader (lean s.r.l.). spot segmentation and intensity calculation of the microarray image was performed by overlaying a virtual grid over the microarray image using the corner features as reference points. spot images detected within the grid were computed for background and signal values [ ] . a signal-to-noise ratio of [ . was defined as a positive result. recombinant plasmids bearing synthetic pandemic influenza a (h n ) ha and na genes with t promoter sequences at their ' ends were obtained from integrated dna technology, inc. (idt inc, coralville, ia). recombinant plasmids bearing ha and na genes from clinical isolates of influenza a h n , h n and influenza b (atcc vr ) viruses were generated in-house. the plasmids were linearized by restriction digestion using sali (new england biolabs inc, ipswich, ma) for the na gene and sacii (promega, madison, wi) for the ha gene. in vitro transcripts (ivts) were generated using riboprobe Ò in vitro transcription system (promega) and quantified using an agilent bioanalyzer rna nano kit (agilent technology, santa clara, ca). ivts of ha and na gene segments from clinical isolates of influenza a h n and h n viruses and an atcc isolate of influenza b virus (vr ) were used for the determination of analytical sensitivity. the rna concentration was measured using a nanodrop nd- spectrophotometer nanodrop technologies, wilmington, de). the rna copy number was calculated using the following formula: rna concentration [x g/ll rna] / (transcript length [bp] x ) x . x , and tenfold serial dilutions of the ha and na rna transcripts were made. the lod and cutoff values were determined in triplicate for the rna transcripts. the lod was the lowest concentration of rna that could give a % detection rate for that virus type for all triplicates. the cutoff value for detection was defined as the lowest rna concentration that would yield a positive assay result % of the time. the analytical specificity of the vereflu tm assay was evaluated using a panel of nine commonly found noninfluenza respiratory viruses purchased from the american type culture clinical samples were subjected to nucleic acid extraction using an ez biorobot (qiagen), and ll of the extracted nucleic acid was used in each vereflu tm assay reaction. the evaluations were carried out at three local hospitals, and the results were collated and presented as a single table (table ) . quantified viral stocks of influenza a (h n ) virus, influenza b virus and negative samples were used for assessing intra-assay and inter-assay reproducibility. low, moderate and strong positives were prepared from influenza a (h n ) and influenza b virus rna. the ct values for h n low, moderate and strong positive samples were , and , respectively, whilst the ct values for influenza b low, moderate and strong positive samples were , , and , respectively. lod and cutoff rna copy number concentrations provided the basis for low and medium positives. testing was carried out at each concentration using influenza virus samples in triplicate and negative samples also in triplicate. studies were conducted with two runs per day, at two local test sites by two operators, over a test period of days. the percentage of agreement with the expected result was calculated. the sensitivity and specificity of the vereflu tm assay were calculated by comparison to a combination of reference methods, which included rt-pcr, sequencing of the ha segment, virus culture and immunoassays. for sensitivity and specificity results, the % confidence interval ci for a proportion was calculated using the wilson score method without continuity correction [ ] . the lod is defined as the rna concentration that would yield a positive assay result % of the time. for all the virus types tested, the lod was determined to be copies/reaction ( table ). the cutoff detection was defined as the rna copy number that could be detected % of the time, and this was determined to be copies for both influenza a (h n ) virus and influenza b virus. for pandemic and seasonal h n , the cutoff was determined to be not higher than copies/reaction. the vereflu tm assay was tested with a panel of noninfluenza viruses, different influenza a virus subtypes (h n , h n , h n ) and influenza b virus. no crossreaction was observed with the influenza viruses tested. with the non-influenza respiratory viruses, cross-reactions were noted for the pandemic influenza capture probe with human coronavirus oc and influenza b capture probes with human adenovirus type . these were considered minor cross-reactions, as their probe signalto-noise ratios were weakly positive. also, in both cases, only one of the triplicate samples was positive. hence, the overall analytical specificity for the vereflu tm assay was calculated to be %. clinical evaluation of the vereflu tm assay, singapore the vereflu tm assay was validated at three sites in singapore using a total of respiratory samples ( table ) . the assay showed good sensitivity ([ %) and excellent specificity ( %) for all the influenza viruses tested. evaluation of the vereflu tm assay at who, melbourne the system was evaluated using clinical and cell-cultured virus samples. additionally, avian influenza a (h n ) virus testing was also performed. overall, out of the samples tested, the vereflu tm assay results for specimens ( %) were concordant with the reference methods. all (n = ) influenza-negative samples tested negative. specifically, the vereflu tm assay detected the influenza viruses in positive samples as follows: / ( . %) for pandemic h n , / ( %) for seasonal h n , / ( %) for h n , / ( . %) for h n and / ( %) for influenza b. observations by the research investigators indicated that the assay was able to detect both lineages of influenza b viruses (yamagata and victoria lineages) and influenza a viruses of different clades, including h n . the reproducibility of the vereflu tm assay is presented in table . when tested at rna copy numbers that would give moderate and strong positives for h n and influenza b, the assays were highly reproducible ([ %). as expected, when seasonal influenza a (h n ) influenza a (h n ) influenza b low rna copy numbers were tested, the reproducibility dropped to almost % for h n and % for influenza b. here, we report the development and evaluation of a portable and easy-to-use diagnostic platform, the vereflu tm assay, for the typing and subtyping of human influenza virus. the test is based on an on-chip multiplex rt-pcr amplification followed by detection using a microarray, and it can be performed fairly rapidly, taking about h to complete. each tcs or thermal reactor accommodates a maximum of five chips, which can be run either simultaneously or independently using different protocols. the modular nature of the system allows the linkup of more than one tcs unit to the computer, hence enabling the system to be adapted to the throughput needs of either large-or small-scale diagnostic settings. laboratory reproducibility data from our study suggest that the assay is easy to handle, which is an important consideration during outbreak situations. the overall clinical sensitivity for all influenza a viruses tested was . %, and the specificity was . %. these values were comparable to those obtained in similar microarray based influenza virus typing assays [ , ] . it is known that microarray-based detection and typing of influenza viruses is generally less sensitive than performing a real-time pcr [ , ] . our analytical sensitivity was determined to be copies/reaction for each influenza type. these figures are similar to those reported by huang et al. [ ] , at rna copies/reaction for seasonal h n , h n , h n and influenza b virus. the detection limit for a multiplex pcr assay has been reported to be copies for influenza a and influenza b viruses [ ] , which explains why some of the samples that were typed and confirmed by the reference methods of cell culture and real-time rt pcr were missed by the vereflu tm assay. in our laboratory, we use real-time pcr to detect influenza a virus and then follow up with a second subtyping pcr when influenza a positives are detected. we anticipate that the vereflu tm assay will ease that process, since detection and subtyping is done simultaneously onchip. multiplexing capabilities of real-time pcr reactions are typically limited to a fourplex as restricted by the number of fluorescence acquisition channels present in the pcr instrument. in contrast, the detection format in the vereflu tm assay is low-density-array-based, allowing tens to hundreds of capture probes to be spotted and correspondingly allowing a similar number of amplicons to be detected. hence, the arrays have a much greater detection capability for robust parallel testing than real-time pcr. given the high mutational frequency of influenza virus genomes [ ] efforts taken to increase the specificity of the vereflu tm assay included the use of short capture probes of to nucleotides [ ] as well as the incorporation of probe redundancy. the capture probes were designed to detect between and sections of the targeted gene and may be useful in overcoming the problem of misdiagnosis due to capture probes failing to hybridize with the amplicon as a result of gene polymorphisms. in our assay, capture probe cross-reactions were observed for the pandemic influenza a (h n ) probe with human coronavirus oc and the influenza b capture probe with human adenovirus type . during the testing of clinical samples, false positives of this nature will be unlikely, as hybridization to only one capture probe (instead of both probes for influenza b virus or all three probes for pandemic h n ) is insufficient to permit the vereflu tm software to call a positive result. instead, the operator will be alerted that the particular influenza gene segment has been detected, and a re-test will be advised. we are working on applying a more stringent hybridization wash, which may solve the problem of cross-reactions. in summary, we have developed a lab-on-chip device that has been clinically evaluated both locally and at who, melbourne. the system was found to be reliable and accurate for the identification of clinically significant influenza viruses and could potentially prove to be a valuable method for routine influenza surveillance as well as outbreak events. characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls the h n influenza outbreak in its historical context global patterns of influenza a virus in wild birds the pandemic threat of avian influenza viruses use of the dna flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses validation of a fully integrated microfluidic array device for influenza a subtype identification and sequencing simultaneously subtyping of all influenza a viruses using dna microarrays experimental evaluation of the fluchip diagnostic microarray for influenza virus surveillance multiplex assay for simultaneously typing and subtyping influenza viruses by use of an electronic microarray subtype identification of the novel a h n and other human influenza a viruses using an oligonucleotide microarray detection and subtyping of influenza a virus based on a short oligonucleotide microarray development of a realtime reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification reassortants in recent human influenza a and b isolates from south east asia and oceania adamantane resistance in influenza a(h ) viruses increased in in south east asia but decreased in australia and some other countries spot detection and image segmentation in dna microarray data two-sided confidence intervals for the single proportion: comparison of seven methods comparison of the mchip to viral culture, reverse transcription-pcr, and the quickvue influenza a ? b test for rapid diagnosis of influenza design and validation of a microarray for detection, hemagglutinin subtyping, and pathotyping of avian influenza viruses multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution experimental optimization of probe length to increase the sequence specificity of high-density oligonucleotide microarrays acknowledgments the work that was carried out at the national university hospital, singapore, was supported by the ministry of health, singapore (hsdp /x ).the melbourne who collaborating centre for reference and research on influenza is supported by the australian government department of health and ageing. the author(s) declare that they have no competing interests. key: cord- -ifdjyp b authors: sato, k.; inaba, y.; tokuhisa, s.; miura, y.; kaneko, n.; asagi, m.; matumoto, m. title: detection of bovine coronavirus in feces by reversed passive hemagglutination date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ifdjyp b a reversed passive hemagglutination (rpha) method was developed for the detection of bovine coronavirus in fecal specimens. sheep erythrocytes fixed with glutaraldehyde, and then treated with tannic acid were coated with anti-bovine coronavirus rabbit antibodies purified by affinity chromatography using bovine coronavirus linked to sepharose b. the rpha test was carried out by a microtiter method. erythrocytes coated with purified specific antibodies were agglutinated by bovine coronavirus, but not by bovine rotavirus or enterovirus. the reaction was inhibited by antiserum to bovine coronavirus, confirming the specificity of the reaction. the rpha test detected bovine coronavirus in of fecal specimens ( per cent), from natural cases of diarrhea, while the positive rates were only per cent ( / ) and per cent ( / ) for immunofluorescent staining of primary cultures of calf kidney cells infected with the specimens, and immune electron microscopy respectively. the advantages of the rpha method are its simplicity, high sensitivity and rapidity. mebus and his associates ( , ii, ) have demonstrated an agent with the morphologic features of a coronavirus by electron microscopy of the feces of calves with neonatal diarrhea, and have proved it to be a causative agent of the disease. the virus multiplied in bovine embryonic kidney cell cultures, but failed to induce a readily recognizable cytopathic effect. therefore although the virus may be assayed in these cultures, the method is rather cumbersome, as the presence of virus is detected either b y the microscopic examination of stained cultures, or b y immunofluorescence with a specific antiserum ( ) . i~aba et al. ( ) have d e m o n s t r a t e d t h a t the virus replicates readily producing a m a r k e d cytopathic effect in cultures of the continuous cell lines. b e k - , derived from bovine embryonic kidney, thus providing a sensitive, practical assay m e t h o d for the virus and neutralizing antibody. a hemagglutination-inhibition test was also developed ( , ) . more recently taxahasm et al. ( ) have isolated a bovine coronavirus strain from feces in an o u t b r e a k of diarrhea a m o n g adult cows in p r i m a r y cultures of calf kidney cells. h o w e v e r a cytopathic effect was not detected until the th blind passage. the demonstration of the virus in diarrheal feces b y virus isolation and electron microscopy is therefore time consuming and the procedures are elaborate. sa~ekata et al. ( , ) have reported t h a t the reversed passive hemagglutination ( r p t t a ) test is a useful and practical m e t h o d for the detection of rotavirus in the feces of infants with acute gastroenteritis. the advantages of the r p h a m e t h o d are its simplicity, high sensitivity compared with the electron microscopy, and its rapidity. these observations p r o m p t e d us to investigate the application of r p h a to the detection of bovine coronavirus in the feces of cattle with diarrhea. primary bovine kidney (bk) cells were grown at °c in eagle's minimum essential medium (mem) containing per cent tryptose phosphate broth (tpb) (difco), per cent calf serum, units/ml penicillin, ~g/ml streptomycin and ~g/ml fungizone. the maintenance medium was mem containing per cent tpb. . per cent yeast extract, . per cent sodium glutamate, . per cent glucose and antibiotics. the kakcgawa strain of bovine coronavirus ( ) was used. this strain was isolated in our laboratory from a cow with diarrhea and is identical to the bovine coronavirus of mebus et al. ( ) by neutralization and immunofluorescence. the shimane strain of bovine rotavirus ( ) and the bfi strain of bovine enterovirus ( ) were also used. both were propagated in bk cells. this was carried out in bk cell cultures prepared in × mm tubes. serial -fold dilutions of the virus-containing material were made in maintenance medium, and each dilution was inoculated ( . ml/tube) into tube cultures. the inoculated cultures were incubated in a roller drum apparatus at °c for days and were examined for any cytopathic effect. the per cent infectious dose (tcids ) of virus was calculated. bovine eoronavirus was purified from the culture fluid of b e cells infected with the kakegawa strain which had been passaged eleven thnes in b e cells. infectious culture fluid, clarified by centrifugation at , × g for minutes was centrifuged at , ×g for hours. the deposit was resuspended in . volumes of pbs ( . m nacl, . ~ phosphate buffer, pi . ) and centrifuged at , ×g for minutes. the supernatant fluid from this centrifugation was mixed with a cscl solution to a density of . g/ml and centrifuged in a beckman sw . rotor at i , xg for hours. the gradient was collected in . ml volumes and fractions with a buoyant density of . to . were pooled, and dialyzed against pbs at ° c for hours. the resulting material was layered on to a -- per cent sucrose density gradient and centrifuged in a beckman sw rotor at i , × g for hours. fractions ( . ml) were collected and those containing i ~g/ml or more protein as determined by optical density were pooled and dialyzed against pbs at °c for hours. the resulting virus suspension was tested for infectivity, and for protein content by the method of lowry et al. ( ) . l~otavirus and enterovirus were concentrated by centrifugation of infectious culture fluid at i , ×g for hours. after resuspension in . volume of pbs the purified virus was clarified by centrifugation at × g for minutes. a n t i s e r a a g a i n s t t h e k a k e g a w a s t r a i n was p r e p a r e d in rabbits. virus grown in b k cell cultures was purified u p to t h e step of csc density gradient c e n t r i f u g a t i o n as described above. f r a c t i o n s w i t h a b u o y a n t d e n s i t y of . to . g/ml were pooled a n d dialyzed a t ° c for hours a g a i n s t pbs. sufficient p b s was a d d e d to t h e resulting m a t e r i m to m a k e a suspension c o n c e n t r a t e d i -fold from t h e original culture fluid. e a c h r a b b i t received one i n t r a v e n o u s dose of ml of virus suspension, followed after weeks b y a n i n t r a m u s c u l a r dose of ml of equm volumes of virus suspension a n d f r e u n d ' s complete a d j u v a n t . a f u r t h e r i n t r a m u s c u l a r dose was given at weeks, a n d s e r u m was o b t m n e d weeks later. t h e a n t i s e r u m used in t h e present s t u d y h a d a n e u t r a l i z i n g a n t i b o d y titer of : , a n d a h e m a g g l u t i n a t i o n -i n h i b i t i n g a n t i b o d y titer of : . specific antibodies to t h e k a k e g a w a s t r a i n were purified b y affinity c h r o m a t og r a p h y from a r a b b i t a n t i s e r u m using c n b r -a c t i v a t e d sepharose b coupled to b o v i n e coronavirus, purified as described above. t h e m e t h o d was essentially t h a t of a x~n et al ( ) a n d cuatrecasas et al ( ) . three g r a m m e s of sepharose b ( p h a r m a c i a f i n e chemicals, sweden) a c t i v a t e d b y c n b r was a d d e d to ml of d i l u e n t ( . nac , .i ~ nahco~) c o n t a i n i n g ~g/ml of purified virus protein. the m i x t u r e was stirred a t room t e m p e r a t u r e for hours, m i x e d w i t h ml of . ~ glycine, p h . (wako chemicals, j a p a n ) a n d stirred for one additionm hour. this m a t e r i m was p a c k e d into a -ram d i a m e t e r column a n d w a s h e d r e p e a t e d l y w i t h a solution conraining . ~ naci and .i ~ nahco until the ph of the eluate was . . the column was h~rther washed with . m naci in .i ~ acetic acid, pi-i . (until the ph of the filtrate became . ) and then equilibrated with pbs. the column was stored at ° c after fi]]ing with . per cent nan . for the purification of specific antibodies, rabbit antiserum against the kakegawa strain was inactivated at °c for minutes and centrifuged at , ×g for minutes. eight ml of the supernatant fluid was applied to the column, which was then washed with pbs until buffer passing through had an optical density of zero. specific antibody to bovine coronavirus was eluted with ~ sodium thiocyanate solution in . ml fractions, which were tested for optical density. fractions containing i ~g/ml or more of protein were pooled and dialyzed against :pbs at ° c for ] hours. sheep blood was collected in alsever's solution. after washes w i t h p b s the e r y t h r o e y t e s were fixed b y mixing equal volumes of a per cent suspension and per cent g l u t a r a l d e h y d e (iwai chemicals, j a p a n ) in p b s . the m i x t u r e was i n c u b a t e d at r o o m t e m p e r a t u r e for hours w i t h occasional shaking. the fixed cells were w a s h e d times w i t h pbs, s u s p e n d e d in p b s at a c o n c e n t r a t i o n of per cent a n d m i x e d w i t h an equal volume of ~g/ml tannic acid (merck, w e s t germany) in pbs. after incubation at ° c for m i n u t e s the cells were w a s h e d times w i t h pbs, s u s p e n d e d in p b s at a c o n c e n t r a t i o n of per cent and m i x e d w i t h an equal volume of p b s containing specific antibodies to bovine eoronavirus purified as described above. after incubation at room t e m p e r a t u r e for hour, t h e cells were w a s h e d twice a n d a per cent suspension was prepared. the diluent used for washing a n d p r e p a r a t i o n of the suspension was p b s containing per cent calf serum a n d . per cent nan . the calf serum used was s h o w n to be free of antibodies to bovine eoronavirus and was absorbed w i t h glutarmdehyde-fixed e r y t h r o c y t e s at ° c for hour to r e m o v e antibodies to sheep red blood cells. the test was carried out by a microtiter m e t h o d , using t h e diluent described above. this was carried out b y the indirect m e t h o d using r a b b i t a n t i s e r u m against the k a k e g a w a strain. i n f e c t e d b k cells in t u b e cultures were i n c u b a t e d at °c for days in a roller d r u m a p p a r a t u s and t h e n scraped off the glass using a r u b b e r policeman. the cells, s u s p e n d e d in pbs, were smeared onto glass slides, air-dried a n d fixed in acetone at -- ° c for minutes. the fixed preparations were t r e a t e d at ° c for m i n u t e s w i t h specific r a b b i t antiserum, stained at ° c for m i n u t e s w i t h fluorescein i s o t h i o c y a n a t e -c o n j u g a t e d goat a n t i b o d y to r a b b i t igg (miles, u.s.a.) a n d e x a m i n e d for fluorescence. a per cent suspension of feces in p b s was centrifuged at , x g for m i n u t e s a n d t h e s u p e r n a t a n t was filtered t h r o u g h ~o m e m b r a n e filter w i t h a pore size of nm. one m] of the filtrate was m i x e d w i t h ml of a : dilution in p b s of t h e r a b b i t a n t i s e r u m against bovine coronavirus a n d i n c u b a t e d at °c for hours. this m a t e r i a l was centrifuged at , × g for hours, a n d the pe]lets r e s u s p e n d e d in . ml of p b s were e x a m i n e d b y the negative staining t e c h n i q u e w i t h p h o s p h o t u n gstate in a jeol je /i- cx electron microscope (jeol ltd., j a p a n ) . this was carried out b y a microtiter m e t h o d using chicken e r y t h r o c y t e s ( ) . serum to be tested was inactivated at ° c for minutes and then treated with kaolin and packed chicken erythroeytes. the serum-antigen mixtures were incubated at room temperature for hour, then mixed with chicken erythroeyte suspension and incubated at room temperature for a further hour. the hi titer was expressed as the reciprocal of the highest serum dilution showing complete inhibition of hemagglutination by ha units. sheep erythrocytes were fixed with glutarmdehyde, treated with tannic acid and coated with various amounts of purified antibodies to bovine coronavirus. using these erythrocytes the r p h a titer was determined for a concentrated suspension of purified bovine coronavirus with a titer of l s. tcids / . ml. the results are summarized in table . erythrocytes treated with antibody concentrations from to izg/ml gave an r p h a titer of : whereas those treated with to tzg/ml antibody gave an r p h a titer of : . coated erythrocytes did not agglutinate in the absence of the virus, and virus did not agglutinate erythrocytes fixed with glutaraldehyde or fixed erythrocytes and treated with tannic acid. based on these results erythrocytes coated with ~g/ml of the purified antibodies were used in the following experiments. a concentrated suspension of bovine rotavirus with a titer of s. tcids / . ml and a similar suspension of bovine enterovirus with a titer of . tcids / . ml gave negative results in the i~pha test providing further confirmation of the specificity of the test. the fecal specimen from which the kakegawa strain of bovine coronavirus was isolated ( ) , gave an r p h a titer of : . in the virus dilution method of r p h a inhibition, a -fold dilution of the rabbit antiserum against bovine coronavirus completely inhibited hemagglutination produced by the concentrated suspension of bovine coronavirus as well as that due to the fecal specimen which yielded the kakegawa strain. the pre-immunization serum as well as antisera against bovine rotavirus and enterovirus did not inhibit the r p t i a reaction. in the serum dilution method of r p h a inhibition the antiserum had a titer of : . i~pha tests were carried out on fecal specimens from cattle with diarrhea. of the specimens tested, were collected from calves, . to table amount of antibody for coating rpi-ia titer~ tzg/ml < tzg/ml bg/ml ~ bg/ml ~-~ ~zg/ml r p t t a titer of a purified coronavirus suspension with s-~-tcids / . ml and ~g/ml protein, using erythrocytes coated with indicated amount of purified antibody month of age during an outbreak of acute gastroenteritis in the fukushima prefecture in march . the remaining specimens were obtained during an outbreak of diarrhea among milking cows in the miyagi prefecture in april . all the specimens were also tested by immune electron microscopy and immunofluorescence. acute and convalescent serum samples were available from of the animals and these were tested for • h i antibody against the kakegawa strain. the results are summarized in table . in r p h a tests of the fecal specimens, or per cent, were positive and the remaining were negative ( < / : ) . the positive specimens did not agglutinate erythrocytes fixed with glutara]dehyde or fixed erythrocytes treated with tannic acid. of the positives, had titers equal to or higher than : , had a titer of : and the remaining had a low titer of : . the fecal specimens from calves had a higher positive rate ( / , per cent) than those from milking cows ( / , per cent). in the virus dilution method of r p h a inhibition a fold dilution of the rabbit antiserum against bovine coronavirus inhibited the reaction of the positive specimens, whereas no inhibition was shown with the pre-immunization s e r u m . i~pha tests were carried out on fecal specimens from healthy cattle. the specimens gave invariably negative results, supporting the specificity of the test. serum from of the animals were tested for h i antibody against the kakegawa strain, and significant rises in titer were demonstrated in of these ( per cent). the remaining animals had h i antibodies, but showed no rise in titer. of the fecal specimens from the animals showing a rise in h i titer, were positive in rpi-ia tests and were negative, whereas of the fecal specimens from the remaining animals which did not have rises in titer, only one was positive in r p h a tests. however, the difference in the positive rate of r p h a reaction observed between the two groups was not statistically significant (fisher's exact test). by the i f staining method only specimens, or per cent, were positive, while electron microscopy gave a positive rate of / or per cent. the specimens positive by the if staining method were also positive in the g p h a test and the electron microscopy. all the specimens positive by electron microscopy were also positive in the g p h a test. this observation, that the positive rate obtained by g p h a test was higher than those by the other methods, was not statistically significant. in the present study the i~pha method was shown to be useful and practicm for the detection of bovine coronavirus in the feces of cattle with acute gastroenteritis, although the number of fecal specimens examined in the present study was small. bovine coronavirus has been demonstrated in fecal specimens by the immunofluorescent staining of cell cultures inoculated with the specimen ( , , ) , and by electron microscopy ( , , , , ) . however, these methods are time consuming and elaborate procedures. on the other hand, the r p h a method is simple and the results are obtained in a short time. the r p h a method developed in this study was shown to be specific for bovine coronavirus. the r p h a reaction occurred only with bovine coronavirus, not with bovine rotavirus or enterovirus, and the reaction was inhibited by antiserum against bovine eoronavirus, whereas no inhibition could be demonstrated with pre-immunization sera or antisera against bovine rotavirus and enterovirus. furthermore the fecal specimen which yielded the kakegawa strain of bovine coronavirus ( ) , had a r p h a titer of : and the reaction was specifically inhibited by antiserum against the virus. bovine coronavirus was detected by r p h a more frequently in the feces of those cattle which had a rise in h i antibody to the virus than of those which did not. this observation provides further evidence of the specificity of r p h a test. however, the observed difference was not statistically significant, and further confirmation of this finding is required. the concentration of purified specific antibodies to bovine coronavirus used for the coating of erythrocytes was important in obtaining a high sensitivity for the test. the rate of detection of bovine coronavirus in the fecal specimen from natural cases was higher ( per cent) by the r p h a method than by the i f staining technique ( per cent) or electron microscopy ( per cent). however, these results require further confirmation as the differences observed were not statistically significant. the r p h a test could find wide application in the study of bovine coronavirus infections in cattle. r p h a may also be applicable to other viral infections such rotavirus infection of cattle and transmissible gastroenteritis of swine, in which virus is excreted in abundance in the feces. chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides isolation of coronaviruses from neonatal calf diarrhea in great britain and denmark selective enzyme purification by affinity chromatography rotavirus and coronavirus associated diarrhea in domestic animals a coronavirus-like agent present in feces of cows with diarrhea bfi virus: a new cytopathogenic virus isolated from cattle. i. isolation and properties replication of bovine eoronavirus in cell line bek- culture protein measurement with the folin phenol reagent neonatal calf diarrhea: results of a field trial using a reovirus-like virus vaccine neonatal calf diarrhea: propagation, attenuation and characteristics of a eoronavirus-like agent pathology of neonatal calf diarrhea induced by a eoronavirus-like agent detection of rotavirus from feces by reversed passive haemagglutination method detection of rotavirus from infantile gastroenteritis patient's stools by means of reversed passive haemagglutination hemagglutination by calf diarrhea coronavirus isolation of a reovirus-like agent (rotavirus) from neonatal calf diarrhea in characterization of a calf diarrheal eoronavirus neonatal calf diarrhea: purification and electron microscopy of a coronavirus-like agent epizootic diarrhea of adult cattle associated with a coronavirus-like agent authors' address: dr. y. i~aba, national institute of animal health, tsukuba, ibaraki, , japan.i~eeeived june , key: cord- - wefykbi authors: deng, xiaoyu; zhang, jiali; su, jiazi; liu, hao; cong, yanlong; zhang, lei; zhang, kemeng; shi, ning; lu, rongguang; yan, xijun title: a multiplex pcr method for the simultaneous detection of three viruses associated with canine viral enteric infections date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: wefykbi the aim of this study was to establish a multiplex pcr (mpcr) method that can simultaneously detect canine parvovirus (cpv- ), canine coronavirus (ccov) and canine adenovirus (cav), thereby eliminating the need to detect these pathogens individually. based on conserved regions in the genomes of these three viruses, the vp gene of cpv- , the endoribonuclease nsp gene of ccov, and the k gene of cav were selected for primer design. the specificity of the mpcr results showed no amplification of canine distemper virus (cdv), canine parainfluenza virus (cpiv), or pseudorabies virus (prv), indicating that the method had good specificity. a sensitivity test showed that the detection limit of the mpcr method was × ( ) viral copies. a total of rectal swabs from dogs with diarrheal symptoms were evaluated using mpcr and routine pcr. the ratio of positive samples to total samples for cpv- , ccov, and cav was . % ( / ) for mpcr and . % ( / ) for routine pcr. thirty-five positive samples were detected by both methods, for a coincidence ratio of %. this mpcr method can simultaneously detect ccov (ccov-ii), cav (cav- , cav- ) and cpv- (cpv- a, cpv- b, cpv- c), which are associated with viral enteritis, thereby providing an efficient, inexpensive, specific, and accurate new tool for clinical diagnosis and laboratory epidemiological investigations. recently, pet dogs have become more common in people's daily lives. pet dogs often bring joy and abate loneliness and, more importantly, provide some people with emotional sustenance. viral enteritis is a major threat to pet dogs. canine parvovirus (cpv- ), canine coronavirus (ccov) and canine adenovirus (cav) are the main pathogens that cause canine viral enteritis. cpv- is a member of the genus protoparvovirus, family parvoviridae, and is an important, highly contagious and potentially lethal enteric pathogen in dogs. it can infect dogs of any age, resulting in enteritis symptoms that include hemorrhagic diarrhea, vomiting, loss of appetite, and depression [ , , ] . ccov, a member of the family coronaviridae, often causes mild diarrhea and is characterized by high morbidity and low mortality in canids; however, in puppies, handling editor: roman pogranichniy. xiaoyu deng and jiali zhang contributed to the work equally and should be regarded as co-first authors. all the authors have agreed that xijun yan may act on their behalf regarding any subsequent processing of the paper. * xijun yan tcsyxj@ .com the disease is more serious, with high morbidity and high mortality. if ccv and cpv- coinfection occurs, it often leads to lethal diarrhea [ , ] . cav is a member of the genus mastadenovirus, family adenoviridae. although cav is not primarily responsible for enteritis, it can also cause diarrheal clinical symptoms [ , ] . these three viral infections are often mixed in the clinic, and their clinical symptoms of diarrhea are similar; therefore, it is often difficult to distinguish between them during clinical diagnosis and epidemiological investigations [ , ] . pcr combined with sequencing is the most widely used detection method in the field of pathogen detection. due to its specificity and sensitivity, this method is gradually becoming the gold standard for pathogen identification. the multiplex pcr (mpcr) method is based on a single pcr that can simultaneously detect a variety of pathogens and simplify the operation steps, thereby saving time and expense [ ] . recently, mpcr has been widely used in the field of pathogen detection because it has advantages of speed, specificity and efficiency [ , , ] . in this study, an mpcr method was established and applied for the detection of cpv- , cav and ccov. the mpcr method was validated as an easy and effective method for the detection of these three viruses in rectal swab samples. the ccov (ccov-ii), cav (cav- , cav- ), cpv- (cpv- a, cpv- b, cpv- c), pseudorabies virus (prv), canine distemper virus (cdv), and canine parainfluenza virus (cpiv) strains used in this study were maintained in our laboratory. primers for the conserved regions of cpv- , ccov, and cav were designed using oligo . software. the specificity of the primers was verified using blast (https ://www. ncbi.nlm.nih.gov/tools /prime r-blast /). the optimal combination of the three pairs of primers ( nucleic acids were extracted from cpv- , cav and ccov using a minibest viral rna/dna extraction kit (takara, japan) according to the manufacturer's protocol. the rna extracted from ccov was reverse transcribed to cdna using the specific primer ccov-r (table ) and moloney murine leukemia virus (m-mlv) reverse transcriptase (promega, usa) according to the manufacturer's protocol prior to its use as a template. in contrast, the nucleic acid extraction products of cpv- and cav were used directly as pcr templates. viruses obtained from cell culture were used directly for nucleic acid extraction, but clinical samples were first diluted and centrifuged at rpm for min before nucleic acid extraction to remove impurities. a mixture containing proviral dna and cdna in equal amounts was used as a template to optimize the annealing temperature and primer concentration. the reaction was performed in a -μl volume containing . μl of ex taq (takara, japan), μl of × pcr buffer ( mm mg + plus), μl of dntp mixture ( . mm each), ng of both template proviral dna and cdna, . μl each of ccov-f and ccov-r, . μl each of cav-f and cav-r, . μl each of cpv-f and cpv-r, and distilled water. the reaction procedure was as follows: initial denaturation at °c for min; cycles of denaturation at °c for s, annealing at °c for s, and extension at °c for s; and a final extension at °c for min. the mpcr products were evaluated by . % agarose gel electrophoresis. to determine the sensitivity of the mpcr and to obtain positive controls, the specific pcr products of ccov ( bp), cpv- ( bp) and cav ( bp) were ligated to the pmd- t vector (takara, japan) to construct the recombinant plasmids pmd-c (ccov), pmd-p (cpv- ) and pmd-a (cav), respectively. the plasmid copy number was calculated according to the following formula: copy number (copies/μl) = na (copies/mol) × concentration (g/μl)/mw (g/mol) [ ] . the mpcr method was used to detect ccov, cpv- , cav, prv, cdv and cpiv. nucleic acids were extracted from cav, cpv- , cpiv, cdv, ccov and prv, and the extraction products of cdv, ccov and cpiv were reverse transcribed into cdna using the special primer ccov-r (table ) and m-mlv reverse transcriptase (promega, usa) according to the manufacturer's protocol. the nucleic acid extraction products of cpv- , prv, and cav were used directly as pcr templates. in contrast, the viral rna extraction products of ccov, cdv, and cpiv required reverse transcription prior to use as templates. deionized water was used as a negative control. the concentrations of the three plasmids (pmd-c, pmd-p and pmd-a) were measured using a nanodrop spectrophotometer (thermo scientific, usa). the copy number was calculated based on the plasmid concentration. the mixture of the three plasmids (pmd-c/pmd-p/pmd-a) and each individual plasmid (pmd-c, pmd-p and pmd-a) were diluted from × to × copies/μl and used to determine the minimum detection limit of the mpcr method. this experiment was used to verify the broad-spectrum ability of this method to detect the different genotypes of the three viruses. in this experiment, we detected cpv- a, cpv- b, cpv- c, ccov-ii, cav- and cav- , respectively. the mpcr products were evaluated by . % agarose gel electrophoresis. a nested pcr method for the detection of ccov [ ] , a pcr method for the detection of cpv- [ ] , and a pcr method for the detection and differentiation of cav [ ] were used as the routine pcr methods for comparison with the mpcr method. the details of the primers used are provided in table . the protocols were the same as the protocols described in the references. during the period from april to september , a total of rectal swabs ( ml) from dogs with diarrheal symptoms were collected from five pet hospitals in jilin province, china. all swab samples were used for the primary application of the mpcr method. each swab sample was also evaluated using the routine pcr methods for the three viruses. the viral swab storage solutions were centrifuged at , rpm for min, and the nucleic acids in the supernatant were extracted for detection via mpcr and routine pcr in triplicate. to confirm the results, all specific fragments amplified by mpcr were sequenced by the sangon biotech company (beijing, china). the best reaction conditions were determined by optimizing the annealing temperature, primer concentration, extension time, and cycle number. seven mixed combinations of the three viral templates were used to verify the mpcr, and the products were visualized by . % agarose gel electrophoresis (fig. ) . the specificity of the mpcr method was determined by detecting ccov, cpv- , cav, prv, cdv and cpiv. the results showed amplified bands only in the three lanes for ccov, cpv- and cav, whereas the lanes for prv, cdv and cpiv did not show amplified bands (fig. ) . these findings indicated that the mpcr method has good specificity. the sensitivity test results showed that the minimum detection limit for mpcr was × viral copies of each virus when the mixed plasmids (pmd-c/pmd-p/pmd-a) were used as the template (fig. ). when only a single plasmid was used for detection, the minimum detection limits for pmd-c, pmd-p and pmd-a were × , × , and × viral dna copies, respectively (fig. ) . the broad-spectrum ability of this method to detect specific genotypes of these three viruses was tested (fig. ) , and it was found that cpv- a, cpv- b, cpv- c, ccov-ii, cav- and cav- all can be detected by the mpcr method. the statistics of the clinical samples evaluated using mpcr and routine pcr are shown in table . the ratio of positive samples to total samples for cpv- , ccov and cav was recently, animal hospitals have experienced more cases of viral enteritis in dogs, and these cases have primarily been caused by ccov, cav and cpv- . the clinical symptoms of these three viruses are similar, and cases of illness are often caused by a mixed infection. several detection methods for these three viruses exist [ , , ] , but no single method can detect the presence of all three viruses together. as a result, the ccov, cpv- and cav must be detected individually, which is not only cumbersome but also a waste of time and reagents. in this study, an mpcr method for cpv- , ccov and cav was established and applied. this method can detect all three viruses together, therefore simplifying the operation steps, shortening the detection time, and lowering the cost. primer design is the most important step in the process of establishing the method and must satisfy the following conditions: the use of conserved sequences, the appropriate combination of amplicons, similar annealing temperatures, and the lack of dimers or card structures. in this method, the primer combination produced amplicons of bp, bp and bp, which were easy to distinguish from one another. in addition, the three pairs of primers had similar annealing temperatures, which needed to be sufficiently high to avoid nonspecific bands, and °c was found to be sufficient. the specificity of the primers was verified using ncbi primer-blast and, the dnastar software was used to check whether dimers or hairpin structures could form between the three primer pairs. the specificity of the mpcr method was evaluated using other common canine viruses (prv, cdv and cpiv) and found not to produce cross-reactions or nonspecific reactions with these viruses. the mpcr method used a specific primer as the reverse transcription primer, which improved the specificity of the method. the sensitivity of the mpcr results was × viral copies, which is less than that of the routine pcr method. the mpcr method is based on a single pcr method. therefore, although this method has the advantages of simple operation, time savings, and low cost, there are inherent weaknesses of sensitivity due to the amount of competitive inhibition between the amplicons, which renders the sensitivity of the mpcr lower than that of single pcr [ , , ] . therefore, our expectation of the mpcr effect was that when the viral titer of each virus in the sample exceeded the sensitivity of the mpcr method, all viruses would be detected. a total of rectal swabs from dogs with diarrheal symptoms were evaluated using mpcr and routine pcr; the results obtained by sequencing are shown in table . the ratio of positive samples to total samples for cpv- , ccov and cav was . % ( / ) by mpcr and . % ( / ) by routine pcr. thirty-five positive samples were detected by both methods, for a coincidence ratio of %, indicating that the specificity and sensitivity of the method was sufficient for testing clinical specimens. in summary, the mpcr established in this study is an efficient, inexpensive, specific, and accurate method for detecting ccov (ccov-ii), cav (cav- , cav- ) and cpv- (cpv- a, cpv- b, cpv- c). this study provides a new tool for clinical diagnosis and laboratory epidemiological investigations. a multiplex pcr for viruses associated with exanthematic and vesicular disease in cattle pathogenesis of canine parvovirus- in dogs: haematology, serology and virus recovery multiplex pcr: optimization and application in diagnostic virology development and application of a multiplex pcr method for rapid differential detection of subgroup a, b, and j avian leukosis viruses association of a type- canine adenovirus with an outbreak of diarrhoeal disease among a large dog congregation detection and differentiation of cav- and cav- by polymerase chain reaction isolation of canine adenovirus- from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping canine parvovirus: the worldwide occurrence of antigenic variants recent epidemiological status of canine viral enteric infections and giardia infection in japan pathogenesis of feline panleukopenia virus and canine parvovirus severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type and canine coronavirus development of a nested pcr assay for the detection of canine coronavirus detection by pcr of wild-type canine parvovirus which contaminates dog vaccines canine coronavirus infection in the dog following oronasal inoculation co-circulation of canine coronavirus i and iia/b with high prevalence and genetic diversity in heilongjiang province establishment and application of a multiplex pcr for rapid and simultaneous detection of six viruses in swine a duplex real-time reverse transcription polymerase chain reaction for the detection and quantitation of avian leukosis virus subgroups a and b acknowledgements this work was supported by the national key research and development program of china ( yfd ). we express our sincere gratitude to jia-li zhang at the institute of animal hospital of jinlin agricultural university for assistance with clinical sample collection. funding this study was funded by the national key research and development program of china ( yfd ). ethical approval this article does not contain any studies with human participants or animals performed by any of the authors.informed consent informed consent was obtained from all individual participants included in the study. key: cord- -rpv oy authors: park, jung-eun; cruz, deu john m.; shin, hyun-jin title: receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: rpv oy porcine epidemic diarrhea virus (pedv) infection in vero cells is facilitated by trypsin through an undefined mechanism. the present study describes the mode of action of trypsin in enhancing pedv infection in vero cells during different stage of the virus life cycle. during the viral entry stage, trypsin increased the penetration of vero-cell-attached pedv by approximately twofold. however, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon pedv infection. trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. furthermore, we also show that the pedv spike (s) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. these findings indicate that trypsin affects only cell-attached pedv and increases infectivity and syncytium formation in pedv-infected vero cells by cleavage of the pedv s protein. these findings strongly suggest that the pedv s protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion. porcine epidemic diarrhea virus (pedv), a member of the family coronaviridae, is an economically important pathogen of swine. pedv causes acute watery diarrhea, resulting in approximately % mortality among suckling piglets and reduced weight among fattening pigs [ ] . although the structural and pathological properties of pedv are similar to those of other group coronavirus, including human coronavirus e (hcov- e), transmissible gastroenteritis virus (tgev) and feline infectious peritonitis virus, many biological issues, such as the role of trypsin in infection, remain unresolved [ , , ] . the first successful propagation of pedv in cell culture was done by supplementing the vero cell culture medium with trypsin [ ] . the addition of trypsin was shown to induce fusion of the infected vero cells, resulting in the formation of multiple syncytia, and produced a significant increase in virus titer after several passages. soon afterwards, several other groups performed pedv infection in vitro using the same conditions and reported similar findings [ , ] . on the other hand, another study reported the successful propagation of the p- v strain in porcine enterocyte cell lines without trypsin supplementation of the medium, suggesting that the proteolytic processing of pedv in enterocytes may have occurred during maturation or prior to virus release [ ] . the spike (s) glycoprotein is the dominant surface protein in coronaviruses. the protein is responsible for virus attachment and fusion. the requirement of proteinase-cleaved s glycoprotein has been reported for almost all group and coronavirus. for example, infection by severe acute respiratory syndrome coronavirus (sars-cov) and murine hepatitis virus strain (mhv- ) requires proteolytic cleavage in their target cells, which is mediated by trypsin-like proteases [ , , ] . the situation for group coronavirus is unclear. recently, the s protein of the group coronavirus hcov- e was reported to be cleaved by treatment with cathepsin l and trypsin, which prompts the fusion of the viral envelope and the cell membrane, similar to sars-cov [ ] . the present study reports the putative role of trypsin in cell-adapted pedv infection of vero cells. trypsin treatment was performed in the early and late stages of viral infection, and its influence on viral titer and syncytium formation was examined. furthermore, the effect of trypsin on the s protein was compared in free and receptor-bound virions. the results suggest that trypsin activity is involved mainly with receptor-bound s protein of pedv, leading to the conclusion that the effect of trypsin on pedv is similar to that of the group coronaviruses, sars-cov and mhv. african green monkey kidney cells (vero, ccl- ) were prepared in minimum essential medium (mem, gibco) supplemented with % fetal bovine serum (fbs, gibco). the cell-adapted strain of the korean pedv isolate, kpedv- , was propagated as described elsewhere [ ] , with some modifications. briefly, vero cells were inoculated with kpedv- at a multiplicity of infection c and cultured in serum-free mem at °c, % co for - h. the supernatant was harvested and then clarified by centrifugation at , g for min at °c. concentration and partial purification were performed by ultracentrifugation under a % sucrose cushion at , rpm for . h. the pellet was resuspended in mm phosphate-buffered saline (pbs, ph . ) and stored at - °c. mouse polyclonal antibodies against pedv were generated by immunizing -week-old female balb/c mice (samtako) intraperitoneally with focus-forming units (ffu) of purified kpedv- emulsified in an equal volume of complete freund's adjuvant (sigma-aldrich) on day and incomplete freund's adjuvant (sigma-aldrich) on days , and . whole blood was collected from the retro-orbital sinus on days and and centrifuged at g for min to separate the sera. cultured vero cells were inoculated with kpedv- as described above and allowed to adsorb for h at °c. the vero cells were washed twice with pbs and cultured in serum-and trypsin-free mem or mem containing trypsin ( lg/ml, sigma-aldrich). at , , , and h postinoculation (hpi), culture supernatants were collected for titration in a focus-formation assay, and cells were fixed with % formaldehyde in pbs for min and permeabilized with % np- (sigma-aldrich) in pbs, followed by immunocytochemistry using mouse anti-pedv polyclonal sera [ ] . clusters of infected cells staining dark gray were counted under an inverted microscope and reported as ffu. trypsin treatment at various stages of virus infection cells or viruses were treated with trypsin at various stages of virus infection as described in fig. . to investigate the effects of proteolytic cleavage of the surface protein of vero cells and free virions by trypsin, vero cells or kpedv- were pre-treated with trypsin prior to infection. trypsin treatment was performed for min at °c prior to inoculation, and enzyme activity was neutralized with lg/ml aprotinin (sigma-aldrich). trypsin-pretreated vero cells were inoculated with kpedv- , and untreated vero cells were inoculated with trypsin-pretreated kpedv- . after a -h incubation to allow adsorption, cells were washed three times with pbs and then cultured in serum-free mem without trypsin for h. in another experiment, trypsin treatment was carried out during the virus adsorption stage to determine whether trypsin is involved in the entry of kpedv- . vero cells were inoculated with kpedv- in the presence of various concentrations of trypsin ( , , , and lg/ml) during the adsorption period and were cultured in serum-free mem at °c for h. to investigate the effect of trypsin on the budding stage of pedv infection, kpedv- -infected vero cells were prepared by inoculating them with purified kpedv- and then cultured in mem for h. prior to trypsin treatment, the cell monolayer was washed three times with pbs to remove residual fbs and released virions, prior to treatment with trypsin for min. after neutralization of trypsin by the addition of aprotinin, cells were cultured for an additional h. the culture supernatants were harvested for virus titration, and cells were fixed for immunocytochemistry at the indicated times. virus-infected cells were detected by probing with mouse anti-pedv polyclonal antisera and biotinylated rabbit antimouse igg and visualized by treatment with streptavidinbiotinylated horseradish peroxidase (vector labs) followed by , '-diaminobenzidine tetrahydrochloride dihydrate (dab, vector labs). all specimens were observed under an inverted microscope. ultrapurified kpedv- was treated with various concentrations of trypsin at room temperature (rt) for min and then analyzed by western blotting. vero cells were infected with kpedv- in the absence of trypsin for h, and kpedv -infected vero cells then were harvested and treated with trypsin for min at rt. mock-infected trypsin-treated vero cells were used as a negative control. samples for western blot analysis were treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoresed in an % sds-page gel system. the separated proteins were electrically transferred onto a polyvinyl difluoride membrane (amersham bioscience). the antibodies used in this study were mouse anti-pedv polyclonal antibodies against pedv s protein, and monoclonal anti-b-actin-peroxidase (sigma-aldrich). the bands were visualized using supersignal west dura (pierce) with las- plus (fujifilm). statistical analysis was performed using spss, version . , for windows. correlation coefficients were calculated using pearson's correlation coefficient. error bars represent the standard deviations from at least three replicates. trypsin is not essential for pedv infection pedv propagation in vero cells results in low infection rates, even after subsequent passages in the absence of trypsin supplementation [ ] . however, with the addition of trypsin, virus adaptation to vero cells increased, and a prominent cytopathic effect (cpe) marked by formation of syncytia was observed in subsequent passages. following this observation, the growth rate of kpedv- in vero cells in the presence or absence of trypsin supplementation was compared. as shown in fig. , detectable levels of progeny virions were observed from hpi in both trypsin-and nontrypsin-supplemented media. at hpi, the titer was higher ( . ffu/ml) in trypsin-supplemented samples than in non-trypsin-supplemented samples ( . ffu/ml). the rate of virus production in trypsin-supplemented cultures was also significantly higher than in trypsin-free cultures (virus titer of . and . ffu/ml at hpi, respectively). even at hpi, the titer in the trypsinfree cultures only reached peak titer levels of . ffu/ml, which was significantly lower than the peak titer attained at hpi in trypsin-supplemented cultures. these results are consistent with the suggestion that trypsin is not absolutely essential for vero-cell-adapted pedv infection, as reported for other group coronaviruses, but the titer increases during infection with trypsin-treated virus. trypsin mediates the penetration of cell-attached pedv to investigate how trypsin enhances pedv infectivity of vero cells, trypsin was added during various stages of infection. trypsin treatment of kpedv- prior to inoculation did not significantly differ from non-trypsin-treated virus after hpi ( . and . ffu/ml, respectively) (fig. ) . this suggests that proteolytic processing of the surface glycoprotein by trypsin prior to receptor binding does not have a significant effect on enhancing infectivity. to determine whether trypsin interaction with vero-cell-surface proteins contributes to enhanced pedv infectivity, vero cells were pre-treated with lg/ml trypsin for min before inoculation. this treatment did not significantly alter virus titer when compared to the untreated cells. interestingly, addition of trypsin immediately after inoculation during the absorption to investigate the mechanism of trypsin in more detail, vero-cell-bound pedv was treated with trypsin. vero cells were inoculated with kpedv- in serum-and trypsin-free media for h and then washed twice to remove un-bound kpedv- . the cell-bound kpedv- was treated with different concentrations of trypsin for min, and the titers of penetrating and produced virus were determined. when the concentration of trypsin in the medium was increased from lg/ml to lg/ml, the number of pedv penetrating the vero cells also increased from ffu/ml to ffu/ml. the enhanced trypsin-mediated penetration during initial infection resulted in an increase in virus titer at hpi, from . ffu/ml to . ffu/ml (fig. ) . these findings are consistent with the notion that trypsin activity during the initial stage of virus infection enhances the efficiency of virus penetration into vero cells, thereby increasing viral infectivity. although the penetration of cell-attached virions was facilitated by trypsin treatment, virions treated with trypsin before cell attachment did not show any difference when compared to the results obtained in the absence of trypsin. based on these findings, it is appropriate to suggest that trypsin might only affect the receptor-bound spike, inducing fusion between the cell membrane and the virus envelope, leading to increased virus penetration. to investigate the role of trypsin on the late stage of infection and syncytium formation, kpedv- -infected vero cells were prepared by inoculation for h. the cells were washed extensively and treated with various concentrations of trypsin for min prior to continuing cell cultivation in fresh serum-and trypsin-free medium. kpedv- -infected vero cells did not show visible signs of syncytium formation in the absence of trypsin, while kpedv- -infected vero cells treated with , and lg/ml trypsin at hpi contained multiple syncytia (fig. a) . without trypsin treatment, the virus titer at hpi was . ffu/ml while kpedv- -infected vero cells treated with , , , and lg/ml trypsin showed virus titers of . , . , . , . and . ffu/ml, respectively (fig. b) . in virus budding stage, trypsin also activated syncytium formation of infected vero cells and consequently increased virus infectivity. newly packaged virions budding from infected vero cells could be activated by trypsin, which caused the infected vero cells to form syncytia. this finding was consistent with the previous results shown in figs. and . the collective results supported the idea that trypsin acts on cell-attached virions, both during virus attachment and during virus release and induces membrane fusion between the host-cell membrane and the virus envelope, and also between host-cell membranes. cleavage of receptor-bound s protein by trypsin the s protein from ultrapurified virions and receptor-bound virions was treated with trypsin and analyzed by western blotting. s protein from both ultrapurified virions and kpedv- -infected vero cells that had not been treated with trypsin was apparent as a species of about kda, which represented the glycosylated native s protein (fig. ) . in ultrapurified virus, only this protein species was detected, even after trypsin treatment, while -kda and -kda proteins, likely trypsin-cleaved s protein, were detected in trypsin-treated kpedv- -infected vero cells (fig. ) . the findings supported the suggestion that pedv s protein has a site that is highly sensitive to trypsin cleavage to produce two fragments, as has been reported for other coronaviruses [ , , ] . however, this fig. enhancement of cellattached kpedv- penetration by trypsin. vero cells were inoculated with kpedv- for h and then washed to remove unbound kpedv- . only cellattached kpedv- was treated with trypsin for min, and penetrated virus (j) and progeny virus were titrated after h (h). increasing the amount of trypsin added during virus adsorption resulted in increased virus penetration into vero cells during initial entry and higher virus titers after hpi cleavage only occurred when the virus was associated with receptor protein. several enterotropic or pneumotropic viruses, such as those belonging to the families orthomyxoviridae and paramyxoviridae, undergo proteolytic cleavage of their surface glycoprotein prior to entry into host cells to facilitate virus penetration by activating the fusion domain of the surface glycoprotein [ , ] . the activated fusion protein undergoes conformational changes that induce fusion of the viral envelope and host membranes [ , , ] . this process usually occurs during the period between virus maturation and virus attachment to the host receptors [ , ] . after the fusion process, the viral core, including the viral genome, is transported into the cytoplasm where uncoating and replication ensue [ ] . in natural infections, the viral surface glycoproteins that are not cleaved during maturation are subsequently cleaved by exogenous proteases secreted from host pancreas, liver and bronchiolar epithelia [ ] . in cell culture, the protease cleavage that is required for virus propagation is carried out by exogenous proteases such as trypsin or pancreatin [ , , ] . these exogenous proteases induce syncytium formation by activating the fusion domain of the viral glycoprotein expressed on the surface of infected cells [ , ] . several studies of different coronaviruses have shown that proteolytic cleavage of the s protein enhances viral infectivity. for mhv, separation of the s and s subunits enhances the fusion activity of the s subunit and increases viral infectivity [ , ] . mutations that alter the furin protease recognition sequence (rxr/kr) located at the junction of the s and s subunits as well as treatment with a peptide furin inhibitor prevent the proteolytic cleavage of the s protein, resulting in reduced cell-cell fusion activity, although viral entry is not significantly affected [ , , ] . conversely, the addition of trypsin to the culture medium can enhance the fusion of mhv-infected cells [ ] . in contrast to mhv, the s protein of sars-cov does not show any evidence of proteolytic maturation to cleaved s and s subunits in mature virions [ ] . instead, proteolytic cleavage of the s protein on the surface of infected cells occurs by exogenous proteases, mediating cell-cell fusion [ , ] . similar to sars-cov, the s protein in most group coronaviruses also does not exhibit cleaved s and s subunits during virus maturation and biogenesis [ ] . several studies on tgev and pedv have used trypsinsupplemented culture media to induce cpe by syncytium formation in st cells and vero cells, respectively [ , ] . furthermore, in the case of pedv, trypsin facilitates successful propagation in vero cells as well as other primate cell lines [ , ] . however, the role of cellular and exogenous proteases on the cell entry of group coronaviruses, particularly pedv, as well as the putative proteolytic cleavage site on the s protein, remains unclear. based on the present results summarized in figs. , and , enhancement of virus penetration and cellcell fusion induced by the addition of trypsin suggests that the pedv s protein may also be cleaved into s and s subunits during the course of infection. electrophoretic examination of purified virions resulted in the detection of the pedv s protein as a monomer of about kda in the absence of trypsin, while the protein was detected as two fragments of kda and kda in the presence of trypsin (fig. ) . it may be that the pedv s protein on native virions adopts a conformation that protects it from various exogenous proteases, but the s protein attached to the host receptor protein may undergo a conformational change that exposes a trypsin cleavage site. previous reports have described the formation of syncytia by pedvinfected cells only upon addition of trypsin in the culture medium, and sequence analysis of the pedv s protein has revealed the absence of the rrx(r/h)r motif, which is associated with cleavage into the s and s subunits [ ] . this suggests that the pedv surface glycoprotein does not undergo proteolytic processing upon maturation and release. conversely, the observation that trypsin can induce temperature for min. pedv s was detected using anti-pedv polyclonal antibodies raised in mice. uncleaved s protein and cleaved s protein are indicated by black and white arrows, respectively cell-cell fusion in pedv-infected cells suggests that proteolytic processing of the s protein by exogenous trypsin may augment viral entry by facilitating fusion of the viral membrane with the host membranes [ , ] . it seems that the timing of the cleavage of the s protein by trypsin is critical for the activation of fusion activity. as shown in fig. , early activation of the s protein before binding to cellular receptors did not enhance viral entry into the host cell, while addition of trypsin shortly after receptor binding increased the efficiency of virus entry. while the s protein in infected cell lysates and receptorbound virions was cleaved into two fragments, the s protein cleavage in pedv was different from that of other coronaviruses, as pedv s protein was only cleaved when associated with its host cell. this implies that cleavage of the s protein by trypsin occurs only when it is bound at the surface of host cells to the host receptor protein, which presumably induces a conformational change in the bound s protein. this conformational change might expose a trypsin cleavage site. cleavage of the s protein could result in membrane fusion. it would be of interest to determine the nature of the conformational change that is involved and the location of the s protein cleavage site. in summary, the present results reveal the role and importance of trypsin in pedv infection of vero cells. trypsin is not essential for pedv infection but enhances its infectivity and cpe formation. trypsin cleaves pedv s protein only when it bound its cell receptor and in the later stages of infection. the association of the s protein with the host receptor could induce conformational changes that expose a trypsin cleavage site(s). the resulting cleavage might expose or 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entry changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion transmissible gastroenteritis (tge) of swine: effect of age of swine testes cell culture monolayers on plaque assays of tge virus proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cellfusing activity of virions by trypsin and separation of two different k cleavage fragments evolution and ecology of influenza a viruses nucleotide sequence and expression of the spike (s) gene of canine coronavirus and comparison with the s proteins of feline and porcine coronaviruses the sars-cov s glycoprotein: expression and functional characterization porcine epidemic diarrhea virus (cv ) and feline infectious peritonitis virus (fipv) are antigenically related key: cord- -h smg w authors: takano, tomomi; yanai, yoshitomo; hiramatsu, kanae; doki, tomoyoshi; hohdatsu, tsutomu title: novel single-stranded, circular dna virus identified in cats in japan date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: h smg w we detected a novel feline stool-associated circular dna virus (fescv) in fecal samples from cats with diarrhea using consensus primers matching those of circovirus and cyclovirus. fescv is a circular dna virus containing a genome with a total length of , nt encoding open reading frames. phylogenetic analyses indicated that fescv is classified into a clade different from that of circovirus and cyclovirus. since the fescvs detected in several cats in the same household had genetically similar genomes, these viruses are most likely derived from the same origin. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. rcr iii) are commonly present [ ] . circoviruses include porcine circovirus , which causes post weaning multisystemic wasting syndrome in pigs [ ] , beak and feather disease (bfd) virus, which causes bfd in psittacines [ ] , and canine circovirus (cacv), which causes vasculitis and gastroenteritis in dogs [ ] . cycloviruses include human cyclovirus, which is associated with human acute flaccid paralysis [ ] , and bat cyclovirus isolated from bat feces [ ] . in cats, no circoviridae family other than feline cyclovirus has been reported. feline cyclovirus was identified by next-generation sequencing analysis in which the viral gene was detected in a pooled fecal sample collected from - healthy cats. its pathogenicity in cats has not been clarified [ ] . in this study, we performed nested pcr using circoviridae family consensus primers and detected novel cress dna viruses in several cats with diarrhea symptoms. the full genomic sequences of these viruses were clarified, and a phylogenetic analysis was performed. fecal samples were collected from cats at a private cattery in japan. these cats were maintained under conditions in which they had contact with each other. fourteen of the cats developed diarrhea and did not respond to anthelmintics or antibiotics. the remaining cats were healthy. the cats with diarrhea were separated from the other cats, and newly-collected feces were subjected to experiments. feline coronavirus was detected in fecal samples from healthy cats and cats with diarrhea. in addition, feline bocavirus was detected in fecal samples from cats with diarrhea ( we named the virus detected in this study feline stoolassociated circular virus (fescv). to determine the complete genomic sequence of fescv, inverse primers (cv f '-ttc tcc cga cct gga cat ag and cv r '-aca gag atg ata gcg tcc gg) were prepared based on a section of the base sequence of the acquired rep gene, and inverse pcr was performed. fig. s shows the schematic position of the inverse pcr primers and other primers used for the fescv genome. prior to inverse pcr, circular dna was multiplied using templiphi tm amplification kit (ge healthcare, usa). viral dna was amplified by inverse pcr, which was performed in a total volume of μl using the following mixture: μl of multiplied circular dna mixed with μl of x primestar buffer (takara, japan), μl of dntp mixture (takara, japan) containing . mm of each dntp, μl of μm primer mix (fc f), . μl of primestar hs dna polymerase ( . u/ml; takara, japan), and . μl of distilled water. using a thermal cycler, the dna was amplified at °c for min followed by cycles of denaturation at °c for sec, primer annealing at °c for sec, and synthesis at °c for min with a final extension at °c for min. from all samples, approximately , -bp amplicons were acquired. the sequences of the viral genomes were obtained by sequencing the overlapping pcr products (amplicons of and , bp in size). phylogenetic trees based on rep were analyzed using mega software (version ). phylogenetic relationships were evaluated using the neighbor-joining algorithm, and branching order reliability was analyzed by , replications of bootstrap resampling. all strains of fescv were circular dna viruses containing a genome (fig. b) . of the viruses that infect eukaryotic organisms, those most similar to fescv were rodent stool-associated circular viruses (rodscv). of note, the putative rep and putative cap of fescv were approximately % homologous with rna helicase and the hypothetical protein of giardia intestinalis, respectively. the fescv genomes were submitted to genbank under accession numbers lc (ku ), lc (ku ), lc (ku ), and lc (ku ), respectively. based on the base sequence of fescv clarified in this study, specific primers to detect the fescv gene (fescv f '-gct aag gtc tgc ctc agg tg and fescv r '-cta tgt cca ggt cgg gag aa) were prepared. pcr was performed as described above except the reaction temperature and time were modified as follows: dna was amplified at °c for min followed by cycles of denaturation at °c for sec, primer annealing at °c for sec, and synthesis at °c for sec with a final extension at °c for min. when an amplicon with the expected size of bp was detected, the sample was regarded as fescv-positive. of the cats with diarrhea, were fescv-positive ( . %). three of the healthy cats were fescv-positive ( . %) ( table ; see fescv-specific primer). to detect feline circovirus or feline cyclovirus in feline fecal samples, nested pcr using circoviridae consensus primers was performed. the degenerate primers used in table highly conserved motifs of rep detected in fescv based on the report by rosario et al. [ ] x: any amino acid u: hydrophobic amino acid (f, i, l, v, or m) this nested pcr assay were prepared based on the consensus sequence of the rep gene in strains of circoviruses and one strain of cyclovirus [ ] since we expected only circoviruses and cycloviruses to be detected. however, a novel cress dna virus, fescv, was detected in the fecal samples. phylogenetic analysis based on rep revealed that fescv belongs in a clade different from that of circoviruses and cycloviruses for which the reason is unclear. very few known cress dna viruses, including circoviruses, have been confirmed to have pathogenicity in the host. moreover, many studies on novel cress dna viruses analyzed only viral genes, that is, only a few studies analyzed whether the viral infection is manifested in the host. the fescv found in this study was detected in of the cats. based on this result, cats may be the natural host of fescv. furthermore, the fescvs (ku strain, ku strain, ku strain, and ku strain) are genetically similar and share approximately % of their genomes. this data suggests that these fescvs have the same origin. the fescv infection rates in healthy cats and cats with diarrhea were . and . %, respectively and demonstrate that although the infection rate was slightly higher in cats with diarrhea, half of the healthy cats were also infected with fescv. thus, it is unclear whether fescv is pathogenic. furthermore, the rate of co-infection with fescv and other viruses was approximately -fold higher in cats with diarrhea ( . %) than in healthy cats ( . %). in dogs, a relationship between cacv and gastroenteritis has been suggested. dowgier et al. [ ] reported that cacv infection and the development of acute gastroenteritis were correlated in dogs co-infected with other enteric viruses. the incidence of enteritis may increase when fescv-infected cats are co-infected with other viruses. further investigation of fescv pathogenicity in cats is necessary. the putative rep of fescv was % homologous with rna helicase of giardia intestinalis in blastx analysis, and the putative cap of fescv was approximately % homologous with a cysteine protease (hypothetical protein) of giardia intestinalis. we first suspected that the fescv gene identified in this study was derived from giardia intestinalis. however, we concluded that fescv is a circular dna virus based on the following: ) no giardia intestinalis was detected in the fecal test, and ) the complete genome of fescv was amplified using the rolling-circle amplification and inverse pcr assays. similar with fescv, rodscv is reported to have rep with < % homology with rna helicase of giardia intestinalis [ ] . moreover, both the putative rep and putative cap of fescv are similar to proteins derived from giardia intestinalis. to our knowledge, no study has reported a gene encoding cap or putative cap of cress dna virus generated by recombination with parasitic or bacterial genes. further investigation is necessary for the origin of the putative cap of fescv. a region presumed to be a classical nls was detected in the putative cap of fescv. as circoviruses lack dna polymerase, virus particles have to transfer into the nucleus for viral dna replication [ ] . in bfdv and duck circoviruses, viral dna may by transported into the host cell nucleus via nls-containing viral cap [ , ] . it is unclear whether the putative cap of fescv functions similar to the cap of bfdv and duck circoviruses. further studies regarding the intracellular localization of fescv putative cap is required. we detected a novel cress dna virus, fescv, in fecal samples from cats. although it was detected using consensus primers of circovirus and cyclovirus, fescv was phylogenetically positioned in a clade different from that of these viruses. fescv was detected in several cats that were housed together, suggesting that fescvs are derived from the same origin. in addition, fescv is considered to cause diarrhea in cats via co-infection with other enteric viruses. therefore, it is necessary to perform a large-scale epidemiological survey and clarify whether diarrhea is associated with fescv infection. consensus statement: virus taxonomy in the age of metagenomics time-dependent rate phenomenon in viruses characterization of the genomic sequence of a novel cress dna virus identified in eurasian jay (garrulus glandarius) identification and genetic characterization of a novel circular singlestranded dna virus in a human upper respiratory tract sample plasma virome of cattle from forest region revealed diverse small circular ssdna viral genomes pervasive chimerism in the replication-associated proteins of uncultured singlestranded dna viruses revisiting the taxonomy of the family circoviridae: establishment of the genus cyclovirus and removal of the genus gyrovirus molecular evolution of porcine circovirus type genomes: phylogeny and clonality evidence for specificity of psittacine beak and feather disease viruses among avian hosts circovirus in tissues of dogs with vasculitis and hemorrhage novel cyclovirus in human cerebrospinal fluid bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses faecal virome of cats in an animal shelter multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces a molecular survey for selected viral enteropathogens revealed a limited role of canine circovirus in the development of canine acute gastroenteritis the fecal viral flora of wild rodents replication of porcine circoviruses structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal identification of two functional nuclear localization signals in the capsid protein of duck circovirus acknowledgements this work was in part supported by mext/jsps kakenhi grant number jp k . conflict of interest all authors declare that have no conflict of interest.ethical approval all applicable international, national, and institutional guidelines for the care and use of animals were followed by the authors. samples were obtained from an animal hospital, and this study does not include animal experiments. key: cord- - yafd d authors: tsunemitsu, h.; saif, l. j. title: antigenic and biological comparisons of bovine coronaviruses derived from neonatal calf diarrhea and winter dysentery of adult cattle date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: yafd d the antigenic and biological properties of strains of bovine coronavirus (bcv) derived from neonatal calf diarrhea (cd) and strains of bcv from winter dysentery (wd) of adult cattle, propagated in hrt- cells, were compared to determine if cd and wd strains belong to distinct serotypes or subtypes of bcv. all strains hemagglutinated both mouse and chicken erythrocytes at °c, but the ratios of hemagglutination titers with mouse erythrocytes compared to chicken erythrocytes showed diversity for both cd and wd strains. some cd and wd strains did not hemagglutinate chicken erythrocytes at °c and showed receptor-destroying enzyme activity against chicken erythrocytes. hyperimmune antisera were produced in guinea pigs against and strains of bcv from cd and wd, respectively. no significant differences in antibody titers against these strains were observed by indirect immunofluorescence tests. however, in virus neutralization tests, antisera to cd and wd strains had -fold or lower antibody titers against wd and cd strains than against the homologous strains, and this variation reflected low antigenic relatedness values (r= – %), suggesting the presence of different subtypes among bcv. in hemagglutination inhibition tests, some one-way antigenic variations among strains were also observed. these results suggest that some antigenic and biological diversity exists among bcv strains, but these variations were unrelated to the clinical source of the strains; i.e. cd or wd. which were concentrated approximately -to -fold. serial -fold dilutions of bcv were prepared in . ml of veronal buffered saline containing . % bovine serum albumin and . % gelatin and mixed with . ml of . and . % suspensions of mouse and pooled adult chicken erythrocytes, respectively, in the same buffer. the mixtures were then incubated for h at either or °c and the ha titers were determined. the plates incubated at °c were moved to °c for h to measure inactivation of receptors reflected by the disaggregation of the bcv-erythrocyte complexes mediated by the receptor destroying enzyme (rde) activity [ ] . antigenic comparisons of bcv strains were done by indirect if, virus neutralization (vn) and ha inhibition (hi) tests. the indirect if tests were performed as previously described [ ] . virus titration and vn tests were conducted with hrt- cells in microplates as previously described [ ] . virus infectivity titers were expressed as median tissue culture infective doses (tcids )/ml. the vn antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited cytopathic effects (cpe). the antigenic relatedness (r) between the strains was calculated using the formula [ , ] : in which rl is heterologous titer (strain )/homologous titer (strain ), and r is heterologous titer (strain )/homologous titer (strain ). the hi test was done using standard techniques with mouse erythrocytes [ ] and sera treated with kaolin and mouse erythrocytes. the antibody titers were expressed as the reciprocal of the highest serum dilutions producing complete hi. cytopathic effects were evident in hrt- cells inoculated with each of the strains of bcv. cytopathic effects were characterized by enlarged, rounded, and densely granular cells that occurred in clusters at to postinoculation days [ ] , and no differences were observed in cpe among these strains. syncytia were also clearly observed in hrt- cells at days after inoculation with these strains following staining with fluorescein isothiocyanate-conjugated anti-bcv serum. infectivity titers of bcv reached ° to - tcidsjml at the th to th passages on hrt- cells. the ha and rde titers of purified bcv strains are summarized in table . all strains agglutinated mouse erythrocytes and no differences were observed in ha titers against mouse erythrocytes at ° and °c. all strains also agglutinated chicken erythrocytes at °c, but the ha titers varied among the bcv strains. this diversity was reflected in variations of the ratios of ha titer with mouse erythrocytes to ha titer with chicken erythrocytes (m/c ha titer ratio) at °c. however there was no relation between m/c ha titer ratio and the clinical source (cd or wd) of the strains. at °c, the mebus and db strains of cd bcv and the dba and sd strains of wd bcv agglutinated chicken erythrocytes with the same ha titers as at °c. however, the other strains of bcv did not agglutinate chicken erythrocytes at °c, and showed rde activity against chicken erythrocytes. receptor-destroying enzyme activity with mouse erythrocytes was not uexpressed as the reciprocal of the highest dilution of virus causing complete disappearance of ha patterns at °c after h incubation at °c ° cd calf diarrhea, wd winter dysentery of adult cattle dm/c ratio of ha titer with mouse erythrocytes to ha titer with chicken erythrocytes detected for any strain of bcv. according to these results, bcv strains were classified into groups. the first group (cd isolates, mebus, db ; and wd isolates, dba, sd) showed low m/c ha titer ratios(<_ ), no differences in ha titers against chicken erythrocytes at and °c and no rde activity against chicken erythrocytes. the second group (cd isolate, xf; and wd isolates, cn, be, aw)showed low m/c ha titer ratios (< ), no ha against chicken erythrocytes at °c and rde activity with chicken erythrocytes. the third group (cd isolates, ohc, sdc, jaz; and wd isolates, ts, bm, bw) showed high m/c ha titer ratios (> ), no ha against chicken erythrocytes at °c and rde activity with chicken erythrocytes. these variations in ha and rde activities were unrelated to the clinical source of the isolates (cd or wd). in indirect if tests, all of the antisera reacted to each virus strain with high titer ( to ), and each antiserum showed no significant differences in reactivity with the homologous and heterologous strains (not greater than twofold differences). the results of vn tests are shown in table . all of the antisera neutralized the heterologous strains, showing that the strains were closely related antigenically. however, antisera to the mebus cd, and sd and bm wd strains showed -fold or lower vn antibody titers against the dba, ts, be and bw wd strains and the xf, jaz, ohc and sdc cd strains than against the homologous strains. these differences were reflected in the r% values: the mebus, sd and bm strains generated r % values of to against the dba, ts, be and xf strains. the hi antibody titers are shown in table . all of the strains showed crossreactivity, but differences in antibody titers were observed. the db strain of cd bcv and the sd strain ofwd bcv were closely related in the hi tests, and antisera to these strains distinguished most other strains with -fold or greater differences in the hi antibody titers. bovine coronavirus causes neonatal cd [ ] and is also associated with wd of adult cattle [ ] . based on epidemiological data, these disease syndromes often occur as separate and distinct outbreaks in herds [ , , ] ; hence antigenic or biological differences between cd and wd bcv might be expected. calf diarrhea bcv isolates belong to a single serotype [ , , ] , but minor antigenic and biological variations among them have been revealed in limited studies [ , , , , ] . in this study, we compared the antigenic and biological diversity of a variety of wd and cd bcv isolates. storz et al. [ ] reported that variations in the ratios of ha titers with mouse erythrocytes to those with chicken erythrocytes (m/c ha titer ratio in this report) ~expressed as the reciprocal of the highest dilution of serum inhibiting ha activity. homologous titers are in bold. titers which differed by -tbld or greater with homologous titers are underlined bcd calf diarrhea, wd winter dysentery of adult cattle were observed among cd bcv strains. the l strain of cd bcv at the high cell culture passage level (the th passage) showed a low mtc ha titer ratio ( ) whereas the wild type cd bcv strains at low cell culture passage levels (the rd to th passages) showed high m/c ha titer ratios ( to ). in this study, the high cell culture-passaged mebus strain of cd bcv (the th passage) showed a low m/c ha titer ratio ( ), but some low cell culture-passaged cd strains (db , xf) and wd strains (cn, dba, sd, be, aw) of bcv (the th to th passages) also showed low m/c ratios ( to ). the differences in ha titers against chicken erythrocytes at ° and °c showed good agreement with the rde titers for chicken erythrocytes. this suggests that comparison of ha titers obtained at ° and °c may provide an alternative method for evaluting rde activity. on the basis of ha and rde patterns, bcv strains were classified into groups. however, each group contained both cd and wd bcv and no relationship between each group and the clinical (cd or wd) or geographic origin of the strains was observed. all strains of both cd and wd bcv examined in this study were related antigenically. specially, each antiserum showed no significant difference between the homologous and heterologous strains in indirect if antibody titers. however, some antigenic diversity among bcv strains was observed by vn and hi tests. in our previous report [ ] , hyperimmune antiserum prepared in a gnotobiotic calf to the mebus strain of cd bcv had an -to -fold lower vn antibody titer against the dba strain ofwd bcv than against the homologous strain. in this study, guinea pig hyperimmune antiserum to the mebus strain showed similar vn antibody titer differences between homologous and the dba strains. in addition, this serum also distinguished three other strains ofwd bcv and four strains ofcd bcv from the homologous virus by -to -fold differences. moreover, hyperimmune antisera to the sd and bm strains ofwd bcv also distinguished the same strains which were distinguished by the anti-mebus serum, form homologous strains by -to -fold differences in antibody titers in vn tests. although these variations were recognized only in one-way reactions and all strains examined could be classified into a single serotype, the strains showing these variations might be further divided into subtypes. the mebus, sd and bm strains which belong to the same potential subtype could be distinguished from the dba, ts, be and xf strains, which constitute another possible subtype (r% values of to ). the bw, jaz, ohc and sdc strains also appeared to belong to the latter subtype. the db , cn and aw strains comprised an intermediate group that cross-reacted with both subtypes. interestingly, antiserum to the mebus strain of bcv which had been prepared in guinea pig in japan showed only a -fold lower vn antibody titer against the xf strain than against the homologous strain [ ] . in the present study, antiserum to the mebus strain prepared in the u.s.a. had -fold vn antibody titer differences against the homologous and xf strains. the reason for this discrepancy is unknown, but differences in the passage level of the mebus strain at the preparation of antiserum might affect the virus antigenicity [ ] . alternatively, contamination of cultures with other bcv might occur after import and propagation in japan. bovine coronavirus strains examined in this study showed minor antigenic and biological variations, but this diversity was unrelated to the geographic origin or affected animal age groups (wd and cd) from which these strains were recovered. based on preliminary data, gnotobiotic and colostrum-deprived calves inoculated orally and nasally with wd bcv shed bcv rectally and nasally and developed diarrhea, which was indistinguishable from disease symptoms in calves inoculated with cd bcv [ ] . also, a cow inoculated via a duodenal cannula with cd bcv developed diarrhea and shed bcv (h. tsunemitsu et at. ' unpublished data). these results suggest that the differences in these disease syndromes (wd and cd) are not related to viral factors, but to host and environmental factors; e.g. the immunological status of animals, environmental temperatures, secondary or coinfections with other pathogens, etc. [ , , ] . further studies are in progress to compare the antigenicity of bcv strains using monoclonal antibodies and in vivo cross-protection tests. persistent antigenic variation of influenza a viruses after incomplete neutralization in vivo with heterologous immune serum cell culture propagation ofa coronavirus isolated from cows with winter dysentery the enigma of winter dysentery bovine coronavirus comparison ofdifferent antigen preparations as substrates for use in passive hemagglutination and enzyme-linked immunosorbent assays for detection of antibody against bovine enteric coronavirus antigenic variations among calf diarrhea coronaviruses by immunodiffusion and counterimmuno-electrophoresis monoclonal antibodies to bovine coronavirus: characteristics and topographical mapping of neutralizing epitopes on the e and e glycoproteins a serological comparison of bovine coronavirus strains infection and cross-protection studies of winter dysentery and calf bovine coronavirus strains in gnotobiotic calves antigenic variation of porcine transmissible gastroenteritis virus detected by monoclonal antibodies comparison of bovine coronavirus (bcv) antigens: monoclonal antibodies to the spike protein distinguish between vaccine and wildtype strains an epidemiotogical study ofwinter dysentery in fifteen herds in france neonatal calf diarrhea: propagation, attenuation, and characteristics ofcoronavirus-like agents characterization of monoclonal antibodies to bovine enteric coronavirus and antigenic variability among quebec isolates studies on the relationship between coronaviruses from the intestinal and respiratory tracts of calves immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine a review of evidence implicating bovine coronavirus in the ethiology of winter dysentery in cows: an enigma resolved winter dysentery in dairy herds: electron microscopic and serological evidence for an association with coronavirus infection hemagglutination by calf diarrhea coronavirus the s protein of bovine coronavirus is a hemagglutinin recognizing -o-acetylated sialic acid as a receptor determinant coronaviruses: structure and genome expression monoclonal antibodies differentiate between the haemagglutinating and the receptordestroying activities of bovine coronavirus comparison ofhemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains epizootic diarrhea of adult cattle associated with a coronavirus-like agent isolation of bovine coronavirus from feces and nasal swabs of calves with diarrhea serologic evidence of coronavirus infection in new york and new england dairy cattle with winter dysentery human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses received january , key: cord- -kvcqayl authors: wei, zhan-yong; wang, xue-bin; ning, xiao-dong; wang, ya-bin; zhang, hong-ying; wang, dong-fang; chen, hong-ying; cui, bao-an title: nitric oxide inhibits the replication cycle of porcine parvovirus in vitro date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: kvcqayl this study investigated the inhibitory effect and mechanism of nitric oxide (no) on porcine parvovirus (ppv) replication in pk- cells. the results showed that two no-generating compounds, s-nitroso-l-acetylpenicillamine (snap) and l-arginine (la), at a noncytotoxic concentration could reduce ppv replication in a dose-dependent manner and that this anti-ppv effect could be reversed by the no synthase (nos) inhibitor n-nitro-l-arginine methyl ester (l-name). by assaying the steps of the ppv life cycle, we also show that no inhibits viral dna and protein synthesis. this experiment provides a frame of reference for the study of the anti-viral mechanism of no. compounds, and n-nitro-l-arginine methyl ester (l-name) was used as an no synthase (nos) inhibitor. these reagents were purchased from sigma chemical company. virus titer was determined using a plaque assay. the culture supernatants were harvested and diluted serially tenfold using rpmi medium. for passage in host cells, the cells were inoculated in -well plates with ll of diluted virus per well. the plate was then incubated for h at °c, the medium was removed, and the cells were washed twice with hank's medium. one milliliter of . % agar (about - °c) was added to each well. the plaques were counted after a -h incubation at °c. all experiments were done in triplicate. the concentration of no in the culture medium was determined by using an no reagent kit (jiangcheng co., china). in brief, -ll samples were used for assaying the stable end product, no -. all of the media were mixed in -well microplates, and the plates were incubated at room temperature for min. the reaction produced a pink color, which was measured at nm using a micro-elisa reader (model uv- , unico). viral dna was extracted from the culture supernatant using a dna extraction kit (takara, china) according to the manufacturer's protocol. the ppv vp gene was quantified using a real-time pcr that had already been established in our laboratory [ ] . a double-antibody (sandwich) elisa was used to quantify ppv antigen in the culture supernatants. the elisa procedure was performed as described by dong et al. [ ] . a sample was considered positive if the od was more than the mean background ? standard deviations. optimal dilutions of affinity-purified rabbit antibodies and mouse antibodies were determined for each batch prepared by box titration. results are presented as means ± standard error (se). the se was multiplied by an index that was determined by the degree of freedom for % confidence. statistical significance at p \ . was determined by either t test or rank analysis. to explore the inhibitory effect of no donors (snap or la) on ppv replication, different concentrations of snap (or la) were added to the cultures, which were infected with ppv at an moi of . as shown in fig. a , snap and la have the ability to inhibit ppv replication in pk- cells, and there is a dose-dependent relationship between the inhibitory effect and the snap (or la) concentration. a slight decrease in virus titer was observed with both lm snap and la (p [ . ), while the number of pfus in pk- cells was clearly reduced after treatment with lm of snap or la (p \ . ). however, in samples with lm of snap (or la) plus lm of l-name (a competitive inhibitor of nos), the number of pfus was higher than with snap (or la) treatment alone, although the virus titer was still lower than that of the control, indicating that l-name partially reversed the inhibitory effect of snap (or la) on ppv replication. to verify that the antiviral effect was caused by no released from the snap or la in the cultures, no figure b shows that the antiviral effects of snap or la appears to correlate with the amounts of no produced in the cultures with increasing snap or la concentration. treatment with snap or la plus l-name resulted in an inhibition of no production, with a rate of up to %. to exclude the possibility that the detected antiviral effect might have resulted from the toxicity of snap or la for the cells, we performed an mtt cell proliferation test. the results clearly excluded the possibility that the antiviral effect was due to general cytotoxicity of snap (or la) itself (fig. c) . based on the above results, we selected lm as the optimal snap or la concentration for further experiments. the inhibitory effect of snap or la on ppv infection of pk- cells was further investigated by determining the kinetics of ppv reproduction. the infected cells (moi = ) were treated with lm snap or la at different time points. as shown in fig. , the pfu value increased with later addition of snap (or la), and adding snap (or la) or h prior to viral infection resulted in the highest level of inhibition. to investigate whether no inhibits ppv dna replication, viral dna was isolated, and the partial vp gene was quantified by real-time pcr. as shown in fig. a , the amounts of viral dna in snap-or la-treated cells were reduced significantly compared to mock-treated samples (p \ . ). adding snap or la or h prior to infection resulted in a greater inhibition of the amount of viral dna produced than adding snap or la at the time of infection. there was less reduction when lm l-name was added. the kinetics of protein synthesis in infected pk- cells was analyzed. from the elisa results, we can see that snap or la ( lm) inhibited viral protein synthesis at each time point. the inhibitory rate was high (up to %) when snap or la was added h prior to infection (fig. b) , and the inhibitory rate became gradually lower with later addition of the drug. in addition, there was less reduction of protein synthesis when lm l-name was added. this study demonstrates that the antiviral effect of no was generated by the organic donors snap and la in the pk- cell line, reaffirming the antimicrobial capacity of no against a wide range of intracellular pathogens. furthermore, we also provide the first direct evidence that the inhibitory effect of no on the ppv life cycle occurs at the step of viral dna and protein synthesis. these results are important for the understanding of the pathogenesis of ppv, and the use of pk- cells to study the inhibitory our data demonstrated that the addition of snap and la inhibited ppv replication in pk- cells in a dose-dependent manner. this finding is consistent with previous reports in which severe acute respiratory syndrome coronavirus (sars-cov) and vesicular stomatitis virus (vsv) were studied [ , ] . although this reduction in viral replication is seen with no itself, it is not certain whether the antiviral effects of snap or la are actually due to some other norelated species. to address this question, three lines of evidence from our experiments indicate that the inhibition of ppv replication in pk- cells is most likely caused by no and not by other factors. firstly, snap and la could induce the release of no and inhibit ppv replication in a dosedependent manner (fig. b) . secondly, the addition of l-name, a nos inhibitor, could reduce no production in stimulated cultures, and consequently, the inhibition of ppv replication was reversed (fig. a) . finally, the result of the mtt assay clearly excluded the possibility that the antiviral effect of snap or la was due to the general cytotoxicity of snap or la itself (fig. c) . taken together, these results strongly suggest that no, generated from snap or la, could inhibit the replication of ppv in pk- cells. by investigating the time-of-addition effects of snap and la on anti-ppv in pk- cells, our data confirmed that when pk- cells were pretreated with snap or la h prior to infection, there was a maximal inhibitory effect on ppv replication (fig. ) . these results were similar to those of a previously study by rimmelzwaan et al. [ ] , which showed that addition of no donor h prior to infection significantly reduced the synthesis of both vrna and mrna. in contrast, some papers have indicated that pretreatment of n and sw cells with snap did not enhance anti-jev and anti-sindbis virus inhibition, respectively [ , ] . these differences in antiviral responses were probably due to the different natures of the viruses and cell lines in the experiments. more importantly, these phenomena are possible because of the different infection mechanisms of different viruses. our experiments demonstrate that no can inhibit ppv replication by blocking viral dna synthesis. this finding is consistent with previous studies [ , , ] . harris et al. [ ] found that no affects the late stages, including viral dna replication, viral protein synthesis, and virion maturation, of vv in macrophages. sara et al. also showed, using a real-time pcr assay, that snap significantly inhibited sars-cov viral rna production in vero e cell [ ] . these inhibitory effects suggest that no inhibits cellular enzymes necessary for viral dna or rna synthesis, such as eif- g. no typically interacts with ironcontaining proteins and interferes with the function of ribonucleotide reductase [ , ] . no might act upon certain viral targets that are necessary for protein synthesis and processing, either by directly acting on host translation enzymes or by reducing the levels of high-energy phosphate compounds. for example, no inhibits enzymes implicated in diverse metabolic processes, such as glyceraldehydes- -phosphate dehydrogenase [ ] , cis-aconitase [ ] and nadph-ubiquinone reductase [ ] , reducing the production of atp. our results demonstrate that no specially inhibits the ppv replication cycle during the step of viral protein synthesis (fig. b) . the rates of inhibition by snap and la on viral protein synthesis were high-up to %-when these compounds were added h prior to infection, whereas the inhibition rates of snap and la on viral dna synthesis were about %, showing that the inhibitory effect on dna replication was less than that on the viral protein synthesis. the reason for this discrepancy is uncertain but may reflect an inhibition by no of one or more steps of the ppv life cycle. these results are consistent with a previous report by lin et al., who showed that no is able to reduce the amount of viral glycoprotein and packaged virion rna secreted from jev-infected cells into the medium, implying that no may interfere with the release and/or maturation of virions [ ] . however, since our data are unable to furnish us with information on how no inhibits ppv at the molecular level, more studies are required to elucidate the potential viral and cellular targets of no. in conclusion, we have demonstrated that no inhibits ppv replication in pk- cells by inhibiting synthesis of viral dna and protein. however, further study is needed to identify the host and viral targets of no, and the exact mechanism by which no inhibits viral replication in vitro and in vivo remains to be determined. suppression of herpes simplex virus type (hsv- )-induced pneumonia in mice by inhibition of inducible nitric oxide synthase (inos, nos ) inhibitory effect of nitric oxide on the replication of a murine retrovirus in vitro and vivo experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus vaccinia virus-induced inhibition of nitric oxide production inhibition of vesicular stomatitis virus infection by nitric oxide picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for la utilization nitric oxide inhibition of coxsackievirus replication in vitro a taqman-based real-time polymerase chain reaction for the detection of porcine parvovirus nitric oxide and nitric oxidegenerating compounds inhibit hepatocyte protein synthesis establishment of monoclonal antibodies against porcine parvovirus nucleocapsial vp protein and development of double sandwich elisa gamma interferoninduced, nitric oxide-mediated inhibition of vaccinia virus replication inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites nitric oxide and viral infection: no antiviral activity against a flavivirus in vitro, and evidence for contribution to pathogenesis in experimental infection in vivo disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus inhibition of japanese encephalitis virus infection by nitric oxide: an antiviral effect of nitric oxide on rna virus replication macrophage oxidation of la to nitrite and nitrate: nitric oxide is an intermediate inhibition of vaccinia virus dna replication by inducible expression of nitric oxide synthase the effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance posttranslational modification of glyceraldehydes- -phosphate dehydrogenase by s-nitrosylation and subsequent nadh attachment effect of porcine parvovirus vaccination on the development of pmws in segregated early weaned pigs coinfected with type porcine circovirus and porcine parvovirus viral infections of pigs: trends and new knowledge resistance to murine hepatitis virus strain is dependent on production of nitric oxide inhibition of influenza virus replication by nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus induction of nitric oxide synthase during japanese encephalitis virus infection: evidence of protective role the structure of porcine parvovirus: comparison with related viruses effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes nitric oxide synthases: biochemical and molecular regulation stimulation of ire-bp activity of ire by tetrahydrobiopterin and cytokine dependent induction of nitric oxide synthase inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of marek's disease virus key: cord- - m dh hu authors: nomura, naoki; sakoda, yoshihiro; endo, mayumi; yoshida, hiromi; yamamoto, naoki; okamatsu, masatoshi; sakurai, kenji; hoang, nam van; nguyen, long van; chu, huy duc; tien, tien ngoc; kida, hiroshi title: characterization of avian influenza viruses isolated from domestic ducks in vietnam in and date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: m dh hu in the surveillance of avian influenza in vietnam, h n , h n , h n , h n , h n , and h n viruses were isolated from tracheal and cloacal swab samples of domestic ducks in april , and h n virus from bird samples in march . out of the h virus isolates, the hemagglutinins of strains were genetically classified as belonging to the sublineage g , and the other nine belonged to the korean sublineage. phylogenetic analysis revealed that one of the h viruses was a reassortant in which the pb gene belonged to the korean sublineage and the other seven genes belonged to the g sublineage. three representative h n viruses were intranasally inoculated into ducks, chickens, pigs, and mice. on the basis of experimental infection studies, it was found that each of the three viruses readily infected pigs and replicated in their upper respiratory tracts, and they infected chickens with slight replication. viruses were recovered from the lungs of mice inoculated with two of the three isolates. the present results reveal that h avian influenza viruses are prevailing and genetic reassortment occurs among domestic ducks in vietnam. it is recommended that careful surveillance of swine influenza with h viruses should be performed to prepare for pandemic influenza. avian influenza viruses of various subtypes are circulating in poultry in asian countries [ , , , , ] . in particular, h n influenza virus is present in poultry in eurasian countries [ ] [ ] [ ] ] . since h n viruses were isolated from quails in hong kong in , they have become prevalent in live-bird markets and poultry farms in asia [ , ] . h n virus infections have greatly affected not only the poultry industry but also public health [ , ] . the hemagglutinin (ha) genes of eurasian h n viruses have been phylogenetically divided into g , y , and korean sublineages [ ] . h n viruses do not usually cause severe disease in poultry, but co-infection of h n viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine exacerbates the disease [ , ] . h n viruses were also isolated from domestic pigs in china [ ] and korea, and from humans with febrile respiratory illness in hong kong in kong in , kong in , kong in , kong in , and . thus, it is postulated that h n virus may cause pandemic influenza in humans. in our laboratory, avian influenza has been surveyed in japan, alaska, siberia, mongolia, and australia since [ , , , , , ] . the isolates were antigenically and phylogenetically analyzed and assessed for pathogenicity in birds and mammals by experimental infection [ , , , ] . in the present study, a surveillance of avian influenza was carried out in vietnam in domestic ducks and wild birds in and , and the isolates were antigenically and phylogenetically analyzed and their pathogenicity in birds and mammals was assessed. viruses a/duck/hong kong/y / (h n ), a/chicken/hong kong/ g / (h n ) , and a/duck/hong kong/w / (h n ) of the y sublineage and a/quail/hong kong/g / (h n ) of the g sublineage were provided by dr. k. f. shortridge, the university of hong kong, china. a/turkey/wisconsin/ / (h n ) of the north american lineage was provided by dr. r. g. webster, st. jude children's research hospital, united states of america. a/duck/hokkaido/ / (h n ) and a/duck/ hokkaido / / (h n ) of korean sublineage were isolated from ducks under surveillance in our laboratory [ , ] . viruses isolated from domestic ducks in vietnam in and were grown in -day-old embryonated chicken eggs, and infectious allantoic fluids were stored at - °c until use. one hundred tracheal and cloacal swab samples that were viral gene positive from domestic ducks and wild birds (night heron, nycticorax nycticorax; grey heron, ardea cinerea; purple heron, ardea purpurea; chinese pond heron, ardeola bacchus; chinese egret, egretta eulophotes; little egret, egretta garzetta; intermediate egret, egretta intermedia; cormorant, phalacrocorax carbo; little cormorant, microcarbo niger; japanese bush warbler, cettia diphone; black-browed reed warbler, acrocephalus bistrigiceps; olive bulbul, iole virescens; black capped kingfisher, halcyon pileata; collared kingfisher, halcyon chloris; racket tailed treepie, crypsirina temia; oriental magpie robin, copsychus saularis; tiger shrike, lanius tigrinus; yellow bittern, ixobrychus sinensis; indian cuckoo, cuculus micropterus; common koel, eudynamys scolopacea; and black collared starling, sturnus nigricollis) in april and march in southern vietnam were inoculated into the allantoic cavities of -day-old embryonated chicken eggs. viral rna was detected by the reverse transcription loop-mediated isothermal amplification (rt-lamp) method described previously [ ] as a screening test for virus isolation. viral rnas were extracted from the allantoic fluids of chicken embryos infected with viruses by trizol ls reagent (invitrogen, ca, usa) and reverse-transcribed using the uni primer [ ] and m-mlv reverse transcriptase (invitrogen). polymerase chain reaction for amplification of the viral genes was performed using a ptc- thermal cycler (bio-rad, ca, usa). direct sequencing of the viral genes was performed using an autosequencer ceq xl (beckman coulter, ca, usa). for phylogenetic analysis, sequence data for these genes together with those from public database were analyzed by the neighbor-joining method [ ] using mega . software (http://www.megasoftware.net/). accession numbers of the gene sequences of the isolates in the present study are as follows: ab , ab , ab -ab (oie- ), ab , ab -ab (oie- ), ab , ab , ab -ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), ab -ab , ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), ab -ab (oie- ), and ab -ab (oie- ). subtypes of influenza virus isolates were identified by hemagglutination-inhibition (hi) and neuraminidase-inhibition tests using chicken antisera to the reference strains of influenza viruses [ ] . four-week-old chelly valley ducks were purchased from takikawa shinseien (hokkaido, japan). four-week-old boris brown chickens were purchased from hokuren co. (hokkaido, japan). three-week-old crossbred (landrace duroc yorkshire) specific-pathogen-free pigs were purchased from yamanaka chikusan (hokkaido, japan). four-week-old female balb/c mice were purchased from japan slc, inc. (shizuoka, japan). all procedures were performed according to the animal experiment guidelines of graduate school of veterinary medicine, hokkaido university. (dk/vn/oie- ) were inoculated intranasally into three ducks ( ll/duck), six chickens ( ll/chicken), two pigs ( ml/pig), and ten mice ( ll/mouse) at a % egg infectious dose (eid ) of . eid , . eid , . eid , and . eid , respectively. after the inoculation of each influenza virus into three ducks, laryngopharyngeal and cloacal swabs were collected in minimal essential medium (mem; nissui, tokyo, japan) with antibiotics (penicillin g potassium, streptomycin sulfate, gentamicin sulfate, and nystatin) daily from to days post-infection (d.p.i.). all ducks were clinically observed for days after inoculation with influenza viruses. after the inoculation of each influenza virus into six chickens, three chickens were sacrificed at d.p.i., and the brain, trachea, lung, and colon were collected and homogenized to make % (w/v) suspensions in mem. the remaining three chickens were clinically observed for days after inoculation. after the inoculation of each influenza virus into two pigs, nasal swabs from these pigs were collected in mem from to d.p.i. daily, and two pigs were clinically observed for days after inoculation. after the inoculation of each influenza virus into ten mice, five mice were sacrificed at d.p.i., and the lungs were collected and homogenized to make % (w/v) suspensions in mem. the other five mice were clinically observed and their body weight was monitored for days after inoculation. virus titers in the supernatants of the swabs and the tissue homogenates were determined in -day-old embryonated chicken eggs and expressed as the eid /ml and g of tissue, respectively. antibody responses to the inoculated viruses in ducks, chickens, and pigs at d.p.i. were examined by hi test or enzyme-linked immunosorbent assay (elisa) [ ] . in the present study, surveillance of avian influenza was carried out in vinh loi district, bac lieu town, and hoa binh district in vietnam in april and march . twelve strains ( h n , h n , h n , h n , h n , and h n ) were isolated from rt-lamppositive tracheal and cloacal swab samples from domestic ducks in vinh loi district. nine strains ( h n and h n ) were isolated from rt-lamp-positive swab samples from domestic ducks in bac lieu town. nineteen strains ( h n , h n , and h n ) were isolated from rt-lamp-positive swab samples of domestic ducks in hoa binh district ( table ). all of the viruses were isolated from domestic ducks in households, live-bird markets, and slaughterhouses in vinh loi district, bac lieu town, and hoa binh district in vietnam ( the sequence data of the ha genes of h isolates, including reference strains of three different sublineages, were phylogenetically analyzed by the neighbor-joining method (fig. ) . all of the h ha genes were classified as belonging to the eurasian lineage, and the ha genes of and isolates were grouped into the g and korean sublineage, respectively. the partial nucleotide sequence of each gene segment of the isolates was analyzed phylogenetically (fig. ) . gene constellations of the h n virus isolates were divided into three patterns. h n viruses belonging to the g sublineage were isolated from domestic ducks in households a and e. on the other hand, h n viruses belonging to the korean sublineage were isolated in live-bird market g. furthermore, one of these h n viruses of the g sublineage of the ha gene was isolated in live-bird market g, and this virus also possessed a pb gene of the antigenic analysis of the has of h influenza viruses h influenza viruses isolated from domestic ducks in vietnam were analyzed by the hi test ( table ). all of the h isolates tested reacted with antisera against the h viruses of the korean and g sublineages. however, the isolates of the korean sublineage showed low cross-reactivity to antisera against the h viruses of the g sublineage, and all isolates in this study showed moderate and low cross-reactivity to antisera against the h viruses of the y sublineage and north american lineage, respectively. this suggests that the antigenicity of the h isolates of the korean sublineage is different from that of viruses of the g sublineage. it was also found that reactivity patterns of h isolates belonging to the g and korean sublineage in the present study were the same as those of the reference strains. clinical signs were not observed during the experiment in any of the pigs. viruses were recovered from the nasal swabs of pigs inoculated with each of the three h n isolates, and anti-h ha antibodies were detected in the sera of pigs at d.p.i (table ). antibodies were not detected in the sera of pig # inoculated with dk/vn/oie- / , indicating that the pig was not infected with influenza viruses. body weight fell in mice inoculated with dk/vn/oie- / , with - % loss from to d.p.i (fig. ) . viruses were recovered from the lungs of mice inoculated with dk/vn/oie- / and dk/vn/oie- / at d.p.i. (table ) , and anti-h ha antibodies were detected in the sera of all mice at d.p.i (table ). recently, h n viruses of the g , y , and korean sublineages have been isolated from wild birds and poultry worldwide [ , , , , ] . h n viruses have been isolated from pigs and humans in china [ , ] and korea, suggesting that the h n virus is a candidate to cause pandemic influenza in humans. live-bird markets provide an ideal environment for genetic reassortment and interspecies transmission of influenza viruses [ , , , ] . in asia, h n influenza viruses had been isolated only from feral ducks until [ ] , but since then, h n viruses have been isolated from domestic ducks and chickens [ ] . h n viruses belonging to the korean sublineage have been isolated from domestic ducks in vietnam [ ] . in the present study, it was found that h viruses belonging to the korean and g sublineages are circulating in domestic ducks in vietnam, and one of these h n viruses, belonging to the g sublineage but possessing the pb gene of the korean sublineage, was isolated from domestic ducks. thus, genetic reassortment has occurred between viruses of the g and korean sublineages in the poultry population in vietnam. in this study, h n viruses did not replicate well in chickens and ducks. it has been reported that h n viruses isolated from ducks replicate slightly in chickens [ ] , suggesting that the similar results in this study were due to the low susceptibility of chickens to h n viruses. it has also been reported that h n viruses isolated from ducks replicate in only some of the organs in ducks, and viruses of low titer are recovered from tracheal and cloacal swabs [ , ] . the present results were similar to those of the previous reports. in this animal experiment, we collected these factors might affect the titer of recovered virus. in mice, h n viruses replicate in the lungs, and body weight losses are observed [ , ] . in this experiment, viruses replicated efficiently in the lungs of mice the genetic analysis suggested that the pb genes may be responsible for the higher replication rate in mice, since the sublineages of the pb gene are different in these two viruses. further study is needed to clarify the pathogenicity of h n viruses in mice. experimental infection studies revealed that pigs are highly susceptible to infection with avian influenza viruses of each of the known ha subtypes, and genetic reassortment can take place in pigs [ ] . thus, pigs have been suggested to serve as intermediate hosts to generate genetic reassortants [ ] . three h n viruses were recovered from swabs from pigs in this experiment, and the results were similar to those of previous reports [ , ] . especially, viruses were recovered efficiently from nasal swabs from pigs inoculated with dk/vn/oie- / and dk/vn/oie- / , which belong to the g sublineage and replicate efficiently in mice. in addition, h n viruses isolated from humans in hong kong were genetically classified as belonging to the g sublineage [ , , , , ] , suggesting that h n viruses belonging to the g sublineage have the potential to replicate efficiently in mammals. the findings indicate that h n virus is one of the candidates for pandemic influenza in humans. surveillance of influenza in wild birds, domestic birds, and pigs is important in order to prepare for pandemic influenza in humans. sequence and phylogenetic analysis of h n avian influenza viruses isolated from poultry in pakistan a review of avian influenza in different bird species h n influenza viruses from israeli poultry: a five-year outbreak human infection with an avian h n influenza a virus in hong kong in h n subtype influenza a viruses in poultry in pakistan are closely related to the h n viruses responsible for human infection in hong kong continuing evolution of h n influenza viruses in southeastern china phylogeography and evolutionary history of reassortant h n viruses with potential human health implications genetic evolution of h subtype influenza viruses from live poultry markets in shanghai molecular characterization of h n influenza viruses: were they the donors of the ''internal'' genes of h n viruses in hong kong? h n influenza viruses possessing h n -like internal genomes continue to circulate in poultry in southeastern china characterization of the pathogenicity of members of the newly established h n influenza virus lineages in asia coinfection of avian influenza virus (h n subtype) with infectious bronchitis live vaccine universal primer set for the full-length amplification of all influenza a viruses perpetuation of influenza a viruses in alaskan waterfowl reservoirs genetic characterization of h avian influenza viruses isolated from migratory birds and domestic ducks in korea phylogenetic and molecular characterization of h n influenza isolates from chickens in northern china from isolation and characterization of influenza a viruses from wild free-flying ducks in hokkaido biological activity of monoclonal antibodies to operationally defined antigenic regions on the hemagglutinin molecule of a/seal/massachusetts/ / (h n ) influenza virus potential for transmission of avian influenza viruses to pigs genetic relatedness of h subtype avian influenza viruses isolated from wild birds and domestic ducks in korea and their pathogenicity in animals co-infection of staphylococcus aureus or haemophilus paragallinarum exacerbates h n influenza a virus infection in chickens h n influenza virus isolates from terns in australia: genetic reassortants between those of the eurasian and american lineages avian-to-human transmission of h n subtype influenza a viruses: relationship between h n and h n human isolates phylogenetic analysis of the hemagglutinin genes of twenty-six avian influenza viruses of subtype h n isolated from chickens in china during h n influenza viruses prevalent in poultry in china presumed to be the donor of the internal protein genes of the h n hong kong/ virus the influenza virus gene pool in a poultry market in south central china phylogenic analysis of the m genes of influenza viruses isolated from free-flying water birds from their northern territory to hokkaido active reassortment of h influenza viruses between wild birds and live-poultry markets in korea isolation and pathotyping of h n avian influenza viruses in indian poultry isolation and characterization of avian influenza viruses, including highly pathogenic h n , from poultry in live bird markets in hanoi precursor genes of future pandemic influenza viruses are perpetuated in ducks nesting in siberia rapid evolution of low-pathogenic h n avian influenza viruses following poultry vaccination programmes human infection with influenza h n role of quail in the interspecies transmission of h influenza a viruses: molecular changes on ha that correspond to adaptation from ducks to chickens the neighbor-joining method: a new method for reconstructing phylogenetic trees characterization of h n highly pathogenic avian influenza virus strains isolated from migratory waterfowl in mongolia on the way back from the southern asia to their northern territory pandemic influenza: a zoonosis? genotypic evolution and antigenic drift of h n influenza viruses in china from isolation and identification of swine influenza recombinant a/swine/shandong/ / (h n ) virus the genesis and evolution of h n influenza viruses in poultry from southern china characterization of a non-pathogenic h n influenza virus isolated from a migratory duck flying from siberia in hokkaido evaluation of the reverse transcription loopmediated isothermal amplification (rt-lamp) as a screening method for the detection of influenza viruses in the fecal materials of water birds genetic diversity of h n influenza viruses from pigs in china: a potential threat to human health? acknowledgments we thank dr. kennedy f. shortridge for providing h n influenza viruses. we are also grateful for the support of the programme on surveillance of wild birds and domestic animals along migratory flyways under the oie/jtf project for strengthening hpai control in asia. this work was supported by j-grid; the japan initiative for global research network on infectious diseases of ministry of education, culture, sports, science and technology of japan. this work was also supported by japan science and technology agency basic research programs. we are grateful for the support of the global center of excellence (gcoe) program of hokkaido university. key: cord- -y a jf authors: akashi, h.; inaba, y.; miura, y.; sato, k.; tokuhisa, s.; asagi, m.; hayashi, y. title: propagation of the kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: y a jf the kakegawa strain of bovine coronavirus was easily propagated in suckling mice. infected animals died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. the rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain by the neutralization test. w'ith figure accepted november , summary the kakegawa strain of bovine coronavirus was easily propagated in suckling mice. infected animals died with nervous symptoms, and serial passage was readily accomplished by intraeerebral inoculation with brain emulsions. the rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain by the neutralization test. a bovine coronavirus (bcv) has been recognized as one of the causative agents of diarrhea in calves ( ) . l~ecently the kakagawa strain isolated from the feces of a cow with cpizootic diarrhea has been identified as bcv ( , ) . however, the study of bovine diarrheal disease produced by coronavirus has been greatly hampered by difficulty in isolating virus in cell cultures and the lack of experimental hosts other than cattle. recently many members of the eorona~drus family" have been found to grow in suckling mice ( -- ). the present paper describes briefly our recent observation that the kakegawa strain of bcv readily propagates in suckling mice, rats and hamsters. the kakegawa strain of bcv used for mouse inoculation was at, the th passage level in primary bovine kidney cell cultures. conventionally reared oneday-old mice (strain ddy), rats and syrian hamsters were each inoculated with . -ml of the viral materials by the intracerebral (ic) route and with . -ml by the subcutaneous (s.c.) route. virus recovery and infectivity assay were carried out. by inoculation into cell cultures of bek- ecll, derived from bovine embryonic kidney ( ). the a:nti-kakegawa strain immune serum having a homologous neutralizing antibody titer of was prepared in specific germ-free rabbits with virus grown in bek- cells as described previously ( ), and the anti-mouse hepatitis virus (mhv) strain rabbit immune serum having a neutralizing antibody titer of about , by per cent plaque reduction neutralization test was kindly supplied by dr. k. fujiwara, university of tokyo, japan. neutralization (nt) test was carried out by the method described previously ( ) . viruses recovered from the brains of affected animals containing tcids were mixed with an equal volume of two-fold serum dilutions. each mixture was assayed for infectivity by using two tubes of bek- cell cultures per dilution. the antibody titer was expressed as the reciprocal of the highest serum dilution that showed no cytopathic changes in at least, one of the two tubes. fig. . four-day-old mouse days after intraeerebral inoculation with the rd mouse brain passaged kakegawa strain (left) and an uninoeulated -day-old mouse (right) four litters ( to /litter) of suckling mice were inoculated intracerebrally with infected tissue culture fluid containing - tcid /ml. at daily intervals, four animals were sacrificed and their brains taken. the inoculated animals developed an illness days post-inoculation (p. i.) and began to die at days p. i. the affected animals became anemic and showed mild neural symptoms such as weak limbs and sluggish staggering gait. the virus titers of infected brains rose at days p. i. with a subsequent gradual rise to a maximum of s.~ tcids /g at days p. i. serial passage in suckling mice was readily accomplished by the i. c. route with per cent, (w/v) brain emulsions (fig. ) . the incubation period fell to to days by the rd passage. uninoculated mice and mice inoculated with normal brain emulsions remained healthy. virus at the rd passage level in suckling mice containing . tcids /ml also produced a clinical illness with the same symptoms described above. it caused death in all animals when ineoulated into one litter of suckling mice ( /litter) by the s.c. route, and into rats ( or /litter) and hamsters ( or /litter) by the i.e. or s.c. route. table neutralizing antibody titer the results of one way cross nt test are summarized in table . the homologous nt titers were much higher than the heterologous liters. these findings showed that the viruses recovered from the brains of affected animals were antigenieally the same as the cell culture passaged virus and could be clearly differentiated from the mttv strain . a histological study of affected animals showed inflammation in the brains and vacuolar degeneration of the intestinal villi only in the case of s.c. inoculation. however, there was no evidence of inflammation in the liver and other organs (dr. /[. kubo, personal communiea, tion). kaye et al. ( ) reported that they had adapted the american strain of calf diarrhea coronavirus to suckling mouse brain. on ~he other hand, dea et al. ( ) reported that the american strain and their new isolates of calf diarrhea coronavirus were not virulent for suckling mice, hamsters and guinea pigs. our present results support kaye's findings. nt tests and histological studies have ruled out contamination with mhv. further studies, however, wil] be necessary to investigate the immunological and pa~thological differences between bcv and mi-iv. properties of a coronavirus isolated from a cow with epizootic diarrhea physicochemical and biological properties of neonatal calf diarrhea eoronaviruses isolated in quebec and comparison with the nebraska calf eoronavirus replication of bovine coronavirus in cell line ben-i culture calf diarrhoea coronavirus antigenic relationship between human eoronavirus strain oc and hemagglutinating encephalomyelitis virus strain n of swine: antibody responses in human and animal sera coronaviruses: a comparative review feline infectious peritonitis virus. ii. propagation in suckling mouse brain neonatal calf diarrhea: purification and electron microscopy of a coronavirus-iike agent epizootic diarrhoea of adult cattle associated with a coronavirus-like agent authors' address: dr. h. akas~i, national institute o~' animal ttealth a- wien. fiir den text~eil verantwortlieh: dr. wilhelm schwabl, m kerbastei , a- wish. fiir den anzeigenteil verantwortlieh: niag. bruno schweder, m kerbastei , a- wien key: cord- -g jfnxja authors: falcone, valeria; bierbaum, sibylle; kern, winfried; kontny, udo; bertz, hartmut; huzly, daniela; panning, marcus title: influenza virus a(h n )pdm hemagglutinin polymorphism and associated disease in southern germany during the / influenza season date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: g jfnxja a novel influenza a virus emerged in early to cause the first influenza pandemic of the (st) century. understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. in this study, we investigated the molecular evolution of influenza virus a(h n )pdm in the / influenza season in southern germany by sequence analysis of the influenza virus hemagglutinin gene from patients with mild, moderate, and severe disease. phylogenetic analysis revealed co-circulation of different genetic groups. the d g mutation, which had previously been observed in severe cases, was not detected. immunocompromised patients were not affected more severely than non-immunocompromised patients (p> . ), although longer shedding was observed in some of them. interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. the h y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient. the first influenza pandemic of the st century emerged in mexico in march and was caused by a novel influenza a(h n ) virus (a(h n )pdm ). in general, the course of the pandemic was moderate from a public-health perspective. however, a particular proportion of the human population including immunocompromised patients, children and pregnant women was at risk to develop severe disease. hemagglutinin (ha) and neuraminidase (na) represent two major surface glycoproteins of influenza virus. the ha determines host-receptor tropism and constitutes the key immunogenic site for the human immune response. since the beginning of the pandemic, a(h n )pdm rapidly evolved, and seven different clades/groups characterized by distinct molecular markers in the ha gene have been reported [ ] . the d g mutation in the receptor-binding domain of the ha gene has been linked to severe cases [ ] . of note, another mutation involving the same residue (d e) was also observed but could not be associated with more severe cases [ ] . understanding the molecular evolution of influenza virus is therefore crucial to identify mutations that might be associated with a more virulent phenotype. the second surface glycoprotein, na, facilitates the release of newly synthesized virions from infected cells [ ] . neuraminidase can be blocked by antiviral drugs such as oseltamivir and zanamivir, which act by interfering with the release of progeny virus, thereby preventing new rounds of infection. a single point mutation at position in the neuraminidase glycoprotein, resulting in a histidine-to-tyrosine shift, can confer resistance to oseltamivir [ ] . aim of this study was to investigate the molecular evolution of a(h n )pdm in the / influenza season in southern germany by sequence analysis of the ha gene of mild, moderate, and severe influenza cases. particular attention was paid to immunocompromised patients. moreover, the occurrence of resistance to oseltamivir, the only drug currently recommended for prophylaxis in high-risk groups, was determined. respiratory specimens were obtained from paediatric and adult patients hospitalized at the freiburg university medical centre as well as from outpatients. all had influenza-like illness, including fever, cough, and/or sore throat, as judged by the treating physician. respiratory specimens comprising nasopharyngeal aspirates (npa), tonsillo-pharyngeal flocked swabs collected in . ml viral transport medium (copan, brescia, italy), or bronchoalveolar lavage fluids (bal) were analysed at the department of virology. testing of patient samples was approved by the institutional review board of freiburg university. in brief, nucleic acids were extracted using a qiaamp minelute virus spin kit (qiagen, hilden, germany) on a qiacube robot (qiagen) according to the manufacturer's instructions. in / , a broadly reactive multiplex pcr approach was chosen to detect influenza viruses a and b as well as other relevant respiratory viruses. samples were analysed using ftd respiratory pathogens version / (fast-track diagnostics, junglinster, luxemburg) as recommended. the assay utilizes 'nuclease technology (taqman) and employs a -tube multiplex one-step realtime rt-pcr approach. in tube , real-time rt-pcr for influenza virus a, a(h n )pdm , influenza virus b, and rhinovirus are combined. tube contains reagents for parainfluenza viruses , , and , and brome mosaic virus (bmv) as a pcr inhibition control; tube , for coronavirus e, coronavirus nl , coronavirus oc , and coronavirus hku ; tube , for respiratory syncytial virus a/b, adenovirus, parechovirus, and enterovirus; and tube , for parainfluenza virus , human metapneumovirus a/b, human bocavirus, and mycoplasma pneumoniae. the bmv inhibition control was added to each patient sample before nucleic acid extraction. an agpath-id one-step rt-pcr-kit (invitrogen, karlsruhe, germany) was used for rt-pcr on an abi real-time machine (applied biosystems, wiesbaden, germany). cycling conditions were as follows: °c for min, °c for min followed by amplification cycles of denaturation at °c for s and combined annealing/extension at °c for s. for the detection of the oseltamivir-resistance-associated mutation h y, a commercially available assay (tib-molbiol, berlin, germany) was used according to the manufacturer's instructions. the ha gene was amplified directly from clinical samples using the superscript tm iii one-step rt-pcr system (invitrogen, karlsruhe, germany). in brief, a reaction volume of ll contained x reaction buffer, mm mgso , . lm each primer, ll enzyme mix, and ll of purified nucleic acids. the primer sequences were as follows: h n _ha_f , ccg caa atg cag aca cat ta; h n _ha_r , ccc att aga gca cat cca gaa [ ] . cycling conditions in a veriti -well thermal cycler (applied biosystems, weiterstadt, germany) were °c for min and °c for min, followed by amplification cycles at °c for s, °c for s, and °c for s. pcr products were purified using a qiaquick pcr purification kit (qiagen) as recommended. purified pcr products were sequenced directly using primers h n _ ha_f (ccgcaaatgcagacacatta) and h n _ha_ fseq_ (cagacacccaagggtgctat). sequences were aligned using bioedit (mega . ). nucleotide sequence alignments were done using a clu-stalw method with mega . . phylogenetic trees were constructed using the maximum-parsimony method. ncbi genbank accession numbers for the sequences determined here are jx to jx . amino acids were numbered starting after the dtlc signal peptide. potential n-linked glycosylation sites were predicted using the free software netnglyc . . data were analyzed using spss software version (spss, chicago, usa). data were compared by fisher's exact test, and p-values were deemed significant at the . level. in order to detect molecular changes in the ha gene of influenza virus, a(h n )pdm ha sequences representing individual patients were analysed. as shown in table , of the patients ( %) were immunocompromised or immunosuppressed, suffering from different underlying diseases. virus isolates on mdck siat- cells were obtained from of the respiratory samples (data not shown) [ ] . the median age of the patients was years ( % confidence interval [ci], . - . years]. based on clinical criteria, / ( %) were regarded as mild (median age, years; range, - years), ( %) as moderate (median age, years; range . - years) and ( %) as severe cases (median age, years; range, . - years) according to zarychanski et al. [ ] . all severe cases were hospitalized patients known to be at risk for severe disease ( of these patients were immunocompromised, and one of them was obese). only of the immunocompetent patients and of the immunocompromised patients developed severe disease (fisher's exact test, p= . ). in ( %) of the samples (corresponding to [ %] of the patients), co-infection with another respiratory virus was detected. coronavirus oc and/or nl were detected in of these patients, rsv and human bocavirus were co-detected in the npa of one, and rsv alone was detected in a pharyngeal swab from another patient. clinically, the patient with rsv and bocavirus coinfection presented with mild disease; the remaining patients displayed moderate (n= ) to severe (n= ) symptoms. none of the patients showed co-infection with mycoplasma pneumoniae as assessed by multiplex pcr. prolonged shedding (i. e., c weeks) of influenza virus in the respiratory tract was observed in of the patients ( %; with severe, with moderate, and with mild disease). a total of out of patients were immunocompromised, and of the patients ( of them immunocompromised) received oseltamivir therapy. the ha sequences were closely related to each other and to the reference strain a/california/ / (a/cal/h n / ). the differences between german sequences and a/cal/h n / ranged from to amino acids ( table ). all of the viruses analysed displayed the amino acid changes p s and s t in the ha region as well as i v and e k in the ha region. phylogenetic analysis showed simultaneous co-circulation of influenza virus of groups , , , and in southern germany ( figure ). genetic groups were named according to the ecdc technical document of august/september . in detail, of the sequences ( %) belonged to genetic group , characterized by the double mutation d n and s t. moreover, of these showed an additional s i mutation (as influenza a/delaware/ af / ), and one of them had a p s mutation. of note, this particular strain was isolated from an influenzavaccinated individual. ten of the sequences ( %) belonged to genetic group , characterized by the mutations d n, r k, i v and v l. in this group, three also had the h q mutation. as observed by piralla et al. [ ] , two sub-clusters were seen within this group. group , characterized by amino acid mutations n d included four sequences, all obtained from the same patient. finally, of the sequences ( %) clustered within group and were characterized by the mutations s t, s g, and a t. double mutations characteristic of group (a t, s p), and group (n d, s n) were not observed. severe, moderate, and mild cases were scattered throughout the phylogenetic tree. some of the observed amino acid substitutions involved the major antigenic sites of the ha molecule (table ) . five potential n-glycosylation sites, typical of a/cal/ h n / , corresponding to residues , , , , and , were also detected in the german sequences. however, in two patients, two additional glycosylation sites were detected at residue (lysine to asparagine) in one case and at residue (serine to asparagine) in the other case. as shown in table , jx and jx correspond to sequences obtained from the upper and lower respiratory tract of the same patient. interestingly, the k n mutation, representing an additional potential n-glycosylation site, was present only in the bal sample. moreover, the cycle threshold (ct) value of the h n similarly, in another severe case, the ct value for the bal sample obtained days after onset of disease was lower (ct ) than that for the nasopharyngeal swab (ct ). also in this case, two additional amino acid changes (k r and m l, genbank accession number jx ) were detected in the bal sample. treatment with oseltamivir was reported in of the patients ( immunocompromised patients and obese patient). the oseltamivir-resistance-associated mutation h y was detected in ( %) of the treated patients, a one-year-old child with a genetic disorder, who was treated for days with oseltamivir. he recovered fully from ca s t t t t t t t t t t t t t t t t t t t t t t ca s t t t t t t t sb a t sb s g a ca s t t t t t t t t t t t ca influenza despite development of oseltamivir resistance and prolonged shedding of resistant virus for [ weeks. in this molecular study, we were able to show that influenza viruses circulating in southern germany in the first post-pandemic season differed relatively little from each other and from the vaccine strain a/cal/h n / . some of the amino acid substitutions that were observed involved the major antigenic sites of the ha molecule. five classical antigenic sites (sa, sb, ca , ca , and cb) have been described in the ha of seasonal influenza h n virus [ , ] , all located in the globular head of ha. recent studies have also described some important antigenic sites in the stem region of ha [ ] . all viruses analysed here had the amino acid changes p s and s t in the ha region and i v and e k in the ha region. as reported by others [ , , ] , and as published in the ecdc report released in august/september [ ] , co-circulation of different genetic groups was observed. group , characterized by mutations d n and s t, was the dominant h n lineage ( %), followed by group ( %; d n, r k, i v), group ( %; n d) and group ( %; s t, s g, a t). although a number of mutations have been reported in circulating a(h n )pdm , they have not significantly affected virus antigenicity and pathogenicity as demonstrated by in vitro studies [ ] . clinically, the mutations d g and n have been associated with a more virulent phenotype [ ] . however, recent studies show that within the current a(h n )pdm ha framework, the effect of the mutation on receptor binding appears to be less dramatic when compared to the influenza a(h n ) virus ha framework, since the binding preference for a - sialylglycans is still maintained [ ] . also, other mutations, such as the double mutation n d and e k, have been associated with a more virulent phenotype. this double mutation has been associated with several breakthrough infections despite influenza vaccinations and was identified in some fatal cases [ ] . moreover, it was associated with decreased antibody recognition in vaccinated individuals [ ] . the german ha sequences carrying this double mutation originated from an unvaccinated immunocompromised individual with severe illness. due to the lack of serum specimens, we could not analyze the ability of antibodies to recognize the hemagglutinin of a(h n ) pdm in our study. however, immune escape from the vaccine strains might be an issue of concern. no d g change was observed here. in our study, no association of a specific amino acid change with severe illness could be observed. however, in two patients, differences between the ha sequence could be detected in viruses isolated either from the upper respiratory tract (urt) or the lower respiratory tract (lrt). in particular, two additional mutations (k r-m l in one case and k n-i k in the other case) were identified only in the lrt. moreover, analysis of potential glycosylation sites revealed that the k n mutation provides an additional potential glycosylation site. human influenza viruses carrying the k n mutation show improved growth in eggs and appear to exhibit enhanced virulence in the mouse model [ , ] . glycosylation at position is essential for improved virus protein yield in eggs [ ] . egg adaptation of human influenza viruses is known to increase their affinity for the , -sialic acid (sa) receptor and concomitantly impairs their ability to bind to , -sa [ ] . although it is clear that influenza virus tropism depends on several viral and host factors and not only on ha specificity, the presence of the k n mutation exclusively in the virus isolated from the lrt might indicate a more efficient binding/replication of viruses carrying this mutation to/in cells expressing , -sa. importantly, , -sa is known to be found in abundance in the lower respiratory tract [ ] . of note, the ct value of the a(h n )pdm real-time rt-pcr from the nasopharyngeal swab was higher than that of the bal sample carrying the mutation (ct versus ct ) . since bal samples are usually significantly more diluted than nasopharyngeal swabs, the observed difference suggests a real replication advantage of the strain harboured in the lrt. virus histochemistry studies could be used to analyze the pattern of binding of mutants to human respiratory tissue, as already suggested [ , ] . moreover, analysis of growth curves of the two isolates in different cell lines may reveal a possible replication advantage, at least in cell culture. many patients with influenza have more than one viral agent, with reported co-infection frequencies as high as % [ ] . we detected co-infection with another respiratory virus in out of samples ( %), corresponding to of the patients ( %). the most frequent co-infecting agents were coronaviruses ( of samples), followed by rsv ( of samples) and human bocavirus ( / ). disease resulting from co-infection with influenza virus and coronaviruses has been reported to be more severe [ ] , although the number of influenza virus and coronavirus co-infections is low [ , , ] . here, patients co-infected with coronaviruses showed moderate to severe disease, whereas co-infection with rsv and human bocavirus in one child resulted in mild disease. finally, the oseltamivir-resistance-associated mutation h y was detected only once in our study population. although oseltamivir has been widely used in the pandemic and thereafter, cases of resistance have remained scarce to date. however, prolonged shedding of high levels of resistant influenza virus in individual cases poses the threat of spread into populations that are at risk and finally into the general population as resistant viruses retain fitness [ ] . in concordance with other studies, our results underline the importance of monitoring influenza virus evolution and development of resistant viruses. of note, the lrt might harbour more virulent variants than the urt. immune escape of influenza virus in subsequent influenza seasons is an issue of concern, making continuous surveillance essential. severe outcome of influenza a/h n / v infection associated with g/n polymorphisms in the haemagglutinin: a multicentre study a new pandemic influenza a(h n ) genetic variant predominated in the winter influenza season in australia the antigenic structure of the influenza virus a/pr/ / hemagglutinin (h subtype) rate and influence of respiratory virus co-infection on pandemic (h n ) influenza disease influenza hemagglutinin and neuraminidase membrane glycoproteins influenza virus receptor specificity: disease and transmission characteristics of a widespread community cluster of h y oseltamivir-resistant a(h n ) pdm influenza in australia genetic diversity of the pandemic influenza a(h n ) viruses in finland adaptation of pandemic h n influenza viruses in mice observed association between the ha mutation d g in the pandemic influenza a(h n ) virus and severe clinical outcome early alterations of the receptor-binding properties of h , h , and h avian influenza virus hemagglutinins after their introduction into mammals first sequence-confirmed case of infection with the new influenza a(h n ) strain in germany neuraminidase inhibitors for influenza an additional oligosaccharide moiety in the ha of a pandemic influenza h n candidate vaccine virus confers increased antigen yield in eggs frequency of detection of upper respiratory tract viruses in patients tested for pandemic h n / viral infection mdck-siat cells show improved isolation rates for recent human influenza viruses compared to conventional mdck cells streptococcus pneumoniae coinfection is correlated with the severity of h n pandemic influenza segregation of virulent influenza a(h n ) variants in the lower respiratory tract of critically ill patients during the - seasonal epidemic genetic characterization of the influenza a pandemic (h n ) virus isolates from india avian flu: influenza virus receptors in the human airway minor changes in the hemagglutinin of influenza a(h n ) virus alter its antigenic properties seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses nucleotide sequence of the haemagglutinin gene of a human influenza virus h subtype screening of random peptide library of hemagglutinin from pandemic a(h n ) influenza virus reveals unexpected antigenically important regions structure and receptor binding properties of a pandemic h n virus hemagglutinin correlates of severe disease in patients with pandemic influenza (h n ) virus infection acknowledgments we are grateful to nadja besazza for excellent technical assistance. we are grateful to dieter neumann-haefelin and markus hufnagel for critical reading of the manuscript. this work was partially supported by the german bundesministerium für bildung und forschung (bmbf, contract # es ). key: cord- -dje qbps authors: macnaughton, m. r.; madge, m. hilary title: the polypeptide composition of avian infectious bronchitis virus particles date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: dje qbps egg grown avian infectious bronchitis virus (ibv) centrifuged on sucrose density gradients was found to consist of a major virus peak of density . to . g/cm( ) and occasionally two minor virus peaks of density . to . g/cm( ) and . g/cm( ). three different ibv strains were examined and no morphological differences were detected between virus particles of different densities or from different strains. the polypeptides of the different density virus particles from the three ibv strains were analysed on polyacrylamide gels. in all cases polypeptides were observed, although there were differences in the proportions of these polypeptides in particles of different densities and those from the different strains. the polypeptides have been called vp (molecular weight , ), vp ( , ), vp ( , ), vp ( , ), vp ( , ), vp ( , ) and vp ( , ). additional polypeptides were produced if slightly harsher treatments were used. avian infectious bronchitis virus (ibv) is a member of the eoronavirus group and typiemly exists as pleomorphie, although generally spherical, virus particles to nm in diameter with coronas of widely spaced club shaped surface projections about nm in length ( , ) . however, the presence or absence of the surface projections varies with different strains ( ) . previous studies on ibv have shown the presence of several virus peaks in sucrose gradients ( , ) -with buoyant densities varying from. . to . g/cm a, although there usually seems to be a main peak of virus activity with respect to infectivity ( , ) . it has been observed that ibv particles have to t polypeptides ( , ) . other studies on the polypeptide composition of eoronaviruses have shown polypeptides in the virus particles of human coronavirus (hcv) strain oc ( ) a n d t r a n s m i s s i b l e g a s t r o e n t e r i t i s v i r u s ( t g e v ) ( ), a n d p o l y p e p t i d e s in t h e v i r u s p a r t i c l e s of h c v s t r a i n e ( ) i b v strains b e a u d e t t e (ibv ), connecticut (ibv ) a n d massachusetts (ibv ) were used. these strains h a v e h a d a long h i s t o r y of passage in e m b r y o n a t e d chicken eggs before their use in the p r e s e n t studies. the b e a u d e t t e strain is serologically similar to t h e massachusetts strain as it is a massachusetts strain t h a t has been highly passaged in eggs. to a tcds of virus was i n o c u l a t e d b y t h e allantoic route into d a y old e m b r y o n a t e d chicken eggs which were t h e n i n c u b a t e d a t ° c for hours. the allantoic fluid was chilled a t ° c overnight, h a r v e s t e d a n d i m m e d i a t e l y clarified b y eentrifugation a t × g for m i n u t e s a t ° c. all t h e purification steps were p e r f o r m e d at. ° to ° c. t h e virus was pelleted a t , × g for i h o u r a n d t h e n r e s u s p e n d e d in ml dulbeceo's p h o s p h a t e buffered saline 'a' (pbsa). t h e resuspended virus was overlaid on to a linear to per cent (w/w) sucrose g r a d i e n t in p b s a a n d centrifuged for hours at , × g. t h e virus peak(s) were collected, diluted in p b s a a n d again layered on to linear to per cent (w/w) sucrose gradients in p b s a a n d centrifuged for hours at , × g. virus samples were e x a m i n e d after negative staining w i t h per cent (w/v) p o t a s s i u m p h o s p h o t u n g s t a t e , p i t . , in a phillips e m electron microscope. virus peak(s) from the second sucrose g r a d i e n t were collected a n d sodium dodeeyl s u l p h a t e (sds) a n d -merca.ptoethanol were a d d e d to c o n c e n t r a t i o n s of a n d p e r cent respectively a n d t h e samples were i n c u b a t e d in a boiling w a t e r b a t h for t. minutes. i n some cases t h e samples were dialysed o v e r n i g h t a t room t e m p e r a t u r e against mm tris, m~i glycine buffer (ph . ) c o n t a i n i n g p e r cent sds, . per cent -mereaptoethanol, . ~ u r e a a n d per cent, sucrose ( ) . before get electrophoresis a trace a m o u n t of b r o m o p h e n o l blue dye was a d d e d a n d in some eases t h e virus solution was c o n c e n t r a ted using u l t r a -t h i m b l e s (sehteieher a n d sehfilt). i n a modified procedure of polypeptide p r e p a r a t i o n t h e virus solutions were boiled for m i n u t e s before o v e r n i g h t dialysis a n d for m i n u t e after dialysis. eleetrophoresis was carried out on cylindrical p o l y a c r y l a m i d e gels. t h e a c r y l a m i d e c o n c e n t r a t i o n was . per cent (w/v) a n d t h e a e r y l a m i d e : bis-acrylamide ratio was . : b y weight. the gels also c o n t a i n e d . per cent sds, . n urea, . per cent n , n , n ' , n ' -t e t r a m e t h y l e t h y l e n e d i a m i n e , . per cent a m m o n i u m p e r s u l p h a t e a n d . ~ tris-ttc buffer, p h . . the gels were pre-electrophoresed at v for hours in a n electropboresis buffer consisting of m~ tris, mn glycine, . per cent sds, . ~ urea a n d . per cent -mercaptoethanol, pi~ . . after pre-electrophoresis, samples were layered directly on top of the gels a n d eleetrophoresis was after eleetrophoresis the gels were removed from their supporting tubes and stained for proteins. staining was with . per cent coomassie brilliant blue in per cent methanol, . per cent acetic acid for hours and destaining was carried out over to days with several changes of per cent methanol, . per cent acetic acid. the mobilities of the virus polypeptides were measured relative to the bromophenol blue dye and compared with the mobilities of proteins of known molecular weight that had been reduced, electrophoresed as above and stained with coomassie brilliant blue. the proteins used as standards were obtained from sigma chemical co. ltd., and comprised bovine serum albumin (dimer and monomer), ovalbumin, trypsin and lysozyme. the gels were scanned at nm using a joyee loebl chromoscan. centrifugation of i b v particles on sucrose density gradients produced a relatively sharp peak of material ranging in density from . to . g/em a depending upon the strain of i b v used (table ). i n per cent of cases an extra peak, forming up to per cent of the total material, was found sedimenting at a density of . to . g/em a. material also sedimented as a diffuse b a n d in sucrose gradients at a density of . g/cm in per cent of preparations (table ) . there was no difference in the relative amounts of these peaks over a series of experiments for the different i b v strains. all the virus peaks were infectious, although comparative studies on the infectivity of these peaks were difficult as i b v is e x t r e m e l y labile ( ) and large drops in infectivity were observed even after minimal t r e a t m e n t of the virus particles. figure shows electron micrographs of i b v b e a u d e t t e virus particles from the major peak (fig. a) and from the high density peak (fig. b) of sucrose density gradients. there was never enough virus material from the low density peak to s t u d y the morphology of these virus particles b y electron microscopy. arch, virol. / -- essentially, there was little morphological difference between the two forms of the virus particles although the high density form of the virus appeared slightly larger than the major density form. virus particles from beaudette were the same size and shape as connecticut and massachusetts : all the strains had overall diameters ranging from to nm. virus particles in most preparations contained only partial coronas of surface projections and in a few eases almost no surface projections, although in no ease did the virus particles contain complete coronas. we only used virus particles that contained partial coronas as they occurred more frequently and appeared more typical of eoronaviruses generally. the virus particles with no coronas will be discussed in a later paper. virus polypeptide analysis figure shows the polsteptides of the purified major virus species of the ibv strains beaudette, connecticut and massachusetts, on polyaerylamide gels. seven polypeptides, vp to vp , were observed in each of the strains. i{owever, polypeptides vpzi and vp could not in all cases be clearly distinguished. the proportions of these polypeptides were similar, apart from the proportion of vp which varied considerably in the strains. there was more vp in massachusetts (fig. c) and connecticut (fig. b ) than in beaudette ( fig. a) . figure shows a comparison between the polypeptides of the major (fig. a ) and high density (fig. b) virus species of massachusetts. the polypeptide profiles were similar except that there was considerably more vp in the major species than in the high density species. similar results have been obtained with beaudette and comleetieut. the approximate molecular weights of the virus polypeptides have been determined by comparison with the relative mobilities of bovine serum albumin (dimer and monomer), ovalbumin, trypsin and lysozyme. table shows the molecular weights of the viral polypeptides determined from experiments. previous papers on the pol~peptide composition of ibv have shown the presence of i to polypeptides in the virus particles ( , ). when we prepared virus polypeptides for eleetrophoresis using per cent dithiothreitol to reduce the polypeptides ( ) or by boiling the polypeptides before and after overnight dialysis ( ), we obtained polypeptide patterns similar to those leported previously ( , ) . figure shows a profile of ibv beaudette polypeptides prepared using our modified preparative procedure involving boiling of the polypeptides after overnight dialysis (see methods). ~!dthough the pattern varied from experiment to experiment, at least polypeptides were seen compared with the polypeptides observed using our standard procedure (fig. a) . the new polypep~ides have been designated vpa, vpb, vpc, vpd and vpe and have molecular weights of approximately , , , , , , , and , respectively. however, in all cases the proportions of the new polypeptides increased with more degradation of the polypeptides vp to vp . there was essentially no difference between the polypepgide profiles of the major and high density virus species. generally, even harsher preparative procedures (such as boiling the samples for minutes before dialysis and minutes after overnight dialysis) removed most in purified ibv preparations run on sucrose density gradients we saw ~ major virus peak and up to two others of different densities. the peaks were seen with three dilferent ibv strnns and electron microscope studies showed that the viruses from these peaks were morphologically similar and there was little or no contaminating cellular debris. the polypeptide compositions of the virus particles isolated from the different peaks were similar in that they all contained polypeptides, although there was considerably less vp in the high density peak than in the major virus peak. it is interesting to note that the proportion of vp also varied considerably between the three ibv strains examined: there was more vp in connecticut and massachusetts than in beaudette. as mentioned above, the beaudette strain is serologically similar to massachusetts from which it has been derived by multiple passages in embryonated chicken eggs. however, our results suggest that the peptide composition of beaudette now appears to be more similar to connecticut than to massachusetts. harsher conditions of polypeptide preparation produced polypeptide profiles containing more than polypeptides, instead of the polypeptides that were usually obtained. furthermore, considerable variation occurred in the amount, proportion and resolution of these polypeptides with this harsher treatment. from such observations we suggest that these new polypeptides are degradation products and that ibv particles contain only polypeptides. six or seven polypeptides have been observed in the virus particles of hcv oc ( ) and e ( ) and tgev ( ) . a similar number of polypeptides has been identified in mouse hepatitis virus (mac~avg~to~ and madge, unpublished results). thus the polypeptide composition of these coronaviruses resemble that of ibv, ~lthough none is identical. we are indebted to dr. d. a. j. tyrrell for his advice and encouragement throughout these studies. we wish to thank dr. s. patterson and mrs. heather a. davies for the electron microscopy. the morphologicm and biological effects of various antisera on avian infectious bronchitis virus the polypeptide composition of avian infectious bronchitis virus heterogeneity of infectious bronchitis virus grown in eggs avian infectious bronchitis pococ~:, i). h. : the polypeptide structure of transmissible gastroenteritis virus the polypeptides of ibv morphological variation among avian infectious bronchitis virus strains protein composition of coronavirus oc purification and biophysical properties of human coronavirus e coronaviruses: a comparative review the nucleic acid of infectious bronchitis virus key: cord- -vwyny vt authors: zhang, meng-jia; liu, de-jian; liu, xiao-li; ge, xing-yi; jongkaewwattana, anan; he, qi-gai; luo, rui title: genomic characterization and pathogenicity of porcine deltacoronavirus strain chn-hg- from china date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vwyny vt porcine deltacoronavirus (pdcov) was first detected in hong kong and has recently spread to many countries around the world. pdcov causes acute diarrhea and vomiting in pigs, resulting in significant economic losses in the global pork industry. in this study, a chinese pdcov strain, designated chn-hg- , was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central china. subsequently, the virus was identified by an indirect immunofluorescence assay and electron microscopy. a nucleotide sequence alignment showed that the whole genome of chn-hg- is . %- . % identical to other pdcov strains. analysis of potential recombination sites showed that chn-hg- is a possible recombinant originating from the strains ch/sxd / and vietnam/hanoi / . furthermore, the pathogenicity of this recombinant pdcov strain was investigated in -day-old piglets by oral inoculation. the challenged piglets developed typical symptoms, such as vomiting, anorexia, diarrhea and lethargy, from to days post-inoculation (dpi). viral shedding was detected in rectal swabs until dpi in the challenged piglets. interestingly, high titers of virus-neutralizing antibodies in sera were detected at dpi. tissues of small intestines from chn-hg- -infected piglets at dpi displayed significant macroscopic and microscopic lesions with clear viral antigen expression. our analysis of the full genome sequence of a recombinant pdcov and its virulence in suckling piglets might provide new insights into the pathogenesis of pdcov and facilitate further investigation of this newly emerged pathogen. porcine deltacoronavirus (pdcov), a member of the family coronaviridae, order nidovirales, is an enveloped, single-stranded positive-sense rna virus [ ] . the genome of pdcov is . kb in length, making it the smallest genome among the known coronaviruses (covs) [ ] . the coding region of pdcov is flanked by short ′ and ′ untranslated regions (utrs), and the gene order is ′-utr-orf a-orf b-s-e-m-ns -n-ns - ′-utr. pdcov encodes at least four structural proteins, including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, as well as two accessory proteins, ns and ns [ ] [ ] [ ] [ ] . the s protein is responsible for receptor binding and membrane fusion and acts as the major antigen inducing neutralizing antibodies [ ] . the n protein is highly conserved and plays a critical role in viral rna encapsidation [ ] . the m and e proteins are important for virion assembly and budding [ ] . during a molecular surveillance study performed by yuen and coworkers in , pdcov was first identified in swine specimens in hong kong [ ] . following the first detection of pdcov in pigs with diarrhea in ohio, usa, in [ , , [ ] [ ] [ ] [ ] [ ] [ ] . piglets infected with pdcov are characterized by watery diarrhea, variable vomiting, and dehydration [ ] . notably, pdcov-positive piglets are frequently co-infected with other enteric viruses, such as porcine epidemic diarrhea virus (pedv), rotavirus, transmissible gastroenteritis coronavirus (tgev), and porcine respiratory coronavirus (prcv), which considerably increases the morbidity and mortality of piglets [ ] . pdcov outbreaks have been reported in many countries worldwide and have caused significant economic losses in the pork industry. moreover, due to the unique genome replication strategy of coronavirus, pdcov undergoes rna mutation and recombination events at high frequency, giving rise to new variants, which makes it extremely difficult to control and eliminate [ ] . in , saif and coworkers reported that a pdcov strain named oh-fd caused severe atrophic enteritis accompanied by severe diarrhea and vomiting in gnotobiotic piglets [ ] . macroscopic examination revealed that the jejunum and ileum are the major targets for pdcov infection, and the pathological lesions were similar to, but milder than, those caused by pedv [ ] . subsequently, the pathogenicity of several american strains, including usa/il/ , michigan/ , and ohio cvm i, was investigated in piglets [ ] . these american strains exhibited pathogenic characteristics similar to those of oh-fd . in addition, it has been reported that newborn piglets inoculated with two recent chinese pdcov isolates, named chn-hn- and chn-gd- , developed clear clinical signs, including severe diarrhea, vomiting, and dehydration [ , ] . in this study, we successfully isolated a high-growth pdcov strain, hereafter referred to as chn-hg- , from feces of a pig on a farm located in huanggang, hubei province. analysis of the genomic sequence suggested that chn-hg- is a potential recombinant virus derived from chinese and vietnam pdcov strains. furthermore, the pathogenicity of chn-hg- in -day-old piglets was investigated by clinical assessment, the quantification of viral shedding and distribution, histology, and immunohistochemistry. feces (n = ) from pigs experiencing diarrhea were obtained from farms in huanggang, hubei province. they tested pdcov-positive by n-gene-based rt-pcr as reported previously [ ] . these clinical samples were subsequently subjected to virus isolation. briefly, g of pdcovpositive feces was suspended in ml of dulbecco′s modified eagle′s medium (gibco, grand island, ny, usa), vortexed for minutes, and centrifuged at ×g for minutes at °c. the supernatants were filtered through a . -μm filter (millipore, billerica, ma) and stored at - °c for pdcov isolation. llc-pk cells (atcc cl- ) were obtained from the american type culture collection (rockville, md, usa) and were used for pdcov isolation. llc-pk cells were cultured in dmem supplemented with % fetal bovine serum (fbs, gibco). the maintenance medium for pdcov propagation was dmem with % tryptose phosphate broth (tpb) and μg of trypsin (sigma) per ml. cells were cultured in a -well plate until they reached - % confluence and were then washed three times with pbs. filtered inoculum ( μl) in . ml of maintenance medium was added to the cell monolayers, and the cells were kept at °c in % co for h. the cells were then washed three times with pbs, and ml of maintenance medium was added to each well. the inoculated cells were cultured continuously at °c in % co . when a cytopathic effect (cpe) was obvious in the infected cells, the plates were frozen and thawed twice. subsequently, the supernatants were stored at - °c as seed stocks for plaque purification and for the next passage. the isolates were plaque-purified as described previously [ ] . the virus titer was determined based on the % tissue culture infectious dose (tcid ) on llc-pk cells in -well plates as described previously [ ] . the n gene of chn-hg- was amplified by rt-pcr, and the pcr product was cloned into the pet a vector. the recombinant plasmid was expressed in escherichia coli rosetta (de ) under iptg induction. subsequently, female balb/c mice were immunized with the purified recombinant n protein. after hybridoma cell fusion and selection, one strain of hybridoma cells that secreted an anti-n protein monoclonal antibody (mab), named a , was selected. llc-pk cells were seeded on sterilized coverslips placed in -well plates and then mock-infected or infected with plaque-purified pdcov at a multiplicity of infection (moi) of . . at h postinfection (hpi), cells were fixed with % paraformaldehyde for min at room temperature (rt) and then permeabilized with . % triton x- for min. subsequently, cells were blocked with % skimmed milk in pbs for h at room temperature and incubated with mab a for h. the cells were treated with fluorescein isothiocyanate (fitc)-labeled goat anti-mouse igg secondary antibodies (invitrogen) and then stained with ′, -diamidino- -phenylindole (invitrogen) for min at room temperature. fluorescence was examined using a fluorescence microscope (olympus ix , japan). llc-pk cells were infected with pdcov and harvested with lysis buffer ( mm tris-hcl [ph . ], % sodium dodecyl sulfate, % dl-dithiothreitol and % glycerol) supplemented with pmsf. proteins isolated from the lysate were separated by % sds-page and then transferred to a polyvinylidene difluoride membrane. the membrane was blocked with % dry milk and then incubated with the pdcov-n-specific mab a . after washing three times with pbst, the membrane was incubated with horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (abclonal) for min at room temperature. signals were detected using a supersignal west pico luminal kit (pierce). after inoculation with chn-hg- , llc-pk cells were harvested when more than % cpe was observed. cells were frozen and thawed three times and then centrifuged at , ×g at °c for min. the supernatants were filtered and ultracentrifuged at , ×g for h at °c. the pellets were resuspended in pbs and centrifuged in a non-linear %- % sucrose gradient at , ×g for h at °c. the virions that sedimented in the interphase between the % and % sucrose layers were diluted with sterile pbs. the sucrose was then removed by centrifugation for h at , ×g. the virions were resuspended in pbs, stained with % phosphotungstic acid, and examined using a transmission electron microscope (hitachi h- fa, japan). viral rna was extracted from chn-hg- -infected llc-pk cells using trizol reagent (invitrogen, usa) according to manufacturer′s instructions. cdna was synthesized from the extracted rna using random primers and a takara rna pcr kit (takara, japan). the whole genome of chn-hg- was sequenced using pairs of primers reported by lorsirigool et al. [ ] . based on the sequence that was obtained, a pair of primers (pdcov- ′gsp, ′-gat tac gcc aag ctt tag atc gca agg tgg gtt ggg ctc c- ′; pdcov- ′gsp, ′-gat tac gcc aag ctt cgt cgt aag acc caa cat caa gct - ′) was designed to verify the ′-and ′-terminal sequences of chn-hg- using a smarter race ′/ ′ kit (takara) according to manufacturer′s instructions. the sequences were combined using dnastar lasergene . , and the assembled genome sequence was submitted to the genbank database under the accession number mf . multiple sequence alignments and phylogenetic analysis were performed using the neighbor-joining method with mega software (http://www. mehas oftwa re.net/). the aligned sequences were scanned for recombination events, using the recombination detection program (rdp). animal experiments were carried out strictly according to the guide for the care and use of animals in research of the people′s republic of china. all experimental procedures were approved by the scientific ethics committee of huazhong agricultural university (hzausw- - ). twelve -day-old piglets were purchased from a commercial pig farm and were randomly divided into two groups, which were housed in separated containments. prior to pdcov inoculation, piglets were confirmed to be negative for pdcov, pedv, tgev, and rotavirus by testing rectal swabs by rt-pcr. neutralizing antibodies against pdcov were not detected in sera of the piglets or sows. after acclimation for h, the challenged group was inoculated orally with a chn-hg- preparation with a titer of × tcid /ml ( ml per pig), while the mock-infected group was inoculated with ml of maintenance medium. clinical symptoms, including vomiting, diarrhea, lethargy, and appetite loss, were observed and recorded daily. rectal swabs were collected daily from each piglet from the first day postinoculation (dpi) to the end of the experiment. swabs were submerged into ml of pbs immediately after collection. randomly selected piglets from each group (n = ) were subjected to necropsy at dpi, and prolonged pdcov shedding was monitored in the remaining pigs until dpi. at necropsy, tissues of duodenum, jejunum, ileum, cecum, colon, rectum, liver, spleen, lung, kidney, and blood were collected for analysis by real-time rt-pcr. tissues of duodenum, jejunum, ileum, cecum, and colon were also fixed with % formalin for histopathology and immunohistochemistry analysis. sera collected at , , , dpi from piglets were tested for neutralizing antibodies against pdcov. the number of viral rna copies in fecal swabs, sera, and tissue samples was determined by real-time rt-pcr. briefly, fecal swab samples diluted in dmem were homogenized and centrifuged at ×g for min. an aliquot ( μl) of supernatant was extracted for viral rna isolation. one gram of various tissue samples was mechanically homogenized in ml of dmem, and μl of the supernatant was used for rna extraction. two hundred μl of serum was also used for rna extraction. viral rna was extracted using trizol reagent (invitrogen, usa) according to the manufacturer′s protocols and was treated with dnase i. one μg of total rna was used for cdna synthesis by reverse transcription using an iscript cdna synthesis kit (bio-rad). cdna amplification was performed using the itaq universal probes supermix (bio-rad) with primers and probes targeting the n gene of pdcov, as described by ma et al. [ ] . the recombinant plasmid pet a-n containing the n gene of pdcov was serially diluted tenfold to generate a standard curve for each plate. real-time rt-pcr was carried out using an applied biosystems fast real-time pcr system (life technologies, usa). the thermal cycling parameters were as follows: min at °c, min at °c, and cycles of s at °c and min at °c. the number of copies of pdcov viral rna in the tested samples was calculated based on the standard curve. the presence of pdcov-specific neutralizing antibodies in serum samples was determined using a virus neutralizing (vn) test. briefly, llc-pk cells were grown in -well plates until they reached % confluence. the chn-hg- stock was diluted in dmem to make tcid viral stocks. fifty μl of the diluted virus was then mixed with an equal volume of twofold serial dilutions of individual inactivated sera in -well plates and incubated at °c for h. after washing twice with maintenance medium, llc-pk cells were incubated with the mixture at °c for h. the mixture was then removed, and the cell monolayers were washed three times with pbs and cultured in maintenance medium at °c in a % co incubator for days. the neutralization antibody titers were calculated as the reciprocal of the highest dilution of serum at which pdcov infection was blocked. at necropsy, tissues of duodenum, jejunum, ileum, cecum, colon, and rectum collected from the inoculated and control groups were separated and fixed in % formalin for h at room temperature and then dehydrated, embedded, sectioned, and mounted on glass slides. the samples were stained with hematoxylin and eosin (h&e), and the slides were examined and analyzed by conventional microscopy. three representative villi and crypts with integrated longitudinal sections were randomly selected from each h&estained jejunum and ileum. villus-height-to-crypt-depth (vh/cd) ratios were calculated using a computerized image system (nis-elements . ). five-micron-thick sections obtained from formalin-fixed paraffin-embedded tissues were placed onto positively charged glass slides, and the slides were air dried for min. the tissue sections were deparaffinized, and the slides were rinsed and then incubated with target retrieval solution (sigma-aldrich, usa). the sections were blocked with % bsa, incubated with pdcov-n-specific monoclonal antibodies for min, and then with hrp-conjugated goat anti-mouse igg secondary antibodies (zhongshan biotechnology co., beijing, china). the samples were visualized using a substrate-chromogen reagent ( -amino- -ethylcarbazole/h o , solarbio, china). tissues from piglets in the negative control group were used as controls. of the pdcov-positive feces inoculated onto llc-pk monolayers, only one caused obvious cpe in the form of enlarged, rounded and clustered cells at hpi and rapidly detached from the monolayers at hpi (fig. a) . this pdcov isolate, designated as chn-hg- , tested positive for pdcov and negative for other enteric viruses by rt-pcr (data not shown). chn-hg- was further passaged in llc-pk cells for a total of passages, indicating that the pdcov chn-hg- strain could be stably propagated in llc-pk cells. to confirm that virus propagation had occurred, the infectivity of the plaque-purified chn-hg- strain in llc-pk cells was tested by ifa and western blot with an mab against the pdcov n protein. as shown in fig. a , pdcov-specific immunofluorescence was detected in most cells at hpi, and the n protein was mainly localized in the cytoplasm. n protein was also detected in pdcov-infected llc-pk cells by western blot (fig. b) . the morphology of the pdcov particles purified from the supernatants of chn-hg- -infected llc-pk cells was examined by electron microscopy (em) using negatively stained preparations. as shown in fig. , typical crown-shaped coronavirus particles with spiky surface projections and an average diameter of - nm were observed. these results clearly showed that a strain of pdcov had been successfully isolated and identified. the complete nucleotide sequence of the genome of chn-hg- was determined and deposited in the genbank database under the accession number mf . similar to other pdcov strains, the complete genome sequence of chn-hg- is , nt in length, consisting of ′ utr- ab-s-e-m-ns -n-ns - ′ utr, and predicted transcription regulatory sequences (trss) were located at the ′ end of each gene with a highly conserved core sequence of ′ -accac- ′ (table ) . moreover, chn-hg- shared . %- . % nucleotide sequence identity with the other pdcov strains in the database. phylogenetic analysis indicated that the pdcov strains from the united states and south korea clustered into a large clade, whereas chn-hg- clustered with other pdcov strains detected in china since . these data imply that the chinese strains might share a more recent common ancestor with the us and south korean strains than the ancestor of the strains from thailand, laos, and vietnam (fig. a) . notably, phylogenetic trees based on the s gene displayed a similar clustering pattern (fig. b) . the rna genome of coronaviruses can undergo highfrequency recombination due to its large size [ ] . we therefore analyzed the genome sequence of chn-hg- using rdp. as depicted in fig. a , two pdcov strains, vietnam/ hanoi / (genbank accession number: kx ) and ch/sxd / (genbank accession number: kt ), might represent the parent lineages of chn-hg- . similarity plot and bootscanning analyses predicted the potential breakpoints in chn-hg- to be at nt and . we therefore divided the aligned sequences into three separate segments: one from nt up to the breakpoint at position , another from the breakpoint at position to the breakpoint at position , and the last one from the breakpoint at position to the end. a phylogenetic analysis of these segments using the maximum-likelihood algorithm suggested the ch/sxd / strain from china to be its major parent and the vietnam/hanoi / strain from vietnam to be its minor parent (fig. b) , providing strong evidence that recombination events had occurred. to investigate the pathogenicity of chn-hg- , -dayold piglets were challenged with chn-hg- via oral table ). all piglets from the negative control groups were in good health throughout the course of study with no observed clinical signs (fig. a and b) . as expected, three of the six piglets in the challenged group showed diarrhea at dpi, and more piglets developed varying degrees of diarrhea, vomiting, lethargy and anorexia at - dpi ( fig. c and d) . lethargy, anorexia and the progression to watery diarrhea were most severe at - dpi, and the piglets gradually recovered thereafter (table ) . notably, no pdcov-associated deaths were observed in this study. these results demonstrated that chn-hg- is pathogenic to newborn piglets. to determine the duration of fecal shedding and to monitor the changing load of chn-hg- , we attempted to detect viral rna in rectal swab samples from to dpi by quantitative real-time rt-pcr. the viral rna was detected in six out of six rectal swab samples collected from the challenged group from dpi to dpi. the pdcov rna copy number in homogenized rectal swab supernatants reached a peak of over copies/ml at - dpi (fig. a) . notably, no pdcov rna was detected in samples from piglets in the negative control throughout the study. the distribution of chn-hg- in various tissues was also examined by quantitative real-time rt-pcr in three pigs from each group sacrificed at dpi. viral rna was detected in two of three duodenums (average . copies/g), three of three jejunums (average . copies/g), three of three ileums (average . copies/g), three of three ceca (average . copies/g), three of three colons (average . copies/g), and three of three rectums (average . copies/g). it was also detected in one of three livers ( . copies/g), one of three spleens ( . copies/g), one of three kidneys ( . copies/g) and one of three lungs ( . copies/g), but no viral rna was detected in the blood (fig. b) . to investigate when pdcov neutralizing antibodies were detectable in the infected piglets, serum samples collected at , , , dpi were tested for pdcov-neutralizing antibodies. virus-neutralizing (vn) antibodies in sera were detected as early as dpi in sera of pdcovinoculated piglets, and the titers increased gradually and peaked at dpi (table ). pdcov-specific neutralizing antibodies were not observed in sera from the control piglets. to determine the gross pathological and histological changes in piglets infected with chn-hg- , three of the six pigs in each group were selected randomly for necropsy at dpi. the small intestines, where yellow watery contents had accumulated, were clearly transparent, thin-walled, and gas-distended in the pdcov-challenged pigs (fig. a) . no other lesions were observed in any other organs of the challenged groups or in any of the organs of the negative control pigs (fig. b) . these results showed that the small intestine is the major target of chn-hg- infection. microscopic lesions in the small intestine were also observed by microscopy. compared to those of the negative control piglets, the intestinal villi of the pdcov-infected piglets were blunted and fragmented and exhibited atrophy and vacuolation, especially in the jejunum and ileum (fig. c and d and table ). moreover, aggregation of lymphocytes and hyperemia in the intestinal lamina propria were also observed ( fig. c and d) , but the intestines of the negative control were normal ( fig. e and f) . consistent with the histopathological results, pdcov antigen was detected by immunohistochemical staining in the cytoplasm of the infected villous enterocytes ( fig. g and h) . no pdcov antigen was detected in the negative controls ( fig. i and j) . these results show that chn-hg- infection can cause intestinal lesions in newborn piglets, especially in the jejunum and ileum. pdcov is a newly emerged enteropathogenic coronavirus that causes diarrhea, dehydration and death in neonatal piglets. since pdcov was first reported in hong kong, it has spread to many countries around the world, leading to significant economic losses in the pork industry. in mainland china, the prevalence of pdcov in pigs was reported to be . % in jiangxi province [ ] , . % in guangdong province [ ] , and . % in hebei province [ ] . although the prevalence of pdcov in china has been widely reported, only a few pdcov strains could be successfully isolated in cultured cells until now. in the present study, feces from pdcov-infected pigs on farms located in central china were used to isolate pdcov in llc-pk cells. typical cpe was observed in infected llc-pk cells at h after inoculation. following plaque purification, the pdcov strain was confirmed by ifa and electron microscopy. notably, only one pdcov strain was isolated from a total of pdcov-positive feces samples, indicating that the success rate for virus isolation was still relatively low. this low rate of pdcov isolation might be attributed to a small amount of infectious virus in the fecal samples collected for virus isolation. in addition, the time at which the sample is collected might also contribute to the success of pdcov isolation. to characterize the virus isolate, the complete genome of chn-hg- was sequenced and analyzed. while the genome organization is similar to those of other reported pdcovs, the genome size of , nt is somewhat different from the others. moreover, we identified predicted trss at the ′ end of each gene of chn-hg- , each with a highly conserved core sequence of ′-accac- ′, which appears to be unique to members of the genus deltacoronavirus. multiple sequence alignment and phylogenetic analysis showed that all of the known pdcov strains share a high level of nucleotide sequence identities. however, chn-hg- was most closely related to other chinese pdcov strains. our phylogenetic analysis suggests that pdcov strains from china, the united states and south korea are clustered in the same clade, whereas the pdcov strains from thailand, laos, and vietnam are clustered in another clade. like other coronaviruses, pdcov has the potential to undergo recombination at a very high frequency. in this study, we employed the recombination detection program (rdp) to analyze the genome of chn-hg- in search of any recombined fragments. intriguingly, chn-hg- was found to be a possible recombinant virus derived from parental strains from china and vietnam. the major parent and minor parent were identified as strain sxd from china and strain hanoi from vietnam, respectively. the potential breakpoints in chn-hg- were located at nt and in the nsp region. deletion mutations in the nsp region in pdcov have been reported recently [ , ] , suggesting that nsp which encodes a papain-like protease, may be a hotspot for genetic modification. to our knowledge, this is the first time that a recombination event has been identified in the nsp region of pdcov. the emergence of recombinant strains from china and vietnam will probably bring new challenges to the prevention and control of pdcov. to investigate the pathogenicity of this putative recombinant strain, -day-old piglets were inoculated orally with chn-hg- . as expected, severe diarrhea with vomiting and lethargy in piglets was observed from to dpi, suggesting that chn-hg- is pathogenic to newborn piglets. the viral rna distribution in various tissues was also examined. viral rna levels were higher in intestines than in other tissues. previously, it was reported that coronavirus hku may be able to spread through the respiratory route, in addition to fecal-oral transmission [ ] . in line with other reports, viral rna of chn-hg- was also detected in the lungs of some animals, albeit with no gross lesions observed, suggesting that pdcov may also cause respiratory infection. it is noteworthy that we only observed microscopic lesions in the jejunum and ileum in piglets challenged with chn-hg- . in striking contrast, microscopic lesions were observed in all sections of small intestine infected with pedv [ ] . moreover, unlike pedv, pdcov infection usually does not result in the death of newborn piglets despite the fact that these animals exhibit lethargy and anorexia and have watery diarrhea. these results indicated that the virulence of pdcov is milder than that of pedv. recently, it was shown that vn antibodies could be detected in -day-old piglets inoculated with pdcov oh-fd at about dpi and that vn antibody titers increased gradually and remained high at the end of the experiment [ ] . however, to our knowledge, there are currently no reports on the development of pdcov-specific antibodies in sera of pigs infected with chinese pdcov. our study showed that serum pdcov vn antibodies could be detected as early as dpi in pdcov-infected piglets. vn antibody titers increased and remained high until dpi, when all pigs in the challenged groups had completely recuperated. in conclusion, we successfully isolated, characterized and obtained a high titer of chn-hg- in cultured cells. we showed that chn-hg- is probably a recombinant virus derived from ch/sxd / and vietnam/hanoi / . pathogenicity testing of chn-hg- revealed that this virus is still pathogenic in piglets despite multiple passages in cultured cells. high levels of serum vn antibodies were also detected in sera of infected animals. not only will our findings be useful for understanding the molecular epidemiology and pathogenesis of pdcov, but they also provide insights for the development of effective vaccines against this important pathogen. complete genome characterization of korean porcine deltacoronavirus strain kor/knu - comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c coronavirus origin, evolution, and virulence of porcine deltacoronaviruses in the united states complete genome sequence of strain sdcv/usa/illinois / , a porcine 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diarrhoea in hebei province complete genome characterization of novel chinese porcine deltacoronavirus strain sd coronavirus hku in respiratory tract of pigs and first discovery of coronavirus quasispecies in ′-untranslated region pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain oh-fd ethical approval necessary approvals from the scientific ethics committee of huazhong agricultural university (hzausw- - ) were obtained prior to the start of animal experiments. this article does not contain any studies with human participants performed by any of the authors. key: cord- -y rjgbui authors: veretnik, s.; gribskov, m. title: rna binding domain of hdv antigen is homologous to the hmg box of sry date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: y rjgbui the delta antigen of hepatitis delta virus exhibits sequence specific binding to its own rna and is essential for viral replication. using statistical methods we have detected significant similarity between the rna-binding domain of the hepatitis delta antigen and the hmg box of sry. our analysis suggests that the rna-binding domain of hdv antigen evolved from the dna-binding domain of the hmg box. sry, or a related protein, is a probable cellular cognate of hdv. hepatitis delta virus (hdv), the satellite of hepatitis b virus (hbv) is unique among animal viruses in that it contains a circular, viroid-like rna genome. while it is fully dependent on the helper virus for its packaging, hdv has no significant sequence similarity to hbv; its replication is independent of hbv and its geographic prevalence is different [ ] . hdv is a single stranded rna virus. it replicates through a double rolling-circle mechanism, probably using rna polymerase ii and cellular transcriptional machinery [ , ] . two detectable hdv antigens (hdag) are encoded by a single open reading frame on the antigenomic strand. the large antigen (l-hdag) is a result of rna editing which extends the protein coding sequence of the small antigen (s-hdag) by residues; l-hdag appears later in infection, suppresses replication and initiates viral packaging [ , ] . the small antigen (s-hdag) is essential for viral replication and is proposed to facilitate the interactions between the cellular transcriptional machinery and the viral template. the central domain of s-hdag, which is involved in binding to the viral genome, contains two arginine-rich motifs (arms) bracketing a leucine zipper. mutational analysis indicates that both the arms and the leucine zipper region are essential for binding to the hdv genome [ ] . using sequence analysis methods we have discovered significant sequence similarity between this central domain of hdag and the hmg (high mobility group) box of the sry gene [ ] , whose product binds to dna and is essential for sex determination. our results indicate a possible evolution of this widely used dna-binding domain into the rna-binding domain of hdag. the organization of the hdv genome suggests two distinct sources for its genomic rna: % of the genome resembles a plant viroid rna (the smallest known self-replicating genome), while the remaining three-quarters of the genome is more like a cellular rna. half of this cellular domain codes for hdag. this unique combination of domains within hdv gave rise to the hypothesis that a small viroid ( - bp long) captured a cellular rna, resulting in a larger virus that retained the ribozyme activities of the viroid (such as self-cleavage and self-ligation) [ , ] . the sequence similarity between the central domain of hdag and the hmg (high mobility group) box of the sry gene suggests that sry may be a cellular cognate of hdag. another candidate for the 'captured' cellular rna was recently proposed: a residue cellular protein referred to as dipa (delta interacting protein a) was identified by co-immunoprecipitation with hdag and by its in vivo ability to inhibit viral replication [ ] . our analysis shows that the level of similarity between hdag and dipa is within noise level; the similarity is likely to be due to compositional biases found in motifs consisting of arginine-rich regions and leucine zippers (bzip motifs). this paper reports the results of the in-depth sequence analysis applied to the sequence of hdv antigen in an attempt to further understand the function and evolution of hepatitis delta virus -a unique entity in the world of animal rna viruses. sequence alignments were performed using programs from the gcg wisconsin package version . -unix ( ) . for multiple sequence alignments, the pileup program (based on a simplified version of the progressive alignment method of feng and doolittle) [ ] under default conditions (gapopen= . , gapextend= . ) was applied. the gap program, based on the global alignment algorithm of needleman and wunsch [ ] was used for alignment of sequence pairs. the best matched subsequences of sry, hdag and dipa were detected by visual inspection; alignments of these regions are referred to as partial alignments throughout this paper. the partial alignments were constructed by aligning selected subsequences using the gap program. the comparison matrix pam [ ] was used for scoring amino acid residue similarities. gap opening and gap extension penalties were varied and are specified for each alignment. monte carlo evaluation of sequence alignments was done using the gap program with the option '-random= '. this evaluation process consists of randomizing the first sequence and realigning it to the second sequence , times, each time calculating the alignment score. the average and standard deviation of , alignments of randomized sequences were then used to calculate the z score for the original alignment. the swiss-prot database release ( / ) was used for database searches. evolutionary profiles [ ] were constructed from groups of sequences aligned with the pileup program. a profile is a two-dimensional weight matrix that describes the alignment of a group of sequences. each row of a profile describes a single position in the alignment; its values estimate the likelihood that each of the possible amino acid residues occurs at this position in the alignment. two additional values in each row represent the position-specific likelihood of gap initiation and extension. evolutionary profiles improve the description of multiple sequence alignments by modeling varying rates of sequence change at each position in the sequences: each position is modeled using the most appropriate scoring matrix to reflect its rate of change. sequence weighting, based on a modification of felsenstein's method [ ] , was performed prior to profile construction, so that the contribution of each sequence to the evolutionary profile is proportional to the amount of unique information it contributes to the multiple alignment. structure-based profiles (to be described elsewhere) are constructed using scoring matrices based on secondary structure information. each position in the alignment is described as a mixture of four distributions -one for each of the secondary structures (helix, sheet, turn and coil). each distribution is weighted by the likelihood of the observed residue distribution at a given position being found in a specific secondary structure. new sequences identified during the database search with the profile of the hdag family were then re-aligned to this profile with the profilegap program using default gap opening and gap extension penalties (gapopen= . , gapextend= . ). the profilegap program is based on an extension of the smith-waterman local similarity algorithm [ , ] . the gcg package framesearch program was used to search for sequencing errors and potential frameshifts during translation. framesearch is based on a local alignment algorithm that allows introduction of the gaps and shifting of the reading frame. framesearch was used under the following conditions: gapopen= . , gapextend= . , scoring matrix: pam . pam matrices at distances , , and were tested [ ] . we found that the pam scoring matrix consistently produced alignments with more significant z scores. thus pam was used as a scoring matrix throughout this work. p-values, the probability of achieving an equal or better score, for the best partial alignments of sry-hdag and dipa-hdag ( fig. a , c) were calculated by the monte carlo procedure described above (see sequence alignments). randomly generated sequences with the same composition as the best locally matching segments were compared to the respective query sequence , , times. this is a very conservative test since the compositions of these segments are biased. theory suggests that sequence alignment scores follow an extreme value distribution (evd) similar to that of maximal segment scores [ ] , i.e. the logarithm of the frequencies of occurrence of scores greater than or equal to specific values were plotted for the top ( . %) random alignments and the p-values calculated from the resulting plot (fig. ) . the similarity between the sry and hdag was noticed during swiss-prot database searches with evolutionary profiles [ ] constructed from a multiple alignment of the rna-binding domain of hdag from nine distinct isolates of hdv. searches with the evolutionary profile identified several target sequences that contained hmg boxes, such as hmgi and hmgy with z score > . . searches with profiles based on the predicted secondary structures identified other hmg products, such as hmg human and hmg mouse (z score > . ), and sry horse (z score> . ). pairwise alignments of these target sequences with the rna-binding domain of hdag identified the hmg box of sry as most closely related sequence. we then constructed profiles from the sry-hdag alignment, which, upon database search, identified a large family of proteins containing the hmg box: sry genes, sry-related genes and sox (sry box) genes. hdag shows the highest similarity to the hmg box of the human sry and sox gene products; for the sake of clarity only the sry sequence is shown in the analysis below. the best alignment results are achieved with the first residues of the hmg box (corresponding to residues - of sry) aligned to the residues of rna-binding motif of hdag (corresponding to the residues - ): % identity and % sequence similarity are observed ( fig. a ). this region covers the first arm and part of the leucine zipper region. this is the only alignment that produces a significant z score (see below). a longer alignment ( fig. b ) covers two arms bracketing a leucine zipper, which together comprise the rna-binding motif in hdag. this longer alignment between hdag and sry proteins extends though the c-terminal region of both proteins; it exhibits % identity and % similarity (fig. b ). the similarity in hdag begins ten residues upstream of the left arm and continues into the leucine zipper, where similarity gradually weakens but is still detectable through the right arm of hdag ( fig. , b ). when hdag was aligned with arm-containing sequences, the best alignment was found with hiv rev gene product. this alignment shows % identity and % similarity across the entire rev gene (fig. f ). the alignment of hdag and dipa, a proposed cellular homolog of hdag [ ] , shows % identity and % similarity across the entire length of the protein sequences (fig. d ). the similarity is distributed across the entire sequence, however, the amino-terminal and central regions show a somewhat higher level of similarity (fig. d ). our analysis is focused on the central region of hdag which shows higher level of sequence similarity and also corresponds to the most similar region of hdag and sry. this -residue long subsequence corresponds to the first arginine rich motif (arm) and most of the leucine zipper of hdag (fig. c ). the levels of residue identity and similarity of this alignment are % and % respectively. as a control, dipa was also aligned with hiv rev -an arm containing protein unrelated to dipa or hdag. this alignment shows % identity and % similarity across entire length of -residue long rev protein (fig. e) . the hmg box of sry belongs to the hmg- subgroup. many of the residues that are conserved between the hmg box of sry and hmg . are also conserved in the alignment between sry and the rna-binding domain of hdag (data not shown). to identify the residues that are conserved among all sequences, we aligned the sequences of sry, sox , hmg . and the rna-binding domain of hdag to the profile of sry and hdag sequences (fig. a) . a total of residues are identical between sox , sry and hmg . ; the alignment of the rna-binding domain of hdag shares of these identities. the identities are concentrated in the first of the helices predicted for the hmg- subgroup (fig. a, b ). hdag, sry and dipa contain arginine rich motifs. this compositional bias can lead to overestimation of the significance of sequence similarity in the pairwise alignments. to verify that the above hdag alignments are not due to compositional bias of the sequences, a monte carlo analysis was performed for each of the pairwise alignments. one sequence was shuffled and then realigned to the other sequence under the initial conditions. in the case of partial alignments ( fig. a , c) the greatest local compositional bias is preserved, which makes it a stringent test. this process was repeated , times and the distribution of the alignment scores was examined. matches merely due to compositional effects alone should occur at equivalent frequencies in the original and scrambled alignments. when the alignment score of the original sequence is compared to the distribution of the scores of the scrambled sequences, z scores larger than . indicate that the original alignment is not simply due to compositional bias. a z score less than . indicates that the similarities between two sequences are unlikely to be significant and are probably due to biased composition. the only potentially significant alignment is a partial alignment between the functional domains of sry and hdag ( fig. a ) (z score = . ). the rest of the alignments -sry-hdag (long alignment), dipa-hdag (both alignments), hdag-rev and dipa-rev have z scores as a function of gap penalties are reported here for the residue long alignment of sry and hdag ( fig. a ) and the residue long alignment of dipa and hdag (fig. c ). z scores were calculated using pam comparison matrix. average and standard deviation values (for z score calculation) were based on , alignments per sequence. the best z score for each alignment is shown in boldface z score < . in the monte carlo analysis (see fig. ). table reports the z scores for the optimized partial alignments of hdag with sry and hdag with dipa. in all cases the sry-hdag alignment is much more significant than the dipa-hdag alignment. for the best partial alignments of sry-hdag and dipa-hdag we also determined the p-value of the alignment. we plotted the log of the observed frequency of occurrence for the top . % of , , random alignments as a function of their alignment score (fig. ) . the p-value for the sry-hdag partial alignment ( fig. a) is estimated to be . × − . the p-value for the dipa-hdag partial alignment (fig. c ) is estimated to be . × − . in this experiment out of , , random alignments had better scores than the proposed dipa-hdag alignment; some scores were significantly better ( alignments had scores over , while the actual dipa-hdag alignment had score . ). in the case of the sry-hdag alignment, none of the , , random alignments exceeded the reported score of . (the highest random score was . ). testing for frameshifting during translation sry protein was aligned to the hdv genome in all reading frames. as expected, the significant alignment is found on the antisense strand. there is no potential the discovery of sequence similarity between the rna-binding domain of hdag and the hmg box of sry suggests a possible evolutionary relationship between the hmg box, a dna-binding domain, and the rna-binding domain of hdag. for the sake of clarity we will discuss the relationship between hdag and sry, but the same argument could apply to sox and, possibly, to other sry-related genes. statistically significant sequence similarities are limited to the functionally essential regions of both genes: in the sry gene the similarity is confined to the hmg (high mobility group) box, a dna binding motif found in the hmg protein superfamily [ , ] , in hdag the similarity is limited to rna-binding domain. sry, hdag and dipa are bzip proteins -they thus possess a strong compositional bias, which influences sequence analyses. this compositional bias may reflect structural and functional constrains placed upon the rna or dna-binding domain, and could therefore suggest sequence convergence rather than a common evolutionary origin. thus the high values for percent identity and similarity in compositionally biased alignments are less significant than in compositionally unbiased sequences. to address the effects of the local compositional bias we used a monte carlo analysis -a process that compares the alignment of interest with alignments of the randomly generated sequences of the same composition. z scores above . indicate that the alignment of interest supports the inference of homology in spite of compositional biases. the high z score (z score = . ) of sry-hdag alignment argues that the hdag sequence is more similar to the hmg domain of sry than to any randomly assembled sequences of the same composition. therefore, the compositional constraints alone upon the functional domain cannot explain the observed level of sequence similarity between hmg domain of sry and hdag. while it is possible that the unusual degree of sequence similarity is due to convergent evolution to an energetically favorable nucleic acid binding structure, one must keep in mind that there are a nearly unlimited number of ways to create a polypeptide that will fold into a given three-dimensional shape. therefore there is no a priori reason that unrelated molecules should show significant similarity or similar spacing of conserved residues in the absence of an ancestral relationship. see patterson [ ] for a more complete development of this argument. none of the alignments between dipa and hdag were found to be statistically significant based on a monte carlo analysis. the best z score for an alignment is . ; it corresponds to the partial alignment between the -residue long central domain of dipa and rna-binding domain of hdag (fig. c) . the p-value of this alignment is estimated to be . × − (fig. ) . the e-value (expected number of comparisons achieving an equal or higher score in an equivalent database search) for this alignment is for a , sequences database (size of the latest swiss-prot database, release ). this e-value indicates that the database search with the rna-binding domain of hdag will identify approximately unrelated proteins that will have alignment scores as good or better than dipa alignment. in fact, the alignment of dipa and the hiv rev protein (fig. e) , which is presumably unrelated to dipa, has a similar z score = . . alignments of other dipa regions or of the entire dipa protein with sry produced even less significant z scores (fig. d ). statistical analysis recently performed by long et al. considered the entire dipa sequence; it also indicated that sequence similarity between hdag and dipa is not significant [ ] . the p-value of the alignment of the entire dipa and hdag proteins is × − as reported by brazas and ganem in their reply to long et al. [ ] . this value is less significant than the pvalue for the dipa fragment reported above. the presence of the bzip domains, which made detection of functional interaction between dipa and hdag possible [ ] , should also serve as a warning during sequence analysis: bzip domains are compositionally biased and this bias makes it more likely to obtain high alignment scores with unrelated proteins. the monte carlo analysis of the partial alignment of the hmg domain of sry and the rna-binding domain of hdag produces a z score of . -a statistically significant value (fig. a) . the p-value for this alignment is . × − , indicating that an alignment this good is unlikely to arise by chance, even when analysis is limited to the compositionally biased hmg box region. e-value of this alignment is . for the latest release of swiss-prot (release ); thus an unrelated protein with an alignment score as good or better as that of the hmg domain of sry and the rna-binding domain of hdag will be expected to be found only after -fold increase in the size of the current database. a cellular gene and its rna viral cognate diverge more rapidly than would be expected for two cellular genes due to the low fidelity of rna replication. because of this rapid divergence, one would expect the similarity between a dnaencoded gene and its rna-encoded counterpart to correspond to the regions that are absolutely essential for the function of the protein. in the case of sry-hdag this is indeed the case: the region of similarity is limited to the rna-binding region of hdag, which as been shown to be functionally important [ ] . two nuclear localization signals (nlss) recently identified within the hmg domain of sry are also conserved within the rna-binding domain of hdag [ ] . these nlss are distinct from an already identified bipartite nls within hdag which is located to the amino terminal side of the rna-binding domain [ ] . our model predicts these new nlss should be functional in hdag. recent experiments demonstrate that the presence of either one of the arm sequences is sufficient for nuclear localization of the antigen, supporting our prediction [ ] . sry is the only gene from the y chromosome known to be involved in testis development. it is most closely related to the sox gene: these genes were proposed at one time to be alleles of a developmental gene on the undifferentiated or partially differentiated proto-x and proto-y [ ] . sry is a member of hmg superfamily; it possesses a -residue long hmg box which binds to a well-defined consensus sequence: -cctttga and probably controls the transcription of other sex-determination genes. the hmg superfamily is ancient -it is present in plants, yeast and animals, and is characterized by highly diverse sequences [ ] . members of the hmg family may contain single or multiple copies of the hmg box; some members bind dna in a sequence-specific manner, others bind dna non-specifically, and yet others recognize and bind to distorted dna -such as -way junctions or cisplatinmodified dna [ ] . in all known cases, the binding of hmg box proteins causes distortion or a bend in the dna [ ] . our model therefore predicts that hdag will induce a similar bend when binding to the hdv genome. hdag exhibits binding specificity which appears to be determined primarily by the structure of the hdv rna. however some sequence specificity is required, since hdag binds specifically to both a rod-like structure comprising the entire hdv genome, and to a multiple stem-and-loop structure formed by the fragment containing the ribozyme domain of hdv, but does not bind to the double-stranded rna derived from an unrelated coronavirus (mouse hepatitis virus) [ ] . hmg proteins can be divided into major groups based on their size, sequence similarities and dna-binding properties: hmg- /- , hmg- /- and hmg-i/y [ ] . the sry gene belongs to the hmg- /- family, which is represented by the hmg- motif and contains hmg boxes referred as hmg . and hmg . . the hmg box of sry is more similar to hmg . , which is the most likely candidate for an ancestral hmg box [ ] . ten of identities in the alignment of the hmg box of sry with hmg . are also present in its alignment with the rna-binding domain of hdag, and three of the remaining show conservative substitutions (k -> r, a -> g, a ->l, fig. ). thus the sequence constraints upon the rnabinding domain can be traced back through the most closely related hmg box (of sry) to the ancestral sequence of the hmg- family. the predicted secondary structure of hmg- and the solution structure of hamster hmg . have been determined [ ] . the structure consists of a stretch of basic residues followed by proline at the n-terminal end, referred by the authors as the 'terminal unit', and ␣-helices (fig. ) . the sequence similarity between hmg- and the rna-binding domain of hdag begins within the terminal unit and continues through first and second ␣-helices (h and h ). together, these structures comprise the side of a flat-shaped arrowhead which is proposed to lie along the minor groove and continue into the major groove of the dna [ ] . interactions between the terminal unit and the dna are absolutely essential [ ] . the sequence of the terminal unit is conserved between sry and hdag, and it is also similar to regions of hmg /y proteins, perhaps explaining why a database search with the rna-binding domain of hdag showed similarity to several hmgi/y sequences (see 'database searches' section in results). helix of hmg- comprises the other side of the arrowhead and is proposed to lie along the phosphodiester backbone of the dna [ ] ; there is little sequence similarity between this helix and hdag -there appears to be a deletion in the corresponding region of hdag. however, the region immediately following helix has a stretch of basic residues that could be aligned with the right arm of the rna-binding domain of hdag (fig. a) . it is notable that the residues proposed to be involved in contacting dna (terminal unit) and those whose substitution is likely to cause gross structural perturbations (e.g. replacement of the small residue at position , or the presence of phenylalanine at position , fig. ) are conserved among the hmg boxes of sry, hmg- and the rna-binding domain of hdag. gly- is proposed to pack closely to phe- in the first helix in the resolved structure for hmg . of hamster [ ] . these two residues are also preserved within the rna-binding domain of hdag. on the other hand, many of the residues involved in tertiary folding of the hmg domain have been replaced or deleted: in the rna binding region of hdag the n-terminal portion of helix consists of polar and charged residues, while the same region in the hmg box is hydrophobic (fig. a) . another notable feature is the lack of tryptophan residues in the rna-binding domain (sry has tryptophan residues, sox has tryptophan residues and hmg- has tryptophan residue) and an overall lower percentage of the hydrophobic residues. the structure of the rnabinding domain of hdag has not yet been determined, but a computer generated model [ ] predicts a helix-loop-helix structure in which the first helix covers regions corresponding to helix and part of helix of hmg- , while the second helix of the rna-binding domain corresponds to helix of hmg- (fig. c) . based on the sequence similarities discussed above we suggest that the dnabinding domain of sry has evolved into the rna-binding domain of hdag. this particular case is hardly unique: many recent publications report proteins that bind to both dna and rna. among such proteins are: dna-binding protein ssap, which contains an rna recognition motif (rrm) and specifically binds to the enhancer region of sea urchin histone h [ ] , human transcriptional activator p nrb which binds both dna and pre-mrna [ ] , mammalian protein h which binds to single stranded dna and to the corresponding rna sequence [ ] , and y-box proteins which recognize both rna and dna [ ] . two rna-binding proteins have been shown to have a similar structure to that of dna binding domains: ribosomal protein s appears to have a pair of helix-turn-helix motifs similar to dna architectural factors [ ] and the ribosomal protein l contains ␣-helices and is similar to homeodomain proteins [ ] . on the reciprocal side, the homeodomain of the drosophila bicoid protein has been shown to interact in a sequence-specific manner with -untranslated region of the caudal gene transcript [ , ] . both dna-binding and rna-binding domains often interact with nucleic acids using ␣-helical structures. while both the major and minor grooves of regular a-form rna are too narrow to permit a direct interaction with an ␣-helix, bulges and mismatches considerably alter the groove dimensions allowing sufficient contact surface with an ␣-helix [ ] . the hmg box has not been reported to bind rna, however it binds both double-and single-stranded dna [ ] . proteins containing an hmg domain have widely diverse cellular roles, indicating that an hmg domain participates in a variety of dna-protein interactions. it is conceivable that the wide dna-binding range of the hmg domain could extend to rna-binding, especially considering the high rates of mutation and selection existing in the virus. the basic stretches of the rna-binding domain are similar to the argininerich motifs (arms) found in the products of the hiv tat and rev genes, bacterial antiterminators and coat proteins from the rna viruses [ ] . short arginine-rich stretches tend to form ␣-helical structures, which can bind in the wide groove of rna close to loops or large bulges. only one arm is usually necessary for binding; an arginine-rich peptide as short as residues was shown to bind specifically to rna [ , ] . hdag is unique in that it requires two arms instead of one; moreover, the spacer between the arms (leucine zipper) is essential as well [ , ] . we therefore think that each arm constitutes only a part of the rna-binding motif, the entire motif being defined by its alignment with the hmg box. it is not clear whether the arm regions within the rna-binding domain of hdag are evolutionarily related to 'classic' arms or whether the sequence similarity is a consequence of the functional and structural constrains imposed on the rna/dna-binding domain. alignments of hdag and arm-containing sequences such as rev and tat show moderately high levels of similarity, but it is interspersed across the entire sequence and no continuous stretches of similarity were found with the arm domains of hdag. the most extended similarity found between the hiv rev gene and the hdag is outside of the rna binding domain of hdag (fig. f) . homology of hdag and the dna-binding domain of hmg box may give insight into the mechanism of neoplastic transformation and progression toward cirrhosis frequently observed in chronic hepatitis infection [ ] . it is possible that the rna-binding domain of hdag has retained some of the dna-binding capability of its ancestral hmg box and thus could activate cellular proto-oncogenes. it has been shown that the hmg box of the sry protein is involved in specific binding and regulation of the expression of the cellular proto-oncogene c-fos -fra [ ] . the rna binding domain of hdag may be capable of similar binding. while the specificity of such dna binding by hdag is likely to be lower than that of a true transcription factor, the sheer abundance and persistence of the antigen during chronic hepatitis infection, as well as the evolution of the hdv genome throughout the course of infection [ , ] might be sufficient to activate a cellular proto-oncogene. in fact, the expression of the proto-oncogene c-myc is a hallmark of chronic hdv infection; c-myc protein is observed only in cells that contain hdag [ ] . binding of hdag to cellular dna has not been explored. it would be interesting to test for specific binding of the rna-binding domain of hdag to c-myc and other proto-oncogenes that could be involved in the onset of cirrhosis observed in hdv patients. analysis of the hdv genome suggests that a large part of the viral genome, including the sequence coding for hdag, is of cellular origin, while the remaining part resembles a plant viroid sequence [ , ] . the ribozyme activities of hdv, such as self-cleavage and self-ligation, further resemble the properties of the viroid. a possible mechanism explaining the origin of hdv -the only known animal virus with a circular rna genome -involves the capture of a cellular transcript by a viroid. the elegant capture mechanism proposed by brazas and ganem [ ] involves the fusion of the cellular and viral transcript, and duplication of the cellular transcript during the synthesis of the antisense strand, followed by the recircularization of the entire transcript (fig. ) . the size of the sry protein matches the hdv size requirements: sry is residues long; duplication of the sry rna would result in a cellular part of hdv over bases long (the exact length will depend on the length of the non-coding regions of the sry transcript). this agrees with the approximately bases of highly self-complementary rna of cellular origin in hdv. the replication of hdv is conducted by cellular machinery, with the addition of hdag. hdag is absolutely required for in vivo hdv replication [ ] and serves as a bridging factor between the viral template and cellular transcriptional complex. coopting a single component of the cellular fig. . possible mechanism of hdv formation. a viroid sense strand joins at the -end of sry transcript. b replication begins at the -end of the viroid and proceeds through sry, synthesizing an antisense of the viroid genome and of the sry transcript. a hairpin structure at the -end of sry template allows transcription to continue synthesizing a complement of the sry-that is a sense version of sry transcript. the resulting construct has an antisense strand of the viroid, followed by the antisense copy of sry followed by the sense copy of sry. c circularization of the linear transcript. d additional round of replication results in the reversal order of the regions: viroid region (sense) is followed by antisense strand of sry, which in turn is followed by the sense strand of sry. e subsequent evolution of the genome results in the organization of domains seen in the hdv today: a viroid region is followed by an antisense of hdv antigen followed by a non-coding region which is highly complementary to the region coding for the hdv antigen transcriptional machinery and adapting its specificity to prefer the viral template is an elegant way to ensure viral replication and survival. it is notable that the tissue-specificity of sry does not match that of hdv: sry is expressed primarily in germ cells and in somatic cells of the testis [ ] , while hdv is restricted to liver tissue. it has been shown experimentally, however, that the replication of the hdv can proceed in the skeletal muscle [ ] . a low level of sry could be expressed in other tissues, including the liver, allowing for transcript capture, or alternatively, hdv could have evolved from an sry-related gene specifically expressed in liver, such as sox- [ ] . finally, it is possible that the expression of original viroid was not confined to liver cells and the initial capture event of cellular rna could have occurred in a stem or germ cell where sry is abundantly expressed [ ] . this would introduce hdv into many cell types; its eventual dependence for replication on hepatitis b virus would limit its range to the liver cells. it may never be possible to conclusively determine the origin of hdag based on sequence similarity alone, since rna-encoded genes evolve rapidly. however several arguments can be made in support of sry as a cellular counterpart of hdag. . the sequence similarity between sry and hdag corresponds to the functional domains experimentally identified. . sequence similarity is restricted to functional domains -as expected for rapidly evolving rna-encoded genes. . both proteins -sry and hdag -bind nucleic acids and are involved in transcription. . sequence and structure similarity between dna-binding to rna-binding motifs is supported by a number of recent publications. the above arguments make the sry protein a strong candidate for a cellular counterpart of hdag. our hypothesis predicts presence of additional nuclear localization signals within arms which was recently confirmed experimentally. it also predicts structural perturbation of the hdv rna upon hdag binding, and possible specific binding between hdag and the regulatory regions of cellular proto-oncogenes. testing these predictions and determining the structure of hdag may shed further light on possible evolutionary relationship between the hmg domain of sry and the rna binding domain of hdag. the intracisternal a-particle proximal enhancer-binding protein activates 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proteins rna recognition and translational regulation by a homeodomain protein sequence and expression of sox- , a new hmg-box transcription factor the intracellular distribution and function group of the high mobility chromosomal proteins phylogeny of viroids, viroidlike satellite rnas, and the viroidlike domain of hepatitis delta virus rna influence of hepatitis delta virus infection on progression to cirrhosis in chronic hepatitis type b maximum-likelihood estimation of evolutionary trees from continuous characters progressive sequence alignment as a prerequisite to correct phylogenetic trees an sry-related sequence of the marsupial x chromosome: implications for the evolution of the mammalian testis-determining gene the rnas of hepatitis delta virus are copied by rna polymerase ii in nuclear homogenates identity of the rna-binding protein k of hnrnp particles with protein h , a sequence-specific single strand dna-binding protein the hmg domain of lymphoid enhancer factor bends dna 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molecular biology replication of the hepatitis delta virus rna in mice after intramuscular injection of plasmid dna solution structure of a dna-binding domain from hmg replication and evolution of viroid-like pathogens a gene from the human sexdetermining region encodes a protein with homology to a conserved dna-binding motif rna-binding proteins as regulators of gene expression comparison in biosequences two independent nuclear localization signals are present in the dna-binding high-mobility group domains of sry and sox rna recognition by an isolated a helix structural variety of arginine-rich rna-binding peptides expression of the c-myc protooncogene product in cells infected with hepatitis delta virus immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies structural and functional properties of the evolutionary ancient y-box family of nucleic acid binding proteins characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex the rna binding domain of ribosomal protein l is structurally similar to homeodomains transcription of circular and noncircular forms of sry in mouse testes we are grateful to d. w. smith and t. l. bailey for suggestions and critical reading of the manuscript. this work was supported by nih/ncrr grant rr and by nsf grant asc- . received june , key: cord- -vmnj b authors: saikruang, wilaiporn; khamrin, pattara; suantai, boonpa; okitsu, shoko; hayakawa, satoshi; ushijima, hiroshi; maneekarn, niwat title: detection of diarrheal viruses circulating in adult patients in thailand date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vmnj b a total of fecal specimens collected during january-december from adult patients with diarrhea were screened for group a and c rotaviruses, noroviruses gi and gii, sapovirus, aichi virus, human parechovirus, enterovirus, adenovirus and astrovirus by rt-multiplex pcr. the detection rate for diarrheal viruses was . %. adenovirus and enterovirus were equally detected as the most predominant viruses, with prevalence of . %, followed by aichi virus ( . %) and norovirus gii ( . %). mixed infection with norovirus gii and human parechovirus was also detected ( . %). this study provides epidemiological data for a wide variety of diarrheal viruses circulating in adult patients with diarrhea in chiang mai, thailand. acute gastroenteritis (age) is one of the most common diseases in children and adults and continues to be a significant cause of morbidity and mortality worldwide. the most common etiology is diarrheal viruses. among various types of diarrheal viruses, norovirus (nov) and rotavirus (rv) are considered to be the major cause of diarrhea [ ] . moreover, associations with other viruses such as adenovirus (adv), sapovirus (sav) and astrovirus (astv) have also been reported in sporadic and outbreak cases of diarrhea [ , , , ] . nov is now recognized as the main cause of epidemic gastroenteritis in all age groups [ ] . among adults and elderly patients, nov is responsible for . to . % of age cases [ ] . rv is more common in children less than years of age. studies conducted in adults with gastroenteritis in several countries from europe, america, asia, and australia have demonstrated that the prevalence of rv ranges from to % [ , , , ] . enteric adv can also cause age in adults, but at a lower rate than those by rv or nov infections, ranging from . to . % [ , ] . astv has also been shown to associate with age, with a frequency ranging between and % [ ] . for sav, although it is known to cause diseases primarily in children, it has also recently been reported to affect young adults to the elderly [ ] . in addition, there are several reports of newly discovered enteric viruses that are associated with age in humans, including aichi virus (aiv), human parechovirus (hpev), enterovirus (ev), and human cosavirus (hcosv) [ , , , , ] . in thailand, there have been far fewer epidemiological studies of diarrheal viruses in adults than in children. therefore, it is of interest to investigate the molecular epidemiology of diarrheal viruses in adults with diarrhea in chiang mai, thailand. a total of fecal specimens were collected from adult patients with diarrhea, with the ages ranging from to years, who attended chiang mai university hospital, chiang mai province, thailand, during the period of january to december . the specimens were stored at - °c until used. the study was conducted with the approval of the ethical committee for human rights related to human experimentation, faculty of medicine, chiang mai university (no. / ). the viral genome was extracted from a % fecal suspension using a geneaid viral nucleic acid extraction kit ii (geneaid, taipei, taiwan). then, the specimens were tested for the presence of sav, aiv, group a rotavirus (rva), group c rotavirus (rvc), hpev, nov gi and gii, ev, adv, and astv by rt-multiplex pcr using the protocol described previously by khamrin et al. [ ] . positive and negative controls were also concurrently included along with the test samples. the oligonucleotide primers for the detection of each virus are shown in table . the pcr products obtained from the specimens that were positive for diarrheal viruses were subjected to direct sequencing using a bigdye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa). the sequences obtained were compared with reference sequences by searching for closely related reference sequences in the ncbi genbank database using the blast server (http://www.ncbi.nlm.nih.gov/blast). the nucleotide sequences of diarrheal viruses described in the present study have been deposited in the genbank database. the accession numbers are as follows: kj -kj for advs, kj -kj for evs, kj for hpev, and kj -kj for novs gii. screening by rt-multiplex pcr showed that out of ( . %) samples were positive for five types of diarrheal viruses. among these, adv, ev, aiv, nov gii, and hpev were detected, while rva, rvc, sav, nov gi, and astv were not found in this study. adv and ev were detected as the most predominant viruses ( . %, out of for each virus), followed by aiv ( . %, out of ) and nov gii ( . %, out of ). in addition, a mixed infection with nov gii and hpev was also detected in one fecal specimen ( . %), as shown in table . based on nucleotide sequence analysis, all three nov gii specimens detected in the present study belonged to the gii. genotype. for adv, three different genotypes were identified, including adv , adv , and adv . in addition, four strains of ev found in this study belonged to two different species, enterovirus b and enterovirus c. interestingly, aiv of both genotypes a and b were also detected in this surveillance. furthermore, one hpev strain detected in this study belonged to genotype ( table ) . in this study, . % of fecal specimens collected from adults with diarrhea were positive for diarrheal viruses. five types of viruses out of a total of were detected in this study. monoinfections with one type of virus were found for adv, ev, aiv, and nov gii. in addition, a mixed infection with nov gii and hpev was found in one case, but the impact of the mixed infection on clinical severity was not determined for this patient. it had been reported previously that no significant difference was found in the clinical symptoms of patients with multiple viral infections as compared to monoinfection [ ] . the prevalence of diarrheal viruses detected in the present study is consistent with the findings reported previously in all age groups in thailand, where the prevalence was reported at % [ ] . adv infection occurs worldwide and may involve several systems and organs, including the upper and lower respiratory tract, the gastrointestinal (gi) tract, the urinary tract, and the eyes [ ] . the adv types that commonly infect the gi tract, the so-called enteric adenoviruses, are adv and adv in subgroup f. sequence analysis showed that adenovirus detected in this study belonged to two distinct species (d and f) with three genotypes (ad , ad , and ad ). when the adv sequences detected in this study were compared to those from previous studies, the data clearly demonstrated that the adv genotypes identified in children and adults are different. the adv strains found in adults were adv and adv of subgroup d and adv of subgroup f, while the strains identified previously in children were adv of subgroup d, adv of subgroup b, and adv of subgroup f [ ] . it is interesting to note that in the present study we detected adv and adv , in addition to adv , in adult patients with diarrhea. the adv types and commonly infect the eyes, causing conjunctivitis and epidemic keratoconjunctivitis [ ] . to our knowledge, this is the first report of adv and adv in adult patients with diarrhea in this area. in this study, the ev detection rate in adult patients was . %, which is somewhat lower than that reported previously for children in thailand in ( . %) [ ] . nucleotide sequence analysis of four ev strains detected in this study revealed that they belonged to species b and c. for aiv, the prevalence in adults is similar to that in children, where the detection rate is as low as . %. molecular genetic analysis of the only aiv strain detected previously in a child with diarrhea in chiang mai, thailand, revealed that it was genotype a [ ] . it is interesting, however, to note that both genotype a and b of aivs were detected in the present study. these data clearly demonstrate that the aiv strains circulating in this area are genetically diverse. several epidemiological reports of nov infections have shown that nov gii, particularly gii. , is the most predominant genotype in all parts of the world. most recently, surveillance of nov in thailand revealed that the prevalence of nov infection in all age groups is as high as . %. nov gii was shown to be the most predominant genotype and accounted for . % of cases [ ] . however, the nov gii detection rate in adults with diarrhea in the present study was as low as . %. it is possible that nov gii is not the major pathogen causing diarrhea in adults in this area. in addition, this study clearly demonstrates that hpev is an unusual cause of acute gastroenteritis in adults compared to other viruses. the prevalence of hpev infection in thailand has only been reported in children with acute gastroenteritis at an infection rate of . % in [ ] and . % in - [ ] . the hpev genotype found in adults in this study is genotype , which was the predominant genotype detected in diarrheal children. however, hpev genotypes identified in children with diarrhea were highly diverse, including hpev - , , and [ , ] . it should be pointed out that rotavirus, which is the most important cause of diarrhea in pediatric patients, was not detected in adult diarrheic patients in the present study. the low prevalence and lack of detection of rotavirus infection in adult patients observed in this study might be due to immunity to rotavirus resulting from natural infection. the relatively low rate of diarrheal virus detection in adults with diarrhea in this study suggests that acute gastroenteritis in adults in this area may be caused by other pathogens. further investigation for bacterial or parasitic infections may help to clarify this point. nevertheless, several other diarrheal viruses that may cause diarrhea, including saffold virus, pestivirus, coronavirus, picobirnavirus, and torovirus [ , ] , were not included in the screening protocol in this study. in addition, the sensitivity limit of the multiplex pcr method or low amount of target viruses may also affect the detection rate. in conclusion, this study demonstrates that a wide variety of viral pathogens that are associated with diarrhea are circulating in adult patients in chiang mai, thailand. since epidemiological information about gastroenteritis viruses in adults is limited, it is important to 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stool samples collected from thai children with acute gastroenteritis identification of enteroviral infection among infants and children admitted to hospital with acute gastroentritis in ho chiminh city. vietnam serotype g rotavirus infections in adults in sweden molecular detection and characterization of aichivirus a in adult patients with diarrhea in thailand genetic diversity of sapovirus in non-hospitalized adults with sporadic cases of acute gastroenteritis in shanghai viruses causing gastroenteritis adenoviruses application of a reverse transcription-pcr for identification and differentiation of aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans detection of norovirus (gi, gii), sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex pcr development of rt-multiplex pcr assay for detection of adenovirus and group a and c rotaviruses in diarrheal fecal specimens from children in china general primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections the authors declare that they have no conflict of interest. key: cord- -adbxzgvs authors: du, juan; zhu, teng; zhuang, lu; zhang, pan-he; zhang, xiao-ai; lu, qing-bin; liu, wei title: identification and complete genome characterization of human enterovirus from a child with pneumonia in china date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: adbxzgvs in this study, human enterovirus c (ev-c ) was detected in a -month-old boy diagnosed with pneumonia in china. a phylogenetic analysis showed that this strain was genetically closer to the lithuanian strain than to the usa strain. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. -month-old child [ , ] . since then, ev-c has been identified in four children from two other countries, china and mongolia [ ] . here, we report the identification and complete genomic sequence of ev- in a child with pneumonia in china, which may provide more information for understanding ev-c infection. a -month-old boy (patient cq ) from guizhou province was admitted to the children's hospital of chongqing medical university in june and diagnosed as having pneumonia. he had a -month history of phlegm in the throat, especially in the morning and at night, and tachypnoea. he occasionally regurgitated milk along with mucous sputum, and some medium and coarse moist rales could be heard in both lungs. transient rhinorrhoea, paroxysmal cough, and diarrhoea without fever were apparent during the course of the disease. laboratory examinations performed at admission revealed a normal white blood cell (wbc) count ( . × /l) and c-reactive protein level (< mg/l), with a mildly elevated platelet count ( × /l), and a high percentage lymphocytes ( %). two days before admission, an x-ray radiograph showed slight interstitial changes in both lungs, and routine blood examination revealed elevated wbc ( . × /l) and platelet ( × /l) counts. cephalosporin antibiotics for treating the infection for more than days had no obvious effect. a nasopharyngeal aspirate was collected from the child at admission, and the parents gave written informed consent for their child to participate in the study. we extracted rna and dna from the nasopharyngeal aspirate sample using aseptic techniques and tested the viral nucleic acids for evidence of respiratory viruses. using molecular methods described previously, we tested for influenza virus (a, b and c), ev, metapneumovirus, respiratory syncytial virus (rsv), adenovirus, human bocavirus, parainfluenza virus (types - ), and coronaviruses ( e and oc ) [ , ] . the study protocol was reviewed and approved by the ethics review committee of children's hospital of the chongqing medical university. the sample was positive for ev and parainfluenza virus type . igg antibodies against cytomegalovirus and epstein-barr virus were also detected, while the sputum culture was negative for these viruses. to determine the enterovirus type, we amplified a portion of the 'ncr-vp /vp region of the enterovirus by nested pcr as described previously [ , ] . the -bp amplicon was sequenced, and basic local alignment search tool (blast) analysis (http://www. ncbi.nlm.nih.gov) revealed a high degree of similarity to ev-c . using multiple primers (table ) , we determined the full-length viral genome sequence of the strain detected in patient cq (genbank accession no. mk ), which was found to consist of nucleotides, excluding the poly(a) tail. the 'utr contains nucleotides, and the 'utr consists of nucleotides. the cq genome contains a single open reading frame from nt to encoding a -amino-acid polyprotein. the base composition of the full genome is . % a, . % c, . % g, and . % u. the complete genome sequence was compared with those of hev-c strains available in the gen-bank database, which included ev- , ev- , ev- , ev- , and ev- . the cq isolate shares . %- . % nucleotide sequence identity in the coding region with other hev-c strains, including ev- ( . %- . %), ev- ( . %- . %), ev- ( . %- . %), and ev- ( . %- . %), and shares . %- . % nucleotide sequence identity with other ev-c strains ( table ) . after alignment using mega version . , we constructed a phylogenetic tree by the maximum-likelihood method, using the tamura-nei model and bootstrap replicates. the resulting tree showed that cq belongs to genotype ev-c (fig. a) . although it was genetically closer to the lithuanian strain than to the usa strain, the cq strain was separate from both strains, with at least % bootstrap support. the genome of the cq isolate has a typical enterovirus organization, encoding four structural proteins (vp , vp , vp and vp ) and seven non-structural proteins ( a, b, c, a, b, c and d). the vp sequence of the cq isolate is . %- . % identical to those of other ev-c isolates, and other genome regions displayed > % sequence identity to ev-c . conversely, all genome regions of cq showed less similarity to other ev-c strains (including ev-c , ev-c , ev-c , and ev-c ) ( table ) . a phylogenetic tree was constructed based on the vp gene nucleotide sequences using the maximum-likelihood method. this illustrated the differences among the cq strain isolated in this study, the lithuanian strain, the mongolian strains, and the usa strain (fig. b) . in other phylogenetic trees that were constructed based on the 'utr, 'utr, vp -vp region, a- c region and a- d region, more-pronounced genotype clustering was obtained, and the cq strain clustered with ev-c in all cases except for the 'utr tree (supplemental fig. ). in summary, we report the detection of ev-c in a child hospitalized with pneumonia. we demonstrated that the vp -vp , a- c and a- d gene regions of the cq strain were different from those of the other ev-c strains. because parainfluenza virus type and cytomegalovirus were also found in samples from this patient, the causal correlation between ev-c and respiratory disease could not be established. therefore, it is necessary to further investigate the role of ev-c in respiratory illnesses in children. conflict of interest the authors declare that they have no conflict of interest. the study protocol was reviewed and approved by the ethics review committee of children's hospital of chongqing medical university. we obtained informed consent from the child's parents. complete genomic sequencing shows that polioviruses and members of human enterovirus species c are closely related in the noncapsid coding region complete genome sequence of a novel human enterovirus c (hev-c ) identified in a child with communityacquired pneumonia novel human enterovirus c infection in child with community acquired pneumonia real-time rt-pcr detection of respiratory viral infections in four triplex reactions complete genome characterization of enterovirus circulating in northern italy shows recombinant origin of the p region development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses newly identified enterovirus c genotypes, identified in the netherlands through routine sequencing of all enteroviruses detected in clinical materials from respiratory infection with enterovirus genotype c , china and mongolia key: cord- -gyb mjb authors: chai, weidong; burwinkel, michael; wang, zhenya; palissa, christiane; esch, bettina; twardziok, sven; rieger, juliane; wrede, paul; schmidt, michael f. g. title: antiviral effects of a probiotic enterococcus faecium strain against transmissible gastroenteritis coronavirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: gyb mjb the enteropathogenic coronavirus transmissible gastroenteritis virus (tgev) causes severe disease in young piglets. we have studied the protective effects of the probiotic enterococcus faecium ncimb (e. faecium), which is approved as a feed additive in the european union, against tgev infection. e. faecium was added to swine testicle (st) cells before, concomitantly with, or after tgev infection. viability assays revealed that e. faecium led to a dose-dependent rescue of viability of tgev-infected cells reaching nearly to complete protection. virus yields of the e. faecium–treated cultures were reduced by up to three log( ) units. western blot analysis of purified tgev revealed that the levels of all viral structural proteins were reduced after e. faecium treatment. using transmission electron microscopy, we observed attachment of tgev particles to the surface of e. faecium which might be a means to trap virus and to prevent infection. increased production of nitric oxide in the cells treated with e. faecium and elevated expression of interleukin and pointed to stimulated cellular defense as a mechanism to fight tgev infection. transmissible gastroenteritis virus (tgev) infects enteric and respiratory tissues and causes severe gastroenteritis with a mortality rate close to % in newborn piglets [ , ] . the appearance of the closely related tgev variant porcine respiratory coronavirus (prcov) has been found beneficial in preventing tgev infections, possibly through induction of neutralizing antibodies that can provide crossprotection against tgev infection [ , ] . however, tge prevalence is still being reported, and some tgev strains have been isolated from domestic pigs in different parts of the world [ ] . commercially available vaccines, either inactivated or attenuated, have failed to provide full protection to piglets [ ] . it is likely that the parentally applied inactivated viruses do not induce the local immune response in the small intestine that is required for protection. therefore, the discovery and development of new, highly potent anti-tgev agents and effective approaches for controlling the emergence of tgev infection remains an important mission. probiotics are defined as live microbial food supplements with health-promoting attributes. potent mechanisms of beneficial action include the production of antimicrobial agents, modulation of immune responses and promotion of host innate defense mechanisms [ , , , , ] . enterococcus faecium ncimb (e. faecium) is authorized in the eu for use as a probiotic feed additive for sows and piglets and several other farm animal species. beneficial effects of the probiotic e. faecium such as immune modulation and improvement of nutrient transport have previously been reported in several studies [ , , , , ] . in vitro studies have also demonstrated that e. faecium could reduce the rate of invasion of pathogens-for instance, salmonella in intestinal cell lines [ , ] . but detailed knowledge on the impact of e. faecium on viral infections in vivo and in vitro is lacking. the purpose of the present study was to establish an in vitro model to investigate the antiviral potential of e. faecium. we used an established swine testicle (st) cell line to assess the protective effects of e. faecium on tgev infection in terms of viral replication and cell survival. to gain insight into its possible mechanisms of action, the effects of e. faecium on viral protein synthesis as well as the induction of inducible nitric oxide synthase (inos) and selected cytokines were investigated. our results described here suggest that this probiotic e. faecium strain exhibits antiviral activity against tgev and may possibly serve as a useful antiviral agent against coronavirus infections in vivo. the epithelial swine testicle (st) cell line was maintained in dulbecco's modified eagle's medium (dmem, pan biotech) supplemented with % fetal bovine serum (hyclone), and % penicillin/streptomycin (biochrom), growing at °c in a % co humidified incubator. the tgev strain purdue -mad (kindly provided by dr. c. schwegmann-wessels, institut für virologie, tierärztliche hochschule hannover) was used in this study. stock virus was propagated in st cells to a titer of . e? pfu/ml. all infections were done at a multiplicity of infection of . . enterococcus faecium ncimb isolated from a commercial product used in animal nutrition (cylactin Ò , cerbios-pharma sa, lugano) was used in this study and cultivated in todd-hewitt-bouillon (thb, roth). the number of viable bacteria in ml of bacterial culture was determined by plating bacteria on agar. bacterial cultures were then centrifuged at rpm for min, and bacteria were washed twice to remove excess thb. finally, the viable e. faecium particles were resuspended in dmem to a stock concentration of . e? cfu/ml. heat inactivation of bacteria was performed by heat treatment with e. faecium ( . e? , . e? , . e? cfu/ml) in dmem in a water bath at °c for min. bacterial culture supernatants were obtained from growing bacterial cultures in thb. bacteria were removed by centrifugation at rpm for min, and supernatants were collected. assessment of cellular toxicity of e. faecium suspensions of ll containing different amounts of e. faecium ranging from . e? to . e? were added to st cell monolayers in a -well plate (greiner bio-one) for . h before washing away. at the end of the incubation period, a methylthiazolyl-diphenyl-tetrazolium bromide (mtt) viability assay was carried out as described previously [ ] . the cell survival rate was determined as bacteria average od value/control average od value. the % cytotoxic concentration (cc ) was defined as the concentration that inhibited cell proliferation by %, and a non-cytotoxic concentration of e. faecium was used for antiviral assays. four different experimental protocols were applied to investigate the antiviral activity of e. faecium. three setups focused on the effect of e. faecium on the cells by varying the treatment period in relation to infection with tgev. a fourth setup assessed the direct effect of the probiotic on virus particles. in brief, monolayers of st cells were treated with e. faecium for . h, which was washed away before infection with tgev for h (pretreatment assay), e. faecium and tgev were added to the cell layer together during the -h infection period (competition assay), or e. faecium was added for . h right after the infection period (post-infection treatment assay). after probiotic treatment as well as after infection with tgev, cells were washed twice and kept in medium containing % penicillin/streptomycin to kill any viable bacteria that were left. to assess direct effects of e. faecium on tgev without cells being involved, the virus was mixed with different concentrations of e. faecium and incubated for . h at °c. after centrifugation for min at rpm to sediment bacterial cells, the virus containing supernatants were used to infect st cells (cell-free preincubation assay). the antiviral effects of heat-inactivated e. faecium as well as serially diluted e. faecium supernatants were also tested in the competition assay. virus-infected st cells and cells without addition of e. faecium served as controls from which samples were collected at and h after infection (hpi) for the % tissue culture infective dose (tcid ) and the mtt viability assay, respectively. relative survival of cells was calculated as follows [ ] : percent viable cells = [(od value of e. faecium group -od value of infection control)/(od value of blank control -od value of infection control)] . transmission electron microscopy (tem) in order to examine possible direct binding of virus by e. faecium, the cell-free preincubation assay was performed by mixing e. faecium with tgev at a bacteria-tovirus ratio of for . h. after centrifugation for min at rpm to sediment bacterial cells, the pellet was resuspended in ml karnovsky's fixative. the samples were centrifuged for min at rpm and a drop ( ll) was taken from the bottom of the tube and negatively stained with % phosphotungstic acid for min. finally, the samples were evaluated with a transmission electron microscope (zeiss cr). virus yield reduction assay st cell monolayers were infected with tgev with or without probiotic bacteria treatment according to the experimental design. at hpi, aliquots of the supernatants were taken, and serial tenfold dilution steps were performed. infectivity was determined by endpoint dilution titration on st cells in a -well plate. the plate was incubated for hpi, and infectivity was determined by recording the virus-induced cytopathic effect (cpe). virus titer was calculated by the method of reed and muench, which is usually used for the calculation of ld [ ] and documented as tcid values. st cells were infected in culture dishes with -cm growth areas under competition assay conditions with e. faecium. at hpi, virus from cell culture fluids and cells were collected by ultracentrifugation in a l - ultracentrifuge (beckman coulter) at , rpm for . h using a sw rotor. the pellet containing virus particles, with equal amounts of total protein, underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were electro-transferred to hybond lfp (pvdf) membranes (ge healthcare) using a feline anti-tge polyclonal antiserum (natutec) at a dilution of : and an anti-feline igg polyclonal antiserum (rockland) at a dilution of : , . antigenantibody complexes were detected using a western blotting substrate (pierce Ò ecl plus). immunodetectable protein bands on the membrane were visualized using the fusion sl imaging system (vilber lourmat), and protein amounts were estimated by densitometric analysis using the fusion-capt software (vilber lourmat). three independent experiments and appropriate gel exposures yielded very similar results for each treatment modality. detection of nitric oxide (no) release at hpi, in different assays, no release was determined by measuring the amount of released no using the griess-assay (promega) according to the manufacturer's protocol. lps ( . mg/ml)-stimulated cells were used as a positive control. samples from untreated cells with or without prior infection served to define basal values. total rna from st cells was isolated using a gene matrix rna purification kit (eurx) as described by the manufacturer. reverse transcription (rt) was performed using a revertaidtm first strand cdna synthesis kit (fermentas) according to the manufacturer's instructions. pcr reactions were performed in a total volume of ll in an icycler iq detection system (bio-rad laboratories). data analysis was based on the measurement of the cycle threshold (c t ). the differences in the ct values of untreated samples versus treated samples were calculated by using the delta-delta-ct method [ , ] . each sample was measured in triplicate from three independent experiments. the names of genes, the genbank accession number, the primer sequences, the annealing temperatures, and the sizes of the amplification products are listed in table . all calculations were performed with ibm spss . statistical analysis of virus titers and no detection was performed by two and one factorial anova, respectively, followed by scheffé's post hoc test. cytokine expression data analysis was performed by paired t-test. p-values less than . were considered statistically significant. all data are given as the mean ± sd. before the probiotic e. faecium can be used for interference studies, the concentration range in which its addition to cells is non-toxic was defined. the results from cell viability assays (fig. ) show that e. faecium was non-toxic at concentrations up to . e? cfu/ml. the viability rate of st cells was %, and no morphological differences were observed between bacteria-treated and mock-treated cells at this concentration. therefore, the highest concentration of e. faecium chosen for the interference study with tgev was . e? cfu/ml. the cc of e. faecium in st cells was calculated to be . e? cfu/ml. (fig. a) show that e. faecium provided protection from tgev infection in a dose-dependent manner. up to % protection was achieved at the highest concentration of e. faecium ( . e? cfu/ml) when the probiotic was added to the cells together with the virus during the infection period (competition assay). to find out whether e. faecium inactivates tgev particles by direct physical interaction with virus, a cell-free preincubation assay was performed. the results show that the infectivity of tgev was also reduced in a concentration-dependent manner. these results from the mtt assay ( fig. a) were confirmed by flow cytometry using propidium iodide staining in an independent experiment (data not shown). furthermore, as illustrated by electron microscopy (fig. ) , virus particles seemed to be bound by e. faecium and attached to the e. faecium surface. because the competition assay exhibited the most pronounced antiviral activity in terms of cell survival (fig. a) , the antiviral effect of heat-killed e. faecium and e. faecium supernatant in the competition assay was also tested. the results (figs. b, c) show that heat-killed e. faecium and e. faecium supernatant still had antiviral activity, but it was much less pronounced, suggesting that live e. faecium is necessary to exhibit the observed virusreducing effects. the anti-tgev activities of e. faecium were confirmed by measuring released infectious virus in the culture medium using a tcid assay. as expected, the results (fig. ) are consistent with those from the cell viability assays, since tgev yields were found to be reduced by treatment with e. faecium. again, the inhibition of virus production was most effective in the competition assay, when cells had been exposed to the highest concentration of e. faecium ( . e? cfu/ml), amounting to a three-log reduction. reduced virus titers were also found in the cell-free preincubation assay, which indicates that the probiotic e. faecium also has antiviral capacity at the level of direct physical interaction with virus particles. tgev produced on a large scale under e. faecium interference conditions (competition assay) was enriched by ultracentrifugation and subjected to sds-page followed by western blot analysis. protein assays revealed strongly reduced amounts of total viral protein when virus from probiotic treated cells was analyzed (fig. ) dilution of e.faecium metabolic products after h, an mtt assay was carried out. results are plotted as percent viability, with uninfected cells without e. faecium taken as %. results are given as mean ± standard deviation from at least three independent experiments the probiotic during the infection period, tgev protein levels were reduced by more than %. more importantly, after western blotting with antibodies raised against total tgev protein, densitometric inspection failed to show any major aberrations of the relative polypeptide compositions of the virus particles, indicating that the levels of all viral proteins were evenly reduced. in a first approach to elucidating the mechanism of the effect of probiotic treatment on tgev production, the synthesis of antiviral no was measured. as shown in table the levels of the positive control lps, which is a strong inducer of no release. cytokines are important components of cellular defense mechanisms against microbial infection. treatment of st cells with the probiotic could modulate the cellular expression patterns of cytokines and thereby reduce the efficiency of tgev multiplication. as a first step to test this hypothesis, we studied the production of selected cytokines under the influence of e. faecium in tgevand mock-infected st cells at h, h, h, h and hpi (competition assay). a clear increased expression of cytokines was observed, reaching the highest levels of expression at hpi. the results show that administration of . e? cfu/ml e. faecium together with the virus significantly increases mrna expression levels of the pro-inflammatory cytokines interleukin (il- ) and il- (an approximately -and -fold increase, respectively) when compared with tgev-infected st cells that had not been exposed to the probiotic (fig. ) . tumor necrosis factor-alpha (tnf-a), interferon a (ifn-a) and toll-like receptor- (tlr- ) mrna expression showed a less pronounced increase when compared with tgevinfected cells that had not been exposed to the probiotic. administration of . e? cfu/ml e. faecium alone increased similar mrna expression levels of those cytokines. il- b, il- and il- levels were apparently below the detection limit of the pcr assay applied in this study. to assess the potential prophylactic or therapeutic effect of the probiotic bacteria e. faecium on tgev infection, increasing concentrations of probiotic e. faecium bacteria were added to st cells before, concomitantly with, or after tgev infection for a short period of time, and cell viability as well as virus titers in the culture medium were quantitatively assessed later after long-term incubation. a low moi of . was chosen in order to allow multiple infection cycles, as this more closely reflects natural infection. by pre-treatment of st cells with e. faecium (pretreatment), the viability of tgev-infected cells was protected, and virus yields were reduced (figs. and ). it appears that e. faecium can interfere with virus attachment and/or entry into cells. several studies have demonstrated that probiotics can block viral attachment by competitive inhibition if they are able to bind viral receptors at the surface of cells. freitas and coworkers [ , ] reported that the lactobacillus casei strain dn and a strain of bacteroides thetaiotaomicron produce a soluble compound that partially protects epithelial cells from rotavirus infection in vitro by modulating the apical glycosylation pattern of the cells. the post-infection treatment assay suggested that the antiviral activity of e. faecium also contributed to the stimulation of pro-inflammatory factors (i.e., increased mrna expression levels of il- and il- ). pagnini and coworkers have shown that the multiple probiotic formulation vsl# could stimulate the epithelial production of tnf-a and activate nf-jb in vitro [ ] . probiotic bacteria may also indirectly interfere with virus by altering the state of cells, stimulating innate and/or adaptive immunity [ , ] . in this study, the expression of antiviral cytokines il- and il- may alter the state of cells, eventually leading to an antiviral response. in our cell-free pre-incubation assay, improved survival and a significant drop in virus titer were also observed ( figs. and ) . in theory, the virus could also fail to infect the host cells if it is trapped by adsorption to the bacterial surface, and from the tem result (fig. ) , we did observe that virus particles were trapped by e. faecium. there may be some molecular mimicry between a bacterial surface molecule (such as glycoprotein with sialic acid) and a eukaryotic cellular receptor used by a virus for attachment [ ] . in this study, the competition assay in which virus and probiotic bacteria are present in the culture medium side by side, exhibited the most pronounced antiviral activity in terms of cell survival (figs. and ) . this most pronounced antiviral activity most likely resulted from the sum of overlapping mechanisms at different time points before and after virus infection, as shown before, including the finding that the levels of all of the viral structural proteins were equivalently reduced after e. faecium treatment (fig. ) indicates that indeed fewer tgev particles were released from these infected cells. likewise, reduced synthesis of tgev proteins may decrease the amount of virus-induced damage and subsequently also ameliorate the cytopathic effect in virus-infected st cells, which logically must lead to a rescue of cell viability. as the competition assay was the most effective antiviral approach, we looked for possible direct mechanisms for the effect of probiotic treatment by looking for no release from infected cells. we found that e. faecium could significantly induce no release ( table ). the antiviral effects of no have been well studied for several viral infections [ , , [ ] [ ] [ ] . although no production is believed to be released mainly in macrophages, we did detect an increase of no release in st cells upon treatment with e. faecium and tgev, and this release of no was dose dependent. this indicates that an induction of inos could indeed play a role in the mechanisms for our observation that e. faecium inhibits tgev infection in st cells. for the competition assay, we also compared expression of the antiviral cytokines il- and il- . tgev infection of st cells without e. faecium did not significantly increased expression when compared to mock control. however, e. faecium treatment significantly increased the production of pro-inflammatory factors il- and il- in tgev-infected st cells. this result is consistent with those of other authors [ , , , , ] , who showed il- or il- production following the interaction of probiotics with the intestinal epithelium. because il- and il- responses in intestinal epithelial cells play important roles in the pathogenesis and immune defense against enteric pathogens [ ] , the increased level of those cytokines could also possibly indicate an enhanced innate response. according to scientific opinion, probiotic concentrations between and cfu/g of intestinal contents are required to elicit potential benefits to the host [ , ] . in feeding trials with piglets, concentrations of - cfu e. faecium/g digesta could be detected in the intestine [ ] . thus, although not directly comparable, the effective concentrations of e. faecium used in the present study are in a similar range, which adds to the relevance of these data. in conclusion, the results of the present study show that e. faecium inhibits tgev replication in st cells and that possibly overlapping mechanisms lead to the observed reduction of virus growth: direct interference with virus attachment, adsorptive trapping or inactivation of virus particles through surface components of the probiotic bacteria, and the stimulation of pro-inflammatory cytokines il- and il- as well as no production. the data suggest that e. faecium may serve as a useful antiviral agent against infection with tgev and possibly other viruses. challenge experiments with different porcine viruses in piglets are under way to substantiate this hypothesis. nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus field evaluation of the efficacy of a probiotic containing bacillus licheniformis and bacillus subtilis spores, on the health status and performance of sows and their litters nidovirales: a new order comprising coronaviridae and arteriviridae development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic lactobacillus and bifidobacterium species in the upper human gastrointestinal tract molecular interactions between bacteria, the epithelium, and the mucosal immune system in the intestinal tract: implications for chronic inflammation prevention and treatment of enteric viral infections: possible benefits of probiotic bacteria immunomodulatory effects of probiotics in the intestinal tract macrophages and cytokines in the early defence against herpes simplex virus escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies a heat labile soluble factor from bacteroides thetaiotaomicron vpi- specifically increases the galactosylation pattern of ht -mtx cells host-pathogens cross-talk. indigenous bacteria and probiotics also play the game enterococcus faecium ncimb does not protect interleukin- knock-out mice from chronic gut inflammation probiotics to enhance anti-infective defences in the gastrointestinal tract non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus th /th balance: the hypothesis, its limitations, and implications for health and disease receptors for binding measles virus on host cells and erythrocytes effect of probiotic strains on interleukin production by ht / a cells host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens porcine small intestinal epithelial cell line (ipec-j ) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method effects of enterococcus faecium ncimb as probiotic supplement on intestinal transport and barrier function of piglets uptake and toxic effects of heavy metal ions: interactions among cadmium, copper and zinc in cultured cells functional modulation of enterocytes by gram-positive and gram-negative microorganisms probiotics promote gut health through stimulation of epithelial innate immunity new scientific paradigms for probiotics and prebiotics action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus analysis of differential dna damage in the mitochondrial genome employing a semilong run real-time pcr approach innate mechanisms for bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats interaction of probiotics and pathogensbenefits to human health? influence of a probiotic enterococcus faecium strain on development of the immune system of sows and piglets impact of the probiotic bacteria enterococcus faecium ncimb (sf ) and bacillus cereus var. toyoi ncimb on the development of serum igg and faecal iga of sows and their piglets transmissible gastroenteritis virus infection: a vanishing specter an interdisciplinary study on the mode of action of probiotics in pigs transgenic mice secreting coronavirus neutralizing antibodies into the milk influence of a probiotic strain of enterococcus faecium on salmonella enterica serovar typhimurium dt infection in a porcine animal infection model immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) alive and dead lactobacillus rhamnosus gg decrease tumor necrosis factor-alphainduced interleukin- production in caco- cells acknowledgements the study was funded by the deutsche forschungsgemeinschaft (dfg) through grant sfb / to sub-project a (mfgs). key: cord- - cdfhi authors: campalto, m.; carrino, m.; tassoni, l.; rizzo, g.; rossmann, m. c.; cocchi, m.; de benedictis, p.; beato, maria serena title: divergent minute virus of canines strains identified in illegally imported puppies in italy date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: cdfhi minute virus of canines (mvc) belongs to the family parvoviridae, genus bocaparvovirus, and has been mainly described during enteritis episodes in young dogs. this study reports the characterization of four divergent mvc strains detected between and , three of which were from dogs illegally imported into italy, most probably from eastern europe, that cluster together phylogenetically but share low genetic similarity with the fourth mvc from an autochthonous dog and other available mvc sequences. our data indicate that the introduction of genetically distinct mvc strains occurred through the illegal movement of dogs from a geographic area where a distinct mvc lineage was most likely circulating. enforced surveillance of mvc in the dog population of eastern europe and its neighboring countries may shed light on, and eventually trace back to, illegal animal movements. minute virus of canines (mvc) belongs to the family parvoviridae, genus bocaparvovirus, and is antigenically distinct from canine parvovirus (cpv ), which belongs to the genus protoparvovirus and is a major causative agent of gastroenteritis [ ] . canine bocaparvoviruses (cbovs) are non-enveloped single-stranded linear dna viruses with a genome size of about . kb with three open reading frames (orfs) coding for two non-structural proteins, ns and np , and two overlapping capsid proteins, vp and vp [ ] . orf , located at the ′ end, is , bp long and encodes ns ( amino acids [aa]) [ ] , which is involved in replication, regulation of viral expression, and cytotoxicity [ ] . orf , located at the ′ end, is , bp long and encodes vp ( aa) and vp ( aa) [ ] . the vp protein is critical for mvc infection, while vp mediates receptor recognition and nuclear translocation [ ] . orf ( bp long) partially overlaps with orf ( bp) and orf ( bp) and encodes the np protein ( aa) [ ] , which plays an essential role in accumulating capsid mrnas and proteins [ ] . genetic differences in the coding genes for ns , np and vp /vp allow three genotypes to be distinguished: cbov , cbov , cbov . mvc corresponds to cbov [ ] and was first isolated in from the feces of a clinically healthy military dog [ ] . mvc is distributed worldwide in domestic dogs of different ages. its clinical significance and virulence are uncertain. it produces mild to unapparent infections, mainly enteritis, in puppies and is weakly pathogenic in adults [ ] . pneumonitis, myocarditis, lymphadenitis and hepatitis have been reported in dogs with mvc infection [ ] [ ] [ ] . mvc may cross the placenta, causing early fetal death and birth defects [ , ] . the undefined pathogenic role of mvc compared to cpv may account for the limited information available on mvc. in italy, only one report is available describing a high-mortality episode in puppies of a shelter in southern italy in [ ] . novel cases of mvc infection have been detected in italy at the istituto , and sterile ultrapure water to volume. the following thermal profile was used: taq polymerase activation at °c for min followed by cycles of denaturation at °c for s and annealing at °c or °c for s for mvc and cpv , respectively. all of the archived mvc-positive diagnostic samples were re-analysed by real-time pcr, and those with cycle threshold (ct) values below were sequenced by the sanger method. positive mvc samples identified during the diagnostic activity were subjected to virus isolation attempts. five hundred µl of tissue homogenate was passed through a . µm filter and used to infect a confluent madin-darby canine kidney (mdck, nbl- , atcc cll- ) cell monolayer, maintained at °c in a % co atmosphere, and monitored daily. after three blind passages, mvc real-time pcr was carried out on cell supernatants to confirm the isolation of the virus. tissues and cell cultures that were confirmed to be positive for mvc were used to determine the full mvc genome sequence. six different endpoint pcrs were performed using primer pairs described by shan et al. [ ] covering the entire mvc genome with the exception of a small gap of nucleotides (nt) in the ns gene (from nt to , ). to cover this gap, an additional primer pair was designed ad hoc (mvc f, ′-gga tgc ctg gtc ccg ata g- ′; mvc r, ′-ata agt ttg ttc ccg ccc ga- ′). the endpoint pcrs were conducted with µl of primestar gxl buffer ( x) (takara, japan), . µl of each primer ( µm), µl of dntps ( . mm), . µl of primestar gxl taq polymerase ( . u/µl) (takara, japan), and µl of sterile ultrapure water to µl. the conditions for pcr were as follows: denaturation at °c for s, annealing at the primer annealing temperature for s, and extension at °c for min, for cycles. pcr products were subjected to electrophoresis in % acrylamide gels. positive samples were subjected to sanger sequencing using the same primers used for amplification. complete sequences of cbov available in the genbank database were aligned, and a phylogenetic tree was constructed by the maximum-likelihood (ml) method, using the software iqtree- . . , with the tmp u + f + r nucleotide substitution model and of replicates of bootstrap analysis. further phylogenetic analysis was conducted for the individual orfs in the mvc genome. for each analysis, the orf sequences of the mvc strains identified in the present study (completely or partially sequenced) were aligned with those of the twelve complete genome sequences available in the genbank database. additional partial sequences were also included in the analysis orf . the nt substitution models for orf (ns ), orf (vp /vp ), and orf (np ) were hky + f + i, tvm + f + r , and hky + f + g , respectively. the matrices of nt distances for the whole genome sequences and the vp / vp sequences were calculated using mega software . . (fig. a) . in detail: the complete orf , encoding the ns protein, of three of the four mvc isolates (italy/ / , bio-crime/ and italy/ / , which had a gap of nt) was sequenced and was bases in length, corresponding to aa (fig. a) . the complete orf , bp long, encoding the vp /vp proteins ( -aa vp and -aa vp ), was fully sequenced for two out of four mvc isolates: italy/ / (with a small gap of nt) and italy/ / . partial orf sequences were obtained for italy/ / and bio-crime/ : missing nt at ′ end of vp and nt at the ′end and nt at the ′ end of vp (fig. a) . a full orf sequence of nt, encoding the np protein ( aa), was obtained for three out of the four mvc isolates: italy/ / , italy/ / and bio-crime/ , but not for italy/ / (fig. a) . the nearly complete genome sequences of italy/ / , italy/ / and bio-crime/ , the two partial sequences of strain italy/ / , and the whole mvc genome sequences available in the genbank database were used for phylogenetic analysis. bio-crime/ and italy/ / were found to cluster together in a distinct monophyletic branch; by contrast, italy/ / and italy/ / clustered independently (fig. b) . to test the robustness of our analysis, we also generated phylogenetic trees using whole genome sequences trimmed to the shortest sequence length possible and including or excluding the region corresponding to the gap in the italy/ / sequence. although the topologies of the phylogenetic trees generated were slightly different, bio-crime/ and italy/ / strains always clustered together, and the corresponding nodes were supported by high bootstrap values (data not shown). the matrix of nt distances (table ) revealed a high similarity between bio-crime/ and italy/ / ( . %) and showed less than % sequence identity to all of the full mvc sequences available in genbank (table ) . conversely, italy/ / and table ). the topologies of the phylogenetic trees based on each individual orf (fig. ) was similar to that observed for the whole genome (fig. b) , with italy/ / and bio-crime/ , both of which were obtained from smuggled puppies, clustering together in a separate new branch and the other two mvc strains falling in different branches (fig. ) . the phylogenetic tree based on the orf nt sequences encoding the vp /vp proteins (fig. c) , was constructed using all mvc sequences available in the genbank database and included the only italian mvc sequence available, italy/ _ / (jq _canine_ minute_virus_strain_ _ _italy_ ), for which only the partial vp /vp sequence is present [ ] . interestingly, the phylogenetic analysis based on orf sequences, highlighted that, among the mvc strains identified in the present study, the italy/ / strain, from the autochthonous dog, is the only one that shares the highest nt sequence identity with italy/ _ / ( . %) and is % identical to two chinese strains identified in (mh and mh ) ( table ) . moreover, the aa sequences encoded by the orf , orf and orf regions of the four mvcs identified were compared with those of the twelve complete mvc genome sequences available in the genbank database. the aa sequence of the italy/ / strain was analyzed for orf and orf , but the orf was not available. several aa mutations open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. animal bocaviruses: a brief review the canine minute virus (minute virus of canines) is a distinct parvovirus that is most similar to bovine parvovirus a novel recombinant genome of minute virus of canines in china recovery and characterization of a minute virus of canines pathogenicity of minute virus of canines (mvc) for the canine fetus a fatal case of pup infection with minute virus of canines (mvc) a serological survey of minute virus of canines (mvc; canine parvovirus type- ) in dogs in the tokai area of japan molecular characteristics of a novel strain of canine minute virus associated with hepatitis in a dog molecular characterization of canineminute virus associated with neonatal mortality in a litter of jack russell terrier dogs a minute virus of canines (mvc: canine bocavirus) isolated from an elderly dog with severe gastroenteritis, and phylogenetic analysis of mvc strains sequence analysis of an isolate of minute virus of canines in china reveals the closed association with bocavirus acknowledgements this study was partially funded by the european union through the bio-crime project-animal diseases (zoonoses) and illegal trade of young animals in the alps-adriatic region (animal welfare)-(itat ), interreg italia-osterreich. the authors thank francesca ellero for english revisions and anna toffan for providing strain mn _minute_virus_of_canines_ _italy_ (italy/ / ). key: cord- -l ugjou authors: yaling, zhou; ederveen, j.; egberink, h.; pensaert, m.; horzinek, m. c. title: porcine epidemic diarrhea virus (cv ) and feline infectious peritonitis virus (fipv) are antigenically related date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: l ugjou using gut sections from pigs infected with porcine epidemic diarrhea virus (strain cv ) and ascitic fluid from cats which had succumbed to feline infectious peritonitis (fip), a weak cross reaction was found by immunofluorescence. its specificity was confirmed when detergent-treated purified cv showed a prominent reaction with fipv antibodies in elisa; no reaction was obtained with intact virions, which indicated common determinants on an internal component of the particle. antigenic cross-reactions at the nucleocapsid level were found in western blot elisa performed both ways (cv /fipv antibodies; fipv/cv antibodies). in immunoprecipitation using [( )s]methionine labelled fipv, anti-cv sera recognized exclusively the nucleocapsid protein. the significance of these findings for the classification of coronaviruses is discussed. porcine epidemic diarrhea is an acute viral disease in swine of all ages. particles of the cv strain of porcine epidemic diarrhea virus possess typical coronavirus morphology [- , ] and morphopoiesis [ ] . however, antigenic relationships between cv and established coronaviruses have hitherto not been detected [ ] . coronaviruses show a characteristic pattern of structural polypeptides: a peplomer protein with apparent molecular weight (mr) of , to , , a nucleocapsid protein of mr , to , and an envelope protein of mr , to , [ , ] . recent studies from our laboratory have demonstrated that cv is similar in this respect: it possesses a glycosylated surface protein of mr , to , , an envelope protein of mr , to , and an unglycosylated rna-binding protein of mr , [ ] . [ ] , c o m p l e m e n t fixation [ , ] , gel diffusion [ ] , immunofluorescence (ift) [ , , ] , enzyme linked imm u n o s o r b e n t assay (elisa) [ ] or, recently, immuno-electron microscopy (iem) [ ] . using i e m and ift, an antigenic relationship between cv and several coronaviruses had not been found in a previous study [ ] . in the present paper, the problem is re-examined with the aid of m o r e sensitive techniques such as immunoblotting and immunoprecipitation. as will be shown, cv is indeed antigenically related to a coronavirus at the nucleocapsid level. for ift, frozen sections gm thick were prepared from the jejunum of a cesarean-derived colostrum-deprived (cdcd) piglet which had been experimentally infected with the cv isolate; frozen sections from the jejunum of a non-infected cdcd piglet were prepared in the same way to serve as a control. the cv antigen for the elisa and immunoblot assay was prepared from faecal material collected from an experimentally infected cdcd piglet. twenty ml of the faecal suspension was clarified by centrifugation at , x g for minutes. the virus was then concentrated by centrifugation at , × g and °c for hours. the supernatant was discarded and the peliet was resuspended in ml of tes buffer ( mm tris-hc , ph . , mm edta, ram nac ). after another centrifugation at , × g for minutes, the virus-containing supernatant was saved and kept frozen until use. feline infectious peritonitis virus (fipv) strain - was grown in cells of the norden laboratories feline kidney (nlfk) line. for immunoblot assays, fipv infected nlfk cell lysate was used, with mock infected lysate serving as a control. monolayers of nlfk cells were inoculated with fipv as described before [- ] . after to hours, the medium was removed from the infected and mock-infected cells, respectively, and the monolayers were washed once with phosphate buffered saline (pbs, . m phosphate buffer ph . , . m nac ). the cells in a cm costar flask were lysed by adding ml of lysis buffer ( . % triton x- , . % naphthalene disulfonic acid-na and . m phenylmethyl sulfonyl fluoride in tes buffer: . m tris. hc ph . , mm edta, . m nac ), and the lysate were clarified by centrifugation at , x g for minutes. anti-cv hyperimmune sera had been prepared in cdcd piglets. the ascitic fluids used as a source of fipv antibody (a , a , a ) had been collected from naturally infected cats. a specified pathogen-free cat serum had been obtained from the centraal proefdierenbedrijf tno, zeist, the netherlands. for direct blot-elisa, the anti cv hyperimmune serum was purified by standard ammonium sulphate precipitation and deae-sephacel (pharmacia fine chemicals ab, uppsala, sweden) chromatography; the purified immunoglobulins were conjugated with horseradish peroxidase (hrp, boehringer mannheim gmbh, federal republic of germany) according to wilson and nakane [ ] . the ift was performed on frozen sections of cv -infected and non-infected jejunum samples. the tissues were fixed onto glass slides with precooled acetone at - °c. after cycles of washing with pbs ( minutes each) the tissues were overlaid with : dilutions of pig antiserum and cat ascitic fluids, respectively, followed by incubation in a moist chamber at °c for minutes. after more washes, the fitc-conjugated rabbit antiswine igg or rabbit anti-cat igg (nordic immunological laboratories, titburg, the netherlands) diluted in pbs were pipetted onto the tissues and the slides were incubated as before and rinsed again. they were mounted with % glycerol in pbs and examined with a fluorescence microscope. the elisa was carried out in -well flat bottom microtiter plates (bioreba f). the wells were coated overnight at °c with gl-volumes of triton x- ( %) pretreated and native cv antigen ( . - . gg), respectively, diluted in coating buffer. after cycles of washing with a . m nac solution containing . % tween , gl-volumes of antiserum diluted in a buffer consisting of . m naci, mm edta, . m tris ph . , . % tween and % foetal calf serum (fcs) were pipetted into the wells, and the plates were incubated for hours at °c. they were then washed as described above and the proteins in the respective antigen preparations were separated by electrophoresis in . % polyacrylamide gels in the presence of sodium dodecyl sulphate (sds-page) and were electrophoretically transferred to nitrocellulose sheets (ba , . lain, schleicher und schuell, dassel, federal republic of germany) using a lkb novablot electrophoretic transfer unit (lkb-produkter ab, bromma, sweden) at . ma/cm for hour in continuous transfer buffer ( mm glycine, mm tris, . % sds and % methanol). after transfer, the nitrocellulose filters were blocked overnight at room temperature using gelaton buffer ( . % pig skin gelatin, . % triton x- in pbs). the filters were incubated at °c for hour on a rocker platform with the antisera diluted in gelatin buffer. after washings with gelatin buffer ( minutes each at room temperature), the filters were incubated with hrp-conjugated anti-species igg at °c for another hour. they were then washed times as above and once with pbs alone. the enzyme reaction was visualized by soaking the filters in freshly prepared substrate solution ( . mg/ml , -diaminobenzidine tetrahydrochloride from sigma, st. louis, mo, u.s.a. and . % h in pbs). the reactions were stopped by submerging the filters in % trichloroacetic acid. mock-and fipv-infected monolayers of nlfk cells in c m costar flasks were labelled with [ s]methionine starting at hours after infection. the medium was removed, the monolayers were rinsed once with pbs and ml of methionine-free eagle's minimum essential medium (gibco) supplemented with % foetal fcs and gci [ s]methionine (specific activity, , ci/mm; amersham international) was added. arer another hour of incubation, cell lysates (in gl) were prepared as described above. label incorporation was determined after precipitation with % tca using a liquid scintillation counter. for rip, ~tl of [ s]methionine-labelled fipv or mock infected cell lysate containing approximately , cpm was diluted with gl lysis buffer and mixed with ~d-volumes of undiluted serum or ascitic fluid. immune complexes were allowed to form at room temperature for hour, and subsequently kc (final concentration . m) and gl of a % suspension of formaldehyde-fixed staphylococcus aureus cells were added. after incubation for minutes at room temperature, the immune complexes were pelleted at , x g, washed times with tes buffer containing . % triton x- and resuspended in laemmli sample buffer ( mm tris-hc ph . , % glycerol, % sds, % -mercaptoethanol). the proteins were analyzed in . % gels by sds-page and fluorography as described previously [ ] . both pig anti cv hyperimmune serum and cat f i p ascitic fluids showed fluorescence in cv infected epithelial cells of the small intestinal villi. with heterologous antibody, the fluorescence was m u c h weaker than with homologous serum; however, distribution of fluorescent cells in the tissue was identical to the positive control (results not shown). n o fluorescence was seen when pig antiserum or fip ascitic fluids were tested on normal gut sections or when a negative pig serum and an spf cat serum were tested on cv -infected sections ( table ) . since these results suggested a cross-reaction between fipv antibodies and cv antigen, we attempted to confirm the reaction by elisa. in order to detect possible cross-reactions also at the level of internal virion proteins, cv was disrupted with triton x- and the results were compared with those obtained with intact virus. the positive/negative (p/n) ratios were calculated by dividing the absorbance readings obtained with the positive sera or ascitic fluids (p) by the values obtained with the negative sera (n). a quotient of . would mean that no specific reaction has occurred. we obtained p / n ratios in the homologous reaction of . - . for disintegrated virus and of . + . for the native cv preparation, respectively. a n average p / n ratio of . - . (p< . ) was found when fip ascitic fluid a had reacted with triton x- treated cv whereas a ratio of . - . obtained with native cv antigen. we concluded from these observations that the heterologous reaction involved internal components of cv . as a next step, immunoblotting experiments were designed to identify the viral subunit responsible lbr the cross reaction. figure shows the western blot-elisa of cv proteins with homologous and heterologous antibodies. when homologous antiserum was used, prominent bands were seen at positions corresponding to mr of , and , ; they probably relate to the rnabinding protein of mr , found in % gels [ ] and its degradation product. at the same positions bands were seen in the lane with fip ascitic fluid was used. at these mr locations, bands were absent in the lanes where a preimmunization pig serum or a spf cat serum had been used. in the second series of experiments, we studied the inverse reaction, i.e. fipv antigen with homologous and cv antibodies (fig. ) . using fip ascitic fluid, a strong band with a calculated mr of , and three additional bands between mr , and , (representing the nucleocapsid and envelope proteins, respectively, of fipv) [ , ] can be seen. when an anti cv hyperimmune serum is used, a band is seen occupying the same place as the fipv nucleocapsid protein. porcine epdidemic diarrhea virus immune sera from belgium and the netherlands gave identical patterns (results not shown). the specificity of the reaction was confirmed by the empty appearance of the lanes where negative cat and pig serum had been used. no reaction was found at the nucleocapsid protein position when blots of a mock infected n l f k cell lysate had been incubated with anti-cv antibodies. immunoblotting would not detect reactions with conformation-dependent antigenic determinants. to show the reaction between homologous or heterologous antibodies and fipv antigens under non-denaturing condition, immunoprecipitation was employed using [ s]methionine labelled infected and non-infected cell lysates. the inverse experiment was not performed since cv virus cannot be grown and labelled in culture. as shown in figure , results of the rip confirmed the cross-reaction between pig anti-cv sera and the , protein of fipv found in western blots; the homologous reaction reveals the typical pattern of fipv structural polypeptides with mr of , (peplomer), , (nucleocapsid) and , to , (envelope). no precipitation was seen in this mr range using a negative pig serum or a mock infected lysate. porcine epidemic diarrhea virus, strain cv has been tentatively classified as a member of the family coronaviridae based on its morphologic, morphogenetic and physico-chemical properties [ , ] . characteristics of the viral structural polypeptides supported this taxonomic proposal [ ] . we now present evidence for an antigenic relatedness between cv and fipv which provides a decisive argument in favor of the classification of cv as a coronavirus: in figs. to , a two-way cross reaction is shown between the nucleocapsid at the present time, coronaviruses are assigned to four antigenic clusters. in , pedersen et al. studied eight mammalian coronaviruses and established two groups on the basis of cross-reactivities by immunofluorescence; the authors found that viruses within one group were antigenically unrelated to members of the second group [ ] . we have confirmed the finding of cross reactions within one group, namely between transmissible gastroenteritis virus (tgev) of swine, fipv and canine enteric coronavirus, and showed that common determinants are present on all three structural polypeptides [ ] . surprisingly, another study at the virion subunit level revealed an antigenic relatedness between mouse hepatitis virus type (mhv) and human coronavirus (hcv) e which had been assigned to different clusters [ - . also, the hcv e nucleocapsid protein can be detected by antisera directed against tge, mhv and hemagglutinating encehalomyelitis virus (hev) of swine [- ]. we have obtained preliminary evidence in immunoblotting assays that mhv strain a and avian infectious bronchitis virus (ibv) strain m cross-react at the nucleoprotein level [niesters , unpublished observations], as do also fipv and hev [yaling , unpublished observations]. these observations, together with the finding that amino acid sequences are conserved between coronaviruses from different antigenic clusters [ ] , indicate that common antigenic determinants may exist which are suitable for the identification of many, if not all, members of the family. zhou yaling et al. we have reported earlier that antigenic cross-reactions between cv and other coronaviruses could not be detected by ift [ ] . using elisa, immunoblotting and rip in the present study, however, we did find a cross reaction of cv with fipv. upon re-examination, a reaction was also seen by ift, which--when assessed by itself--would have appeared too weak to be considered as specific. several explanations can be offered for the discrepancies obtained with the different techniques. the selected polyclonal antiserum may be crucial. it has been demonstrated that, e.g., fip ascitic fluids may lack antibodies against one or two of the three viral proteins [ ] . since a cross reaction may exist at the level of only one of them--as found in this study--the antigenic relationship may thus escape detection. cross-reactivities may vary with different serological variants of the viruses compared (e.g., m h v and hcv); the existence of antigenic differences between fip viruses should also be taken into account [ , ] . due to its low sensitivity, the fluorescent antibody technique is not ideal for the examination of viral cross reactivities and contradictory results have indeed been obtained [ , , , ] . in view of the high frequency of mutation and recombination of coronaviruses, the establishment of antigenic clusters within the family may be of only limited value. antigenic relationships amongst coronaviruses some structural and antigenic properties of a new porcine coronavirus, cv . enterites virales/viral enteritis evidence for a coiled-coil structure in the spike proteins of coronaviruses in vivo morphogenesis of a new porcine enteric coronavirus, cv characterization of the structural proteins of porcine epizootic diarrhea virus (cv ) serological relationships of the subcomponents of human coronavirus strain e and mouse hepatitis virus strain antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus enzyme-linked immunosorbent assay for coronaviruses hcv e and mhv cleavage of structural proteins during the assembly of the head of bacteriophage t classification and nomenclature of viruses antigenic relationships among the coronaviruses of man and between human and animal coronaviruses antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species a new coronavirus-like particle associated with diarrhea in swine an immuno electron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv , and several coronaviruses translation of three mouse hepatitis virus strain a subgenomic rnas in xenopus laevis oocytes the structure and replication of coronaviruses the molecular biology of coronaviruses recent development in the periodate method of conjugating horseradish peroxidase (hrpo) to antibodies untersuchungen fiber die antigenverwandtschaft der viren der felinen infekti sen peritonitis und der transmissiblen gastroenteritis des schweines comparative antigenic studies on coronaviruses we wish to thank dr. p. callebaut, ghent, belgium, and dr. van nieuwstadt, cdi, lelystad, the netherlands for providing faecal suspensions and gut material from cv infected piglets, and pig anti cv hyperimnmne sera. we are indebted to norden laboratories, lincoln, nebraska, u.s.a. for providing the nlfk cell line. key: cord- -cz bi ym authors: yu, liping; zhang, xiaorong; wu, tianqi; wang, yuyang; meng, jie; liu, qian; niu, xiaosai; wu, yantao title: the papain-like protease of avian infectious bronchitis virus has deubiquitinating activity date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: cz bi ym coronavirus papain-like proteases (plps) can act as proteases that process virus-encoded large replicase polyproteins and also as deubiquitinating (dub) enzymes. like the plps of other coronaviruses (covs), the avian infectious bronchitis virus (ibv) plp catalyzes proteolysis of gly-gly dipeptide bonds to release mature cleavage products. however, the other functions of the ibv plp are not well understood. in this study, we found that ibv exhibits strong global dub activity with significant reductions of the levels of ubiquitin (ub)-, k -, and k -conjugated proteins. the dub activity exhibited a clear time dependence, with stronger dub activity in the early stage of viral infection. furthermore, the ibv replicase-encoded plp, including the downstream transmembrane (tm) domain, is a dub enzyme and dramatically reduced the level of ub-conjugated proteins, while processing both k - and k -linked polyubiquitin chains. by contrast, plp did not cause any reduction of haemagglutinin (ha)-ub-conjugated proteins. in addition, mutations of the catalytic residues of plp-tm, cys ser and his lys, reduced dub activity against ub-, k - and k - conjugated proteins, indicating that the dub activity of the plp-tm wild-type protein is not completely dependent on its catalytic activity. overall, these results demonstrate that the ibv-encoded plp-tm functions as a dub enzyme and suggest that ibv may interfere with the activation of host antiviral signaling pathway by degrading polyubiquitin-associated proteins. avian infectious bronchitis virus (ibv) is the prototype of gamma coronaviruses (cov), a family of enveloped viruses that possess a large continuous positive-stranded rna genome [ ] . the genomic rna is . kb in length, and approximately two thirds of the nucleotide sequence encodes orf , which includes orf a and orf b [ , [ ] [ ] [ ] [ ] . a papain-like protease (plp) is encoded by the region from nucleotides to of orf a [ ] , comprising the catalytic domain of nonstructural protein (nsp ). proteolysis at the gly -gly and gly -gly dipeptide bonds leads to release of the -kda and -kda n-terminal mature proteins and the c-terminal -kda cleavage product [ , , ] . site-directed mutagenesis studies have confirmed that the cys and his residues of plp are essential for proteinase activity [ ] . covs such as the human coronavirus nl (hcov-nl ) [ ] and murine hepatitis virus-a (mhv-a ) [ ] encode two functional papain-like proteases (plp), termed plp and plp . at least two covs, severe acute respiratory syndrome coronavirus (sars-cov) [ ] and ibv [ ] encode only one functional papain-like protease, termed plp. the proteolytic processing mediated by the plp encoded by ibv [ ] is similar to that of plp encoded by other covs [ , ] . plps of covs also perform deubiquitinating (dub) and interferon (ifn) antagonism activities. for instance, plp of mhv-a [ ] can bind to irf , cause its deubiquitination and prevent its nuclear translocation, thus inhibiting cellular irf -mediated type i ifn production and promoting viral infection. the sars-cov plp pro-catalytic core also has dub activity and can deubiquitinate irf , thereby stalling its migration to the nucleus and preventing the host antiviral response [ , , ] . similarly, purified plp of hcov-nl can hydrolyze k linked hexa-ubiquitin (k -ub ) to produce monoubiquitin [ , ] , and therefore negatively regulate antiviral defenses by disrupting the sting-mediated ifn induction [ ] . structural and enzymatic studies have revealed that covs plps can act as both a protease, to process virusencoded large replicase polyproteins, and a dub enzyme, to cleave the isopeptide bonds found in polyubiquitin chains [ , ] . here, we demonstrate that ibv has dub activity, and like other covs, ibv can recognize and process both k -and k -linked polyubiquitin chains. we also demonstrate that the core domain of ibv plp-tm is a coronaviral dub enzyme. we also evaluated the role of plp catalytic activity in dub activity and found that plp-tm does not require catalytic activity to cleave polyubiquitin-linked proteins. this study represents a first step in elucidating the role of plp-tm in ibv pathogenesis and provides new insights on how ibv escapes host antiviral immune mechanisms. chicken embryonic kidney (cek) cells were aseptically generated from -day-old specific pathogen-free (spf) chicken embryos (beijing merial vital laboratory animal technology company). the cell suspension was obtained by trypsinization of kidneys for min at °c and subsequent filtration through a -lm mesh. the cells were then cultured in m medium (hyclone) supplemented with % fetal bovine serum (fbs). the df chicken fibroblast cell line was used for all transfection-based assays. the cells were maintained in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with % fbs. ibv (js/ / strain, genbank accession no. jq . ) was cultured in -day spf chicken embryos or cek cells. the initial ibv stock was inoculated in chicken embryos for six passages (p ). this study was conducted based on this p stock of ibv. the % tissue culture infective dose (tcid ) of the ibv p stock was determined by identifying the cytopathic effect (cpe) of the virus in cek cells. the plasmid pcdna- ' flag was a kind gift from dr. meng (dalian medical university). prk -ha-ub and the mutant derivatives prk -ha-ub-k and prk -ha-ub-k were obtained from addgene (plasmids # , # and # ) [ ] . attachment of ubiquitin (ub) modifiers is a reversible post-translational modification that regulates the fate and function of proteins. the ubiquitin molecule contains a total of seven lysine residues at positions , , , , , (k ), and (k ). these lysine residues potentially mediate ubiquitin chain elongation. the two most common types of polyubiquitin chains are linked through ubiquitin lysine (k ) and lysine (k ). plp is encoded by ibv orf a at the region of nsp between nucleotides and [ ] . biological analysis software (tmhmm server v. . ) was used to analyze the amino acid sequence of nsp to predict the transmembrane (tm) domain near plp [ ] . in figure b , the red area indicates the transmembrane domain; the extracellular protein and short cytoplasmic domain are indicated by pink and blue, respectively (fig. b) . plp and plp-tm constructs were generated using specific primers (table ) to amplify the designated a cysteine residue (cys ) and histidine residue (his ) of plp are the catalytic residues of the proteinase activity [ ] . either of these two sites could play an important role in the dub activity of plp-tm. to explore these possibilities, substitution mutations of the cys residue with ser and the his residue with lys were constructed to obtain the mutant constructs plp-tm cys ser (plp-tm c s) and plp-tm his lys (plp-tm h k) [ ] . to generate specific mutations of the catalytic residues, mutagenic primers (table ) were incorporated into newly synthesized dna using the fast mutagenesis system protocol (trans, fast mutagenesis system, fm ) according to the manufacturer's instructions. the transfections were performed using lipofectamine (invitrogen) according to the manufacturer's instructions. this method resulted in a transfection rate of at least %. briefly, the plasmid ( lg for -well) was diluted into opti-mem, and lipofectamine ( ll for -well) was diluted into opti-mem. the diluted dna was added to diluted lipofectamine ( : ratio), incubated for min, and then added to the cell cultures. df cells were co-transfected with prk -ha-ub, prk -ha-ub-k or prk -ha-ub-k plasmids and the indicated amounts of ibv plp, plp-tm and specific catalytic mutants. the cells were then lysed in ripa lysis buffer (beyotime institute of biotechnology, china). the cell lysates were analyzed for ha-conjugated proteins using western blotting with monoclonal anti-ha antibody ( : , sigma). an anti-n antibody was used to detect the viral replication level. to confirm the expression levels of plp and the mutants, anti-flag antibody ( : , sigma) was used to detect the flag-tagged proteins. actin was detected using b-actin antibody ( : , sigma) as a protein loading control. the effect of ibv on ubiquitinated proteins in cells was assessed as follows. df cells were infected with ibv at a multiplicity of infection (moi) of or mock-infected. the cells were then transfected with prk -ha-ub, prk -ha-ub-k or prk -ha-ub-k for an additional h. to investigate the relationship between ibv replication and dub activity, df cells were infected with ibv at an moi of or mock-infected and then transfected with prk -ha-ub at h, h or h post-infection for an additional h. cell lysates were then prepared and the extent of protein ubiquitination was assessed by western blot as described. assays for the dub activity of plp, plp-tm and the catalytic mutants the effects of plp, plp-tm and the catalytic mutants on ubiquitinated proteins in cultured cells was assessed as follows. df cells were co-transfected with prk -ha-ub/ prk -ha-ub-k /prk -ha-ub-k and the indicated amounts of the constructs encoding plp-tm or the corresponding catalytic mutants. the pcdna- ' flag vector was used to standardize the quantity of dna. the total cells were harvested, and ubiquitinated proteins were assessed as described previously h post infection. to determine if ibv exhibits dub activity against host cellular substrates after infection, df cells were transfected with prk -ha-ub, prk -ha-ub-k or prk -ha-ub-k after infection with ibv or mock infection. the levels of ub- (fig. a, lane ) , k - (fig. a , lane ) and k - (fig. a, lane ) conjugated proteins were reduced dramatically in df cells after infection with ibv. this suggests that ibv exhibits strong dub activity that recognizes and processes both k -and k -linked polyubiquitin chains. to investigate the relationship between ibv replication and dub activity, df cells were transfected with prk -ha-ub at different times post-infection. western blot analysis revealed that ibv decreased the levels of ub-conjugated proteins at different times, and the levels of ub-conjugated proteins were lower at h than at h and h. thus, ibv exhibited stronger dub activity in the early stage of infection (fig. b, lanes , and ; fig. c ). overall, ibv has global dub activity against ubiquitinated proteins, and the dub activity exhibits a clear time dependence in cultured cells. plp is the catalytic domain of ibv, encoded by the genomic region from nucleotides to , and the tm domain is encoded by the region from nucleotides to , downstream of plp (fig. a, b) . to determine if plp and plp-tm have dub activity, plp and plp-tm were separately co-transfected with prk -ha-ub into df cells. we found that expression of plp-tm resulted in a dramatic reduction in the level of ub-conjugated proteins (fig. c, lane ) . by contrast, expression of plp did not result in any significant reduction of ha-ub conjugates (fig. c, lane ) . this result indicates that ibv plp-tm has effective dub activity that can remove ub conjugates from many cellular substrate proteins. the two most common types of ubiquitinated proteins are linked through ubiquitin k and k . to determine if plp-tm of ibv has selectivity for ubiquitinated substrates with k and k linkages, df cells were transfected with plp-tm with or without prk -ha-ub (fig. d, lanes and ) , prk -ha-ub-k (fig. d, lanes and ) or prk -ha-ub-k (fig. d, lanes and ) . the extent of ubiquitinated products was assessed by western blot. consistent with the processing of ub-linked proteins by plp-tm, plp-tm dramatically reduced the levels of both k -and k linked ubiquitin (fig. b, lanes , and ) . this result shows that both major forms of polyubiquitinated proteins can be recognized and degraded by plp-tm. the dub activity of plp-tm does not completely depend on its catalytic activity plp contained within the nsp has been shown to be responsible for the cleavage of orf a at the two gly-gly dipeptide bonds to release mature protein, and the catalytic dyad for this activity was cys and his [ ] . either of the two catalytic residue sites may play an important role in the dub activity of plp-tm. to explore these possibilities, df cells were transfected with plp-tm, plp-tm c s or plp-tm h k, together with prk -ha-ub (fig. a) , prk -ha-ub-k (fig. b) , or prk -ha-ub-k (fig. c) , and the extent of ubiquitinated products was assessed by western blot. we found that the catalytic mutants (c s and h k) of plp-tm exhibited reduced dub activity against ub-, k -and k -linked proteins (fig. a-c) . these results demonstrate that the dub activity of plp-tm is not completely dependent on its catalytic activity. in this study, we characterized the dub activity of ibv plp in df cells and the core domain of the dub activity. we found that ibv exhibits strong global dub activity during infection of df cells, indicating that ibv infection disrupts polyubiquitin modification in host cells or encodes a protein with dub activity [ ] . further experiments indicated that the proteinase plp-tm plays an important role in ibv dub activity and can process both k -and k -linked polyubiquitin chains. moreover, the dub activity of plp-tm was not completely dependent on its catalytic activity. deubiquitinating function of an avian ibv papain-like protease similar to the cov porcine epidemic diarrhea virus (pedv) [ ] , the dub activity of ibv also dramatically reduced ub-conjugated protein levels in virus-infected cells, and the ibv plp plays an important role in this dub activity. plp of pedv has potent dub activity that is dependent on its catalytic activity. in notable contrast to pedv plp , the dub activity of ibv plp is not dependent on its protease activity. another group reported that ibv plp can degrade k -and k -linked polyubiquitin chains to monoubiquitin but cannot degrade linear polyubiquitin [ ] . hcov-nl plp has a dub activity that is dependent on its protease activity [ ] . by contrast, both the plp -tm and catalytic mutants of plp -tm [ ] had dub activity, although the dub activity of the plp -tm catalytic mutants was lower than that of plp -tm, suggesting that the tm domain plays a role in plp dub activity. similarly, our study confirmed that the tm domain downstream of ibv plp is essential for plp dub activity. the ibv plp cleaves the orf a to generate nonstructural proteins that associate with endoplasmic reticulum membranes to generate convoluted membranes and double membrane vesicles (dmvs), which are the site of viral replication. the plps are tethered to the dmvs by a tm domain [ , ] . many reports have demonstrated that the tm domain plays an important role in covs dub activity, possibly by facilitating correct protein folding or interactions with cellular proteins. additional studies are needed to identify the exact functions of the tm domain during ibv replication and the interaction between the host innate immune system and ibv infection. this study raises important questions concerning the role of viral dub activity in cov replication and pathogenesis. coronaviral dub activity may target the ubproteasome pathway to facilitate virus replication and damage host defense mechanisms, including innate immunity [ , ] . some covs may utilize dub activity to escape the host innate antiviral response [ , ] . for example, plp of mhv-a can bind to irf , cause its deubiquitination and prevent its nuclear translocation. coexpression of plp inhibits cardif-, tbk -and irf mediated ifn-b reporter activities [ ] . mhv-a may use dub activity to reduce ifn induction, to promote viral growth and to escape from host innate antiviral responses [ ] . pedv infection suppresses the production of ifn-b and plp acts as a viral dub to interfere with rig-i-and sting-mediated signaling pathways [ ] . however, some covs plp-mediated interferon antagonism is independent on protease and dub activity. the plp of nl has dub activity and antagonizes the induction of type i ifn, whereas plp-mediated ifn antagonism is independent of dub activity [ ] . these data show that covs plps target the activity of type i ifn through dub activity and inhibit the activation of the innate immune system. joeri kint et al. showed that ibv inhibits the synthesis of host proteins, including type iifn, a key component of the antiviral response [ , ] . our study also indicates that ibv plp-tm process both k -and k -linked ubiquitin. k -and k -linked ubiquitin are the most common types of modified polyubiquitin and play key roles in protein degradation and in response to changes in the innate and adaptive immunity systems. further investigations are necessary to determine if ibv plp dub activity inhibits the host antiviral response. overall, the results of our study show that ibv has dub activity and confirm that plp-tm is not only a classic papain-like protease encoded by ibv but is also a multifunctional protein that plays important roles in the regulation of interactions between ibv and host antiviral innate immune response proteins. ubiquitin modifications play key regulatory roles in protein degradation and in innate and adaptive immunity signaling pathways. ibv plp-tm may prevent the activation of host antiviral signaling pathways by degrading polyubiquitin chains associated with ubiquitin proteins. the interactions between ibv and the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity characterization of the leader papain-like proteinase of mhv-a : 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gene of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an -kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products characterisation and mutational analysis of an orf a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus a/ b polyprotein proteolytic mapping of the coronavirus infectious bronchitis virus b polyprotein: evidence for the presence of four cleavage sites of the c-like proteinase and identification of two novel cleavage products a -kilodalton polypeptide encoded by open reading frame (orf) b of the coronavirus infectious bronchitis virus is processed by orf a products coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling ubiquitin and ubiquitin-like proteins as multifunctional signals deubiquitinating function of an avian ibv papain-like protease the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production key: cord- -v gjabp authors: dea, s.; garzon, s.; tijssen, p. title: intracellular synthesis and processing of the structural glycoproteins of turkey enteric coronavirus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: v gjabp pulse labeling of cells with [( )s]methionine or [( )h]glucosamine at different times after infection, followed by sds-page and western immunoblotting analysis using rabbit anti-tcv hyperimmune serum, was used to resolve and identify tcv-induced intracellular proteins. the viral structural proteins (gp , gp /gp , gp /gp , p , and gp /p ) were detected in radiolabeled cell extracts by to hours post-infection, as well as two possible non-structural proteins with apparent mol.wts. of , and , . the predominant , nucleocapsid protein could be detected in cell lysates as soon as to hours after infection; it was initially resolved as a complex of closely migrating species with mol.wts. ranging from , to , . pulse-chase and immunoprecipitation experiments indicated that gp arose from a putative precursor with mol.wt. of , to , , that underwent glycosylation. proteolytic cleavage of gp , in turn, probably yielded the gp and gp species. the unique tcv hemagglutinin protein originated from a primary precursor with mol.wt. of , , which underwent rapid dimerization by disulfide bond formation and glycosylation to yield gp . the peplomeric and matrix proteins were both shown to be n-glycosylated, as indicated by their sensitivity to tunicamycin (tm) and their resistance to sodium monensin (sm). in the presence of tm, proteins with mol.wts. of , , – , , and , accumulated in tcv-infected cells rather than peplomeric glycoproteins, and the matrix protein e was only detected in its unglycosylated form. the addition of tm to the culture medium interfered with the maturation of progeny viral particles, as suggested by the absence of peplomers at the surface of the intravacuolar and extracellular virions, and the accumulation of amorphous material not found in the absence of the glycosylation inhibitor. high yields of virus replication were obtained, in the presence of sm, even at concentrations which greatly affected the cellular functions. the coronavirus virion possesses two or three major envelope glycoproteins [ ] . the latter are synthesized from different virus-specific subgenomic rnas which are translated at the level of membrane-bound ribosomes [ , ] , and then undergo post-translational processing at the level of rough endoplasmic reticulum or golgi apparatus [ , ] . the peplomeric (e ) protein precursors of mouse hepatitis (mhv) and avian infectious bronchitis (ibv) viruses are cotranslationally n-glycosylated and have then an apparent mol.wt, of , [ , , ] . the polypeptides synthesized in vitro or in tunicamycin-treated cells have a mol.wt, of approximately , [ , , ] . this agrees with the mol.wts, of the polypeptide moieties predicted from nucleotide sequences of their e genes [ , , ] . a glycoprotein with a mol.wt, of approximately , has been identified as the intracellular precursor of the e protein of bovine enteric coronavirus (bcv), and hemagglutinating mammalian coronavirus [ , ] . unglycosylated precursors have not been yet identified for bcv, but concomitant with the loss of its surface projections, the virus is neither infectious nor hemagglutinating, after cultivation in the presence of tm [ ] . following further glycosylation, the peplomeric protein of these coronaviruses undergoes proteolytic cleavage to yield two subunits with mol.wts, of - , [ , , ] . this proteolytic cleavage is a host-cell dependent event and is required for activation of the cell-fusing activity, and therefore appears to be an important determinant of viral pathogenesis [ ] . the precursor proteins to the bcv hemagglutinating protein (gp /gp ) are gp (monomer) and gp (disulfide-linked dimer) [ , ] . the n-terminal regions of the matrix e proteins of mhv and bcv bear oligosaccharides chains that lack mannose and fucose, and are o-linked to serine or threonine residues, an unusual feature among viral glycoproteins [ , , ] . therefore, the glycosylation process of the p - intracellular precursors does not depend on transfer of oligosaccharides from dolichol phosphate intermediates and is not inhibited by tunicamycin, but by sodium monensin, an inhibitor of the golgi function [ , ] . the matrix glycoprotein of the avian infectious bronchitis virus (ibv) and the porcine transmissible gastroenteritis virus (tgev), unlike that of mhv and bcv, has n-linked complex oligosaccharides like those found on the peplomeric glycoproteins [ , , ] . indeed, two potential n-glycosylated sites are available near the n-terminus of the sequence of the e gene of both viruses [ , ] . the significance of this diversity of glycosylation patterns among coronaviruses is not known. turkey enteric coronavirus (tcv), a major etiological agent of epidemic diarrhea in turkey poults [ , ] , possesses an hemagglutinating activity, surface projections of two different types, and biochemical characteristics that resemble those of mammalian hemagglutinating coronaviruses [ , , , ] . tcv isolates also can be propagated in hrt- cells, an established cell line originated from a human rectal adenocarcinoma [ , , , ] . in the present study, we have investigated the intracellular synthesis and post-translational modifications of virus-coded polypeptides in cultures of hrt- infected with tcv in the presence or the absence of glycosylation inhibitors. putative intracellular glycosylated and unglycosylated precursors have been identified for glycoproteins associated with both types of surface projections, and the matrix protein of t c v was f o u n d to be glycosylated by a process which was sensitive to tunicamycin, but resistant to sodium monensin. the effects of these glycosylation inhibitors on the viral morphogenesis was confirmed by electron microscopy of tcvinfected cells. glycosylation of the matrix protein was apparently not required for the formation of the tcv virions. the minnesota strain [ ] of tcv and virus stocks were obtained and propagated in hrt- cells, in the presence of trypsin, as previously described [- , ] . after five successive passages, the virus was cloned twice by the limiting dilution method. cloned virus was passaged at a multiplicity of infection of . to tcid per cell. the origin of the rabbit anti-tcv hyperimmune serum was described previously [ ] . to analyse intracellular synthesis of viral polypeptides, hrt- cells were infected at a multiplicity of tcid /cell. at given times, indicated in results and figure legends, the maintenance medium was replaced by medium without methionine. after min of methionine starvation, the cells were pulse-labeled for min by the addition of ml/flask of methionine-free rpmi containing gci/ml [ s]methionine. replicate cultures were chased for hour by replacing pulse-medium with complete rpmi containing % fbs and then harvested in ice-cold pbs. in experiments where tunicamycin ( . to . gg/ ml) or sodium monensin ( . to gm) was used, the inhibitor was added at the beginning of the starvation period and was present throughout the rest of the experiment. for pulse-chase labeling of intracellular polypeptides, the infected cells were starved for methionine for min and then labeled by adding ~tci/ml of [ s]methionine to methionine-free medium, beginning at to hours p.i. for different experiments. after a min pulse period, the cells were rinsed twice in complete rpmi and then further incubated with this medium for rain to h before harvesting. the cell sheets were rinsed twice in pbs, and scraped into ice-cold lysis buffer (ripa) made of mm tris-hc , ph . , mm nac , mm edta, . m kc , . mm mgc , % triton x- , . % sds, % np , units aprotinin/ml and gg pmsf/ml. the cell lysate was immediately passed times through a -gauge syringe needle, heated to °c for rain, clarified by centrifugation at , x g for min, and stored at -- °c. uninfected hrt- cells were similarly radiolabeled, and mock lysates were prepared as control samples. the immunoprecipitation assays were performed according to deregt et al. [ ] . briefly, samples of to gl of clarified radiolabeled cytosol extracts ( × c.p.m.) were mixed with the anti-tcv serum bound to protein a-sepharose and incubated for h at °c replicas were prepared by electrophoresis transfer of proteins separated by sds-page to nitrocellulose sheets ( . ~tm pore size, schteicher et sch/ill) as previously described [ ] . after saturation with bovine serum albumin (grade v), the nitrocellulose sheets were incubated for h at room temperature in rabbit hyperimmune sera diluted : in tbs with . % (v/v) tween (tbs-t). the nitrocellulose sheets were subsequently washed times for min in tbs-t, and then incubated with : dilution of peroxidase-labeled goat anti-rabbit igg (boehringer-mannheim) in tbs-t for rain. after washing, blots were developed by reaction for to min with . % -chloro-l-naphthol (sigma) substrate, prepared in tbs containing % (v/v) methanol. samples were mixed with equal volumes of double strength sample buffer with or without -mercaptoethanol, boiled for min, and clarified at , x g for min before electrophoresis in . or percent sds-polyacrylamide slab gels, as previously described [ ] . high and low molecular weight marker proteins or c-methylated marker proteins were run on each gel to allow molecular weight estimates of viral proteins. gels were analyzed by autoradiography or fluorography after "amplify" (amersham) treatment. dried gels were exposed to kodak x-omat rp films at -- °c. em of infected cells, cultivated in the presence of tunicamycin or sodium monensin, was done by fixation with . % glutaraldehyde followed by post-fixation with % osmium tetroxide, dehydration in graded ethanol and embedding in epoxy , as previously described [ ] . thin sections were stained with lead-citrate and uranyl acetate [ ] and examined on a em philips microscope. tunicamycin (tm), sodium monensin (sm), phenylmethylsulfonyl fluoride (pmsf), aprotinin were purchased from boehringer-mannheim canada ltd., dorval, quebec. stock solutions of tm ( mg/ml) and sodium monensin ( , gm) were prepared in dimethylsulfoxide and in methanol, respectively. these inhibitors were also purchased from calbiochem, la jolta, ca. bovine pancreatic trypsin (tpck treated, , units/mg) and chloro-l-naphthol were purchased from sigma chemical co, st. louis, mo. reagents for sds-page were purchased from bio-rad laboratories, richmond, ca. protein a sepharose cl- b and molecular weight marker proteins were purchased from pharmacia, uppsala, sweden. l-[ s]methionine ( , ci/mmole) and a mixture of c-methylated marker proteins were purchasesd from amersham searle co., oakville, ontario. d-[ - h]glucosamine hydrochloride ( % ci/mmole) was purchased from icn biochemical canada ltd, montreal, quebec. pulse labeling of cells with [ s]methionine at different times after infection, followed by s d s -p a g e was used to resolve and to identify polypeptides designated as specific to tcv-infected hrt- cell cultures (fig. a) . late in infection, it was possible to detect the synthesis of four major tcv-induced polypeptides with apparent mol.wt, of , , , , , , and , which were not present in mock-infected control cultures (lane m). up to four or five minor polypeptides were also consistently resolved and migrated with estimated mol.wt, of , , , , - , , and - , , respectively (fig. a, arrowheads) . the , minor polypeptide was reproducibly resolved as a doublet. the predominant , nucleoprotein could be detected as soon as to hours post-infection and was initially resolved as a group of closely migrating bands; the two other polypeptides migrated with estimated mol.wt, of , and , , respectively. by hours, all four major tcvinduced polypeptides were present; they persisted until virus and host cell protein synthesis was reduced due to extensive cytopathic changes (between and hours post-infection). between and hours, all polypeptides appeared to be synthesized at a relatively constant rate, without any apparent shut-off of host cell protein synthesis. the four major intracellular polypeptide species comigrated with the viral polypeptides (lane v), as well as the , and the - , polypeptide species. the latter co-migrated also with a polypeptide having similar mol.wt, which also was present in mock-infected cells. all these polypeptides, as well as the species that closely migrated with the nucleocapsid protein, were revealed by western-immunoblotting using the anti-tcv rabbit hyperimmune serum (fig. c) . the - , polypeptide was not revealed by the antiserum and may represent a non-structural polypeptide. under reducing conditions ( fig. b) , the major , intracellular polypeptide was not present, but replaced by another major component with mol.wt. of approximately - , . the intracellular polypeptide corresponding to the viral matrix protein was usually resolved as a doublet ( , and , ). to investigate the intracellular processing of the viral structural proteins, tcvinfected and mock-infected hrt- cells were pulse-labeled from to hours after infection for min with [ s]methionine, and the label was chased for various times. polypeptides specific to tcv-infected cell cultures were then immunoprecipitated with the rabbit anti-tcv hyperimmune serum, and the precipitates were analyzed by sds-page. as shown in fig. , pulse-chase experiments conducted at and hours after infection permitted the identification of new immunoprecipitable protein species with mol.wts, of approximately , to , , , , , , , , and - , . no processing of the major p nucleocapsid protein was apparent during the hourchase period, but significant variation was noted in the intensity of the , , , , and , protein bands. the , mol.wt, species was present during the first hour of the chase period and then disappeared from the gel, concomitant with the appearance of a weak band corresponding to a polypeptide with an approximative mol.wt, of , , which later seemed to be converted into the gp species. the , mol.wt, species, present since the beginning of the chase period, was progressively chased to give the final gp species. the , protein species was also present from the beginning of the chase period and increased in intensity without any apparent change in its electrophoretic mobility. the to , mol.wt, species was evident after rain, and remained detectable until the end of the chase period. the , protein band intensified as the infection progressed, concomitant with an increase in the intensity of gp and the , mol.wt, species. the higher mol.wt. polypeptide disappeared later during the infection. processing seemed also to occurred among lower mol.wt, proteins. a , mol.wt, protein, co-migrating with a protein present in mock-infected cell lysates (data not shown), appeared to be chased into a , mol.wt, species. a minor band of , also appeared in the gels after the first hour of the chase period. processing of the small matrix glycoproteins could hardly be demonstrated by immunoprecipitation. however, by sds-page analysis of cell lysates obtained after various chase periods, an increase in the intensity of the , protein band was noted concomitant with a decrease in the - , protein band, presumably due to the glycosylation of the p - to form the gp (fig. b) . the immunoprecipitations of chased lysates with pre-immune serum were all negative, as was the immunoprecipitation of uninfected lysates with the specific hyperimmune serum (data not shown). to determine the nature of the glycosidic bonds present on each tcv glycoprotein, infected and mock-infected cells were maintained in media supplemented with either tunicamycin (tm) or sodium monensin (sm) and pulselabeled with [ s]methionine or [ h]glucosamine to follow the effects of the inhibitors on synthesis and glycosylation of tcv-induced intracellular proteins. no cytopathic changes or polycaryocytosis were observed when tcv-infected cell cultures were grown in the presence of . or . ~tg/ml of tm. in contrast, the addition of . to . ~tm sodium monensin did not interfere with tunicamycin at a concentration of . lag/ml slightly decreased the overall intracellular protein synthesis, as suggested by the intensity of bands of [ s]methionine-labeled proteins resolved by sds-page of cell lysates. polypeptides corresponding to tcv peplomeric glycoproteins showed a drastic decrease in their apparent molecular weights, yielding , , - , , and , polypeptide species (fig. a, arrowheads) . these new intracellular polypeptides were also resolved by western immunoblotting (fig. b) . radioim-munoprecipitation experiments using the anti-tcv hyperimmune serum, after [ s]methionine and [ h]glucosamine labeling, confirmed the absence of the peplomeric glycoproteins from tm-treated cells, whereas the relative amount of the unglycosylated high mol.wt, polypeptide species increased (fig. a, lanes and ) . a major non-glycosylated polypeptide having a mol.wt, of , was also revealed in the presence of tm. in contrast, both types of surface glycoproteins (gpl - /gpl and gp ) were immunoprecipitated from extracts of sm-treated tcv-infected cells and strongly labeled with [ h]glucosamine (fig. a, lane ) . a major polypeptide with an approximate mol.wt, of - , was also immunoprecipitated after treatment with sm, but appeared slightly labeled with [ h]glucosamine (fig. a, lanes and ) . glycosylation of the matrix protein of tcv was also affected by tm. neither the major gp , nor the - , and , mol.wt, species could be immunoprecipitated after tm treatment (fig. a) . in contrast, all these protein species were identified in immunoprecipitates from extracts of untreated and sm-treated tcv-infected cells (fig. a, b) . the maturation of tcv in the absence or presence of glycosylation inhibitors was followed by sequential analysis by em of ultrathin sections of tcv-infected cells. electron microscopic examination of negatively stained untreated tcvinfected cells, at hours after inoculation, revealed budding of coronavirus particles through intracytoplasmic membranes and their accumulation in the lumen of smooth-walled vesicles (fig. a) . progeny viral particles were shown at the outer surface of infected cells, but none have been observed to be budding from the plasma membrane. the intracellular and extracellular viral particles were spherical in shape with an electron-lucent center and possessed large bulbous surface projections (fig. b) . the formation ofvirions was not inhibited by . to . gg/ml of tm (fig. ). complete virions were observed in dilated cytoplasmic vacuoles, but there was also an important accumulation of amorphous material, suggesting at least a partial interference with the viral assembly (fig. a) . virions that were present in the cytoplasmic vacuoles migrating to the cell surface apparently lacked the characteristic large peplomers of coronaviruses (fig. b) . the virions were released from tm-treated cells, but usually none were adsorbed to the plasma membrane. the addition of sm to the medium of tcv-infected cell cultures did not interfere with the normal maturation of the virus (fig. ) . large numbers of progeny viral particles could be observed in the cytosol even at a sm concentration of gm/ml. however, such high concentration of sm seemed to be very toxic for hrt- cells, as suggested by the intensive vacuolisation (fig. b) . the viral particles that accumulated in the lumen of smooth-walled vesicles of (fig. a ). n u m e r o u s viral particles were observed in the intercellular spaces and, m a n y were adsorbed to the plasma membrane. these virions possessed the characteristic surface peplomers. long tubules, to n m in diameter, accumulated in dilated cysternae or in cytoplasmic vacuoles (fig. b) . previous studies on the structural proteins of the tcv virions, produced in cell cultures or in ovo, have revealed a polypeptide pattern described so far only for viruses belonging to the subgroup of mammalian hemagglutinating coronaviruses [ ] . in addition to the peplomeric and matrix glycoproteins, tcv virions contain an envelope glycoprotein, having a mol.wt, of , , which behaves as a disulfide-linked dimer of - , subunits, and which has been associated to the hemagglutinin (accompanying paper). in this paper, we described the identification of virus-induced intracellular polypeptides in tcv-infected hrt- cells, the kinetic of their synthesis, their post-translational processing in the presence of glycosylation inhibitors and their significance with respect to the production of mature virions. we found that tcv had a modest inhibitory effect on cell protein synthesis, making the analysis of virus-specific proteins difficult. nevertheless, new intracellular protein species could be detected as soon as h post-infection either by sds-page analysis of pulse-labeled tcv-infected cell extracts or by western-immunoblotting, using anti-tcv hyperimmune serum produced to the eggadapted minnesota strain. the tcv structural proteins were all detected by h post-infection, in agreement with the one-step growth curve that was determined in a previous study [ ] and the replication cycle of coronaviruses [ ] . at least to polypeptides immunoprecipitated from lysates of tcvinfected cells. six of these polypeptides, with approximate mol.wts, of t - , , , , , , , , , , and - , , did not co-migrate with virion proteins. although a definitive relationship could not be established among these various intracellular polypeptides, pulse-chased experiments and analysis of infected cell extracts after cultivation in the presence of glycosylation inhibitors suggested that some of these products correspond to intracellular precursors, cleavage products or aggregated forms of structural glycoproteins. no processing of the p nucleocapsid (n) protein was apparent. two additional virus-specific binding proteins ( , and , ) which migrated slightly ahead of n were also identified for mhv and ibv [ , , ] . these two species reacted with monospecific anti-n antibody and, in vitro translation studies of the n gene have shown that they may correspond to degradation products of n [ , , ] . the - k polypeptide species present in tcv-infected cell lysates is apparently a virus-induced host cell or non-structural protein, as it was not revealed by western-immunoblotting. a non-structural protein with a similar mol.wt. also has been detected in mhv-a infected cel lysates, and a protein with a mol.wt, of , was also obtained following cell-free translation of the virion genomic rna [ , ] . since p is a basic protein which is most abundant in infected cells late in infection, it has been suggested that it may be involved in the processing of the genomic or subgenomic rnas. pulse-chased studies also revealed that tcv peplomeric glycoproteins undergo considerable post-translational changes before incorporation into the virion. first glycosylation of the primary translation product from the e gene may result in the synthesis of the gp - , species, and further glycosylation yields gp . subsequent proteolytic cleavage of gp to yield gp and gp , as demonstrated in the accompanying paper, appeared to occur quite efficiently, as these two species were the predominant species precipitated from tcv-infected cell extracts. this pattern of processing of e is similar to the patterns proposed for the e of bcv, mhv, and ibv [ , , . a primary glycosylated precursor with apparent mol.wt, of , also has been identified for bcv using anti-e monoclonal antibodies [ , . in case of tcv, proteolytic cleavage of e probably occurs also prior to the final glycosylation step. a , mol.wt, non-glycosylated polypeptide was revealed by immunoprecipitation prior to the appearance of gp and, was apparently chased to give the tcv virion gp . whether these two intracetlular polypeptides are in fact related remains to be demonstrated. it may be possible that a certain amount of a primary e apoprotein is cleaved and then further glycosylated to give the final gp - . a recent study on the intracellular processing of measles virus f and ha proteins demonstrated that glycosylation was essential to the post-translation proteolytic cleavage to proceed [ ] . similarly, results obtained with coronavirus mhv-a have suggested that the proteolytic cleavage of e was a post-translational event that occured only after assembling of the virion and further processing of the e protein in the golgi apparatus [- , , . with tcv, gp and gp species were also demonstrated after treatment with monensin, an inhibitor of the golgi function, suggesting that the first glycosylation acquired in the rer may be sufficient. the k protein may also represent a host cell component similar in mol.wt. to a group of resident proteins of the er identified as putative receptors or carriers involved in the rer-golgi translocation step [ . the "glucose-regulated proteins" grp and grp , for example, have been identified in the lumen of the er of a large number of different types of mammalian cells [ . recently, it has been demonstrated that unassembled or incorrectly assembled ha protein of influenza virus is prevented from leaving er, being complexed to a cellular protein with approximate mol.wt, of , resident in the lumen of er [ ] . a host-cell protein with similar mol.wt, also co-precipitated from lysates of measles virus-infected and tm-treated cells [ ] . propolypeptides or primary-translation products with approximate mol.wts. of , to , have been predicted from the amino acid sequences determined by sequencing analysis of the genes encoding the e of ibv and mhv [ , , ] . non-glycosylated polypeptides with apparent mol.wts, of - , and , were immunoprecipitated from extracts of tcv-infected, tm-treated cells. the new intracellular polypeptides were also identified by western-immunoblotting. although a definitive precursor-final product relationship was not demonstrated with e or e , the latter were not synthesized in the presence of tm. this was confirmed by radiolabeling with glucosamine and by em. both surface glycoproteins of the tcv virions thus possess n-linked oligosaccharides. the observation that the nonglycosylated polypeptides were only detected in the presence of this glycosylation inhibitor indicated that glycosylation of the e and e was initiated at the co-translational level. this is, again, typical for n-glycosidic linkages. the accumulation of amorphous material in the cysternae of tcv-infected, tm-treated cells may result from the rapid degradation of these putative non-glycosylated precursors. monensin has been shown to produce an intra golgi blockade of a number of secretory proteins by collapsing the hydrogen ion gradient across cellular membranes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it thus blocks "o" glycosylation, but not n-glycosylation which occurs in the rer. the k and t k species were heavily labeled with glucosamine in the presence of monensin, which further supported the results obtained with tm. the unglycosylated , and , mol.wt, proteins species immunoprecipitated from extracts of tcv-infected cells, and the virion - , and , mol.wt, glycoproteins appear to be different forms of the same viral protein. the non-glycosylated p may correspond to the precursor protein, which undergoes glycosylation to yield the gp - species, before rapid dimerization by disulfide bonds to yield gp , as suggested by electrophoresis in the presence of -me. a further glycosylation step would result to the final e protein, as described for bcv [ , ] . the fact that e was not detected in lysates of methionine labeled tcvinfected, tm-treated cells, is suggestive of a n-linked glycosylation. the absence in the same gel of the - , mol.wt, species, probably corresponding to an aggregated form of e [ , , , ] , and the presence of e in lysates of tcv-infected, sm-treated cells, further supported this notion. in this respect, tcv differs from viruses of the subgroup of mammalian hemagglutinating coronaviruses [ , ] , but resembles ibv [ , , ] . whereas glycosylation of glycoproteins with n-glycosidic linkages is generally initiated at the co-translational level, glycosylation of e of tcv appears to be exclusively a posttranslational event. this is suggested by the observation that apoprotein precursor of e (p ) was present in tcv-infected cells prior gp . for tcv and ibv, e therefore constitutes an example of a membrane glycoprotein that is targeted to the er and is not subsequently directed further along the secretory pathway. as previously reported for ibv and mhv [ , , ] , e appeared to be the only glycoprotein required for the formation of tcv virions, and its glycosylation is apparently not essential [ , ] . arrest of virus budding, as demonstrated in mhv-infected cells cultivated in the presence of monensin [ ] , was not demonstrated in tcv-infected cells in the presence of tm. this strengthens the notion that glycosylation of e is not necessary for virus budding. 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virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) coronavirus multiplication strategy. i. identification and characterization of virus-specific rna coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins coronavirus proteins: structure and function of the oligosaccharides of the avian bronchitis virus glycoproteins characterization of a coronavirus. ii. glycoproteins of the viral envelope: tryptic peptide analysis isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid the molecular biotogy of coronaviruses proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments infection of att murine pituitary tumour cells by mouse hepatitis virus strain a : virus budding is restricted to the golgi region the authors would like to acknowledge the technical assistance of h. strykowski for preparing the specimens for electron microscopy. authors' address: p. tijssen, centre de recherche en m~decine compar e institut armand-frappier, , boulevard de prairies laval-des-rapides, quebec h v b , canada.received february , key: cord- -tp mlqtw authors: li, yingli; chang, jitao; wang, qian; yu, li title: isolation of two chinese bovine enteroviruses and sequence analysis of their complete genomes date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: tp mlqtw in this study, rna corresponding to bovine enterovirus (bev) was detected in . % of faecal samples ( / ) from diarrheic and healthy cattle in six different areas in china by an rt-pcr screening method. furthermore, two cytopathic agents, designated as bhm and bj , were isolated from the bovine diarrheic fecal samples. during passage in ma cells, ultrathin sections of virus-infected monolayers were examined using a transmission electron microscope, and a large number of symmetrical virus crystals were seen in the cytoplasm, with monomorphic small viral particles of - nm in diameter. the full-length rna genomes were and nucleotides long, respectively, with a genome organization analogous to that of picornaviruses. phylogenetic analysis of the vp and vp capsid protein coding sequences suggested that the viruses bhm and bj belong to genotype of the bev cluster b (bev-b). in addition, sequence comparisons of the ′ and ′ utrs and p , p and p subgenomic regions of the two isolates suggested that there were intergenotypic recombination events occurring during evolution of the bhm and bj isolates. bovine enteroviruses (bevs), which are members of the family picornaviridae, genus enterovirus, form two phylogenetic clusters (designated bev-a and -b), with two and three genotypes, respectively [ ] . bev virions consist of a naked capsid that surrounds a core of positive-sense, single-stranded rna. hydrated native particles are - nm in diameter in electron micrographs, which reveal the virion to be a smooth sphere. the bev genome is approximately . kb long and has a typical picornavirus genome organization, including vpg following the utr, structural (vp , vp , vp and vp ) regions, nonstructural ( a, b, c, a, b, c and d) regions, utr and a poly (a) tail [ ] . bevs were first isolated in the late s [ , ] . they were isolated from (i) faeces of cattle with symptoms of pneumonia, respiratory disease, enteritis, dysentery and fertility disorders, (ii) fetal fluids from an aborted calf, (iii) the faeces of apparently healthy animals, and (iv) sewage, treated effluents, and materials from a farm environment. difficulties in reproducing clinical symptoms following experimental infection of animals led to the conclusion that bevs were of only minor veterinary medical importance. however, they have been shown to be widely detectable in a farm environment and have thus been proposed as markers for fecal contamination from cattle [ ] . it is generally assumed that bevs are avirulent, and if so, it is possible that they would make useful vectors for delivery of small molecules to the gut. additionally, bevs have been shown to have oncolytic properties and may be useful as therapeutic agents [ , ] . for these various reasons, we were interested in learning about the epidemiology and molecular evolution of bevs from china. in this study, a preliminary epidemiological study was performed, the results of which suggested that bevs are present in certain cattle in china. also, the present report described the first isolation, by means of ma cell cultures, of two isolates of bev (designated bhm and bj ) obtained from rectal swabs from diarrheic cattle of dairy herds in china. these viruses were characterized molecularly through sequence analysis of complete genomes, and the bhm and bj isolates were therefore classified into genotype of the bev-b cluster. the complete genome sequences of two chinese bev isolates not only enhance the database of sequences of members of the family picornaviridae, providing data of use to picornavirus virologists, but they also can be used further for construction of a bev infectious clone and development of bev-vectored vaccines based on epitopes. a total of fecal samples were collected from cattle at six farms in china from october to july . out of a total of fecal samples, were from animals with diarrhea and were from non-diarrheic animals. the samples were collected from inner mongolia (n = ), heilongjiang (n = ), anhui (non-diarrheic samples, n = ), liaoning (n = ), xinjiang (n = ) and shanghai (n = ), which are the main production areas of cattle in china. stool specimens were stored at - °c until the testing was performed. the samples were diluted : in mm phosphate-buffered saline (pbs) (ph . ), clarified by low-speed centrifugation ( g for min), and kept at - °c until they were used to isolate viruses. monolayers of ma cells grown for or days were used for virus isolation. the growth medium was dulbecco s minimum essential medium (dmem) supplemented with % fetal bovine serum (fbs) and antibiotics ( u of penicillin per ml and lg of streptomycin per ml). the centrifuged fecal samples were filtered through . -lm filters (millex, millipore). subsequently, ml of filtrate was inoculated onto a ma cell monolayer that had been grown in flasks and washed three times with pbs. after adsorption for min at °c, ml of fresh medium (without fbs) was added, the cultures were incubated at °c in a co incubator. when cytopathic effect (cpe) was not observed one week after inoculation, or the cells showed to % cpe, the cultures were frozen and thawed three times for harvest of cell lysates. subsequent passages were carried out in the same manner by using . ml of cell suspension from the previous passage as the inoculum. total rna from fecal samples and virus-infected ma cells was extracted by using trizol ls reagent (invitrogen, carlsbad, ca, usa) following the manufacturer s instructions. extracted viral rna was used as a template for cdna synthesis using primescript reverse transcriptase (takara). oligonucleotide primers (table ) used in this study were designed according to the consensus sequences of the fully sequenced bev rna genomes available from the genbank database. 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 denaturation at °c for . min, annealing for . min at °c to °c depending on the t m values of the different primers, and extension at °c for . min using a thermal cycler (bio-rad, usa). the final extension step was done at °c for min. the ends of the virus genomes were amplified using a -full race kit (takara, dalian, china) following the manufacturer s instructions. sequences at the end of the virus genomes were determined using rt-pcr, with first-strand cdna synthesis carried out using an oligo (dt) primer, thus taking advantage of the poly (a) tail in bev rna genomes as a priming site. all of the pcr products were detected by electrophoresis in % agarose gels and were purified using a dna gel extraction kit (watson biotechnologies, inc., shanghai, china). they were then sequenced directly in both directions by a commercial service (invitrogen, china). the complete nucleotide sequences of the isolates were assembled from the sequence fragments using the seqman module of the dnastar software package (dnastar inc., madison, wi). amino acid sequences were deduced from the nucleotide sequences using the editseq module. multiple sequence alignments and phylogenetic trees were constructed by the neighbor-joining method using molecular evolutionary genetics analysis (mega, version . ) software, and bootstrap resampling analysis of replicates was performed. a total of bovine diarrheic and healthy faecal specimens from six different areas were examined by rt-pcr using the primer pair u and l , which cover the highly conserved partial d gene and utr of bevs. the results showed that virus-specific rna could be detected directly in faecal material in of samples ( . %). the prevalence rate of bev in the six cattle farms located in inner mongolia, heilongjiang, anhui, liaoning, xinjiang, and shanghai was . % ( / ), % ( / ), . % ( / ), . % ( / ), % ( / ) and % ( / ), respectively, indicating that bevs are present in certain domestic cattle of china. all of the bev-specific-rna-positive faecal samples from inner mongolia and xinjiang were inoculated onto ma cells for isolation of bev. after the centrifuged fecal samples were inoculated onto cell monolayers, cpe was observed in the second-passage culture on the third day in culture plates inoculated with stool specimens (from inner mongolia) and (from xinjiang). the cultivated virus derived from sample was designated bhm , and that from the sample was designated bj . distinctly recognizable cpe was observed until the third passage at to h after infection. the cpe produced by the two isolates consisted of obscure cell borders, cell rounding, piling up of cells, and detachment of cells from the surface of plates (fig. ) . ultrathin sections of the infected monolayers were stained with uranyl acetate and lead citrate and examined using a transmission electron microscope. a large number of symmetrical virus crystals were seen in the cytoplasm, and the monomorphic small viral particles with a diameter of - nm were consistent with viruses of the family picornaviridae (fig. ) . the presence of bevs in cell cultures was further detected by rt-pcr with the primer set u and l , indicating that the two isolates were bev positive. pcr products were sequenced directly in both directions, and sequence alignments were conducted by using mega version . . the results showed that the d/ utr ( nt) of bhm and bj shared % and % nucleotide sequence identity, respectively, to that of bev strain jena / (genbank accession number: dq ), indicating that the two isolates are bovine enterovirus. nucleotide sequences of the complete bhm and bj genomes several overlapping pcr amplifications spanning the entire genomes of the bhm and bj isolates were obtained, and their complete nucleotide sequences were determined. the bhm and bj genomes consist of , bp and , bp, including and a bases, respectively, in the poly (a) tail. the complete genomes of the two isolates have a typical picornavirus genome organization, including a utr, a large single orf, a utr low nucleotide sequence identity of the genes encoding the capsid proteins c and d is the main criterion for the identification of genotypes of bev [ ] . thus, phylogenetic analysis of the c and d encoding regions of the two isolates and other representative bev strains was performed to determine the phylogenetic characteristics of the two bev isolates. it was found that both bhm and bj were classified into genotype of the bev-b cluster and were most closely related to bev strain jena / (fig. ) , an isolate from germany (genbank accession number: dq ). furthermore, similar results were obtained when the c and d encoding regions were used to analyze phylogenetic relatedness among the bev strains, although the branch orders were somewhat different (fig. ) , showing that the c and d coding regions can be used simultaneously to determine the genotypes of the bev strains. the complete genomes of bhm and bj were divided into utr and utr and p , p and p subgenomic regions for sequence comparisons with those of the referenced bev strains. for these subgenomic regions, the two sequenced isolates shared . % ( utr), . % ( utr), . % (p ), . % (p ) and . % (p ) nucleotide sequence identity ( in the present study, we first conducted a preliminary epidemiological survey for detecting bevs from diarrheic and healthy cattle in of china. a total of bovine fecal samples collected from the six different areas of china were tested by rt-pcr, and samples ( . %) in the six areas were positive for bev, indicating that bevs are widespread in dairy farms in china. subsequently, two bev isolates (designated bhm and bj ) were isolated from bovine fecal specimens, and they were then characterized morphologically and genotypically. in addition, the complete sequences of the bhm and bj genomes were determined and were submitted to the genbank database under accession nos. hq and hq , respectively. by phylogenetic analysis of the c and d capsid protein genes, both bhm and bj were classified into genotype of the bev-b cluster. sequence comparisons of the utr and utr and p , p and p subgenomic regions suggested that bhm and bj are intergenotypic recombinant bev isolates. in general, incongruence between phylogenies of different genome regions is considered indicative of recombination events in enteroviruses [ , ] . for example, phylogenetic analysis of german bev isolate d / / yielded incongruent results [ ] in which comparisons of b and c indicated a similarity with genotype of the bev-a cluster; however, in the d comparison, the d / / strain clustered with genotype . another example of intragenotypic recombination is shown by the lc-r strain, which was grouped with the vir / strain in the b phylogeny but with the vg( ) strain in the c and d phylogenies [ ] . these findings are an indication of intergenotypic or intragenotypic recombination in the evolution of the bevs. in this study, for the china bev isolates bhm and bj , sequence analysis based on the utr and utr and p , p and p subgenomic regions yielded some incongruent results, suggesting that there were intergenotypic recombination events occurring during evolution of the bhm and bj isolates. the earliest reports concerning the virulence of bevs suggested that there might be an association between bev infections and a range of diseases in cattle, as viruses were isolated from cattle with clinical signs that varied from respiratory to enteric to reproductive disease and infertility [ , ] . however, bovine enterovirus has also been isolated from apparently healthy cattle and wildlife [ , , ] . interpretation and comparison of these reports are difficult because in many cases other infectious agents were identified or were not investigated. as reviewed by jiménez-clavero and co-workers [ ] , enteroviruses are highly stable in the digestive tract, are shed in large amounts, and persist in the environment for prolonged times, and thus serve as indicators of fecal pollution. in susceptible individuals, enteroviruses cause subclinical infection or mild disease, although occasional infections may cause serious disease, such as the poliomyelitis caused by poliovirus in humans [ ] . the bhm and bj were isolated from bovine diarrheic fecal samples, but their pathogenicity in cattle experimentally infected with the bhm and bj has not been conducted. it is well known that bovine rotavirus and coronavirus are the most important cause of diarrheal diseases in cattle, especially in neonatal calves. if one animal is simultaneously infected by rotavirus and coronavirus, it can cause significant morbidity and mortality. in this study, bev-specific rna not only was detected in diarrheic faecal samples ( of bev-positive samples collected from diarrheic cattle) but also was detected in healthy faecal samples ( of bev-positive samples collected from healthy cattle). at the same time, two other rna viruses that are transmitted by the fecal-oral route (bovine coronavirus and rotavirus) were detected from diarrheic faecal samples. although little is actually known regarding the pathogenesis of the chinese bev isolates within the enteric tract, according to the epidemiological survey in this study, the bevs in the fecal specimens from china are only considered to be a minor cause of diarrhea in these dairy farms. several characteristics of the bevs make them good candidates for development of a vaccine vector. first, they are physically very stable, e.g., bev particles are resistant to lipid solvents (e.g., chloroform) and are stable at ph to . second, bev is currently considered avirulent in cattle, making it safe to use as a vaccine vector. third, since enteroviruses enter their host via the enteric tract, they could be administered orally as vaccine vectors to stimulate mucosal immunity. thus, we are interested in their potential as vaccine vectors for expressing small peptides. we have recently constructed an infectious cdna clone of the bev bhm isolate on the basis of these sequencing data (yingli li et al., unpublished data). our previous studies have finely mapped a conserved type o fmdv neutralizing epitope recognized by a specific monoclonal antibody [ ] . engineering of recombinant bevs displaying the fmdv neutralizing epitope in our laboratory is underway for development of a bev-vectored and epitopebased vaccine. bovine viral diarrhea virus type -induced meningoencephalitis in a heifer the complete nucleotide sequence of a bovine enterovirus isolation and classification of viruses from cattle during outbreaks of mucosal or respiratory disease and from herds with reproductive disorders survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription-pcr fields virology, th edn a serological classification of bovine enteroviruses bovine enteroviruses as indicators of fecal contamination evolution of the genome of human enterovirus b: incongruence between phylogenies of the vp and cd regions indicates frequent recombination within the species molecular characterisation of australian bovine enteroviruses one hundred years of poliovirus pathogenesis evidence of recombination among enteroviruses therapeutic effects of bovine enterovirus infection on rabbits with experimentally induced adult t cell leukaemia bovine enterovirus as an oncolytic virus: foetal calf serum facilitates its infection of human cells bovine enterovirus- : characterization, replication and cytopathogenic effects molecular-based reclassification of the bovine enteroviruses identification of a conserved linear epitope on the vp protein of serotype o foot-and-mouth disease virus by neutralising monoclonal antibody e key: cord- -x mjov l authors: ribeiro, juliane; headley, selwyn arlington; diniz, jaqueline assumpção; pereira, alfredo hajime tanaka; lorenzetti, elis; alfieri, amauri alcindo; alfieri, alice fernandes title: extra-intestinal detection of canine kobuvirus in a puppy from southern brazil date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: x mjov l this study presents the pathological, immunohistochemical, and molecular findings associated with the extra-intestinal detection of canine kobuvirus (cakv) in a -month-old chihuahua puppy, that had a clinical history of bloody-tinged feces. principal pathological findings were interstitial pneumonia, necrotizing bronchitis, and parvovirus-induced enteritis. molecular diagnostic methods identified cakv within the cerebellum, cerebrum, lung, tonsil, and liver. cakv and rotavirus were not identified within the feces and intestine. immunohistochemistry (ihc) assays detected antigens of cdv and cadv- in the lungs. these results confirmed the extra-intestinal detection of cakv in this puppy and represent the first extra-intestinal detection of cakv in a dog. structural polyprotein (vp , vp , and vp ) and nonstructural ( a- c and a- d) proteins [ ] . the d segment of the kobuvirus genome is a conserved region, and the vp protein is the most variable, similar to other picornaviruses [ , ] . kobuviruses were identified in fecal samples from diarrheic and asymptomatic humans as well as in several animals. in brazil, kobuviruses were initially detected in the fecal samples of pigs and sheep, and viral rna was identified in the serum of pigs between and days-ofage [ ] . thereafter, the presence of aichivirus b and c was identified in fecal samples of cattle [ ] and pigs [ ] , respectively. canine kobuvirus (cakv) was first described in fecal samples of dogs with gastroenteritis in the usa in [ ] , with subsequent reports in the uk [ ] , italy [ ] , korea [ ] , and china [ ] . cakv is frequently identified within fecal samples of animals with or without clinical signs of diarrhea [ ] . there are few descriptions of coinfection due to cakv and other viral agents: a recent study identified cakv in association with a several agents including canine herpesvirus type (cahv- ), canine distemper virus (cdv), canine parvovirus (cpv), and canine adenovirus a types (cadv- ) and (cadv- ) in diarrheic and non-diarrheic dogs from korea [ ] . however, none of these studies have identified cakv in the tissues of dogs. consequently, the current study describes the first occurrence of cakv in multiple tissues of a puppy from southern brazil, which was also coinfected with other infectious disease agents. in early march, , an outbreak of diarrhea occurred at a kennel in the city of colorado, paraná state, southern brazil. the owner reported that seven - -day-old chihuahua puppies developed diarrhea, anorexia, and apathy with death occurring between - days after the onset of clinical manifestations. a necropsy on one of these puppies, done elsewhere, revealed interstitial pneumonia and necrotizing hepatitis associated with intranuclear inclusion bodies in hepatocytes, and a diagnosis of infectious canine hepatitis was established. in late july, , the carcass of a -month-old puppy with a clinical history of abdominal pain, anorexia, and blood-tinged feces was submitted for routine necropsy. this puppy had received two doses of a commercial vaccine that contained eight immunogens (cdv, cpv- , canine parainfluenza virus, canine adenovirus and , canine coronavirus, and leptospira canicola and grippotyphosa serovars.); the last dose being six weeks before spontaneous death. several tissue fragments (liver, spleen, lung, cerebellum, cerebrum, mesenteric lymph node, kidney, heart, and intestine) were collected and routinely processed for histopathological evaluation. selected formalin-fixed paraffin embedded (ffpe) tissue fragments of the liver and lung were used in immunohistochemistry (ihc) assays designed to detect the n protein of cdv (vmard, washington, usa), and with a commercial monoclonal antibody designed to identify cadv- and cadv- (vmard, washington, usa), using a previously described procedure [ ] . positive controls consisted of ffpe tissues from previous studies [ ] ; negative controls consisted of the diluents of the primary antibodies. duplicates of the tissues cited above, as well as feces, were collected for parasitological and virological evaluations. for virological investigation the tissues were processed using the magnalyser instrument (roche diagnostics, mannheim, germany) and the fecal samples were submitted to nucleic acid extraction as described elsewhere [ ] . the nucleic acid was eluted in ll of ultrapure rnase-free depc-treated sterile water. negative (sterile water) and positive controls, consisting of viral nucleic acids (cadv- , cadv- , cdv, and cahv- ) from previous cases [ , ] , as well as cpv commercial vaccine, were included in all nucleic acid extraction procedures. the extracted nucleic acids were used in rt-pcr/pcr assays designed to amplify specific genes from viral agents known to infect dogs, including the: cdv n gene [ ] , cahv- glycoprotein b gene [ ] , cpv vp gene [ ] , and the e gene of cadv- and [ ] . the rt-pcr assay to detect cakv is designed to amplify a bp region of the rdrp gene [ ] . the partial region of the vp protein cakv was amplified by using the primer pairs vp -dogf ( '-caahctkgar-aacttcttctc- ') and vp -dogr ( '-gaagttkga-gagcatctgkc- '), which were designed using the complete sequences of human, canine, and feline kobuvirus available in genbank. the intestine and the fecal samples were used in rt-pcr assays designed to identify rotavirus species a, b, and c [ ] . all rt-pcr and pcr products were analyzed by electrophoresis in a % agarose gel in tbe buffer, stained with ethidium bromide, and visualized under uv light. two rt-pcr products of the rdrp gene and the vp region of cakv were purified using the gfx pcr dna and gel band purification kit (ge healthcare), quantified in a qubit Ò fluorometer (invitrogen life technologies), and sequenced in an abi genetic analyzer sequencer with the bigdye Ò terminator v . cycle sequencing kit using the forward and reverse primers used in the rt-pcr assay (applied biosystems). the phylogenetic tree and the nt identity matrix were developed using the mega . and bioedit . . , respectively. the analyses were based on the neighbor-joining method and the kimura -parameter model. bootstrapping was statistically supported with , replicates. the referenced sequences included in this study were acquired from genbank. the most remarkable pathological alterations were observed in the lungs and consisted of discrete necrosis of bronchiolar and bronchial epithelial cells associated with small basophilic intranuclear inclusion bodies within some of these cells, severe diffused interstitial pneumonia, in addition to pulmonary congestion and edema. the intestinal lesions were suggestive of parvovirus enteritis. other significant pathological findings included necrotizing myocarditis and lymphoid depletion of the spleen and tonsils. immunohistochemistry revealed the detection of the antigens of cdv within the bronchiole and bronchiolar epithelium (fig. a) and antigens of cadv- within the cytoplasm of endothelial cells (fig. b) ; antigens of cadv- and - , and cdv were not identified in the liver. examples of giardia spp. were identified by the floating technique from the fecal sample of the puppy. the distribution of the viral nucleic acids identified within multiple tissues and the feces of the puppy are given in table . specific fragments of the genes of cdv, cakv, cpv- , and cahv- were amplified from multiple tissues. mixed infections associated with cakv were identified in the cerebrum, lung, and tonsils. in addition, nucleic acid from cpv- was identified within fragments of the small intestine and a single infection associated with cakv was also detected in the cerebellum and liver. further, the fecal sample of the puppy contained cpv- dna. the nucleic acids of all other viral agents evaluated, including rotavirus species a, b, and c, were not amplified. the partial sequences of cakv vp and rdrp genes identified in this study have been deposited in genbank (accession numbers: vp : brain kx , lung kx ; rdrp: lung, kx ; tonsil kx ). the phylogenetic analysis of the rdrp region (nt ) showed that the cakv strains from the lung and tonsil of this dog grouped with other strains of cakv previously described ( fig. a) , and they exhibited higher nt sequence identity ( to %) with canine than with mouse ( %) and human ( to %) kobuvirus strains. moreover, the phylogenetic analysis of the vp region (nt ) revealed that the cakv strains identified in the lung and cerebrum of this dog clustered with other strains of cakv and mouse kobuvirus. in addition, no phylogenetic difference was observed between strains of aichivirus a, since those derived from dogs, mouse, and humans, clustered within the same group (fig. b) . further, the vp cakv strains from the present study showed . * %, . * %, % nt identity; and . %, . * . %, and . % amino acid similarity with dog, mouse, and human kobuvirus strains, respectively. the unique finding of this investigation was the amplification of the nucleic acid of cakv from multiple tissues of this puppy, using two rt-pcr assays. these results contrast with previous descriptions of cakv, where nucleic acids were detected exclusively in the feces of diarrheic dogs [ , , ] ; however, these studies did not evaluate the presence of cakv in tissues. surprisingly, cakv was not detected in the feces or the intestinal fragment of this puppy using the rt-pcr assay applied during this study. alternatively, a more sensitive assay might be required to detect this virus when there is concomitant infection with other severe intestinal diseases, such as those associated with cpv- and giardia spp. nevertheless, these findings confirm the detection of cakv in this dog, and represent the first description of canine kobuvirus in brazil. it must be highlighted that coinfections of cakv with other viral agents were identified in several tissues/organs, including the cerebrum, lung, and the tonsils (table ) ; and there was concomitant parvovirus enteritis and giardiasis. mixed infections in dogs have been previously related with descriptions of simultaneous viral, bacterial, and parasitic agents [ , ] , as well as mixed infections with multiple viral agents [ ] . coinfections with cakv and other infectious agents in dogs, as observed in this investigation, have been described in diarrheic outbreaks [ ] , although the pathogenic effect of cakv is unclear. interestingly, we have identified cakv with concomitant infections related to cdv (by rt-pcr and ihc) and cadv- (only by ihc) in the lung of this dog and associated with necrotizing bronchiolitis, interstitial pneumonia, as well as pulmonary congestion and edema. interstitial pneumonia is a typical pulmonary detection of canine kobuvirus in southern brazil manifestation of infection due to cdv and cadv- [ , ] . cdv produces interstitial pneumonia due to the viral tropism for epithelial cells of the pulmonary alveoli, while cadv induces the same lesions due to alteration of the integrity of the vascular endothelium of the alveolar wall [ ] . however, interstitial pneumonia was also described in pigs experimentally infected with porcine kobuvirus [ ] , so it is possible that cakv could have been associated with the pulmonary infection observed in this dog. additionally, the presence of kobuvirus is frequently associated with an initial infection induced by other infectious agents. in this case, there were concomitant coinfections caused by cpv- and cdv; these are well known immunosuppressive infectious pathogens [ ] that facilitate the entry and colonization of secondary agents. therefore, we believe that the multiple coinfections observed in this puppy might be related to the immunosuppression induced by cdv and cpv- , and worsened with the concomitant giardiasis. furthermore, widespread viral dissemination was demonstrated in multiple tissues as cakv nucleic acids were detected in the liver, lung, brain, and tonsils of this fig. phylogenetic analysis of a partial nucleotide sequence (nt ) of the rdrp gene (a) and partial region of the vp protein (nt ) of canine kobuvirus (cakv) (b). the strains of cakv identified during this study are marked with a filled circle puppy. these findings complement a previous investigation by our group, where kobuvirus was identified in the sera of pigs of various ages with and without clinical signs of diarrhea [ ] . the phylogenetic analysis of the conserved rdrp region of the cakv described in this study revealed higher nt sequence identity between canine ( %- %), mouse ( %), and human ( %- %) kobuvirus strains. the sequence analysis of the vp protein, considered the most variable protein in this virus, obtained from the brain and pulmonary fragments were identical to each other and had . * %, . * %, % nt identity; and . %, . * . %, and . % amino acid similarity with dog, mouse, and human kobuviruses, respectively. moreover, these strains of kobuvirus grouped within the same cluster during phylogenetic evaluation. in conclusion, cakv genomes were identified in multiple tissues from a puppy that was coinfected with other viral and parasitic agents, confirming the systemic distribution of kobuvirus in this puppy. although the role of cakv in the etiopathogenesis of disease is obscure, there is evidence that pathological alterations might have occurred. finally, the phylogenetic analysis confirmed that the strains of cakv identified during this investigation are similar to those described in other geographical locations. these findings represent the first characterization of cakv in the tissues of a puppy. frequency of group a rotavirus in diarrhoeic calves in brazilian cattle herds first detection of kobuvirus in farm animals in brazil and the netherlands phylogeny and prevalence of kobuviruses in dogs and cats in the uk canine kobuviruses in diarrhoeic dogs in italy detection of canine distemper virus nucleoprotein rna by reverse transcription-pcr using serum, whole blood, and cerebrospinal fluid from dogs with distemper canine viral enteritis. in: greene ce (ed) infectious diseases of the dog and cat canine distemper. in: greene ce (ed) infectious diseases of the dog and cat glial fibrillary acidic protein (gfap)-immunoreactive astrocytes in dogs infected with canine distemper virus molecular detection of canine distemper virus and the immunohistochemical characterization of the neurologic lesions in naturally occurring old dog encephalitis diagnostic exercise: tyzzer's disease, distemper, and coccidiosis in a pup concomitant canine distemper, infectious canine hepatitis, canine parvoviral enteritis, canine infectious tracheobronchitis, and toxoplasmosis in a puppy canine distemper virus with concomitant infections due to canine herpesvirus- , canine parvovirus, and canine adenovirus in puppies from ssouthern brazil occurrence of canine parvovirus type c in the united states detection and differentiation of cav- and cav- by polymerase chain reaction ictv ( ) international comittee on taxonomy of viruses characterization of a canine homolog of human aichivirus prevalence and phylogenetic analysis of canine kobuviruses in diarrhoetic dogs in northeast china porcine rotavirus groups a, b, and c identified by polymerase chain reaction in a fecal sample collection with inconclusive results by polyacrylamide gel electrophoresis canine kobuvirus infections in korean dogs complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus kobuvirus, family picornaviridae kobuviruses -a comprehensive review high frequency of aichivirus c (porcine kobuvirus) infection in piglets detection of canine kobuvirus in southern brazil from different geographic regions of brazil kobuvirus (aichivirus b) infection in brazilian cattle herds canine herpesvirus- (chv- ): clinical, serological and virological patterns in breeding colonies complete nucleotide sequence and genetic organization of aichi virus, a distinct member of the picornaviridae associated with acute gastroenteritis in humans isolation and characterization of a new species of kobuvirus associated with cattle histopathology of porcine kobuvirus in chinese piglets conflict of interest the authors declare that they have no conflict of interest.ethical approval supervision of the ethical committee to conduct this study was not required because this work was conducted with organ fragments of an animal that died spontaneously and was submitted for routine diagnosis by the owner. all applicable international, and national guidelines for the care and use of animals was followed. key: cord- -kq wmavt authors: kasai, h.; morita, e.; hatakeyama, k.; sugiyama, k. title: characterization of haemagglutinin-esterase protein (he) of murine corona virus dvim by monoclonal antibodies date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: kq wmavt we analyzed the characteristics of seven monoclonal antibodies (mabs) raised against purified he (hemagglutinin-esterase) glycoprotein of the murine coronavirus dvim (diarrhea virus of infant mice). immunocrossreaction of these mabs with jhm and/or mhv-s suggest that antigenic epitopes of he of dvim are similar to those of jhm and/or mhv-s. four mabs ( b , aff , c , b ), designated as group a mabs, strongly inhibited both ha and ae activities. on the other hand, three mabs ( aff , aff , aff ), referred to as group b, had a comparatively weak ha inhibition activity. these results indicate that the antigenic epitopes of this glycoprotein can be classified into at least two groups and that the functional sites of ha and ae activities are similar but not identical. neutralizing activity was shown in group a mabs exclusively, suggesting that the ratio of ha and/or ae activities may play important roles in the cell fusion activity of dvim-infected cells. coronaviruses are enveloped viruses containing a single-stranded rna genome of positive polarity [ ] [ ] [ ] . murine coronavirus mhv-dvim is an enteropathogenic coronavirus causing severe diarrhea in newborn mice [ ] [ ] [ ] . as described in a previous report [ ] , by treatment of dvim with the non-ionic detergent np- we isolated the he protein, which carries the functional sites for both acetylesterase (ae) and the haemagglutination (ha) activities. the contribution of these activities to infection by mhv-dvim is not yet known, nor is their relation to the antigenic epitopes. to know the antigenic properties of he, several monoclonal antibodies (mabs) raised against the purified protein were established and used to characterize the he protein. in the present report, we provide evidence that the functional sites for ae and ha activities have separate locations on the he protein. furthermore, mabs that inhibit ae activity also suppress the ability of dvim to cause cell fusion from within. murine coronavirus mhv-dvim was grown in dbt cells using eagle's mem supplemented with % newborn calf serum (gibco) and % tryptose phosphate broth (difco). the virus was purified as described previously [ , ] . the ae activity was determined according to the methods described by vlasak et al. [ , ] . purified virions dissolved in l of pbs (l g/l) were reacted with l of mabs ( . g/l) (dilution rate − to − ) for h at room temperature. after addition of l of esterase substrate (l mm p-nitrophenilacetate: dissolved in % acetonitril), the solution was incubated for min at • c, and hydrolysis of the substrate was monitored at od nm with a spectrophotometer (hitachi - ). the inhibitory effect was expressed as the percentage against a blank that contained pbs instead of the virus. ha assays were performed in v-shaped microtiter plates as described previously [ ] using . % (vol/vol) erythrocytes of balb/c mice (in dulbecco's pbs, ph ) supplemented with . % bovine serum albumin (bsa: sigma). the titer was recorded as the reciprocal value of the highest dilution causing a detectable ha reaction. hai assays were also carried out in microtiter plates. aliquots of l of purified virions ( -hau) were reacted with l of serial two-fold dilutions of mabs in a v plate. after h at • c, l of . % (v/v) erythrocytes were added. after incubation for h at • c, the hi titer was recorded as the reciprocal value of the highest dilution causing a detectable inhibitory effect. the preparation method was the same as that described in a previous paper [ ] . neutralization tests by mabs were carried out by a semimicro reduction assay [ , ] . the virus ( . tcid /ml) was mixed with diluted antibodies, incubated for h at • c, and plated onto -well plates. after adsorption for h at room temperature, the plates were washed with pbs (ph . ), and the culture medium was added to the cells. the neutralization titer was expressed as the highest antibody dilution that prevented virus infection (tcid /ml). the ratio of fusion formation was calculated from the number of nuclei in which the cells formed a syncytium. since gp was identified as the he glycoprotein of dvim [ ] , we attempted to characterize the antigenic sites of this glycoprotein. we obtained seven clones a lgg / + + + a hai was determined as described in materials and methods b aei was determined at the dilution of : of mabs c neutralization activity was expressed as the reciprocal of the highest dilution of mab that showed fusogenic cpe at h post infection. cpe (syncytium) (+ + +) developed overall in the cell sheet fig. . reactivity of mabs with the he protein of dvim shown by western blot analysis. after solubilization in a sample buffer with -mercaptoethanol, dvim virions were subjected to electrophoresis on . % sds-polyacrylamide gels. following transfer to a nitrocellulose membrane, the proteins were incubated with an mab as described in materials and methods. as a negative control, pbs was reacted instead of the st antibody. the molecular weights are indicated on the left. the arrow on the right side points to the he protein of dvim of mabs ( c , b , a , b , a , a and a ) directed against the he glycoprotein. all of them were identified as belonging to the igg ( /) isotype (table ) and reacted with the polypeptide of the gp kda of dvim in western blot analysis, indicating that they recognize an epitope that exists on this glycoprotein (fig. ) . these mabs were also examined for the crossreactivity with other strains of mhv as well as with influenza c virus, which also contains an acetylesterase, by western blot analysis. as shown in fig. , six mabs recognized a major kda protein of mhv-jhm but no crossreactivity was observed with mhv- , mhv- and influenza c/miyagi/ virus (fig. ) . however, mab a also reacted with gp of mhv-s. these results indicate that the he glycoprotein of dvim has antigenic similarity with that of mhv-jhm and to some extent also with mhv-s. to determine the biological properties of these mabs, we examined their inhibitory activities against acetylesterase, hemagglutination, and fusion ( from within) activites, respectively. inhibition of acetylesterase (aei) was analyzed by determining the effect of the mabs on the ability of the viral enzyme to release p-nitrophenol from p-nitrophenylacetate. as shown in table , five mabs ( c , b , a , b and a ) showed high aei titers ( - %). two mabs ( a and a ) were found to have low levels ( - %) of aei titer. high hemagglutination inhibition (hai) titers (> ) were measured with three mabs ( b and c ). with two mabs ( b and a ), intermediate values ( - ) were obtained. the mabs ( a , a and a ) had low hai titers (< ∼ ) (table ) . these data suggest that these mabs were classifiable into at least two groups: group a ( c , b and b ) that has a high titer to both activities and an intermediate titer to the ha activity, and group b ( a , a and a ) that has low titers to both activities ( fig. and table ). to determine the correlations between ae and ha active sites, the inhibitory effects against ha and ae activities by dfp were also tested. inhibitory effects were shown only in the ae activity and not in the ha activity (data not shown). these results suggests that ha and ae active sites that exist on the he glycoprotein of dvim are separated etiologically. we examined the cytopathic effect of mabs with respect to the formation of syncytia in dvim-infected cells. fusion was evident at h postinfection (p.i.). the mabs a , a , and a were unable to prevent this cytopathic effects, and no syncytia formation was observed at h p.i. when the virus was treated with mabs b , a and c prior to infection (fig. ) . it should be noted, however, that these antibodies did not completely neutralize dvim. they only delayed control, v virus control, a b , b a , c c , d a , e a , f a . cytopathic effect (fusion from within) is shown by asteriks virus cell fusion activity, because syncytia were observed by h p.i. (fig. ) . these data indicate the existence of a neutralization factor on the he glycoprotein of dvim. the role of the he protein in virus infection is unclear [ ] [ ] [ ] . for some viruses, such as bovine coronavirus and human coronavirus oc , this protein appears to be essential, because no variants or mutants that lack this protein have been found [ , ] . the esterase activity of he has been suggested to play a role either in virus entry or in virus release [ ] [ ] [ ] . though mhv is serologically related, it differs from the other viruses of this serogroup in several aspects. while the bovine and human counterparts use -o-acetylated sialic acid on the plasma membrane for binding to cells, biliary glycoprotein serves as a receptor for mhv [ , , ] . in addition only some strains of mhv contain an he-protein, while others lack such a protein [ , ] . even strains like dvim that contain an he-protein and thus are able to release the o-acetyl groups from n-acetyl- -o-acetylneuraminid acid use biliary glycoprotein as a receptor [ ] . thus, the importance of the he-protein for mhv is not known. to obtain more information about the he protein, we raised antibodies to this protein and analyzed their effect on the biological activities of he. with respect to inhibition of hemagglutination activity and acetylesterase activity, there were antibodies that had a high titer to both activities or a high titer to one activity and an intermediate titer to the other activity. two antibodies had low titers to both activities. from these results, we conclude that there are several epitopes present on the he protein. the data also indicate that the functional sites of the hemagglutination and acetylesterase activities are similar but not identical. none of the antibodies completely neutralized dvim. however, some of the antibodies delayed the onset of syncytium formation and the cytopathic affect. all these antibodies had high aei activities and high or intermediate hai activities. the antibodies that had low titers to both activities did not delay the fusion of infected cells. therefore, a functional acetylesterase activity and/or hemagglutination activity appear to be necessary for optimal infection by dvim. further investigation is needed to determine the exact role of the he protein. the antibodies described here should be helpful in this respect. genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues interaction of mouse hepatitis virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with an mhv strain that express the hemagglutininesterase glycoprotein coronaviridae and replication ter meulen v ( ) the structure and replication of coronaviruses the molecular biology of coronavirus hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice structural polypeptides of the murine coronavirus dvim haemagglutinin-esterase protein (he) of murine corona virus: dvim human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses the e protein of bovine coronavirus a receptor-destroying enzyme with acetylesterase activity we wish to h. ebina of hirosaki university, faculty of science for his helpful assistance of this work. key: cord- - l pglef authors: percy, d.; bond, s.; macinnes, j. title: replication of sialodacryoadenitis virus in mouse l- cells date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: l pglef sialodacryoadenitis (sda) is a naturally-occurring infection of the laboratory rat raused by the coronavirus, sialodacryoadenitis virus (sdav). the study of sdav has been limited because there is no widely available continuous cell line for the propagation of high titers of the virus. the purpose of this study, therefore, was to compare the ability of sdav to replicate in the permanent cell lines, lbc, of rat origin, and the mouse cell lines. l- and l- . following to repeated passages of sdav in lbc cells, the virus could be readily propagated in lbc and l- cells, but not in l- cells. similarly, sdav adapted to replicate directly in l- cells could be readily propagated in lbc, but not l- cells. in lbc and l- cells, cytopathic effect (cpe), viral antigen, viral particles, and virus infectivity could be demonstrated. titers of up to ( . ) infectious viral particles/ . ml of culture fluid were obtained at hours in l- cells. titers in lbc cells were one to two logs lower. when susceptible rats were inoculated with eighth passage l- cell-adapted virus, they developed typical lesions of sda. virus could be recovered from infected tissues and propagated in l- cells on first passage. the ability to propagate sdav to high titers in the widely available l- cell line should promote the study of this virus and facilitate its comparison with other murine coronaviruses. sialodacryoadenitis virus (sdav) infection is widespread in the laboratory rat. based on reported serological surveys, the incidence of this coronavirus in rat colonies may be as high as % [ , ] . lesions associated with sdav infection include sialoadenitis, dacryoadenitis, rhinitis [ , , , t ] , reproductive disorders [ ] , and tracheobronchitis [ ] . sdav may also accelerate the progression of murine respiratory mycoplasmosis [ ] , and through damage to submandibular salivary glands, may cause depletion of epidermal growth factor [ ] . although the course of the disease is generally short, with rapid recovery [ ] , sda can cause a variety of complications which may affect the results of behavioral, eye, reproductive, cancer and respiratory research [- , , , ] . in an early study by bhatt et al. [ ] , a variety of continuous cell fines including bhk- , vero, and hep- cells were evaluated for their ability to support the replication of sdav. a variety of primary monolayer cultures including primary rat kidney cells (prk) were also tested. bhatt et al. [ ] also tried propagating sdav in explant cultures of rat submaxillary (submandibular), parotid, harderian and exorbital glands; and in trypsin-dispersed mouse brain cell cultures. replication and cytopathic effect (cpe) were observed only in prk cells. maximum titers of . tcid were obtained prk cells at hours post-inoculation (pi) with strain # of sdav [ ] . all attempts to isolate virus from tissue culture systems other than prk cells were negative. however, they found that sdav could be propagated by intracerebral inoculation of suckling mice, and recorded titers of up to . tcids in prk cells [ ] . recently hirano et al. [ ] reported the ability of rat coronaviruses to replicate in the lbc continuous cell line derived from a mammary tumor in a lewis rat. in lbc cells inoculated with strain # of sdav, cpe was first detected in infected cultures hours pi, and syncytia were observed by hours pi [ ] . by the sixth passage of sdav in lbc cells, cpe was extensive by hours. viral antigen and tcids titers of / . ml could be detected in inoculated culture fluid at hours pi by the tenth passage of sdav in lbc cells [ ] . the work by hirano et al. [ -- ] with the lbc cells thus confirmed that it was possible to propagate rat coronaviruses in a continuous cell line. lbc cells are not, however, widely available and they are relatively slow growing. thus, we attempted to find another permanent cell line which would support the production of high titers of sdav. in this report we compare the titers and the in vivo and in vitro infectivity of sdav grown in lbc cells, l- cells, and in l- cells. ce//s the lbc cell line was kindly supplied by dr. k. kai (university of yamaguchi, yamaguchi city, japan). mouse l- cells were obtained from the american type culture collection (rockville, md). the l- cell line, a subline of l- cells [ ] was acquired from dr. v. l. morris (university of western ontario, london, ontario). the characterization of the original cell line has been previously described [ ] . cell cultures were grown in eagle's minimal essential medium (gibco/brl inc., burlington, ontario) containing u/ml penicillin, gg/ml streptomycin and . gg/ml gentamicin supplemented with % (l- ; l- ) or % (lbc) fetal bovine serum. cells were propagated on x mm nunc polystyrene tissue culture dishes (gibco/brl inc.) at °c in a humidified atmosphere containing % co . strain # of sdav was obtained from dr. p. n. bhatt (yale school of medicine, new haven, ct). virus was passaged five times in specific pathogen-free (spf) male wistar rats which were serologically negative for antibodies to rat coronavirus (charles river canada, st. constant, quebec). infected parotid and submandibular salivary glands were harvested and homogenized in tenbroeck tissue grinders to make a % (w/v) suspension in culture medium. aliquots of this stock virus suspension were stored at -- °c. when approximately %-confluent, monolayers of lbc, l- or l- cells were inoculated with (). ml of stock virus suspension or infected tissue culture supernatant. following adsorption at °c for one hour, the monolayers were washed and the culture medium was replaced. inoculated and control cell cultures were incubated at °c and % co for - hours. tenfold serial dilutions of virus were made in cell culture medium and used to inoculate l- or lbc cells. replicate cultures were then fixed and examined by immunofluorescence microscopy as described below. the tcid was determined by the demonstration of viral antigen in % of inoculated cultures. cultures for light microscopy were fixed with methanol and stained with giemsa stain. tissues for immunofluorescence microscopy were fixed with methanol, incubated with antiserum from sdav infected rats, then labelled with fluorescein-labelled goat anti-rat igg (antibodies inc., davis, ca) as previously described [ ] . for electron microscopy, pelleted cells were fixed in . % glutaraldehyde, and stained with % osmium tetroxide. cells were then diluted : in % agar, embedded in epon, processed, stained with uranyl acetate and lead citrate, and examined in a jeol s electron microscope operating at kv. supernatant fluid of infected cultures was negatively stained with % phosphotungstic acid [ ] . two replicates of duplicate cultures were infected with . ml of stock eighth passage virus containing . tcids / . ml and incubated at , , , and °c for hours. cells were fixed, stained and examined, and the titers calculated as previously described. in order to determine the optimal ph for replication of sdav, cells were infected in the presence of culture medium adjusted to ph . , . , . , . , and . with sterile sodium bicarbonate. cells were incubated at °c for hours. using optimal conditions, the time course for production of infectious sdav in l- cells was determined. l- cells infected with . tcids of sdav were harvested at hour intervals for up to hours. virus was collected at each time point and titered as previously described under viral infection. specific pathogen free (spf) male wistar rats approximately four months of age were inoculated intranasally with approximately . ml of eighth or th passage l- cell adapted virus. animals were killed at , , , or days pi by the intraperitoneal administration of pentobarbitone sodium (mtc pharmaceuticals, mississauga, ontario). in the th pass, four rats were used for each time point. blood was collected by cardiac puncture for serology. salivary glands, trachea, and lung were collected from inoculated and control rats for light microscopy. tissues for light microscopy were fixed in % formalin, embedded in paraffin, and stained with hematoxylin and eosin. serum samples from inoculated and d. percy et al. control rats were diluted : , : , and : in phosphate buffered saline (pbs) and evaluated for antibodies to sdav. binding of anti-sdav antibodies to coronavirus-infected cells [ ] was detected using fluorescein-tabelled goat anti-rat globulin. submandibular and parotid salivary glands from animals infected with the eighth pass of sdav were homogenized to make a % (w/v) suspension in pbs, then . ml was inoculated onto l- or l- cells, incubated at °c, and observed for evidence of viral antigen and cpe. repeated attempts to demonstrate sdav in lbc cells after initial infection with either purified # virus or sdav-infected salivary gland material were unsuccessful. following - serial passages of supernatant fluid from infected cell cultures, viral antigen, infectious virus, and cpe could be readily demonstrated. microscopic findings were similar to those previously described [ , ] . in l- cell cultures infected with tissue culture fluid from sdav-infected lbc cells, cpe was evident in the first passage at hours pi. there was extensive cell destruction, with fragmentation and separation of cells from the monolayer and the formation of syncytial giant cells by - hours pi ( fig. a, b) . viral antigen was evident in the cytoplasm of infected single and syncytial giant cells stained for immunofluorescence microscopy ( fig. a) . we also attempted to infect l- cells directly with purified # stock virus and with sdav-infected salivary gland material. in repeated experiments, to serial passages were required before virus could be detected by the indirect fluorescent antibody (ifa) technique at - hours pi. once sdav was adapted to replicate in l- cells, viral antigen could be detected by immunofluorescence microscopy at - hours pi in the cytoplasm of individual cells, and in syncytial giant cells (fig. b) . uninoculated monolayers were consistently negative for viral antigen and cpe throughout the study. viral particles were also demonstrated in sdav-infected l- cells by electron microscopy. in infected cells, reduced electron density, duplication of endoplasmic reticulum, cytoplasmic vacuolation, and fragmentation of cells were observed. typical coronaviral particles to nm in diameter were demonstrated within cytoplasmic vesicles (fig. ) . optimal titers were obtained in cultures at ph . (fig. ) . titers fell to .o tcids at a ph of . and .o tcids at ph . (fig. ) . the drop in titre at a ph greater than . was less marked (fig. ) . temperature also had a marked effect on viral replication. optimal titers were obtained at °c. titers fell by logs at °c and by . logs at °c (fig. ) . titers of up to . tcids were detected at hours pi (fig. ). viral titers rose rapidly within the first hours, reaching a maximum of l ° tcids / . ml at hours pi, with subsequent decline. titers fell rapidly after hours (fig. ) . in our studies, the maximum titers obtained in lbc cells were approximately .o tcids / . ml. characteristic lesions were observed in the salivary and lacrimal glands on gross and microscopic examination (fig. ) . in inoculated rats examined microscopically during the acute stages of the disease at days pi, there was a destructive sdialoadenitis and dacryoadenitis typical of sda [ , ] . typical lesions were also present in the trachea and bronchi in those rats examined at days pi. no lesions were observed in the glands of control rats inoculated with supernatant from uninfected l- cells. antibodies to sdav at dilutions of : - : were readily demonstrated in rats inoculated with sdav-infected l- cells and killed at days pi, but not in control rats. using submandibular gland homogenates prepared from infected animals killed at days pi and inoculated onto l- cells, sda viral antigen and typical syncytial cell formation were detected on the first passage. when virus from the th passage of sdav in l- cells was used to infect spf rats by intranasal inoculation, salivary and lacrimal glands were histologically normal at necropsy. lesions were minimal in the respiratory tract in these animals. by days pi, / of these rats had anti-sdav antibody titers of up to : . non-inoculated control rats and rats killed at days pi were serologically negative for detectable antibodies to sdav. although there have been numerous reports on the characterization of the various strains of the mouse coronaviruses [ , ] , there is little information available on coronaviruses in the rat. until recently, the study and characterization of sdav was hampered by the absence of a continuous cell line capable of supporting the replication of high titers of the virus. the report by hirana et al. of the ability of sdav to grow in lbc cells was an important advancement [ ] . maru and sato [ ] isolated a strain of coronavirus from the submandibular salivary glands of the rat with sialoadenitis, which replicated in mousederived a ( t ) cell culture. however, strain # , acquired from bhatt's original isolate of sdav, failed to replicate in t cells [ ] . although lbc cells do support the replication of sdav, these cells are not widely available and have a doubling time of approximately twice as long as l- cells. in addition, it is not possible to make a direct comparison of sdav grown in lbc cells with other murine coronaviruses cultivated in l- cells. using lbc cell-adapted sdav, we were able to infect l- cells, and virus could be demonstrated in l- cells on the first passage. titers up to .o tcidso/ . ml were obtained at hours pi. bhatt's original strain # sdav was also adapted directly to grow in l- cells. optimal titers were obtained when that virus was propagated at °c for hours in medium at ph . . in the case of both the l- and the lbc cells, or more passages were required before infectious virus and/or viral antigen was detected. serial passage presumably allowed for the selection of virions which could readily replicate in continuous cell cultures. the fact that lbc-adapted sdav could be propagated in l- cells (and vice versa) on the first passage suggested that any alterations in the virion required for the replication in the two cell lines were similar if not identical. adaptation also appears to occur in vivo. for example, sdav has produced encephalitis in newborn rats only after prior intracerebral passage of the virus in suckling mice [- ] . there have been several mechanisms proposed which could explain variations in virulence and adaptation in animal viruses; variation by mutation, hostinduced variation, and genetic recombination are all possible explanations [ ] . the failure to replicate sdav in earle's clone l- cells, while virus replicated in the l- subline, emphasizes the variations that may occur in cells originally derived from the same parent source. in a karyologic study of various sublines of l- cells, rothfels et al. emphasized the marked chromosomal variations observed in these lines [ ] . despite the fact the l- cell-adapted sdav presumably underwent some alteration(s) to allow high levels of expression in cell culture, the passage of the virus grown in vitro retained the ability to produce lesions typical of sda in intranasally-inoculated rats. furthermore, the virus, when recovered from infected animals, could again infect l- cells and viral antigen could be readily demonstrated on the first passage. these data indicate that although the virus was adapted to replicate in l- cells, this strain was still capable of producing lesions typical of sda in susceptible rats. following passages in l- cells, sdav produced in culture failed to produce the typical lesions in salivary and lacrimal glands, although lesions were evident in the respiratory tract. animals infected with th pass l- cell virus did, however, have significant antibody titers against sdav. experiments are currently underway to see if the immune response induced by the high passage virus is protective. although attenuation of virulence following extended passage outside the natural host is a common phenomenon, the precise mechanism is not known. further in vitro and in vivo characterization studies of the high and low passage sdav are currently in progress. epizootiological observations of natural and experimental infection with sialodacryoadenitis in rats characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group a study of the negative staining process production of malignancy in vitro. iv. the mouse fibroblast cultures and changes seen in living cells a subclinical enzootic of sialodacryoadenitis in rats variation in virulence in relation to adaptation to new hosts effects of sialodacryoadenitis virus and rat coronavirus on maternal reproductive parameters and neonatal viability practical methods in electron microscopy. fixation, dehydration and embedding of biological specimens replication of rat coronavirus in rat cell line growth of rat sialodacryoadenitis viruses in lbc cell culture replication of sialodacryoadenitis virus of rat in lbc cell culture characterization of murine hepatitis virus (jhm) rna from rats with experimental encephalomyelitis pathogenesis of sialodacryoadenitis in gnotobiotic rats sialodacryoadenitis in the rat. a light and electron microscopic study prevalence of viral and mycoplasmal infections in laboratory animals in vivo and in vitro models of demyelinating disease: tropism of the jhm strain ofmurine hepatitis virus for cells of glial origin prevalence of natural virus infections in laboratory mice and rats used in canada characterization of a coronavirus isolated from rats with sialoadenitis comparison of strain susceptibility to experimental sialodayryoadenitis in rats central nervous system lesions in suckling mice and rats inoculated intranasally with sialodacryoadenitis virus depletion of salivary gland epidermal growth factor by sialodacryoadenitis infection in the wistar rat replication of sdav in l- cells the origin of altered cell lines from mouse, monkey and man as indicated by chromosome and transplantation studies exacerbation of murine respiratory mycoplasmosis by sialodacryoadenitis virus infection in gnotobiotic f rats an immunofluorescence test for detection of serum antibody to rodent coronaviruses sialodacryoadenitis virus-associated lesions in the lower respiratory tract of rats the authors thank drs. p. n. bhatt received august , key: cord- -np b a o authors: mardani, karim; noormohammadi, amir h.; ignjatovic, jagoda; browning, glenn f. title: naturally occurring recombination between distant strains of infectious bronchitis virus date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: np b a o new variants of infectious bronchitis virus (ibv) have emerged in australia despite its geographical isolation and intensive vaccination programs. in the present study, the ′ terminal . kb of the genome of a recently isolated variant of ibv (n / ) was sequenced and compared with the sequences of classical and novel strains of ibv, the two main groups of these viruses in australia. the comparison revealed that recombination between classical and novel ibvs was responsible for the emergence of the new variant. it was concluded that novel ibvs, which have not been detected since , and which are phylogenically more distant from classical ibvs than turkey coronaviruses, might still be circulating and contributing to the evolution of ibv in australia. infectious bronchitis virus (ibv), the prototype of the family coronaviridae, is an important pathogen in chickens and infects the respiratory tract, kidneys and oviduct, causing reduced performance, reduced egg quality and quantity, increased susceptibility to infection with other pathogens and increased mortality [ ] . the genome of ibv is single-stranded, positive-sense rna of about . kb, encoding four structural proteins, including the spike glycoprotein (s), the membrane glycoprotein (m), the phosphorylated nucleocapsid protein (n) and the small membrane protein (e). new serotypes and genotypes of ibv emerge frequently in different parts of the world [ , , , , , , , ] . a number of factors, including mutation and recombination, and the widespread use of live attenuated vaccines, play an important role in increasing the number of new genetic variants [ , , , , ] . recombination and mutation are two major forces in coronavirus evolution, and polymerase jumping during coinfection may promote recombination events between coronaviruses [ ] . molecular characterization using sequencing and phylogenetic analysis are effective methods for detection and characterization of recombination events among rna viruses [ ] . n / , a nephropathogenic strain of ibv, was the first isolate of ibv obtained in australia, in [ ] . since , a number of distinct strains of ibv have been isolated, including a group of strains that were found to have no epitopes in common with other ibv strains on either their n or m proteins [ ] . in a study on the pathogenicity of australian strains of ibv, a change was detected in the prevalence of ibv strains, from highly nephropathogenic strains in the s and s to strains predominantly pathogenic for the respiratory tract in the s and early s, indicating that ibv strains in australia have undergone a significant change since [ ] . ibv strains in australia have been classified into two groups, classical and novel [ ] , based on their genotypes. the novel ibvs are phylogenetically distant from the classical ibvs, and in fact, classical ibvs and turkey coronaviruses are more similar to each other than they are to the novel ibvs. recently, the isolation of a new variant of ibv in australia, from broiler farms located in new south wales, that had low s gene identity to those of classical and novel ibvs but n gene and untranslated region sequences similar to those of classical ibvs, suggested the emergence of a new subgroup of ibvs in australia [ ] . in this study, the new variant of ibv that circulated in nsw during and was further analysed by comparing the terminal . kb of its genome (all the structural protein genes) with those of the other two ibv groups in australia. the ibv strain n / used in this study has been described previously [ ] . virus propagated in allantoic fluid was used for rna extraction. extraction of rna was performed using an rneasy kit (qiagen, hilden, germany) according to the manufacturer's instructions. approximately ll of allantoic fluid was used for each extraction, and rna was eluted in ll of elution buffer. the extracted rna was used as template in a reverse transcription reaction. for synthesis of cdna, ll of extracted rna was denatured at °c for min, cooled by placing on ice for min, and then mixed with ll of reaction buffer containing ll of diethylpyrocarbonate-treated water, . lm oligo (dt) (promega, madison, wi, usa), . u of rnaguard (amersham pharmacia biotech, sydney, australia), lm each of datp, dttp, dgtp and dctp, ll of reaction buffer (promega), and u of moloney murine leukaemia virus reverse transcriptase (promega). the reaction mixture was incubated at °c for h and subsequently incubated at °c for min to inactivate the reverse transcriptase. the resultant cdna was immediately used or stored at - °c for later use. for the amplification reaction, two primers, poly-f (gattgtgcatggtggacaatg) and utr-r (ctgt accctcgatcgtactc), binding to the end of the polymerase gene (nucleotides , - , , beaudette strain, genbank accession number nc_ ) and the untranslated region (nucleotides , - , , beaudette strain) of the ibv genome, respectively, were used to amplify a . -kb fragment of the ibv genome that contains all of the genes for the structural proteins. the pcr reaction was carried out in -l mixtures containing lm each of datp, dttp, dgtp and dctp, . lm of each primer, ll of high fidelity pcr buffer (invitrogen, carlsbad, ca, usa), mm magnesium sulfate, . u platinum taq high fidelity dna polymerase (invitrogen) and ll of cdna as template. amplification was performed using cycles of incubation at °c for s, °c for s and °c for min, with a final extension at °c for min. the resultant pcr products were separated in a . % agarose gel, and the gel was photographed. pcr product was purified using a pcr purification kit (ultra clean pcr clean-up, mo bio laboratories, solana beach, ca, usa) according to the manufacturer's instructions. the . -kb pcr product from ibv strain n / was cloned in the pgem-t plasmid using a pgem-t vector system cloning kit (promega). the cloned pcr product was sequenced using a big dye terminator v . cycle sequencing kit (applied biosystems), and the reaction products were sent to the australian genomic research facility, walter and eliza hall institute of medical research, melbourne, for analysis on an abi prism genetic analyzer. sequences of different genes of n / were aligned with the same genes from other australian ibvs and the beaudette strain (table ) using clustalw [ ] . evolutionary relationships between australian ibv genes were inferred using the neighbour-joining method [ ] . the evolutionary distances were computed using the maximum composite likelihood method [ ] and measured as the number of base substitutions per site. phylogenetic analyses were conducted in mega [ ] . the -terminal . kb of the genomes of the representative classical and novel australian ibv strains were aligned with the sequences from the same region of the n / isolate, and the multiple alignment results were introduced into simplot version . . to identify likely recombination sites [ ] . bootscanning analysis was performed in simplot to identify and map the putative recombinant ibv genome [ ] . this program was also used to find phylogenetically informative sites, as described previously by robertson et al. [ ] . statistical analysis of the distribution of phylogenetically informative sites was performed using minitab version (minitab inc, us). a two-proportion comparison was performed, and a p value of less than . was considered significant. amplification, sequencing and analysis of the . -kb region of n / the . kb of the n / genome was amplified, cloned and sequenced successfully and compared with the same region of a number of classical and novel strains. phylogenetic trees were constructed for each gene. analysis of the s gene sequences of australian ibv strains showed that, among classical strains, they differed by - . % and, among novel strains, by . - . %. the classical and novel ibvs differed by . - . %, indicating a major difference between the s genes of these two groups of ibvs. interestingly, the s gene sequence of strain n / differed from those of both classical and novel ibvs by . - %. phylogenetic analysis of the s gene grouped n / in a separate cluster from classical and novel ibvs (fig. ) . e gene sequences of classical strains differed by - . %, and those of novel strains by - %. the difference between classical and novel ibvs ranged from . to . %. more interestingly, the e gene sequence of n / was very similar to those of the classical strains (differences of . - . %) and very different from those of novel ibvs (differences of . to . %). in the e gene phylogenetic tree, n / clustered with the classical ibvs (fig. ) . m gene sequences of classical strains differed by - . %, and those of novel ibvs differed by - . %. the m gene sequences of classical and novel ibvs differed by . - . %. the n / m gene clustered with the classical strains (differences of . - . %) (fig. ) . the n gene of classical ibvs differed by . - . %, and those of novel ibvs differed by . - . %. the n gene sequences of classical and novel ibvs differed by . - . %. the n gene of n / was more similar to the n genes of the classical ibvs (differences of . - . %) than of novel ibvs (differences of . - . %) and clustered with the classical strains in phylogenetic analyses (fig. ) . to identify the region likely to have been involved in recombination in n / , similarity plots and bootscan analysis were performed using strains vics and n / as representatives of the two main groups of ibv in australia and the armidale strain as a query. in both the similarity plots and the bootscan graph, n / had greater similarity to n / (a novel ibv strain) in its s gene region (almost kb), while the reminder of the -terminal . -kb region had greater similarity to vics (a classical ibv strain) (fig. , ). data from phylogenetic analyses also suggested that n / had sequence similarity to both main groups of australian ibvs. these findings suggested that there had been a recombination event with a crossover point at the end of the s gene (base number ) of n / (fig. , ) . to obtain a precise picture of this possible crossover point, the distribution of phylogenetically informative sites along the -terminal . -kb sequences of n / and vics were examined. using simplot, informative sites were found that could support one of three possible trees. for almost % of the s gene ( kb), informative sites were found, and % of these informative sites supported a tree clustering n / with the novel ibv strain n / . in contrast, only % of the informative sites supported each of the other two possible trees in this region (p \ . ). for the rest of sequences ( , - , bp), informative sites were found, with % supporting the tree clustering n / with vics and only % supporting the tree clustering n / with n / (p \ . ). this indicated that n / clustered with the classical ibv strain vics throughout the -terminal . kb of its genome and confirmed that a recombination event had occurred at the end of the s gene region (at around position ) and that n / is a recombinant virus that emerged from recombination between these two groups of ibvs in australia. new ibv isolates have been reported in recent years in australia and have been classified as subgroup viruses. the emergence of these new isolates was considered to be unexplained, as was the earlier emergence of subgroup , or novel viruses. n / is one of these new viruses, which have been isolated from broiler farms using one of the four commercial ibv vaccines [ ] . this study was undertaken to explore the mechanism behind the emergence of the subgroup ibvs in australia. based on phylogenetic analysis of the complete . -kb region of the genome, n / was distinct from both classical and novel ibvs. however, the s gene sequence of fig. evolutionary relationships between n genes of australian ibv strains inferred using the neighbour-joining method. the evolutionary distances were computed using the maximum composite likelihood method and are given in the number of base substitutions per site n / was equally different from both the classical and novel ibvs, while the rest of the -terminal . -kb region of the genome clustered closely with those of the classical ibvs and was very different from those of the novel ibvs. the similarity of the n / e, m and n genes to those of classical ibvs suggested that this isolate was closely related to the most commonly used australian ibv vaccine strains. as these strains have been used for a long time in australia, it is possible that they have contributed to the emergence of variant ibvs in the field. the s gene of n / clustered more closely with the novel ibvs, and similarity plotting and bootscan analysis confirmed the close relationship between the s genes of n / and the novel ibvs and suggested that the crossover event occurred near the end of this gene. these analyses provide convincing evidence that recombination has occurred between classical and novel ibvs, leading to emergence of variant ibvs in the field. novel ibvs have not been isolated since , but this finding suggests that novel ibvs are still circulating in the field in australia. the failure to detect them since may be a result of the relatively slow growth of these viruses [ ] . these results confirm the hypothesis of ignjatovic et al. [ ] that recombination may have played a role in the emergence of this new variant of ibv in australia. recombination between ibvs and its role in emergence of new ibv variants has been reported previously [ , , , ] and may occur at multiple sites [ , ] , although crossover events occur more frequently at the end of the s gene [ , , ] , as seen in this study. however, it is particularly notable here because of the very significant phylogenic distance between the novel and classical australian strains of ibv. indeed, the novel strains of ibv are more distinct from the classical strains than is turkey coronavirus, and they lack several of the smaller nonstructural genes found in classical strains. in conclusion, this study showed that the recombination was involved in the emergence of the new variant of ibv in australia and that a rapid and reliable technique is needed to determine whether the novel ibvs are still circulating in poultry farms. it would be appropriate to sequence and analyse the polymerase genes of classical, novel and new variant strains of ibv to obtain further information about the relationships between the different australian ibvs. isolation and characterization of new infectious bronchitis virus variants in hungary comparisons of envelope through b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes recent advances in avian virology severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan isolation of a new serotype of infectious bronchitis-like virus from chickens in england infectious avian nephrosis (uraemia) in australia a recombination event, induced in ovo, between a low passage infectious bronchitis virus field isolate and a highly embryo adaptedvaccine strain variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens a 'new' strain of infectious bronchitis virus infecting domestic fowl in great britain bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt a long-term study of australian infectious bronchitis viruses indicates a major antigenic change in recently isolated strains pathogenicity of australian strains of avian infectious bronchitis virus isolation of a variant infectious bronchitis virus in australia that further illustrates diversity among emerging strains a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus evidence of genetic diversity generated by recombination among avian coronavirus ibv a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in china fulllength human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination infectious bronchitis viruses with a novel genomic organization genotypic and phenotypic characterization of the california (cal ) variant of infectious bronchitis virus coronavirus replication and pathogenesis: implications for the recent outbreak of severe acute respiratory syndrome (sars), and the challenge for vaccine development recombination in aids viruses the neighbor-joining method: a new method for reconstructing phylogenetic trees identification of breakpoints in intergenotypic recombinants of hiv type by bootscanning prospects for inferring very large phylogenies by using the neighbor-joining method mega : molecular evolutionary genetics analysis (mega) software version . evidence of natural recombination within the s gene of infectious bronchitis virus evolutionary aspects of recombination in rna viruses characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens avian infectious bronchitis: characterization of new isolates from italy acknowledgments the senior author would like to thank the department of education, employment and workplace relations of australia for providing him with an endeavour research fellowship award, which enabled this work to be completed. he also thanks urmia university, iran, for granting him leave to go to australia to undertake this work. key: cord- -ttb o lv authors: choi, jeong-won; jung, ji-youl; lee, jae-il; lee, kyoung-ki; oem, jae-ku title: molecular characteristics of a novel strain of canine minute virus associated with hepatitis in a dog date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ttb o lv a -year-old female yorkshire terrier dog died a few days following hernia and ovariohysterectomy surgeries. necropsy performed on the dog revealed that the surgeries were not the cause of death; however, degenerative viral hepatitis, showing intranuclear inclusion bodies in hepatic cells, was observed in histopathologic examination. several diagnostic methods were used to screen for the cause of disease, and minute virus of canines (mvc) was detected in all parenchymal organs, including the liver. other pathogens that may cause degenerative viral hepatitis were not found. infection with mvc was confirmed by in situ hybridization, which revealed the presence of mvc nucleic acid in the liver tissue of the dog. through sequencing and phylogenetic analysis of the nearly complete genome sequence, the strain was found to be distinct from other previously reported mvc strains. these results indicate that this novel mvc strain might be related to degenerative viral hepatitis in dogs. viruses the family parvoviridae are currently categorized into two subfamilies: densovirinae and parvovirinae, members of which infect in-vertebrate and vertebrate hosts, respectively [ , ] . the subfamily parvovirinae is further divided into eight genera: amdoparvovirus, aveparvovirus, bocaparvovirus, copiparvovirus, dependoparvovirus, erythroparvovirus, protoparvovirus, and tetraparvovirus [ ] . the genus bocaparvovirus contains small, non-enveloped, autonomously replicating, single-stranded dna viruses with an icosahedral capsid. bocaparvoviruses are unique among parvoviruses, as they contain a third open reading frame (orf) between the regions encoding the non-structural and structural proteins, and they have a genome length of approximately . kb. the genus was originally named according to its initial two members, bovine parvovirus (bpv) and minute virus of canines (mvc) [ , , , ] . minute virus of canines, also known as canine minute virus or canine parvovirus type , is an autonomous parvovirus of dogs that is genetically and antigenically unrelated to canine parvovirus type , which belongs to a separate genus in the family parvoviridae and is a common agent of canine hemorrhagic gastroenteritis [ ] . mvc was first discovered in germany in in feces samples of clinically healthy dogs [ , ] . initially, mvc was not thought to cause disease; however, several studies, including experimental infections, revealed its role as a pathogen in newborn pups and canine fetuses [ , ] . recently, mvc was reported to be associated with neurological disease in dogs of various ages [ ] and with severe gastroenteritis in an elderly dog [ ] . a seroprevalence study undertaken in showed an . % mvc-positive rate for korean canines [ ] , and an mvc strain was isolated from a korean dog in [ ] . since then, mvc has not been reported in korea for over years, but it has been reported infrequently in other areas of asia, including japan and china [ , ] . the present study describes a previously unreported mvc strain, isolated from a canine in korea, and we have analyzed its genetic relationship to other bocaparvoviruses. a five-year-old female yorkshire terrier dog underwent a hernia operation and ovariohysterectomy on dec , . five days after surgery, the dog began to experience loss of appetite and vomiting. the dog died on jan , , following symptoms of hypothermia, bloody vomiting, and bloody diarrhea. the dog was submitted to the animal and plant quarantine agency for diagnostic examination. a full necropsy was performed; brain, lung, kidney, liver, spleen, lymph node, heart, and intestine were among the tissues collected, which were subsequently fixed in % buffered formalin, dehydrated in graded ethanol solutions, and embedded in paraffin wax. four-micrometer serial sections from the paraffin-embedded organs were stained with hematoxylin and eosin for histological analysis. for the generation of an mvc-specific in situ probe, pcr labeling with a pcr dig probe synthesis kit (roche diagnostics gmbh, mannheim, germany) was performed according to the manufacturer's protocol. briefly, a specific region (nucleotides - ) of the ns gene of the d strain was amplified by pcr, using ns forward ( ' aacgcgatttgcaccttcat ') and ns reverse ( ' catcaaacatttctccggca ') primers. the pcr product was labeled with digoxigenin (dig) and subsequently used as a hybridization probe. using this probe, the tissues of parenchymal organs taken from the infected dogs were investigated for the presence of mvc nucleic acid. to detect mvc dna, additional paraffin-embedded tissues were sectioned at -lm thickness and hybridized in situ using a fully automated system (nexes ihc instrument; ventana medical systems, inc., tucson, az, usa) and a dab detection system (ventana medical systems inc.). the tissue sections were deparaffinized (standard xylene and industrial methanol) and fixed in % paraformaldehyde for min. tissues were then permeabilized by incubation in . m hcl containing mg of pepsin per ml for min at °c, followed by denaturation at °c for min before hybridization with the dig-labeled riboprobes at °c for h. after hybridization, samples were washed twice . ssc at °c for min, followed by pbs at room temperature for min. after hybridization, slides were incubated with qds conjugated anti-dig antibody (invitrogen, carlsbad, ca) for min and then rinsed twice in pbs for min. all tissue sections were mounted with coverslips using % (v/v) glycerol/pbs mounting solution. the dab detection system was then used to detect antibody binding. finally, sections were incubated at °c and then counterstained with hematoxylin and post-counterstained with bluing reagent. polymerase chain reaction (pcr) and reverse transcription (rt)-pcr was performed for genetic diagnosis. extraction of viral rna and dna from organ tissue was performed using an inclone mini extraction kit (inclone biotech co., yongin, gyeonggi, , korea), according to the manufacturer's instructions. canine parvovirus types and (cpv- , ), canine distemper virus (cdv), and canine influenza virus (civ), were examined using a virus detection kit (intron biotechnology, inc., sungnam, gyeonggi, , korea). canine herpes virus (chv) and canine adenovirus types and (cadv- and, - ) were amplified by pcr, using a hotstart pcr premix (imod, hanam, gyeonggi, , korea). canine corona virus (ccv) and canine parainfluenza virus (cpiv) were screened using an rt-pcr one-step kit (imod). pcr products ( ll) were subjected to electrophoresis in . % agarose gels at v for min to visualize the amplified dna. a molecular weight marker was run with the samples to determine the length of the amplified product. bands were visualized under ultraviolet illumination after ethidium bromide staining and photographed using an ultraviolet gel imaging system. for genome sequencing and phylogenetic analysis, sequence-specific oligonucleotides were designed based on the conserved regions of several published mvcs [ ] [ ] [ ] . the amplified pcr products were purified using an agarose gel dna extraction kit (intron biotechnology, korea) and cloned into the pgem-t vector (promega, madison, wi, usa). the cloned plasmids were purified and sequenced using t and sp primers, a bigdye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa), and an automated dna sequencer (abi xl genetic analyzer, applied biosystems). all nucleotide sequences were confirmed by three or more independent, bi-directional sequencing runs. nucleotide and putative amino acid sequence alignments were generated using the bioedit sequence alignment editor (ibis biosciences, carlsbad, ca, usa). the nearly complete sequence of the mvc genome was deposited in the gen-bank database, under accession number kt . this sequence was compared to those of previously reported mvcs and bocaparvoviruses from other species at both the nucleotide and amino acid level, using orthologous sequences available in genbank. phylogenetic analysis was conducted using bioedit and molecular evolutionary genetics analysis (mega) . software, with bootstrap values calculated from , replicates. the neighborjoining phylogenetic algorithm was used to construct phylogenetic trees. from post-mortem examination, the immediate and surrounding areas of the surgical incisions showed no indication of surgical complications. the gastrointestinal tract was sparsely filled with fluid contents, and liver and spleen damage was evident. histopathologic examination revealed massive hepatic necrosis and basophilic intranuclear inclusion bodies in degenerated hepatic cells (fig. ). there were no other macroscopic or significant histological findings in the other organs. on analyzing the in situ hybridization images, hepatic cells surrounding the damaged regions and intranuclear inclusion bodies were found positive for mvc nucleic acid (fig. ) . in situ hybridization in other tissues also revealed cells that were positive for mvc nucleic acid; however, with the exclusion of the liver, there was no evidence of lesions in any other organs. therefore, it was difficult to establish a correlation between the results of in situ hybridization for these organs and the presence of disease in the present case. by using pcr and rt-pcr, cpv- , cadv- , , cdv, civ, chv, ccv, and cpiv were not detected; however, positive pcr products of the expected size of bp, corresponding to mvc, were detected in samples of the brain, lung, kidney, liver, spleen, lymph node, heart, and intestinal tissue. the isolated mvc strain, designated d , was sequenced and aligned with the other mvc strains obtained from genbank, using bioedit. the mvc d strain has a , -nucleotide-long genome. the non-coding region of the d strain on the left-hand side (lhs) terminus, located at the -end of positive-sense ssdna genomes, was nucleotides in length. the right-hand side (rhs) non-coding region of the d strain, found at the end of positive-sense ssdna genomes, was nucleotides in length. however, the very end of the and termini of this genome could not be definitively sequenced, presumably because of its peculiar hairpin structures, which are also present in other parvovirus genomes [ ] . orf of the d strain encodes a -aa non-structural (ns) protein, which is - aa longer than the ns protein of other mvc strains. orf and orf of the d strain encode a -aa protein that overlaps (table ) , which is also lower than the mean nucleotide and amino acid sequence identity values for the vp /vp region of other mvc strains (approximately . % and . %, respectively). phylogenetic analysis based on these ns and vp /vp regions also showed that the d strain formed a distinct branch that was separate from the other previous mvc strains. this indicates that this strain is distinct from these previously isolated strains (fig. ) . however, the np region of the d strain showed greater nucleotide and amino acid sequence similarity to that of the hm- strain (ab ), which was isolated from a korean dog in ( . % and . %, respectively), compared to those of the other mvc strains (mean similarities of . - . % and . %, respectively) ( table ). phylogenetic analysis based on the np region of the mvc strains also showed that the d and hm- strains fell into the same cluster, which is distinct from other mvc strains (fig. ) . the high level of nucleotide and amino acid sequence similarity in the np region between these two korean strains implies that the d strain could be a mutated strain of hm- or an endemic strain from korea that has not been isolated previously. in addition, the coding region of d shared . - . % nucleotide sequence identity to the other mvc strains (table ) , and this value is lower than the mean nucleotide sequence identity of the coding region of the other mvc strains (approximately . %). phylogenetic analysis based on the complete sequence of the d strain indicated that this strain could be correctly classified as an mvc strain, although it forms a distinct branch on phylogenetic trees when analyzed with respect to the ns and vp /vp regions. interestingly, the mvc strains, including the d strain, are more closely related to feline bocaviruses than the novel canine bocaviruses (fig. ) . this observation supports the possibility of a common origin and crossspecies transmission between mvc and feline bocavirus. in conclusion, our results indicate that the mvc d strain is a possible cause of degenerative viral hepatitis in dogs. although liver degeneration in canines has also been described at post-mortem examination of a pup infected with mvc [ ] , and a novel bocavirus (kc ) that was highly divergent from known mvc strains has been isolated from canine liver [ ] , hepatitis with evidence of intranuclear inclusion bodies that correspond to mvc infection has not yet been reported. therefore, the present study suggests two possible mechanisms for the observation of hepatic intranuclear inclusion bodies following infection with the novel mvc d strain. first, there is the possibility of pathogenic variation between the d and other mvc strains, based on differences in nucleotide and amino acid sequences. the d strain showed comparatively low nucleotide and amino acid sequence similarity to other mvc strains, except in the np region of the hm- strain, and the region related to pathogenicity might be altered, resulting in an acquired ability to cause hepatitis. the second possibility is that an immunocompromised state of the host animal might affect virulence. generally, the pathogenic potential of mvc for adult dogs has been considered minimal [ , , , ] . however, the host animal in the present study had surgery a few days before presenting with symptoms, and this procedure may have weakened the host immune system, which can be influenced by factors such as stress, anesthetic effects, or the effects of other drugs. this weakened immunity might explain the occurrence of hepatitis; in fact, simultaneous human bocavirus infection and hepatitis has been reported in a boy with severe t-cell immunodeficiency [ ] . further studies, such as isolation of the d strain and a subsequent animal inoculation tests, are required to clarify the exact relationship between this strain and hepatitis and to determine the distribution, diversity, and pathogenesis of mvc in dogs. recovery and characterization of a minute virus of canines novel canine bocavirus strain associated with severe enteritis in a dog litter the expanding range of parvoviruses which infect humans 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 molecular characterization of canine minute virus associated with neonatal mortality in a litter of jack russell terrier dogs minute virus as a possible cause of neurological problems in dogs virus taxonomy: eighth report of the international committee on taxonomy of viruses seroprevalence of canine calicivirus and canine minute virus in the republic of korea hepatitis and human bocavirus primary infection in a child with t-cell deficiency a novel bocavirus in canine liver animal bocaviruses: a brief review virologic and serologic identification of minute virus of canines (canine parvovirus type ) from dogs in japan sequence analysis of an asian isolate of minute virus of canines (canine parvovirus type ) a minute virus of canines (mvc: canine bocavirus) isolated from an elderly dog with severe gastroenteritis, and phylogenetic analysis of mvc strains the canine minute virus (minute virus of canines) is a distinct parvovirus that is most similar to bovine parvovirus sequence analysis of an isolate of minute virus of canines in china reveals the closed association with bocavirus experimental infection of calves with hemadsorbing enteric (haden) virus parvoviruses associated with diarrhea in calves acknowledgments this research was supported by a grant from the animal and plant quarantine agency, anyang, gyeonggi, republic of korea. conflict of interest the authors declare that they have no conflict of interest.ethical approval this article does not contain any animal studies that were performed by any of the authors. key: cord- -ffex dw authors: escutenaire, s.; isaksson, m.; renström, l. h. m.; klingeborn, b.; buonavoglia, c.; berg, m.; belák, s.; thorén, p. title: characterization of divergent and atypical canine coronaviruses from sweden date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ffex dw field canine coronaviruses (ccvs) identified during a series of outbreaks of gastroenteritis in swedish dogs were subjected to genetic analysis involving the open reading frame b (orf b) and the membrane (m) and spike (s) protein genes. four field viruses originating from the stockholm region presented identical sequences and segregated separately from other ccvs characterized so far and from got/ , the variant recovered in western sweden. a recombinant origin of the fifth virus identified in the stockholm region is suggested. in addition, the five viruses originating from the same geographical area displayed atypical ′ s gene sequences. rna of - kb in length. it contains in its region two overlapping reading frames (orfs), orf a and orf b, encoding polyproteins leading to the replicase complex. downstream orfs code for the structural proteins, i.e. spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins, respectively. canine coronaviruses (ccvs) responsible for gastroenteritis and feline coronaviruses (fcovs) belong to the group coronaviruses. fcovs are subdivided into types i and ii, differing by immunological properties and by orf b and s gene sequences [ , ] . both types include feline enteric coronaviruses, causative agents of unapparent to mild transient enteritis, and feline infectious peritonitis viruses, which are associated with fatal systemic disease [ ] . fcov type ii was suggested to originate from homologous recombination between ccv and fcov type i [ ] . ccvs have also been differentiated into types i and ii, based on genetic similarity with the respective fcov types [ , ] . ccv type ii includes most canine reference strains, and ccv type i constitutes a new genotype represented by italian viruses [ , , ] . both ccv types i and ii have been associated with mild to moderate gastroenteritis in dogs with more severe clinical signs in young animals [ ] . in this study, we characterized field ccvs originating from a series of outbreaks of gastroenteritis in swedish dogs. sequences from three different genes were determined, including the highly variable regions of the m and s genes. most gastroenteritis cases in dogs occurred in december and january , in different regions of sweden including the stockholm area. the animals presented vomiting, inappetence and recurrent diarrhea for several weeks. abundant haemorrhagic diarrhea was also recorded, and more severe clinical signs with occasional fatal outcome occurred in pups. the present analysis was based on six faecal samples that first tested positive for group coronaviruses by real-time reverse transcriptionpolymerase chain reaction (rt-pcr) [ ] . of the six dogs, four originated from the stockholm area: Å kersberga= (akbg= ), s € o odert € a alje= (sdt= ), uppsala = (upps = ) and uppsala = (upps = ). only two of these animals (upps = and upps = ) were epidemiologically related: they had the same owner, a veterinarian whose clinic recorded during the same period - cases of canine gastroenteritis. the fifth dog [g € o oteborg= (got= )] was from gothenburg, a town located in western sweden. all five animals were adults. the sixth sample was collected from a -day-old pup (mora= ) originating from central sweden and which died after severe gastroenteritis. the pup was born to a bitch which also presented diarrhea and vomiting. the bitch had supposedly been infected a few days before in a kennel housing dogs that had participated in the stockholm international dog show. several gastroenteritis cases had been recorded during the exhibition. viruses identified in this study will be given the same reference name as the dog from which they were recovered. total rna was extracted from faecal homogenates by means of the qiaamp viral rna kit (qiagen, hilden, germany) according to the manufacturer's instructions. consensus degenerate primers targeting a region of the orf b and primers specific for ccv and fcov m and s gene se-quences were selected. the reverse transcription and amplification were carried out as described in ref. [ ] . sequences of the primers and cycling conditions are available upon request. dna samples generated from two different rt-pcr runs were sequenced in both directions using the pcr primers. longer pcr products amplified from the s gene were cloned with the pgem-t cloning kit (promega) and sequenced automatically with primers m forward and m reverse. editing and translation of sequences were done with the bioedit package (version . . ). the clustal x program (version . ) was used for sequence alignment. percentages of nucleotide and amino acid identity were determined with the megalign program (version . ) (dnastar inc.). maximum-likelihood trees were constructed from sequence alignments by means of the program package tree-puzzle (version . ) and visualized with the program treeview (version . . ). nucleotide positions refer to fipv - (accession number: dq ) for the orf b sequences and to ccv insavc- (accession number: d ) for the m and s genes. sequences generated in this study were deposited in the genbank database. the designations and genbank accession numbers are as follows: based on the genomic regions studied, akbg= , sdt= , upps = and mora= viruses displayed identical nucleotide sequences and were named swe viruses. the six swedish viruses shared higher identity with ccvs type ii based on alignments of nucleotide and deduced amino acid s. escutenaire et al. sequences of the m gene ( table ). the swe viruses displayed . and . % nucleotide identity with upps = and got= , respectively (table ) . inferred amino acid sequences were mostly divergent within the amino terminus of the m protein where the swedish viruses presented one unique amino acid site (v instead of l or i; fig. ). in addition, the swe viruses displayed one common unique amino acid deletion in the amino terminus ectodomain. these viruses differed from got= and upps = by and amino acids, respectively, and by amino acid deletions. six of the amino acid mutations were non-conservative. in the alignment based on s gene sequences, the swedish field viruses displayed higher ranges of nucleotide and amino acid identity with both ccvs and fcovs of type ii (table ). in contrast with data from the m gene, the s regions of swe viruses and upps = were more closely related. in addition, swe viruses and upps = presented one common unique amino acid site (l instead of v) when compared with all ccvs type ii and differed from got= by amino acids. all five substitutions were conservative. amplification of the highly variable region of the s gene failed in all but one sample (got= ) using ccv type ii-specific primers [ ] . by means of newly designed and broadly reactive primers, a successful amplification was obtained from all six swedish field samples. whereas a pcr product of the expected size ( bp; nucleotide positions: - ) was generated from got= , the frag- in the phylogenetic tree based on partial orf b sequences (nucleotide positions: - ), the swedish viruses constituted with the ccvs a lineage well separated from fcovs, transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and human coronaviruses within the genogroup (data not shown). the viruses from the stockholm region also clustered separately from got= . in the tree based on m gene sequences, the viruses from sweden segregated into two distinct subgroups among the ccvs type ii (fig. a) . got= and upps = branched together with two viruses originating from italy, separately from the swe variants. in the tree based on the region of the s gene, the swedish viruses displayed a different clustering from the m gene tree (fig. b) . whereas got= grouped together with the main ccv reference strains, upps = and the swe viruses constituted a sublineage distinct from those represented by the ccvs and fcovs of type ii. the contradictory clustering of upps = supports the above data on genetic diversity and sequence analysis and suggests that the virus was derived from a recombination between swe and got= -like viruses with a crossover site located between the s and m genes. in the tree based on the s gene, got= branched together with ccv ucd- , separately from the other ccvs type ii (data not shown). given the scarcity of genomic sequences available from ccvs type i, no phylogenetic tree was constructed from the s gene sequences generated from swe viruses and upps = . the genetic relatedness among the field ccvs mirrored their geographical origin: the four swe variants originating from the stockholm region presented identical sequences and grouped separately from got= , recovered in the western part of the country. the clustering pattern of the fifth virus from the stockholm region, upps = , differed according to the genomic region examined, suggesting a recombinant origin of its genome. deduced from phylogenetic data, this hypothesis is supported by amino acid markers both in the s and m proteins. data from the region of the s gene suggested that the swe viruses and upps = constituted a distinct sublineage, well separated from the ccvs type ii identified so far. in a previous analysis of the highly variable s gene, the australian isolate uwsmn- was found as an outlier among the ccvs, with . - . % nucleotide divergence from ccv type ii reference strains [ ] . comparable or even higher percentages of nucleotide variation were observed between got= and the same reference strains, thus suggesting that got= also constitutes a genetically distinct variant among ccvs. in addition, s gene sequences related to ccv type i were identified among the swe viruses and upps = . a dual infection of the dogs with both ccvs types i and ii may not be excluded. however, as there was no evidence of s gene sequence of ccv type ii or m gene sequence of ccv type i in their stools, the dogs were more likely infected with a single viral type presenting an atypical s gene. taken together, these data suggest that the stockholm viruses arose from recombination between ccv type i-and ccv type ii-like or possibly a third type of ccv. results generated lately also strengthen the hypothesis of a recombinant origin of upp = by confirming its relatedness to the swe viruses for the s gene. although the tgev-like s segment of the ccv isolate ucd- has been postulated to originate from recombination [ ] , no such event was previously reported among field ccvs. our data suggest that for the swe viruses and upps = , a template switch would have taken place within the s gene and that for upps = another crossover site would be located between the s and the m genes. the identification of hotspots for recombination within the s gene of mhv or ibv corroborates our assumptions [ , ] . in addition, a recombination event also arose between the s and m genes of fcovs type ii [ ] . sequence analysis from upps = and upps = , recovered from dogs living in the same house, further suggests that viral populations within a same environment may be complex and represented by several distinct variants. the infection of an animal with genetically different viruses allows the occurrence of recombination events. it is unknown whether the recombination associated with upps = took place among the dogs from uppsala or whether the recombinant virus originated from another location. data from the m gene confirm previous observations of accumulation of non-synonymous substitutions within the amino terminus ectodomain of the protein, which is exposed on the outside of the virion [ , ] . as the amino terminus of the m protein is highly immunogenic, these features could result in different antigenic properties among ccvs recovered in sweden, including the ability of the viruses to escape the host immune response. there was no apparent relationship between the severity of the clinical signs in the dogs and the differences observed among the ccvs identified in this study. the death of the pup infected with mora= was most probably related to the age of this animal as a higher susceptibility to develop severe clinical signs has been reported in young dogs infected with ccv [ ] . [ ] divergent and atypical canine coronaviruses from sweden a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus sybr green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses one-tube fluorogenic reverse transcriptionpolymerase chain reaction for the quantitation of feline coronaviruses feline coronavirus type ii strains - and - 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 identification of canine coronavirus strains from feces by s gene nested pcr and molecular characterization of a new australian isolate genetic evolution of canine coronavirus and recent advances in prophylaxis two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea cloning and expression of two fragments of the s gene of canine coronavirus type i genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype molecular characterization of a virulent canine coronavirus bgf strain a comparison of the genomes of fecvs and fipvs and what they tell us about the relationships between feline coronaviruses and their evolution evidence of natural recombination within the s gene of infectious bronchitis virus 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 we thank lihong liu for assisting sequencing and useful advice. the work was supported and financed by project . = - of the swedish research council for environment, agricultural sciences and spatial planning (formas). key: cord- - m q c authors: wo, ying; lu, qing-bin; huang, dou-dou; li, xiao-kun; guo, chen-tao; wang, hong-yu; zhang, xiao-ai; liu, wei; cao, wu-chun title: epidemical features of hadv- and hadv- in pediatric pneumonia in chongqing, china date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: m q c human adenoviruses (hadv) of species b, c, and e (hadv-b, -c, -e) are frequent causative agents of acute respiratory infections worldwide. no specific analysis has been done on the epidemiological and clinical features of hadv in pediatric pneumonia in china. nasopharyngeal aspirates were collected from hospitalized children with pneumonia from june to may . all samples that tested positive for hadv were typed by sequencing the hexon and fiber genes. from a total of samples, ( . %) were positive for hadv, identified as belonging to hadv-b ( , . %), hadv-c ( , . %) and hadv-e ( , . %). hadv- ( , . %) and hadv- ( , . %) were the two major types, followed by hadv- , hadv- and hadv- . there were ( . %) single hadv infections, of which % were hadv- infections. multivariate analysis showed that single infections with hadv- were associated with a higher prevalence of severe pneumonia. temporal patterns showed that, except for a simultaneous outbreak of hadv- and hadv- during the years – , hadv- and hadv- were alternately predominant, and the dominance shifted to hadv- after . identification of the predominant hadv genotypes and their epidemical features is useful for determining preventive strategies. hadv- associated severe pneumonia needs to be considered with high priority in clinical practice. human adenoviruses (hadvs) are a significant cause of acute respiratory disease (ard). they are associated with sporadic infection, as well as with community and institutional outbreaks, particularly among military recruits [ ] . there are at least recognized hadv genotypes (http:// hadvwg.gmu.edu/), which are assigned to seven subgroups (a-g) according to their biophysical, biochemical, and genetic characteristics, with marked differences in tissue tropism and clinical manifestations. depending on the infecting hadv types, a broad spectrum of clinical illnesses can be observed [ ] . the types most commonly associated with respiratory syndromes belong to hadv species c (hadv-c , -c , -c , and -c ), hadv species b, subspecies b (hadv- and hadv- ) and b (hadv- ) [ ] . these viruses rarely cause serious or fatal illness in otherwise healthy individuals, but they can cause severe disease in newborn, elderly, or immunocompromised persons [ , ] . a well-known feature of hadv is frequent recombination between members of the same species and between members of different species, which has acted as an important force driving the evolution of hadv genetics [ ] . for the emerging new types or recombinant strains, there is strong potential for wide spread and even epidemic outbreaks, partially due to the lack of herd immunity. there is therefore a need for continuous surveillance, with extensive molecular characterization, to determine the prevalence and genetic characteristics of the circulating hadvs. the current study was performed to identify the predominant hadv types associated with pediatric pneumonia and to trace the new genetic variants that might be derived from recombination of different types. clinical features were also revealed with regard to hadv genotype and genetic characteristics. the current study was performed as part of an ongoing, integrated surveillance for acute respiratory tract infections (arti) carried out in the respiratory department of children's hospital of chongqing medical university (chcmu). chcmu is the largest children's hospital in southeastern china, serving the population of sichuan province and two neighboring provinces, with beds in the respiratory department. the surveillance project was begun in june , and a total of ari patients were recruited by the end of may . from the cohort, patients with pneumonia were selected to analyze the epidemiological and clinical features of hadv infection. pneumonia was defined by the presence of patchy alveolar opacities in chest radiographs, in addition to symptoms of cough, dyspnea (lower chest wall indrawing), or tachypnea (in infants, [ - breaths/min; in older children, [ breaths/min). severe pneumonia was defined as pneumonia plus hypoxemia (maintained sao \ % in air) or rising respiratory and pulse rates with clinical evidence of respiratory distress and exhaustion with or without raised paco . underlying diseases included asthma, eczema and tuberculosis. nasopharyngeal aspirates (npa) were collected from the patients upon hospitalization and stored in a - °c freezer until they were tested. dna and rna were extracted from each specimen using a qiaamp Ò minelute virus spin kit (qiagen, hilden, germany). molecular assays for hadv detection were performed using pan primers as described previously [ ] .the presence of other viral pathogens, including influenza viruses a and b, respiratory syncytial virus (rsv) subtypes a and b, parainfluenza virus (piv) types , , , and , metapneumovirus (mpv), human rhinovirus (hrv), coronavirus (cov), and human bocavirus (hbov), were tested by rt-pcr or pcr as described previously [ , ] . each pcr run included viral dna or rna as a positive control and water as a negative control. for samples that were positive by generic hadv pcr, the entire fiber gene and highly variable regions of hexon gene were amplified and then sequenced as described previously [ , ] . descriptive statistics were performed for all variables; the continuous variables were summarized as means and standard deviations (sd) or as medians and ranges, and the categorical variables were summarized as frequencies and proportions. to determine the difference between groups, an independent t-test, the v test, fisher's exact test, or a nonparametric test was used where appropriate. the logistic regression model was used to examine the potential risk factors for outcomes such as severe pneumonia. odds ratios (ors) and their % confidence intervals (cis) were estimated using maximum-likelihood methods. a twosided p-value of \. was considered to be statistically significant. all analyses were performed using sas software, version . . (sas institute inc., cary, nc, usa). over a period of six years, a total of npas ( from , from , from , from , from and from ) were collected from children aged month to years (median, months) diagnosed as having pneumonia. of these, . % ( ) were boys. overall, hadv was detected in . % ( / ) of the samples, which were identified as hadv-b ( , . %), hadv-c ( , . %), hadv-e ( , . %) and untyped hadv ( , . %, not sequenced due to inadequate sample volume). the typed hadvs were further classified into six genotypes ( hadv- , hadv- , hadv- , hadv- , hadv- , and hadv- ). the hadv-positive patients were significantly older than the hadv-negative patients ( vs. months, p \ . ) ( table ) . when compared regarding age, the infection rate increased significantly with older age (cochran-armitage trend, - . , p \ . ). the highest positive rate ( . %) was found in patients of - years old. the gender distribution was similar between the hadv-positive group and the negative group as a whole (p = . ) ( table ) . the most commonly seen clinical manifestations of hadv-positive patients were fever ( . %), cough ( . %), moist rale ( . %) and expectoration ( . %), which did not deviate from those of hadv-negative patients. however, hadv infection was associated with more-severe pneumonia and more frequent transfer to the intensive care unit in comparison with hadv-negative patients (p \ . and p = . , respectively) ( table ) . the hadv- and hadv- were the most prevalent types, accounting for . % and . %, respectively of the total hadv positive samples. the comparison between the two hadv types revealed that hadv- infection caused more development of expectoration, dyspnea and diarrhea than hadv- infection (all p \ . ). among the hadv- -infected patients, . % ( ) were diagnosed as having severe pneumonia, significantly higher than the . % ( / ) among the hadv- -infected patients (p \ . ) ( table ). hadv- was similarly common among patients with non-severe and severe pneumonia ( . % vs. . %; p = . ), whereas hadv- was the dominant type among patients with severe versus non-severe pneumonia ( . % vs. . %; p \ . ). the children with single hadv infections were significantly older than those with coinfection ( months vs. months, p \ . ). there was also a higher rate of coinfection ( . %) in children of - months, mostly with hadv and rsv ( / , . %).the gender distribution was comparable between the single-infection and coinfection groups (p = . ) ( table ). the hadv- had a significantly higher proportion of single infections ( . %) than hadv- ( . %, p = . ). severe pneumonia occurred in over % ( / ) of hadv single infections, among which % ( / ) were hadv- infections. coinfection with hadv and other respiratory virus did not result in a difference in clinical manifestation or severe clinical outcomes. the differences for neutrophils, lymphocyte, hemoglobin and platelet count between hadvpositive and negative patients attained a significant level, which was not observed between single hadv infections and coinfections (table ) . severe pneumonia was considered an important dependent variable and analyzed for its risk factors by applying multivariate logistic analysis for hadv infection in general and for hadv- /hadv- specifically. in the hadv-positive vs. negative model, after adjusting for the effects of age, gender, delay from onset to admission, and coinfection with other viruses, hadv infection was demonstrated to significantly increase the risk of severe pneumonia (or, . ; % ci, . - . ). in the hadv- vs. hadv- model, the or of hadv- for severe pneumonia was . ( % ci, . - . ) compared to hadv- after considering the other effects (table ) . temporally, hadv was active all year round but displayed characteristic biennial incidence peaks with alternating low peaks appearing in the winter and summer, respectively (fig. ) . a simultaneous outbreak of hadv- and hadv- occurred during the years - , with the proportion of hadv- increasing significantly to . % during the winter peak and to . % during the summer peak in (p \ . ). in comparison, the incidence peaks of hadv- in winter and summer were less prominent. the proportion of hadv- remained at levels comparable to those of the other years. after a shift to hadv- since , another round of hadv- -dominant circulation emerged after (fig. ). pediatric pneumonia caused by viral infection has always been of great public-health concern worldwide because of its high morbidity and mortality. a wide variety of etiological agents has been documented, among which hadv is thought to be a significant contributor. in china, there have been recent studies investigating the prevalence of hadv subspecies b in respiratory distress, but mostly involving adults. a multicenter surveillance for community-acquired pneumonia that was conducted in northern china between and demonstrated hadv- to be the predominant genotype, followed by hadv- , hadv- , hadv- and hadv- . this study, however, provided no data on children [ ] . in taiwan, china, a large community outbreak of hadv in was shown to be predominantly associated with hadv- , although hadv- began to show an increased circulation [ , ] . similarly, in some studies and outbreaks in which respiratory adenoviruses were identified and typed, in guangzhou during to , in hangzhou during [ ] and in shaanxi [ ] , the most common hadv type was shown to be hadv- [ ] . the prevalence of hadv and the circulating genotypes in pediatric respiratory infection have been investigated in two recent studies. in lanzhou, from to , hadv infections represented . % of arti of viral etiology in children, and among all hadv genotypes, hadv- was most frequently detected, followed by hadv- and other types [ ] . our previous study performed in chongqing during - identified the emergence of hadv- in pediatric patients with arti, but there was no comprehensive analysis of all hadv genotypes in the local region [ ] . in most of the abovementioned studies, no specific analysis was done of the epidemic and clinical features of hadv in cases of pediatric pneumonia. hadv coinfection with another respiratory virus was rarely determined, and differentiation of hadv-associated clinical manifestations from other viral respiratory viral infection was not mentioned. here, in a prolonged surveillance study, we have identified hadv- and hadv- as the two major genotypes causing pediatric pneumonia in southeastern china. these two genotypes alternate as the predominant cause of pediatric pneumonia. although the seasonality of hadv-related pneumonia is less distinct, the notable increase in circulation of hadv- between and might represent an undetected outbreak in local children, which, however, might have been undiagnosed or misdiagnosed. a recent report of an hadv- -associated outbreak in a military camp in shaanxi, china [ ] , could likewise have been caused by the predominant circulation of hadv- in the local area, as demonstrated in the current study. with the advent of hadv- dominance since in chongqing, we propose a high potential of outbreak events caused by hadv- in the local area. in the united states, the hadv- prototype strain accounted for two-thirds of hadv- isolates from to [ ] . changes in serotypes and genome types among geographic regions underscore the potential for new strains to evolve and replace existing strains. in the united states and southern ontario from to , hadv- accounted for . % of hadv respiratory tract infection in civilians and . % among military trainees, while hadv- accounted for only / ( . %) of clinical hadv respiratory isolates in military facilities and / ( . %) of isolates in civilian settings [ ] and . % in zagreb county [ ] . by contrast, hadv- has been a prominent cause of fri in asia. especially in the current study, we observed a high proportion of hadv- and hadv- in pediatric pneumonia, which was similar to that in beijing, china [ ] . the predominant serotypes differ among different countries or regions, and they change over time [ ] . this also might reflect more-severe clinical disease in the current patients than in those of previous studies [ , , ] , where outpatients with ard instead of pneumonia were recruited. we also found the clinical syndromes and symptoms of both hadv single infection and hadv coinfection to be largely nonspecific and less differentiated from those of hadv-negative patients. as a result, ascertaining hadv infection based on the clinical manifestations is challenging. however, we found that pneumonia caused by hadv infection was more severe than that caused by other viral infections, with over half of the cases of hadv-associated pneumonia causes by hadv- . coinfection with other respiratory viruses, on the other hand, did not lead to moresevere disease outcome. these findings have the clinical implication that in patients with severe pneumonia, the contribution of hadv, especially hadv- , should be considered with high priority, regardless of whether another respiratory pathogen has been detected. because we did not conduct a seroconversion study, the exact role of hadvs by themselves in clinical manifestations and pathogenicity remains to be studied in the future. in conclusion, the identification of the predominant hadv genotypes and their temporal pattern could help to achieve a better prediction of seasonal activity, guiding timely preventive strategies if a vaccine is available. the clinical pictures and clinical outcomes of hadv infection alone and in combination with other pathogens might help to inform clinical practice. human adenoviruses in respiratory infections: sequencing of the hexon hypervariable region reveals high sequence variability emergence of community-acquired adenovirus type as a cause of community-onset pneumonia identification and typing of adenovirus from acute respiratory infections in pediatric patients in beijing from molecular epidemiology of adenovirus type in the united states genotype prevalence and risk factors for severe clinical adenovirus infection real-time rt-pcr detection of respiratory viral infections in four triplex reactions the virus watch program: a continuing surveillance of viral infections in metropolitan new york families. ix. a comparison of infections with several respiratory pathogens in new york and new orleans families identification and typing of respiratory adenoviruses in guangzhou, southern china using a rapid and simple method prevalence of adenovirus in children with acute respiratory tract infection in lanzhou adenovirus serotype 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discovery/investigation group, huang lm ( ) community outbreak of adenovirus two adenovirus serotype outbreaks associated with febrile respiratory disease and pharyngoconjunctival fever in children under years of age in hangzhou, china, during species-specific identification of human adenoviruses by a multiplex pcr assay outbreak of acute respiratory disease caused by human adenovirus type in a military training camp in shaanxi conflict of interest regarding this report, the authors do not have any commercial or other associations that would be considered a conflict of interest. key: cord- -ruwxm fd authors: chen, s.-p.; yu, m.; jiang, t.; deng, y.-q.; qin, c.-f.; han, j.-f.; qin, e.-d. title: identification of a recombinant dengue virus type with recombination regions in natural populations in guangdong province, china date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ruwxm fd using recombination analysis, we identified a recombinant dengue virus type strain, namely, gd / , with three recombination regions, located within the sequences of the prm/e junction, ns , and ns , respectively. the recombinant dengue virus was further confirmed by phylogenetic analysis based on its recombination and non-recombination regions. this appears to be the first study to confirm the existence of three recombination regions in a single dengue virus isolate and to report recombination between parent virus strains isolated from the same geographic area (guangdong province, china). it is also the first to report breakpoints within the ns gene of dengue viruses. seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) [ ] . recombination events have been proven to occur in rna viruses such as polioviruses [ ] , feline calicivirus [ ] , western equine encephalitis virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ , , ] , and hepatitis c virus (hcv) [ , , , , ] . recombination events also occur in denv, which are one of the most important mosquito-borne rna viruses in tropical and subtropical areas. in defined recombinant denv, all confirmed recombination breakpoints (''hot spots'') are located just within the c, prm, e, and ns sequences [ , [ ] [ ] [ ] rather than in other regions of the dengue genome. guangdong province is the most severely affected epidemiological area for dengue in china. between and , there were ten major dengue epidemics ( , , , , , , , , , and ) mainly caused by denv in guangdong province. among them, the most severe outbreak occurred in , when , people were infected. in this study, denv isolates from guangdong province were analyzed to investigate the existence of a recombinant virus in the area. the denv strains used in this study are as follows: gz / , / gz, gd / , gd / , gz / , gd / , gz/ , mochizuki, , a , , zj / , nb , fj / , , westpac , arg , br , fga/ , denv - , denv - - , and denv -b . the first seven strains were isolated from guangdong province. gd / was recovered from c / cells (that showed distinct cytopathic effect) infected by the acute serum of dengue patients in guangdong province in . it was identified by serotype assays, nested reverse transcriptionpolymerase chain reaction (rt-pcr) [ ] , and sequence analysis. all the data demonstrated that gd / was a dengue virus type strain. the sequences of the denv isolates from guangdong province (gd / , gz / , / gz, gd / , gd / , gz/ , and gz / ) and those of other reference isolates ( , zj / , nb , and fj / ) were firstly aligned using clustalw as implemented in the mega . (molecular evolutionary genetics analysis, pennsylvania state university, pa, usa) [ ] software with default parameters. the aligned sequences were then analyzed by rdp as implemented in recombinant detection program version (rdp , http://darwin.uvigo.es/rdp/ rdp.html) [ ] with default parameters. the results showed that gd / was a recombinant (daughter) virus and that the major and minor parent viruses were gz / and gd / , respectively. the three regions of recombination (termed regions a, b, and c) between gz / and gd / were distributed in the structural and nonstructuralcoding regions. region a (between nucleotides and , in gd / ), region b (between nucleotides , and , in gd / ), and region c (between nucleotides , and , in gd / ) were located within the sequences of the prm/e junction, ns , and ns , respectively. the p value for each recombination region was significantly lower than . (the p values were . - , . - , and . - for regions a, b, and c, respectively) (fig. a) . the recombination events detected by rdp were further investigated by bootscan [ ] as implemented in rdp . the bootscan analysis also confirmed the three recombination regions between gz / and gd / (the p value was . - , . - , and . - for regions a, b, and c, respectively) ( fig. b) , which were consistent with the rdp results. this is the first study to confirm recombination breakpoints within the ns gene in denv, although they have been confirmed in hcv [ , , ] . to determine the phylogenetic relationships between gd / , gz / , gd / , and other defined reference isolates (gz/ , mochizuki, , a , , west-pac , arg , br , and fga/ ) [ ] , phylogenetic trees, based on the putative recombination and non-recombination regions described above, were generated and visualized by the neighbor-joining method using mega . . bootstrap analysis was performed using , replicates. as seen in fig. , the left panels represent non-recombination regions and the right ones, recombination regions. we compared these phylogenetic trees and found that the topology of the phylogenetic trees in the left panels (nonrecombination regions) was very different from that of the trees in the right panels (recombination regions). for example, with regard to the non-recombination regions (the region upstream of a, the region between a and b, the region between b and c, and the region downstream of c), gd / and gz / were clustered into the same group as westpac , , and a , but gd / fell into the same group as gz . on the contrary, with regard to the recombination regions (regions a, b, and c), gd / and gd / fell into the same group as gz , and gz / were clustered into the same group as westpac , , and a (fig. ) . thus, phylogenetic analysis further confirmed that gd / was a recombinant virus, and that the regions a, b, and c in gd / were inherited from the minor parent virus gd / , while the other regions were inherited from the major parent virus gz / . the recombination events observed in this study are unusual in natural populations. it is unclear whether the recombination events took place in a human host or a mosquito vector co-infected by multiple virus strains. however, when two different virus strains simultaneously infect a single cell, it is theoretically possible for recombination to occur through a copy-choice mechanism [ ] wherein recombination results from template switches during viral genome replication. the existence of six breakpoints (two breakpoints per recombination region) in gd / implies six template switches. recombination might occur during synthesis of the positive strand of the viral genome as observed in the case of polioviruses [ ] and pestiviruses [ ] . during the recombination processes observed in this study, no insertions, deletions, or duplications occurred. however, the precise mechanism underlying the recombination events observed in the present study is unknown. a better understanding of the recombination process requires the development of experimental models for co-infection and the generation of recombinant denv. in conclusion, we confirm a recombinant denv strain, namely, gd / , with three regions of recombination between the parent virus strains gz / and gd / . this appears to be the first study that confirms the existence of three recombination regions in a single dengue virus isolate, that identifies breakpoints within the ns gene, and that reports recombination between parent virus strains isolated from the same geographic area (guangdong province). the present study also contributes toward the understanding of the pathogenesis, evolution, vaccine development, treatment, and diagnosis of denv. molecular epidemiology of dengue- and dengue- viruses evidence of intratypic recombination in natural populations of hepatitis c virus on the nature of poliovirus genetic recombinants recombination of feline calicivirus within an endemically infected cat colony resurgent vector-borne diseases as a global health problem western equine encephalitis virus is a recombinant virus phylogenetic evidence for recombination in dengue virus poliovirus rna recombination: mechanistic studies in the absence of selection a natural inter-genotypic ( b/ b) recombinant of hepatitis c virus in the philippines a natural intergenotypic recombinant of hepatitis c virus identified in st. petersburg mega : integrated software for molecular evolutionary genetics analysis and sequence alignment new natural intergenotypic ( / ) recombinant of hepatitis c virus molecular biology of flaviviruses rdp : recombination detection and analysis from sequence alignments detection of flaviviruses by reverse transcriptase-polymerase chain reaction with the universal primer set molecular characterization of pestiviruses identification of a naturally occurring recombinant genotype / hepatitis c virus novel dengue virus type from travelers to yap state identification of breakpoints in intergenotypic recombinants of hiv type by bootscanning evidence from the evolutionary analysis of nucleotide sequences for a recombinant history of sars-cov mosaic evolution of the severe acute respiratory syndrome coronavirus evidence for recombination in natural populations of dengue virus type based on the analysis of complete genome sequences molecular epidemiology of dengue type virus in venezuela: evidence for in situ virus evolution and recombination widespread intraserotype recombination in natural populations of dengue virus testing the hypothesis of a recombinant origin of the sars-associated coronavirus acknowledgments we thank wei zhao for providing some df epidemiological data of guangdong province. key: cord- -vvo a hp authors: wang, xiaobo; chen, jianfei; shi, da; shi, hongyan; zhang, xin; yuan, jing; jiang, shibo; feng, li title: immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g and g date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vvo a hp porcine epidemic diarrhea virus (pedv) is a coronavirus that infects cells lining the small intestine of swine, resulting in vomiting, diarrhea, and dehydration. the amino acid sequence of the spike (s) protein, which is the principal target recognized by host immune cells, has multiple mutations that distinguish the two pedv genotypes, g and g . to determine whether these mutations lead to changes in antigenicity, as suggested by the failure of pedv vaccines in china, we first optimized the codons of typical s genes of the cv vaccine strain (g subtype) and lnct strain (g subtype) and expressed the recombinant full-length sequence of the s protein in a eukaryotic expression system. the igg antibody levels of serum from mice immunized with purified s protein were markedly high. antigenicity was compared by detection of polyclonal antibodies (pabs) against the virus and s protein using an enzyme-linked immunosorbent assay (elisa), an indirect immunofluorescence assay (ifa), and a serum cross-neutralization (sn) assay. reactivity with the pabs revealed significant cross-reactivity between the two pedv subtypes, although there was a twofold difference in the antigenic responses based on pab titers in the elisa and ifa. consistent with the variation in the s gene sequences, the sn titer suggested differences in the neutralization activity of the s protein between the two subtypes, which could explain the antigenic variation between the pedv subtypes g and g . porcine epidemic diarrhea virus (pedv) is an acute and highly contagious enteric infectious disease characterized by vomiting, diarrhea, and dehydration in pigs of all ages, but especially in newborn piglets [ , ] . pedv was first reported in england in [ ] and then detected in japan in and subsequently confirmed in other southeastern asian countries [ ] . in the usa, pedv was first reported in and has since rapidly spread throughout the country [ , ] . in china, the incidence of pedv outbreaks has rapidly increased since , especially among newborn piglets aged from a few hours to one week, often resulting in death due to watery diarrhea and dehydration [ , ] . although the use of inactivated and attenuated vaccines may have helped to reduce the prevalence of disease, pedv has continually emerged, causing tremendous losses to the swine industry in china [ ] . pedv, a member of the genus alphacoronavirus in the family coronaviridae, has a single-stranded positive-sense rna genome of approximately kb encoding four structural proteins: the spike (s), nucleocapsid (n), envelope (e), and membrane (m) proteins [ , ] . the s protein is a glycoprotein consisting of - amino acids (aa) on the viral surface, which is composed of four regions, a signal peptide (aa , an extracellular region, a transmembrane domain (aa , - , ), and a short x. wang [ ] . unlike other coronaviruses (i.e., murine coronavirus and bovine coronavirus), the s protein of pedv cannot be cleaved into s and s domains after pedv maturation. thus, the s domain (residues - ) and s domain (aa - ) of pedv are artificially defined based on homology to other coronavirus s proteins [ , ] . the s protein is responsible for binding and fusion of the virus to the host cells. it therefore not only determines cellular tropism but also plays a vital role in inducing production of neutralizing antibodies in the host [ , ] . the s gene of pedv is often used to evaluate the genetic diversity of coronaviruses [ , ] . based on phylogenetic analysis of the full-length s sequences isolated in china, pedvs can be divided into two genotypes, g and g [ ] . furthermore, pedv detected in china from to were mostly variant strains (most belonging to subtype g ) that differed genetically from the cv vaccine strain (belonging to subtype g ) [ ] . however, many pig herds vaccinated with inactivated or attenuated cv vaccines still experienced high mortality rates among newborn piglets [ , ] . therefore, further evaluation of the immunogenicity and antigenic relationships of pedv subtypes g and g is of vital importance. in the present study, we expressed the full-length s gene in a eukaryotic expression system, prepared anti-s polyclonal antibodies (pabs), and performed subsequent experiments to investigate differences in antigenicity between the pedv subtypes to determine if mutations in the s gene are the cause of immunization failure. the findings of this study also lay a foundation for further study of the function of the s protein and to facilitate development of pabs against pedv. the pedv g cv vaccine strain (genbank accession number: kt ) was preserved at harbin veterinary research institute (harbin, china). pedv g strain lnct (genbank accession number: kt ) was isolated in vero e cells in our laboratory. vero e cells and hek t cell were cultured in gibco Ò dulbecco's modified eagle medium (dmem; thermo fisher scientific, inc., waltham, ma, usa) with % fetal bovine serum (thermo fisher scientific, inc.) and maintained at °c in a humidified atmosphere of % co . the expression vector paav-ires-hrgfp was kindly provided by professor shibo jiang (fudan university, shanghai, china). x-tremegene hp dna transfection reagent was purchased from roche diagnostics gmbh (mannheim, germany). mouse monoclonal anti-flag Ò antibody produced in mouse and fluorescein isothiocyanate (fitc)conjugated goat anti-mouse igg were purchased from sigma-aldrich corporation (st. louis, mo, usa). anti-dykddddk g affinity resin was purchased from genscript usa, inc. (piscataway township, nj, usa). a bca protein assay kit was purchased from thermo fisher scientific, inc. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg was purchased from zsgb-bio (beijing, china), and irdye@ rd goat anti-mouse igg was purchased from li-cor biosciences (lincoln, ne, usa). full-length s sequences of the pedv cv vaccine strain, the classical chinese strain ch/s, and epidemic chinese strains isolated from to were used for sequence alignments and phylogenetic analysis. homology among multiple aa sequences was analyzed using megalign sequence alignment software (dnastar, inc., madison, wi, usa). a phylogenetic tree was constructed from the aligned nucleotide sequences by the maximumlikelihood method using mega . statistical analysis software (http://www.megasoftware.net/). to improve the expression levels of the s protein, s gene sequences were codon optimized with the strategy of 'one amino acid-one codon', in which the most preferred codon of the host for a given amino acid is used in the target sequence without changing the amino acid sequences [ ] . the nucleotide sequences of the cv and lnct s genes were edited in accordance with the codon table for homo sapiens in the codon usage database (http://www. kazusa.or.jp/codon/) for optimal expression in hek t cells and biochemically synthesized for cv -s and lnct -s by beijing genomics institute (beijing, china). full-length sequences of the s gene were cloned between the bamhi and xhoi restriction sites in the paav-ires-hrgfp plasmid. the recombinant plasmids paav-opti cv s-flag and paav-opti-lnct s-flag were extracted using a plasmid maxi kit (qiagen, hilden, germany). hek t cells were cultured to % confluency in a sixwell tissue culture plate at °c and then transfected with lg of paav-opti cv s-flag and paav-opti lnct s-flag together with ll of transfection reagent. after h, the hek t cells were collected, treated with ip lysis buffer (thermo fisher scientific, inc.), and centrifuged at , rpm for min. next, proteins in the supernatants were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on % gels and transferred to a polyvinylidene difluoride (pvdf) membrane for min. the pvdf membrane was blocked with % skim milk for h at °c, incubated with mouse anti-flag antibody (dilution, : , ) at °c for h, and then further incubated with irdye rd goat anti-mouse igg (dilution, : , ) at °c for min. the protein bands were visualized using an odyssey Ò clx infrared imaging system (li-cor biosciences). hek t cells were cultured in -cm bottles and transfected with lg of paav-opti pedv-s-flag plasmids. at h post-transfection, the cell lysate was harvested as described above, recombinant s protein was purified using anti-dykddddk g affinity resin, the cell lysate was loaded onto a column, and the soluble proteins were immunoprecipitated with anti-flag resin. the purified proteins were concentrated using amicon ultra centrifugal filters (molecular weight cutoff, k; emd millipore, billerica, ma, usa). protein concentrations were measured using the bicinchoninic acid (bca) protein assay, and the final products were analyzed by sds-page to confirm purification of the target protein. all animal procedures were approved by the ethics committee of harbin veterinary research institute. three groups of -week-old female pathogen-free balb/c mice were respectively immunized subcutaneously three times, at two-week intervals, with lg/mouse of recombinant cv s protein, lnct s protein formulated in freund's adjuvant (sigma-aldrich corporation) and phosphate-buffered saline (pbs) which was used as a negative control. blood was collected from the tail vein of three mice from each group, and serum samples were prepared prior to the first immunization and at one-week intervals, then stored frozen until assayed. detection of igg antibodies of immunized mice using an enzyme-linked immunosorbent assay (elisa) igg antibodies of the immunized mouse's serum samples were detected by elisa [ ] . ninety-six-well plates (costar tm ; thermo fisher scientific, inc.) were coated with the purified recombinant cv and lnct s proteins ( ng/well) in coating buffer ( mm na co , mm nahco , ph . ) at °c overnight. the antigencoated plates were washed three times with pbs with tween Ò (pbst) and blocked with ll of % skim milk for h at °c. the blocked wells were washed three times with pbst. anti-s lnct or anti-s cv polyclonal antisera (pabs) collected at different time points were diluted from : to : , with pbs and added to the wells. the plates were incubated at °c for h, washed three times with pbst, and hrp-conjugated goat antimice igg ( : ) was added to each well. the plates were incubated at °c for min and washed three times with pbst. then, ll of abts [ , -azinobis-( -ethylbenzothiazoline- -sulfonic acid)] peroxidase substrate was added to each well, and the plates were incubated for an additional min at °c in the dark. the reaction was stopped by the addition of m naf, and the absorbance was read at a wavelength of nm (od ) using an enzyme-linked immunosorbent assay (elisa) plate reader. confluent monolayers of vero e cells in -well plates were washed three times with pbs. subsequently, each well was incubated with times the median tissue culture infective dose (tcid ) of strain cv and/or lnct in dmem supplemented with lg of trypsin per ml. after incubation at °c for h, monolayers were rinsed with pbs and fixed with % paraformaldehyde at °c for min. they were then washed twice with pbs and incubated with . % tritonx- at °c for min. after washing with pbs, the wells were blocked with % bovine serum albumin at °c for h, washed three times with pbs, and incubated with cv or lnct anti-s pabs at °c for h. to determine titers, serum samples were initially diluted -fold and then serially diluted twofold and rinsed three times with pbs. fluorescein-isothiocyanate-conjugated goat anti-mouse igg at a dilution of : , in pbs was added to each well. after min of incubation in the dark at °c, the plates were washed three times with pbs and incubated with , diamidino- -phenylindole at a dilution of : , for min in the dark at °c. after washing three times with pbs, the samples were examined using a digital inverted microscope (thermo fisher scientific, inc.). the cross-reactivity between two types of virus s protein and anti-s protein pabs were assessed by western blot analysis. briefly, vero e cells in -cm bottles were infected with either the cv or lnct virus variation between porcine epidemic diarrhea virus subtypes (multiplicity of infection = ) for h, treated with mammalian protein extraction reagent (thermo fisher scientific, inc.) for min, and centrifuged at , g for min. the supernatants containing total virus proteins were subjected to % sds-page followed by western blot analysis as described above with anti-s pabs diluted to : , . the sn test was performed according to the fixed-virusdilution serum method described by reed and muench [ ] . briefly, confluent monolayers of vero e cells in -well plates were washed three times with dmem. pabs against the s protein were inactivated at °c for min and then diluted twofold starting at : . they were then mixed with the same volume ( ll) of tcid of virus diluted with dmem supplemented with lg of trypsin per ml and incubated at °c for h. subsequently, . ml of each virus-serum mixture was inoculated onto vero e cell monolayers in -well tissue culture plates. after days, specific cytopathic effects (cpes) of cells were observed under an inverted microscope. sn titers were expressed as the titer of the highest serum dilution resulting in % inhibition of pedv infection. one-way analysis of variance was used to determine statistical differences between groups. all statistical analysis was performed using graphpad prism version . software (graphpad software, inc., la jolla, ca, usa). probability (p) values of . and . were considered statistically significant and highly statistically significant, respectively. the maximum-likelihood phylogenetic relationships inferred from the aa sequences of the s protein confirmed that the two genotypes were pedv strains g and g . the cv vaccine strain was subtype g , while the lnct strain was subtype g (fig. ) expression and purification of full-length recombinant pedv s proteins hek t cells in six-well tissue culture plates were transfected with recombinant plasmids (paav-cv s-flag and paav-lnct s-flag) containing unoptimized s genes and recombinant plasmids (paav-opti cv s-flag and paav-opti-lnct s-flag) containing optimized s genes. as the s protein was co-expressed with the hrgep protein, the fluorescence intensity reflected the expression level of the s protein. we found that the expression levels of the s proteins were significantly increased by codon optimization ( fig. a) . s proteins were detected by western blotting using anti-flag mouse monoclonal antibody (mab) as the primary antibody. the results showed that the recombinant proteins reacted specifically with the primary antibody, and each had a molecular weight of kda (fig. b) . these results were in accordance with those of the sds-page analysis with the purified s protein (fig. c) . specific igg antibodies were detected in serum samples collected at different time points using an indirect elisa, as described previously. a shown in fig. , igg antibody levels against the s protein in serum from immunized mice were significantly increased at days post-immunization (p \ . ) and then highly significantly increased at days, as compared with mice immunized with pbs (p \ . ), while there were no significant differences in titers of igg antibodies induced by cv and lnct s proteins at different time points (p [ . ). the elisa results indicated high cross-reactivity between the pabs and the two types of pedv s protein. the titers of anti-s pabs reacting with same subtype s protein reached : , , while the titers reacting with different subtype s proteins reached : , . as shown in fig. a and b, the od values of anti-s cv protein pabs at different dilutions reacting with the cv s protein were much higher than those reacting with the lnct s protein. the ifa results indicated that pedv s protein was present in the cytoplasm of vero e cells (fig. a ). both pedv cv and lnct could induce obvious cpe: vero e cell fused with each other and formed syncytia. the structure of cells infected by lnct was still clear, while cv -infected cells were all fused and no single cellular morphology was generally observed. the syncytia of cv -infected cells were larger than those of lnct infected cells (fig. a and b) . the ifa results confirmed that the anti-s pabs crossreacted with pedv strains cv and lnct . next, we detected the fluorescence intensity of the antigenic crossreaction between the the anti-s protein pabs and the two pedv subtypes. consistent with the elisa results, which showed twofold differences between different pedv subtypes based on titers of the anti-s pabs, the highest titer of anti-s pabs reacting with pedv within subtypes (observing green fluorescence) reached : , , while the reaction of anti-s pabs and pedv across subtypes only reached : , ( table ) . anti-s pabs bound with the pedv s protein, which was about kda, slightly smaller than the recombinant s protein, as was expected. the results of the western blot assay demonstrated that the anti-s pabs cross-reacted with strains cv and lnct (fig. a and b) , indicating that pedv s proteins had at least one common antigenic epitope across the g and g strains. the titers of anti-s pabs neutralizing pedv were calculated by the kärber method. the average titer of anti-s cv pab, which protected % of cv -infected cells, was : , while the titer cross-neutralizing lnct was : . the average titer of anti-s lnct pabs neutralizing lnct reached : , while the titer of neutralizing cv was : (fig. ) . the sn titer of anti-s cv pabs neutralizing cv strain was more than twofold higher than those neutralizing the lnct strain, while the neutralizing titers of anti-s lnct pabs were less than twofold of those reacting with the two strains. the ongoing epidemic of pedv has resulted in significant economic losses to the swine industry in asia as well as europe and north america. in china, mortality due to pedv infection can reach %- % in piglets less than days old [ ] . in the usa, pedv infection has resulted in a loss of almost % of the domestic pig population after only a -year epidemic period [ ] . located on the surface of pedv, the s protein plays an pivotal role in recognizing receptors of target cells, thereby inducing production of neutralizing antibodies by activated host immune cells [ , ] . although there have been relatively few studies using full-length sequences of the pedv s protein, most investigating the immunogenicity of the pedv s protein have focused on the s region [ ] [ ] [ ] . as pedv s protein cannot be cleaved into s and s domains after virus maturation and s domain may also have potential neutralizing linear and conformational epitopes, the whole s protein with its native conformation might be a better immunogen than the s protein. previously, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) spike proteins have been shown to produce high-titer antibodies in mice [ ] . in the present study, we successfully expressed and purified the full-length s protein using a eukaryotic expression system. western blot analysis confirmed that the s protein was specifically detected by anti-flag antibodies (fig. b) . the ifa indicated that the s protein was expressed at higher levels in hek t cells through codon optimization (fig. a) , as demonstrated with other coronaviruses [ ] . the pedv s protein has - potential glycosylation sites that help to maintain its biological function [ ] . therefore, we chose a eukaryotic expression system that was particularly useful for the production of such a large multi-domain protein requiring complex folding machinery and post-translational modifications [ ] so that the structure of the s protein was close to the naturally occurring conformation. other advantages of choosing a eukaryotic expression system were that the expressed fusion protein was soluble as the biological function was maintained [ , ] . in the present study, we established a method to express and purify the pedv s protein, which can be used not only to evaluate immunogenicity but also to study s protein function. the igg levels of mice immunized with the s protein were significantly increased beginning on post-immunization day , and antibody titers reached : , on post-immunization day according to the elisa results (fig. ) . the high igg antibody titers of immunized mice indicated that purified s protein may be a good candidate as an immunogen for pab production and diagnostic methods. since , pedv outbreaks in several provinces have caused significant economic losses to the swine industry in china [ , ] . although a cv -inactivated vaccine has been used to immunize swine herds against pedv infection, its efficacy has been rather low [ ] . therefore, it is of vital importance to develop a cv vaccine that can cross-protect against epidemic strains of the g subtype. previous studies have found differences in the antigenicity between pedv cv and strains isolated in the usa, and some pig antibodies and mabs showed -to -fold differences between the titers of the homologous and heterologous strains ccif and vn, as at least one epitope on the n-terminal region of pedv/tgev n protein contributed to this cross-reactivity [ ] . however, the mabs used in the previous study were against the n protein, which is not the major antigen that induces production of neutralizing antibodies by host cells. in our previous study, we also found twofold differences in pig antibodies based on sn titers between strains cv and lnct (data not shown). a comparison of sequences of two types of pedv g s protein from recent chinese epidemic strains from to showed that they had . %- . % sequence identity to the cv vaccine strain (table ) , and sequence variations were concentrated in the n-terminal region of the s protein due to a point mutation, a deletion, and an insertion of four amino acids. the lnct strain we chose belonged to the g subtype, and its s sequence was . % identical to that of the cv vaccine strain (g subtype). the neutralizing epitope domain coe (aa - ) had eight different amino acids, and the the luminescence intensity was divided into four degrees: ???, strong; ??, moderate; ?, weak; -, negative epitope motif ss ( lqdgqvki ) had two aa mutations between the two pedv subtypes [ ] . based on these differences in s protein sequences, we investigated whether mutations in the s protein resulted in a change in antigenicity between strains g and g . the elisa and ifa results showed significant antigenic cross-reactivity between the two pedv subtypes. however, there was approximately a twofold difference in the antigenic responses based on the titers of the pabs. especially, the elisa results showed that reactivity of the anti-s cv pabs at different dilutions with the cv s protein was much greater than that with the lnct s protein, indicating that the ability of anti-s cv pabs to crossreact with different subtypes was lower than that of the anti-s lnct pabs. in addition, previous studies have shown that the pedv cv strain induces larger syncytia than korean strain dr and three us strains [ ] [ ] [ ] . comparing the ifa results of the cv and lnct strains, we also found a similar phenomenon (fig. ) . these results indicated that cv and epidemic strains may have differences in their ability to enter cells and induce fusion and may have different effects on cell morphology. the results of western blot analysis indicated that the pedv s proteins had at least one common antigenic epitope, allowing the s protein pabs to cross-react with different pedv subtypes. likewise, the results of the sn assay confirmed the presence of high titers of pabs against the s protein that were able to cross-neutralize other pedv subtypes. however, consistent with the elisa and ifa results, there were obvious differences in serum neutralizing responses across subtypes. the sn titer of anti-s cv pabs neutralizing strain cv was more than twofold higher than the pabs neutralizing strain lnct, while the differences in the neutralizing titers of anti-s lnct pabs across subtypes was less than twofold. these results indicate that the neutralizing epitopes of the lnct s protein had changed or that new neutralizing epitopes had emerged due to mutations in the s protein. in the subsequent experiments, we found that the f monoclonal antibody against the lnct s protein could react with strain lnct, but not strain cv (data not shown). further studies to identify the mab f epitope are underway. in summary, the full-length sequence of the pedv s gene was codon-optimized and successfully expressed in hek t cells. the findings of this study confirmed that the recombinant s protein was highly immunogenic in cross serum neutralization (sn) between the s proteins of the two pedv subtypes and anti-s pabs. reciprocals of pedv neutralizing antibody titers were expressed as the dilution inhibiting pedv infection by %, which was calculated as follows: log % neutralizing titer = l-d (s- . ), where l is the log of the lowest dilution factor, d is the difference between the dilution factors, and s is the sum of the ratios of positive wells variation between porcine epidemic diarrhea virus subtypes mice and could effectively induce production of antibodies, especially neutralizing antibodies. furthermore, we found significant antigenic relationships between the pedv s protein of subtypes g and g , which indicated that the cv vaccine could cross-protect against g epidemic strains, at least to some extent. however, the antigenic and serologic neutralization responses against the s protein also reflected antigenic differences of twofold between the two pedv subtypes, based on anti-s pabs titers. therefore, future development of vaccines against emerging pedv strains should involve multiple s proteins containing neutralizing epitopes from both the g and g subtypes. experimental infection of pigs with a new porcine enteric coronavirus three-dimensional sequential study of the intestinal surface in experimental porcine cv coronavirus enteritis letter to the editor. pig farming suppl an outbreak of swine diarrhea of a new-type associated with corona-virus like particles in japan fighting a deadly pig disease. industry, veterinarians trying to contain ped virus, new to the us emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets 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strains proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture a single point mutation creating a furin cleavage site in the spike protein renders porcine epidemic diarrhea coronavirus trypsin-independent for cell entry and fusion variation between porcine epidemic diarrhea virus subtypes acknowledgments this work was supported by grants from the national natural science foundation of china (grant number ), innovation capability project for research institutions of heilongjiang province (grant number yc d ). key: cord- -uiwg s authors: truong, c.; mahe, d.; blanchard, p.; le dimna, m.; madec, f.; jestin, a.; albina, e. title: identification of an immunorelevant orf epitope from porcine circovirus type as a serological marker for experimental and natural infection date: journal: arch virol doi: . /s sha: doc_id: cord_uid: uiwg s post-weaning multisystemic wasting syndrome (pmws) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (pcv ). we report here the characterization of immunorelevant linear b-cell epitopes of the open reading frame -encoded protein (orf ) from pcv by an enzyme-linked immunosorbent assay (elisa) using experimental antisera collected from pigs inoculated with a pcv isolate. two epitopes spanning residues to and to were specific to pcv . antibodies to the to epitope (b- ) were detected in % and % of specific pathogen-free (spf) pig sera and weeks post inoculation, respectively. cross-sectional studies performed with field sera collected from pmws-affected herds showed b- antibodies in % of to week-old pigs, % of – week-old pigs, % of to week-old pigs, % of to week-old pigs and % of to week-old pigs. all these data suggest that epitope b- is a serological marker of pcv infection that could be used for the detection of pcv antibody response. pmws is now a well-established disease of swine herds in france, other european countries, north america and asia. clinical signs are unthriftiness, pallor, fever and progressive wasting with associated respiratory and digestive disorders [ , , ] . a clinical outcome is observed in to week-old pigs. mortality attained % in some french farms [ ] . a new porcine circovirus (pcv ) has been isolated from pmws-affected piglet lesions [ , , [ ] [ ] [ ] [ ] [ ] [ ] . pcv is a small non-enveloped circular single-stranded dna virus in the circoviridae family which also includes other animal viruses such as porcine circovirus type (pcv ), psittacine beak and feather disease virus (bfdv) and chicken anemia virus (cav). pcv was first described in as a persistent noncytopathic contaminant of the porcine kidney cell subline pk atcc-ccl [ ] . although serological surveys indicated that pcv antibodies were common in the swine population, disease was not observed either in pcv -seropositive farms [ , , , , ] or after experimental pcv inoculation [ ] . porcine circoviruses pcv and pcv share % nucleotide sequence homology with mutations throughout the viral dna. orf encoded protein is more conserved between the two viruses than orf -encoded protein ( % and % homologies, respectively [ , ] ). orf -encoded protein (rep protein) is essential for circoviral replication [ ] whereas the function of orf -encoded protein has not yet been elucidated. pcv infection diagnosis is currently based upon immunohistochemistry [ , ] and in situ hybridization [ , , ] in porcine lymphoid tissues. pcv antibodies can be successfully detected using immunoperoxidase monolayer assay (ipma) [ ] or indirect immunofluorescent assay (ifa) [ , ] of pcv infected pk cells. serotyping is achieved by comparative analysis of the titers obtained in pcv and pcv ipmas [ , , ] . we have previously demonstrated antigenic cross-reactions between pcv and pcv viruses in orf -encoded protein and orf -encoded protein has been evidenced as a good candidate for serotyping [ ] . here, we describe the characterization of an orf peptide as an immunorelevant pcv specific linear b-cell epitope by elisa, using sera from pigs experimentally inoculated with a pcv isolate, and its potential use as a serological marker to detect pcv antibodies following natural infection in swine herds. pig sera (i) experimental pcv antisera four pmws experimental transmission studies (a, b, c, d) were performed on weekold specific pathogen-free piglets (spf) or on conventional farm piglets in our experimental facilities [ ] . the pigs were challenged with tissue homogenates prepared from sick piglets. enlarged lymph nodes collected from a pmws-affected field pig were crushed in minimal essential medium (e-mem from biowhittaker, belgium) then centrifuged ( g, min, • c). the supernatant was ultrafiltered on . m and further subjected to differential pcv and pcv polymerase chain reaction (pcr) using two sets of type-specific primers: pcv forward ( -ggcggcggcatctgtaacggttt- ) and pcv reverse ( -gatggcgccgaaaga-cgggtatc- ), pcv forward ( -gtggagctcctagatctcaggg- ) and pcv reverse ( -taggagctccacactccatcag- ) [ ] . in this inoculum, the pcv titer was . tcid /ml as determined by ipma of pk cells according to kärber [ ] . for ipma analysis, × pk pcv-free cells were seeded into microplate wells before infection with l of tissue homogenate ( . tcid /ml). circoviral replication was boosted hours post-infection with l of mm d-glucosamine (sigma, usa) in earle's balanced salt solution (sigma, usa) and the cells further grown in e-mem with % fetal bovine serum for h at • c in a % co atmosphere. the cells were then fixed and permeabilized with l of % cold acetone in phosphate-buffered saline (pbs) for min at − • c. the wells were quenched with l of pbs containing % of blotting grade blocker non-fat dry milk for min at • c. the plates were then washed three times with l of pbs containing . % tween (pbs-tw). l of serum sample diluted : in pbs containing % blotting grade blocker non-fat dry milk and . % tween (pbs-m-tw) was added and further incubated for min at • c. after three washes with pbs-tw, l of horseradish peroxidase (hrp)-conjugated rabbit anti-swine igg (dako, denmark) diluted : in pbs-m-tw was added and further incubated for min at • c. the plates were washed again with l of pbs-tw. staining was developed with -aminoethyl carbazole (sigma, usa) in dimethylformamide and hydrogen peroxide and was stopped by substrate removal. the animals were inoculated intramuscularly and intratracheally once with ml and ml, respectively of either the above inoculum (infected piglets) or e-mem (control piglets). serial blood samples were taken weekly. d and d end correspond to the pig serum samples collected before experimental inoculation and at the end of the experiment, respectively. circovirus genotyping was also assessed by pcr in tissue samples from necropsied pigs. trials a, b, and c were carried out for weeks. trial a involved spf piglets of which were controls. trial b involved spf and conventional farm piglets of which spf piglets were controls. trial c involved conventional farm piglets of which were controls. study d was performed over a period of weeks on spf piglets which included controls. (ii) control sera the negative control was collected from a piglet in our spf herd. this serum was previously shown by ipma to be free of pcv antibodies. the positive control was a pcv -specific immune antiserum collected from a pig inoculated with a tissue homogenate containing pcv only, as assessed by differential pcr. pcv antibodies were previously detected in this serum by ipma. the variability of the elisa results was evaluated by testing the positive control serum on an entire plate in two different assays. a normal distribution of the positive control od values was observed. this allowed determination of the coefficient of variation (cv%) corresponding to the ratio between standard deviation (sd)/mean (m) × . (iii) antisera to other pig viruses antisera were raised against the following pig viruses: (i) three pestiviruses: bovine viral diarrhea (bvd), border disease (bd) and classical swine fever (csf) viruses (ii) two herpesviruses: pseudorabies virus (prv) and cytomegalovirus (pcmv) (iii) three coronaviruses: transmissible gastroenteritis virus (tgev), epidemic diarrhea virus (pedv), respiratory coronavirus (prcv) (iv) influenza viruses (v) arterivirus: porcine reproductive and respiratory syndrome virus (prrsv) (vi) cardiovirus: encephalomyocarditis virus (emcv) (vii) enterovirus: talfan disease virus (viii) porcine parvovirus (pv). all these antisera were generated from -to -week-old spf piglets. (iv) field serum samples pig sera were collected from french herds. seventeen of these herds were selected for typical clinical signs of pmws while the others were clinically free of the disease. seven hundred and fifty blood samples were taken from the pmws-affected herds and samples from the pmws-free herds. the pigs were to weeks of age. pepscan was assessed on nitrocellulose membranes with a panel of peptides overlapping the sequences of orf pcv , orf pcv , orf pcv and orf pcv using an indirect im-munoassay with experimental antisera according to the manufacturer's guidelines. pepscanselected pcv peptides were used in elisa to determine their reactivities with pcv experimental antisera collected from spf or/and conventional farm pigs before and at various times after inoculation. pcv counterparts were also analyzed with the same antisera in order to identify cross-reacting pcv peptides. forty-eight wells of the microtiter plate (nunc maxisorp) were coated with l of one pcv peptide ( g/ml) in . m bicarbonate buffer ph . and incubated overnight at • c while the other wells were similarly coated with the pcv counterpart peptide. the unoccupied sites of the wells were then blocked with l of pbs containing % of blotting grade blocker non-fat dry milk after incubation for min at • c. the plates were rinsed twice with l of pbs-tw. the serum samples were diluted : in pbs-m-tw. each serum sample was tested against the pcv and pcv peptides. diluted serum sample ( l) was added to a well and incubated for min at • c. the plates were then washed three times with l of pbs-tw. fifty microliters of hrp-labeled rabbit anti-swine immunoglobulins diluted in pbs-m-tw were added and incubated for min at • c after which the plates were washed three times with pbs-tw. hydrogen peroxide o-phenylenediamine ( l) (opd, sigma, usa) was added and incubated for min at • c. color development was stopped by the addition of l of n h so . the optical densities (od) were read at nm. orf gene from pcv and pcv was amplified and cloned into baculovirus transfer vectors as described elsewhere [ ] . proteins were expressed in spodoptera frugiperda cells following infection with previously selected recombinant baculovirus. expression control was checked by western-blot analysis. briefly, cells were separated on a % sodium dodecyl sulfate polyacrylamide gel and then transferred onto a nitrocellulose membrane. the recombinant orf protein was identified using the experimental positive control sera as a kda product and further used for the screening of pcv antibodies from field sera. to ensure that the experimental sera had been raised against one strain of porcine circovirus, pcv and pcv dnas were screened by pcr in the tissue homogenate inoculum as well as in tissue samples from necropsied pigs. as shown in fig. , no pcv or pcv dnas were detected in tissue samples from the control piglet (lanes a and b). no detectable antibody response was evidenced in either preimmune serum or time-end serum from this control pig as assessed by ipma. pcv dna was demonstrated in the tissue homogenate inoculum (lane b) but no pcv dna was present in this sample (lane a). only pcv was detected in the tissue sample from the necropsied pig inoculated with tissue homogenate (lanes a and b) . no detectable pcv antibodies were evidenced by ipma in the preimmune sera from this inoculated pig but seroconversion was observed at the end of the experiment (fig. ) . pcv dna was detected both in the pk atcc-ccl lysate cell (lane a) and in pk -pcv inoculated b, b) . the tissue homogenate inoculum contained only pcv dna suggesting that the elicited antibodies were relevant to the pcv serotype. orf -and orf -encoded protein pepscan analysis was carried out with pcv experimental antisera in comparison with screening of pcv sequences [ ] . all the orf peptides from pcv that reacted with pcv antisera had counterparts in the pcv sequence which were also recognized by the same pcv antisera. table . none of the pre-immune sera (d ) had detectable pcv antibodies that reacted with any of the peptides. time-end serum samples (d end ) had antibodies that reacted with peptides b- , b- , b and a as indicated by an absorbance value three times higher than that of the negative control (od nm = . ). at the end of the experiment, the levels of antibody of these three pcv peptides, as depicted by the ratio d end /d , was to times higher than those from the preimmune samples. these peptides allowed the detection of circovirus seroconversion after inoculation with the tissue homogenate thereby confirming the immunorelevance of these epitopes during experimental infection with pcv . the respective pcv counterparts a- and a- of the pcv peptides, b- and b- , were not recognized by post-infection serum from the two experimentally infected pigs as shown by the ratio value d end b /d end a > , indicating that these latter two peptides are pcv discriminant (table ) . two out of the three orf immunorelevant epitopes were thus pcv specific. the b- epitope was selected for further elisa since a seroconversion was more consistently evidenced with pcv experimental spf as well as with conventional farm pig antisera. non-specific serological cross-reactions between several other porcine virus antigens and the b- epitope were evaluated with a panel of antisera produced in spf pigs by inoculation with other swine viruses. none of these antisera reacted with our pcv peptide (table ) , indicating a lack of crossreactivity between pcv and the other viruses under study. sera collected from trials a, b, c and d were analyzed by elisa (fig. ) . no b- antibodies were detected in the preimmune sera (d ) from control and infected pigs. preimmune serum samples from control piglets in experimental studies a, b, c and d were used as a source of pcv negative sera to define a positive threshold. using our b- elisa, the od nm values of these negative sera ranged from . to . and showed a normal distribution. this indicates that % of the negative sera have od values ranging from the mean od (m) minus standard deviations (sd) to m + sd. the positive threshold determined from m = . and sd = . , was set at . . all control piglets were seronegative at the end of the experiment (d end control piglets) i.e. after weeks for trials a, b, c and weeks for trial d. none of the pigs had detectable antibodies during the first two weeks post-inoculation although signs of disease were observed to days post-inoculation. these signs consisted of a febrile phase which lasted for about days and retarded growth (albina, manuscript submitted). at six weeks post-inoculation % ( out of ) and % ( out of ) of the spf antisera from trials a and b, respectively had detectable b- antibodies whereas % ( out of ) of the challenged piglets were seropositive weeks post-inoculation (trial d), thus demonstrating that the b- epitope was a serological marker of pcv infection. in contrast to spf pigs, % ( out of ) and % ( out of ) of the conventional farm pigs from trials b and c, respectively had detectable b- antibodies six weeks post-inoculation which suggests that the kinetics of pcv seroconversion vary according to the pig's health status. the cvs for our positive control were . % and . % respectively, in two different assays, indicating that the intravariability of this peptide-based elisa was acceptable. serum samples were collected from post-weaning piglets and growing pigs from to weeks of age in herds located in areas of france (bretagne, vallée de la loire, nord, champagne-ardennes and aquitaine). seventeen of these herds showed typical clinical signs of pmws ( serum samples) whereas the other were clinically free of the disease ( serum samples). the distribution of od values in fig. , based on the above-described positive threshold, shows two serological populations within the pmws-affected porcine population. b- antibodies could be detected in forty-five percent ( samples out of ) of the total serum population collected from pmws-affected herds. these serological analyses performed on post-weaning and growing pigs were used to determine the percentage seropositivities of the different age groups. cross-sectional studies showed that b- antibodies were detected in % ( out of ) of the sera from to week-old pigs, % ( out of ) of sera from - week-old pigs, % ( out of ) of sera from to week-old pigs, % ( out of ) of sera from to week-old pigs and % ( out of ) of sera from to week-old pigs ( table ), suggesting that the b- epitope was a serological marker for the later steps of pcv infection. four hundred and six serum samples collected from pmws-free herds were then serologically screened for pcv antibodies. the od distribution is shown in fig. . surprisingly, two serological populations emerged despite the absence of clinical pmws, thus indicating that some asymptomatic pigs were infected by pcv . twenty-six percent ( out of ) of the pigs in the pmws-free herds had detectable b- antibodies. these seropositivities were further confirmed by western-blot assay for all the tested sera and supported the occurrence in swine herds of subclinical pcv infection. we have previously demonstrated that porcine circovirus pcv and pcv have common antigenic determinants located on the orf -encoded protein which are fully consistent with their high degree of sequence similarity while the orf encoded protein has been characterized as a good candidate for serotyping due to its lack of antibody cross-reactivity between the two viruses under study [ ] . in this report, we show that a peptide based on the amino acid sequence of the orf -encoded protein (residues to ) from pcv binds antibodies from pigs experimentally inoculated with tissue homogenates prepared from pmwsaffected pigs. its specificity for pcv antibodies appears to be high since pig blood samples seropositive for several common porcine viruses did not react with the pcv peptide. moreover this peptide is not a pcv cross-reacting epitope since pcv antibodies did not react with its pcv counterpart which shared % of sequence identity. this peptide-based elisa was thus highly specific to pcv in contrast to the differential ipma or ifa reported by others [ , , ] which used cross-reacting virion subunits as detection antigens. by studying the antibody response in experimentally inoculated pigs over a period of weeks, we established that the b- peptide was a serological marker for pcv infection since all pigs had pcv antibodies at the end of the experiment. this serological marker is thus produced or detected relatively late since anti-b- antibody levels were undetectable during the first weeks postchallenge although pcv antibodies were reported with ipma of pcv -infected cells or orf western blot analyses [ , ] in all inoculated pigs from the th day post-inoculation onwards. these data suggest a lower sensitivity of the peptidebased elisa which is assumed to be linked to the availability of antigenic sites. actually virion proteins may exhibit several linear or conformational antibody binding sites compared to the -mer peptide. kinetic studies showed a variability in seroconversion rate dependent on the pig's initial health since % of the conventional farm animals were seropositive weeks post-inoculation compared to only % of the spf animals. these differential antibody kinetics could be explained by the individual backgrounds of the pigs in terms of previous immunological stimulations. we have already observed, for example, that conventional farm pigs generally exhibit higher cellular immune responses than spf pigs (unpublished data). the number (n) of positive sera and the % of seropositive individuals in each group of pig age are reported by studying the reactivity of the b- peptide with field pig sera, we established that it was also a serological marker for natural pcv infection since farm pigs kept in pmws-affected herds had detectable pcv antibodies. furthermore % of the farm pigs in pmws-free herds were also seropositive and no reactivity was ever observed with pcv peptide. this absence of correlation between pcv seropositivity and the outcome of pmws is fully consistent with previous reports [ , ] . it can be assumed that other factors such as housing or management conditions, concurrent infections or even genetic factors are involved in the onset of clinical symptoms in herds. in particular, recent studies have shown that some management failures [ ] or simultaneous infection with porcine parvovirus [ ] may influence disease outcome. to date, the serodiagnosis of pcv infection necessitates cumbersome differential assays based on cell-cultured antigens. to our knowledge, no elisa has so far been developed mainly because of the absence of immunological data concerning the virus. we have characterized certain of the linear b-cell epitopes of the orf -encoded protein from pcv and focused on an epitope designated b- to develop an elisa. the most interesting feature of this easy-to-use assay is its pcv specificity since it seems to be less sensitive in antibody detection than a multiepitopic antigen. we have also identified two other relevant epitopes: a discriminant epitope (b- ) and b- which is apparently common to both circoviral types although no cross-reactivity has ever been observed from orf -encoded proteins. b- and its pcv counterpart share % amino acid identity while b- and a- share more than % sequence identity. these other epitopes could also be of interest in the serodiagnosis of pcv infection for example, in combination with the b- peptide or with the orf -encoded protein. pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material novel porcine circoviruses from pigs with wasting disease syndromes 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 inoculation of conventional pigs with tissue homogenates from pigs with post-weaning multisystemic wasting syndrome bilan de années d'utilisation de porcs exempts d'organismes pathogènes spécifiques (eops) à la station de pathologie porcine in-situ hybridization for the detection of porcine circovirus in pigs with post-weaning multisystemic wasting syndrome post-weaning multisystemic wasting syndrome: preliminary epidemiology and clinical findings porcine circovirus antigens in pk- cell line (atcc-ccl ) and evidence of antibodies to circovirus in canadian pigs evidence of circovirus infection in british pigs nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs recognizing and diagnosing post-weaning multisystemic wasting syndrome (pmws) porcine circovirus: a serological survey of swine in the united states beitrag zur kollektiven behandlung pharmakologischer. reihenversuche porcine circovirus infection in northern ireland circovirus-like viral associated disease in weaned pigs in indiana piglet wasting disease post-weaning multisystemic wasting syndrome (pmws) in pigs in france: clinical observations from follow-up studies on affected farms differential recognition of orf protein from type and porcine circoviruses and identification of immunorelevant epitopes identification of a protein essential for replication of porcine circovirus characterization of pcv isolates from spain detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome detection and characterization of porcine circoviruses associated with postweaning multisystemic wasting syndrome in pigs a comparison of in-situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (pmws) multiplex pcr for detection and typing of porcine circoviruses post-weaning multisystemic wasting syndrome in spain clinical immune responses in young swine to porcine circovirus type infection ) 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 a very small porcine virus with circular single-stranded dna studies on epidemiology and pathogenicity of porcine circovirus distribution of antibodies to porcine circovirus in swine populations of different breeding farms the authors thank all the farmers, technicians and veterinarians who contributed to this study by collecting serum samples and particularly eric eveno, pierre morvan, jean-luc jobert, nicolas rose and pierre-alexandre beloeil. we also thank roland cariolet, bernard beaurepaire, gérard bennevent, andré keranflec'h and jean-pierre jolly for their daily assistance in our experimental facilities. this work was supported by funds from the "comité régional porcin", the "région bretagne" and the "feoga". key: cord- -vt vwo y authors: jung, kwonil; hu, hui; saif, linda j. title: calves are susceptible to infection with the newly emerged porcine deltacoronavirus, but not with the swine enteric alphacoronavirus, porcine epidemic diarrhea virus date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: vt vwo y fecal virus shedding, seroconversion and histopathology were evaluated in - -year-old gnotobiotic calves orally inoculated with porcine deltacoronavirus (pdcov) ( . - . log( ) genomic equivalents [ge] of oh-fd -p ; n= ) or porcine epidemic diarrhea virus (pedv) ( . - . log( ) ge of pc a; n= ). in pdcov-inoculated calves, an acute but persisting fecal viral rna shedding and pdcov-specific serum igg antibody responses were observed, but without lesions or clinical disease. however, no fecal shedding, seroconversion, histological lesions, and clinical disease were detected in pedv-inoculated calves. our data indicate that calves are susceptible to infection by the newly emerged pdcov, but not by the swine coronavirus, pedv. coronaviruses (covs) are enveloped, single-stranded rna viruses of positive-sense polarity. their genomes range from approximately to kb in size [ ] . the family coronaviridae of the order nidovirales is divided into the four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. bats are the projected host reservoir for alphacoronaviruses and betacoronaviruses, while birds are thought to be the host for gammacoronaviruses and deltacoronaviruses [ ] . the two betacoronaviruses, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), were transmitted by civet cats and camels, respectively, to humans, but they have shown limited capacity for adaptation to humans [ ] . some covs can be transmitted to different animal species, and subsequently adapt to and be maintained in the new host, because they can exploit or share a variety of host cell surface molecules or other undefined factors [ ] . the species porcine epidemic diarrhea virus belongs to the genus alphacoronavirus. porcine epidemic diarrhea virus (pedv) causes acute diarrhea, dehydration and high mortality in neonatal piglets [ ] . for the last four decades, since the first appearance of pedv in , pedv infection has resulted in significant economic losses in the european, asian and us swine industries. pedv has been found only within the pig population, indicating that the virus might have adapted only to pigs [ ] . the species porcine deltacoronavirus belongs to the genus deltacoronavirus. porcine deltavoronavirus (pdcov) is a novel enteropathogenic cov infecting pigs and was previously reported in birds [ ] . pdcov was first identified in pigs in hong kong in [ ] and the associated enteric disease was first reported in us swine only in early [ ] . however, the origin of pdcov infection in pigs and also the sudden emergence and route of introduction of this virus in the us remains unclear [ ] . pdcov may have only partially adapted to pigs and still retain some potential to infect different animal species, particularly poultry or other livestock, which can often have frequent contact with pigs in small-scale, backyard farms in the us. therefore, our aim was to determine whether calves are susceptible to infection with either the newly emerged pdcov or the swine enteric cov, pedv. fecal virus shedding, seroconversion and histopathology were evaluated in gnotobiotic (gn) calves orally inoculated with pdcov or pedv. the pdcov oh-fd virus was isolated and then serially passaged five times (p ) in llc porcine kidney (llc-pk) cells (atcc cl- ) [ ] . the virus was orally inoculated and propagated in a -day-old gn pig. the viral rna titer of oh-fd -p used as inoculum in the intestinal contents (ics) was . log genomic equivalents (ge)/ml. the wild-type us pedv strain pc a, propagated in a gn pig [ ] , was also used in this study. all ics were negative for other enteric viruses, such as rotavirus groups a-c, by pcr/rt-pcr [ ] . near-term angus jersey crossbred gn calves were delivered aseptically by caesarean section [ ] . eight -to -day-old calves were randomly assigned to three groups: pdcov infection (n= ; calves # - ), pedv infection (n= ; calves # - ), and mock (minimum essential medium [mem]; n= ; calf # , days of age) ( table ). calves # - were inoculated orally with . - . log ge of the oh-fd -p , and calves # - were inoculated orally with . - . log ge of the pc a (table ) . after viral inoculation, we monitored clinical signs daily. diarrhea was assessed by scoring fecal consistency as follows: =solid; =pasty; =semi-liquid; =liquid, with scores of or more considered diarrheic. calves # (pdcov) and # (pedv) were monitored for long-term clinical signs and virus shedding until post-inoculation day (pid) - . the other inoculated or mock-infected calves were kept for short-term studies and were euthanized for histopathological examination at acute to mild stages (pids , or ) of viral infection (table ) . rectal and nasal swabs or serum samples were collected and prepared as described previously [ , ] . rectal and nasal swabs were diluted : and : , respectively, in mem. virus rna was extracted using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. titers of virus shed in feces were determined by qrt-pcr using the onestep rt-pcr kit (qiagen, valencia, ca, usa) [ , ] . the detection limit of qrt-pcr for pdcov was ge per reaction, corresponding to . , . , and . log ge/ml of pdcov in nasal, rectal swab, and serum samples, respectively [ ] . the detection limit of qrt-pcr for pedv was ge per reaction, corresponding to . log ge/ml of pedv in rectal swab samples [ ] . pdcov oh-fd -p (passage five) and pedv pc -p (passage ) viruses, grown on llc-pk or vero cells, respectively, were used to infect llc-pk or vero cells, respectively, in -well plates. the viral antigens expressed were detected by indirect immunofluorescence assay (ifa) [ , , ] . a multiplicity of infection of . was used for each viral inoculation in llc-pk and vero cells treated with and lg/ml of trypsin, respectively [ , , ] . the cell culture conditions used to infect llc-pk or vero cells with each virus were described previously [ , , ] . characteristic cytopathic effects (cpe) [ , , ] were monitored regularly in inoculated llc-pk or vero cells. when cpe was pronounced, pdcov-inoculated llc-pk (pid ) and pedv-inoculated vero cells (pid ) were fixed with % ethanol at °c overnight for ifa. the fixed cells were washed with . m phosphate buffered saline (pbs) (ph . ). blocking was performed with buffered solution of casein ( power block tm universal blocking reagent, biogenx, ca, usa) in distilled water for hr at room temperature. four-fold serial dilutions, starting at : , of the paired serum samples of calves # and # , obtained at pid and pid - were added to the wells and the plates were incubated overnight. swine hyperimmune antiserum against pdcov ( : ) or monoclonal antibody against the spike (s) protein of pedv ( c - ) ( : ), with the appropriate detection antibodies, were added to the wells as ifa positive controls [ , , ] . goat polyclonal antibodies specific for bovine, swine or mouse igg (whole igg) conjugated to fluorescein (kpl, gaithersburg, md, usa) were diluted : in . m pbs. the plates were incubated for hr at °c. the stained plates were evaluated using a fluorescence microscope. the antibody titers were expressed as the reciprocal of the highest serum dilution in the wells that were positive for pdcov or pedv antigen detection. formalin-fixed or frozen small (duodenum, jejunum and ileum) and large (cecum/colon) intestinal tissues and other major organs were examined macroscopically and histologically and tested by if for pdcov or pedv antigens [ , , ] . tissues from the negative control calf (# ) were tested for histological comparisons and as an if negative control. tissues from pdcov-or pedv-infected gn pigs at pid were also tested as positive controls [ , ] . clinical observations revealed that none of the gn calves inoculated with pdcov (calves # - ), pedv (calves # - ), or mock (calf # ) exhibited diarrhea or other clinical signs throughout the course of the experiment (fig. ) . pdcov rna was first detected in fecal samples at pid (calves # and ) or pid (calves # and ) by qrt-pcr (table ) . fecal pdcov rna titers, ranging from . to . log ge/ml, peaked at pid (calf # ), pid (calf # ), or pid - (calf # ) ( table ) . calf # , was followedup long-term and had prolonged viral rna shedding until pid (fig. ) . the highest fecal pdcov rna titer of calf # was at pid ( . log ge/ml), and the titers decreased progressively thereafter (fig. ) . on the other hand, none of the gn calves inoculated with pedv shed detectable pedv rna in the feces at pids - (calves # and # ) and pids - (calf # ). the negative control (calf # ) did not shed detectable pdcov or pedv rna in the feces during the experiment. nasal swab or serum samples collected from pdcov-inoculated calf # were also tested by qrt-pcr. serum samples were collected at pids and , and nasal swab samples were collected at pids , , , , and . no pdcov rna was detected in the serum (\ . log ge/ml) and nasal swab (\ . log ge/ml) samples tested at the time-points indicated. ifa showed that calf # had serum igg antibodies (titer of , ) against pdcov oh-fd at pid , indicating seroconversion. large numbers of if-stained cells were consistently observed when pdcov-infected llc-pk cells were incubated with the serum diluted : to : , (fig. ) . no pdcov-specific igg antibodies were detected in the prebled serum samples of calf # and negative control calf # (fig. ) . in pedv-inoculated calf # , there were no detectable pedv-specific igg antibodies in the serum samples at pid and pid (fig. ) . light microscopy analysis revealed that none of the pdcov-or pedv-inoculated calves had major histological changes in the intestine. additionally, by if, no pdcov or pedv antigen-positive cells were observed in the small and large intestinal tissues of pdcov or pedv-inoculated gn calves, while the intestinal tissues (mainly villous enterocytes) of pdcov or pedvinfected gn pigs were found to be positive for pdcov or pedv antigen in earlier studies [ , ] . our study demonstrated that gn calves orally inoculated with the pdcov strain oh-fd (ics of cell-culture grown pdcov from infected gn pigs) develop an acute infection with persistent fecal pdcov rna shedding and pdcovspecific serum igg antibody responses, but show no signs of significant intestinal lesions or clinical disease [ ] . these observations are similar to a previous report showing that, human sars-cov strain urbani, prior to its adaptation to mice (ma strain), replicated in the lungs of young mice, as indicated by their lung homogenates testing positive by qrt-pcr. however, the urbani strain-inoculated mice did not show any clinical signs, but only mild histological lesions with low expression of viral antigens [ ] . in our study, there were no detectable fecal viral rna shedding, virus-specific serum igg antibody responses, histological lesions, and clinical disease in gn calves orally inoculated with the pedv strain pc a (ics of wild-type pedv-infected gn pigs) [ ] . for the past years, pedv has been found only within pigs, indicating that pedv may have only adapted to pigs [ ] . in contrast, pdcov may not yet be completely adapted to pigs, and the disease caused by pdcov has emerged only - years ago. pdcov-infected pigs appeared to shed less pdcov rna and had a lower peak of viral titer in the feces, compared with experimental pedv infections [ , ] , suggesting that there is a lower replication of pdcov in the intestine of pigs, possibly due to its incomplete adaptation to pigs. in a molecular surveillance study in china and hong kong conducted in - , deltacoronaviruses (dcovs) were detected only in pigs and wild birds [ ] . however, dcovs were detected previously in rectal swabs of small mammals, such as asian leopard cats and chinese ferret badgers, in live-animal markets in china, in - [ ] . the helicase and s genes of the covs isolated from the wild small mammals were closely related to those of pdcov [ ] , suggesting that a potential interspecies transmission event may have occurred leading to dcov transmission between wild small mammals and pigs. however, further studies are needed to define the potential role of small mammals as an intermediate host of pdcov and the mechanisms of interspecies transmission of dcovs between small mammals and domestic pigs or wild birds. our data also support the fig. persisting fecal viral rna shedding of calf # inoculated with pdcov strain oh-fd -p , but without diarrhea. calf # was inoculated orally with . log ge of the gnotobiotic pig-passaged oh-fd -p . it was monitored for long-term clinical signs and virus shedding at pids to . rectal swabs were collected daily throughout the experiment. the pdcov fecal shedding titers were determined by qrt-pcr. the dotted line indicates the detection limit ( . log ge/ml) of the qrt-pcr potential ability of the newly emerged pdcov to infect another animal species, such as cattle [ ] . however, our study did not demonstrate pdcov's ability of direct interspecies transmission between pigs and cattle. the s protein of covs is critical for regulating interactions with specific host cell receptor glycoproteins to mediate viral entry [ ] . based on our data, it is possible that interactions between pdcov s protein and the host cell receptor or co-receptor binding may occur in the bovine intestine, whereas this may not be the case for pedv s protein. the major cellular receptors of pedv and pdcov in pigs are unknown [ , ] . the cov s proteins can be divided into two functional subunits, s and s . the s is involved in the receptor recognition and binding via the n-terminal or c-terminal virus receptor binding domains. therefore, proteolysis between s and s is a critical determinant for cov tropism and pathogenesis. the cov s proteins can be cleaved by different animal proteases [ ] . the extent of cleavage of pedv and pdcov s proteins might differ in the bovine intestine, possibly depending on their adaptation to the bovine intestinal environment; for example, exogenous proteolytic enzymes of bovine origin may influence pedv or pdcov s proteinhost cell receptor binding. however, these assumptions should be verified by further studies. in our study, pdcov-inoculated calves showed acute fecal viral rna shedding, followed by progressively decreased titers thereafter. there were also no pdcov rnapositive nasal or serum samples from pdcov-inoculated calf # , indicating that pdcov infection may be limited to the intestine, similar to pdcov infections in pigs [ ] , although pdcov-infected pigs also showed viremia (viral rna in serum) [ ] . our study did not identify which type of cells in the intestines of inoculated calves could be the target for pdcov replication, resulting in fecal shedding. our if staining experiments identified pdcov antigen-positive cells in intestinal tissues from pdcov-infected gn pigs used as positive controls. similar to the giii. bovine norovirus (cv -oh strain) that was shown to cause fecal virus rna shedding in infected gn calves, but in the absence of histological lesions or viral antigen/rna in the intestine [ ] , pdcov did not induce major histological changes in the intestine of gn calves, such as necrosis of intestinal epithelium or villous atrophy. this result is also in line with data showing no pdcov-positive cells in the intestinal epithelium. based on these observations, pdcov may have infected the intestine of calves as an attenuated virus that does not cause clinical disease and histological lesions, with low levels of viral antigen detected in enterocytes in inoculated animals. nevertheless, our study did not clearly rule out an abortive infection with limited pdcov replication in calves. in addition, interestingly, no pedv-specific igg antibodies were detected by ifa in the serum of pedv-inoculated calf # at pid . however, a virus neutralization (vn) assay using a pedv strain iowa [ , ] , showed low pedv neutralizing activity in the sera of calf # at pid and prebled prior to pedv inoculation, with vn titers of . (pid ) and . (prebled). additional studies using larger numbers of serum samples are needed to confirm whether bovine sera have pedv antibodies or neutralizing substances. collectively, our data indicate that calves are susceptible to infection with the newly emerged pdcov, but not with the swine enteric cov, pedv. however, the infectivity of pdcov in calves was limited, as manifested by no clinical disease and histological lesions, possibly due to its incomplete adaptation to the bovine intestine. other animal species with frequent contacts with pigs, such as poultry, also need to be monitored for potential infectivity by pdcov. detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain oh-fd altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus pathogenesis of giii. bovine norovirus, cv -oh/ /us strain in gnotobiotic calves pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc a in conventional -day-old nursing piglets vs. -day-old weaned pigs pathogenicity of porcine deltacoronavirus strains in gnotobiotic pigs porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis characterization of porcine epidemic diarrhea virus isolate us/iowa/ / infection in -day-old cesarean-derived colostrum-deprived piglets 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 mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhoea virus, but promotes its infectivity via aminopeptidase activity proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research epidemiology, genetic recombination, and pathogenesis of coronaviruses detection and genetic characterization of deltacoronavirus in pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed.conflict of interest 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. all authors have seen and approved the manuscript. key: cord- -g ntyddb authors: zhu, yu; wang, gui-hua; cui, yu-dong; cui, shang-jin title: establishment of a nanoparticle-assisted rt-pcr assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: g ntyddb porcine epidemic diarrhea virus (pedv) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. open reading frame (orf ) is the only accessory gene in the pedv genome. previous studies have indicated that pedv vaccine strains have a partial deletion in orf . in this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted rt-pcr) assay targeting the orf of pedv was developed to distinguish pedv field strains from attenuated strains by using a specific pair of primers. the pcr products of field strains and attenuated strains were bp and bp in length, respectively. the sensitivity and specificity of this assay were also assessed. the nanoparticle-assisted rt-pcr assay was - times more sensitive than the conventional rt-pcr assay, with no cross-reactions when amplifying porcine pseudorabies virus (prv), porcine circovirus type (pcv ), classical swine fever virus (csfv), porcine parvovirus (ppv), porcine reproductive and respiratory syndrome virus (prrsv), porcine rotavirus (rv), and porcine transmissible gastroenteritis virus (tgev). the nanoparticle-assisted rt-pcr assay we describe here can be used to distinguish field strains from vaccine strains of pedv, and it shows promise for reducing economic loss due to pedv infection. porcine epidemic diarrhea virus (pedv) is a highly pathogenic and lethal virus. it belongs to the genus alphacoronavirus of the family coronavirus. its main clinical features are vomiting, diarrhea, dehydration and high mortality in suckling piglets [ ] . in the s, pedv first appeared in the united kingdom [ ] . subsequently, it was reported in europe and asia [ , ] . since , pedv has caused large-scale diarrhea outbreaks in pigs and a - % fatality rate in suckling piglets in china [ , ] . recently, a pedv outbreak has rapidly swept throughout the united states in less than a year [ , ] . pedv has lead to significant economic losses worldwide, but there has been no specific or effective method available to keep the spread of pedv under control [ ] . pedv is an enveloped, positive single-strand rna virus. it has a large genome of approximately kb in size, which is composed of utr-orf -s-orf -e-m-n- utr, encoding four structural and four non-structural proteins [ ] . open reading frame (orf ) is the only accessory gene in the pedv genome. the orf of pedv has an important role in virulence. some studies have shown that the coronavirus orf -encoded protein forms an ion channel and modulates virus release, and orf gene silencing can reduce viral titers in vero cells [ , ] . further research has shown that deletions are present in orf of cell-adapted pedv strains when compared to field strains [ , ] . in asia, vaccines based on attenuated pedv strains have been approved [ , ] , including the cv and zj strains in china, the p- v strain in japan, and the kped- and dr strains in south korea. these cell-adapted vaccine strains have a -bp deletion in the orf gene. this feature can be used to distinguish natural field strains from attenuated vaccine strains. the current methods of detecting pedv include electron microscopy, virus isolation, reverse transcription polymerase chain reaction (rt-pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), and real-time rt-pcr [ ] [ ] [ ] [ ] . these approaches have their own disadvantages. electron microscopy and virus isolation are highly specific and sensitive, but they are timeconsuming. samples get contaminated easily in the lamp assays, and real-time rt-pcr requires special instruments. in this study, a nanoparticle-assisted rt-pcr assay was developed to distinguish pedv field strains from vaccine strains. similar to conventional rt-pcr assays, this method showed improved sensitivity and specificity. the advanced nanoparticle-assisted rt-pcr uses metal particles to create a better thermal conductivity environment, thereby reducing nonspecific amplification and facilitating specific amplification. moreover, the nanoparticle-assisted rt-pcr assay does not require specialized instruments, in contrast to the real-time rt-pcr assay [ ] . in this study, a pair of primers targeting the orf was designed, with binding sites at both ends of the orf gene deletion. the nanoparticle-assisted rt-pcr described in this study was developed to identify whether the virus strains in the samples are field pedv or vaccine strains, and it was also used to detect the virus in clinical samples. the pedv vaccine strain (zj ), field strain (hebei ), and the following viruses used in this experiment -porcine circovirus type (pcv- ), porcine productive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), transmissible gastroenteritis virus (tgev), rotavirus (rv), porcine parvovirus (ppv), and porcine pseudorabies virus (prv) -were obtained from the animal medical center dbn technology group. clinical samples from the small intestine were collected from diseased piglets in hebei. clinical samples were collected in accordance with the international guiding principles for biomedical research involving animals. dna and rna were extracted from the viruses and clinical samples using a tianamp virus genomic dna/ rna kit (beijing tiangen biotech company, beijing, china) according to the manufacturer's instructions. cdna synthesis was performed using transcript first-strand cdna synthesis supermix (beijing transgen biotech company, beijing, china). the dna and cdna samples were stored at - °c. primer premier . software was used to design the pedv nanoparticle-assisted rt-pcr primers. the full-length orf genes of the pedv vaccine strain (cv ) and wild strain (jsts ) were chosen based on published sequences in the genbank database (genbank accession numbers: gu and kc ) ( table ). the amplified products of primers were bp in size for the field strain and bp in size for the attenuated vaccine strain. the sequences of the orf gene were inserted into the vector pmd -t (takara biotechnology company, dalian, china) as standards. the recombinant plasmids were amplified in e. coli dh a and purified using an axypreptm plasmid miniprep kit (axygen biotechnology company, hangzhou, china). the plasmids were kept at - °c. a nano pcr kit (npk ) was purchased from gredbio (weihai, china). the nanoparticle-assisted rt-pcr system was used according to the manufacturer's instructions. the annealing temperature and primer concentration were optimized for the nanoparticle-assisted rt-pcr assay. the annealing temperature ranged from to °c. the forward and reverse primers (e and e at lm) were tested at volumes ranging from . to . ll in increments of . ll. the cycling conditions were as follows: °c for min, followed by cycles of °c for s, to °c for s, and °c for s, and a final extension at °c for min. amplified products were analyzed on % agarose gels. to assess the sensitivity of the method compared with conventional pcr, the recombinant plasmids containing inserts from pedv vaccine strains were quantified and serially diluted tenfold from . to . copies/ll. plasmids containing inserts from pedv field strains were quantified and serially diluted tenfold from . to . copies/ll. each diluted sample was used as a template and was tested using the optimized nanoparticle-assisted rt-pcr assay reaction parameters. the conventional rt-pcr assay was performed using the same primers and reaction parameters. amplified products were then analyzed on % agarose gels. dna or cdna samples from the following viruses were used to evaluate the specificity of the nanoparticle-assisted rt-pcr assay: ppv, pcv , prrsv, csfv, rv, tgev, and ?prv?. recombinant plasmids were used as positive controls. amplified products were analyzed on % agarose gels. clinical samples were also tested using the nanoparticleassisted rt-pcr assay. the samples of small intestine from piglets from hebei were tested. amplified products were analyzed on % agarose gels. optimizing the nanoparticle-assisted rt-pcr assay recombinant plasmids were used as templates in this nanoparticle-assisted rt-pcr assay. first, the optimal annealing temperature was determined and found to be °c. then, the optimal annealing temperature was used to determine the optimal primer volume, and the best volume of upstream and downstream primers ( lm) was . ll. the final -ll reaction system included ll of nano buffer, . ll each of the upstream and downstream primers ( lm), . ll of taq dna polymerase ( u/ll), ll of template dna, and ddh o to ll. the pcr reaction conditions were as follows: °c for min, followed cycles of °c for s, °c for s, and °c for s, and a final extension at °c for min. the final amplification (fig. ) could clearly distinguish the field and vaccine strains. sequence analysis showed approximately % sequence identity between the products of nanoparticle-assisted rt-pcr amplification and the reference sequence of pedv. the vaccine strain zj had an obvious sequence deletion in orf gene. the sensitivity of the nanoparticle-assisted rt-pcr assay was compared with that of the conventional rt-pcr by using a dilution series of recombinant plasmids as the amplification templates. the detection limit of the nanoparticle-assisted rt-pcr for the attenuated strain was . copies/ll ( fig. a) , and that of the conventional rt-pcr assay was . copies/ll (fig. b) . for the field strains, the detection limit of the nanoparticleassisted rt-pcr was . copies/ ll (fig. c) , and that of the conventional rt-pcr assay was . copies/ ll (fig. d) . these results indicated that the nanoparticle-assisted rt-pcr assay was - times more sensitive than the conventional rt-pcr. the specificity of the upstream and downstream primers was evaluated using other viruses, and the amplification results showed that there was no cross-reaction (fig. ) , indicating that the assay was specific. fourteen pig diarrhea clinical samples in total tested positive in the nanoparticle-assisted rt-pcr assay (fig. ) . the amplification products of four samples had the same size as that of the pedv vaccine strain, and pedv field strains were detected in nine samples. one sample was found to contain both the pedv vaccine strain and a field strain. since the farm animals were immunized with the inactivated pedv vaccine, we had reason to believe that fig. the size of the product of nano pcr amplification of the target gene, analysed by agarose gel electrophoresis. m, dl marker; , attenuated vaccine strain zj ; , negative control; , the field strain hebei nanoparticle-assisted rt-pcr assay for pedv they were infected with pedv after immunization. the results showed that the nanoparticle-assisted rt-pcr assay could distinguish pedv field strains from the attenuated strain in samples from infected pigs. pedv causes serious disease and economic loss in the pig industry [ ] . inactivated vaccines are effective for controlling the disease. however, pedv has been found to appear in immunized pigs since [ ] . since , pedv outbreaks have led to % mortality in newborn piglets throughout southern china, during which time more than million pigs have died [ ] . in diarrheal piglets, the positive rate for pedv is significantly higher than that for porcine transmissible gastroenteritis virus and porcine rotavirus [ , ] , suggesting that pedv is the main pathogen causing the diarrhea in china. attenuated strains are applied for vaccination in many asian countries, including china. the traditional identification methods may not provide accurate distinction of the attenuated vaccine strain for the field strain. the nanoparticle-assisted rt-pcr assay has been used for rapid and accurate detection of other pig viruses [ ] [ ] [ ] . the present study demonstrated that an effective nanoparticle-assisted rt-pcr assay targeting the orf gene of pedv was able to distinguish field strains from attenuated vaccine strains. this method was more sensitive than the conventional rt-pcr assay and suitable for testing clinical samples with low viral loads. the assay was specific for pedv, exhibiting no cross-reactivity against other viruses. the method could effectively distinguish the pedv field strains and the attenuated vaccine strains in the clinical samples. when testing clinical samples, in one case, a vaccine strain was found to coexist with a virulent strain. the reason for this phenomenon might be the failure of vaccine immunization, suggesting that there were potential threats of pedv infection after vaccination. in addition to this, there are concerns about the safety of the live attenuated ped vaccine for use in pregnant gilts and sows because a live attenuated vaccine virus might be able to revert to virulence under field conditions. nanoparticle-assisted rt-pcr assay (b) for detection of the attenuated vaccine strain zj . a serial tenfold dilution was used and analyzed by agarose gel electrophoresis. m, dl marker; , negative control; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll. sensitivity of the conventional rt-pcr assay (c) and nanoparticle-assisted rt-pcr assay (d) for detection of the field strain hebei . a serial tenfold dilution was used and analyzed by agarose gel electrophoresis. m, dl marker; , negative control; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll; , . copies/ll the application of the nanoparticle-assisted rt-pcr amplification technique is reported here for the first time to distinguish pedv field strains from vaccine strains. this approach will facilitate the identification of piglets that are infected with pedv field strains, the detection of latent infections, and the prevention of pedv outbreaks. prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds an apparently new syndrome of porcine epidemic diarrhoea chinese-like strain of porcine epidemic diarrheavirus an immunohistochemical investigation of porcine epidemic diarrhoea outbreak of porcine epidemic diarrhea in suckling piglets porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines fighting a deadly pig disease. industry, veterinarians trying to contain ped virus, new to the us isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the us receptor usage and cell entry of porcine epidemic diarrhea coronavirus binding characterization of determinants in porcine aminopeptidase n, the cellular receptor for transmissible gastroenteritis virus severe acute respiratory syndrome-associated coronavirus a protein forms an ion channel and modulates virus release characterisation of a recent virulent transmissible gastroenteritis virus from britain with a deleted orf a differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf pedv orf encodes an ion channel protein and regulates virus production molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate rapid and sensitive detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (pedv) to assess pedv transmission in growing pigs effect of temperature on the detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in fecal samples by reverse transcription-polymerase chain reaction nanopcr observation: different levels of dna replication fidelity in nanoparticle-enhanced polymerase chain reactions porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis molecular epidemiology of porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in south china a nanoparticleassisted pcr assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and genedeleted vaccine strains a new nanopcr molecular assay for detection of porcine bocavirus development of a nanoparticle-assisted pcr assay for detection of porcine epidemic diarrhea virus acknowledgements this work was supported by the agricultural science and technology innovation program (astip-ias ). conflict of interest none 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. key: cord- -zu ns yq authors: fennestad, k. l.; macnaughton, m. r. title: pleural effusion disease in rabbits. properties of the aetiological agent date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: zu ns yq the size and heat sensitivity of pleural effusion disease (ped) agent or virus (pedv) propagated in rabbits were examined. the infectious particles were estimated to be between and nm by filtration. residual infectivity of infectious serum was . per cent after heating at ° c for hours. pedv and the stockholm agent appeared identical concerning pathogenic and immunogenic properties by infection experiments and protection tests in rabbits. two of the three pedv isolates were less pathogenic but appeared immunogenically identical to pedv. the third isolate, obtained from the laboratory, which several years previously had supplied material for demonstration of the stockholm agent, differed from pedv in pathogenic and immunogenic properties. serological examinations of paired rabbit sera did not indicate any antigenic relationship between pedv and representative members of the two mammalian coronavirus antigenic groups. it is concluded that the aetiological agent of ped is a virus not belonging to the coronaviridae. pleural effusion disease (ped) agent is found as a passenger in rabbit testieular suspensions of treponema pallidum and causes subclinical to fatal infections in rabbits ( , , , ) . ped has been reported as a new disease ( ) and another name proposed for it is rabbit infectious cardiomyopathia ( ) . the disease has been known since the s ( , ) , but as yet the agent or ~arus (pedv) has not been convincingly demonstrated by tissue culture or electron microscopy. a k . l . pennestad and m. ~. macnaughton: remarkable feature of p e d is the long persisting viraemia ( , ) ; nevertheless, the infection induces clinical protection a n d there is also evidence indicating t h a t protective igg antibodies are produced as a result of the infection ( ) . during a survey in / , isolates were obtained from several laboratories. all these isolates induced clinical protection to challenge with pedv, except one (sbl) from stockholm which induced only partial protection ( ) . s~all et at. ( ) have suggested t h a t the stockholm agent, which was brought to the j o h n s hopkins university school of medicine, baltimore in ( ) , might be a coronavirus antigenically related to h u m a n coronaviruses (hcvs) e and oc . this concept was supported by oster~aus et al. ( ) who observed coronavirus-like particles in rabbits infected with p e d v . however, in this s t u d y we show t h a t p e d v appear to be smaller and more heat resistant t h a n eoronaviruses, and t h a t p e d v and related isolates are antigeniemly unrelated to representative m a m m a l i a n coronaviruses by enzyme-linked i m m u n o s o r b e n t assay (elisa). stock pedv consisted of pooled rabbit sera obtained hours after subcutaneous inoculation with pleural fluid or infectious serum. a low virulent variant of pedv from the blood of a rabbit, six months after infection was also used ( ). stock of this variant was infectious serum h'om an l tth serial rabbit passage, obtained hours after inoculation. the stockholm agent was kindly supplied in by dr. j. d. small, nih, bethesda, u.s.a. as freeze dried infectious serum . three other isolates used were from different laboratories and designated sill, paris i and minneapolis (table ) . stocks of the stockholm agent and the three isolates were from the first or second rabbit passage, prepared in the same manner as for pedv. all stocks were stored at ~- ° c. the number of rabbit-infectious doses per ml of the virus stocks was determined by making -fold dilutions in pbs (ph . ) using one to four rabbits per dilution. the term rabbit-infectious dose refers to a dose capable of producing typical clinical responses, i.e. fever together with uveitis or death with characteristic necropsy findings and/or clinical protection to challenge with pedv days after inoculation. an exception to this procedure was the titration of sbl, where the homologous stock virus was used for challenge. lzepresentative m e m b e r s of t h e two m a m m m i a n eoronavirus antigenic groups were used. these were hcvs e ( ) a n d cv paris ( ) , t h e l a t t e r being closely r e l a t e d to i-icv oc ( ) . all m a m m a l i a n coronaviruses, apart, from one or two possible exceptions t h a t h a v e n o t b e e n fully charaeterised, are antigenicmly r e l a t e d b y e l i s a to one or t h e o t h e r of these strains ( , , , ) . h c v e was g r o w n in m r c c o n t i n u o u s cells ( ) a n d cv paris was g r o w n in i t r t cells ( ) . t h e cells were frozen a n d t h a w e d once a n d t h e resulting" suspensions were clarified a t × g for m i n u t e s . t h e p r e p a r a t i o n s of h c v e a n d cv p a r i s c o n t a i n e d b e t w e e n a n d s particles p e r ml, as d e t e r m i n e d b y electron microscopy. t h e e l i s a was b a s e d on m e t h o d s described previously for d e t e c t i n g t t c v antibodies in r a b b i t ( ) a n d h u m a n ( , ) sera. samples of p e d v stock were d i l u t e d : in p b s (ph % ) a n d passed serially t h r o u g h millipore filter m e m b r a n e s g r a d u a t e d in porosity from to n m . samples of filtrates were t a k e n for t i t r a t i o n in r a b b i t s a f t e r passage t h r o u g h , , a n d n m filters. similar e x p e r i m e n t s were done w i t h i-icv e a n d polio virus, t y p e , s t r a i n s a u k e t t . t h e i n f e c t i v i t y of t h e i-icv e samples were d e t e r m i n e d b y fluorescent focus assay (t ). t h e s a u k e t t s t r a i n was grown a n d t i t r a t e d in p r i m a r y m o n k e y k i d n e y cells. t h e culture was c o n c e n t r a t e d a n d purified, b u t was diluted : . (the e n t e r ovirus d e p a r t m e n t a t s t a t e n s s e r u m i n s t i t u t supplied ~/he polio virus a n d p e r f o r m e d t h e i n f e c t i v i t y titrations). two ml c r y o t u b e s c o n t a i n i n g p e d v stock, u n d i l u t e d a n d d i l u t e d t : in p b s (ph . ), were s u b m e r g e d in a stirred w a t e r b a t h at o a n d o c. a t selected i n t e r v a l s t h e r e a f t e r t h e t u b e s were r e m o v e d a n d t h e c o n t e n t s i m m e d i a t e l y t i t r a t e d in r a b b i t s using chilled p b s diluent. albino rabbits, aged -- m o n t h s , from t h e closed colony at s t a t e n s s e r u m i n s t i t u t (ssc : cpti), were used. t h e m a j o r i t y of these animals h a d b e e n used once for p y r o g e n t e s t i n g of p r o t e i n fractions of h u m a n blood. all inoculations were m a d e s u b c u t a n e o u s l y . f o r serological e x a m i n a t i o n paired sera were o b t a i n e d before a n d days after p r o v e n infections of groups of fore" r a b b i t s w i t h p e d v , s t o c k h o l m agent, sbl, paris i, or minneapolis. t h e sera were stored a t -- ° c until examined. u n i f o r m groups of r a b b i t s were infected w i t h t h e same dose of t h e various isolates. t h e s t o c k h o l m a g e n t was passaged serially in r a b b i t s a t i n t e r v a l s of -- days a n d days as described for p e d v ( ). t h e results of t h e p e d v passages are s h o w n in table for comparison. passages of t h e t h r e e p e d v isolates were done in t h e same w a y b u t only a t i n t e r v a l s of ~ days. t h e i n o c u l u m for t h e first passage was t h e original m a t e r i a l while ml of blood or pleural fluid was used for t h e s u b s e q u e n t passages. t h e low v i r u l e n t v a r i a n t of p e d v was also passed serially in rabbits, w i t h a n i n t e r v a l of days b e t w e e n passages. infectious serum diluted : and with a titre of passed the nm filter without loss in infectivity. the virus also passed through nm membrane, but with a to -fold loss in titre. no detectable infectivity passed nm filter. infectious ttcv e particles did not pass the nm filter, and polio virus particles passed the and nm filters with a -fold loss in infectivity. these findings indicate that p e d v particles are between and nm, smaller than coronavirus particles and larger than polio virus particles. the rate of inactivation of undiluted and diluted infectious serum at ° c was about the same and the calculated half life of infectivity of the two preparations was . hours (fig. ). there was a to -fold loss in infectivity of the diluted preparation after days at ° c, but after days residual infectivity was . per cent. stock virus kept at -- ° c for months showed no loss of infectivity. the pathogenic properties of the isolates were compared directly (table ) and by passage in rabbits to simulate the "natural" infection, i.e. a concomitant infection of rabbits used for propagation of pathogenic treponemes (table ) . both the stockholm agent and pedv produced high mortality with almost the same incidence of fever and uveitis among the survivors. when passed at an number of rabbit-infectious doses b all survivors failing to show fever or uveitis were protected when challenged with pedv, except the sbl-infeeted animals i n t e r v a l of days, there was no m o r t a l i t y a n d the clinical signs of p e d were reduced. this indicates t h a t the two isolates are similar in their pathogenesis and persistence in blood. minneapolis a n d the low virulent v a r i a n t of p e d v produced a febrile response, b u t rarely detectable uveitis. the clinical response by the sbl or paris i was intermediate between these responses. a distinguishing feature of the sblinfection was the delayed onset of uveitis, i.e. t h --l l t h day post-inoculation as compared with r d -- t h days for the other isolates. as measured by clinical response only p e d v appeared to increase in virulence during passage. when rabbits were challenged with pedv days after infection, they were nearly always chnically protected, the only exception being the sbl-infected animals. pedv and the stockholm agent, which both caused high mortality in rabbits, were not used for active immunization. minneapolis was used for challenge in only one experiment since this isolate does not cause typical p e d ( table ). the immunizing infections of the various groups of rabbits produced the same clinical syndromes as observed in the infection experiments. when challenged all the rabbits were protected except for the sbl-infected rabbits challenged with pedv or the stockholm agent. the majority of animals in these two groups developed f e d symptoms, although none died. the sbl-infected rabbits challenged with paris i did not produce ped symptoms, but several showed a transient ephemera] fever. challenge with sbl did not cause clinical signs of ped. these results together with those of the infection experiments demonstrate a similarity in immunogenicity between pedv and the stockholm agent, and between paris i and minneapolis. the immunogenicity of sbl is evidently different from pedv and the stockholm agent. a inoculum expressed as number of rabbit-infectious doses, given subcutaneously b numerator equals number of protected rabbits and denominator number of challenged rabbits c not done hcv e and cv paris were used as antigens in e l i s a to detect antibody rises to coronaviruses in paired sere from rabbits infected with the isolates. a ratio of or more in the absorbance values obtained with postinocnlation serum compared to preinocnlation serum at the same serum dilution was considered to represent a significant antibody rise. using this criterium no antibody rises to hcv e were detected in any of the paired sere, whereas only one paired sere from an sbl-infected rabbit showed an antibody rise to cv paris, i.e. it had a ratio of . . the same results were obtained when the same or other pairs of sera were examined by a neutralisation test with hcv e (kindly performed by dr. sylvia e. reed, central public health laboratory, london, england) and in a cf test with hcv c antigen (kindly performed by dr. t. hovi, university of helsinki, finland). i t has been suggested that the stockholm agent and p e d v are similar on the basis of history and pathology ( , , ) , but they have never been compared directly. this study shows that they are very similar if not identical. the results also indicate that the less pathogenic paris i and minneapolis are similar to pedv, and that sbl is probably closely related to pedv, but differs in its pathogenic and immunogenic properties. this agrees with the observations at isolation, although paris i did not cause mortality ( ) . sbl was isolated in from treponema-infected rabbits from the laboratory, that had supplied the rabbit material from which the stockholm agent was isolated in ( ) . attempts in this laboratory to remove the treponemes from the stockholm agent were made on several occasions. this was done by passaging contaminated treponemal suspensions through hamsters, which are not susceptible to pedv, but which allow the multiplication of treponemes ( , ) . these efforts appeared to be successful since intercurrent rabbit mortality fell to less than one per cent after . however, the isolation of sbl two years later suggests this was a failure and that the reduction in intercurrent mortality was due to a mutational change in the stockholm agent. the apparent stability in the virulence of the isolates during the to rabbit passages may well be due to the number of passages. pedv had rabbit passages over a -year period and during this time the annual mortality increased from to per cent. sh~all et al. ( ) and oster~a~-s et al. ( ) suggested a role for coronaviruses in ped, while the latter and lapierre et al. ( ) also reported the association of coronavinls-like agents with intestinal infections in rabbits. both s ~ali~ et al. and ostei~i~avs st al. detected coronavirus-like particles in infectious serum. however, in our hands no coronavirus-like particles were detected by electron microscopy in rabbits infected with p e d v (fe~nestad et al., unpublished results), and by filtration infectious pedv particles were clearly smaller than infectious hcv e particles. the heat stability of pedv, which is in agreement with s~ai~l et al. ( ) , was greater than that reported for coronaviruses ( , ) . small et al. used a cf assay to show that antigen(s) :in infectious serum displayed a cross-reactivity with hcvs e and c and that hyperimmune sera from convalescent rabbits contained antibodies to hcv e. these results are surprising as these hcvs have been shown to be antigcnically unrelated ( , t , ) . our elisas show that p e d v does not generally produce antibodies related to hcv e and cv paris. one of rabbits showed a positive reaction to cv paris, but this is probably not due to infection with pedv, as normal rabbit sera contains antibodies to both cv paris and hcv c , but not hcv e, indicating the existence of a possible rabbit coronavirus antigenically related to cv paris and hcv oc (maonaughto~¢, unpublished results). k . l . fennestad and m. r. i~¢iacnaughton: b o t h s~l l et al. ( ) a n d steri~aus et al. ( ) left open t h e possibility t h a t t h e cause of p e d m i g h t be a virus different f r o m coronavirus. our findings agree w i t h this i n t e r p r e t a t i o n . pleural effusion disease in rabbits. clinical and post morton observations pleural effusion disease agent as passenger of treponema pallidum suspensions from rabbits pleural effusion disease in rabbits. observations on viraemia, i m m u n i t y and transmissibility f e v e r after inoculation of rabbits with treponema pallidum d e m o n s t r a t i o n of a virus-like agent contaminating material containing the stockholm substrain of the nichols pathogenic treponema pallidum screening out a virus-like agent from the testicular suspension of the nichols pathogenic t. pallidum. w i t h observations on certain characteristics of the agent a new virus isolated from the h u m a n respiratory tract prevalence of h u m a n coronavirus a n t i b o d y in the population of southern iraq spontaneous deaths a m o n g rabbits inoculated with treponema pallidum less t h a n weeks before ~.: e n z y m e -l i n k e d i m m u n o s o r b e n t assay for coronaviruses icicv e and m h v e n z y m e -l i n k e d immunosorbent assay for detection of antibody in volunteers experimentally infected with h u m a n eoronavirus e group viruses preliminary report on the observation of a eoronavirus in the intestine of the laboratory rabbit structural and antigenic relationships between human the genome of h u m a n eoronavirus strain e two antigenic groups of h u m a n coronavirus d e t e e t e d by using enzyme-linked i m m u n o s o r b e n t assay. infect. i m m u n ik~-i i n t e r c u r r e n t death of rabbits after inoculation with treponema pallidum in the netherlands. th clas syrup. u t r e c h t coronavirus-like particles in laboratory rabbits with different syndromes in the netherlands rabbit cardiomyopathy associated with a virus antigenically related to h u m a n coronavirus strain e une dpid mie d'ent rocolites ulc ron crosantes en maternit coronaviridae : second report the biology and pathogenesis of coronaviruses received september , key: cord- -ubrqy zf authors: payne, h. r.; storz, j. title: analysis of cell fusion induced by bovine coronavirus infection date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ubrqy zf polykaryon formation in bovine fetal spleen (bfs) cells infected with bovine coronavirus l occurred only in media supplemented with trypsin. a single to h trypsin treatment h and later after infection induced formation of polykaryons. trypsin treatment at ph . and . induced polykaryons while treatments at lower or higher ph levels did not. cell fusion activity was partially suppressed by the presence of antibody. infection of animal cells by enveloped viruses involves fusion between the viral envelope and a cell membrane. the fusogenic potential of viruses was studied in vitro with systems using erythrocytes, cultured cells, and liposomes as target membranes [ , , , , ] . membrane fusion is induced by paramyxoviruses at neutral ph, which implies that these viruses enter the host cell by fusion of the viral envelop with the plasma membrane at the cell surface [ , ] . in contrast, cell fusion activities of semliki forest virus, influenza virus, and vesicular stomatitis virus strictly depend on acidic ph levels [ , , , - . low ph is used experimentally to simulate conditions existing within the acidic intracellular vesicles where penetration by these viruses is thought to occur. cell fusion and polykaryon formation in cultures infected with bovine coronavirus (bcv) occur late in the virus replication cycle. in some types of host cells, polykaryocytosis is dependent on the presence of trypsin during the course of bcv replication [ ] . the membrane events of virus-induced cell fusion and virus-cell fusion during entry appear to be based on the same principle [ ] . the mode of bcv penetration of the host cell has not been clearly defined. we analyzed the trypsin-dependent cell fusion of bcv-infected bfs cells and found that the range of ph . to . was optimal for polykaryon formation. stock preparations of the mebus strain l of bcv [ ] were propagated in the human rectal tumor cell line hrt- [ ] . virus stocks were generated from cells infected at a multiplicity of . pfu per cell, incubated for to days at °c in dulbecco's modified eagle medium (dmem) buffered with mm nahco , and harvested by freeze-thawing. viral titers in these preparations ranged from l to pfu per ml. the d strain of bfs cells was maintained in minimal essential medium (mem) buffered with mm hepes and supplemented with % fetal calf serum. cell monolayers were grown in c m flasks to analyze the effects of trypsin and trypsin inhibitors on bcv replication. the cells were inoculated with bcv at a multiplicity of pfu per cell, incubated in mem containing trypsin ( . ug per ml; sigma chemical company) treated with l-l-tosylamide- -phenylethyl chloromethyl ketone, an inhibitor of chymotrypsin activity. trypsin-treated and untreated cultures were also exposed to soybean trypsin inhibitor ( . gg per ml; sigma chemical company) during the period of trypsin treatment. after h, the cultures were subjected to two freeze-thaw cycles and disrupted on ice by sound treatment (branson sonic power company) for sec to release the virus. virus yields were determined by plaque assays and hemagglutination assays. monolayers of bfs cells were grown in -well plates for analysis of virus-induced polykaryon formation. cultures infected at a multiplicity of pfu per cell were maintained in mem with and without the addition of . ~tg of soybean trypsin inhibitor per ml. the cultures were incubated for h of infection, fixed for min in bouin's fixative and stained with giemsa. the cells were examined microscopically with a x objective, and the extent of cell fusion was scored by counting the number of polykaryons with or more nuclei in randomly selected fields. experiments were conducted to identify the time period during which fusion of bcvinfected bfs cells is mediated by trypsin. each infected monolayer was exposed to trypsin for -, -, or -h intervals during the period of virus replication. after h, the monolayers were fixed, stained, and scored for polykaryon formation. the effects of various ph levels on polykaryon formation were determined. the monolayers were infected as described above and incubated in mem buffered with mm hepes and mm mes at ph . , . , and . for h at °c without trypsin. the cells then were treated for h at °c with . ~tg of trypsin per ml of mem at various ph levels. the trypsin treatment was terminated and the monolayers were incubated for an additional h in trypsin-free medium at ph . before fixation and staining. antibody suppression of cell fusion was tested with igg fractions of rabbit anti-bcv serum and of normal rabbit serum that were obtained by protein a-sepharose column chromatography. the ability of anti-bcv igg to affect polykaryon formation at °c was tested by two procedures: (i) infected cell monolayers were incubated for h in trypsin-free mem then exposed to mem that contained antibody freshly diluted with trypsin containing medium. these monolayers were fixed and stained after h of infection. (ii) monolayers were infected for h and then treated with antibody for h in the absence of trypsin. the cells were covered with medium containing trypsin after the antibody treatment, incubated for an additional h, and then fixed. polykaryon formation in bfs cells required the presence of trypsin at some time during virus replication. cell fusion failed to occur in infected cultures without trypsin or when soybean trypsin inhibitor was present during trypsin (fig. ) . a single -to -h trypsin treatment h and later after infection induced formation of polykaryons. these polykaryons generally were smaller and contained fewer nuclei per cell than the fusion products of continuous trypsin treatment. intervals of trypsin treatment that ended prior to h after infection produced less than % polykaryocytosis (fig. ) . the period of maximal enhancement by trypsin treatment coincided with the observed onset of cell fusion in cultures treated continuously with trypsin. the optimal time may be related to the appearance of a critical level of the bcv fusion proteins on the cell surface. trypsin treatments that began at or h induced fewer polykaryons, possibly, because insufficient time was available for the fusing cells to coalesce and become visibly multinucleated. the greatest number of polykaryons ( to per field) developed in monolayers treated for a or h interval during the period between and h after infection. the brief trypsin treatment was used to test for the optimal activity level to avoid possible fluctuations in ph that might occur during a prolonged treatment. trypsin treatment at ph . and . resulted in % and % polykaryocytosis, respectively, while treatments with media at lower or higher ph levels failed to support polykaryon development. this result indicated that the fusion function of bcv did not depend on acidic conditions. fusion suppression by anti-bcv antibody was demonstrated by two different experimental protocols. exposure at h after infection to medium containing both trypsin and antibody reduced polykaryon formation by %. greater reduction occurred when infected monolayers were pretreated with the antibody. pre-incubation with the antibody before trypsin treatment suppressed polykaryon formation by % over similar infected monolayers incubated with normal rabbit serum igg. the suppression by antibody indicated that coronaviral components were involved in cell fusion. trypsin treatments of bcv-infected bfs cells during the time of maximal fusion enhancement were effective only with media of ph levels . to . . trypsin was added in this experiment at . gg per ml, a level fold greater than that needed for cell fusion with continuous treatment at ph . . the apparent ineffectiveness of treatments at acidic ph levels may be related to the optimal ph range for trypsin activity. further investigation is necessary to determine the full range of ph levels at which the viral fusion protein is active. our results thus indicate that bcv-induced cell fusion occurs at alkaline ph levels. therefore, the fusion protein of this bcv is not strictly dependent on ph levels of or less for activity. trypsin-treatment has a pronounced effect on bcv infectivity when limiting viral dilutions are employed [st. cyr-coats et al., submitted for publication]. this amplification of bcv infectivity is apparently unable to account for the trypsin dependence of cell fusion activity. treatment with trypsin or trypsin inhibitor under our conditions of single cycle virus replication increased the hemagglutination titer but had only moderate effects on the yield of infectious virus. trypsin modification of bcv proteins appears to activate the viral fusion factor without producing a net change in the total number of infectious particles under these conditions. the viral component responsible for trypsin-activated cell fusion is most likely expressed at the plasmalemma of the infected hrt- cell [payne et al., submitted for publication]. cells infected with bcv fused in slightly basic environments. in this respect, the fusogenic potential of bcv resembles the paramyxovirus type of fusion activity. the trypsin requirement of the bcv fusion factor may involve a maturational cleavage of a coronaviral component analogous to the f protein h.r. payne and j. storz of paramyxovirus [ , , ] . although the fusion protein of bcv has not been clearly identified, recent work revealed that trypsin treatment of bcv-infected bfs cells or virions purified from this system modifies the kd protein of the bcv envelope with a concommitant emergence of the kd protein, a change observable only under reducing conditions [ ] . the membrane fusion activity of another coronavirus, mouse hepatitis virus, also occurs at neutral and slightly alkaline ph levels [ - . like paramyxoviruses, coronaviruses may be capable of penetrating the host cell by fusion with the plasma membrane during infectious entry. the fusion activity of alphaviruses, rhabdoviruses and orthomyxoviruses strictly requires exposure of the virus-cell complex to acidic conditions [ , , t , ] . cell fusion can be induced at physiological ph by paramyxoviruses, either among cells treated under conditions of fusion from without, or among infected cells with fusion from within [ , ] . a specific glycoprotein component (f) of the paramyxovirus envelope is known to be involved in this membrane fusion activity. the precursor form of the f protein which is expressed at the surface of nonpermissive cells can be activated by treatment with an exogenous protease that cleaves the precursor at a specific site [ , ] . the role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses initial stages in infection with animal virus sindbis virus-mediated cell fusion from without is a twostep event la crosse bunyavirus can mediate ph-dependent fusion from without fusion of sendai virus with liposomes: dependence on the viral fusion protein (f) and the lipid composition of liposomes enhancement of membrane fusion activity of sendal virus by exposure of the virus to basic ph is correlated with a conformational change in the fusion protein influenza viruses cause hemolysis and fusion of cells membrane effects of cytopathogenic viruses in vitro culture of bovine enteritic coronavirus (bec) molecular basis of infectivity and pathogenicity of myxoviruses identification of biological activities of paramyxovirus glycoproteins. activations of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus characterization of a calf diarrheal coronavirus bovine enteropathogenic coronavirus: the effect of the host cell and trypsin modification on the virus structure, cytopathic expression and infectivity enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments fusion and hemolysis of erythrocytes caused by three togaviruses: semliki, sindbis, and rubella fusion of semliki forest virus with the plasma membrane can be induced by low ph cell fusion by semliki forest, influenza, and vesicular stomatitis viruses membrane fusion proteins of enveloped animal viruses this work was supported by grants -crsr- - and -crsr- - from the united states department of agriculture. this paper contains parts of a dissertation submitted by h. r. payne to louisiana state university in partial fulfillment of the requirements for the ph. d. degree. received august , key: cord- -dzmvjh j authors: tupper, g. t.; evermann, j. f.; russell, r. g.; thouless, m. e. title: antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: dzmvjh j antigenically related feline coronaviruses cause two distinct disease manifestations in infected cats. the diseases are feline infectious peritonitis (fip), in which the virus is widely disseminated, and feline enteric coronavirus (fecv), a mild disease in which the virus is usually limited to the villi. these two viruses were found to differ in their growth in cell culture. fipv grows to higher titer, forms larger plaques and switches off host cell protein synthesis more effectively than fecv. cross neutralization studies showed antigenic differences between the strains. there also appeared to be a difference in the nucleoprotein molecular weight of the viruses causing these two different disease syndromes. antigenically related i~line coronaviruses (fcv) cause two different disease manifestations in cats ( , , , ) . the first is known as feline infectious peritonitis (fip), and is characterized by peritonitis and/or pleuritis with occasional central nervous system and ocular involvement ( ) . the inflammatory infiltrate consists of lymphoeytes, plasma cells, and macrophages resulting in either nonsuppurative or a granulomatous inflammarion. the second disease is caused by feline enteric eoronavirus (fecv) and is a subclinical or mild enteric infection in which, lesions are located in the upper third of the villi of the small intestine ( ) . although these viruses are associated with very different disease syndromes, evidence has suggested that the viruses are antigenicmly and biologiemly similar ( ) . it was reported that both virus strains produce relatively large plaques in cell culture and grew to fairly high titers ( ) . in addition, their polypeptides appeared to be the same molecular weight by immunoblotting ( ) . although the strains eausing peritonitis and enteritis in eats were antigenieally and biologicmly similar; neither protected against infection with the other ( ) . therefore, we investigated the antigenic biological and biochemical properties of these viruses in more detail. the viruses were grown in crandell feline kidney (crfk) cells. the cells were mycoplasma free by the method of kenny ( ) . cells were grown at ° c in eagle's minimum essential medium (auto-pow, flow lab., mcclean, va) supplemented with l percent heat inactivated fetal calf serum, mm l-glutamine, my~ sodium bicarbonate, m:~ hepes, tlg/ml sm~ptomycin, and units/ml of penicillin (mem- ). the calf serum was reduced to percent for virus propagation (mem- ). the fip strains wsu -i and nor , and the fecv strains wsu - isolates of fcv were studied. the viruses were cloned by endpoint titration times in microtiter plates. the isolation and in vivo pathogenicity of these strains has been previously reported ( , , ) . virus titers were measured by a plaque assay or a tcids endpoint. the plaque assay was done in mm tissue culture petri dishes (corning, ny). confluent monolayers of crfk cells were inoculated with ~i of a tenfold virus dilution. after adsorption for hour at ° c an overlay media of ml of warmed . percent, carboxyraethyl cellulose in mem- was added. monolayers were fixed with formalin and stained with percent crystal violet five days post infection (pi). plaques were counted using a light box and a plaque counting device (seientifiea). the tcids assay tbr virus infectivity was done in well microtiter plates (falcon) by infection of wells with txl of a tenfold virus dilution. the monolayers were fixed and stained after days. wells were examined for cybopathie effects (cpe) and the formula of reed and muench was used to calculate the tcid~ ( ) . photographs of infected monolayers in mm plates containing approximately distinct plaques were enlarged . times to determine the average plaque size. the diameters of the plaques were measured with a ruler. crfk cells were grown in roller bottles at ° c and infected with the feline coronavirus strains at a multiplicity of infection (moi) of . to . . the supernatant was harvested and pooled at hours pi. all subsequent steps were carried out at ° c similar to the procedure used by schmidt and kenny ( ) . the suspension was clarified by centrifhgation at × g for minutes. polyethylene glycol was added to make a final concentration of percent (w/v). after a hour incubation the precipitate was collected by clarification at , × g for minutes. the pellets were resuspended in hepes bus fer ( . rn.~( hepes-- . ~ nac -- . mm edta-na , ph . ) layered onto a and percent (w/v) sucrose step gradient, and centrifuged at , x g for . hours in a beckman sw rotor. the virus bands at the interface were collected, diluted with an equal volume of hepes buffer and pelleted by centrifugation at , x g for minutes in a beckman sw . . the virus was resuspended in the hepes buffer for inoculation into rabbits to obtain hyperimmune sera. further purification by isopycnic banding was undertaken to obtain purified virus for page. the virus bands were concentrated on a ml cushion of . gm/ml renografin (squibb) in a sw . rotor. the concentrated virus was again diluted in the ttepes buffer and layered onto a continuous . to . (gm/ml) renografin gradient for isopyenie banding using a beckman type rotor at , × g for hours. the visible bands were collected (density . gm/ml) and then pelleted using a beckman sw . rotor. virus pellets were resuspended in hepes buffer and frozen until further use. viral infectivity was measured by tcid. and the protein concentrations were determined by the lowry method ( ). rabbits were hyperimmunized with fipv - or fecv - purified by sucrose rate zonal centrifugation as described above. the rabbit immunization schedule was an intramuscular injection of × t plaque forming units (pfu)/ml in freund's incomplete adjuvant, followed by four small intravenous boosts using doses of . to . ml of inocula containing × l s pfu/ml ( ) . the rabbits were bled l days after the last boost. virus neutralization was carried out in well microtiter plates ( ) . virus, ( tcids virus in ~ ), was added to ~ of serial twofold dilutions of rabbit hyperimmune sera against fipv - or fecv - . the virus-serum mixture was incubated at room temperature for hour after which . × crfk cells in ~! mem- was added. plates were placed in a . percent c incubator at ° c for to hours. monolayers were fixed with formalin, stained with percent crystal violet stain. page was performed in . mm thick slab gels by the method of laemmli ( ) . purified virus samples ( l~g) in buffer ( . ~i tris-hct, pig . , percent mercaptoethanol, percent sodium dodecyl sulfate [sds] and l percent glycerol plus bromophenol blue) were boiled for minutes and placed into each well. proteins were migrated through a stacking gel containing . percent polyacrylamide and resolved using a to percent continuous polyacrylamide gradient gel. a modified silver stain was used to stain protein ( ) . the molecular weight of virus structural proteins was determined by using molecular weight standards (sigma, st. louis, mo). the procedure was similar to that used to label rotavirus polypeptides ( ) . monolayers were prepared in well (costar, cambridge, ma) tissue culture plates by adding × cells/well. the monolayers were washed once and infected with a moi of . virus was allowed to adsorb for hour and then t~g actinomycin d (sigma) in ml mem- was added. the infected cells were incubated for the desired time, washed three times and i ml of t~ci/ml s-methionine in methionine free mem~i was added. the plates were incubated for hour at ° c at which time the media was removed and the monolayers washed once. disruption mixture ( m~ tris-hc , ph . , percent mercaptoethanol, percent sds, and percent glycerol with bromophenol blue) in a . ml volume at ° c was added. samples were treated with an ultrasonic probe and kept at - ° c until used. g.t. qsapper et al.: cell controls of uninfected cells were treated in the same manner. an amount of sample containing to , counts was used per lane for page. some of the gels were stained with . percent coomassie blue in percent isopropano percent acetic acid and destained in percent isopropanol i percent acetic acid. dried gels were exposed to kodak film for autoradiography. virus titers were reproducible throughout the study with fipv strains - and nor having virus titers of - × l pfu/ml and fecv - having a lower titer of n × pfu/ml. the cytopathic effect produced by the three feline coronavirus isolates was characterized by syncytial formation. the plaques of the - and nor strains measured . mm with a + . mm standard deviation while the - strain produced smaller plaques of . mm with a _+ . mm standard deviation ( fig. ) in days in crfk. the plaque size of the fecv - strain were significantly smaller than those of the other two fipv strains by the t test (p = . ). the - and the nor fip isolates were distinct from the - fecv isolate by virus neutralization using homologous and heterologous antisera prepared in rabbits against - and - (table ). the neutralizing titers for fipv -i and nor were similar irrespective of the antiserum used. by comparison, the fecv - strain had a fold difference in neutralization titer using antisera against - and a fold difference when antisera against - was used. these results suggest that - and noi~ strains are antigenically similar by virus neutralization and are distinct from the - strain of feline eoronavirus. each of the three virus strains had the structural protein profile characteristic of that reported for the eoronavirus family ( ) . the peplomer (p) surface protein band measured , molecular weight (mw). the mere- (figs. and ) . this indicated the differences were consistent and were not due to maturation artitsct. there was a cell protein band just above the nucleoprotein of the fecv - of the radiolabelled virus. this band was not present in the purified virus preparation or in radiolabelled - where host cell synthesis was switched off. virus proteins appeared at hours pi and continued to be synthesized throughout the hour study period. in the presence of actinomyein d, fipv - shutoff protein synthesis of crfk cells at to hours post infection, nor a,t to hours. celtula, r protein synthesis was reduced at hours pi by fecv - but was not completely shutoff (fig. ) . the feline coronavirus strains did not shutoff eellula, r protein synthesis in the absence of actinomyein d. the - , nor , and - isolates of feline coronavirus have previously been reported to be similar by their relatively high titer in cell culture, large plaque size, and by indirect immunofluorescenee with antibody-to canine coronavirus ( ) . this study showed similarities between the - and the nor isolates of fipv and difference with the - fecv isolate. the fipv strains produced larger plaques in crfk cells and half a log higher titer of virus than the fecv strain. use of a different cell line (fcwl- cells) and time that the plaques were left to develop may have accounted for the fact that boyle et al. ( ) did not find a difference. however, differences in plaque size and virus titers have been reported for human, murine and porcine eoronaviruses grown in different cell lines ( , , , ) . the fipv strains could be distinguished from the fecv strain by cross neutralization. the low level of cross reactivity with heterologous serum compared to homologous serum suggests considerable antigenic variation between the two groups. however the sera were not very high titer despite immunization of rabbits with biologiemly cloned purified virus, perhaps due to the fragility of the peplomer protein which induces neutralizing antibody ( ) . no differences were seen in the molecular weights of the peplomer but this might be undetectable in such a high molecular weight protein where the relevant epitope may be only a very small part of the entity. previous studies with convalescent sera from naturally and experimentally infected cats did not show these differences in vitro ( , ) but did in vivo where fipv strains did not protect against fecv infection and prior fecv infection even seemed to enhance the pathogenicity of fipv. the molecular weight of the nucleoproteins differed by about , with the fecv strain nueleoprotein being smaller than that of the fipv strains. this was determined by co-running purified virus and pulse radiolabelled polypeptides of both strains. this is considered to be a more sensitive technique than immunoblotting used by boyle et al. ( ) , and may explain the fact that they did not observe this difference. the significance of the dit; ferenee in the molecular weight and any possible relationship to pathogenicity is unknown at the present time. however, differences in molecular weight of the nueleoproteins have been found in strains of murine coronaviruses and a high degree of homology was found between them by hybridization kinetics and peptide mapping ( ) . in vitro infection with the fipv strains reduced the production of host cell proteins. this effect was enhanced by actinomycin d. the fecv strain did not switch off host cell synthesis even in the presence of aetinomyein d. host cell protein synthesis was also shut offby infection with murine coronavirus and different strains vary in the extent to which they do it, ( ) . highly lyric strains of mcv synthesize m structurm polypeptides synchronously whereas the nucleoprotein appears earlier than the other two polypeptides in less lytic infections ( , ) . in this study, all st, ruetural polypeptides appeared synchronously in cells infected with fipv or fecv strains. it is interesting that the more vigorously growing strains which produce more varied and severe disease (fipv) shut off host cell synthesis more effectively than the less pathogenic fecv strain. despite the aforementioned differences, there is currently no evidence that the in vitro differences between the virus strains studied are associated with or linked to the differences in the pathogenicity of fipv and fecv strains in vivo. plaque assay, poiypeptide composition, and immunochemistry of feline infectious peritonitis virus and feline enteric coronavirus isolates rna and polypeptide homology among murine coronavirus characterization of a feline infectious peritonitis vires isolate replication ofmurine coronaviruses in somatic cell hybrids foltned between a mouse fibroblast cell line and either a rat schwannoma line or a rat glioina line defective replication of porcine transmissible gastroenteritis virus in a continuous cell line isolation of subviral components from transmissible gastroenteritis virus the virology and pathogenesis of feline infectious peritonitis hnmunogenicity of myeoplasma pneumoniae contamination of mammalian cells in culture with myeoptasmata. in: fogh j (ed) contamination in tissue culture cleavage of structural proteins during the assembly of the head of bacteriophage t protein measurement with the folin phenol reagent isolation of feline coronavirus from two eats with diverse disease manifestations a simplified uttrasensitive silver strain for detecting proteins in potyaerylamide gels feline infectious peritonitis and feline enteric coronavirus intbction ii. feline infectious peritonitis pathogenic differences between various feline eoronavirus isolates an enteric eoronavirus infection and its relationship to feline infectious peritonitis pathogenicity studies of feline eoronavirus isolates - and - muench tt (i ) a simple method for estimating fifty percent endpoints viral protein synthesis in mouse hepatitis virus strain a -infected cells: effect oftunieamyein application of a microtechnique to viral serological investigation plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line immunogenicity and antigenicity of human coronaviruses e and oc coronavirus jhm: intracellular protein synthesis the structure and replication of coronaviruses the molecular biology of coronaviruses thouless me ( ) i~otavirus polypeptides ter meulen v ( ) the biology and pathogenesis of coronaviruses the authors wish to expi~ss their appreciation to a. j. mekeirnan tbr assistance with feline eoronavirus strains. t~eceived august , key: cord- -q mbngqy authors: ge, junwei; gu, shanshan; cui, xingyang; zhao, lili; ma, dexing; shi, yunjia; wang, yuanzhi; lu, taofeng; chen, hongyan title: genomic characterization of circoviruses associated with acute gastroenteritis in minks in northeastern china date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: q mbngqy mink circovirus (micv), a virus that was newly discovered in , has been associated with enteric disease. however, its etiological role in acute gastroenteritis is unclear, and its genetic characteristics are poorly described. in this study, the role of circoviruses (cvs) in mink acute gastroenteritis was investigated, and the micv genome was molecularly characterized through sequence analysis. detection results demonstrated that micv was the only pathogen found in this infection. micvs and previously characterized cvs shared genome organizational features, including the presence of (i) a potential stem-loop/nonanucleotide motif that is considered to be the origin of virus dna replication; (ii) two major inversely arranged open reading frames encoding putative replication-associated proteins (rep) and a capsid protein; (iii) direct and inverse repeated sequences within the putative ʹ region; and (iv) motifs in rep. pairwise comparisons showed that the capsid proteins of micv shared the highest amino acid sequence identity with those of porcine cv (pcv) ( . %) and bat cv (batcv) ( . %). the amino acid sequence identity levels of rep shared by micv with batcv ( . %) and dog cv (dogcv) ( . %) were broadly similar to those with starling cv ( . %) and pcvs ( . %). phylogenetic analysis indicated that micvs were more closely related to mammalian cvs, such as batcv, pcv, and dogcv, than to other animal cvs. among mammalian cvs, micv and batcv were the most closely related. this study could contribute to understanding the potential pathogenicity of micv and the evolutionary and pathogenic characteristics of mammalian cvs. circoviruses (cvs) are small nonenveloped icosahedral viruses measuring - nm in diameter and possessing a single-stranded circular dna genome with a length of . - . kb and a major structural protein [ ] . in the current taxonomy release, the family circoviridae is divided into two genera: circovirus and cyclovirus [ , ] . the most recent release of the universal virus database of the international committee on taxonomy of viruses reported that the genus circovirus includes recognized species whose members infect mammals and birds (ictv, https ://talk.ictvo nline .org/taxon omy/p/taxon omy-histo ry?taxno de_id= , released on july ). cvs have been identified in numerous species associated with various clinical disorders, including asymptomatic infections and lethal diseases [ ] [ ] [ ] . in birds, cv infections are associated with lethargy, weight loss, respiratory distress, diarrhea, and poor race performance [ ] [ ] [ ] . in geese, these infections cause death. in other animals, these infections induce different clinical signs, including respiratory, vesicular, hemorrhagic, and gastroenteritic manifestations and reproductive failure [ , ] . cv infections are also related to lymphoid depletion and immunosuppression, which likely increase the severity of secondary infection [ ] . mink circovirus (micv) is a novel pathogen that was found in cases of minks with diarrhea in dalian city, liaoning province, china, in [ ] . despite the discovery of cv infection in minks, the pathogenic role of micv in single or polymicrobial infections has not been determined, and the prevalence and economic importance of this virus have yet to be elucidated. in this study, micv was detected and molecularly characterized from the collected feces of minks with acute gastroenteritis in harbin city, heilongjiang province, in northeastern china in march . the complete genome sequences of micv strains were compared with those of other cv strains, including recent cvs detected in birds and animals, to enhance our understanding of their genetic and biological differences. in march an outbreak of acute gastroenteritis occurred on three farms under one cooperative in harbin, northeastern china. minks had a sudden onset of gastrointestinal symptoms, and their clinical signs included severe diarrhea, vomiting, lethargy, anorexia, dehydration, and rough fur. their stool was thin or soft and watery. more than minks were owned by the cooperative in the reproduction period, and the same feed and immune procedures were used. a -year vaccination protocol against mink enteritis virus (mev) and canine distemper virus (cdv) was completed. fewer than minks died after week of illness, and most of those that died were malnourished. the other minks completely recovered within - days after showing clinical signs. local veterinary clinicians sampled three or five diarrheic fecal samples from each farm and immediately submitted them to our laboratory in the animal teaching hospital of the college of veterinary medicine, northeast agricultural university. one mink carcass (after flaying) was frozen at − °c to − °c after days of storage at °c and sent to our department for necropsy and laboratory investigations. samples from the heart, liver, spleen, lung, kidney, and intestine of the carcass were collected and homogenized in dulbecco's modified eagle's medium supplemented with % fetal calf serum and iu of penicillin, mg of streptomycin, and mg of amphotericin b per ml. tissue homogenates were clarified by centrifugation at ×g for min. the fecal samples were immediately examined for common enteric bacterial, protozoan, and viral pathogens, including salmonella spp., clostridium spp., campylobacter spp., shiga-toxin-producing escherichia coli and enterotoxigenic e. coli, coccidium spp., cryptosporidium spp., mink aleutian disease virus [ ] , mev [ ] , cdv [ ] , caliciviruses [ ] , rotaviruses [ , ] , coronaviruses [ ] , and astroviruses [ ] . standardized procedures were carried out to isolate bacteria commonly associated with enteritis in vitro. the samples were plated on % sheep blood agar (biocell biotech., co., zhengzhou, china) and cultured aerobically or anaerobically at °c for h for pathogen detection. bacteriological investigations were conducted in accordance with standard biochemical procedures, and bacterial strains were identified using the biolog microbial id system (geneiii, biolog inc. usa). feces or intestinal contents were examined to detect intestinal parasites by zinc sulfate flotation. stools and intestinal sections were subjected to ziehl-neelsen staining to identify cryptosporidium spp. dna/rna extracts were screened to detect enteric viral pathogens that are common in minks. tissue homogenates or fecal suspensions of each sample were prepared by diluting the feces in . m phosphate-buffered saline at a proportion of : and a ph of . . the suspensions were then vortexed for s and centrifuged at ×g for min. the supernatant ( μl) was used for dna/rna extraction using a tianamp virus dna kit (tiangen biotech. co., beijing) or trizol ls (gibco-brl, life tech.) in accordance with the manufacturer's protocol. nucleic acid templates were stored at − °c until use. nucleic acid templates were also extracted from healthy mink feces as a negative control. the primers and the pcr and rt-pcr conditions used to detect viral pathogens have been described elsewhere (table ) . pcr was performed as described previously to determine the full-length genome sequence of the newly identified micv strain, which we have named "heb " [ , ] . the pcr product was analyzed via electrophoresis using an agarose gel and visualized by staining with ethidium bromide. the amplified product from the positive sample was bp long. the complete genome was amplified from the positive sample obtained via pcr using the primers cv- , cv- , repf, and repl. the target dna bands were extracted from the gel using a qiagen gel extraction kit in accordance with the manufacturer's instructions. the purified pcr products were cloned into the pmd -t simple vector (takara, japan), and the plasmids were introduced into e. coli dh a using a standard transformation technique. at least three independent plasmid clones were analyzed, confirmed, and sequenced by comate biotech. co., changchun, china. sequence reads were assembled using seqman (dnastar inc., madison, wi, usa), and the genome sequence of micv was analyzed with the aid of ncbi (http://www.ncbi.nlm.nih.gov) and embl (http://www. ebi.ac.uk) analysis tools. multiple alignments of nucleotide and predicted amino acid sequences were performed and compared with those of other members of the family circoviridae, which were extracted from the genbank database using clustalw in mega . [ ] . putative open reading frames (orfs) were identified using orf finder (ncbi; http://www.ncbi.nlm.nih.gov/ gorf/gorf.html). the nucleotide sequences of different orfs were aligned with the cognate sequences of the reference micv strain dl and other cvs through translation-based alignment. the hairpin and stem-loop structures were identified using the mfold web server (http://unafo ld.rna.alban y.edu/?q=mfold ). furthermore, direct and inverted repeat sequences within the intergenic regions were determined using an oligonucleotide repeat finder (http://wwwmg s.bione t.nsc.ru/mgs/progr ams/oligo rep/inpfo rm.htm). phylogenetic trees based on the full-length genome sequence and the nucleotide sequences of replicase and capsid protein genes were constructed in mega . using neighbor-joining method, which provided statistical support with bootstrapping of over replicates. other tree-building methods, including maximum parsimony and maximum likelihood, were used to confirm the topology of the neighbor-joining tree. the following reference cv strains were used in the phylogenetic analysis: micv isolate dl (nc_ . of the samples tested, ( . %) were positive for micv. no evidence of mixed infection with different pathogens was found in the analyzed samples. the heart, kidney, and gut samples of the deceased mink tested positive in the pcr targeting the replicase gene of micv. e. coli isolated from the liver of the mink carcass harbored iuta, sfa virulence genes and were pathogenic to mice. the complete sequence was deposited in the gen-bank database under accession no. kx . the complete genome of micv strain heb comprises nucleotides as a covalently closed circular dna and has a nucleotide composition of . % a, . % t, the genome sequence of micv isolate heb was compared individually to those of other cvs, and was found to be . % identical to that of cocv and . % identical to that of batcv . by contrast, micv was more closely related to batcv ( . % nucleotide sequence identity) than to dogcvs and gocvs ( . . %- %). nucleotide sequence analyses indicated that the micv heb strain exhibited a genome organization similar to that of previously characterized cvs, such as pig cvs (pcvs) and bfdv [ , ] . sequence features included a distinctive inverted repeat sequence to form a thermodynamically stable stem-loop with a highly conserved nonanucleotide motif tag tat tac at its top, where rolling-circle replication (rcr) of the virus dna strand has been postulated to initiate. accordingly, the convention for nucleotide numbering and labeling of orfs has been adopted from previous reports [ ] . thus, the a residue, which is found in the eighth position in the nonamer and lies immediately downstream of the rcr cleavage site, is regarded as nucleotide position . the micv genome was found to contain seven orfs potentially encoding proteins containing more than amino acids. similar to pcv , pcv , and bfdv, the micv genomes possessed two major orfs, one in the viral sense orientation (v , encoding the viral replicase) and one on the complementary strand in the opposite orientation (c , encoding the capsid protein). fig. presents the genome locations of the major orfs in micv. the largest orf, v , is located at nucleotide position - and is postulated to encode the replication-associated protein (rep), with a predicted length of aa. these values were within the range of amino acids (bfdv) to amino acids (pigeon cv, [picv]) observed in other cvs. the rep gene of heb contained one nucleotide mismatch when compared with dl , and the substitution resulted in a nonsynonymous codon change. the predicted rep of micv shared . % (gucv) and . % (batcv ) amino acid sequence identity with those of other cvs. pairwise comparisons showed that the amino acid identity levels of the putative v orf product shared by micv with batcv ( . %), dogcv ( . %), and swan cv ( . %) were broadly similar to those with pcvs ( . %). a sequence alignment of the putative rep of micv with those of known members of the genus circovirus identified several highly conserved amino acid motifs, including wwdgy (aa - in micv), ddfygw (aa - ), and dryp (aa - ) [ , ] . a closer examination of the v orf amino acid sequences showed that the amino acid motifs associated with rcr and the p-loop were present in the sequences of micv and those of bfdv and pcvs. the motifs related to rcr activity [ ] include ftinn, which occurs at aa to in micv, and tphlqg, which appears at aa to in ficv. csk is the sequence of the third rcr motif located at aa to in the micv rep. g*gks is the fourth motif, which is putatively associated with dntpase activity [ ] , at aa to in the micv rep. the c orfs, which were the putative cap genes of micv (nt - ), encode proteins with amino acids. this value was the smallest and was below the range of (bfdv) to amino acids (picv) observed in other cvs. the predicted cap of micv shared . % (ducv) and . % (pcv and batcv) amino acid identity with those of other cvs. in addition to the high level of identity shared by pcv and batcv ( . %), the highest amino acid sequence identity was shared by pcv ( . %). micv also shared higher levels of identity with mammalian cvs, such as pcvs ( . % and . %) and batcv ( . %), than with avian cvs ( . %- . %). similar to other animal cvs, fig. diagram of the circovirus genome isolated from mink. the nucleotide position is indicated for each orf. rep, replicase; cap, capsid protein. the hairpin-like palindromic structure, the origin of replication, is shown on the right micv had a putative capsid protein with an amino terminus containing an arginine (r)-rich stretch with a length of aa. the cap nucleotide sequences of micv heb and the two other strains of micv, namely, dl and hebei , were . % and . % identical, respectively. furthermore, a high degree of identity ( . %- . %) was observed between the nucleotide sequences of micv strains. the cap gene contained five nucleotide mismatches compared with the dl isolate. three of the five substitutions were transversions, and all of the substitutions resulted in nonsynonymous codon changes. the alignment of the entire cap protein sequence of micv heb with the published sequences of dl and hebei indicated that the sequences were generally highly conserved, showing no variation at all. moreover, the five nucleic acid substitutions did not result in changes in amino acids. database searches with the micv sequence revealed that none of the minor orfs shared significant amino acid sequence similarity with any of the orfs specified by other cvs. however, a putative orf, postulated to use an alternative ttg start codon, occurred on the complementary strands (nt - ), which shared weak homology with the c orf of ducv ( %) and was in a similar location to those of previously characterized cvs. like other cvs, the heb genome was found to contain two intergenic noncoding regions located between the replicase and the capsid protein genes ( table ). the ′ and ′ intergenic regions of micv were and nt in length, respectively ( table ). in the ′ intergenic region of micv, the conserved nonanucleotide motif of micv was flanked by a potential stem-loop with base pairs in the stem. in addition, the intergenic region of micv possessed two tandemly repeated decamers ggc aca cctc (nt - and - ) adjacent to the potential stem-loop. in the ′-intergenic region, a -nt stretch within the ′ intergenic sequence showed . % ( / ) nt sequence identity to its counterpart in batcv (isolate xor), which was recently detected in bats by metagenomic analysis of tissue samples [ ] . potential poly(a) signals appropriately located for use with possible transcripts containing the v and c orfs were identified in the micv genomes by analogy to similar sequences in the pcv and bfdv genomes [ , ] . potential poly(a) signals for the v orf were determined at nt - (aat aaa ) and nt - (att aaa a). in the complementary strand, three possible signals for the c orf were identified at nt - (aat aaa a), nt - (gat aaa aa), and nt - (ctt aaa a). a phylogenetic analysis based on the complete genome sequences of the two micv strains and representative cvs was performed to investigate the genetic relationship between micv and other cvs (fig. ) . in the neighbor-joining tree, the micv strains were grouped with mammalian cv, which comprised batcv , dogcv, and pcvs, and these were clearly separated from bird cvs. micvs were more closely related to pcv and pcv than to dogcv (fig. a) . a phylogenetic analysis based on rep sequences (fig. b) demonstrated that the two micv strains were grouped with batcvs and were more closely related to dogcv than to pcv and pcv . in trees constructed based on the cap gene, the micvs were tightly intermingled with batcv isolate and were close to pcv and pcv , but dogcv tag tat tac ggc aca cctc pcv tag tat tac cgg cag c pcv aag tat tac cgg cag cac ctc bfdv tag tat tac ggg gca ccg picv tag tat tac gga ccc ac cacv cag tat tac gga gcc ac ficv tag tat tac tgg aac c gucv tag tat tac ggg gcc at gocv tat tat tac gta ctc cg ducv tag tat tac tac tcc g dogcv tag tat tac cacag batcv tag tat tac cac ttc ggca stcv cag tat tac gga gcc a swcv tat tat tac actac racv gag tat tac gga gcc was grouped into a separate cluster (fig. c) . these results were verified using the maximum-likelihood and maximumparsimony methods, which yielded the same tree topology (data not shown). micv circulated on mink farms in dalian city, liaoning province, china, in [ ] . farmers observed disease in mev-vaccinated minks in . clinical signs included diarrhea, lethargy, anorexia, pale muzzle, and unkempt fur. on farms with diseased minks, all of the animals were affected, and %- % showed clinical signs. however, most of the animals recovered, and fewer than % of the affected minks died. micv was found in the liver, digestive tract, and fecal specimens of minks and was shown to be responsible for diarrhea [ ] . however, its pathogenic role remains unknown, particularly its infection or coinfection process. in the present study, we investigated a major outbreak in a large population. samples were tested for other causative agents, including madv, mev, cdv, coronavirus, calicivirus, rotavirus, bacterial pathogens, and parasites. after these pathogens were excluded, micv was detected alone, and was not found in combination with other key pathogens, such as madv, mev, and cdv. thus, our findings suggested a possible association between micv and mink acute enteritis. consistent with the detection results presented by lian et al. [ ] , our findings showed that the virus was located in the gut and feces. the virus was also found in the heart and kidney, but not in the liver. the sites where carcasses and feces were collected in this study were approximately km away from where lian et al. obtained their samples [ ] , and the isolates were therefore of different geographical origin. moreover, the mink breed, disease symptoms, and disease duration differed. as such, the sustained direct transmission of micv among minks was excluded, and micv heb was considered a new strain. the data gathered in this study expanded the known geographical distribution of micv. unfortunately, the carcass of the micv-infected mink was not stored properly by the resident veterinarians and underwent repeated cycles of freezing and thawing, making it unsuitable for histopathological examination. for this reason, valuable information on tissue localization and alterations of micv could not be collected. the present study could contribute fig. phylogenetic analysis of the genome sequences of mink circovirus and other circoviruses, using the neighborjoining method, with , bootstrap replicates (mega . ). a analysis of the whole genome sequence. b analysis of the rep gene sequence. c analysis of the cap gene sequence. only bootstrap support values greater than % are shown. the bar indicates the genetic distance. the sequence of the circovirus isolated in this study is indicated by a star. other sequences were obtained from genbank; accession numbers of those sequences are included in the tree to our knowledge of the pathogenic potential of micv and its association with mink enteritis if our results were corroborated by further reports. the presence of micv might help to explain the different disease outcomes and severity often observed in madv-/mev-infected animals. our results indicate that minks with acute gastroenteritis should be screened for micv and established pathogens, such as e. coli and mev. this study was conducted to identify the viral agents associated with acute diarrhea in minks, and no attempt was made to determine the primary cause of death. to date, attempts to isolate micv have been unsuccessful. current surveillance methods are limited to viral detection using molecular assays, and no seroprevalence information is available. as a consequence, the identification and characterization of the virus depends on genetic approaches such as pcr and sequence determination and analysis. in this study, the nucleotide sequence of the micv genome was determined and analyzed. our results showed that the genome sequence of micv isolate heb was highly similar to that of micv dl in terms of size ( nt), nucleotide sequence ( . % identity), and orf analysis. the examination of other micv sequences from different regions will help to assess the level of genetic diversity. in our study, sequence analysis confirmed that micv genomes displayed the characteristics of members of the genus circovirus, and the common features included their genome organization, the presence of a potential stem-loop and conserved nonanucleotide motif postulated to be the origin of viral dna replication, and major orfs and repeats [ , ] . conserved amino acid motifs, including wwdgy, ddfygwlp, and dryp, which have unknown functions, were also recognized within rep associated with rcr and dntpase activity. these motifs could be utilized to design primers that could amplify cv-specific dna for discovery of new cvs in other hosts. additional micv surveillance in animals is required to clarify cv epidemiology. further efforts are necessary to identify and characterize the infection or coinfection processes. future studies should investigate experimental infections to obtain conclusive information on the pathogenic role of micv and to monitor the circulation of these viruses and their effects on mink populations. this study could contribute to our knowledge about the pathogenic potential of micv and its association with acute gastroenteritis in minks. the complete genome sequence of micv strain heb is reported here to help elucidate the epidemiological, evolutionary, and pathogenic characteristics of mammalian cvs. the availability of the micv genome sequence also provides a basis for the development of molecular reagents that could be used to identify other novel cvs that infect other mammalian species. ictv virus taxonomy profile: circoviridae circovirus in tissues of dogs with vasculitis and hemorrhage genomic characterization of a circovirus associated with fatal hemorrhagic enteritis in dog porcine circovirus type (pcv ) infections: clinical signs, pathology and laboratory diagnosis avian circovirus diseases: lessons for the study of pmws circovirus-like infection in a pigeon particles resembling circovirus in the bursa of fabricius of pigeons animal circoviruses porcine circoviruses-small but powerful circoviruses: immunosuppressive threats to avian species: a review novel 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analysis of the n gene of porcine epidemic diarrhea virus ljb/ mega : molecular evolutionary genetics analysis version . psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus genome sequence determinations and analyses of novel circoviruses from goose and pigeon identification of a novel circovirus in australian ravens (corvus coronoides) with feather disease conserved sequence motifs in the initiator proteins for rolling circle dna replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria rep protein of tomato yellow leaf curl geminivirus has an atpase activity required for viral dna replication virome profiling of bats from myanmar by metagenomic analysis of tissue samples reveals more novel mammalian viruses conflict of interest the authors declare no conflicts of interest.ethical approval this article does not contain any studies with human participants performed by any of the authors. this study was performed in accordance with the recommendations in the guide for the care and use of laboratory animals of the ministry of health, china. prior to experiments, the protocol of the current study was reviewed and approved by the institutional animal care and use committee of northeast agricultural university (approved protocol number - key: cord- -mryiqmsw authors: kumar, binod; asha, kumari; khanna, madhu; ronsard, larance; meseko, clement adebajo; sanicas, melvin title: the emerging influenza virus threat: status and new prospects for its therapy and control date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: mryiqmsw influenza a viruses (iavs) are zoonotic pathogens that cause yearly outbreaks with high rates of morbidity and fatality. the virus continuously acquires point mutations while circulating in several hosts, ranging from aquatic birds to mammals, including humans. the wide range of hosts provides influenza a viruses greater chances of genetic re-assortment, leading to the emergence of zoonotic strains and occasional pandemics that have a severe impact on human life. four major influenza pandemics have been reported to date, and health authorities worldwide have shown tremendous progress in efforts to control epidemics and pandemics. here, we primarily discuss the pathogenesis of influenza virus type a, its epidemiology, pandemic potential, current status of antiviral drugs and vaccines, and ways to effectively manage the disease during a crisis. influenza viruses belong to the family orthomyxoviridae and are the leading cause of severe respiratory illness across the world. they are enveloped viruses containing a single-stranded, negative-sense rna genome, and they account for a large number of deaths each year. in an electron microscope, influenza a and b viruses look similar and are virtually indistinguishable. they are either spherical ( nm in diameter) or filamentous (often in excess of nm in length) in form [ ] . of the four influenza virus types (a, b, c and d), influenza a virus (iav) causes the most severe disease and infects a variety of animals, including humans, pigs, horses, sea mammals, and various bird species (reviewed in reference [ ] ). type a mutates more rapidly and exhibits a higher degree of variability in its antigenicity and virulence than the other influenza types [ , ] . it can cause zoonotic infections and adapts easily to humans, leading to a sustained human-to-human transmission, which favors the emergence of novel strains. in this review, we have focused primarily on the contemporary aspects of influenza a virology and new prospects for its treatment and prevention. the genome of influenza a and b viruses consists of eight single-stranded viral rna (vrna) segments, while influenza c virus has a seven-segment genome. each segment codes for one of the viral proteins, which include the major surface glycoproteins hemagglutinin (ha) and neuraminidase (na), the nucleocapsid protein (np), three subunits of the viral rna-dependent rna polymerase (rdrp) (pa, pa-x, pb , pb , pb -f ), the matrix proteins (m , m ) and the nonstructural proteins ns and ns [ ] . all influenza a viruses are classified based on their surface glycoproteins, ha and na. ha is responsible for binding to sialic acid (sa) (n-acetyl neuraminic acid) at the termini of glycans, which act as receptors on the host cell plasma membrane, while the na, a type ii integral membrane glycoprotein with sialidase enzymatic activity, is involved in the final step of the replication cycle and helps in release of mature virions. the two surface glycoproteins, ha and na, are present in a ratio of : [ ] . co-evolutionary adaptation between ha and na allows them to perform the complimentary functions of sa binding (by ha) and sa removal (by na). the segmented nature of the genome and the high frequency of mutations during replication in multiple hosts is responsible for regular epidemics and occasional pandemics. the two major factors in influenza epidemics and pandemics are genetic drift and genetic shift [ ] . genetic drift occurs due to point mutations in the influenza virus genome, as the viral rna polymerase, unlike dna polymerase, lacks a proofreading function making coding errors and multiple mutations more likely. a genetic shift occurs when two or more different influenza virus strains infect the same cell in a host, leading to recombination of genetic materials, an event that occasionally generates a new strain with a novel combination of hemagglutinin and neuraminidase. these genetic shifts lead to pandemics when the novel strain acquires the capacity for sustained efficient human-to-human transmission. to date, novel hemagglutinins (h -h ) and neuraminidases (n -n ) have been identified [ ] . most of the combinations of h and n types ( ) are found in wild birds, which serve as reservoirs for influenza viruses and pose a severe risk, because they can be infected with multiple strains and serve as potential mixing vessels. h - and n - have not been detected in birds but have been found in bats [ , ] . iavs that infects birds have an ha receptor-binding specificity for α - sa, while has from iavs that infect humans have a higher specificity for α - sa, with the major exception of the highly pathogenic avian influenza (hpai) strain h n , which has a preference for α - sa. the differences in preferred cellular binding sites allow different strains of influenza virus to infect either birds or humans, thereby creating lineages that are host specific, and so far, only h n , h n , h n , h n , h n and h n viruses are known to infect humans. however, the respiratory epithelial cells of pigs (swine) express both α - and α - -linked sialic acids and can therefore support infections with both avian and human influenza virus strains. this makes pigs a mixing vessel for producing novel strains with the ability to infect humans, and some of these strains can cause fatal infections ( fig. ) [ ] . these novel reassortant viruses, due to a lack of existing immunity in human population, can lead to pandemic situations, as witnessed in the year . the human population remains at risk of an influenza pandemic each year due to the high mutation rate of the virus. influenza pandemics have occurred several times, with inter-pandemic intervals averaging approximately years [ ] . type a has been responsible for several widespread fig. mechanisms for the emergence of pandemic influenza virus strains. the virus keeps circulating among own species and sometimes jump the species barrier to generate a novel strain of pandemic potential pandemics since the th century. the three major pandemics were the spanish flu , the asian flu ( ), and the hong kong flu , which resulted in a large number of deaths [ ] (fig. ) . the (h n ) pandemic has been recorded as the worst pandemic in history. it infected million people globally, caused approximately , deaths in the united states [ ] , and killed up to - million people worldwide [ ] . the viral genome reconstructed from the lung tissues of several victims demonstrated that it was an avian-descended h n virus [ ] . waterfowl, of the order anseriformes, such as ducks, swans and geese, serve as reservoirs of all iavs. charadriiformes, including shore birds, gulls, and terns, also harbor influenza virus, but of a different gene pool from those of the anseriformes, and the two remain the most important orders for the transmission and spread of hpai [ ] . influenza viruses from these birds are able to infect other bird species, such as chickens, as well as mammals, and they adapt to a new host by accumulating mutations through genetic drift or genetic shifts [ ] . due to the unavailability of any iav sequences from prior to , the possibility of involvement of an intermediate host in the emergence of the virus in humans during the pandemic remains an unresolved mystery [ ] . however, the virus was readily transmitted to pigs, as was also observed during the pandemic of h n [ ] . most of the deaths resulted from respiratory complications, such as bronchopneumonia with bacterial invasion and progressive cyanosis and collapse. scientists believe that the pathogenicity of the h n virus was amplified by concomitant infection of influenza virus with bacteria such as s. pneumoniae and s. pyogenes [ ] . the pandemic spread in three rapid waves within an approximately -month period. the large number of deaths could also be attributed to several other factors, such as unpreparedness for an influenza virus strain of pandemic potential and the lack of effective vaccines to prevent influenza and antibiotics to treat secondary bacterial pneumonia. after the pandemic period, the virus kept accumulating mutations for several years and disappeared in , only to reappear in circulation in [ ] . following the spanish flu in , another influenza pandemic occurred in and was called the asian flu. the pandemic was caused by the h n strain of iav and resulted in ~ , excess deaths. the overall impact on mortality was one-tenth of that observed during the spanish flu (h n ) pandemic [ ] . this new influenza strain was detected in february in yunnan province of china, and by april, the virus had spread to hong kong, followed by singapore, taiwan, japan and the rest of the world by the summer of [ ] . in the usa alone, this strain caused [ , ] . "original antigenic sin" is a phenomenon where a prior exposure to an antigen leads to an optimal immune response to the related antigen. thus, during the asian flu pandemic, individuals, except those who were years and older, had no prior exposure to the h n strain and therefore had no previous immunity, leading to a large susceptible population in the united states becoming infected [ ] . the effectiveness of an influenza vaccine may decrease if the antigenic distance between the vaccine and circulating strains increases. also there is a possibility that the original antigenic sin could make people who are vaccinated, more susceptible to the virus than those who are not vaccinated [ ] . in the year , a new influenza virus strain (h n ) that differed from the asian pandemic strain (h n ) by its ha glycoprotein but had the same na glycoprotein, replaced the h n strain that had been circulating in all countries since , and this led to the third pandemic causing a large number of deaths [ ] . the h n strain, which was first isolated in hong kong in july [ ] , was highly transmissible but caused disease milder than the asian flu. the virus mainly spread due to international air travel and resulted in an increase in the mortality rate in united states during the pandemic season ( / ), especially in persons < years old [ ] . the h n strain caused an estimated , excess deaths over the -year period - [ ] . the (h n ) and (h n ) influenza pandemic viruses were avian-human reassortants in which avian gene segments were introduced into a human-adapted virus that was already in circulation [ ] . the spring of again marked the emergence of a novel subtype of influenza a virus (pandemic h n - ), which caused the first pandemic of the st century. the newly emerged virus subtype spread worldwide with unprecedented speed and proved its ability to be transmitted from human to human [ ] . the health authorities gained momentum, and strict surveillance programs started globally to combat the threat by this novel virus [ ] , which was a fourth-generation descendant of the h n virus [ ] . the world health organization (who) declared a pandemic in june , and the phase ended by august . according to who, . % of subtyped influenza a viruses collected globally from july - , and reported on july were the pandemic h n - strain [ ] . by october , around countries had reported more than , laboratory-confirmed cases of pandemic (h n ) and more than deaths [ ] . the rates of hospitalization and death varied among countries. according to a study done from april through january in the usa, pediatric deaths were found to be associated with laboratory-confirmed pandemic h n - [ ] . while the rate of hospitalization was higher in children, the adult population aged years of age or older showed the lowest rate [ ] . according to one study, the death toll due to this novel pandemic h n - strain was , , as declared in the who reports. however, they reported that the actual mortality burden due to the pandemic was substantially higher and that the number of cases was underreported in africa and asia [ ] . the influenza pandemic came to an end, and just like any other pandemic strain, the pandemic h n - strain has been considered a seasonal strain since july . iavs are also widely distributed in avian species (ducks, geese, swans, gulls, terns, etc.) around the world and are predominantly maintained in asymptomatic infections termed "low-pathogenic avian influenza" (lpai). around different species of birds have been documented to harbor iavs [ ] . the virus predominantly infects the epithelial cells of the intestinal tract [ ] and is subsequently excreted in the faeces. iavs are known to cross species barriers and be transmitted to other species. a recent example could be seen in the harbour seals (phoca vitulina) of the north-european coastal waters, where h n (lpai) infection caused high mortality [ ] . there are also instances where lpai has jumped from birds to long-finned pilot whales (globicephala melas) and balaenopterid whales [ ] . influenza viruses circulating in mammalian species including dogs (canis lupus familiaris) and horses (equus ferus caballus) are also thought to be derived from avian influenza viruses [ ] . strains of subtypes h n and h n are currently circulating amongst dogs [ ] while h n virus has long been circulating amongst horses [ ] , and bactrian camels (camelus bactrianus) [ ] . the lpai subtypes h and h can subsequently evolve into highly pathogenic avian influenza (hpai) viruses by insertion of a multi-basic cleavage site in the viral ha [ ] . recent years have seen the occurrence of two such hpai strains of subtypes h n and h n in asian countries that resulted in a high fatality rate and hospitalization [ ] . an h n strain is currently in circulation and is the cause of significant public health concern in china [ , ] . another hpai strain of subtype h n was first reported to infect humans in april in sichuan province and again in december in guangdong province, followed by four more cases in december in china [ ] . the recent presence of h n and h n infections in various duck species also poses a threat of evolution of this lineage of hpai h viruses in the future [ ] . hpai viruses have also been documented in some non-primate mammals living in captivity, such as tigers (panthera tigris), cats (felis catus), leopards (panthera pardus), and owston's palm civets (chrotogale owstoni) [ ] . influenza virus primarily spreads from one person to another through respiratory droplets when the infected person comes in close contact with a healthy person (generally within a distance of a meter). the virus can survive for to hours on hard, non-porous surfaces and thus may also spread when a person comes in contact with any such surface or item contaminated with the respiratory droplets from an infected person [ ] . a typical influenza infection is often characterized by sudden onset of fever, chills, headache, malaise and myalgia, followed by prominent upper respiratory tract symptoms, such as rhinorrhea, cough, sore throat and inflammation of the upper respiratory tract. apart from these, gastrointestinal symptoms such as nausea, vomiting and diarrhea are very common [ ] . however, the duration of illness in cases of pandemic h n - infections were found to be slightly longer than that of seasonal influenza infections [ ] , and gastrointestinal symptoms, especially diarrhea, appeared to be more prominent than in seasonal influenza [ ] [ ] [ ] . the incubation period of influenza virus from the time of infection to appearance of symptoms typically varies from to days [ ] , but it may extend up to days in some cases [ , ] , and weakness and fatigue can sometimes last for weeks. an infected person typically sheds virus one day prior to the appearance of symptoms, which spreads infection before the sick can be isolated, and the virus continues to be shed until the symptoms resolve. the peak viral load is generally observed on the day of the onset of symptoms and gradually decreases with time. children and younger adults often shed the virus for days or more [ ] , while an immunocompromised person may shed the virus for weeks [ ] . the virus can be detected in easily clinical specimens such as nasal/throat swabs and nasopharyngeal aspirates. there are also reports of viral load detection in urine and stool of infected patients [ , ] . accurate diagnosis and prompt treatment with antiviral drugs can have positive effects on human health and reduce the economic burden of influenza illness each year. however, because several other respiratory viruses, including adenoviruses, rhinoviruses, respiratory syncytial virus (rsv), coronaviruses, metapneumoviruses and parainfluenza viruses, can cause common symptoms of influenza-like-illness (ili), many cases are misdiagnosed as influenza [ ] . proper specimen collection is of paramount importance, regardless of the diagnostic method used. nasopharyngeal specimens are always preferred over throat swabs or other specimens [ ] . the best time to collect the clinical specimen is on the second or third day of symptoms (when viral shedding is at its peak), as the results obtained will be more reliable than when samples are obtained earlier or later in the course of disease [ , , , , ] . there are a number of methods available for influenza diagnosis including rapid antigen tests, viral culture, serology, conventional reverse transcription polymerase chain reaction (pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), real-time reverse transcription polymerase chain reaction (rt-pcr) and immunofluorescence assay. for rapid antigen (influenza) tests, the preferred specimens are nasopharyngeal or nasal swabs or throat swabs collected within - days of infection for more accurate testing. these tests provide results in less than minutes with - % sensitivity [ ] when compared with viral culture ( - days) or rt-pcr, which has greater than % specificity and is moderately fast. therefore, false negative results are more common than false positive results during influenza seasons when bedside rapid antigen tests are used [ , ] . these rapid antigen tests can differentiate between seasonal influenza a and b types, but they are unable to detect pandemic h n - viruses exclusively [ ] . health care professionals, during the time of year when outbreaks of ili are common, can perform tentative diagnosis of influenza using various commercially available rapid immunoassay kits, but due to the limitations of rapid viral tests, confirmatory laboratory testing should be done to determine the treatment of choice [ ] . while virus culture is believed to be one of the most accurate methods for identifying viral strains and subtypes, it can sometimes be an impractical choice for physicians who usually need to initiate antiviral drug therapy within hours of the onset of symptoms [ ] . the virus culture method also becomes a secondary choice during pandemic situations when a large number of infected people rush to hospitals for diagnosis and treatment [ , ] . the most sensitive diagnostic tool available to date is the real-time reverse transcription polymerase chain reaction (rrt-pcr) test [ , ] . rrt-pcr detects the viral rna with high sensitivity in a few hours and requires relatively little effort. it targets the matrix gene to detect influenza viruses and the ha gene, not only to broadly distinguish between influenza a from b types but also to detect different strains of influenza a viruses (h n , h n , h n pdm , etc. with high sensitivity and specificity [ ] . the taqman chemistry is the most commonly used, as it gives high accuracy and specificity; however, it also comes with the burden of slightly higher costs when compared to the sybr green chemistry. the sybr green chemistry is cost effective, as it does not require dual-labelled probes like the taqman chemistry and is highly sensitive. the sybr green chemistry however, has not been widely used for clinical diagnostics, as it uses an intercalating dye that can produce fluorescence with any mis-amplified dna, thus compromising the specificity of the test [ ] . a new diagnostic method named rt-smartamp assay was developed in japan during the h n pandemic to reduce the time required for detection. the rt-smartamp assay includes reverse transcription and isothermal dna amplification in one step, and rna extraction and pcr are not required. an exciton-controlled hybridizationsensitive fluorescent primer specifically detected the ha segment of the pandemic h n - influenza a virus within minutes without cross-reacting with seasonal a (h n ), a (h n ), or b-type virus. it was found to be an efficient method for detection of iav in patient's swab samples in early stages of infection [ ] . a recent study demonstrated the diagnostic potential of recombinant scfv antibodies generated against the hemagglutinin protein of influenza a virus for diagnosis and treatment of human influenza a virus infections. in that study, an elisa was developed that demonstrated . % sensitivity and % specificity for h n influenza a viruses and promised to be a cheaper alternative to the costly rrt-pcr test [ ] . at research institutes and in reference or hospital laboratories, where sophisticated equipment is available, electron microscopy, cytology and histology may also be used to diagnose influenza virus infections. effective management of influenza lies in following good health practices and preventive measures laid down by health authorities. appropriate treatment of the patients can be done after accurate and timely diagnosis, and this can further reduce the inappropriate use of antibiotics and antiviral therapy. usually, ??antiviral?? therapy is preferred, as bacterial co-infection usually occurs only after viral infection. the first line of antiviral therapeutics that are chosen are inhibitors of viral proteins. the antiviral drugs currently available against influenza viruses are adamantane derivatives (amantadine and rimantadine) and neuraminidase (na) inhibitors (zanamivir, oseltamivir and peramivir). a viral infection can be inhibited at several crucial steps, such as entry, signaling, assembly, and egress (fig. ) . adamantane derivatives inhibit virus multiplication by interfering with the transmembrane domain of the matrix protein (m ) of influenza type a viruses and also interferes in viral assembly during viral replication [ , ] (fig. ) . amantadine was approved for clinical use in , and rimantadine was approved in [ , ] . in the united states, three fda-approved neuraminidase inhibitor antiviral drugs are currently recommended by the us centers for disease control and prevention (cdc): oseltamivir (available under the trade name tamiflu), zanamivir (trade name, relenza), and peramivir (trade name, rapivab). the recently circulating influenza a and b viruses in the usa are susceptible to neuraminidase inhibitors, but amantadine and rimantadine are not recommended because of resistance to these drugs and also because they are not effective against influenza b viruses. all of these drugs are partially licensed against influenza in various countries. controlled clinical trials have shown sufficient effectiveness of these classes of drugs, which also prevent influenza-related illness. oseltamivir and zanamivir are recommended for all individuals with suspected or confirmed influenza requiring hospitalization and patients in high-risk groups, such as children under the age of two years, adults years or older, pregnant women, immunosuppressed individuals, and ??women who have given birth within the previous two weeks??. in addition to the antiviral drugs that are available for treating influenza infections, there are new alternatives with better therapeutic potential, which studies suggest may prove to be beneficial in the near future. the longacting inhaled neuraminidase inhibitor (nai) cs- (also known as r- ) has shown promising results in murine models of influenza treatment [ ] . a polymerase inhibitor, t- (toyama chemical), whose mechanism of action is the inhibition of the viral rna polymerase, has not only been found effective against all three influenza virus types (a, b and c) but is also effective to some extent against other rna viruses, including hemorrhagic fever viruses [ ] . another drug, das , which is a fusion construct that includes the sialidase from actinomyces viscosus, targets the viral attachment process during the early stages of replication of influenza virus [ ] . another recent study showed that chlorogenic acid (cha) has antiviral properties and shows an inhibitory effect on a/puertorico/ / (h n ) (ec = . μm), a/ beijing/ / (h n ) (ec = . μm), and oseltamivirresistant strains in the late stage of the infectious cycle by blocking neuraminidase activity [ ] . cha ( mg/kg/d) administered as an intravenous injection, showed % and % protection from death against the h n and h n strains, respectively, by reducing the viral titers and alleviating virus-associated inflammation in lungs of infected mice [ ] . there are several other novel influenza antiviral drugs under clinical development in the united states, such as avi- , which is a -mer phosphorodiamidate morpholino oligomer (pmo) iv formulation that hampers the translation and splicing of mrna derived from the matrix gene [ ] . cr and cr are monoclonal antibodies that bind to the conserved stalk region of ha and inhibit the entry and fusion stages [ ] . ev- is a dual thromboxane receptor antagonist and thromboxane synthase inhibitor that inhibits virus replication by inhibiting the increase of prostanoids that is associated with influenza virus infections. the drug prevents inhibition of the host immune response by the virus, thus increasing virus replication [ ] . a recent study also showed the anti-influenza activity of a natural product, aureonitol, a compound obtained from fungi that has shown inhibitory effects against both influenza a and b virus replication. aureonitol inhibits influenza virus hemagglutination and thus impairs virus adsorption [ ] . the use of antiviral drugs is preferred during pandemic situations until an effective and sufficient vaccine is available. however, these drugs have a number of side effects, and viruses tend to develop resistance against these drugs over the course of time. the emergence of antiviral-drug-resistant seasonal influenza a viruses is a major concern. initially both amantadine and rimantadine were successful in inhibiting iav infection, and the efficacy was around % [ ] . the first adamantine-resistant viruses were reported during the epidemic, and since then, the number has continued to increase [ ] . surveillance for adamantane resistance among a (h n ) viruses from to revealed that the global frequency of resistance was as low as . % [ ] . another study conducted in showed that this global resistance frequency increased to . %, and a year later it reached %, %, and . % in china, south korea, and the united states, respectively [ ] . the alarming increase in drug-resistant h n strains in the usa in led the us-cdc to issue a public health warning recommending clinicians not to prescribe adamantanes for the remainder of the and season [ ] . most of the adamantineresistant h n isolates ( . %) were found to contain an s n mutation in the m transmembrane domain, while l f, v a, and a t mutations accounted for the rest ( . %) [ ] . starting in , the number of cases of resistance increased exponentially, and from to , almost . % of h n strains and . % of h n were adamantane resistant [ ] . in the usa alone, . % of h n isolates and % of h n isolates were adamantane resistant [ , ] , and the resistance-conferring mutation was s n in the m gene in both h n and h n strains. the pandemic h n - strain [ ] as well as the h n and h n strains that caused fatal zoonotic infections in humans in and , respectively, were also observed to have the same s n mutation in the m gene [ , ] . by , almost % of all the iav isolates were resistant to adamantanes [ ] . the neuraminidase inhibitors (nais) are the second class of anti-influenza drugs, and the only one currently being used worldwide. these drugs target the surface protein na to produce an antiviral effect [ ] . the nais oseltamivir (tamiflu) and zanamivir (relenza) are most effective if administered within - hours of the onset of symptoms [ ] . zanamivir was approved for prophylaxis and treatment of iav infection in humans in july , followed by oseltamivir in october [ ] . the global neuraminidase inhibitor susceptibility network (nisn), established in , reported that during the period - , all human influenza isolates were found to be susceptible to nais; however, in the later years and , the frequency of oseltamivir resistance in global h n isolates increased slightly by . % and . %, respectively [ ] . in - , there was a significant % global rise in oseltamivir-resistant h n , but not h n strains, and all oseltamivir-resistant h n isolates from that season were sensitive to zanamivir [ ] . in the - season, more than % of the globally circulating h n subtypes were found to be oseltamivir resistant [ ] . in the year , the circulating nai-resistant h n strains were replaced by the novel pandemic h n - strain, which, fortunately, was sensitive to nais [ ] . these influenza a (h n and h n ) viruses have shown resistance due to mutation of a histidine to a tyrosine at residue of the na (h y), which confers a high level of resistance to oseltamivir but has no effect on susceptibility to zanamivir or to the adamantanes [ ] . although antiviral drugs against influenza are readily available worldwide, the administration of vaccines remains at the forefront for managing influenza virus infections because prevention is still better and more cost-effective than cure. the administration of influenza and pneumonia vaccines is one of the highest priorities in primary-care medicine [ ] . since their first introduction in the s, influenza vaccines have come a long way [ ] . these early vaccines were inactivated whole-virus vaccines that were generated in embryonated chicken eggs and inactivated by treatment with formalin. the genetic drift in viral genome has made it necessary to formulate new vaccines each year. although separate vaccines are now available for individual influenza viruses, a universal influenza vaccine has not yet been developed due to the highly variable nature of the surface glycoproteins ha and na. who maintains surveillance of circulating strains of influenza viruses in both the northern and southern hemisphere, and influenza vaccines are formulated annually to be administered to healthy individuals and those at higher risk of complications prior to the start of the flu season. there are currently several types of influenza vaccines available, of which the major types are conventional inactivated influenza vaccines and live attenuated influenza virus (laiv) vaccines. the conventional inactivated influenza vaccines consist of purified virus particles that have been inactivated by treatment with formalin or β-propiolactone. live attenuated influenza vaccines are made using virus strains that are cold adapted, temperature sensitive, and attenuated to prevent them from causing illness. laiv vaccines have been successfully made that can be administered via nasal spray (flumist). they have shown high efficacy in children when compared to inactivated vaccines [ ] , as the laiv activates mucosal, systemic humoral, and cellular immunity, just like natural influenza viruses. in the usa and canada, an laiv vaccine is licensed under the trade name flumist, while in europe it is licensed under the trade name fluenz. since laiv vaccines have been observed to be less effective in adults, inactivated split vaccines are recommended for adults. traditional trivalent vaccines containing two influenza a strains (h n and h n ) and one influenza b strain sometimes show limited protection due to a lineage mismatch between the vaccine b strain and the circulating b strain. to minimize the limitation in protection by trivalent vaccines, the fda, for the first time in , considered the inclusion of an additional influenza b strain, thus making a quadrivalent vaccine. both live-attenuated quadrivalent influenza vaccines and inactivated quadrivalent influenza vaccines are known to confer significant protection against the drifted circulating influenza b viruses [ ] . apart from the traditional vaccine approaches, other approaches includes the development of dna vaccines against different influenza virus antigens [ ] , the development of a possible universal influenza vaccine targeting the ha stalk domain [ , ] , and the use of influenza-virus-like particles as vaccines [ ] . the long delivery time frame for egg-based vaccines can be a critical factor during a pandemic, and therefore, cell-culture-based vaccines (e.g., optaflu, flucelvax, preflucel, and celvapan) are also being used to overcome this issue [ ] . due to the increasing burden of vaccine formulations and cases of antiviral-drug-resistant influenza virus isolates turning up every year, it has become necessary to search for alternatives to the current treatment and prevention strategies. the last few decades have seen a tremendous effort being made to develop inhibitors and blockers of vital genes of influenza viruses. novel drugs have been formulated against the viral nucleoprotein [ ] and non-structural proteins [ ] . several studies have also been performed using sirna and antisense oligonucleotides as gene silencing tools to inhibit influenza virus replication in cell lines, chicken embryonated eggs, and mice [ ] [ ] [ ] . the potential of sirnas as antivirals was first recognized in , when this approach was used against the viral transcription factor, p (phosphoprotein), and viral f (fusion) protein of rsv [ ] . in , chen's laboratory published the first report of the use of sirnas against np, pa, pb , pb , m, and ns genes that showed various degrees of inhibition of multiple subtypes of influenza viruses [ ] . a study conducted using antisense oligonucleotides against the ' ncr of vital segments of the iav genome showed significant inhibition of viral replication. the designed antisense molecules were tested against the a/pr/ / (h n ), a/ udorn/ / (h n ), and a/new caledonia/ / (h n ) strains of iav and were found to reduce the cytopathic effect caused by these viruses for almost hours postinfection in cell lines and to increase the survival of experimental mice [ ] . ribozymes (rz) and dnazymes (dz) are yet another class of gene-silencing tools that have been demonstrated to control iav replication [ , ] . a study conducted on the a/pr/ / (h n ) strain showed that rz and dz along with antisense molecules accomplish a synergistic cleavage of the matrix (m ) gene of influenza virus, thus inhibiting virus replication in host cells [ ] . another recent study conducted on influenza b virus also confirmed the role of dz in inhibiting iav replication [ ] . the designed dz showed a significant % inhibition of the b/yamagata/ / strain of influenza b virus [ ] . in another recent study, the authors revealed that self-assembling influenza nanoparticle vaccines could elicit broadly neutralizing h n antibodies. they genetically fused the viral hemagglutinin to ferritin, a protein that naturally forms nanoparticles, and showed that this influenza nanoparticle vaccine generated more than tenfold higher ha inhibition antibody titres than those induced by the licensed inactivated vaccine [ ] . another prospective approach to achieving high virus-neutralizing activity is the use of monoclonal antibodies and recombinant antibody fragments [ , ] . several host-cell molecules have been known to play a crucial role during influenza virus infection, thereby representing targets for designing inhibitors of virus-cell interactions. one such target is the vacuolar proton-atpase, which when inhibited, renders viral m ion channels inactive [ ] . a few other studies have also focused on inhibitors of cellular proteases [ ] that block the proteolytic activation of ha and blockers of the cellular ubiquitin-proteasome system [ ] . in the past few years, there has been a remarkable increase in the number of studies describing the use of a new class of influenza-virus-neutralizing antibodies targeting conserved sites in the ha stem. these molecules have shown varying levels of cross-reactivity toward group [ , ] , group [ , ] and group & viruses [ , ] . despite these efforts, antibodies that can react with the stem region of both group and subtypes are rare and do not cover all subtypes. in view of this, in a recent study, a broad spectrum human monoclonal antibody (mab-medi ) was developed, which unlike other stem-reactive antibodies, used a rare heavy chain vh (vh - ) gene and carried a low level of somatic mutations [ ] . medii was effective in mice and ferrets and was better than oseltamivir. its broad neutralizing capability makes this molecule a good candidate for development as an immunotherapy for influenza-virusinfected humans [ ] . these alternative approaches, which, when backed up with clinical trials, will provide promising tools for managing influenza virus infections effectively. influenza viruses have successfully evolved with striking survival strategies. they circulate worldwide with established lineages in avian and mammalian species. of the four influenza virus types (a, b, c and d), influenza a viruses are the most virulent and have the potential to cause both epidemics and pandemics due to genetic drift and genetic shift, respectively. types b, c and d are not known to cause pandemics. influenza a virus has a wide range of hosts, which provides opportunities to cross species barriers, thus increasing the chances of an influenza pandemic. the virus circulates around the world and causes annual outbreaks resulting in about - million cases of illness and up to , deaths [ ] . two classes of antiinfluenza drugs (adamantanes and nais) are available, of which only the nais are currently effective against circulating strains of iav. vaccination is one of the best approaches to prevent influenza infections annually. however, due to frequent mutations in the surface glycoprotein ha, iav acquire enough mutations each year to escape the protectivity of the annually formulated vaccines, and some of them show a high level of resistance against antiviral drugs [ ] . alternative approaches such as the use of sirna, antisense nucleotides, dz and rz have gained importance in the past few decades and have shown promising results in cell lines and mouse models [ , , , , , ] . several other studies are still being performed to develop a universal influenza vaccine that can neutralize all types of iav. influenza viruses have evolved in parallel to humans to establish successful infections and continue to pose a significant threat to both life and economy. the health authorities invest large amounts of money into annual vaccine formulations, and the virus acquires several mutations to render those vaccines ineffective within a year. thus, new alternative approaches to combating antiviral resistance and the development of universal vaccine formulations are currently needed in order to manage future influenza threats. influenza viruses have been the cause of annual epidemics throughout the world. the a subtypes occasionally cause pandemics that lead to the death of millions of people. the history of influenza suggests that the virus is highly unpredictable in its ability to jump species barriers and cause threatening situations for mankind. health authorities across the world have influenza preparedness plans that are based on combined surveillance data received from both the southern and northern hemisphere. advancements in science have brought together several antiviral therapeutic strategies combined with novel drugs that can be used to manage influenza during annual epidemics. recent attempts to produce a universal influenza vaccine have also shown promise for combating future flu pandemics. disclosure of potential conflicts of interest all of the authors declare that they have no conflict of interest (financial or non-financial). the authors further declare that this is a review article and it did not require research involving human participants and/or animals. informed consent all of the authors declare that this is a review article and did not require research involving human participants; thus, no informed consent was needed. filamentous forms associated with newly isolated influenza virus influenza virus evolution, host adaptation, and pandemic formation fields virology, th edn 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influenza a virus challenge protective immunity based on the conserved hemagglutinin stalk domain and its prospects for universal influenza vaccine development influenza virus vaccine based on the conserved hemagglutinin stalk domain an intranasal virus-like particle vaccine broadly protects mice from multiple subtypes of influenza a virus traditional and new influenza vaccines development of an anti-influenza drug screening assay targeting nucleoproteins with tryptophan fluorescence quenching novel influenza virus ns antagonists block replication and restore innate immune function small interfering rna targeting the nonstructural gene transcript inhibits influenza a virus replication in experimental mice cross-protective effect of antisense oligonucleotide developed against the common ' ncr of influenza a virus genome rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering rna and its application in the reverse genetics of wild type negative-strand rna viruses gene silencing: a therapeutic approach to combat influenza virus infections design of deoxyribozymes for inhibition of influenza a virus nucleic acid-mediated cleavage of m gene of influenza a virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza a h hemagglutinin in mice potent neutralization of influenza a virus by a singledomain antibody blocking m ion channel protein concanamycin a blocks influenza virus entry into cells under acidic conditions aprotinin and similar protease inhibitors as drugs against influenza the clinically approved proteasome inhibitor ps- efficiently blocks influenza a virus and vesicular stomatitis virus propagation by establishing an antiviral state heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine broadly cross-reactive antibodies dominate the human b cell response against pandemic h n influenza virus infection preexisting human antibodies neutralize recently emerged h n influenza strains a highly conserved neutralizing epitope on group influenza a viruses a potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza a virus a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins structure and function analysis of an antibody recognizing all influenza a subtypes epidemiology and pathogenesis of influenza influenza virus resistance to neuraminidase inhibitors key: cord- -r c sx r authors: gaertner, diane j.; smith, abigail l.; paturzo, f. x.; jacoby, r. o. title: susceptibility of rodent cell lines to rat coronaviruses and differential enhancement by trypsin or deae-dextran date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: r c sx r cell lines of rodent origin were tested for susceptibility to infection with rat coronavirus (rcv), including sialodacryoadenitis virus (sdav) and parker's rat coronavirus (prcv). lbc rat mammary adenocarcinoma cells were susceptible only if the cells were treated with diethylaminoethyl-dextran (deae-d). a recent report that rcvs grow well in l mouse fibroblast cells was confirmed and expanded. rcv infection of l cells was substantially enhanced by treatment of cells with trypsin but not by treatment with deae-d. primary isolation of sdav from experimentally infected rats was accomplished using trypsin-treated l cells. one of additional cell lines tested (rat urinary bladder epithelium, rbl- ) supported growth of rcvs, and growth was slightly enhanced by deae-d, but not by trypsin. these refinements of in vitro growth conditions for rcvs should facilitate further studies of their basic biology and improve options for primary isolation. rat coronaviruses (rcvs), notably sialodacryoadenitis virus (sdav) and parker's rat coronavirus (prcv), infect laboratory rats at high prevalence, cause clinically and economically significant disease and interfere with research using rats [ ] . diagnostic and experimental studies of rcvs have been limited by the lack of a reliable cell line in which to grow the viruses [ ] . rcvs have traditionally been grown in primary rat kidney (prk) cells or infant mouse brain but these techniques require the use of animals and are labor intensive. although many coronaviruses have fastidious growth requirements, replicating only in organ cultures or cell lines derived from the animal or tissue of origin, cell lines supporting growth of most coronaviruses have been identified [ , ] . attempts to grow sdav in a variety of rodent cell lines, including mouse neuroblastoma (clone n ) cells, c rat glial cells, and mhv-susceptible cell lines such as nctc cells failed (l. e. garlinghouse and a. l. smith, unpubl, data) . recently h i r a n o et al. reported that a rat m a m m a r y adenocarcinoma cell line (lbc) supported r c v growth [- ] , and percy et al. reported that l cells supported r c v s [ ] . in studies reported here, l b c cells, l cells and other cell lines o f rodent origin were tested for their susceptibility to rcvs. a rat bladder epithelial cell line (rbl- ) was identified which supports r c v growth. m e d i u m containing trypsin or diethylaminoethyl-dextran ( d e a e -d ) , which have been used to enhance the growth of other coronaviruses [ , ] , also differentially enhanced r c v growth in selected cell lines. the cell lines tested and media used are listed in table . hh cells were obtained by in vitro transformation of prk cells [- ] by high multiplicity infection with sv- [ ] . cell cultures were grown in cm polystyrene flasks (corning glassworks, corning, ny) at °c in a humidified % co, atmosphere and were maintained in media containing gg/ ml streptomycin and units/ml penicillin. all media and supplements were purchased from gibco brl (gaithersburg, md), except mcdb- and f- media (sigma chemical co., st. louis, mo). cells were transferred when confluent using . % trypsin and . mm edta. two sublines of l cells obtained from different laboratories were used (table ) . cell lines received from extramural sources except the american type culture collection were tested for mycoplasma spp. and were culture-negative. stocks of sdav, strain (sdav- ), and parker's rat coronavirus (prcv) were prepared in infant mouse brain [- , ] . titration of prcv and sdav- stocks in primary rat kidney cells [ , ] yielded titers of tcids and . tcids per ml, respectively. the sdav stock used for concurrent l and mouse titration was one infant mouse passage removed from the stock used for cell culture inoculations. cr: orl sencar dams with litters were purchased (animal genetics and production branch, national institutes of health, bethesda, md) and housed under conditions which met or exceeded standards of the u.s. public health service. when cell monolayers were approximately % confluent, growth medium (table ) was removed and cultures were exposed to . ml of a % suspension of virus-infected mouse brain or . ml of diluent. fetal calf serum (fcs) was omitted from medium used for trypsin-or deae-d-treated cells. following virus absorption at °c for h, ml of medium were added to all flasks. cultures were observed daily. cytopathic effect (cpe) was typified by syncytium formation. the percentage of the monolayer that consisted of syncytia was estimated, and all infected and mock-inoculated cultures were harvested at h post-inoculation. air-dried, fixed cells were incubated with sdav-immune mouse ascitic fluid or with pooled sdav-immune serum from experimentally infected rats followed by incubation with fluoresceinated goat anti-species immunoglobulin (antibodies, inc., davis, ca) containing . % evans's blue [ ] . cells were examined at a magnification of x with a transmitted light fluorescence microscope. concentrations of porcine parvovirus-free, mycoptasma-free trypsin (gibco brl) from . gg/ml to gg/ml were added to the absorption and incubation media for l (percy) and rbl- cells. since some tissue homogenates in combination with ~tg/ml trypsin were toxic to l (percy) cells, . gg/mt of trypsin was used. trypsin-treated uninoculated cells were included at each concentration to control for toxicity. four ml of trypsincontaining medium were added for incubation. concentrations of , , , and ~tg/ml of deae-d (pharmacia fine chemicals, uppsala, sweden) were added to the pre-absorption and absorption media of lbc, l (percy) and rbl- cells to allow sufficient time for action on cell membranes. duplicate cultures were pre-treated with deae-d-containing medium for h, and virus or diluent was added for h at °c [ ] . deae-d containing medium was replaced with growth medium for incubation. nasal washes, lungs and salivary and lacrimal glands from control rats or rats experimentally infected with sdav- were provided by dr. eleanor c. weir, yale university. the tissues had previously been tested for the presence of infectious virus by intracerebral inoculation of infant mice [ ] and had been stored at - °c until use in this study. one ml of a - % (w/v) tissue homogenate in trypsin-containing medium ( . ~tg/ml) was used for inoculation of l (percy) cells. because lacrimal and salivary gland inocula were toxic to l (percy) cells, they were removed following absorption. nasal wash and lung inocula were not removed. cells were examined daily for cpe by an observer uninformed of their status in the prior mouse inoculation study. cells were subsequently processed and stained for viral antigen. serial ten-fold dilutions of stock sdav- virus were inoculated intracerebrally into day old sencar mice and into flasks containing l (percy) cells in trypsin ( . gg/ml)supplemented medium. mouse mortality at each dilution was recorded for days. l (percy) cells were observed daily for cpe and were stained for viral antigen h after inoculation. titers expressed as lds or tcids per ml were calculated [ ] . tibility of l (percy) cells to prcv was erratic. less than % of the monolayer formed syncytia, and antigen-positive cells ( < %) were seen on the first passage in half of the attempts, whereas prcv growth was not detected in the remaining attempts. seven additional passages did not enhance prcv growth. rbl- cells sdav- and prcv grew in the first passage in rbl- cells. syncytia involving % of the monolayer formed within h (fig. ) and antigen-positive cells (about %) were seen by if staining (fig. ) . cpe was not seen in the second passage, and fewer cells contained antigen. virus growth was not detected in the third through seventh passages. repeated attempts, including blind passages, failed to reveal growth of sdav- or prcv in lbc cells. cpe was not seen, and virus antigen was not detected. other cell lines rcv growth was not detected in other cell lines listed in table . at least passage attempts of both sdav- and prcv were completed in all cell lines. trypsin enhanced growth o f r c v s in l (percy) cells in the first passage ( table ) concentrations o f - gg/ml of d e a e -d substantially enhanced growth of r c v s in l b c cells, enhanced growth marginally in r b l - cells, and did not affect growth in l (percy) cells (table ). viral antigen was not detected in mock-treated, inoculated l b c cells, b u t a b o u t % of non-adherent cells were virus antigen-positive in treated, inoculated cultures following exposure to in a single attempt to isolate sdav- from the lung of an experimentally infected rat, syncytia and viral antigen were first seen after passages in untreated l (percy) cells. in contrast, syncytia and antigen were observed in the first passage in trypsin-treated l (percy) cells. based on this finding, a small study was initiated to ascertain the sensitivity of trypsin-treated l (percy) cells for primary isolation of rcvs. virus isolations were attempted using tissue homogenates previously tested for sdav- by infant mouse inoculation (table ). cpe and virus antigen were observed in % and %, respectively, of cultures exposed to viruspositive tissues. the highest concordance between mouse and l (percy) cell inoculation ( %) was obtained using nasal wash as the inoculum. a single stock of sdav was quantified concurrently in infant mice and trypsin-treated l (percy) cells. the titer of stock sdav- w a s . ldso per ml in infant mice and . tcids per ml in trypsin-treated l (percy) cells. limited rcv growth was demonstrated in untreated l (percy) and rbl- cells, but not in other celt lines tested, including lbc cells and an l subtine obtained from another source. a recent observation of sdav antigen in transitional epithelium of experimentally infected athymic rats [ ] led us to assess rat bladder epithelial cell lines for growth of rcvs, and rbl- cells supported rcv growth on initial passage. of the cell lines and treatments tested, l (percy) cells treated with trypsin supported maximal growth of rcvs as evidenced by syncytium formation and viral antigen. rcv growth in l (percy) cells was not enhanced by trypsin treatment of later passages when the enzyme was added at the time of virus inoculation. adding trypsin at a different time in the rcv replicative cycle might enhance rcv growth in successive passages. as previously reported by percy etal. [ ] , repeated passage of sdav- in untreated l (percy) cells increased viral growth. repeated passage of other animal-adapted rcv strains in l (percy) cells may accomplish adaptation and increased virus yield. trypsin, but not deae-d, enhanced rcv growth in l (percy) cells. the mechanism by which trypsin increases rcv growth in these cells is not known. however, trypsin enhances mouse hepatitis virus (mhv) plaque formation [ , -] and cleaves the k e glycoprotein of mhv a into two k fragments [ ] . cleavage is associated with a two-fold increase in infectivity and with activation of the cell-fusing activities of virions in vivo [ , ] . therefore, trypsin could enhance rcv infectivity by cleaving its e protein analogue. trypsin treatment of l (percy) cells permitted primary isolation of sdav from tissues of infected rats without repeated passage. the use of trypsin-treated l (percy) cells to screen tissues for rcvs would significantly reduce animal use and expense. however, l (percy) cells were about log less sensitive than infant mice based on comparative titration of sdav- . this may have been due to adaptation of stock sdav- to infant mouse brain by virtue of multiple prior passages in mice. if trypsin-treated l (percy) cells are inherently less sensitive than infant mice, tissues that are virus-negative by assay in these cells should be re-tested in mice to eliminate false negatives. uninfected l (percy) cells are morphologically heterogeneous and sensitive to tissue toxicity; therefore, antigen detection which is less subjective than interpretation of morphologic changes should be the definitive test used after cell culture inoculation. using trypsin-treated l (percy) cells, virus in lacrimal gland was more difficult to detect than virus in other tissues tested. a possible explanation for low isolation rates from lacrimal gland is the presence of a trypsin-inhibitor that is not present in other tissues. however, litters of mice inoculated with lacrimal gland homogenates tended to have lower mortality rates and deaths that occurred later than in groups inoculated with other tissues (data not shown). this suggests that lacrimal gland samples may simply have contained lower concentrations of virus. rcv growth was increased in lbc and rbl- cells treated with deaedextran, a chemical that enhances virus binding and uptake by altering electrostatic interactions [ ] . this result suggests that virus attachment and/or penetration are the limiting steps for rcv growth in these cell lines. deae-d also enhances infection of cells with mhv- [ ] , although the mechanism is unknown. studies of binding and internalization with m h v -j h m have shown, however, that several rat cell lines share a common deficiency at the level of virus internalization [ ] . d e a e -d may act by enhancing rcv binding to lbc and rbl- cells, thereby facilitating viral entry into cells that otherwise support rcv growth. little is known about the replication and biochemistry of rcvs largely due to their fastidious in vitro growth requirements. our described refinements of in vitro growth conditions for rcvs should facilitate both in vitro and in vivo studies. repeated passage of rat-adapted rcv isolates and rcv growth in l (percy) and rbl- ceils may increase virus yield and permit more sophisticated studies of antigenic relatedness among rcv isolates. use of trypsin-treated l (percy) cells should facilitate in vivo studies of rcv pathogenesis requiring rcv isolation. effects of deae-dextran on infection and hemolysis by vsv. evidence that nonspecific electrostatic interactions mediate effective binding of vsv to cells experimental infection of adult axenic rats with parker's rat coronavirus respiratory infection in mice with sialodacryoadenitis virus, a coronavirus of rats replication of rat coronavirus in a rat cell line mouse hepatitis virus: molecular biology and implications for pathogenesis several rat cell fines share a common defects in their inability to internalize murine coronaviruses efficiently viral and mycoplasmal infections of laboratory rodents: effects on biomedical research radial gradient culture on the inner surface of collagen tubes: organoid growth of normal rat bladder and rat bladder cancer cell line nbt-ii cell culture techniques for diagnostic virology correlations between cell surface protease activities and abnormalities of occludens junctions in rat bladder carcinoma in vitro replication of sialodacryoadenitis virus in mouse l- cells a simple method of estimating fifty percent endpoints cellular effects of haematoporphyrin derivitive photodynamic therapy on normal and neoplastic rat bladder cells an immunofluorescence test for detection of serum antibody to rodent coronaviruses enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment characterization of a coronavirus. ii glycoproteins of the viral envelope: tryptic peptide analysis proteolytic cleavage of the e glycoprotein of murine coronavirtls: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments enhanced growth of a murine coronavirns in transformed mouse cells an improved method for titration of mouse hepatitis virus type in a mouse cell line reciprocal productive and restrictive virus-cell interactions of immunosupressive and prototype strains of minute virus of mice persistence of sialodacryoadenitis virus in athymic rats the authors thank dr. dean received october , key: cord- -ygomb oi authors: zakaryan, hovakim; arabyan, erik; oo, adrian; zandi, keivan title: flavonoids: promising natural compounds against viral infections date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: ygomb oi flavonoids are widely distributed as secondary metabolites produced by plants and play important roles in plant physiology, having a variety of potential biological benefits such as antioxidant, anti-inflammatory, anticancer, antibacterial, antifungal and antiviral activity. different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. this review summarizes the evidence for antiviral activity of different flavonoids, highlighting, where investigated, the cellular and molecular mechanisms of action on viruses. we also present future perspectives on therapeutic applications of flavonoids against viral infections. throughout human history, thousands of biologically active plants have been identified and used in medicine. virtually all cultures around the world continue to rely on medicinal plants for primary health care. according to the world health organization report, about % of the world's population depend on medicinal plants to satisfy their health requirements [ ] . furthermore, there are currently hundreds of modern drugs based on active compounds isolated from plants. plants have the ability to produce a wide range of compounds including flavonoids, phytoalexins, lignans, and tannins, which are responsible for key functions in plant growth and development. flavonoids or polyphenolics comprise the largest group of secondary metabolites found in vegetables, fruits, seeds, nuts, spices, stems as well as in red wine and tea (table ) [ ] . these compounds are synthesized in response to various abiotic stress conditions such as ultraviolet radiation and play an important role as defense agents against plant pathogens and insects [ , ] . the first evidence of a biological activity of flavonoids was reported by albert szent-gyorgyii in , who showed that citrus peel flavonoids prevent capillary bleeding and fragility associated with scurvy [ ] . since then, a broad spectrum of biological activities such as anti-inflammatory, antioxidant, antibacterial, antiviral, anticancer, and neuroprotective has been described for flavonoids [ , , , , ] . research for antiviral agents isolated from plants started in s, when the activity of plants against influenza a virus was evaluated in embryonated eggs [ ] . during the last years, several plants and plant-derived compounds with antiviral properties were identified. in this article, we review the results of both in vitro and in vivo experiments demonstrating the antiviral activity of flavonoids, especially focusing on those classes of flavonoids that have been extensively investigated. there are now more than varieties of flavonoids that have been structurally identified [ ] . all these compounds comprise a flavan nucleus and a fifteen-carbon skeleton consisting of two benzene rings (a-and b-rings, as shown in fig. ) connected via a heterocyclic pyrene ring (c-ring, as shown in fig. ). flavonoids are divided into several classes such as anthocyanidins, flavones, flavonols, flavanones, flavan, isoflavanoids, biflavanoids, etc (table ) [ ] . the various classes of flavonoids differ in the level of oxidation and pattern of substitution of the pyrene ring, whereas individual compounds within the classes differ in the pattern of substitution of benzene rings. while in flavonoids the b-ring links to the c-ring at the c position, the b-ring of isoflavonoids is substituted at position c (fig. ). biflavonoids comprise of two identical or non-identical flavonoid units conjoined through an alkyl-or alkoxybased linker (fig. ) . in plants, flavonoids generally occur as aglycones, glycosides and methylated derivatives. they are biosynthesized through the phenylpropanoid pathway, transforming phenylalanine into -coumaroyl-coa, which then enters the flavonoid biosynthesis pathway [ ] . depending on the plant species, a group of enzymes, such as hydroxylases and reductases, modify the basic flavonoid skeleton, resulting in the different flavonoid classes. finally, transferases modify the flavonoid skeleton with sugar, methyl groups and acyl moieties. these modifications alter the solubility and reactivity of flavonoids [ ] . a large body of evidence supports the role of light in the regulation of flavonoid biosynthesis [ ] . flavones constitute a major class in the flavonoid family based on a -phenyl- -benzopyran- -one backbone. natural flavones include apigenin, baicalein, chrysin, luteolin, scutellarein, tangeritin, wogonin and -hydroxyflavone. the antiviral activity of flavones is known from the s, when it was showed that the simultaneous application of apigenin with acyclovir resulted in an enhanced antiviral effect on herpes simplex virus types and (hsv- and hsv- ) in cell culture [ ] . apigenin is most commonly isolated in abundance from the family asteraceae. the organic and aqueous extracts from asteraceae plants with apigenin as a major compound were found to be active against hsv- , poliovirus type and hepatitis c virus (hcv) [ , ] . apigenin isolated from sweet basil (ocimum basilicum) showed a potent antiviral activity against adenoviruses (adv) and hepatitis b virus in vitro [ ] . besides these dna viruses, apigenin was found to exert antiviral effect against african swine fever virus (asfv), by suppressing the viral protein synthesis and reducing the asfv yield by log [ ]. apigenin is also active against rna viruses. for picronaviruses, it has been shown that apigenin is able to inhibit viral protein synthesis through suppressing viral ires activity [ , ] . furthermore, apigenin affects enterovirus- (ev ) translation by disrupting viral rna association with trans-acting factors regulating ev translation [ ] . shibata et al. [ ] showed that apigenin has antiviral effect on hcv through the reduction of mature microrna , a liver-specific microrna which positively regulates hcv replication. among flavones, baicalein and luteolin have been also extensively investigated with respect to their antiviral activity. baicalein significantly reduced the levels of human cytomegalovirus (hcmv) early and late proteins, as well as viral dna synthesis, although it had no effect on viral polymerase activity [ , ] . baicalein impaired avian influenza h n virus replication in both human lung epithelial cells and monocyte-derived macrophages by interfering with neuraminidase activity [ ] . other studies showed that oral administration of baicalein to balb/c mice infected with influenza h n virus decreased the lung virus titer and increased the mean time to death [ ] . similar effects were recorded on mice infected with sendai virus [ ] . these inhibitory effects in vivo were mediated by serum baicalin, a metabolite of baicalein which has a glucose residue [ ] . baicalin alone exerts its anti-influenza activity by modulating the function of ns protein, which down-regulates ifn induction [ ] . further studies indicated that baicalin can directly induce ifn-c production in human cd ? t cells and cd ? t cells and act as a potent inducer of ifn-c during influenza virus infection [ ] . recently, novel baicalein analogs with b-rings substituted with bromine atoms demonstrated extremely potent activity against influenza h n tamiflu-resistant virus, indicating that baicalein and its analogs can be favorable alternatives in the management of tamiflu-resistant viruses [ ] . in vitro replication of hiv- was suppressed by baicalin when infected cells were treated during the early stage of the virus replication cycle [ ] . hiv- envelope protein was found to be the target site of baicalin's antiviral action via the interference of interactions between the virus structural protein and specific host immune cells [ ] . baicalein and baicalin were also investigated against dengue virus (denv). they exerted a significant virucidal effect on extracellular viral particles and interfered with different steps of denv- replication [ , , ] . in silico studies revealed that baicalein has strong binding affinity with denv ns /ns b protein (- . kcal/mol), and baicalin may interact closely with the virus ns protein at a binding affinity of - . kcal/mol [ ] . for baicalin, computational studies also showed a high binding affinity (- . kcal/mol) against chikungunya virus (chikv) nsp protein, suggesting that baicalin can potentially interfere with chikv infection [ ] . it was found that luteolin has antiviral effect on hiv- reactivation by blocking both clade b-and c-tat-driven ltr transactivation [ ] . luteolin also showed significant inhibition of epstein-barr virus (ebv) reactivation in cells [ ] ; it suppressed the activities of the immediate-early genes zta and rta by deregulating transcription factor sp binding. xu et al. [ ] tested highly purified natural compounds for inhibition of ev and coxsackievirus a infections and found that luteolin exhibited the most potent inhibition through disruption of viral rna replication. besides these antiviral activities, luteolin or luteolin-rich fractions showed antiviral effects against severe acute respiratory syndrome coronavirus (sars-cov), rhesus rotavirus, chikv and japanese encephalitis virus (jev) [ , , , ] . flavonols are characterized by a -hydroxy- -phenylchromen- -one backbone. among flavonols the antiviral effect of quercetin was the most extensively investigated. early in vivo studies showed that oral treatment with quercetin protected mice from lethal mengo virus [ , ] . furthermore, an enhanced protection was observed when quercetin was administered in combination with murine type i interferon (ifn) [ ] . quercetin also demonstrated a dose-dependent antiviral activity against poliovirus type , hsv- , hsv- , and respiratory syncytial virus (rsv) in cell cultures [ , ] . epimedium koreanum nakai, which contains quercetin as the major active component, has been shown to induce secretion of type i ifn, reducing the replication of hsv, newcastle disease virus (ndv), vesicular stomatitis virus (vsv) in vitro, as well as influenza a subtypes (h n , h n , h n and h n ) in vivo [ ] . hung et al. [ ] have suggested possible mechanisms whereby quercetin may exert its anti-hsv activity. they revealed that quercetin inhibits the infection of hsv- , hsv- and acyclovirresistant hsv- mainly by blocking viral binding and penetration to the host cell. they also reported that quercetin suppresses nf-jb activation, which is essential for hsv gene expression. recent investigations also pointed out the antiviral activity of quercetin against a wide spectrum of influenza virus strains. it interacts with influenza hemagglutinin protein, thereby inhibiting viralcell fusion [ ] . in addition, in silico analysis revealed that quercetin may be a potential inhibitor of the neuraminidase of influenza a h n and h n viruses [ , ] . molecular docking analysis also found that quercetin may interact with hcv ns helicase, ns b polymerase and p proteins [ , ] . these results correlate with experimental studies showing the anti-hcv activity of quercetin through inhibition of ns helicase and heat shock proteins [ , ] . besides these viruses, the inhibitory activity of quercetin and its derivatives have been reported for other viruses, including advs, arthropod-borne mayaro virus, porcine reproductive and respiratory syndrome virus, canine distemper virus, jev, denv- , porcine epidemic diarrhea virus, and equid herpesvirus [ , , , , , , , ] . quercetin also possesses anti-rhinoviral effects by inhibiting endocytosis, transcription of the viral genome and viral protein synthesis [ ] . in mice infected with rhinovirus, quercetin treatment decreased viral replication and attenuated virusinduced airway cholinergic hyper-responsiveness [ ] . kaempferol is another flavonol extracted from different medicinal herbs. kaempferol and its derivatives bearing acyl substituents have shown inhibitory activity against hcmv [ ] . kaempferol derivatives isolated from ficus benjamina leaves were more effective against hsv- and hsv- than their aglycon form [ ] . kaempferol derivatives with rhamnose residue turned out to be potent inhibitors of the a channel of coronavirus, which is involved in the mechanism of virus release [ ] . one of the kaempferol derivative, kaempferol -o-a-lrhamnopyranoside, obtained from zanthoxylum piperitum was shown to significantly inhibit the replication of influenza a virus in vitro [ ] . behbahani et al. found that kaempferol and kaempferol- -o-glucoside have strong hiv- reverse transcriptase inhibitory activity [ ] . these compounds exerted their effects, at a concentration of lg/ml, on the early stage of hiv replication in target cells. recently, kaempferol- , -bisrhamnoside isolated from chinese medicinal taxillus sutchuenensis was shown to have potent in vitro activity on hcv ns protease function [ ] . antiviral activity of kaempferol on the influenza viruses h n and h n were mentioned in a study conducted by a group of researchers in south korea. mechanistic and structural studies suggested that the compound acts on the virus neuraminidase protein and specific functional groups are responsible for kaempferol's efficacy [ ] . a study comparing the antiviral activities of kaempferol and an isoflavone, daidzein, showed that kaempferol exerted more potent inhibitory activities on jev replication and protein expression, than daidzein. jev's frameshift site rna (fsrna) has been proposed as the target site for kaempferol's inhibitory activity against this flavivirus [ ] . seo et al. conducted a study comparing the potency of different classes of flavonoids against two rna viruses, namely murine norovirus and feline calicivirus. their findings demonstrated that, among the flavonoids tested, kaempferol exhibited the most potent inhibitory activity against these two viruses [ ] . there are number of other flavonols and derivatives acting as antivirals. for example, sulfated rutin, which is modified from glycoside rutin, demonstrated significant activity against different hiv- isolates [ ] . this compound inhibited hiv- infection by blocking viral entry and virus-cell fusion, likely by interacting with hiv- envelope glycoproteins. rutin at lm concentration was shown to inhibit ev infection by suppressing the activation of mek -erk signal pathway, which is required for ev replication of [ ] . rutin and fisetin also inhibited the replication of ev-a by affecting the enzymatic activity of the c protease [ ] . fisetin treatment caused a dose-dependent decrease in the production of chikv nonstructural proteins and inhibition of viral infection [ ] . moreover, zandi et al. showed that denv- rna copy number was significantly reduced following addition of fisetin to infected cells [ ] . yu et al. found that myricetin may serve as chemical inhibitor of sarscoronavirus because it affects the atpase activity of the viral helicase [ ] . flavans are characterized by a -phenyl- , -dihydro- hchromene skeleton. these compounds include flavan- -ols, flavan- -ols and flavan- , -diols. among flavan- -ols, the antiviral activity of catechin and its derivatives epicatechin, epicatechin gallate, epigallocatechin (egc), and epigallocatechin gallate (egcg), which are found in tea, has been largely investigated [ ] . among different viruses studied as potential targets, influenza virus has received the most attention after an initial report by nakayama et al. showing that tea catechins, particularly egcg, are able to bind to the haemagglutinin of influenza virus, preventing its adsorption to madin-darby canine kidney cells [ ] . furthermore, it has been suggested that egcg may be able to damage the physical properties of the viral envelope, resulting in the inhibition of hemifusion events between influenza virus and the cellular membrane [ ] . recently, colpitts and schang reported that egcg competes with sialic acid for binding to influenza a virus, thereby blocking the primary low-affinity attachment to cells [ ] . another tea catechin, egc, exerted the inhibitory effect on the acidification of endosomes and lysosomes, thereby reducing viral entry via clathrin-mediated endocytosis [ ]. a structure-function relationship analysis of tea catechins revealed the important role of the -gallolyl group of the catechin skeleton for its antiviral activity [ ] . the results also showed that modification of the -hydroxyl position significantly affected the antiviral activity. catechin derivatives containing carbon chains at -hydroxyl position demonstrated potent anti-influenza activity in vitro and in ovo [ ] . several reports have demonstrated that tea catechins have an antiviral effect against hiv infection. among tea catechins, egcg is the most effective because it exerts its antiviral effect throughout several steps of the hiv- life cycle. it directly binds to cd molecules with consequent inhibition of gp binding, an envelope protein of hiv- [ , ] . these studies identified trp , arg and phe of cd as potential sites for interaction with the galloyl moiety of egcg. the same residues are involved in interaction with viral gp [ ] . furthermore, early studies from nakane and ono showed that egcg and ecg were effective at inhibiting hiv- reverse transcriptase in vitro [ , ] . tillekeratne et al. modified the molecular structure of egcg to determine the minimum structural characteristics necessary for hiv- reverse transcriptase inhibition [ ] . in their study, the gallate ester moiety was found to be important for inhibition. besides these effects, egcg has the ability to reduce viral production in chronically infected monocytoid cells [ ] . the inhibitory effect was increased by approximately %, when egcg was modified with lyposomes. tea catechins are also effective against herpesviruses. egcg has been shown to block ebv lytic cycle by inhibiting expression of viral genes including rta, zta and ea-d [ ] . further studies indicated that one of the mechanisms by which egcg may inhibit ebv lytic cycle involves the suppression of mek/erk / and pi -k/akt signaling pathways, which are involved in the ebv lytic cycle cascade [ ] . isaacs et al. found that egcg can inactivate hsv virions by binding to the envelope glycoproteins gb and gd, which are essential for hsv infectivity [ ] . the egcg digallate dimers theasinensin a, p , and theaflavin- , '-digallate inactivated hsv- and hsv- more effectively than did monomeric egcg [ ]. these dimers are stable at vaginal ph, indicating their potential to be antiviral agents against hsv infections. the inhibitory effect of green tea extracts against hbv has been reported [ ] . in hepg . cells, egcg inhibited hbv replication through impairing hbv replicative intermediates of dna synthesis, thereby reducing the production of hbv covalently closed circular dna [ ] . in contrast, huang et al. found that egcg decreased hbv entry into immortalized human primary hepatocytes by more than % but had no effect on hbv genome replication [ ]. furthermore, egcg is able to enhance lysosomal acidification, which is an unfavorable condition for hbv replication [ ] . besides these viruses, egcg has been found to exert antiviral activity against hcv by preventing the attachment of the virus to the cell surface and suppressing rna replication steps [ , ] . a recent study also showed inhibitory activity of egcg against another flavivirus, zika virus (zikv): in this study, foci forming unit reduction assays were performed to evaluate the antiviral activity of egcg on zikv at different stages of virus replication. foci observed showed more than % inhibition when the cells were treated with egcg during virus entry [ ] . similarly, egcg is able to block chikv attachment to target cells, but has no effect on other stages of infection [ ] . naringenin, which belongs to the flavanones class, has been shown to reduce the replication of a neurovirulent strain of sindbis virus in vitro [ ] . it also reduced sindbis virus-and semliki forest virus-induced cytopathic effect in virus yield experiments [ ] . interestingly, naringin, the glycoside form of naringenin did not have anti-sindbis virus activity, indicating that the rutinose moiety of this flavanone blocks its antiviral effect. naringenin is also able to block the assembly of intracellular hcv particles and long-term treatment leads to . log reduction in hcv [ , ] . the alphavirus chikv was effectively inhibited when infected vero cells were treated with naringenin at the post-entry stage. in the same study, hesperetin, another flavanone which is found richly in citrus fruits, was found to exert most potent anti-chikv effect during the virus intracellular replication, with an ic of . lm [ ] . molecular docking and molecular dynamics studies by oo et al. also revealed strong and stable interactions between hesperetin and chikv nonstructural protein (nsp ) as well as non-structural protein (nsp ), suggesting that these proteins may be the target of hesperetin's anti-chikv activity [ ] . genistein is an isoflavonoid found in a number of plants including soybeans and fava beans. as a tyrosine kinase inhibitor, genistein reduced bovine herpesvirus type and new world arenavirus pichinde replication, by preventing the phosphorylation of viral proteins [ , ] . kinase inhibitor cocktails containing genistein displayed a broadspectrum antiviral activity against arenaviruses and filoviruses [ ] . genistein was shown to inhibit hiv infection of resting cd t cells and macrophages through interference with hiv-mediated actin dynamics [ ] . furthermore, it may act against hiv ion channel since it has the ability to block the viral vpu protein, which is believed to form a cation-permeable ion channel in infected cells [ ] . genistein also exerted its antiviral effects on the replication of hsv- , hsv- , and avian leucosis virus subgroup j, by inhibiting virus transcription [ , ] . the antiviral activity of other flavonoids is presented in table . in spite of the wide range of biological health benefits which flavonoids possess, in addition to their high availability in humans' daily diets, there are challenges ahead for researchers before these natural compounds can be applied as therapeutic options in the clinical setting. bioavailability, defined by the us food and drug administration as ''the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action'', has been the main stumbling block to further advances in the potential use of flavonoids in the medical community. intake of metabolic derivatives of flavonoids from various food sources leads to relatively large differences in the final amount being successfully absorbed and utilized by humans [ ] . factors such as molecular sizes, glycosylation, esterification, lipophilicity, interactions with the enteric microorganisms, pka, and other metabolic conjugations along the alimentary tract, affect the absorption and bioavailability of flavonoids in humans [ , , , , , , ] . hence, efforts in enhancing the bioavailability of flavonoids upon intake by humans are vitally necessary in order to develop these natural compounds into potential antiviral drugs. the following are a few examples of efforts being carried out to tackle this issue which can be used as platforms for further successes in the future. in the past, researchers have looked into alternative methods to improve the compounds' solubility or to switch the site of absorption in the gut, with the aim of enhancing their bioavailability. a structural modification to hesperetin- -glucoside, which resulted in a change in site of absorption from the large to the small intestine, has successfully yielded a higher plasma level of hesperetin in healthy subjects [ ] . wang et al. [ ] formulated a way to increase the oral bioavailability of flavonols extracted from sea buckthorn, by forming a phospholipid complex via solvent evaporation method. relative to the parent compounds, oral bioavailability of the tested flavonols was % - % higher when the phospholipid complex was administered into rats [ ] . flavonoids loaded in engineered nanoparticles have also been tested for their bioavailability following oral consumption. improved stability of catechin and egcg in chitosan nanoparticles have been shown to result in a higher rate of intestinal absorption [ ] . poly (d, l-lactide) (pla) nanoparticles and polymeric micelles contributed to a more sustainable release of quercetin, which has poor bioavailability and undergoes substantial first-pass metabolism, as well as of the poorly absorbed apigenin [ , , ] . self-microemulsifying drug delivery system (smdds) is another technology which has been used to overcome the problem of low bioavailability of hydrophobic molecules. upon entering the lumen of the intestine, an oil-in-water microemulsion containing the drug will be formed. the microemulsion increases the intestinal absorption of the drug or compound by avoiding the dissolution process [ , ] . puerarin, an isoflavone isolated from the root of pueraria lobata, exhibited . -fold higher bioavailability when prepared using smdds [ ] . however, it is worth noting that while the bioavailability of flavonoids can be increased via different methodologies, it is vital that their biological efficacies are not affected, but maintained or enhanced. for instance, phosphorylated icariin has been found to inhibit duck hepatitis virus a more effectively than the parent compound [ ] . isorhamnetin is a methylated flavonol derived from the structure of quercetin. dayem et al. investigated the antiviral potency of isorhamnetin against influenza a h n virus and discovered that the methyl group on the b ring enhances its antiviral activity compared with the other tested flavonoids [ ] . the efficacy of isorhamnetin against influenza virus was also shown when in vivo and in ovo models were tested [ ] . improvement in bioavailability will definitely enhance the efficacy of different biological effects of all classes of flavonoids. hence, in addition to discovering the hidden potentials of flavonoids, scientists should also aim to identify ways to increase the amount of flavonoids available for the health benefits of human beings. natural compounds have been the center of attention among researchers working in various fields, including those related with antiviral drug development, due to their high availability and low side effects. the phytochemicals flavonoids, which are abundantly found in our daily diets of fruits and vegetables, have been actively studied as potential therapeutic options against viruses of different taxa in the past decade. numerous positive findings have been reported on the in vitro efficacy of flavonoids, but less promising results have been obtained for most compounds in in vivo studies. multiple factors contributed to this scenario, and in vivo studies must be prioritized by researchers. it is well-known that flavonoids possess enormous potential to be included in the daily prescriptions by physicians treating illnesses ranging from infectious and oncogenic to inflammatory and chronic degenerative diseases. however, it is time for researchers worldwide to take the initiative in making these compounds a success not only in the in vitro stage of research, but also in animal models, as well as in subsequent clinical studies. biochemistry and mechanistic studies on the flavonoids' inhibitory activities can improve our understanding of how these natural compounds work and, on the other hand, identify the stumbling block that is hindering further improvements in flavonoids antiviral research. ( ) inhibition of chikungunya virus replication by hesperetin and naringenin effect of genistein on replication of bovine herpesvirus type antiherpes evaluation of soybean isoflavonoids suppression of hepatitis c virus by the flavonoid quercetin is mediated by inhibition of ns protease activity in vitro anti-hiv- activities of kaempferol and kaempferol- -o-glucoside isolated from securigera securidaca glycosyltransferases: managers of small molecules structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (pim- ) kinase epigallocatechin- -gallate is a new inhibitor of hepatitis c virus entry flavonoids and melanins: a common strategy across two kingdoms the green tea molecule egcg inhibits zika virus entry in vitro inhibition of canine distemper virus by flavonoids and phenolic acids: implications of structural differences for antiviral design effect of naringenin, hesperetin and their glycosides forms on the replication of the d strain of yellow fever virus inhibition of epstein-barr virus lytic cycle by (-)-epigallocatechin gallate the action of plant extracts on a bacteriophage of pseudomonas pyocyanea and on influenza a virus epigallocatechin- -gallate inhibits the replication cycle of hepatitis c virus in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids antiviral activities of extracts and selected pure constituents of ocimum basilicum epimedium koreanum nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state role of baicalin in anti-influenza virus a as a potent inducer of ifngamma inhibitory effects of flavonoids on moloney murine leukemia virus reverse transcriptase activity synthesis and anti-influenza activities of novel baicalein analogs a small molecule inhibits virion attachment to heparan sulfate-or sialic acid-containing glycans eight flavonoids and their potential as inhibitors of human cytomegalovirus replication the chemistry and biological effects of flavonoids and phenolic acids antiviral effect of methylated flavonol isorhamnetin against influenza antiviral activity of baicalin against influenza a (h n / h n ) virus in cell culture and in mice and its inhibition of neuraminidase quercetin and quercetin -o-glycosides from bauhinia longifolia (bong.) steud. show anti-mayaro virus activity effects of baicalein on sendai virus in vivo are linked to serum baicalin and its inhibition of hemagglutinin-neuraminidase chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (?)-catechin and (-)-epigallocatechin gallate the growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety human cytomegalovirus-inhibitory flavonoids: studies on antiviral activity and mechanism of action flavonoids: biosynthesis, biological functions, and biotechnological applications antiviral activity ofl uteolin against japanese encephalitis virus docking studies of pakistani hcv ns helicase: a possible antiviral drug target structure and function of enzymes involved in the biosynthesis of phenylpropanoids inhibition of infectivity of potato virus x by flavonoids quercetin inhibits rhinovirus replication in vitro and in vivo inhibition of hsp reduces porcine reproductive and respiratory syndrome virus replication in vitro naringenin inhibits the assembly and long-term production of infectious hepatitis c virus particles through a ppar-mediated mechanism polyphenols as mitochondria-targeted anticancer drugs in vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus genistein interferes with sdf- -and hiv-mediated actin dynamics and inhibits hiv infection of resting cd t cells anti-hepatitis b virus activity of wogonin in vitro and in vivo effect of quercetin on the course of mengo virus infection in immunodeficient and normal mice. a histologic study antiviral effect of flavonol glycosides isolated from the leaf of zanthoxylum piperitum on influenza virus antiviral activity of flavonoids anti-h n virus, cytotoxic and nrf activation activities of chemical constituents from scutellaria baicalensis antiviral activity of baicalein and quercetin against the japanese encephalitis virus development of self-microemulsifying drug delivery systems (smedds) for oral bioavailability enhancement of simvastatin in beagle dogs antiviral effect of flavonoids on human viruses epigallocatechin gallate, the main component of tea polyphenol, binds to cd and interferes with gp binding development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target divergent antiviral effects of bioflavonoids on the hepatitis c virus life cycle fisetin: a dietary antioxidant for health promotion inhibition of influenza virus internalization by (-)-epigallocatechin- -gallate an evaluation of the inhibitory effects against rotavirus infection of edible plant extracts inhibition of lassa virus and ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin chemistry and biological activities of flavonoids: an overview development of biodegradable nanoparticles for delivery of quercetin updated knowledge about polyphenols: functions, bioavailability, metabolism, and health antiviral activity of selected flavonoids against chikungunya virus -o-arylmethylgalangin as a novel scaffold for anti-hcv agents flavonoid baicalin inhibits hiv- infection at the level of viral entry fisetin and rutin as c protease inhibitors of enterovirus a structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities epigallocatechin- -gallate inhibition of epstein-barr virus spontaneous lytic infection involves erk / and pi -k/akt signaling in ebv-positive cells computational screen and experimental validation of anti-influenza effects of quercetin and chlorogenic acid from traditional chinese medicine molecular docking of potential inhibitors for influenza h n quercetin: bioflavonoids as part of interferon-free hepatitis c therapy? apigenin inhibits enterovirus replication through suppressing viral ires activity and modulating cellular jnk pathway antiherpetic activities of flavonoids against herpes simplex virus type (hsv- ) and type (hsv- ) in vitro phenolic acids act as signaling molecules in plant-microbe symbioses identification and evaluation of antihepatitis c virus phytochemicals from eclipta alba computational docking study of p ion channel from hcv genotype and genotype and its interaction with natural compounds a flavonoid, luteolin, cripples hiv- by abrogation of tat function the effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer evaluation of the antiviral activity of kaempferol and its glycosides against human cytomegalovirus metabolomics view on gut microbiome modulation by polyphenol-rich foods baicalin, a metabolite of baicalein with antiviral activity against dengue virus combined effects of flavonoids and acyclovir against herpesviruses in cell cultures absorption, excretion and metabolite profiling of methyl-, glucuronyl-, glucosyland sulpho-conjugates of quercetin in human plasma and urine after ingestion of onions anti-chikungunya activity ofl uteolin and apigenin rich fraction from cynodon dactylon neuroprotective effects of chrysin: from chemistry to medicine differential inhibition of hiv-reverse transcriptase and various dna and rna polymerases by somecatechin derivatives differential inhibitory effects of some catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and cellular deoxyribonucleic and ribonucleic acid polymerases inhibition of the infectivity of influenza virus by tea polyphenols antiviral activity of baicalin against influenza virus h n -pdm is due to modulation of ns -mediated cellular innate immune responses bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial silico study on anti-chikungunya virus activity of hesperetin anti-sindbis activity of flavanones hesperetin and naringenin anti-hiv- activity of flavonoid myricetin on hiv- infection in a dual-chamber in vitro model plant derived compounds having activity against p and l leukemia cells inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays genistein inhibits the replication of avian leucosis virus subgroup j in df- cells apigenin restricts fmdv infection and inhibits viral ires driven translational activity anti-inflammatory effect of quercetin-loaded microemulsion in the airways allergic inflammatory model in mice drugs of natural origin genistein as antiviral drug against hiv ion channel absorption and metabolism of polyphenols in the gut and impact on health kaempferol derivatives as antiviral drugs against the a channel protein of coronavirus comparison of the antiviral activity of flavonoids antiviral activity of flavonoids against murine norovirus and feline calicivirus computational approach towards exploring potential anti-chikungunya activity of selectedflavonoids the flavonoid apigenin inhibits hepatitis c virus replication by decreasing mature microrna levels differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin a and baicaleinin h n influenza a virus-infected cells antiviral activity of chrysin derivatives against coxsackievirus b in vitro and in vivo quercetin -rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species silymarin efficacy against influenza a virus replication antiviral effect of catechins in green tea on influenza virus biological evaluation of anti-influenza viral activity of semi-synthetic catechin derivatives tea catechins as a potential alternative anti-infectious agent in vitro anti-hiv and -hsv activity and safety of sodium rutin sulfate as a microbicide candidate simplified catechin-gallate inhibitors of hiv- reverse transcriptase synergistic action of quercetin and murine alpha/beta interferon in the treatment of mengo virus infection in mice genistein treatment of cells inhibits arenavirus infection in vitro antiviral activity of plant extracts from asteraceae medicinal plants multiple effects of silymarin on the hepatitis c virus lifecycle saururus chinensis (lour.) baill blocks enterovirus infection by hijacking mek -erk signaling pathway a phospholipid complex to improve the oral bioavailability of flavonoids antienterovirus effects of chrysin and its phosphate ester the green tea catechin, epigallocatechin gallate inhibits chikungunya virus infection colonic metabolites of berry polyphenols: the missing link to biological activity? epigallocatechin gallate, the main polyphenol in green tea, binds to the t-cell receptor, cd : potential for hiv- therapy kinetic and structural analysis of mutant cd receptors that are defective in hiv gp binding quercetin as an antiviral agent inhibits influenza a virus (iav) entry. viruses biological activities of polyphenols from grapes determine the structure of phosphorylated modification of icariin and its antiviral activity against duck hepatitis virus a inhibitory effects of baicalein on the influenza virus in vivo is determined by baicalin in the serum green tea extract and its major component epigallocatechin gallate inhibits hepatitis b virus in vitro tangeretin from citrus reticulate inhibits respiratory syncytial virus replication and associated inflammation in vivo identification of luteolin as enterovirus and coxsackievirus a inhibitors through reporter viruses and cell viability-based screening inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type (hiv- ) activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns serine protease potent antiviral flavone glycosides from ficus benjamina leaves small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp extract of scutellaria baicalensis inhibits dengue virus replication antiviral activity of four types of bioflavonoid against dengue virus type- novel antiviral activity of baicalein against dengue virus preparation and in vitro evaluation of apigenin-loaded polymeric micelles anti-japanese-encephalitis-viral effects of kaempferol and daidzin and their rna-binding characteristics apigenin inhibits enterovirus- infection by disrupting viral rna association with trans-acting factors characterization and evaluation of self-microemulsifying sustained-release pellet formulation of puerarin for oral delivery epigallocatechin- -gallate opposes hbv-induced incomplete autophagy by enhancing lysosomal acidification, which is unfavorable for hbv replication light-controlled flavonoid biosynthesis in fruits acknowledgements the authors acknowledge all reviewers whose detailed comments improved the review and apologize to those authors whose contribution to flavonoids antiviral research may have been inadvertently missed. ethical standards this study is supported by the ra mes state committee of science, in the frames of the research projects rf- and yr- f . all authors declare that they have no conflict of interest. this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- -zhywlf r authors: wu, jiaqi; chi, heng; fu, yali; cao, aiping; shi, jingxuan; zhu, min; zhang, lilin; hua, deping; huang, jinhai title: the antiviral protein viperin interacts with the viral n protein to inhibit proliferation of porcine epidemic diarrhea virus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: zhywlf r in the early stage of virus infection, the pattern recognition receptor (prr) signaling pathway of the host cell is activated to induce interferon production, activating interferon-stimulated genes (isgs) that encode antiviral proteins that exert antiviral effects. viperin is one of the innate antiviral proteins that exert broad-spectrum antiviral effects by various mechanisms. porcine epidemic diarrhea virus (pedv) is a coronavirus that causes huge losses to the pig industry. research on early antiviral responses in the gastrointestinal tract is essential for developing strategies to prevent the spread of pedv. in this study, we investigated the mechanisms of viperin in pedv-infected ipej-c cells. increased expression of interferon and viperin and decreased replication of pedv with a clear reduction in the viral load were observed in pedv-infected ipec-j cells. amino acids – of porcine viperin contain an endoplasmic reticulum signal sequence that allows viperin to be anchored to the endoplasmic reticulum and are necessary for its function in inhibiting pedv proliferation. the interaction of the viperin s-adenosylmethionine domain with the n protein of pedv was confirmed via confocal laser scanning microscopy and co-immunoprecipitation. this interaction might interfere with viral replication or assembly to reduce virus proliferation. our results highlight a potential mechanism whereby viperin is able to inhibit pedv replication and play an antiviral role in innate immunity. porcine epidemic diarrhea virus (pedv), the causative agent of ped, is a member of the genus alphacoronavirus, family coronaviridae [ ] . in pigs, pedv first infects the peyer's patch, a small area of intestinal lymph nodes [ ] . it then proliferates and spreads to intestinal epithelial cells, eventually leading to infection of the entire small intestine [ ] . injury of intestinal organelles causes cell dysfunction and a reduction or loss of related enzyme activities. impaired nutrient absorption due to enzyme inactivation can lead to osmotic diarrhea, dehydration, and death [ ] . pedv is a common coronavirus that has caused huge economic losses to the pig industry and its related peripheral industries worldwide [ ] . research on the natural immune responses to pedv is still in its infancy, and there have been few reports in the literature about this topic [ ] . viperin is a broad-spectrum antiviral protein that has important antiviral effects, and its full potential remains to be explored. its role in pedv infection remains unclear, but its possible involvement in preventing ped is potentially significant for the stable and healthy development of the pig industry. previous studies have shown that virus-infected cells activate various signaling pathways to produce interferons [ ] . when type i interferon is released, it binds to specific receptors on the cell surface and activates more than downstream ifn-stimulated genes (isgs) through a signal cascade reaction [ ] . many isgs have been found to significantly limit viral replication and participate in a variety of antiviral processes, including presentation of viral antigens, apoptosis, and interference with viral replication and assembly [ ] . products of interferonstimulated genes with antiviral activity are also known as "innate immune factors". at present, only a small number of proteins encoded by interferon-stimulated genes have been reported, such as protein kinase r (pkr), ribonuclease l (rnase l), and viperin [ ] . host stress or overexpression of certain proteins can inhibit virus proliferation during viral infection [ ] . the antiviral effect of ifn is usually indirect. it induces host cells to produce antiviral proteins and then exerts antiviral effects on transcription and translation through protein kinases, '- 'a synthase, and -phospholipase [ ] . viperin is a broad-spectrum antiviral protein that is usually overexpressed at a low level in many types of normal healthy cells [ , ] . however, when induced by interferon, double-stranded dna, double-stranded rna, lipopolysaccharide, poly(i:c), or various viruses, the expression of viperin increases significantly [ , ] . there are two main pathways by which the expression of viperin is induced. sendai virus, pseudorabies virus, and sindbis virus are all capable of inducing the expression of interferon-stimulated genes [ , , ] . these viruses are first recognized by pattern recognition receptors (prrs), such as the toll-like receptors tlr and tlr , the retinoic-acid-inducible gene rig- , and the cytoplasmic dna sensor. the interferon regulatory factors irf and irf are then activated to produce ifn-β, which binds to type i interferon receptors on the cell surface through autocrine or paracrine pathways, resulting in the synthesis of the complex isgf , which binds to the viperin promoter to activate its expression [ ] . in addition, there are some other viruses, such as vesicular stomatitis virus and human cytomegalovirus, whose dsrna stimulates rlrs and interaction with the adaptor protein mavs can activate the production of irf and irf , which in turn can induce viperin expression [ ] . viperin plays an important role in the production of type i interferon in plasmacytoid dendritic cells (pdcs) [ ] , which are immune cells derived from bone marrow. these cells are capable of rapidly activating responses to non-self nucleic acids to produce interferons in large amounts [ ] . the main reason for this ability is that pdcs continuously produce the endogenous toll-like receptors tlr and tlr . activated tlr / , combined with irak and traf , can induce viperin expression [ ] . in pdcs, viperin is necessary for producing type i interferon [ ] . in viperin-deleted mouse cells, activation of the toll-like receptors tlr and tlr does not lead to the production of interferon. recruiting irak and traf in lipid droplets can activate irf , and viperin is also crucial for this process. thus, the induction of viperin in pdcs depends on the activation of the tlr and tlr signaling pathways. irak and traf need to be recruited to lipid droplets through viperin, and irf is then activated to induce the expression of type i interferon [ ] . the role of porcine viperin in pedv infection is still not well understood. in this study, the mechanisms by which porcine viperin regulates pedv proliferation were investigated in detail. the study was approved by tianjin university institutional animal care and use committee (tjiacuc) (protocol number: syxk-jin - ). all balb/c mice were kept in well-ventilated cages and were anesthetized for blood collection. the guide for the care and use of laboratory animals of the tianjin government authority was strictly followed. balb/c mice were obtained from the tianjin laboratory animals center. cells of the porcine alveolar macrophage (pam) cell line crl -cd ( d / cells) were kindly donated by professor han jun of china agricultural university. hek- t cells were cryopreserved in our laboratory. cells of the porcine intestinal epithelial cell line ipec-j and human embryonic kidney (hek) t cells were cultured in high-sugar dmem medium supplemented with % (v/v) fetal bovine serum (fbs) and antibiotic-antifungal solution. both cell lines were cultured in a humidified % co incubator at °c. pedv strain cv was used in our study, and the titer was pfu/ml [ ] . polyclonal antibodies against viperin were prepared by immunizing balb/c mice with recombinant his-viperin with a mineral oil adjuvant. labeled antibodies were purchased from cell signaling technology (cst, danvers, ma, usa) and applied biological materials inc. (abm, vancouver, canada). the internal reference antibody and hrp-conjugated antibody were purchased from invitrogen (thermo fisher scientific, waltham, ma, usa). total rna was extracted from pams using trizol reagent (takara, china) and transcribed into cdna using reverse transcriptase (takara). viperin was amplified using oligonucleotide primers (table ) designed based on a porcine viperin sequence (nm_ . ) obtained from the genbank database. the amplified fragment was cloned into pgem-t easy vector (transgen, beijing). a flag-viperin construct for expression in eukaryotic cells was constructed as follows: the viperin gene was amplified using a specific primer pair (table ) with a sequence in common with the vector was ligated to the pflag-cmv vector using a one-step cloning kit (vazyme, nanjing, china). the prokaryotic expression plasmid pet- a-viperin was constructed using the same method to express the recombinant protein his-viperin. the % tissue culture infective dose (tcid ) method was used to determine virus titers. ipec-j cells were seeded in -well plates ( × cells/well). after the cells had adhered to the plate and grown to about % confluency, a serial dilution ( - − ) of pedv was inoculated onto the cells. mock-infected cells were used as a control. the cells were incubated at °c for days, and tcid values were calculated by the reed-muench method [ ] . primers were designed based on available sequences in the genbank database (table ) . relative gene expression was analyzed using transstart top green qpcr supermix (dye i + dye ii) reagents on a real-time pcr instrument (abi ) with the following cycling conditions: °c for min, cycles of °c for s, °c for s, and °c for s; °c for s, °c for min, °c for s, and °c for s. the cdna was obtained by rna extraction and reverse transcription. three replicates were performed for each sample. the relative transcription level of each gene was calculated using graphpad prism software with β-actin as an internal reference. the procedure used for confocal microscopy was described previously [ ] . ipec-j cells were cultured overnight at °c in -well plates (corning inc., corning, ny, usa) and transfected either with plasmids containing the viperin gene or with empty vector ( . g). after the cells had adhered to the plate and grown to about % confluency, pedv was inoculated onto the cells ( ml/well), and an uninfected blank control (nc) was also included. twenty-four hours later, cells were fixed with % paraformaldehyde solution and permeabilized with pbs containing . % triton x- . five hundred microliters of . % triton x- was then added to each well to permeabilize the cells for minutes at room temperature. the cells were blocked with % bovine serum albumin (bsa) at room temperature for minutes and incubated with anti-flag and anti-myc or anti-gfp antibody at °c overnight. secondary antibodies (fluorescein-isothiocyanateconjugated anti-mouse igg or phycoerythrin-conjugated anti-rabbit igg) were used, and nuclear dna was stained with , -diamidino- -phenylindole (dapi). the cells were examined under an inverted fluorescence and phase-contrast microscope (olympus) to determine the subcellular localization of viperin protein. cells were lysed and homogenized in ripa cell lysate in the presence of % phenylmethylsulfonyl fluoride (pmsf) to inhibit proteases. the protein samples were separated by sds-polyacrylamide gel electrophoresis, transferred to nitrocellulose (nc) filter membranes (expro), and blocked. the nc membrane was treated overnight at °c with antibodies against flag ( : ), myc ( : ), viperin ( : ) or β-actin ( : ) and then washed and incubated with hrp-conjugated antibody for h at room temperature. immunoreactive bands were visualized using a chemiluminescence detection kit (thermo scientific, waltham, ma, usa) and a gel doc xr imaging system (bio-rad, usa). ipec-j cells cultivated in -mm-diameter plates were transfected with flag-viperin and myc-n, or related expression plasmids. twenty-four hours after transfection, the cells in each plate were treated with μl of lysis buffer containing % protease inhibitor (pmsf). the supernatant of the cell lysate was incubated overnight with anti-myclabeled or anti-flag-labeled beads (sigma) at °c. the beads were washed three times for min with lysis buffer and boiled for min. the proteins bound to the beads were separated by sds-page and detected by western blot. a small interfering rna (sirna) against viperin was synthesized by genepharm (table ) . a tube containing the rna oligo dry powder was centrifuged at rpm for min and diluted with diethyl pyrocarbonate (depc)-treated water. when the cells in a -well plate had grown to % confluency, they were transfected with the sirna using lipofectamine at a final concentration of nmol. after transfection with sirna-viperin for hours, the cells were collected to measure the level of interference and then infected with pedv. the cells were then collected for realtime quantitative rt-pcr and western blotting (table ). statistical analysis was performed using graphpad prism software, and the significance of changes in gene expression was analyzed by two-way anova. statistical results are presented as mean ± standard deviation (mean ± sem). p < . (*) indicates a statistically significant difference. through cell culture and inoculation, we found that pedv can stably proliferate in porcine intestinal epithelial cells (ipec-j ), providing a cell model for further studies. at h postinfection, the cells gradually became detached from the culture vessel, indicating that infection had occurred (fig. a) . fluorescence-based quantitative analysis also showed that the number of pedv virus particles stabilized and peaked at - h postinfection (fig. b) . we selected the structural protein n and the non-structural protein nsp of pedv for immunofluorescence experiments and observed their expression to reflect the replication of the virus. in the early stage of pedv replication, genes encoding viral non-structural proteins were transcribed first, which could reflect viral replication to some extent. the amount of nsp protein gradually increased with time (fig. c) . in the late stage of pedv replication, the viral structural protein genes began to be transcribed and expressed. expression of the structural protein n was also be used to trace the proliferation of pedv (fig. d) , and the pedv cv strain was found to stably proliferate in ipec-j cells, reaching a peak at hours postinfection. the innate immune system is evolutionarily conserved to defend against pathogens [ ] . after virus infection, pattern recognition receptors recognize released viral nucleic acids and activate downstream signaling pathways to promote the production of specific transcription factors and type i interferons [ ] . to measure interferon levels during pedv infection, we infected ipec-j cells with pedv at a multiplicity of infection (moi) of . . using quantitative reverse transcription pcr (qrt-pcr), we found that pedv infection resulted in a significant increase in the level of ifn-β mrna compared to mock-infected cells. interferon production was lower in pedv-infected cells than in cells that were stimulated with poly(i:c) (fig. a) . when ipec-j cells were infected with pedv, the transcription level of viperin increased significantly and reached a peak after hours. after that, as the virus continued to replicate, the cells began to die and the transcription level of viperin began to decline. however, compared to control cells, the transcription level of viperin remained high in the infected cells (fig. b ). an immunofluorescence assay also showed that the expression of viperin protein increased significantly infection (fig. c) . these results indicate that pedv infection upregulates the expression of interferon and viperin in ipec-j cells. we constructed a flag-viperin eukaryotic expression plasmid and verified the expression of this protein in t cells (fig. a) . to investigate the effect of viperin on pedv replication, the viperin gene was overexpressed in ipec-j cells, and the cells were infected with pedv h later. the viral load was measured to investigate the effect of viperin overexpression on pedv proliferation. overexpression of the viperin gene was found to reduce the levels of pedv produced (p < . ) (fig. b) . for further validation, a specific sirna targeting viperin was designed. transfection of j cells with this sirna resulted in a decrease in the expression of viperin (fig. c) , which was accompanied by an increase in pedv proliferation that was proportional to the degree of interference of viperin expression (fig. d) . this suggests that viperin inhibits the proliferation of pedv in ipec-j cells. it has been reported that human viperin can be localized in the endoplasmic reticulum [ ] . it has been proposed that viperin forms a dimer through its c-terminal domain and its n-terminal alpha helical domain to mediate the endoplasmic reticulum crystallization [ ] . since the amino acid sequences of porcine and human viperin are % identical, we investigated the location of porcine viperin in ipec-j cells. fluorescence microscopy showed that the viperin protein was distributed in the cytoplasmic region (fig. a) and had significant aggregation and colocalization with an endoplasmic reticulum marker protein. at the same time, no co-localization of viperin with golgi and lysosome markers was observed (fig. b) . when pedv infects cells, structural proteins and nonstructural proteins encoded by the virus use the endoplasmic reticulum for their synthesis, processing, and modification, which makes us suspect that viperin interacts with certain proteins in pedv. we therefore chose the major structural protein n and two non-structural proteins, nsp and nsp , for experiments. immunofluorescence images of ipec-j cells hours after infection with pedv showed that viperin and n showed clear co-localization, but no obvious co-localization with nsp and nsp was observed (fig. a) . considering that the antibodies used in previous experiments were polyclonal antibodies, we cotransfected ipec-j cells with recombinant plasmids encoding viperin and n, nsp or nsp and carried out the immunofluorescence experiments again. the results showed that there was significant co-localization between viperin and n, but no colocalization between viperin and nsp or nsp (fig. b) . a co-immunoprecipitation assay further demonstrated that viperin interacted with n ( fig. c) but not nsp or nsp (fig. d) . these experiments showed that porcine viperin interacts with the n protein, but not with the non-structural proteins nsp and nsp . studies have shown that the n-terminal alpha-helical structure of human viperin protein helps to anchor it to the endoplasmic reticulum. in order to explore the specific mechanism of interaction between viperin and the pedv n protein, the domains of viperin was analyzed and a truncated form of the gene was constructed. viperin is approximately kda in size and can be divided into three distinct domains [ ] , an n-terminal alpha-helical domain, a c-terminal conserved domain, and a middle s-adenosyl methionine domain (sam domain) (fig. a) . we made three ??truncated forms of the viperin gene, vip - , vipΔ - , and vipΔ - ??. these truncated genes were overexpressed in ipec-j cells, which were then infected with pedv. the transcription level of n was measured by fluorescence quantification (fig. b) . as described previously, viperin inhibited the proliferation of pedv. ??this inhibition was not observed with the n-terminal fragment vip - alone, but inhibition was still observed with the deletion mutants vipΔ - , and vipΔ - ?? (fig. c) , suggesting that the interaction region should be the middle part, and this was confirmed by immunoprecipitation (fig. d) . these data indicated that aa - of viperin play an important role in its anchoring to the endoplasmic reticulum, while the middle sam domain of viperin the specific region interacts with n protein. this study showed that the expression of type i interferon increases and that of viperin is also upregulated in ipec-j cells infected with pedv. overexpression of viperin and interference with its expression showed that viperin inhibits pedv proliferation. porcine viperin was found to be localized in the endoplasmic reticulum, and aa - of this protein were found to be indispensable for its ability to anchor itself to the endoplasmic reticulum. furthermore, there was colocalization and interaction between porcine viperin and the structural n protein of pedv. the s-adenosyl methionine domain in the middle of the viperin protein was found to be the key region for its interaction with the n protein. viperin may interfere with the assembly and proliferation of the virus by interacting with the n protein, thereby exerting an antiviral effect. viperin is a broad-spectrum antiviral protein that can inhibit the proliferation of multiple viruses [ ] . previous studies have shown that human viperin relies on its n-terminal amphipathic alpha-helical domain to anchor itself to the endoplasmic reticulum membrane, with its c-terminus protruding into the cytoplasm. overexpression of viperin results in the formation of homodimers that crystallize the endoplasmic reticulum and destroy its structure [ ] . viperin can also be localized on lipid droplets [ ] . it is noteworthy that lipid droplets have been shown to be sites of replication of many viruses, such as hepatitis c virus (hcv) and dengue virus (denv). the amphiphilic alpha-helical domain at the n-terminus of viperin is also necessary for its localization to lipid droplets [ ] . consistent with previous studies, we demonstrated that porcine viperin was also localized in the endoplasmic reticulum. after entering the host cell by membrane fusion, pedv releases its positive-strand rna genome in the cytoplasm [ ] . the viral polymerase uses the positive-strand genomic rna as a template to generate negative-strand rna, which in turn is used as a template for synthesis of subgenomic rna and subgenomic mrna of different lengths by the polymerase. mrnas of different lengths are translated to produce viral proteins of different sizes [ ] . the newly synthesized structural n protein forms a nucleocapsid by binding to the genomic rna in a helical pattern [ ] . the nucleocapsid then binds to the e and m proteins and to the modified s protein to form a complete mature virus particle in the endoplasmic reticulum-golgi interaction between truncated viperin and the pedv n protein in ipec-j cells transfected with constructs with a truncated viperin gene and pedv n overexpression plasmids, detected by immunoprecipitation intermediate compartment (ergic), which is then released by exocytosis [ ] . our experiments showed that viperin can inhibit the proliferation of pedv. in order to investigate the specific mechanism of this inhibition, we analyzed interactions between viperin and different viral proteins of pedv. the experiments showed that viperin colocalized and interacted with the n protein, but there was no obvious co-localization or interaction with the nsp or nsp protein. viperin differs slightly among different species although it is highly conserved. in porcine viperin, the n-terminal amphipathic alpha-helical domain consists of approximately aa - . the highly conserved c-terminal domain consists of approximately aa - . the middle part consists of aa to in which there are four relatively conserved modules (m , m , m , m ). the m module contains a conserved cxxxcxxc sequence, a feature of enzymes that use s-adenosyl methionine as a cofactor that binds to iron-sulfur clusters [ , ] . viperin decreases flavivirus virulence by promoting the secretion of unproductive noninfectious virus particles via a gbf -dependent mechanism [ ] . the iron-sulfur clusters are also important for its antiviral function. in order to identify the specific site of interaction between viperin and n, we constructed truncation forms of viperin. coimmunoprecipitation assays showed that the loss of the n-terminus and c-terminus did not affect the interaction between viperin and the n protein, indicating that the key residues responsible for interaction between viperin and n are in the middle region of the s-adenosyl methionine domain. in conclusion, this study showed that viperin regulates the proliferation of pedv, and our results suggest a possible role of viperin in pedv infection, and its antiviral activity was confirmed. this provides new ideas for research on the natural immune response to pedv infection and strategies for prevention of ped. however, the molecular mechanism by 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viperin protein in epithelium to enhance host antiviral defense evaluation of reed-muench method in determination of activity of biological preparations grouper viperin acts as a crucial antiviral molecule against iridovirus transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j cells conflict of interest the authors declare no competing interests. key: cord- -bl r v authors: miguel, b.; pharr, g. t.; wang, c. title: the role of feline aminopeptidase n as a receptor for infectious bronchitis virus : brief review date: journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: bl r v feline aminopeptidase n (fapn) has been shown to serve as a receptor for feline, canine, porcine and human coronaviruses. our objective was to determine if fapn can serve as a receptor for infectious bronchitis virus (ibv). feline kidney cells that express fapn and hamster kidney fibroblasts that do not express fapn were inoculated with ibv and monitored for replication by indirect fluorescent assay and confocal microscopy and in chicken embryonated eggs. the results showed that the feline cells were permissive to ibv but the hamster cells were not. the hamster cells became permissive to ibv after transfection with a fapn cdna suggesting that the feline apn molecule plays a role in ibv entry. members of the family coronaviridae infect a wide range of hosts and have been classified into three groups. one major group (group i) was recently shown to use aminopeptidase n (apn) as its cell surface receptor [ , , , , , - , , ] . aminopeptidase n, also called cd in humans [ ] , is a zinc metalloprotease. it is a -kda glycoprotein with zinc located in its globular region that is believed to be the active enzymatic site playing a major role in the cleaving of peptides [ , , , , ] . apn is expressed on the plasma membranes of granulocytes [ , ] , lymphocytes and monocytes [ , , , ] . it is also expressed on non-hematopoetic tissue including fibroblasts [ ] , synaptic membranes in the central nervous system [ ] , epithelial cells from the renal proximal tubules and the intestinal brush border, and endothelial and epithelial cells of the respiratory tract [ , , , ] . most recently apn has been isolated from chicken embryo yolk [ ] and chicken intestine [ ] . the feline aminopeptidase n has been shown to serve as a receptor for feline, canine, porcine and human coronaviruses in group i [ , ] . the coronaviruses from group i cause disease only in their usual target host species. however, it has been reported that some strains of canine coronavirus (ccv) and human coronavirus e can infect other non-target species without causing disease [ , ] . cats inoculated with human coronavirus e or ccv seroconverted and in many cases shed the virus with no signs of clinical disease [ , ] . infectious bronchitis virus (ibv) is a member of the group iii of coronaviridae. this virus causes severe respiratory disease in chickens, and its receptor has not been identified. the objectives of this study were to: ( ) determine if feline cells were permissive to arkansas (ark ) serotype of ibv, ( ) evaluate if the ibv can replicate in feline cells, and ( ) determine the contribution of apn to permissiveness of feline cells. a cell line derived from a normal kidney cortex of a -week-old female feline (felis catus) (crfk-ccl- ) was purchased from american type culture collection (manassas, va) and is henceforth designated as fek. the cells were cultured in dulbecco's modified eagle medium (dmem) (sigma chemical co., st. louis, mo) consisting of % fetal bovine serum (fbs), iu/ml penicillin and µg/ml streptomycin (sigma). the cells were incubated in % co , % humidity at • c until they were confluent. for the ifa assay, × cells/ml were seeded into -well tissue culture plates (becton dickinson labware, franklin lakes, nj) containing × mm coverslips. the cells were used once attachment had occurred ( h). a cell line of normal hamster kidney fibroblasts (bhk- ) was used as control cells for the indirect immunofluorescence assay (ifa). the medium and culture conditions for bhk cells were identical to those used for fek cells. a field strain of ark of ibv provided by dr. j. gelb jr. (dept. of animal and food sciences, university of delaware, newark, de) was propagated by passage in specific pathogen free (spf) -day-old embryonated chicken eggs (hy-vac, adel, iowa). the allantoic fluid was harvested h after infection, clarified by centrifugation at low speed ( , × g for min), and the pellet was discarded. the virus was collected from the supernatant and concentrated by overnight high-speed centrifugation ( , × g) at • c. the supernatant was discarded and the pellet was resuspended in ml of tne buffer ( mm tris-hcl ph . , . m nacl, mm edta) [ ] . the virus suspension was aliquoted and stored at − • c until used. for the calculation of % embryo infectious dose (eid ) an aliquot was titrated in spf embryonated chicken eggs [ ] . the feline aminopeptidase cdna (pbk-fapn) subcloned into the plasmid pbk-cmv vector (stratagene) was kindly provided by dr. andreas kolb, hannah research institute, ayr, scotland, uk. bhk- cells were transfected with pbk-fapn or pbk-cmv (control plasmid) using lipofectamine reagent (stratagene). transfected cells were seeded in -cm flasks for h, at which time the medium was changed. the cells incubated for an additional h before evaluation by flow cytometry. to evaluate the permissiveness of cells to ibv, monolayers of transfected (pbk-fapn and pbk-cmv) bhk- , non-transfected bhk- and fek on coverslips were infected with a . eid of ark/ibv virus. the cells were washed, fixed in ethanol, rinsed again with pbs (ph . ) and incubated with a monoclonal antibody to the s glycoprotein of ark serotype ibv monoclonal (dr. s. a. naqi, dept. of microbiology and immunology, cornell university, ithaca, ny) at a : dilution for min at • c. the coverslips were washed in pbs three times for min each and then incubated with a goat anti-mouse igg conjugated to fluorescein isothiocyanate (fitc) (southern biotechnologies associates inc., birmingham, al) at a dilution of : for min at • c. the coverslips were washed three times for min in pbs, then mounted on microscope slides with mounting medium (dako corporation, carpinteria, ca). slides were viewed using epifluorescent microscopy (olympus america, inc., lake success, n.y.), and confocal microscopy (leica, heidelberg, germany). fek grown on -well tissue culture plates were infected with a . eid of ark/ibv virus. the supernatant of these infected cell cultures was collected at , , and d post infection. the media in this last culture was collected twice, pooled and frozen at − • c until titrated. virus titer was calculated using the method of reed and munch described in [ ] . expression of fapn on the surface of transfected bhk- cells was determined using a monoclonal antibody to human cd clone wm- (accurate antibodies, westbury, ny) that cross-reacts with the feline apn (personal communication with dr. k. v. holmes). bhk- cells were washed two times with phosphate buffered saline (pbs), incubated with anti-human cd or an isotype matched control, washed, and then incubated with fitc conjugated goat anti-mouse igg. an additional control included cells with the secondary antibody only. all incubations were done for min on ice. after the final wash, all cells were suspended in µl of pbs and analyzed by flow cytometry (calibur flow cytometry system, san jose, ca). ifa and confocal analyses of fek cells inoculated with ark/ibv demonstrated that the fek cells were susceptible to the virus. fluorescence was detected on the surface of inoculated cells and the accumulation of viral antigen in the cytoplasm, the site of replication for ibv, was detected (figs. a and e ). viral antigen was detected as soon as h post infection (pi). the bhk- cells, which were shown to be non-permissive to ibv infection by ifa, were used as a negative control cell line (figs. b and if) . to determine if ibv could replicate in the fek cells, cell cultures were infected with ark/ibv and the supernatants from infected cells were collected at , , and d pi. the cell supernatants were titrated in chicken embryos and the eid was calculated. the results indicated that ark/ibv does replicate in the fek cells in vitro. the eid /ml values were . , . , . and . for the , , , and d incubation, respectively. the pathological lesions, including dwarfed and hemorrhagic embryos with curled toes typically present in ibv infected embryos [ ] were produced in spf embryonated chicken eggs infected with virus passaged in fek cells (fig. ) . previous reports have shown fapn to be the molecule that allows entry of the coronavirus from other species into the feline cells [ , ] . to test the hypothesis that the feline apn had a role in permissiveness of fek cells to ark/ibv, bhk- cells were transfected with pbk-cmv plasmid or pbk-fapn. surface expression of fapn was assayed by flow cytometry. the bhk- cells transfected with pbk-fapn were shown to express the fapn receptor on the cell surface (fig. ) , and were permissive to ark/ibv (figs. c and g). viral antigen labeled with fluorescein was detected in the cell cytoplasm, and further examination of the cells with confocal microscopy also showed that the virus was intracytoplasmic and not membrane bound (fig. g ). however, fluorescence was not detected on the surface or in cytoplasm of bhk cells transfected with pbk-cmv (figs. d and h). this study showed that fek cells in culture were permissive to ark/ibv and the virus was capable of replicating within the cells. previous studies have shown that cats may become infected with coronavirus from other animal species and seroconvert without developing clinical signs of disease [ , ] polymerases, and therefore may undergo recombination [ , , ] in cats coinfected with different coronaviruses. the resulting recombinant viruses may have properties different from either parent, infect different hosts and have different tissue tropism, antigenicity and virulence, possibly resulting in the emergence of a new disease [ ] . because feline cells can harbor coronaviruses from several species, this may explain why recombination of coronaviruses occurs somewhat frequently. cats may serve as a vehicle for the recombination event and may contribute to new ibv emerging serotypes. there have been more than serotypes of ibv identified and new ibv variants continue to emerge. recombination has been experimentally proven to occur in ibv [ , , , , , ] particularly in the hypervariable region of s [ ] . this region of the s glycoprotein of ibv has also been postulated to be one of the major elicitors of the immune response [ , , ] . consequently, current vaccines generally do not cross protect against the different serotypes, resulting in a serious problem in the control and prevention of this disease. bhk- cells are not naturally permissive to this virus but became permissive when transfected with the fapn cdna. this result indicates that the fapn plays a role in ibv entry. previous work demonstrated that fapn serves as the receptor for group i coronaviruses 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characterization of determinants involved in the feline infectious peritonitis virus receptor function of feline aminopeptidase n feline coronavirus type ii strains - originate from a double recombination between feline coronavirus type i and canine coronavirus production and immunogenicity of multiple antigenic peptide (map) constructs derived from the s glycoprotein of infectious bronchitis virus (ibv) a novel variant of infectious bronchitis virus resulting from recombination among three different strains antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions characterization of functional domains in the human coronavirus hcv e receptor identification of residues critical for the human coronavirus e receptor function of human aminopeptidase n molecular analysis of the coronavirus-receptor function of aminopeptidase n first experimental evidence of recombination in infectious bronchitis virus. recombination in ibv experimental evidence of recombination in coronavirus infectious bronchitis virus aminopeptidase n (cd , ec . . . . ) occurs on the surface of resting and concanavalin a-stimulated lymphocytes sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus molecular cloning and sequence comparison of the s glycoprotein of the gray and jmk strains of avian infectious bronchitis virus evidence of genetic diversity generated by recombination among avian coronavirus ibv expression of the aminopeptidase n (cd ) gene in the human t cell lines hut and h human myeloid plasma membrane glycoprotein cd (gp ) is identical to aminopeptidase n isolation and characterization of cdna encoding chicken egg yolk aminopeptidase ey structure and expression of aminopeptidase n an enhancer with cell-type dependent activity is located between the myeloid and epithelial aminopeptidase n (cd ) promoters induction of aminopeptidase n/cd on human lymphocytes after adhesion to fibroblast-like synoviocytes, endothelial cells, epithelial cells, and monocytes/macrophages look at ( ) separate promoters control transcription of the human aminopeptidase n gene in myeloid and intestinal epithelial cells induction of protective immunity in chickens vaccinated with infectious bronchitis virus s glycoprotein expressed in a recombinant baculovirus feline aminopeptidase n is a receptor for all group i coronaviruses feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i isolation and identification of avian pathogens, th edn fapn as a receptor for infectious bronchitis virus evidence of natural recombination within the s gene of infectious bronchitis virus experimental confirmation of recombination upstream of the s hypervariable region of infectious bronchitis virus human aminopeptidase n is a receptor for human coronavirus e we thank dr. andreas kolb key: cord- -h sfd wa authors: mciver, david j.; silithammavong, soubanh; theppangna, watthana; gillis, amethyst; douangngeun, bounlom; khammavong, kongsy; singhalath, sinpakone; duong, veasna; buchy, philippe; olson, sarah h.; keatts, lucy; fine, amanda e.; greatorex, zoe; gilbert, martin; lebreton, matthew; saylors, karen; joly, damien o.; rubin, edward m.; lange, christian e. title: coronavirus surveillance of wildlife in the lao people’s democratic republic detects viral rna in rodents date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: h sfd wa coronaviruses can become zoonotic, as in the case of covid- , and hunting, sale, and consumption of wild animals in southeast asia increases the risk for such incidents. we sampled and tested rodents ( ) and other mammals and found betacoronavirus rna in rodents. the sequences belong to two separate genetic clusters and are closely related to those of known rodent coronaviruses detected in the region and distantly related to those of human coronaviruses oc and hku . considering the close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. severe acute respiratory syndrome-related coronavirus (sars-cov- ), the causative agent of the global covid- outbreak in humans, was first detected in the city of wuhan, hubei province, in the people's republic of china in late [ ] . the suspected index case probably contracted the virus at a local seafood and wildlife market in the city, yet the exact species of animal that hosted the virus remains unknown. phylogenetic analysis of the sars-cov- genome indicates a strong likelihood that the reservoir species is a bat, as in the case of the related betacoronaviruses sars-cov- and middle east respiratory syndromerelated coronavirus (mers-cov) [ ] . an involvement of another intermediate host between bats and humans in the transmission of sars-cov- remains unknown. wildlife and bushmeat markets are common across southeast asia, and represent a significant risk for the transfer of zoonotic pathogens between wildlife and humans. indeed, % of all emerging infectious diseases in the past decades have their origin in wildlife, including highly pathogenic influenza viruses (h n ), ebolaviruses, henipaviruses, and hantaviruses, among others [ ] . the covs most closely related to sars-cov- were isolated from bats living in yunnan province, in the south of china, not far from the -km-long border with the landlocked lao people's democratic republic (laos) [ ] . both countries were involved in the united states agency for international development's (usaid) emerging pandemic threats predict program, and surveillance of bats in wildlife markets in rural areas in laos using family-level pcr assays revealed the presence of cov rna in animals [ ] . in addition to bats, rodents are recognized as significant hosts of viral zoonoses and represent an important potential host for zoonotic viral spillover in laos through their frequent incidental and intentional interaction with humans [ , ] . multiple groups in laos are at high risk of zoonotic viral spillover from wildlife, including from rodents, due to their occupation or economic or geographic circumstances. people contact rodents incidentally and intentionally in various ways. in traditional-style homes, especially in rural areas, rodents are often able to easily enter the houses in search of food and shelter. these circumstances promote incidental contact with rodent urine and feces during everyday life when household members clean their houses. rodents also commonly raid food storage areas, including rice storage huts near paddies. in terms of more direct and intentional contact, some species of rodents, including the indian giant flying squirrel (petaurista philippensis), finlayson's squirrel (callosciurus finlaysonii), red-cheeked flying squirrel (hylopetes spadiceus), and others, are hunted or trapped in rural forested areas using traps, guns, sticks, or other implements. designated for food or for medicinal purposes, depending on the species, the rodents are consumed within the hunter's village or enter the value chain to reach markets. the value chain involves a series of intermediaries that transport animals from small villages to progressively larger populated areas. at the market, animals are sold to locals or to lao people visiting from other areas of the country, and often to foreign visitors from neighbouring thailand, china, and vietnam [ , ] . even though the sale of these animals is illegal, laos attracts many wildlife trade tourists simply because wildlife products are more available. throughout this value chain, people are exposed to blood, viscera, feces, and saliva of rodents and can be further exposed to these materials during the butchering process, where butchers can accidentally cut themselves with knives, allowing efficient transmission of viruses from rodents to humans to occur [ ] . considering the significant interactions of wildlife and especially rodents with humans in laos, we were interested in investigating the presence of covs in these animals, which can be primary or intermediate hosts for covs with zoonotic potential. samples were collected from both live and freshly killed animals, either trapped in or around village homes or voluntarily provided by local hunters upon their return to the village following hunting forays. with the permission of market operators and vendors, samples were also collected from freshly killed animals for sale in markets. oral and rectal swab specimens were collected in duplicate into individual . -ml screw-top cryotubes containing either µl of trizol reagent (invitrogen) or universal viral transport medium (bd), respectively. samples were then placed directly in liquid nitrogen dry shippers and, upon arrival at the laboratory, transferred to a - °c freezer. staff wore n masks, nitrile gloves, dedicated clothing, washable shoes or shoe covers, and protective eyewear during sampling of both live and dead animals. rna was extracted using a zymo direct-zol rna kit and stored at - ºc until analysis. rna was converted into cdna using a maxima h minus first strand cdna synthesis kit (thermo scientific) and was stored at - °c until analysis. two conventional nested broad-range pcr assays, both targeting conserved regions in the rna-dependent rna polymerase gene (rdrp), were used to test the samples for cov cdna. the first pcr amplifies a product of approximately nt between the primer binding sites. the first-round (cov-fwd : cgt tgg iac waa ybt vcc wyt ica rbt rgg and cov-rvs : ggt cat kat agc rtc avm asw wgc nac atg) and secondround (cov-fwd : ggc wcc wcc hgg nga rca att and cov-rvs : ggw awc ccc ayt gyt gwa yrt c) primers of this pcr were specifically designed for the detection of a broad range of covs [ ] . the second pcr was used in two modified versions, one of them specifically targeting a broad range of covs in bats and the other broadly targeting covs of other hosts. in both cases, the semi-nested pcr primers cov-fwd (ggt tgg gay tay cch aar tgt ga) and cov-rvs (cca tca tca swy raa tca tca ta) were used for the first round. in the second round, either cov-fwd /bat (gay tay cch aar tgt gay aga gc) or cov-fwd /other (gay tay cch aar tgt gau mgw gc) was used as the forward primer, while the reverse primer was again cov-rvs [ ] . both versions amplify nt between the primer binding sites. cov-rna-positive samples were subjected to a cytochrome b pcr assay to verify the host species. the primers cytb_f (gag gmc aaa tat cat tct gag g) and cytb_r (tag ggc vag gac tcc tcc tag t) were used to amplify a primer-flanked -nt fragment of this highly conserved mitochondrial gene [ ] . pcr products were subjected to electrophoresis in a . % agarose gel, and products of the expected amplicon sizes were excised. dna was extracted using a qiagen qiaquick gel extraction kit and was sent for commercial sanger sequencing ( st base). all results from sequencing were analyzed using geneious . software and primer trimmed, and the consensus sequences were compared to the genbank database (blastn, ncbi). all sequences were deposited in the genbank database under accession numbers mt , mt , mt -mt , mt -mt and mt . maximum-likelihood phylogenetic trees were constructed including members of different genera (alphacoronavirus, betacoronavirus, and gammacoronavirus) and species of known covs as well as other covs detected in laos during the predict project. only a single sequence was included for isolates with more than % nucleotide sequence identity. multiple sequence alignments were made in geneious (version . . , muscle alignment), and regions supported by less than % of the sequences were excluded. bayesian phylogeny of the polymerase gene fragment was inferred using mrbayes (version . ) with the following parameters: datatype=dna, nucmodel= by , nst= , coavion=no, # states= , rates=equal, runs, chains of , , generations. the sequence of a whale gammacoronavirus served as outgroup to root the trees, with sampling after every , steps during the process to monitor phylogenetic convergence [ ] . the average standard deviation of split frequencies was below . for the watanabe pcr-based analysis and below . for the quan pcr-based analysis (mrbayes recommended final average < . ). the first % of the trees were discarded, and the remaining ones were combined using treeannotator (version . . ; https ://beast .bio.ed.ac.uk) and displayed with figtree ( . . ; https :// tree.bio.ed.ac.uk/) [ ] . during the sampling phases of the project ( - and - ) rodents, carnivores, primates, eight tree shrews, and one colugo (galeopterus variegatus) were sampled and tested for the presence of cov rna (fig. ) . the rodents belonged to the families sciuridae ( ), muridae ( ), diatomyidae ( ), and hystricidae ( ); the carnivores belonged to the families viverridae ( ), mustelidae ( ), and felidae ( ); the primates belonged to cov rna was detected in rodents, which corresponds to . % of the rodents sampled. all of them were sampled in the south of laos, and all but one were oral swab samples (fig. , supplement ) . no cov rna was detected in any of the carnivores, primates, tree shrews, or the colugo. in all positive animals, it was the watanabe pcr that amplified cov nucleic acid. eleven of the isolates were identical or very similar to each other, and all were similar to covs found in different rodents in neighboring china and vietnam. nine of the cov-rna-positive animals were caught in the same area within a time window of days (fig. , table ). nine of the positive animals, all rattus exulans, were found in and around human dwellings, while the other were three squirrels (one dremomys rufigenis, two menetes berdmorei), sampled at a wet market not far from pakse, the capital city of champasak province, where they were being sold for consumption. eleven of the positive animals were sampled in the dry season (december), and one was sampled in the wet season (june). since there are abundant contact opportunities for wildlife pathogens and humans in laos, and considering that coronavirus-zoonotic events can involve intermediate hosts, as in the cases of sars and mers, we focused our screening on non-bat species potentially capable of playing that role. we found cov rna in swab samples from several of the rodents examined in the study. earlier, we noted a relatively high number of diverse covs detected in various species of bats all over laos (supplements and ) [ ] . this corresponds to similar findings in other countries with a tropical climate, and a hypothesis has been suggested that bats may serve as a seeding host for zoonotic cov infections [ , ] . the . % prevalence of cov rna in rodents was much lower than what had been detected in bats in laos; however, such observations have been made repeatedly, re-emphasizing the role of bats as a primary cov source [ , [ ] [ ] [ ] [ ] . it is worth noting that we targeted rodents most likely to be in contact with humans and transmit virus and found fewer cov-rna-positive animals than in other studies of rodents in the region or elsewhere. a variety of factors may explain the lower incidence of coronavirus-positive animals in this study, including the sample types tested. studies in which cov rna was more frequently detected in rodents have used intestine or fecal matter for their studies, while we tested oral and rectal swab samples [ ] [ ] [ ] . we employed swab sampling to minimize harm to live animals and to avoid any damage to rodents possessed by hunters or market vendors during sampling. organ collection was rarely feasible, even from dead rodents in market settings, since the size and mass of the animals are used to determine their selling price. while potentially underestimating the actual cov circulation, swab sampling has the advantage of being minimally invasive, quick, and applicable to all species. the rodent cov sequences we found fall into two clusters, with of them differing by only one nucleotide. therefore, these likely belong to the same strain that may have been circulating at that time, since they were obtained from rodents in the same southern region of laos during december (table ). both cov strains detected here cluster with other betacoronaviruses previously detected in rodents in the region (fig. , table ). this suggests that the viruses had a longer evolutionary history within rodent hosts and probably did not originate from a recent cross-species transmission event. none of the covs detected in laos wildlife, neither the ones described earlier in bats nor the ones described here in rodents, have a very close relationship to covs currently known to cause human disease. nevertheless, the rodent covs do fall into the same cluster as human covs oc and hku , and these two human covs probably originated from direct or indirect spillover events of rodent viruses sometime in the past [ ] . however, we still do not know enough about the molecular mechanisms and drivers of zoonotic events to determine the level of risk with certainty. we conclude that laos' wildlife harbors diverse covs and that a potential for interspecies transmission of viruses and novel diseases exists. human contact with wildlife such as bats and rodents is common throughout the country, with many rural households consuming bushmeat as a main source of protein and utilizing it as a trade commodity, and this risk potential is particularly relevant. therefore, behavioral risk reduction, vigilance, ongoing surveillance and research are important to help mitigate the risks of coronavirus zoonotic disease emergence and transmission in the region, especially in the aftermath of covid- . fig. maximum-likelihood phylogenetic tree of coronaviruses presented as a proportional cladogram, based on the -nt rdrp region targeted by the pcr by watanabe et al. [ ] . the tree includes the sequences detected here (red boxes) and those described previously in laos (grey boxes) and indicates the number of isolates with less than % difference in brackets for isolates. genbank accession numbers are listed for published sequences from outside of laos, while sequences obtained during the predict project are identified by cluster names (compare table a novel coronavirus from patients with pneumonia in china genomic characterization and epidemiology of novel coronavirus: implications for virus origins and receptor binding global trends in emerging infectious diseases discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus genetic diversity of coronaviruses in bats in lao pdr and cambodia rodent reservoirs of future zoonotic diseases host and viral traits predict zoonotic spillover from mammals wildlife trade and human health in lao pdr: an assessment of the zoonotic disease risk in markets newton p ( ) toward a quantification of risks at the nexus of conservation and health: the case of bushmeat markets in lao pdr identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria bat coronaviruses and experimental infection of bats, the philippines identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences mrbayes . : efficient bayesian phylogenetic inference and model selection across a large model space wild animal surveillance for coronavirus hku and potential variants of other coronaviruses global patterns in coronavirus diversity discovery, diversity and evolution of novel coronaviruses sampled from rodents in china detection of potentially novel paramyxovirus and coronavirus viral rna in bats and rats in the mekong delta region of southern viet nam detection of alpha-and betacoronaviruses in rodents from yunnan identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs origin and evolution of pathogenic coronaviruses the authors would like to thank the government of key: cord- - i ld d authors: nefedeva, mariia; titov, ilya; malogolovkin, alexander title: molecular characteristics of a novel recombinant of porcine epidemic diarrhea virus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: i ld d porcine epidemic diarrhea (ped) is a contagious viral disease in pigs, caused by the coronavirus porcine epidemic diarrhea virus (pedv). pedv infection results in significant mortality in piglets in unvaccinated herds. like many others rna viruses, pedv has high evolutionary rate and is prone to genetic mutations. in this study, we analyzed the complete genome sequence of the recently sequenced isolate pedv/belgorod/dom/ . a recombination event in s gene of pedv/belgorod/dom/ was detected. pairwise identity analysis of the whole genome sequences revealed that pedv/belgorod/dom/ is an intermediate between pedv and transmissible gastroenteritis virus (tgev) strains. these results can be used for further analysis of the evolutionary variability, prevalence, and epidemiology of the porcine epidemic diarrhea virus. the spike (s) protein of pedv is the viral protein that is subjected to the greatest immunological pressure and variability. deletions (s-indels) or small insertions have been observed in the s gene nucleotide sequences of many pedv isolates [ ] . the pedv strains that are currently circulating in the european union are similar to the american s-indel strains [ ] [ ] [ ] . the phylogenetic classification of the pedv strains is based on the analysis of complete genomes sequences obtained worldwide [ ] or individual genes such as s, m, n, or orf [ , , ] . in this study, we analyzed the genome sequence of recently sequenced pedv isolate, pedv/belgorod/ dom/ (genbank accession number mf ) [ ] . pathological samples from the intestine and stomach were taken from one-month-old sick piglets from the belgorod region of russia in [ ] . total rna was extracted from a % organ suspension using trizol reagent (ther-mofisher scientific) according to the manufacturer's instructions. next-generation sequencing was done using an illumina miseq instrument with a miseq reagent kit v in -× -bp pe mode (illumina, san diego, ca, usa) [ ] . the pedv/belgorod/dom/ isolate was subsequently isolated from the small-intestine tissue in vero cell culture. prediction of homologous recombination events was carried out using rdp (recombination detection program) and simplot [ , ] . pairwise identity analysis [ ] . phylogenetic trees were constructed based on pedv m and s gene sequences using the maximum-likelihood method in mega . . [ ] . bootstrap values were estimated for replicates. the complete coding sequence of the pedv/belgorod/ dom/ is , nucleotides (nt) in length (genbank accession number mf ) [ ] . two putative recombination sites were detected in the genome of recombinant pedv/belgorod/dom/ at nt in orf b and nt in the s gene ( fig. ). pedv strain lzc (ef ) and pedv strain slo/jh- / (ku ) were identified as the major and minor parental viruses, respectively. the recombinant event was identified by six modules (rdp, maxchi, chimaera, geneconv, bootscan, siscan) with high confidence (average p-value, . × − ). a similarity plot showed high overall sequence similarity between the pedv/belgorod/dom/ strain and the parental pedv strains, but with a marked drop in the nucleotide sequence similarity in the s gene region (fig. ) . phylogenetic analysis of the complete genomes showed that pedv/belgorod/dom/ has a distant relationship to known pedv strains. the pedv/belgorod/dom/ isolate does not belong to any groups formed by the american or chinese strains and forms a separate cluster together with the secov-ita recombinant strain isolated in italy (fig. ) . since only m gene sequences are available in the gen-bank database for the russian pedv isolates, we rebuilt the phylogenetic tree to refine the analysis. based on the phylogenetic analysis of the m gene, the pedv/belgorod/ dom/ isolate belongs to the same clade as other virulent russian pedv strains, indicating a high degree of sequence homogeneity in the m gene (fig. a) isolate is genetically distinct and does not belong to any group (fig. b ). this robust incongruence between the mand s-gene-based trees may be explained by a recombination event within the genome of the pedv/belgorod/dom/ isolate. such variability can lead to dramatic changes in viral virulence, pathogenicity, and antigenicity. pairwise identity analysis based on the spike amino acid sequences revealed that pedv/belgorod/dom/ is an intermediate between pedv and tgev and is also distantly related to other pedv strains (fig. ) . pedv/belgorod/dom/ has a unique spike protein sequence and low similarity to other pedv isolates. changes in the s glycoprotein gene play an important role, since this protein is important for tissue tropism and virulence [ ] . a preliminary animal study with pedv/belgorod/dom/ demonstrated that this recombinant is highly virulent in unvaccinated suckling piglets [ , ] . recombination events are possible and can sometimes be observed in cases where pigs have been vaccinated or infected with a mixture of tgev and pedv. such recombination events can potentially result in a loss of vaccine efficacy. boniotti et al. reported a virus possessing a tgev genome sequence in which the s protein sequence was identical to that of a pedv isolate (secov-ita ) [ ] . this chimeric virus was probably generated by recombination between tgev and pedv. similar chimeric viruses have been found by other research groups in germany [ ] and eastern europe [ ] . the genome sequence of one pedv isolate (ch/hnqx- / ) from china shows that this strain appeared due to naturally occurring recombination of the attenuated strains cv and dr with the circulating field strain ch/ zmdzy/ . the recombination events occurred in the s, orf , and n-structural protein-coding region and the replicase orf a region [ ] . the results of phylogenetic and recombination analysis revealed a discrepancy between the s gene sequence of pedv/belgorod/dom/ and the sequences of other isolates available in the genbank database. our results indicate that pedv/belgorod/dom/ is a new recombinant strain. interestingly, pedv/belgorod/dom/ and secov-ita (a recombinant strain from italy) form a unique phylogenetic group. pairwise identity analysis demonstrated that the amino acid sequence of the s gene of pedv/belgorod/dom/ is % identical to the s gene of other pedv strains and % identical to those of tgev strains. these data argue that pedv/belgorod/dom/ occupies an intermediate position between tgev and pedv. the identification of recombinant regions in pedv/belgorod/dom/ can be useful for further analysis of evolutionary variability, epidemiology, and development of a new diagnostic gene-based assay for porcine epidemic diarrhea virus. porcine epidemic diarrhea isolation and characterization of a variant porcine epidemic diarrhea virus in china nc_ . _secov_strain_italy/ / kr kr . _pedv_isolate_ v /bel/ mf . :pedv_isolate_kupe _ kx . :pedv_isolate_chsd kc . _pedv_strain_ch/fjzz- ef . _pedv_strain_lzc_from_china_ kc . _pedv_isolate_js jq . _pedv_strain_aƩenuated_dr _ jq . _pedv_strain_virulent_dr _ mf . _pedv_strain_pedv/belgorod/dom/ kc kx . _tgev_strain_tgev_ahhf_ dq . _tgev_strain_ts_ mf . :pedv_isolate_kupe _ kx . :pedv_isolate_chsd kc . _pedv_strain_ch/fjzz- mf . _pedv_strain_nw _ kp . _pedv_strain_ukraine kj . _pedv_strain_usa/minnesota af . _pedv_strain_cv _ ef . _pedv_strain_lzc_from_china_ kc . _pedv_isolate_js jq . _pedv_strain_aƩenuated_dr _ jq . _pedv_strain_virulent_dr _ mf . _pedv_strain_pedv/belgorod/dom kx . _tgev_strain_tgev_ahhf_ dq . _tgev_strain_ts_ new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in asia sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea detection of porcine epidemic diarrhea virus using polymerase chain reaction and comparison of the nucleocapsid protein genes among strains of the virus porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis 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 new variant of porcine epidemic diarrhea virus comparison of porcine epidemic diarrhea viruses from germany and the united states genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus distinct characteristics and complex evolution of pedv strains complete genome sequence of a porcine epidemic diarrhea virus rdp : detection and analysis of recombination patterns in virus genomes full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination sdt: a virus classification tool based on pairwise sequence alignment and identity calculation muscle: multiple sequence alignment with high accuracy and high throughput isolation and identification of porcine epidemic diarrhea virus in pigs under the outbreak at a large farm biological characteristics of an epizootic isolate bs- of porcine epidemic diarrhea virus porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china acknowledgements we thank olga strizhakova for providing pedv/ belgorod/dom/ . we also acknowledge yegor bazykin and alexey neverov for fruitful discussions of virus evolution and recombination analysis. we appreciate sandra blome and dennis hanke for pedv/ belgorod/dom/ complete genome sequencing. funding our work was supported by the ministry of science and higher education of the russian federation. the authors declare no competing interests. key: cord- -yirs kjg authors: mueller, andreas; simon, arne; gillen, julia; schildgen, verena; tillmann, ramona liza; reiter, karl; schildgen, oliver title: polyomaviruses ki and wu in children with respiratory tract infection date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: yirs kjg the polyomaviruses ki (kipyv) and wu (wupyv) have recently been discovered in specimens from patients with respiratory tract infections. to analyze the frequency and clinical impact in a cohort of pediatric patients in a german university children’s hospital. nasopharyngeal aspirates or bronchoalveolar lavage specimens of children with acute respiratory tract infection were screened for kipyv and wupyv using polymerase chain reaction-based methods. kipyv was detected in ( . %) and wupyv in ( . %) patients, without co-infections with other respiratory viruses but with co-detection of cmv, ebv and hhv in one immunocompromised patient. only a very small proportion ( . %) of positive samples for kipyv and wupyv was documented in this study; the clinical relevance of these viruses remains unclear and requires further evaluation. since , an increasing number of ''new'' respiratory viruses have been detected in children with respiratory tract infection (rti), including the human metapneumovirus (hmpv) and the human bocavirus (hbov) [ , ] . recently, two novel polyomaviruses were detected from respiratory secretions. allander et al. [ ] discovered a previously unrecognized human polyomavirus in ( %) of nasopharyngeal aspirates (npas) and ( . %) of fecal samples and proposed a new ki polyomavirus (kipyv). the second newly identified polyomavirus was described by gaynor et al. in respiratory samples from brisbane, australia, and st. louis, usa. the authors amplified and cloned the genome of wu polyomavirus (wupyv) from npa of a -year-old patient with pneumonia without detection of other respiratory pathogens. screening of , patients with rti yielded additional positive patients [ ] . since the first description of kipyv and wupyv, a number of prevalence studies from various areas have been published, but until now, the clinical relevance for rti in children remains an unresolved issue [ , , ] . in this study, we retrospectively investigated npas from children with acute rti for the presence of kipyv and wupyv dna. the major aim was to elucidate the frequency of the novel viruses and to identify cases that may be of clinical interest. nasopharyngeal aspirates or bronchoalveolar lavage specimens from children admitted to the hospital with acute respiratory tract disease were sent to the department of virology at the university of bonn between january and december . samples were aliquoted for preparation of dna/rna and stored at - °c. the following clinical pictures and course were defined as acute respiratory tract disease: bronchitis, bronchiolitis, central pneumonia, lobar pneumonia. a lower respiratory tract infection was documented as pneumonia only if a chest x-ray had confirmed the clinical diagnosis. all samples were prospectively examined for rsv, influenza viruses a and b, coronavirus nl , hku , oc , and e, rhinovirus, adenovirus, hmpv, and hbov by antigen detection, pcr or rt-pcr, as described previously [ , ] . dna for pcr detection of kipyv and wupyv was prepared from nasopharyngeal aspirates by using a qiamp viral dna kit (qiagen) following the manufacturer's instructions, for rna virus detection, rna was extracted using a qiamp viral rna kit (qiagen, hilden, germany). pcr amplifications were performed in -ll reaction mixtures under the following conditions: pmol of each primer, lm each deoxynucleoside triphosphate, mm tris-hcl (ph . ), mm kcl, . mm mgcl , and . u of taq dna polymerase (roche, germany). reactions were run in a biometra t thermocycler using a step cycle programme. after initial denaturation of dna at °c for min, cycles were run: °c for min, annealing temperature of °c for min, and °c for min with a -min °c extension after the cycles. a -ll aliquot from each reaction was run on a % nusieve : electrophoresis-grade agarose gel (fmc bioproducts, rockland, maine, usa) in tae buffer ( . mol/l tris acetate, . mol/l edta) with ethidium bromide ( . g/ml) to visualize the amplified pcr products under uv illumination. primer sequences were taken from the original descriptions of kipyv and wupyv [ , ] . procedures for avoiding contamination were strictly followed. dna/rna isolation, preparation of reaction mixtures, and amplification and analysis were physically separated and performed in three different rooms. positivedisplacement tips were used for all manipulations, and negative controls containing reaction mixtures without dna were always done. kipyv and wupyv dna was detected in npa specimens of ( . % of ) patients (kipyv n = ; wupyv n = ), with co-detection of cmv, ebv and hhv in one immunocompromised patient. other common respiratory viruses were found in children (rsv, n = ; hmpv, n = ; influenza a virus, n = ; coronavirus, n = , hbov, n = ). kipyv case description a -year-old girl was admitted to the hospital in november with fatigue, polydypsia, polyuria and anogenital candidiasis. a primary manifestation of diabetes type i was diagnosed, and insulin treatment was started immediately. the treatment was complicated by fever and signs of an acute rti on day . no viral respiratory pathogens were detected in a nasopharygeal aspirate (except kipyv, retrospectively). on day , the patient was discharged in good condition without further respiratory symptoms. kipyv case description a -year-old girl with high-risk acute myeloid leukemia was admitted for bone marrow transplantation (bmt). graft-versus-host prophylaxis was done with cyclosporine a and was switched to mycophenolate mofetil and tacrolimus because of suspected severe acute graft-versus-host disease of the skin and the gastrointestinal tract on day after transplantation. colonoscopy revealed crohn-likedisease, and treatment with mesalazine, etanercept and cortisone was started. at day (may ), the patient displayed respiratory distress; a chest x-ray examination showed left-sided basal pneumonia. antimicrobial treatment including antifungals was started. in addition, ganciclovir was added because of the detection of cytomegalovirus, ebv and hhv- in a bronchoalveolar lavage specimen. the number of copies in quantitative dna testing was found to be very low for all of these pathogens. no other respiratory viruses were detected (except kipyv, retrospectively). symptoms of the acute rti improved during the following days, and the patient was discharged from the hospital in good clinical condition months after bmt. wupyv case description a . -year-old girl was admitted to the hospital in october because of acute bronchitis. no viral pathogens were detected in a nasopharygeal aspirate (except wupyv, retrospectively). the patient received inhalative salbutamol, ipatropium bromide and budesonide as well as montelukast as oral medication. the patient was discharged on day with salbutamol and montelukast continued. the very low prevalence of polyomavirus ki and wu dna in npa specimens of out of pediatric patients with acute rti in this series does not confirm or exclude a pathogenic role of these newly described viruses. this, on the one hand, may be due to the relatively low sensitivity of our methods, which detected only copies per ml npa, but on the other hand, may also be caused by the lack of kipyv and wupyv in our patient cohort. prevalence studies of kipyv and wupyv reported a worldwide distribution, with an incidence for kipyv of % in sweden, . % in the uk, % in thailand and . % in australia [ , , , , ] . the prevalence of wupyv was highest, with %, in south korea and . % thailand [ , ] . from australia, the us and the uk, a prevalence of . , . and . %, respectively, has been reported [ , , ] . the age distribution of kipyv-and wupyv-infected patients showed two peaks, with the highest rates in children under years and in patients older than years [ , ] . a seasonal variation of kipyv and wupyv has not been consistently demonstrated. most infections with kipyv were detected during the winter months in thailand, but not in a study conducted in australia. in australia, the incidence of wupyv infections showed the highest peak in december; this was not confirmed in a study from thailand [ , , ] . the detection of kipyv and wupyv is frequently associated with co-infections with other respiratory viruses. high co-detection rates were found in australia, with % for kipyv, % for wupyv; the most common copathogens were human rhinovirus and hbov. co-detection of kipyv and wupyv occurred in patients, and only in one of these cases were no other respiratory virus present [ ] . norja et al. [ ] detected kipyv and wupyv in patients and viral co-infection in patients, with predominant co-detection of adenovirus. the overall frequencies of detection of kipyv and wupyv were equal in patients with upper and lower respiratory tract infection, and in asymptomatic patients. eight of the patients were immunocompromized [ ] . in our study, we did not find co-infections with respiratory viruses in the three patients with kipyv or wupyv infection, and all patients had symptoms of arti. the clinical relevance of other viruses (cmv, ebv, hhv ) co-detected in one severely immunocompromised patient in a bronchoalveolar lavage specimen is unclear. nevertheless, coinfections still remain possible, as we were not able to test for all putative copathogens, and the amounts of viral nucleic acids present in the samples may have fallen below the detection limits for some pathogens. hence, we carefully suggest that kipyv and wupyv are more than a viral colonization, with the limitation of our study being that we did not examine children without acute respiratory symptoms. it is unknown whether kipyv and wupyv persist in the host and may be reactivated in cases of immunosuppression, similar to jcv and bkv. furthermore, whether these viruses display an oncogenic potential, as described for other human and animal polyomaviruses, remains unclear and needs further evaluation [ ] . however, koch's modified postulates have not yet been fulfilled, and so far, it is controversial whether kipyv and wupyv are indeed respiratory pathogens rather than innocent bystanders. further studies are required to evaluate the clinical relevance of the detection of kipyv and wupyv in respiratory specimens. wu polyomavirus in children age-related pattern of ki and wu polyomavirus infection identification of a third human polyomavirus cloning of a human parvovirus by molecular screening of respiratory tract samples identification of the novel ki polyomavirus in the respiratory tract of an italian patient presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection a newly reported human polyomavirus, ki virus, is present in the respiratory tract of australian children identification of a novel polyomavirus from patients with acute respiratory tract infections wu polyomavirus in children with acute lower respiratory tract infections wu polyomavirus in children with acute lower respiratory tract infections wu polyomavirus infection in children no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in thailand human metapneumovirus rna in encephalitis patient osterhaus ad ( ) a newly discovered human polyomaviruses ki and wu pneumovirus isolated from young children with respiratory tract disease prospective study of human bocavirus (hbov) infection in a pediatric university hospital in germany novel human polyomaviruses-re-emergence of a well known virus family as possible human carcinogens conflict of interest statement none of the authors reports any conflict of interest. key: cord- -r jqmes authors: althani, asma; bushra, sumbul; shaath, noor; sattar, hisham a. title: characterisation of winter respiratory viral infections in patients with asthma and copd in qatar date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: r jqmes respiratory viruses in patients with chronic obstructive pulmonary disease (copd) or asthma have not been characterised in qatar. this study aimed to identify the most common viral strains responsible for respiratory tract infections in asthma/copd patients (without exacerbations) in qatar during the winter season ( - ). nasal swabs from patients with asthma/copd and respiratory symptoms were evaluated for common viruses. adult patients ( with asthma and with copd) were enrolled. viral infections were present in out of patients ( %). cough and wheezing were the most common symptoms. rhinovirus was the most common causative agent, followed by coronaviruses. our findings confirm previous reports of rhinovirus prevalence in respiratory tract infections in asthma/copd. a countrywide survey to confirm our findings is warranted. abstract respiratory viruses in patients with chronic obstructive pulmonary disease (copd) or asthma have not been characterised in qatar. this study aimed to identify the most common viral strains responsible for respiratory tract infections in asthma/copd patients (without exacerbations) in qatar during the winter season ( ) ( ) . nasal swabs from patients with asthma/copd and respiratory symptoms were evaluated for common viruses. adult patients ( with asthma and with copd) were enrolled. viral infections were present in out of patients ( %). cough and wheezing were the most common symptoms. rhinovirus was the most common causative agent, followed by coronaviruses. our findings confirm previous reports of rhinovirus prevalence in respiratory tract infections in asthma/copd. a countrywide survey to confirm our findings is warranted. respiratory viruses in patients with underlying lung diseases such as chronic obstructive pulmonary disease (copd) or asthma are associated with exacerbations and excess morbidity and mortality. murphy et al. reported that influenza in patients with asthma can cause acute exacerbations, whereas in patients with copd, it can lead to respiratory distress [ ] . other common respiratory viruses, especially rhinoviruses, cause the majority of exacerbations in children and adults with asthma [ ] . johnston et al. carried out a study in the uk to investigate the role of viral infections in acute exacerbations of asthma in schoolchildren, and they reported that the most commonly identified virus type in this population was rhinovirus [ ] . in one of the earlier studies using pcr to detect respiratory viruses in adults, a respiratory virus was found in % of exacerbations ( % rhinoviruses) [ ] . rhinovirus infection has also been associated with nearly half of all chronic obstructive pulmonary disease (copd) exacerbations [ ] . furthermore, the presence of two or more viral agents may contribute to the severity of exacerbations. wilkinson et al. have reported that a total of % of copd exacerbations in the uk were associated with the bacterial pathogen haemophilus influenzae, and rhinovirus was identified in % of exacerbations [ ] . however, higher bacterial loads were observed in exacerbations with both rhinovirus and h. influenzae, thus suggesting that interaction between these pathogens may contribute to exacerbation severity [ ] . other viruses have also been implicated in exacerbation of asthma and copd symptoms. for example, ko et al. reported that the most prevalent viruses detected during acute exacerbations of copd in hong kong were influenza a virus and coronavirus [ ] . much of the morbidity, mortality, and excess health-care utilisation associated with asthma and copd are related to exacerbations [ ] . identifying viral aetiology associated with respiratory tract infections in asthma and copd is useful for the development of strategies for the prevention and treatment of infections leading to exacerbations in this vulnerable population. there is little data on the frequency of respiratory viruses in the middle east, particularly in qatar. in this study, we sought to determine the burden of respiratory viruses in adult patients with asthma/copd in qatar, using real-time reverse transcription polymerase chain reaction (rt-pcr). the objective of this study was to identify viral strains responsible for respiratory tract infection in asthma/ copd adult patients who visited the chest clinic in qatar during the winter season from october to march (corresponding to the annual peak in respiratory infections in qatar) in order to identify the viral pathogens involved. during october -march , adults with copd or asthma, seeking care at the chest clinic of the hamad medical corporation, qatar, with symptoms of upper respiratory tract infection were eligible for participation. all patients were outpatients at the time of recruitment. patients with lower respiratory tract infections were excluded. all of the patients were assessed clinically by the recruiting physician to ensure the absence of other significant respiratory diseases. all patients had at least five symptoms of respiratory tract infection (table ) but otherwise were stable and had no exacerbation of copd/asthma at the time of sample collection. the most common symptoms in all patients were dry cough and wheezing. medicines taken for asthma/ copd by the patients were not recorded in our study. routine testing for bacteria was not performed, because the primary objective of the study was to examine the frequency of viral pathogens that cause respiratory tract infections. nasal swabs were collected from the sample population (to detect any upper respiratory tract viruses) and sent to the health science department at qatar university for processing. the study protocol was approved by the research committee of hamad medical corporation. all study participants signed informed consent forms according to the recommendations of the hamad medical corporation research committee, which approved the study. nasal swabs were collected from the patient population. an aliquot of processed samples was frozen at - °c for subsequent rna extraction and polymerase chain reaction (pcr). rna extraction from the frozen samples was performed using a standard extraction kit ( the primers and probe were used at a concentration according to the manufacturer's instructions (ftd respiratory pathogen samples, mikrogen tm ). for all pcr amplifications positive and negative controls were included. as positive controls, the positive controls provided in the kit were used. negative controls were carried out with water instead of rna. pcr runs were carried out according to the standard taqman Ò pcr profile. amplification of target dna and detection of pcr products were performed using an abi machine. amplification of the target sequence was detected as an increase in fluorescence above a baseline with no or little change in fluorescence. in order to analyse the data, the reporter (fam) fluorescence was automatically normalised to a passive reference to avoid the measurement of non-pcrrelated fluorescence. a threshold was set above the baseline, and the threshold cycle value (ct) was defined as the cycle number at which the fluorescence passes the fixed threshold and a statistically significant increase in fluorescence is first detected. data analysis was performed using statistical software (spss version . ; spss; chicago, il, usa) to calculate correlations and/or significance of the data. a value of p \ . was considered statistically significant. a total of adult patients ( females and males) were enrolled: patients of arab origin and patients of non-arab origin. all patients were aged c years (average age of asthma patients = yr [ranges; n = for - yr; n = for - yr; n = for [ yr]; average age copd patients = yr [ranges n = for - yr; n = for - yr; n = for [ yr). the majority of the patients had asthma ( asthma patients vs. patients with copd). none of the copd patients and of the asthma patients had received an influenza vaccine over the last two years. fourteen of the asthma patients and five of the copd patients were smokers. table both groups of patients with viral infections and those without viral infections shared the same symptoms, with cough and wheezing being the most common in both groups (table ). however, among the patients suffering from viral infection, those infected with rhinovirus had the most severe symptoms compared with other viruses (results not shown here). dry cough was the predominant symptom among patients with rhinovirus (n = ) and coronavirus infections (n = ). chi square analysis demonstrated that the correlation between the symptoms and the presence of the virus was non-significant (p c . ). no significant relationship was observed between specific symptoms and age or ethnic origin. rhinoviruses and coronaviruses usually cause self-limited and harmless upper respiratory illnesses. however, these viruses have been associated with exacerbations in patients with asthma and copd. our study is the first in qatar to analyse the clinical aetiology of respiratory tract viral infections in adult patients from all age groups with asthma or copd. however, it is important to note that our study did not investigate the viral aetiology in patients with exacerbations. all patients were stable at time of recruitment and sample collection. to our knowledge, there are no other studies in which the frequency of respiratory viruses in adults with asthma/copd in the middle east has been reported. rhinovirus was the most common viruses identified, followed by coronaviruses. the copd sample was too small to allow any specific conclusions regarding viral aetiology. however, in the copd population, the corona viruses were the most common viral infectious agents. viral infections in our sample population were identified in out of patients. the low detection rate could reflect lower sensitivity of detection in older age groups or with nasal swabs compared with paediatric groups or with nasopharyngeal samples. it may also be that other pathogens are involved in respiratory tract infections affecting middle eastern populations. for example, metapneumovirus has been shown to affect adults with respiratory tract infections and wheezing in this region [ ] [ ] [ ] . cough and wheezing were the most predominant clinical manifestations in the present study in patients at baseline, with and without viral infections. we found that the distribution of viral infections did not correlate with the age and ethnic background of the patient. however, the effects of smoking were difficult to evaluate due to the small number of smokers in the population. in the last several years, respiratory virus infections have been identified in [ % of wheezing episodes in adults [ , , ] . furthermore, in several studies, rhinovirus has been identified as the most common respiratory virus associated with asthma exacerbations, and coronavirus is the second most frequent [ , ] . our findings in the qatari population corroborate data from previous studies and present the first of such studies in qatar, albeit in adult asthma and a minority of copd patients with no exacerbations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is possible that the aetiology of viral infection would differ if samples were analysed during an exacerbation. furthermore, while most studies have focused on the paediatric population, this is one of the few studies that examines viral aetiology in adults. there are several potential shortcomings of the current study. the sample size is quite small and may not be an accurate representation of the qatari asthmatic population. larger studies are needed to verify the current data. the number of copd patients was markedly smaller than that of asthma patients. this was mainly because patients with asthma may be more concerned for their health, especially when they suffer from upper respiratory tract infections (urtis), and go to the hospital, while copd patients may not consider their symptoms very serious. indeed, there is very poor education and awareness of copd in qatar among physicians and patients. in addition, there is a large population of copd smoker patients in qatar who are not aware of their disease status. larger numbers of copd patients must be enrolled in order to determine viral aetiology more accurately in this population. furthermore, an equal number of females and males also need to be included in future studies. another potential shortcoming is that a control population was not included in the study; therefore, the frequency of rhinovirus or coronavirus infections in populations hospitalised with non-respiratory conditions or in asymptomatic populations or non-asthmatic or non-copd populations with respiratory tract infections cannot be determined. finally, this study was limited to the ''winter season'' in qatar, and it may be that the infection pattern changes throughout the year. longer-term studies are needed to determine this. the morbidity associated with rhinovirus and coronavirus infections, especially in high-risk populations such as patients with asthma or copd, suggest that these infections should be a target for prevention or treatment strategies. in addition, the relatively low number of viruses found in our sample population may point towards other pathogens (viral or bacterial pathogens not investigated here) that may be implicated in causing urtis in patients in qatar. in patients with and without viral infections, dry cough and wheezing were the most common symptoms efficacy and safety of inhaled zanamivir for the treatment of influenza in patients with asthma or chronic obstructive pulmonary disease: a double-blind, randomized, placebo-controlled, multicentre study acute exacerbations of asthma: epidemiology, biology and the exacerbation-prone phenotype community study of role of viral infections in exacerbations of asthma in - year old children ireland, dc respiratory viruses and exacerbations of asthma in adults respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of copd a -year prospective study of the infectious etiology in patients hospitalized with acute exacerbations of copd how viral infections cause exacerbation of airway diseases respiratory tract viral infections in inner-city asthmatic adults study of human metapneumovirus-associated lower respiratory tract infections in egyptian adults human metapneumovirus in hospitalized children in amman prevalence of human metapneumovirus (hmpv) in children with wheezing in shiraz-iran acknowledgments the authors would like to thank all nurses in the chest clinic of hamad general hospital in qatar for their help in sample collection; dr. r. singh, biostatistician at hamad medical corporation; and ehssan othman for coordinating the project. the authors would like to acknowledge support of dr. sabah allawati, from medcommz ltd., in providing editorial support for this article. the medical research department at hmc and the research office of qatar university provided financial support to carry out this study. no competing financial interests exist for any of the authors. key: cord- -msk p yy authors: lee, c.-w.; jackwood, m. w. title: evidence of genetic diversity generated by recombination among avian coronavirus ibv date: journal: arch virol doi: . /s sha: doc_id: cord_uid: msk p yy previously, we demonstrated that the de strain of ibv is a recombinant which has an ibv strain d -like sequence in the s gene. herein, we analyzed the remaining . kb ′ end of the genome, which includes gene , gene , gene , gene , and the ′ non-coding region of the de and d strains. those two viruses had high nucleotide similarity in gene . however, the other individual genes had a much different level of sequence similarity with the same gene of the other ibv strains. the genome of five ibv strains, of which the complete sequence of the ′ end of the genome has been determined, were divided at an intergenic (ig) consensus sequence (ctgaacaa or cttaacaa) and compared phylogenetically. phylogenetic trees of different topology indicated that the consensus ig sequences and the highly conserved sequence around this regions may serve as recombination ‘hot spots’. phylogenetic analysis of selected regions of the genome of the de serotype field isolates further support those results and indicate that isolates within the same serotype may have different amounts of nucleotide sequence similarity with each other in individual genes other than the s gene. presumably this occurs because the consensus ig sequence serves as the template switching site for the viral encoded polymerase. infectious bronchitis virus causes a highly contagious upper-respiratory disease in chickens. the disease is characterized by increased ocular and nasal secretions, excess mucus in the trachea, decreased weight gain and feed efficiency in broilers, and declines in egg production and egg quality in layers. although live attenuated vaccines are available, ibv continues to be a severe economic problem in commercial chickens because many different serotypes of the virus exist and do not cross protect [ ] . [ ] . members of the nidovirales order have a single stranded positive sense rna genome and produce a nested set of subgenomic mrnas when they replicate [ ] . coronaviruses are divided into three antigenic groups based primarily on their structural proteins. infectious bronchitis virus is the type strain of coronaviruses and is the only virus placed in antigenic group three. characteristics of this group are a cleaved spike (s) glycoprotein, an n-glycosylated membrane (m) protein, and no hemagglutinin/esterase protein [ ] . the genome of ibv is approximately kilobases in length [ ] . it is organized into six regions, each containing one or more open reading frames (orf's), which are separated by intergenic sequences (ig) that contain the signal for transcription of subgenomic mrnas [ , ] . the viral rna-dependent rna polymerase is encoded in the two thirds of the viral genome by two overlapping open reading frames (orf a and orf b) [ ] . the structural protein genes are located to the viral polymerase gene and are in order from to , the s glycoprotein gene (gene ), the small envelope (e) gene (gene ), the m glycoprotein gene (gene ), and the nucleocapsid (n) gene (gene ) [ , ] . evolution in ibv has been observed through the occurrence of variant viruses and analysis of known serotypes. more than twenty serotypes within ibv have been recognized worldwide and are thought to be generated by insertions, deletions, point mutations and rna recombination [ , , , ] . evidence of natural recombination for several ibv strains has been reported [ , , ] . however, because of the limited sequence information, recombination has only been described for a small part of the genome. so far, the complete sequence of the end of the genome (from the end of the polymerase gene to the poly a tail) of only three strains, beaudette, kb and cu-t have been determined [ , , ] . the de strain was first isolated in in the delmarva peninsula region of the usa and initial characterization of this virus indicated this virus was serologically distinct from any other ibv serotypes in north america [ ] . previously, we demonstrated that the de strain is a recombinant which has a d -like sequence in the s and s genes [ ] . d is an ibv vaccine strain of the d serotype from the netherlands [ , , ] . herein, we describe the sequences of the remaining genes of the de and d strains with the exception of gene (the polymerase gene). we conducted phylogenetic analysis by dividing the genome in the ig sequence to elucidate possible role of this sequence in the homologous recombination in ibv. further, we conducted sequence analysis of six isolates of the de serotype in order to determine if recombination is frequently occurring in this region in field isolates of ibv. viruses used in this study are listed in table . the viruses were propagated in -day-old embryonated specific-pathogen-free (spf) chicken eggs (select laboratories, gainesville, ga, usa). the d strain of ibv was obtained as phenol-inactivated allantoic fluid using usda import permit # . viral rna from ibv grown in embryonating eggs was extracted using the high pure pcr template preparation kit (boehringer mannheim, indianapolis, in, usa) according to the manufacturers recommendation. rna from the phenol-inactivated allantoic fluid of d was extracted with a modification in first several step of the high pure pcr template preparation kit. briefly, . ml of the infectious allantoic fluid was placed into a microcentrifuge tube and centrifuged at , ×g for min. the aqueous top layer, approximately l, was transferred to new tube. binding buffer ( l) and l of proteinase k ( mg/ml) was added and incubated for min at • c. then l of chloroform/isoamyl alcohol ( : ) was added, vortexed gently for - sec and then placed on ice for min. the mixture was centrifuged at , ×g for min. the upper phase was transferred to a clean . ml tube and l of chloroform/isoamyl alcohol ( : ) was added. the mixture was vortexed gently for - sec. this was centrifuged for min at , ×g, and the upper phase was transferred to a clean . ml tube. remaining steps were followed sequentially as described by the manufacturer. gene , gene , gene , gene , and a bp hypervariable region (hvr) of the s gene were amplified separately using the titan one tube rt-pcr system (boehringer mannheim). primer sets used to amplify gene , gene , and the hvr in s are listed in table . the primers utilized for amplification of gene and gene have been reported [ , ] . the reaction conditions for rt-pcr were previously described [ , ] . pcr products were cut from % agarose gels and purified using the qia quick gel extraction kit (qiagen, santa clarita, ca, usa). purified pcr products were either sequenced directly or cloned into the ta cloning vector (invitrogen, carlsbed, ca, usa), and automated sequencing with the prism dyedeoxy terminator cycle sequencing kit (perkin elmer, foster city, ca, usa) was conducted at the molecular genetics instrumentation facility, university of georgia. sequencing primers to various regions of the gene for de and the relative primer positions were calculated using the atg start site of gene as for primers gene and , and atg start site of s gene as for primers hvr in s d were designed using oligo version . software (national bioscience, plymouth, mn, usa) and are available upon request. assembly of sequencing contigs, translation of nucleotide sequence into protein sequence, and initial multiple sequence alignments were performed with the clustal v method in megalign software versin . (dnastar inc., madison, wi, usa). phylogenetic trees for each gene were generated using the maximum parsimony method with bootstrap replicates in a heuristic search using the paup . software program [ ] . the nucleotide sequences reported here have been deposited with the genbank. the accession numbers are as follows: de (gene ), af ; de (gene ), af ; de (gene ), af ; de (gene ), af ; de ( end non-coding region), af ; d (gene ), af ; d (gene ), af ; d (gene ), af ; d (gene ), af ; d ( end non-coding region), af ; - (hvr in s ), af ; - (hvr in s ), af ; - (hvr in s ), af ; - (hvr in s ), af ; - (hvr in s ), af ; - (hvr in s ), af the complete sequence of the end of the genome of three strains, beaudette, kb and cu-t and gene of holl strain have been previously reported [ , , , ] . a total of nucleotide and nucleotide were found, respectively, in a region beginning from the end of gene to the end of de and d genome. the intergenic sequence ctgaacaa or cttaacaa was found immediately upstream of the start site for each gene of both strains. the sequences were identical to those found in the corresponding genomic areas of the beaudette, kb , and cu-t strains (fig. ). fig. . the nucleotide sequence alignment of gene , gene , gene , and gene , and noncoding region. dots indicates nucleotide identical to that of de strain. the conserved nucleotide sequences ctgaacaa or cttaacaa, which is located at the starting site of each gene, is in bold character. heavy underlines indicate the putative start codons, asterisks above the sequence indicate the stop codons gene of both strains contained three orfs, a, b, and c. gene consisted of the m protein gene with a single orf and a non-coding region between the end of the m protein gene and gene . gene contained two orfs ( a and b). gene consisted of the n protein gene with a single orf and a non-coding region. downstream from the stop codon of the n gene, a base insertion was found in the d genome which also occurs in the genome of the holl (fig. ) . the -terminal . kb of the genome of five strains and gene of the holl strain were compared. the nucleotide sequence similarities among coding regions of gene , m, gene , and the n protein gene of de and other strain were between . - . %. those of d and other strains were between . - . % identical. d showed only . % nucleotide difference with holl in gene . gene c and gene b were relatively more conserved than the other genes (table ) . genes were divided by ig sequences (ctgaacaa/cttaacaa) and phylogenetic analysis was conducted. the de strain clustered with the cu-t (fig. ) . kb , which is only the nephropathogenic strain, was solely placed in all genes compared. in order to demonstrate the genetic heterogeneity of the same serotype isolates of ibv, we conducted phylogenetic analysis using six de serotype field isolates. phylogenetic analysis of the hypervariable region (hvr) in s , clustered all the de serotype isolates in one group with the prototype strain of the de serotype of ibv. this group was far from other serotypes of ibv strains in tree length. however, phylogenetic tree of gene and gene showed differences in tree topology among six isolates. in gene , only one isolate, - , clustered with de . in gene , no isolates clustered with de and formed groups randomly with other serotypes of ibv (fig. ) . de is a recent isolate made in [ ] . in a previous study of the s gene, we demonstrated that this virus was closely related to d which is an ibv vaccine strain of the d serotype from the netherlands [ , , ] . analysis of gene also reveals a high sequence relatedness between de and d (table ) . however, in the other genes analyzed in this study, de shares high sequence similarity with the cu-t strain which has also been reported to be a recombinant between arkansas and massachusetts strains [ ] . considering the fact that both strains were isolated in the northeastern usa, it is possible that they had undergone similar selection pressure. on the other hand, d shows high similarity with beaudette and holl strains in genes other than the s gene. the percent similarity in the n gene and a base insertion in the non-coding region suggests that both d and holl are closely related (table , fig. ). the holl strain has been extensively used as a live vaccine in europe [ ] . this finding provides more convincing evidene that vaccine strains are contributing to the emergence of variants in the field. based on these results, we suggest that de and d had the same origin, but diverged a long time ago and evolved independently in different geographical locations. since recombination in coronaviruses is thought to occur by a template switching mechanism [ , ] , we speculate that ig sequences may serve as 'hot spots' for homologous recombination. so far, recombinations suggested in ibv have been used on a small part of the genome [ , , ] . examining only a small part of the genome may result in misleading conclusions because of point mutations or conserved regions of the gene. we conducted phylogenetic analysis by dividing . kb of the end of the genome among five ibv strains at the ig sequences. phylogenetic trees of this sequence data had very different topology (fig. ) , which indicates that recombination had occurred. it has been reported that rna recombination in ibv can occur randomly in non-localized sites in vitro [ ] . however, considering the selection pressure in vivo recombination in the ig sequences should be advantageous to virus in two aspects. first, since crossovers occur at the site of consensus ig sequences, there would be no shift in the codon reading frame. second, since whole genes are substituted, there would be no drastic change in the conformation of proteins encoded by individual genes. further, cross-overs at each of the five ig sequences would generate tremendous genetic diversity. this amount of diversity may contribute to persistence and to the continuing emergence of new variants of ibv despite vaccination efforts. finally, we conducted sequence analysis of isolates of the de serotype to demonstrate how random recombination occurs within the same serotype. phylogenetic analysis of the hvr in s shows that these isolates cluster together because they are the same serotype. however, these isolates had a much different level of nucleotide sequence similarity with each other in gene and gene , and clustered randomly with other serotypes of ibv (fig. ) . based on this result, it is clear that isolates of the same serotype can differ substantially in individual genes. thus, every field isolate of ibv could be unique in each gene sequence because of recombination. completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus location of the amino acid differences in the s spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus infectious bronchitis nidovirales: a new order comprising coronaviridae and arteriviridae occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the netherlands variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens antigenic and s- genomic characterization of the delaware variant serotypes of infectious bronchitis virus infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a dna probe poliovirus rna recombination: mechanistic studies in the absence of selection a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains sequence analysis of gene , gene and gene of avian infectious bronchitis virus strain cu-t experimental evidence of recombination in coronavirus infectious bronchitis virus molecular epidemiology of infectious bronchitis virus in the netherlands phylogeny of antigenic variants of avian coronavirus ibv sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction-fragment-lengthpolymorphism analysis coronavirus: how a large rna viral genome is replicated and transcribed spike gene analysis of the de strain of infectious bronchitis virus: origin and evolution the coronaviridae cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus paup: phylogenetic analysis using parsimony. version . illinois natural history survey evidence of natural recombination within the s gene of infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis viruses and other coronaviruses we express appreciation to dr. yoram weisman for providing d virus and deborah hilt for technical assistance. thanks are also extended to drs. bruce seal, maricarmen garcia, and holly sellers for the review of this manuscript. received december , key: cord- -ct rvqtd authors: mohamed, fakry f.; mansour, shimaa m. g.; el-araby, iman e.; mor, sunil k.; goyal, sagar m. title: molecular detection of enteric viruses from diarrheic calves in egypt date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ct rvqtd neonatal calf diarrhea (ncd) is a major cause of morbidity, mortality and economic losses in the beef and dairy industries. this study was conducted to investigate the existence of enteric viruses in two egyptian farms with a history of recurrent diarrhea. fecal samples were collected from diarrheic calves. rna was extracted and tested by reverse transcription polymerase chain reaction (rt-pcr) for the presence of rotavirus, norovirus, astrovirus, torovirus, coronavirus and bovine viral diarrhea virus. overall, % ( / ) of samples tested positive for one or more viruses. rota-, noro- and astroviruses were detected in %, % and % of tested samples, respectively. about % ( / ) of positive samples had two different viruses. one-month-old calves were the group most vulnerable to infections. based on phylogenetic analysis, bovine rotaviruses were of genotypes g and g , bovine noroviruses were in giii. , and bovine astroviruses were in the bastv lineage . astrovirus sequences showed a high level nucleotide sequence similarity with the brazilian bastv sequences available in genbank. we believe this is the first report of bovine norovirus and bovine astrovirus circulating among calves in egypt. further epidemiological studies are recommended to investigate their presence on a wider scale, to predict their association with ncd, and to design appropriate diagnostic and control methods. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. neonatal calf diarrhea (ncd) is one of the most common causes of morbidity and mortality in cattle. in the dairy industry, approximately % of deaths in one-month-old calves have been attributed to diarrhea caused by bacterial, viral or parasitic pathogens [ ] . bovine rotavirus and bovine coronavirus (brv and bcv) are the leading causes of viral diarrhea [ ] , although bovine viral diarrhea virus (bvdv), bovine norovirus (bnov), bovine astrovirus (bastv) and bovine torovirus (btov) have also been implicated. co-infection with two or more viruses is also common and often aggravates the diarrheal symptoms [ ] . both bcr and btov are members of the family coronaviridae. they are frequently associated with acute diarrheal and respiratory infections in calves of different ages in many countries [ ] . bvdv is a member of the genus pestivirus, family flaviviridae. bvdv is classified molecularly into two species (bovine viral diarrhea virus , and bovine viral diarrhea virus ). it was first identified in egypt in , and bvdv- b was circulating in the ismailia province of egypt during [ ] . members of the genus rotavirus, family reoviridae, are medium-sized, non-enveloped viruses. their dsrna genome consists of rna segments that encode six structural (vp - , vp and vp ) and six non-structural proteins (nsp - ) [ ] . based on their common group antigen (vp ), rotaviruses are classified into eight antigenically distinct groups (from a to h). group a rotaviruses are usually incriminated in human and animal cases of gastroenteritis [ ] . since capsid proteins vp and vp autonomously trigger neutralizing antibodies, group a rotaviruses are further classified into p (for protease sensitive) and g (for glycoprotein) types, respectively. at least p-types and g-types have been reported (http://rotac. regatools.be/classificationinfo.html), of which g , g , g , p , p and p are usually associated with most cases of bovine diarrhea [ ] . norwalk-like viruses (noroviruses) are known to infect humans and animals and cause epidemic and sporadic gastroenteritis. as members of the family caliciviridae, they are small, non-enveloped, positive-sense, ssrna viruses. their viral genome is . to . kb in size, excluding the poly(a) tail and consists of three open reading frames (orfs). orf encodes the non-structural polyprotein, which is cleaved by c-like protease into six proteins, including the rna-dependent rna polymerase (rdrp). orf and orf encode the structural capsid proteins vp and vp , respectively [ ] . based on phylogenetic analysis, noroviruses are classified into at least six genogroups (gi to gvi). a tentative genogroup vii has also been proposed. the bovine noroviruses are clustered in genogroup giii, which is further divided into two distinct genotypes (giii. and giii. ) [ ] . initially giii. (prototype bo/jena/ /de) was identified in germany in while giii. (prototype bo/newbury- / /uk) was discovered in england in [ , ] . the lack of a culture system for in vitro propagation of noroviruses, recombination events, and the presence of the virus in fecal samples of both diarrheic and healthy animals are major obstacles to determine their impact on the cattle industry, especially in developing countries. astroviruses are small, star-shaped, non-enveloped viruses with a positive-sense ssrna. the family astroviridae consists of two genera, mamastrovirus and avastrovirus, whose members infect mammals and birds, respectively [ ] . the genome of astroviruses is . - . kb long and consists of three orfs in addition to a utr, a utr and a polyadenylated tail. orf a encodes the nonstructural polyprotein, orf b encodes the rdrp, and orf encodes the structural capsid protein. orf is the most divergent part of the genome, while orf b is the least divergent [ ] . bastv was first reported in england in from calves suffering from acute enteritis. initially, it was considered avirulent [ ] , but subsequently, it was proven to be a pathogen, especially in calves that are coinfected with brv or btov. the astroviruses have also sometimes been detected in association with bovine encephalitis [ ] . based on serological assays, bovine astroviruses are classified into two serotypes, bastv- and bastv- [ ] . a total of fecal samples were randomly collected from two private cattle farms in sharkia and cairo provinces in early . the calves had diarrhea, fever and variable degrees of dehydration and weakness. diseased calves did not respond to antibiotic therapy. no mortalities were found at the time of sample collection. the average age of animals ranged from three weeks to ten months. the calves on one farm were vaccinated against brv and bcv. the viral rna was extracted from a % fecal suspension using a blood/liquid sample total rna rapid extraction kit (bioteke corporation, china) according to the manufacturer's instructions. the rna extracts were screened by rt-pcr for the detection of brv, bcv, bvdv, btov, bnov and bastv. reactions were performed in a ll volume using a one step rt-pcr kit (qiagen, valencia, ca, usa). reverse transcription was done at °c for min, followed by pcr activation at °c for min and cycles of °c for min for denaturation, the appropriate temperature and time for annealing (table ) and °c for min for extension. the final extension was done at °c for min. for the g and p typing of brv-positive samples, rt-pcr reactions were performed using specific primers for vp and vp , respectively. the primers used in this study are listed in table . the pcr products were confirmed by analysis in an ethidium-bromide-stained agarose gel followed by visualization under a uv transilluminator. the pcr products were purified using a qiaquick pcr purification kit (qiagen, valencia, ca, usa). the purified dna was sequenced at the university of minnesota genomics center (umgc) using the same forward and reverse primers as were used for rt-pcr. forward and reverse sequences were aligned together to generate a consensus sequence using sequencher software version . (http://genecodes.com/). also the newly obtained sequences were compared with existing sequences in the gen-bank database using online blast search tool (http:// www.ncbi.nlm.nih.gov/). the newly obtained nucleotide sequences were deposited in the genbank database under accession numbers kx to kx for brv, kx to kx for bnov, and kx to kx for bastv. using mega . software, a comparative alignment was done using the clustal w method. the evolutionary distances were calculated using the pairwise (p) distance method (percent of nucleotide [nt] and amino acid [aa] sequence identity and divergence). the phylogenetic trees were constructed using the neighbor-joining method in mega . software [ ] , and the tree topology was evaluated by , bootstrap replicates. the rt-pcr results of the tested rna showed that % ( % ci: . % to . %) were brv positive, % ( % ci: . % to . %) were bnov positive, and % ( % ci: . % to . %) were bastv positive. about % ( / ) of positive samples had two different viruses ( table ). products of bp, bp, bp, and bp were visualized on a . % agarose gel for brv-vp , brv-vp , bnov-rdrp and bastv-rdrp, respectively ( supplementary fig. a-d) . when data were correlated to the age of the tested calves, one-month-old calves showed the highest rate of infections (fig. ) . results were negative for bcv, bvdv and btov. the phylogenetic analysis of the brv-vp coding sequences revealed that they clustered with the g and g genotypes of group a rotaviruses (http://rotac.regatools.be/) [ ] (fig. ) . at both the nt and aa level, the g sequences were % identical and the g sequences showed . - % identity to each other. a comparison of the g sequences with published sequences in the genbank database showed overall identities of . - . % and . - . % in nt and aa, respectively. the same comparison using g sequences showed identities of . - . % at the nt level and . - . % at the aa level. on p typing, only one isolate was found to be p , while all remaining samples were non-typable. the norovirus sequences of this study showed identities of . - . % and . - % at nt and aa levels, respectively. a phylogenetic tree illustrated that all samples were related to genotype giii. of bnov (bovine/ newbury /uk strain-like) (fig. ) . three sequences (bnov- , bnov- , and bnov- ) grouped together with . - . % nt and % aa identity with each other and maximum identity with previously published sequences from the usa (nov- and nov- ), the uk (penrith ) and tunisia (monastirb ). one of the study sequences (bnov- ) grouped differently from the remaining sequences, with . % and . % nt and aa identity, respectively, and showed maximum identity to isolates from italy (bec ), the uk (aberystwyth ) and belgium (florennes-b ). the bastv study sequences were compared with each other as well as with previously published bastv sequences available in genbank (fig. ) and clustered with bastv lineage . except bastv- , all study sequences grouped together with . - % nt and . - % aa identity to each other. these sequences showed maximum sequence similarity to and ishikawa (japan). the bastv- showed . % nt and . % aa sequence identity to the rest of the study sequences and maximum identity to kagoshima - and hokkaido - (japan). calves within their first month are highly susceptible to viral diarrhea probably due to suckling milk, which neutralizes the acidic ph of their digestive tract and in turn allows several pathogens to survive [ , ] . several improvements in vaccination, medication and management have been implemented to reduce the incidence of ncd. however, ncd is still persistent because it is complex and multifactorial and can be triggered by several different infectious and non-infectious causes [ ] . ncd has a devastating economic impact on cattle-raising businesses worldwide [ , , ] . direct losses are due to dehydration, reduced growth rate, and high morbidity and mortality. indirect losses are due to the imposition of trade restrictions and increased costs of management and veterinary care as well as animal suffering. in egypt, the diarrhea control programs are completely dependent on mass vaccination of animals using commercially available inactivated vaccines. data on the detection, prevalence and typing of infectious diarrheal causes are not available, and this study was undertaken in an attempt to fill that gap. rotaviruses are the main causes of diarrhea in many animal and human species. the g , g and g types of group a rotaviruses are usually found in cases of ncd. we detected rotaviruses in % of the tested samples ( . % for g and . % for g ). this could be a preliminary indication of the types of bovine rotaviruses currently circulating in egypt. nearly the same rates of g and g were recorded by caruzo et al. [ ] , while the overall rotavirus detection rate was . %, probably due to a larger sample size. only one of the brv isolates in our study was p typable (brv- /sharkia/ ) and was found to be p , which is the most dominant p type. also, the g -p genotype is a common combination worldwide [ ] . in egypt, brv was first diagnosed in the early s. the brv-g type was detected by merwad et al. [ ] . some studies have found evidence of viral reassortment and the possibility of interspecies transmissions, including to humans [ ] . our results might help in future studies on rotaviruses in egypt. bnov was molecularly detected ( %) either alone or as a co-infection with other enteric viruses (brv and bastv). all bnov sequences were found to be phylogenetically related to genotype giii. , which is known to be distributed globally. ferragut et al. [ ] discussed an interesting finding about the argentinean strain b (tentatively classified as giii. ), which grouped with giii. based on partial orf sequence analysis but showed a significant divergence from previously described genotypes based on orf / sequence analysis. molecular investigations in several countries have revealed a number of potential recombinant strains, which emphasizes the importance of studying complete genome sequences; the use of only a single primer set (cbecu-f and r) may limit the opportunity to detect novel divergent/recombinant strains. the connection between bovine astroviruses and enteric diseases in not obvious; some researchers maintain that bastv is not associated directly with severe diarrhea under natural conditions [ ] , while others believe that it can happen [ ] . we reported bastv in feces of calves with clinical diarrhea ( %), which clustered with bastv lineage [ ] . lineage involves one and nine strains of genogroup g and g , respectively [ ] , in addition to five korean strains within genogroup g [ ] . the egyptian bastv strains were closely related to isolates from brazil and japan. this further supports the assumption of nagai et al. [ ] that lineage might be the most prevalent genogroup of bastv in the world. efforts should be made to develop specific criteria for the classification of bastvs as lineages or genogroups. our bastv sequences showed . - % nt sequence identity, in contrast to those in a brazilian bastv study in which the identity was . - . % [ ] . this may be due to the small sample size from two egyptian provinces, while the brazilian study was based on samples from seven different states. to the best of our knowledge, this is the first report on the existence of bovine noroviruses and bovine astroviruses in egypt. the association of these viruses with the pathogenesis of severe diarrhea is still inconclusive. the genetic diversity and evolution of these enteric viruses should be monitored regularly in domestic and wild animal populations as well as in humans. our results will contribute to better understanding of these challenging brv/g /australia/ (gq ) human-rotavirus/g /p /vietnam/ (ab ) brv/g /strain-b (m ) brv/g /uk/ (x ) brv/g /morocco/ (kt ) /g /sharkia/egypt/ (kx ) brv/g /strain-kk /japan (d ) g /egypt/ (kf ) brv/g /egypt/ (kf ) human-rotavirus/g /p /egypt/ (af ) brv/g /india/ (jx ) brv- /g /cairo/egypt/ (kx ) brv- /g /cairo/egypt/ (kx ) brv- /g /cairo/egypt/ (kx ) brv- /g /cairo/egypt/ (kx ) /monastirb /tunisia/ (jn ) bnov/bv /belgium/ (eu ) bec/bec /italy/ (hm ) bnov/florennes-b /belgium/ (ay ) /b /belgium/ (eu ) bnov/bv /belgium/ (eu ) sharkia/egypt/ (kx ) astrovirus/bovine/bastv- /cairo/egypt/ (kx ) astrovirus/bovine/boastv- -bra/brazil/ (kj ) astrovirus/bovine/boastv- -bra/brazil/ (kj ) astrovirus/bovine/hokkaido - /japan/ (lc ) bastv lineage calf health from birth to weaning. ii. management of diarrhoea in pre-weaned calves an overview of calf diarrhea-infectious etiology, diagnosis, and intervention high prevalence and diversity of bovine astroviruses in the faeces of healthy and diarrhoeic calves in south west scotland detection and molecular characterisation of bovine corona and toroviruses from croatian cattle circulation of bovine viral diarrhea virus- (bvdv- ) in dairy cattle and buffalo farms in ismailia province rotaviruses: from pathogenesis to vaccination uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) diversity and zoonotic potential of rotaviruses in swine and cattle across europe caliciviridae: the noroviruses advances in laboratory methods for detection and typing of norovirus zackenvirus'' (jena agent / )-a new diarrhea pathogen in calves isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves astrovirus infections in humans and animals-molecular biology, genetic diversity, and interspecies transmissions genomic analysis of closely related astroviruses characterization of a calici-like virus (newbury agent) found in association with astrovirus in bovine diarrhea detection of a novel bovine astrovirus in a cow with encephalitis serotypes of bovine astrovirus mega : molecular evolutionary genetics analysis version . rotac: a web-based tool for the complete genome classification of group a rotaviruses the dairy calf mortality: the causes of calf death during ten years at a large dairy farm in korea perinatal death in production animals in the nordic countries-incidence and costs molecular characterization of g and p-types bovine rotavirus strains from goiás, brazil: high frequency of mixed p-type infections close relationship of group a rotaviruses between bovine and human based onvp gene sequence in egypt detection of the first g p [ ] human rotavirus strain from a child with diarrhea in egypt molecular detection of bovine noroviruses in argentinean dairy calves: circulation of a tentative new genotype phylogenetic analysis of bovine astrovirus in korean cattle full genome analysis of bovine astrovirus from fecal samples of cattle in japan: identification of possible interspecies transmission of bovine astrovirus molecular detection and phylogenetic analysis of bovine astrovirus in brazil two out of the genes of an unusual human g p [ ] rotavirus isolate are of bovine origin development and application of one-step multiplex reverse transcription pcr for simultaneous detection of five diarrheal viruses in adult cattle amino acid substitution within the vp protein of g rotavirus strains associated with failure to serotype identification of group a rotavirus gene types by polymerase chain reaction rediscovery and genomic characterization of bovine astroviruses reverse transcription-pcr assays for detection of bovine enteric caliciviruses (bec) and analysis of the genetic relationships among bec and human caliciviruses experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons analytical sensitivity of assays used for detection of bovine viral diarrhea virus in semen samples from the southeastern united states rotavirus diarrhea in bovines and other domestic animals enteric viruses from diarrheic calves in egypt acknowledgments the authors thank the ministry of higher edu- the authors confirm that this article content has no conflict of interest. key: cord- - wfx wey authors: li, renfeng; qiao, songlin; yang, yanyan; su, yunfang; zhao, pu; zhou, enmin; zhang, gaiping title: phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf gene and the main neutralization epitopes date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: wfx wey since , porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central china. to investigate the epidemic characteristics of pedv, the complete orf genes of pedv field strains from central china during to were cloned, sequenced and compared with reference strains. phylogenetic analysis based on the complete orf gene showed that the pedvs in central china and the reference strains could be divided into three groups: g , g , and g . the pedv isolates were classified as g and showed a close relationship to some chinese strains isolated previously in central china and differed genetically from recent isolates from southern china, korean strains (sm and db , ), the chinese lzc strain ( ), and the vaccine strain (cv ) being used in china. our findings suggested that the pedvs circulating between and in central china might have evolved from earlier strains in the local region. to determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the s gene. the results showed that the neutralizing epitopes at aa - were highly conserved, but most of the amino acid changes occurred in the epitope regions aa - and - . we speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of pedv and consequently result in vaccination failure. together, these findings may be useful for understanding the epidemiology of pedv and may be relevant for designing of new and more efficacious vaccines. porcine epidemic diarrhea (ped) is an acute, highly contagious viral enteric disease of swine caused by ped virus (pedv), a member of the genus alphacoronavirus [ ] . ped was first reported in belgium and the united kingdom in [ ] . since then, the disease has been recognized in many european countries, such as germany, france and switzerland, and more recently in korea and thailand [ ] [ ] [ ] . in china, pedv was first confirmed in [ ] , and since , it has caused enteric disease with a devastating impact on the swine-raising industry. this disease is characterized by high morbidity and mortality among preweaning piglets, causing serious economic losses to the swine industry in china [ ] . pedv is an enveloped virus possessing an approximately -kb genome. the pedv genome contains at least seven open reading frames (orfs), encoding four structural proteins (spike [s], envelope [e] , membrane [m], and nucleocapsid [n] ) and three non-structural proteins (replicases a and b and orf ) [ ] . the orf gene is an accessory gene of pedv and has been demonstrated to be associated with the virulence of pedv [ ] . a region (nt to ) that is crucial for pedv pathogenicity, is deleted in all live vaccine strains, and this could be a marker of adaptation to cell culture and attenuation of the virus. thus, the orf gene could be used as a valuable tool for differentiation of wild-and attenuated-type pedvs and molecular epidemiology studies of pedv [ ] . the spike protein (s) is the major structural proteins of pedv and consists of amino acids. similar to those of other coronaviruses, the s protein of pedv consists of three domains, including a large outer domain, a transmembrane domain and a short cytoplasm domain at the carboxyl end. it has been demonstrated to have four neutralizing epitopes (aa - , - , - , and , - , ) on the surface of the s protein [ ] [ ] [ ] , which are important for receptor binding and virus entry, the induction of neutralizing antibodies, and host-cell fusion [ , , ] . therefore, the s protein is also essential for understanding the epidemiological status of pedv in the field, diversity of pedv isolates, and the association between genetic mutations and viral antigenicity [ , ] . recent studies have shown that the ped caused by pedv is becoming increasingly serious in china, and current vaccines are ineffective on most swine-raising farms. molecular epidemiology and virus isolation of pedv have been undertaken in some regions of china [ , [ ] [ ] [ ] [ ] . these studies are essential for identifying the epidemic characteristics of pedv and have the potential to provide information about its pathogenesis. nevertheless, little is known about the antigenic variability of pedv prevailing currently in the field. in this study, we aimed to investigate the molecular epidemiology and antigenic variability of pedv field strains based on analysis of orf and a portion of the s gene encoding three neutralizing epitopes (aa - , - , and - ) of the s protein. this study provides new information about the prevalence of pedv strains currently circulating in china. intestinal and fecal samples were collected from piglets suffering from severe diarrhea from september to june in central china. the scraped-off mucosa and the contents of the small intestine were pooled, diluted : in phosphate-buffered saline (pbs; . m nacl, . m phosphate buffer [ph . ]), homogenized by ultrasonication, and clarified by centrifugation for min at , g after freezing and thawing three times. the supernatants were collected for rt-pcr. pedv rna was extracted using trizol reagent (invitrogen), dissolved in ll of % diethylpyrocarbonate-treated water, and stored at - °c. the complete orf gene was amplified using previously published primers [ ] , and the size of expected product was bp. the primers for amplifying the partial s gene were designed based on the genome of pedv cv (genbank no. af ), and the size of the final fragments, containing three neutralizing epitopes of pedv, was bp. two sets of primers were synthesized by sangon biotech, china. the rt-pcr primers are listed in table . synthesis of the first-strand cdna for the n gene was carried out by reverse transcription using reverse transcription reagents from promega. the viral rna ( ll) was mixed with . ll of pm antisense primer, incubated at °c for min, and placed on ice for min. after that, ll of rt buffer, ll of . mm dntp mixture, ll of rnase inhibitor ( u/ll), ll of reverse transcriptase m-mlv ( u/ll), and . ll h o were added with gentle mixing. the reaction mixture was incubated for h at °c, and the reaction was terminated by heating for min at °c. the cdna was either stored at - °c or amplified immediately. for pcr, ll of cdna was mixed with a reaction mixture containing . ll of taq dna polymerase buffer (promega, madison, wi), mm mgcl , . ll of dntps ( . mm), . ll of each specific primer ( pmol), ll of taq dna polymerase (promega, madison, wi) and autoclaved, filtered ( . lm) distilled water in a total volume of ll. amplification was performed as follows: one cycle at °c for min, followed by cycles at °c for min, °c for min, and °c for min, and a final extension at °c for min. the rt-pcr products were visualized by electrophoresis in a . % agarose gel containing ethidium bromide. purified rt-pcr products were identified by electrophoresis in a . % agarose gel and cloned into pmd Ò -t. the recombinant vector was identified by pcr and enzyme digestion. the positive clones were sent to sangon biotech, china, for sequencing. all sequencing reactions were performed in duplicate. the expected sizes of the pcr products were and bp. the former contained the complete orf gene and its flanking sequences, while the latter contained the partial s gene. the nucleotide sequences of the pedv isolates were deposited in the genbank database, and the corresponding accession numbers are listed in table . the nucleotide sequences were assembled and proofread using contigexpress software. multiple sequence alignments were generated by the clustal w method using the megalign . program in dnastar (dnastar inc. usa) (version . ). phylogenetic trees were constructed by [ ] . in addition, reference strains with complete s gene sequences were chosen for the analysis of the region containing neutralizing epitopes ( - ). b-cell epitope prediction was performed using the bepipred . b server (http://www.cbs.dtu.dk/services/bepipred/) [ ] . the reference strains used for phylogenetic analysis and the samples strains are listed in table . fourteen (fig. ) . fourteen partial s genes amplified from the clinical samples (genbank accession nos. kf -kf ) had a size of nt, encoding a -amino-acid polypeptide, corresponding to nt - of the complete s gene of the cv strain, containing three neutralizing epitopes ( - , - , - ), with . - . % nucleotide and . - . % deduced amino acid sequence similarity to each other, and . - . % nucleotide and . - . % amino acid sequence identity to the reference strains. a phylogenetic tree based on the partial s nucleotide sequence showed that the isolates could be clustered into three groups. all isolates belonged to g , together with most chinese strains and six foreign strains, including two american strains (usa/colorado/ and - ), three strains from thailand (vn , ku rb , and th/np- / ) and one strain from japan (mk). the samples in g were clearly separated into two different subgroups: ch/fch- , ch/lh, ch/ly- , ch/jch, ch/zhz- , and ch/heb formed a unique subgroup and differed clearly from the other pedv isolates, and the unique subgroup was adjacent to mk, a strain from korea. in addition, g included two korean strains (ad and nj ). g included one european strains (br / ), four korean strains (dr , kh, nk, and sm ), two early domestic strains (lzc and ch/s), and the vaccine strain cv ). these strains differed genetically from the sample strains because of their distant location in phylogenetic tree (fig. ) . in addition, the deduced amino acid sequences from the partial s gene analysis showed that there was no amino acid change in the epitope at aa - in any of the isolates. however, when compared with the reference strains, some variants were observed in the epitope regions at table . only one deletion was observed at aa in the epitope region aa - of ch/ xip- . in addition, b-cell epitope in the partial s protein (aa - ) was predicted using the bepipred . b server. the results showed that there were three b-cell epitopes with higher scores between aa and , located at positions - , - , and - . in the current study, the amino acid mutations at aa , , and were within the epitopes predicted above (aa - , - , - , respectively; fig. ; table ), indicating that these changes might result in antigenicity differences. pedv has been detected frequently in many provinces in china, including central china, since it was first identified in , and has become one of the most important viral enteric diseases [ ] . despite the current vaccination strategy, the losses caused by pedv infection are continuous and serious. it is necessary to understand the epidemiology of pedv and to explore the antigenic variation of the virus. during the epidemiologic investigation, we confirmed that pedv was the predominant causative pathogen contributing to outbreaks of clinical diarrhea in central china and determined that the positive rate of pedv in all diarrhea samples tested was . %, followed by rotavirus ( . %) co-infections with multiple pathogens in diarrheal disease were very frequent, especially mixed infections with pedv and porcine group a rotavirus (garv). these findings are consistent with those reported by zhang et al. [ ] . the samples used in this study were mostly from neonatal piglets ( . %), and they were obtained mainly in winter. to further investigate the molecular epidemiology of this virus, we chose the orf gene and a partial s gene encoding a region including three neutralization epitopes for studying the evolutionary characteristic and antigenic variation of pedv. orf is an important virulence gene that can be used to differentiate highly cell-adapted viruses and field isolates of pedv and is a potential tool for studying the molecular epidemiology of pedv [ , ] . in the present study, orf genes of pedv field strains collected in central china between and were amplified by rt-pcr, cloned, and sequenced to determine the genetic characteristics of viruses causing ped outbreaks in central china. the results confirmed that none of the strains had the -nt deletion in orf . sequence comparison with other pedv reference strains selected from the genbank database indicated that the orf genes from the sample strains had a high degree of homology to most chinese strains. for example, ch/ly- , ch/fch- , ch/heb, and ch/lh were distributed in the same subgroup with ch/zmdzy/ ; ch/yf- and ch/kf- were in the same subgroup with ah ; and both ch/zmdzy/ and ah were collected previously in central china. these results revealed that the pedv strains prevailing in central china might originate from local regions in central china. in addition, although relatively conserved when compared with the reference strains, variable sites were found in the orf nucleotide sequences of these isolates (alignment data not shown), leading to the following single amino acid changes: l ? we speculate that these mutations may cause differences in the virulence of pedv in the field and consequently intensify the epidemic. moreover, the results in this study did not show a -bp deletion in the complete orf nucleotide sequence, indicating that the pedv prevailing on swine farms of central china were wild-type strains. as a glycoprotein on the viral surface, the s protein of pedv is closely associated with its pathogenicity and immunogenicity [ ] . in this study, partial s genes from field pedv strains were amplified by rt-pcr, cloned, sequenced, and analyzed to study their antigenic variation. alignment of the deduced amino acid sequences of the partial s regions showed relatively high sequence identity among the pedv strains. in particular, the samples in this study were highly conserved in the neutralizing epitope region aa - , but most of the changes occurred in the core epitope regions of aa - and aa - . of these, there were samples with amino acid sequences changes at position : from a to v (ch/yf- , ch/qx- , ch/ kf- , ch/lshan) and a to s (ch/ly- , ch/fch- , ch/lh, ch/xip- , ch/zhz- , ch/heb). the amino acid sequences in four samples (ch/yf- , ch/qx- , ch/kf- , ch/ly- ) changed from h to r at aa , and (fig. ) . these changes might alter the antigenicity of pedv and consequently result in vaccination failure. further study is needed to confirm the relationship between amino acid mutations in epitope regions and antigenicity, which would help to improve our understanding of the prevalence of pedv in china. phylogenetic analysis based on the partial s gene showed that the isolates were separated into two groups ( fig. ) , with ch/fch- , ch/lh, ch/ly- , ch/jch, ch/zhz- , and ch/heb forming a unique subgroup. due to amino acid changes occurring mostly in epitope regions, these results indicated that the antigenicity of these strains differed from that of other strains, and these might be new pedv variants prevailing in central china. interestingly, although isolated from different geographical areas, two american strains (usa/colorado/ and - ), three thai strains (vn , ku rb , and th/np- / ) and one korean strain (mk) were clustered within the same subgroup with some recent chinese strains, indicating that these strains from different geographic regions might have similar antigenicity. in addition, one european strain (br / ), two japanese strains (kh and nk), two korean strains (dr and sm ), two early domestic strains (lzc and ch/s), and the vaccine strain (cv ) differed genetically from the other strains and clustered in the group g in the phylogenetic tree based on the partial s gene (fig. ) . these results are consistent with those reported previously [ ] [ ] [ ] . based on the sequence analysis of orf and partial s genes of pedv, molecular epidemiology was conducted using field strains from central china. our results revealed that the pedvs prevailing in the central china are still wild-type strains rather than the vaccine strain, and they mainly originated from the earlier strains in local regions. the amino acid mutations that occurred in the s epitope region might be associated with vaccination failure and the emergence of new pedv variant strains. further study should be done to examine the pathogenicity, antigenicity, and epidemiology of pedv. sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus e and porcine transmissible gastroenteritis virus a new coronavirus-like particles associated with diarrhea in swine genetic characterization of porcine epidemic diarrhea virus pedv isolates from southern vietnam during - outbreaks molecular detection of porcine kobuviruses in pigs in korea and their association with diarrhea chinese-like strain of porcine epidemic diarrhea virus study on the culture of porcine epidemic diarrhea virus adapted to fetal porcine intestine primary cell monolayer phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china 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 diagnostic notes: update on porcine epidemic diarrhea identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein major receptor-binding and neutralization epitope are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein spike protein region (aa - ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea molecular epidemiology of porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian new variants of porcine epidemic diarrhea virus outbreak of porcine epidemic diarrhea in suckling piglets mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods improved method for predicting linear b-cell epitopes occurrence and investigation of enteric viral infections in pigs with diarrhea in china role of proteases in the release of porcine epidemic diarrhea virus from infected cells molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china key: cord- - aqc e authors: ito, y. title: induction of interferon by virus glycoprotein(s) in lymphoid cells through interaction with the cellular receptors via lectin-like action: an alternative interferon induction mechanism date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: aqc e when animals and cells are infected with a virus, interferon is produced. viral-nucleic acid is considered to be one of actual components for interferon induction. in addition, viral glycoproteins trigger interferon induction in lymphoid cells by membrane-membrane interaction via a lectin-like activity. a biological significance of lectin-like activity of viral glycoproteins is discussed. there are different levels of host defence mechanisms against viral infection. viruses are unable to replicate on their own but must enter a host cells and used the host-cell macromolecular machinery and energy supplies to replicate. therefore, reactions of hosts to virus-infected cells are more characteristic of anti-viral mechanisms than those to virus particles. furthermore, since lymphoid cells have the principal role in anti-viral mechanisms of a host, analysis of interactions between lymphoid cells and infected cells expressing virus genes is one of the main subjects of research on anti-viral mechanisms. interferon is also known as anti-viral factor and has been proven to play an important role in the anti-viral defence mechanism. we found that mouse spleen cells produced interferon through interaction with virus-infected cells in vitro and in vivo [ , ] . consequently, mechanisms by which interferon is induced should be clarified for better understanding of cellular and humoral events of the host defence mechanism against virus infection. when animals and cells are infected with a virus, interferon is produced. a large number of interferon genes have been cloned and regulation mechanisms on interferon gene expression have been extensively investigated [ ] . however, what component(s) of the virus triggers interferon induction remains unclarified. by using temperature-sensitive mutants [ , , , ] , defective interfering particles [ ] , uv-inactivated viruses [ , , ] or cell lines nonpermissive for virus replication, a close relationship between interferon induction and double-stranded rna was revealed: for instance, the fact that prolonged ultraviolet irradiation disrupts the interferon-inducing ability of a virus suggests that the interferon induction is related to the function of virus nucleic acid. in addition, some of the cellular proteins induced by interferon such as - a synthetase and a protein kinase are specifically activated by double-stranded rna, indicating that the interferon system is intimately related to doublestranded rna. furthermore, since the interferon inducing activity of a man-made double-stranded rna, poly i: c, is found to be high potent, virus nucleic acid, especially double stranded rna, is thought to be the actual component for interferon induction by viruses. this conception is drawn from the experimental results in virus-fibroblast cell systems, however. whether the mechanism of virus-interferon production in lymphoid cells is identical with that in fibroblasts has not been elucidated. this nucleic acid mediated triggering pathway of interferon induction found in fibroblast is defined as a classical and major pathway. in this review, i propose that there is another, alternative, triggering pathway of interferon induction in lymphoid cells, in which virus glycoprotein(s) is involved in interferon induction. interferon-~/ is found in the culture fluid of mouse spleen cells cocultivated with bhk-sv cells, a baby hamster kidney (bhk) cell line persistently infected with sendai virus, but not in the medium with normal bhk cells [ , ] . no interferon is detected when either l cells, a mouse fibroblast cell line, or mouse liver cells are cocultivated with bhk-sv cells [ ] . these findings suggest that mouse lymphoid cells have a capacity to produce interferon-~/ when cocultivated with virus-infected cells, whereas nonlymphoid somatic cells lack this capacity [ ] . interposition of a millipore filter between bhk-sv monolayer and mouse spleen cells, or pretreatment of bhk-sv cells with anti-sv antiserum, results in a blockage of interferon production [ ] . these findings suggest that the following sequence is necessary for the mouse spleen cells cocultivated with bhk-sv cells to produce interferon: first, attachment of the spleen cells to bhk-sv cells and, second, recognition by the former of virus antigen(s) present on the surface of the latter. therefore, interferon production in" this system is considered to be initiated by membrane-membrane interaction between lymphoid cells and virus-infected cells [ ] [ ] [ ] . the bhk-sv cell membrane is found to be an active inducer of interferon in mouse spleen cells, but not in l cells [ ] . however, sonication of bhk-sv cells causes a loss of capacity for interferon induction, suggesting that some structural integrity of membranes is necessary for the interferon triggering [ ] . when mice are inoculated intraperitoneally with bhk-sv cells, interferon is detected viral glycoproteins and interferon induction in the peripheral blood [ ] . however, sonication of bhk-sv cells also suppresses their in vivo interferon-inducing ability [ ] . naturally, uninfected bhk cells have no ability to induce interferon in mice [ ] . these findings indicate that the interferon inducing mechanism by membrane-membrane interaction functions in vivo. prolonged ( - min) uv-irradiation results in complete loss of the interferoninducing ability of sendai virus in mouse l cells [ ] . in contrast to this result, sendai virus irradiated for h can induce interferon in mouse spleen cells as efficiently as untreated sendai virus [ ] , showing that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but some other viral component(s). when sendai virus is treated with potassium periodate ( . m) at °c for i h, infectivity for eggs and the hemolytic and neuraminidase activities of the virus are not detectable, but a considerable portion of its hemagglutinating activities is retained [ ] . although this inactivated sendai virus shows no interferon-inducing ability in either l cells or mouse spleen cells, the binding of this inactivated virus to erythrocytes restores an interferoninducing ability in mouse spleen cells but not in l cells [ ] . these results indicate that hemolytic and neuraminidase activities are not essential for interferon and that hemagglutinating activity may be closely related to interferon induction in mouse spleen cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the ceils. la sota and ulster strains of ndv possess uncleaved fo glycoprotein and are characterized by an apparent lack of hemolytic and cell fusion activity and infectivity for tissue culture cells. these viruses cannot induce interferon in l cells, whereas a high titer of interferon is induced in mouse spleen cells [ ] . treatment of la sota and ulster strains with trypsin results in cleavage of f protein and restores the hemolytic activity and infectivity [ ] . an appearance of interferon-inducing activity in l cells correlates to the cleavage of fo protein. in addition, hela cell-grown sendai virus, which has a similar property to the la sota strain of ndv, that is, it is characterized by its inability to penetrate into tissue culture cells, is found to stimulate interferon production in mouse spleen cells but not in l cells [ ] . these findings also indicate that penetration of the virus into mouse spleen cells is not needed for interferon induction and simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon triggering in mouse spleen cells. furthermore, uv-irradiated influenza virus can induce interferon in mouse spleen cells but not in l cells [ ] . periodate treatment of influenza virus destroys its interferon-inducing ability in both l cells and mouse spleen cells, but binding of the inactivated virus to erythrocytes restores its interferoninducing activity in mouse spleen cells but not in l cells [ ] . these results suggest that the mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that by paramyxoviruses. furthermore, when mouse spleen cells are incubated with sendai virus envelope with virus glycoprotein(s) such as hn + f or hn + fo, interferon-~/ is induced [ ] . even when mouse spleen cells are incubated with membranes containing hn glycoprotein alone, they produce interferon [ ] . however, l cells have no capacity for interferon production in response to any stimulation of subviral components [ ] . it is concluded from these findings that hn glycoprotein is the active component of sendai virus responsible for interferon induction in mouse spleen cells and that viral rna and f glycoprotein are not required. the results confirm that the interaction between hn glycoprotein and receptors on the cell surface triggers production of interferon in lymphoid cells. when mice are given an intravenous injection of isolated viral glycoproteins of purified sendai virus, circulating interferon is detected [ ] , indicating that isolated viral glycoproteins per se have the ability to induce interferon in vivo. to further determine whether the actual inducer of interferon in mouse spleen cells was hn glycoprotein, a recombinant plasmid was constructed by inserting the cdna of the hn gene of parainfluenza virus type (piv- a) into pcdl-sr~ expression vector. interferon activity cannot be detected in culture fluids of cos cells expressing hn protein (cos/hn cells), mouse spleen cells or cos cells [ ] . mouse spleen cells produced interferon when cocultured with cos/hn cells, but do not produce it when cocultured with cos cells transfected within or without the vector alone [ ] . in addition, we established hela cell lines constitutively expressing piv- a hn (hela- ahn cells) or f protein (hela- af cells). mouse spleen cells produce interferon-~/ when cocultured with hela- ahn cells, but not when cocultured with hela- af cells [ ] . therefore, it is concluded that hn glycoproteins of paramyxovirus on the cell surface are sufficient for interferon induction in mouse lymphoid cells. francis and meltzer [ ] have recently reported that hiv- virions and hiv- infected cells both induce interferon-~ production in monocytes through interaction between envelope gp and cell surface cd molecule and that induction of interferon-~ by hiv- does not require virus replication. interferonwas induced by (a) heat-inactivated hiv- , (b) virions from e cells, a cell line that releases noninfectious hiv- , (c) hiv-l-infected cells fixed in paraformaldehyde, and (d) t cell-tropic hiv- that binds to but does not infect monocytes, indicating a similarity to induction of interferon by paramyxovirus in mouse spleen cells. furthermore, capobianchi et al. [ a] have recently reported that recombinant glycoprotein is a potent interferon inducer. when mouse spleen cells are cocultured with bhk-sv cells for - h, and further incubated without bhk-sv cells, interferon is produced, indicating that viral glycoproteins and interferon induction short-period stimulation is sufficient for interferon triggering and the following process of interferon production progresses without further stimulation [ , ] . on the contrary, interferon-y-producing cells adhere closely to target antigen until interferon production begins. mouse spleen cells stimulated by virusinfected cells cannot produce interferon in the presence of either cytochalsin or colchicine [ ] . however, when cytochalasin or colchicine is added to culture fluid of mouse spleen cells or h, respectively, after mix-culture with bhk-sv cells, these drugs show no inhibitory effect [ ] . when mouse spleen cells are cocultured for h with bhk-sv cells in the presence of cytochalasin and then mouse spleen cells are further incubated without bhk-sv cells and cytochalasin, interferon production is not found, showing that interferon induction is not triggered in the presence of cytochalasin [ ] . on the other hand, bhk-sv cells show adsorption of spleen cells to their surfaces in the presence of cytochalasin [ ] . these findings show that cytochalasin does not inhibit the celt-to-cell contact between spleen cells and bhk-sv cells and therefore contacts alone of spleen cells to hn proteins are not sufficient, but active interactions between these membranes are necessary for triggering interferon production. intriguingly, neither cytochalasin nor colchicine suppresses interferon production of l cells stimulated with ndv [ ] . from these findings, early steps (triggering) of interferon production by lymphoid cells stimulated with viral glycoprotein progresses as follows: binding of viral glycoproteins to lymphoid cells --, cytochalasin sensitive step (microfilament-related step) ~ colchicine sensitive step (microtubulus-related step) -~ further steps unrequired for stimulation by viral glycoprotein. further identification of the signals transduced by the viral glycoproteins-cellular receptor interaction is an obvious future goal. the interferon induction system, in which attachment of virus glycoprotein to the cellular receptor on the cell surface triggers interferon induction in lymphoid cells, is similar to a system in which plant lectins such as concanavalin a (con-a) and phytohemagglutinin (pha) stimulate lymphocytes and consequently induce interferon. con-a and pha are mitogenic lectins that have an ability to stimulate dna synthesis and to induce interferon- , [ , ] . however, glycoprotein of sendai virus shows no mitogenic activity under our experimental conditions, although its low mitogenicity is reported by other investigators [ ] . therefore, it is very likely that non-mitogenic lectins are able to induce interferon in mouse spleen cells. when twenty-two sorts of lectin are tested for interferon-inducing ability in mouse spleen cells, all the mitogenic lectins, con-a, succinylated-con-a (s-con-a), lens culinaris type a (lch-a), lch-b and poke weed mitogen (pwm), induced interferon- ,, and out of nonmitogenic lectins, wheat germ agglutinin (wga), ulex europeus ii (uea-ii), lotus tetragonolobus seed tectin, salanum tuberosame (sta), and bandeiraea simplicifolia ii (bs-ii) prove to be capable of inducing interferon-j [ ] . when wga, a non-mitogenic lectin, is administered intraperitoneally to mice, y. ito interferon is induced in the circulation [ ] . from these results, it is found that interferon inducing ability is not limited to mitogenic lectins. it is conceivable that virus glycoproteins induce interferon, one of the cytokines, through their interaction with the cellular receptors via lectin-like action. a lectin is a sugar-binding protein or glycoprotein of non-immune origin that agglutinates cells (most commonly erythrocytes) [ ] . because we think of a conception that virus glycoproteins have a lectin-like function, we try to arrange functions of viral glycoproteins from this point of view. significantly, the glycoproteins of parainfluenza and influenza viruses used mainly in the above studies bind to sialyl-oligosaccharides [ ] . since hirst [ ] discovered the hemagglutinating activity of influenza virus in , a large number of viruses have been found to have hemagglutinating activity and a large number of virus glycoproteins have proved to possess this activity. when cells are infected with a virus, viral hemagglutinin functions as a viral attachment factor and its binding to cellular receptors is the first step of infection. lectins were discovered in the form of hemagglutinin originating from plants and the most universal property of lectins is hemagglutinating activity [ ] . the finding that hemagglutinating activity is detected in a large number of virus glycoproteins indicates a certain similarity in the properties of virus glycoproteins and lectins. important components on cell surface membranes such as major histocompatibility complexes (mhc) act as lectin receptors. and important constituents of host cell membranes have been found to be also used as viral receptors. for example, sialyl-oligosaccharides are receptors for parainfluenza and influenza viruses [ ] , human membrane cofactor protein (cd ) for measles virus [ , ] , mhc class i for semliki forest virus (sfv) [ ] , mhc class ii for lactose dehydrogenase virus (ldh virus) [ ] , phosphatidylserine and phosphatidylinositol for vesicular stomatitis virus (vsv) [ ] , the cd for human immunodeficiency virus (hiv) [ , ] , the c d receptor cr for epstein-barr virus [ , , ] , acetylcholin receptor for rabies virus [ ] , the intercellular adhesion molecule- (icam- ) for the major subgroup of human rhinoviruses [ , , ] , another member of the immunoglobulin superfamily for poliovirus [ , - , a basic amino acid transporter for gibbon ape leukemia virus and feline leukemia virus [ , , , , ] , aminopeptidase n [ , - or a member of the carcinoembryonic antigen family of proteins for the coronavirus virus [ ] , a high-affinity laminin receptor for sindbis virus [ ] , ~ subunit of human vla- for echoviruses and [ ] , erythrocyte p antigen for b parvovirus viral glycoproteins and interferon induction [ ] , and cd (human aminopeptidase n) for cytomegalovirus [ ] . most of these virus receptors are involved in cell-cell adherence and in cell-cell recognition. therefore, it is highly probable that binding of viral (glyco)protein to the cellular receptor influences some cell functions. infection with viruses often results in shut-off of cellular macromolecular syntheses, although degrees of the suppression are varied. in some cases (for example, reovirus [ ] , vsv [ ] , mumps virus [ ] infection), attachment of virus glycoproteins to cellular receptors leads to suppression of cellular macromolecular synthesis. intriguingly, binding of hiv-gp to cd molecule suppresses various cellular functions [ , , ] . these findings show that inhibition of the cellular macromolecular syntheses is mediated through an interaction at the cell surface. in nowell [ ] found that when human lymphocytes were incubated with phytohemagglutinin (pha), a lectin originating from phaseolus vulgaris, lymphocytes transformed into lymphoblastoid cells. this mitogenic function is the most noteworthy property of lectins. recently, some virus gtycoproteins have been found to show a mitogenic effect, although the activity is rather weaker than that of plant lectins [ , , , , , , , , , ] . hn glycoprotein of sendai virus used in our studies shows weak mitogenic effect under some conditions [ ] . therefore, an interaction between viral virus glycoproteins and virus receptors of host cells at the cell surface can induce or enhance cellular macromolecular syntheses. the finding that some viral glycoproteins possess mitogenic activity is a basis of evidence that virus glycoprotein can be considered as a viral-tectin. as demonstrated above, viral glycoproteins (cell attachment proteins) have a viral lectin-like property. in other words, some viral glycoproteins are simply lectins, so it is not surprising that stimulation of lymphocytes with the viral lectin triggers production of interferon, one of the cytokines. interferon is induced by interaction between nk and tumor cells or by mixedlymphocyte culture. this interferon induction is also triggered by the membranemembrane interactions. recently, some cytokines such as tumor necrosis factor (tnf) and platelet derived growth factor (pdgf) have been found to induce interferon [ , ] . furthermore, interferon per se has been reported to induce interferon [ , ] . binding of the cytokines to the cellular receptors triggers interferon induction. these findings show that interferon triggering mechanisms unrelated to double-stranded rna function particularly in lymphoid cell systems. in this review, i show clearly that there are two interferon induction systems in virus infection: one is that viral-nucleic acid is involved in interferon induction, and the other is that viral glycoproteins trigger interferon induction by the membrane-membrane interaction via a lectin-like activity. some kinds of viral glycoproteins can be regarded as viral lectins and their attachment to the cellular receptors influences cellular functions, for instance, suppression or enhancement (induction) of cellular macromolecular synthesis and therefore stimulation of cellular gene expressions resulting in production of cellular factors including interferon. cell-to-cell interactions are mediated by receptorligand (counter-receptor) system found on the cell surface membrane, that is, a membrane-membrane interaction. most virus receptors are considered to be adhesion or adhesion-related molecules, and consequently interactions between lymphoid cells and virus-infected cells are similar to the cell to cell interactions mediated by adhesion molecules. judging from this point of view, the recently reported embryonic interferon (interferon-c ) [ , ] that is thought to be induced during differentiation and to play important roles in embryogenesis intrigues us. although interferon was first discovered as an antiviral substance [ ] , it has since been shown to affect a wide variety of cellular functions such as cell-multiplication-inhibitory activity, immune regulatory function, and the enhancing activity of multiple cellular genes. therefore, it is inferred that interferon may be originally regulatory-molecules that are induced by interaction between different cells and function in differentiation and maturation. when we first discovered an interferon induction system triggered by virus glycoprotein in lymphoid cells [ ] , the meaning of this phenomenon remained obscure. however, when interferons and the interferon induction are interpreted as described here, the interferon induction triggered by viral glycoprotein can be categorized within general biological phenomena, though it appears puzzling at first sight. a putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules mitogenicity of influenza hemagglutinin glycoproteins and influenza viruses bearing h -hemagglutinin induction of interferon in chick cells by temperature-sensitive mutants of sindbis virus infection by echoviruses and depends on the ~ subunit of human vla- viral glycoproteins and interferon induction erythrocyte p antigen: cellular receptor for b parvovirus interferon production and viral ribonucleic acid synthesis in chick embryo cells recombinant glycoprotein of human immunodeficiency virus is a potent interferon inducer synthetic peptides homologus to hiv transmembrane glycoprotein suppresses normal human lymphocyte blastogenic response high homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and alpha-interferons relationship between the ribonucleic acid synthesizing capacity of ultraviolet-irradiated newcastle disease virus and its ability to induce interferon the cd (t ) antigen is an essential component of the receptor for the aids retrovirus aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev richardson cd (t ) the human cd molecule is a receptor for measles virus (edomonston strain) cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv epstein-barr virus receptor of human b lymphocytes is the c d receptor cr induction of ifn-a by hiv- in monocyte-enriched pbmc requires gp -cd interaction but not virus replication stimulation of interferon production in human lymphocytes by mitogens virus rna synthesis by ultraviolet-irradiated newcastle disease virus and interferon production direct activation of resting t lymphocytes by human t-lymphotropic virus type i what should be called a lectin? mcsharry jj (t ) the glycoprotein isolated from vesicular stomatitis virus is mitogenic for mouse b lymphocytes mitogenic activity of sindbis virus and it's isolated gtycoproteins the major human rhinovirus receptor is icam-t human (hla-a and hla-b) and murine (h- k and h- d) histocompatibility antigens are cell surface receptors for semliki forest virus adsorption of influenza hemagglutinin and virus by red blood cells mouse ia antigens are receptors for lactate dehydrogenase virus interferon-like substance of ovine trophoblast protein secreted by embryonic trophectoderm virus interference: i the interferon production of interferon-like substance by mouse spleen ceils through contact with bhk cells persistently infected with hvj interferon induction in mice by bhk cells persistently infected with hvj active component of hvj (sendal virus) for interferon induction in mice mechanism of interferon induction in mouse spleen cells stimulated with hvj component(s) of sendai virus that can induce interferon in mouse spleen cells the effects of cytochalasin and colchicine on interferon production suppression of interferon production in mouse spleen cells by cytochalasin d interferonproducing capacity of germfree mice interferon induction in mouse spleen cells by mitogenic and nonmitogenic lectins interferon production in mouse spleen cells and mouse fibroblasts (l cells) stimulated by various strains of newcastle disease virus hn proteins of human parainfluenza type a virus expressed in cell lines transfected with a cloned cdna have an ability to induce interferon in mouse spleen cells transport of cationic amino acids by the mouse ecotropic retrovirus receptor in vitro mitogenic stimulation of murine spleen cells by herpes simplex virus sendai virus glycoproteins are t cell-dependent b cell mitogens t-lymphocyte t molecule behaves as the receptor for human retrovirus lav viral glycoproteins and interferon induction a cytokine network in human diploid fibroblasts: interaction of -interferons, tumor necrosis factor, platelet-derived growth factor, and interleuken- the poliovirus receptor protein is produced both as membrane-bound and secreted forms lymphocyte activation by hiv- envelope glycoprotein is the acetylcholine receptor a rabies virus receptor? viral events necessary for the induction of interferon in chick embryo cells htlv-iii large envelope protein (gp ) suppresses pha-induced lymphocyte blastogenesis defective interfering particles with covalently linked [ + / -] rna induce interferon characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic ph biological properties of the vsv glycoprotein effects of the isolated glycoprotein on host macromolecular synthesis production of interferon by ultraviolet radiation inactivated newcastle disease virus cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily regulated expression of a gene encoding a nuclear factor, irf- that specifically binds to ifn-~ gene regulatory elements the interaction of herpes simplex virus with murine lymphocytes i mitogenic properties of herpes simplex virus temperature-sensitive phenomenon of viral maturation observed in bhk cells persistently infected with hvj human membrane cofactor protein (cd ) acts as a cellular receptor for measles virus identification and characterization of the epstein-barr virus receptor on human b lymphocytes and its relationship to the c d complement receptor (cr ) phytohemagglutinin: an initiator of mitosis in cultures of normal human leukocytes synergistic interactions ofinterleuken interferon-g, tumor necrosis factor in terminally differentiating a mouse myeloid leukemia cell line (m ) viral glycoproteins and interferon induction restoration of specific myxovirus receptors to asialoerythrocytes by incorporation of sialic acid with pure sialytransferases direct role of viral hemagglutinin in b-cell mitogenesis by influenza viruses reovirus inhibition of cellular dna synthesis: role of the s gene a temperature-sensitive event in interferon production cd (human aminopeptidase n) mediated human cytomegalovirus infection a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses feline leukemia virus subgroup b uses the same cell surface receptor as gibbon ape leukemia virus cdna cloning reveals that the major group rhinovirus receptor on hela cells is intracellular adhesion molecule virus receptors as permeases cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter high-affinity laminin receptor is a receptor for sindbis virus in mammalian cells hiv- gp mediated immune suppression and lymphocyte destruction in the absence of viral infection inhibition of host cellular ribonucleic acid synthesis by gtycoprotein of mumps virus human aminopeptidase n is a receptor for human coronavirus e two color immunofluorescence studies on the association between ebv receptor and complement receptor on the surface of lymphoid cell lines received october , key: cord- -wpeac lk authors: hu, hui; jung, kwonil; vlasova, anastasia n.; saif, linda j. title: experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain oh-fd date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: wpeac lk porcine deltacoronavirus (pdcov) is a novel enteropathogenic coronavirus in pigs. we have isolated and passaged the pdcov strain oh-fd in an llc porcine kidney (llc-pk) cell line. our study investigated the pathogenicity of the tissue-culture-grown pdcov (tc-pdcov) oh-fd at cell passages , and in llc-pk cells, in eight -day-old gnotobiotic (gn) pigs. pigs (n = ) were euthanized for pathologic examination at post-inoculation day (pid) , and the remainder were monitored for clinical signs, virus shedding, and serum antibody responses until pid . all inoculated pigs developed watery diarrhea and/or vomiting at pid - and shed the highest amount of viral rna in feces at pid - , accompanied by severe atrophic enteritis. they developed high titers of pdcov-specific igg/iga and virus-neutralizing antibodies in serum at pid - . histologic lesions were limited to the villous epithelium of the jejunum and ileum at pid . two inoculated pigs tested at pid - had small to moderate numbers of pdcov antigen-positive cells in the intestinal lamina propria and mesenteric lymph nodes, but not in enterocytes. an analysis of full-length s and n genes of tc- and gn-pig-passaged oh-fd revealed a high genetic stability in cell culture and pigs. tc-pdcov oh-fd (cell passages , and ) was enteropathogenic, and the pathogenicity was similar to that of the original field virus. the tc-pdcov oh-fd will be useful for further pathogenesis studies and for evaluating if higher-level cell-culture passaged virus becomes attenuated for vaccine development. porcine deltacoronavirus (pdcov), belonging to the genus deltacoronavirus of the family coronaviridae [ ] , is a porcine diarrheal pathogen that was initially reported in hong kong in [ ] and emerged in the united states in [ , , ] . infected herds had clinical signs of acute watery diarrhea in sows and nursing pigs, but mortality was reported only in nursing pigs [ , , ] . pdcov disease was reportedly milder than that caused by porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) in seronegative herds [ , ] . molecular surveillance studies indicated that pdcov coinfections are common, especially with rotavirus group c (rota c) and pedv [ , ] . to date, outbreaks have been documented in more than states in the united states and in canada, china, south korea, and thailand [ , , , ] . a report from jiangxi province, china, indicated that the prevalence of monoinfection by pdcov was high ( . %), and coinfection by pdcov and pedv was . % in diarrheic pigs [ ] . several investigators have described the molecular detection and genetic analysis of pdcovs [ , , , , ] . other studies confirmed that pdcov is enteropathogenic in young pigs [ , , ] . our previous study showed severe watery diarrhea and/or vomiting and severe atrophic enteritis in -day-old gnotobiotic (gn) pigs inoculated with the pdcov oh-fd original fecal sample [ ] . another study reported that a cell culture isolate of pdcov, usa/il/ , caused clinical disease in conventional -day-old piglets, accompanied by macroscopic and microscopic lesions in the small intestine [ ] . it was also reported that a cell culture isolate of pdcov, strain michigan/ / (mi), caused severe gastrointestinal diseases in gn and conventional -day-old piglets [ ] . in our recent publication, we focused on the isolation of pdcov in cell culture. the pdcov oh-fd strain originated from intestinal contents from a diarrheic pig, and it was successfully isolated in two cell lines of swine origin, swine testicular (st) and llc porcine kidney (llc-pk) cells. it was serially passaged in llc-pk cells [ ] . the aims of our current study were to investigate i) the pathogenicity of the tissue-culture-grown pdcov (tc-pdcov) strain oh-fd at passage (p ), p , and p on llc-pk cells in -day-old gn pigs compared with that of the wild-type parent virus [ ] ; ii) fecal pdcov shedding, viremia, and pathology tested at post-inoculation day (pid) to - or pid ; iii) possible attenuation of tc-pdcov oh-fd p , the highest passage in cell culture among the passages tested; iv) serum-pdcov-specific igg and iga and virus neutralization (vn) antibody titers and any differences in the titers among oh-fd p , p , and p -inoculated pigs; and v) the genetic stability of tc-and gn-pig-passaged pdcov oh-fd p , p , and p by analysis of the complete spike (s) and nucleocapsid (n) gene sequences. the llc-pk cell line (atcc cl- ) was used to serially passage the pdcov oh-fd strain, which was isolated from the intestinal contents collected from a diarrheic pig from ohio [ ] . the tc-pdcov oh-fd p , p , and p strains were propagated on llc-pk cells and harvested when the cpe was [ %. virus titer was determined by using taqman real-time quantitative rt-pcr (qrt-pcr) and by plaque assay as described previously [ ] . the propagated pdcov culture was confirmed negative for other swine enteric viruses, including tgev, pedv, rota a to rota c, and caliciviruses (noroviruses, sapoviruses, and st-valerien-like viruses), by using rt-pcr as reported previously [ , , , , ] . the tc-pdcov strains oh-fd p , p , and p in llc-pk cells were used for inoculation of gn pigs. all animal studies were performed as approved by the institutional animal care and use committee (iacuc) of the ohio state university. the gn pigs were delivered aseptically by hysterectomy from specific-pathogen-free sows. prior to inoculation, pigs were confirmed negative for the major swine enteric viruses by testing of fecal samples using rt-pcr. six -day-old gn pigs (n = for each passage) were inoculated orally with tc-pdcov oh-fd p ( . log genomic equivalents [ge] per pig), p ( . log ge per pig), and p ( . log ge per pig). two pigs of the same age were used as negative controls. clinical signs were monitored at least twice daily [ ] . for each virus passage group, one of the two pigs was euthanized for pathologic examination at - h after onset of clinical signs [ ] . large-intestinal contents (lic) and small-intestinal contents (sic) were collected and tested by qrt-pcr for pdcov and by rt-pcr for other enteric viruses. the remaining pigs were monitored for longer-term clinical signs, fecal virus rna shedding, and virus-specific serum antibodies until pid . diarrhea was assessed by scoring fecal consistency as follows: = solid; = pasty; = semi-liquid; = liquid, with scores of or more considered diarrheic [ ] . rectal swabs were collected daily from each pig throughout the experiment. viral rna was extracted from the intestinal content suspensions, rectal swab fluids, serum, and cell culture samples by using a magmax- virus isolation kit (ambion by life technologies, usa) and the rna extraction robot magmax express (applied biosystems, foster city, ca) according to the manufacturer's instructions. viral rna titers were determined by qrt-pcr (qiagen, valencia, ca, usa) as reported [ ] . the detection limit of the qrt-pcr was ges/reaction, which corresponded to . and . log ge/ml of pdcov in fecal and serum samples, respectively. small (duodenum to ileum) and large (cecum and colon) intestinal tissues and other major organs (lung, liver, heart, kidney, spleen, and lymph nodes) were examined grossly and histologically and fixed in % neutral formalin for - days at room temperature [ ] . they were embedded, sectioned, and stained with mayer's hematoxylin and eosin (h&e) for light microscopy examination. mean jejunal ratios of villous height and crypt depth (vh:cd) were measured by using pax-it software (paxcam, villa park, il, usa) as described previously [ ] . the prepared tissues were tested by immunofluorescence (if) staining for the detection of pdcov antigen using a swine hyperimmune antiserum (oh-dc ) against pdcov [ , ] . tissues from age-matched control pigs were tested for histological comparisons and as a negative control for if. neutralizing antibodies in the sera of pdcov-inoculated pigs were tested by prnt using st cells in -well plates. the swine hyperimmune antiserum oh-dc and the mock-infected pig's serum were used as positive and negative serum controls, respectively. all sera were inactivated at °c for min prior to testing and prepared as -fold serial dilutions in mem. each sample was mixed with an equal volume of tc-pdcov oh-fd ( plaqueforming units [pfu]/ ll in each well) and then incubated at °c for min. the growth medium for st cells ( % confluent) was replaced with maintenance medium (without trypsin) as described [ ] . following h of incubation at °c, cells were washed once with maintenance medium. the virus-serum mixtures were added to the st cells, and the virus control wells were inoculated with ll of pdcov oh-fd ( pfu/well). the negative control wells received the maintenance medium only. after adsorption for h at °c in an atmosphere of % co , the inoculum was removed, and cells were washed twice with dulbecco's phosphate-buffered saline (dpbs) without mg ? and ca ? (sigma, st. louis, mo). two ml of overlay medium ( mem [gibco, usa] containing % antibiotic-antimycotic, hepes, neaa, and % pancreatin [sigma, usa] and an equal volume of % seaplaque agarose [lonza, rockland, me]) was added to each well. the plates were incubated for days in an incubator ( °c, % co ), and the plates were then stained with . % neutral red (sigma) for h at °c. the plaques were counted, and the prnt was determined as the reciprocal of the highest serum dilution that neutralized % of the plaques as compared to that in the virus-only control wells. an indirect enzyme-linked immunosorbent assay (elisa) was used to detect pdcov-specific iga and igg antibodies in the sera of pdcov-inoculated pigs. the protein concentration in the tc-pdcov oh-fd was determined by a bradford protein assay using bovine serum albumin (bsa) as a standard [ ] . the -well maxisorp plates (nunc, san diego, ca) were coated with the tc-pdcov oh-fd ( ng/well) in coating buffer ( mm na -co , mm nahco , ph . ) at °c overnight. the mock-infected llc-pk cells were processed in parallel as above, and a similar concentration of antigen was used to coat the negative control wells. the wells were washed with phosphate-buffered saline (pbs) containing . % tween (pbst) and then blocked with % non-fat dry milk in pbs. one hundred ll of serially diluted serum ( fold dilutions) was added to the antigen-coated or mock antigen-coated wells and incubated for h at °c. the plates were washed with pbst, and ll of horseradish peroxidase (hrp)-conjugated anti-pig iga (abd serotec, raleigh, nc, usa) was added to each well at a dilution of : , and incubated at °c for h. after washing with pbst, , -azino-bis ( -ethylbenzthiazoline- -sulfonic acid) (abts) substrate (kpl, gaithersburg, md, usa) was added. finally, the plates were read at an absorbance of nm using a spectramax elisa reader (molecular devices, union city, ca). for igg detection, plates were prepared as above, the -fold serially diluted sera were incubated for h, and the plates were washed with pbst. biotinylated anti-pig igg (kpl, gaithersburg, md, usa) was added at ll per well at a dilution of : , and incubated for h at °c. the plates were washed, and peroxidase-conjugated streptavidin ( : , ) (roche, indianapolis, in, usa) was added and incubated at room temperature for h. plates were washed again, and abts substrate was added. finally, the plates were read at an absorbance of nm by using a spectramax elisa reader. six controls were included: a coating-buffer-only group; a mock llc-pk cell lysis diluted with coating buffer group; a negative-serum (serum obtained from the mockinfected pig of the same age and serum samples collected pre-inoculation from all pigs) group; a positive-serum (hyperimmune pdcov serum oh-dc ) group; a secondary-antibody-free group; and a blocking buffer group. mock-antigen-coated wells did not show a background reaction. positive samples were those with an absorbance equal to or greater than the cutoff that was determined as the average value of the absorbance of negative control samples plus three times the standard deviation. the antibody titers were calculated and expressed as the reciprocal of the highest serum dilution that was positive for pdcov iga or igg antibodies. the lic of gn pigs inoculated with tc-pdcov oh-fd p , p , and p were designated gn pig oh-fd p (dc ), gn pig oh-fd p (dc ), and gn pig oh-fd p (dc ), respectively. the complete s and n genes of tc-pdcov oh-fd p and p as well as the gn-pig-passaged tc-pdcov oh-fd p , p , and p in the lic were amplified, cloned and sequenced, and the sequence data were assembled and subjected to phylogenetic analysis according to our previously reported methods [ ] . the complete s and n genes of tc-pdcov oh-fd p , p , gn pig oh-fd p (dc ), gn pig oh-fd p (dc ), and gn pig oh-fd p (dc ) were deposited in genbank under accession numbers kt , kt , and kt to kt . clinical observations and pdcov rna titers in the feces and sera of tc-pdcov oh-fd inoculated gn pigs all gn pigs that were orally inoculated with tc-pdcov oh-fd developed typical clinical disease, characterized by acute and severe watery diarrhea, vomiting, and mild dehydration. clinical signs developed at pid - , regardless of the cell culture passage level of the viral strains tested ( table ). the pigs monitored for long-term clinical signs showed moderate to severe diarrhea for about - days (fig. a ). all inoculated pigs exhibited an onset of clinical disease that was similar or later by day than that after inoculation of gn pigs with the original pdcov oh-fd (table ) [ ] . the negative control pigs showed no clinical signs during the study period. the rt-pcr tests showed no contamination of the diarrheic fecal samples from the gn-pig-passaged tc-pdcov with other detectable enteric viruses (pedv, tgev, rota a/c, and caliciviruses). the kinetics of fecal rna shedding are shown in table and fig. b . as reported [ ] , the original pdcov oh-fd -inoculated pigs showed clinical signs at about pid , and fecal viral rna was detected on pid , whereas for the tc-pdcov oh-fd -inoculated pigs, fecal viral rna was detected later on pid , and the pigs showed clinical signs at pid to . fecal viral rna peaked on pid to and then decreased gradually thereafter. similar to the original pdcov oh-fd inoculation, fecal virus shedding in the tc-pdcov oh-fd p -or p -inoculated pigs was still detectable at pid and , and then negative at pid and , respectively. the tc-pdcov oh-fd p -inoculated pig had pdcov-rna-positive feces until pid . the negative control pigs did not shed detectable pdcov viral rna in the feces throughout the experiment. pdcov rna was detected in the inoculated pigs' sera by qrt-pcr (table ) . only the pid sera of the tc-pdcov oh-fd -inoculated pigs were pdcov positive, but the viral rna titer was very low, with a range of . - . log ge/ml. the original pdcov oh-fd -inoculated pigs were negative for pdcov rna in the sera [ ] . no infected pigs had detectable viral rna prior to inoculation in the prebled serum samples. two duodenal, - proximal, middle, and distal jejunal, two ileal, and two cecal/colonic tissues and other major organs were collected and tested for all pigs. by macroscopic examination, inoculated gn pigs (p ), (p ) and (p ) tested at pid exhibited extensive 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. the other inoculated pigs, pigs (p ), (p ) and (p ), tested at pid - or , and negative control pigs and did not show gross lesions. histologic lesions were limited to villous epithelial cells, but not the crypts, of the small and large intestines, and mainly, the jejunum and ileum, as tested at the early stage of infection at pid , but not at pid - or ( table ) . tc-pdcov oh-fd p -inoculated pig and oh-fd p inoculated pig tested at pid ( days of age) had diffuse, severe villous atrophy in the jejunum (mean vh:cd ratio ± sdm, . ± . for pig and . ± . for pig ) and ileum, with frequent fusion of atrophied villi and diffuse, mild cytoplasmic vacuolation of enterocytes located at the upper half to the tip of the severely atrophied villi. other major histologic changes in pigs (p ) and (p ) included a diffuse, mild vacuolation of superficial cecal and colonic epithelial cells. on the other hand, tc-pdcov oh-fd p -inoculated pig tested at pid ( days of age) and oh-fd p -inoculated pig tested at pid ( days of age) had no histologic lesions or evident villous atrophy in the jejunum (mean vh:cd ratio ± sdm, . ± . for pig and . ± . for pig ) and ileum, similar to the negative control pig euthanized at days of age (mean jejunal vh:cd ratio ± sdm, . ± . ) or negative control pig euthanized at days of age (mean jejunal vh:cd ratio ± sdm, . ± . ). no villous atrophy or histologic lesions were evident in the remainder of the small intestine, duodenum, and other organs of the inoculated pigs - and negative control pig at the time points tested. tc-pdcov oh-fd p -inoculated pig tested at pid ( days of age) had diffuse, moderate villous atrophy in the jejunum (mean vh:cd ratio ± sdm, . ± . ) and ileum, with frequent fusion of adjoining atrophied villi and diffuse, mild to moderate cytoplasmic vacuolation of enterocytes lining the epithelium of atrophied jejunal and ileal villi ( fig. a) . pig had no histologic lesions in the large intestine. the tc-pdcov oh-fd p -inoculated pig tested at pid ( days of age) had no histologic lesions or villous atrophy in the jejunum (mean vh:cd ratio ± sdm, . ± . ) and ileum compared to negative control pig euthanized at days of age (mean jejunal vh:cd ratio ± sdm, . ± . ) ( table ). no villous atrophy or other histologic lesions were evident in the remainder of the small intestine, duodenum, and other organs of inoculated pigs and and negative control pig . as determined using formalin-fixed, paraffin-embedded tissues, if-stained cells were observed mainly in the atrophied villous epithelium of the small intestine (fig. b) , proximal jejunum to ileum, and occasionally, in the duodenum and cecum/colon of pigs (p ), (p ) and (p ) tested at pid (table ) , as reported previously [ , ] . the fluorescence was confined to the cytoplasm of the villous epithelial cells. pigs (p ), (p ) and (p ) also had a few if-stained cells in the mesenteric lymph nodes. no other internal organs of the infected pigs tested at pid showed ifpositive staining. when tested at pid - , no if-stained cells were detected in the villous epithelium of the small or large intestine of inoculated pigs (p ) and (p ). however, small numbers of if-stained cells were observed in the lamina propria of the villi and crypts of the proximal jejunum to ileum (fig. c) , and to a lesser extent, in duodenum and cecum/colon. moderate numbers of if-stained cells were also detected in peyer's patches (fig. c ). pigs (p ) and (p ) also had small to moderate numbers of ifstained cells in the mesenteric lymph nodes (fig. d) ; however, no other internal organs of infected pigs showed if-positive staining. no if-stained cells were detected in the negative control pigs and ( fig. e and f) . serum samples were collected at pid , , , and / to test for pdcov-specific igg and iga antibodies by indirect elisa (table ). all inoculated pigs developed pdcov- oh-fd -p pdcov-specific iga antibodies in sera were also determined by indirect elisa ( table ) . titers of iga antibodies were detected at pid in all the inoculated pigs' sera, with a lower iga antibody titer of for the tc-pdcov oh-fd p and p groups, and higher ( ) for the tc-pdcov oh-fd p and the original inoculated pigs. the highest iga antibody titers were detected at pid / . tc-pdcov oh-fd p elicited the highest iga antibody titer ( ) at pid , whereas tc-pdcov oh- to investigate if pdcov-neutralizing antibodies were induced, a plaque reduction pdcov neutralization assay (prnt ) was performed. serum samples at pid , , , and / were tested for pdcov neutralizing antibody titers (table ). all inoculated pigs tested at the later stage of infection (pid / ) exhibited high pdcovneutralizing titers. regardless of the cell culture passage number of the virus strains used, serum virus neutralization (vn) antibodies were first detected in all of the original and tc-pdcov oh-fd -inoculated pigs at pid ( - ), and thereafter, the titers increased gradually and peaked at pid / . tc-pdcov strain oh-fd p induced the highest vn titer at the time points tested (pid , and ), which was times higher than that of the other groups. there were no differences in vn antibody titers among the gn pigs inoculated with the tc-pdcov strains oh-fd p and p , and original pdcov oh-fd . no neutralizing antibodies were observed for the control pig serum. to examine if genetic changes occurred in the major structural genes of pdcov oh-fd during serial passages in llc-pk cells and gn pigs, the complete s and n genes of tc-and gn-pig-passaged pdcov oh-fd p , p and p were sequenced and compared with those of the original pdcov oh-fd . the s genes of gn-pig-passaged pdcov oh-fd p , p and p strains shared . % to % nucleotide sequence identity, and they shared . % to % nucleotide sequence identity with the original pdcov oh-fd and tc-pdcov oh-fd (p , p , p and p ). all of the pdcov oh-fd -related s genes shared . % to % nucleotide sequence identity with other pdcov strains available in genbank (fig. ) . when comparing the tc-pdcov oh-fd p strain with the original oh-fd strain, only one nucleotide change was found in the s gene at position nt (nucleotides and amino acids are numbered according to the s gene of the pdcov oh-fd sequence [genbank accession no. kp ]), which resulted in an amino acid (aa) change (val changed to phe at position aa). this was subsequently found in the following sequenced tc-pdcov oh-fd passages (p and p ) [ ] (table ). the tc-pdcov oh-fd p (passage in llc-pk cells) and p had five nucleotide changes (at positions , , , , and nt) in the s gene when compared to the original oh-fd strain, which also resulted in amino acid changes [ ] . in the current study, the s genes of tc-pdcov oh-fd p , pig-passaged oh-fd p and p had the same changes (table ) . when comparing the s gene of tc-pdcov oh-fd p with the original virus and tc-pdcov oh-fd (p and p ), one nucleotide change was found in the s gene at position nt, which resulted in an aa change (asn changed to asp at position aa). the gn-pig passaged tc-pdcov oh-fd p (dc ) had the same change in this position (table ). only one nucleotide change was found in the s gene of pig-passaged pdcov (oh-fd p [dc ] and oh-fd p [dc ]) when compared with the corresponding tc-pdcov oh-fd p and p at position , nt of the s gene. this nucleotide change resulted in an amino acid change (ala changed to thr at table detection of pdcov-specific iga, igg and neutralizing antibody titers in the sera of gn pigs inoculated with original and tissueculture-grown pdcov oh-fd * pid, postinoculation days; vn, virus neutralization # see reference [ ] . pig was inoculated with the original pdcov oh-fd as reported note: pdcov-specific igg and iga antibodies in serum were determined by indirect elisa using cell culture pdcov oh-fd as the coating antigen. the neutralizing antibody titers were tested by the % plaque reduction neutralization test (prnt ). prnt was determined as the reciprocal of the highest serum dilution that neutralized % of the plaques as compared to that in the virus-only control wells. the iga, igg and vn antibody titers of hyperimmune pdcov serum oh-dc (positive control serum) were , and , respectively experimental infection with porcine deltacoronavirus position ) ( table ). phylogenetic analysis of the s genes showed that all of the us pdcov strains (including the oh-fd strains) clustered in one group, which had a more distant relationship to the hong kong pdcov strains (hku - and hku - ) and the mainland china strains. furthermore, all pdcov oh-fd strains clustered into a subclade, while other us pdcov strains were clustered separately (fig. ) . the n genes of the pig-passaged and the corresponding tc-pdcov oh-fd shared % nucleotide sequence identity and were also % identical to the original pdcov oh-fd n gene. they shared . % to . % nucleotide sequence identity with the other pdcov strains available in genbank. phylogenetic analysis of the n genes of the pigpassaged and corresponding tc-pdcov oh-fd revealed that they belonged to the same group (fig. ) . our study demonstrated similar clinical diseases and pathologic lesions in all -day-old gn pigs inoculated with the tc-pdcov oh-fd p , p , and p strains when compared with previous observations in gn pigs of the same age inoculated orally with the original field pdcov oh-fd [ ] . however, the following differences were identified: i) earlier onset of clinical signs and fecal virus shedding by pid in original virus-inoculated pigs (table ) [ ] ; ii) frequent detection ( / ; %) of transient viremia (viral rna) in serum in tc-pdcov oh-fd -inoculated pigs on pid , while no viremia was detected in the original virus-inoculated pigs at pid and other time points (table ) [ ] ; and iii) in addition to pdcov antigen-positive enterocytes in the small and large intestines at the early stage of infection (fig. b) , a few to moderate numbers of pdcov-antigen-positive cells were also detected by if in the intestinal lamina propria and mesenteric lymph nodes of the tc-pdcov oh-fd - table ). in this study, no attenuation of the tc-pdcov oh-fd p strain (cell passage ) in pigs was evident, since severe clinical disease and lesions were reproduced in the two inoculated gn pigs. further studies are needed to investigate if higher levels of cellculture passaged tc-pdcov oh-fd are attenuated. in our study, -day-old gn pigs inoculated with . - . log ge (& log pfu per pig [ ] ) of tc-pdcov oh-fd p , p , and p all ( / , %) showed severe diarrhea and/or vomiting at pid , which coincided with the first detection of viral rna in feces ( fig. b and table ). fecal pdcov shedding titers peaked at pid to ( fig. b and table ). another study using conventional -day-old pigs and a cell-culture-adapted pdcov usa/il/ strain (passage ) reported onset of mild diarrhea (soft feces) at pid in five pigs that were orally inoculated with tcid of the virus, which was later than or coincided with the first detection of viral rna in feces at pid ( / pigs tested) or ( / pigs tested) [ ] . in another study of a cell-culture-grown pdcov michigan/ / strain and an ohio wild-type field cvm strain using -day-old conventional or gn pigs, moderate to severe diarrhea occurred in pigs orally inoculated with genomic rna copies or pfu/pig of each strain at pid - . the gn pigs inoculated with the cvm field strain exhibited more-severe clinical disease or histologic lesions compared to gn or conventional pigs inoculated with the cell-culture-adapted michigan/ / strain (cell passage ) [ ] . these discrepant observations may be due to: i) differences in the pdcov strains used, inoculation doses, and cell passage level; ii) conventional vs. gn pigs used and different ages of pigs used; and iii) selective pig euthanization that could influence fecal virus shedding titers in the remaining pigs at the later stages of infection. in our study, we detected pdcov rna in the feces of tc-pdcov oh-fd p or p -inoculated gn pigs collected until pid - , indicating prolonged fecal virus rna shedding in pdcov-infected pigs. however, a shorter duration of fecal viral rna shedding by - days was noted in a tc-pdcov oh-fd p -inoculated pig that shed until pid (fig. b) . since the pig exhibited moderate to severe clinical signs and gross and histologic lesions (intestinal villous atrophy) and high serum antibody titers to pdcov, based on a single pig, it is unclear if the shorter duration of fecal rna virus shedding is related to any reduced virulence of tc-pdcov oh-fd p . higher passage levels of the tc-pdcov oh-fd strain should be tested for attenuation in additional pigs in a future study. in addition, low pdcov rna titers were detected in sera collected in the acute stage from the tc-pdcov oh-fd inoculated pigs at pid , which coincided with previous studies, showing frequent detection of table nucleotide and amino acid changes in the s gene/protein of pdcov oh-fd passaged in cell culture and in gn pigs posiƟon* virus * nucleotides and amino acids are numbered according to the s gene of the pdcov oh-fd sequence (genbank accession no. kp ) [ ] ** nt, nucleotide; aa, amino acid # see reference [ ] pdcov rna in serum collected at early stages of infection [ , ] , although sera from the parental pdcov oh-fd inoculated pigs were pdcov rna negative at pids - [ ] . in the previous study, the infectious titer of the wildtype pdcov in the fecal sample was unknown because it does not grow in tissue culture. however, the viral rna titer of the wild-type pdcov oh-fd was determined by qrt-pcr. three -day-old gn pigs were inoculated orally with . log ge of the virus [ ] , which was approximately log ge lower than that of the inoculum of tc-pdcov oh-fd used in the current study. based on titer differences in the virus inocula, we speculate that the lower titer of the inoculation dose might be an explanation for lack of detection of viremia (viral rna) in the wild-type pdcov oh-fd inoculated pigs. relative to pdcov infection, higher titers of pedv rna in serum were detected during the acute stage of infection [ , ] . further studies are needed to determine whether pdcov present in serum remains infectious in pigs. the severity of clinical disease and histologic lesions (intestinal villous atrophy) in all tc-pdcov oh-fd p , p , and p -inoculated gn pigs was similar to that in gn pigs inoculated with the original field pdcov oh-fd in our previous study conducted under similar experimental conditions [ ] . at the early stage of infection (pid ), the majority of pdcov antigen-positive cells were in intestinal villous epithelial cells in jejunum and ileum. a few macrophage-like cells located in the lamina propria and peyer's patches of the jejunum and ileum were also positive for pdcov antigen by if. in contrast, after recovery at pid - , when no fecal virus rna shedding or clinical disease was detected, moderate to large numbers of pdcovantigen-positive cells were found by if in the intestinal lamina propria, peyer's patches and mesenteric lymph nodes, in what appeared to be macrophage-like cells. because there was no detectable fecal virus rna shedding at this time, pdcov antigens on the surface or within the cells may not indicate replicating virus, but possibly antigen that was taken up. in pedv-infected pigs, serum pedv-specific antibody was first detected at about pid to [ , ] . there is no information on the development of pdcov-specific antibodies in serum of pdcov-infected pigs, and to our knowledge this is the first report of development of an assay for pdcov serology. in our study, -day-old inoculated gn pigs had detectable serum igg/iga antibodies at pid and vn antibodies at pid (table ) . thereafter, pdcov-specific igg, iga and vn antibody titers increased and remained high at the end of the experiment at pid / , when the pigs were fully recovered from clinical disease and fecal virus rna shedding. compared to tc-pdcov oh-fd p -or p -inoculated pigs, the tc-pdcov oh-fd p -inoculated pig had higher serum pdcov-specific vn antibody titers at the time points tested. because of the limited number of pigs tested, it is unclear whether these observations were related to the shorter duration (by days) of fecal shedding of virus rna. covs have high mutation rates and can easily undergo recombination and deletion events, leading to altered tissue tropism, transmission routes, and host specificity [ ] . in our study, a comparative analysis of the sequenced s genes of tc-and gn-pig-passaged tc-pdcov oh-fd showed that only one nucleotide change occurred in the s gene of the gn-pig-passaged tc-pdcov oh-fd p (dc ) and p (dc ) when compared with the corresponding tc-pdcov oh-fd p and p . because tc-pdcov oh-fd p or p -inoculated pigs showed severe clinical disease and histologic lesions, the single nucleotide change likely does not reflect changes related to the pathogenicity of tc-pdcov oh-fd . however, a nucleotide change in the tc-pdcov oh-fd p and five subsequent nucleotide changes in the tc-pdcov oh-fd p and the conservation of these changes in passages (p and p ) might reflect changes related to adaptation of pdcov to cell culture. in conclusion, our study reproduced and confirmed the enteropathogenicity of tc-pdcov oh-fd p , p and p strains. the virulence appeared to be similar to that of the original field pdcov oh-fd strain, as evident by severe diarrhea/vomiting, atrophic enteritis, and high levels of serum igg, iga and vn antibodies in the gn pigs. these findings suggest that the tc-pdcov oh-fd strains tested in this study will be useful for pdcov pathogenesis studies and evaluation of higher-cell-culture passaged tc-pdcov oh-fd strains to verify attenuation and vaccine potential. ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. conflict of interest 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. molecular detection and genetic characterization of kobuviruses and astroviruses in experimental infection with porcine deltacoronavirus asymptomatic local pigs in east africa prevalence and genetic heterogeneity of porcine group c rotaviruses in nursing and weaned piglets in ohio, usa and identification of a potential new vp genotype a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in -day-old neonatal piglets mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus porcine deltacoronavirus in mainland china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states the effects of transplacental porcine circovirus type infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets pathogenesis of giii. bovine norovirus, cv -oh/ /us strain in gnotobiotic calves pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs pathogenicity of porcine deltacoronavirus strains in gnotobiotic pigs porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs complete genome characterization of korean porcine deltacoronavirus strain kor/knu - 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hwq jfv authors: erles, k.; brownlie, j. title: investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: hwq jfv two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (cird). tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. additional samples were collected during outbreaks of cird. the swabs were examined by virus culture and pcr for canine parainfluenza virus, canine adenovirus, canine herpesvirus (chv) and canine respiratory coronavirus (crcov). furthermore the prevalence of antibodies to chv and crcov was determined. during this study cird was reported mainly in one of the two kennels investigated. in that kennel antibody responses to crcov indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. chv antibody responses were detected throughout the year. in the other kennel, which reported few cases of cird a high prevalence of antibodies to crcov was detected on entry but only sporadic seroconversions to crcov or chv. by pcr three dogs were found positive for crcov in one kennel whereas all pcr tests for other viruses were negative for both kennels. virus culture failed to detect any viruses in either kennel. canine infectious respiratory disease (cird) or "kennel cough" is a disease complex, which occurs frequently in densely housed dog populations especially after new dogs have been introduced. [ ] . the problem is well known in rehoming and boarding kennels however it is also an important disease of working dogs because it disrupts their training and can even result in temporary closure of kennels. many factors contribute to the aetiology of cird including viruses and bacteria as well as stress due to mixing and housing in an unfamiliar environment. vaccines are available for some of the microorganisms involved in the disease such as canine parainfluenza virus (cpiv), canine adenovirus type (cav- ) and the bacterium bordetella bronchiseptica. the vaccines have been shown to prevent disease upon experimental challenge with these agents [ ] nevertheless cird has been reported in many kennels that are using regular vaccination. this may suggest that other microorganisms are present in this disease complex. canine herpesvirus (chv) has been detected in dogs with cird [ ] the importance of this infection in the pathogenesis of the disease however has not yet been determined. in other species such as cats, horses and cattle the involvement of herpesviruses in respiratory disease is well established [ , , ] . chv has been shown to be able to replicate in the respiratory tract of dogs [ ] and has also been detected by pcr in a variety of canine tissue samples [ ] . in a study of the english pet dog population by reading et al. [ ] using an elisa % of dogs were found to be positive for chv igg. the prevalence of antibodies to chv in the adult dog population in belgium was found to be . % [ ] and a similar prevalence was detected in the netherlands ( . %) [ ] . chv infections therefore appear to be common amongst adult dogs. whereas rijsewijk et al. did not find an increase in the number of seropositive dogs after a stay at a boarding kennel, an investigation involving dogs at a rehoming centre in london showed that the percentage of positive dogs increased distinctly amongst dogs that had stayed at the kennel for five weeks or longer [ ] . experimental infection of dogs with chv has been shown to cause mild clinical symptoms of rhinitis and pharyngitis [ ] or to result in "kennel cough" [ ] . recently a new canine coronavirus was detected in dogs with cird housed at a rehoming centre [ ] . this canine respiratory coronavirus (crcov) was shown to be most similar to bovine coronavirus and human coronavirus oc and therefore to be distinct from the canine coronavirus that causes enteritis. in that study, dogs that were positive for antibodies to crcov on arrival in the kennel were less likely to develop cird. coronaviruses are known to cause respiratory disease in humans, cattle and poultry [ , , ] however the role of crcov in the aetiology of cird requires further investigation. the study presented here aimed to identify viruses present in dogs at two training centres for working dogs over a period of one year. both kennels had experienced several outbreaks of cird during recent years. the investigation was carried out at two training centres located in southeast england (kennel a) and central england (kennel b). all dogs were from the same breeding centre from where they had been placed into families at six to eight weeks of age. all dogs were vaccinated against distemper, cav- , canine parvovirus and leptospira interrogans. at approximately one year of age the dogs then were transferred into the training centres. day one or day of entry in this investigation was defined as the day dogs entered the training centre, at which time they underwent a routine examination by a veterinary surgeon. dogs at kennel a received vaccines (nobivac dhppi and nobivac lepto, intervet, uk) containing distemper, cav- , canine parvovirus, cpiv and leptospira interrogans. dogs at kennel b were given a vaccine against distemper, cav- , canine parvovirus, cpiv and leptospira interrogans (nobivac dhppi and nobivac lepto, intervet, uk) as well as an additional intranasal vaccine against cpiv and bordetella bronchiseptica (nobivac kc, intervet, uk). the target was to follow dogs from each kennel for a period of one year. all dogs were therefore taken on to the study on their day of entry. dogs that left the kennel during the study were replaced by new dogs entering the kennel on the following intake date. furthermore samples were also collected from additional dogs at these kennels during outbreaks of cird. all sampling was approved by the ethics committee responsible for the kennels involved in this study. a blood sample was obtained from all dogs on the study on the day of entry and every four weeks thereafter. tonsillar swabs were taken on the day of entry and every three months thereafter. from dogs that were not on the study but showed clinical symptoms of cird, both a blood sample and a swab were taken at the time of disease as well as four weeks after. all samples were transported on ice to the laboratory within h. blood samples were centrifuged to obtain serum samples that were stored at − • c. virus transport swabs (bibby sterilin, stone, uk) were used to take swabs from the pharyngeal tonsil of the dogs. cells were detached from the swab and suspended in the transport medium by vigorous mixing. the transport buffer containing the cells was then split into three parts and stored at − • c for subsequent rna extraction, dna extraction and virus culture. in total serum samples and tonsillar swabs were collected from a total of dogs at kennel a from june to july . in december after the study had finished a further outbreak of cird occurred at kennel a and samples were obtained from dogs during the outbreak as well as follow-up samples from dogs four weeks later. at kennel b in total serum samples and swabs were collected from a total of dogs from july to july . the elisa for crcov antibodies was performed using a bovine coronavirus antigen as described previously [ ] . briefly, the serum samples were routinely tested at a dilution of : . for samples an endpoint titration was performed starting at : followed by two-fold dilutions until a dilution of : . the immunofluorescence method for the detection of antibodies to chv using chv infected mdck cells has been described elsewhere [ ] . serum samples were tested at a dilution of : . for dna extraction using the qiamp tissue kit (qiagen, crawley, uk) the transport buffer obtained from tonsillar swabs was centrifuged at rpm and the resulting cell pellet was resuspended in µl of its transport buffer. the extraction was performed as recommended by the manufacturer's protocol for cultured cells. rna extraction was performed on swab samples centrifuged as described above. the cell pellet was resuspended in ml of trireagent (sigma, poole, uk) and the extraction was performed according to the manufacturer's protocol for cultured cells. reverse transcription was performed using impromii reverse transcriptase (promega, southampton, uk) with the provided buffer and µl of rna in a total volume of µl. for pcr, µl of cdna or dna respectively was used. the nested pcr for crcov was carried out using the primer pairs sp /sp and sp /sp directed to the coronavirus spike gene as described previously [ ] . the primers and methods for the pcr detection of cpiv, cav and chv were the same as described in [ ] . virus isolation was attempted on canine adult lung fibroblasts (passages three to seven) that were derived from a tissue sample obtained in a previous study [ ] and mdck cells. all cells were maintained in mem (sigma, poole, uk) containing u/ml of penicillin (sigma), . mg/ml of streptomycin (sigma) and % fetal calf serum for canine adult lung fibroblasts and % fetal calf serum for mdck cells. canine adult lung fibroblasts and mdck cells were inoculated with µl of tonsillar swab sample and incubated for h at • c. the inoculum was then removed and maintenance medium was added to the cells. the cultures were passaged weekly for up to three weeks and examined daily for cytopathic effect. analysis of categorical data was performed using fisher's exact test. for comparison of two means a t-test was used. p values below . were considered to be statistically significant. at kennel a, out of ( . %) female study dogs developed cird in contrast to out of ( . %) male dogs on the study (p = . ). at kennel b three out of ( . %) female and four out of ( . %) male dogs showed clinical signs of cird during the study period (p = ). dogs that developed cird had stayed at kennel a on average for . days ( to days). the average length of stay of dogs that were present during the same month as the dogs that developed cird but remained healthy was . days ( to days). the difference between the mean stay of dogs with and without cird was found to be statistically not significant (t-test, p = . ). due to the low number of cases of cird at kennel b the comparison of the mean length of stay was not performed for this kennel. in total twelve out of serum samples ( . %) obtained from dogs on the day they entered kennel a were positive for antibodies to crcov. table shows the (fig. ). only one dog showed a seroconversion to crcov during the study period. serum samples taken from the same dogs at different time points were analysed by titration to assess if the titres declined over time ( table ) . paired serum samples from seven dogs at kennel a consisting of the first crcov antibody positive sample (at seroconversion) and a follow-up sample three to five months later were analysed by crcov antibody titration. the titres at seroconversion ranged from to . of the seven dogs five remained positive, two titres remained stable whereas in three dogs the titres decreased two-fold. two dogs with titres of and at seroconversion were negative when tested three months later. titration of seven serum samples from dogs that had been positive for antibodies to crcov on entry into kennel b revealed titres ranging from to . follow-up samples from six to eight months later showed that the titres had reduced four-fold in two dogs, in three dogs the titres had decreased two-fold and in two dogs they remained unchanged. overall the percentage of chv antibody positive dogs was . percent ( out of ) on the day of entry into kennel a. dogs joining the kennel from june to september were more likely to be positive than those joining during the following months (table ) . seroconversions to chv occurred throughout the study period with two peaks in november and may (fig. ) . due to antibody positive dogs leaving the study in february and march the percentage of positive dogs declined but increased back to more than percent in may following a high number of seroconversions. at kennel b four out of dogs ( . %) had antibodies to chv on their first day at the kennel. the overall prevalence of chv positive dogs over the study period was . percent and in total only four dogs seroconverted to chv (fig. ) . out of crcov antibody negative dogs at kennel a that developed cird, ( . %) showed a seroconversion to crcov within eight weeks after showing clinical signs. of healthy dogs that were present at the same time as the dogs with cird ( . %) subsequently showed a seroconversion (p = . ). out of chv antibody negative dogs with cird at kennel a five ( . %) produced antibodies to chv within eight weeks of showing clinical signs whereas of healthy dogs present at the same time four ( %) showed a seroconversion (p = . ). dogs that were from the same litter but were placed into different kennels for training were paired to compare their antibody status for crcov and chv. analysis of pairs of dogs for antibodies to crcov showed that in eight pairs the dog going into kennel a was negative whereas its littermate going to kennel b was positive. in one pair both dogs were positive and in two pairs both dogs were negative. the analysis of antibodies to chv revealed that in eight pairs both dogs were negative, in one pair both dogs were positive, in one pair the dog going into kennel a was positive and in one pair the dog going into kennel b was positive. male dogs showed seroconversions to crcov more frequently ( . %) than female dogs ( . %) and were also more likely to become antibody positive for chv ( . %) compared to female dogs ( . %) but the differences were statistically not significant (p = . for crcov, p = . for chv). nested rt-pcr for crcov was performed on samples obtained from dogs housed at kennel a and three samples were found to be positive. one of the positive samples had been taken during an outbreak of cird in september but was from a dog with no clinical symptoms at that time. two further samples were derived from dogs with cird during the outbreak in november . all samples tested for cpiv (n = ), cav (n = ) and chv (n = ) were negative. none of the samples obtained from dogs at kennel b tested by pcr for crcov (n = ), cpiv (n = ), cav (n = ) or chv (n = ) were found positive. in total throat swab specimens from dogs at kennel a were used to inoculate canine adult lung fibroblasts or mdck cells, of these were from dogs without clinical signs of respiratory disease and were from dogs suffering from cird. furthermore samples from dogs at kennel b taken from healthy dogs and seven dogs with cird were tested for viral growth on both cell lines. none of the cultures from either kennel a or kennel b yielded any cytopathic effect. in this study outbreaks of cird occurred mostly in one of the two study kennels and most cases there were recorded in autumn and winter. similar to the common cold in humans cird appears to be seasonal indicating that environmental factors such as low outside temperatures may play a role. although it has not been proven that cooling of the body surface increases susceptibility to respiratory disease it is feasible that cold temperatures may affect the immune defences of mucosal surfaces of the upper respiratory tract permitting facilitated entry of infectious agents [ ] . alternatively cold temperatures may be beneficial for a prolonged survival of infectious agents outside a host thereby increasing the likelihood of transmission. male dogs were more frequently affected by cird at one of the kennels while there was no difference at the other kennel possibly due to the low number of cases there. however in a recent investigation we carried out at a rehoming centre the prevalence of cird was the same in male and female dogs (unpublished results). the mean length of stay at the kennel did not have an influence on the development of cird but dogs developing the disease had stayed at the kennel for at least days indicating that the dogs were generally healthy on arrival and became infected whilst staying at the kennel. at kennel a two outbreaks of cird, which occurred in november and december were followed by major increases in the number of seroconversions to crcov. few seroconversions to crcov were detected after the cird outbreak in october and none after the small outbreaks from may to july . similarly infections with coronaviruses in humans have been reported to be seasonal with peaks in late autumn to winter but also in early summer [ , ] . crcov may have contributed to the outbreak of cird in november however almost all dogs present at the kennel at that time seroconverted to crcov but not all dogs developed respiratory disease. other infectious agents are likely to have been present in the dogs that developed cird. infections with crcov therefore may not cause any symptoms of clinical respiratory disease. nevertheless it is possible that the virus may predispose dogs to infections with other viruses or bacteria. future studies using in situ hybridisation or immunohistochemistry will be necessary to determine if crcov is associated with lesions in the respiratory epithelium. interestingly the percentage of crcov antibody positive dogs entering kennel b was much higher than at kennel a. all dogs were bred at the same breeding facility and dogs from the same litter did not have the same antibody status indicating that the dogs entering kennel b became infected with crcov after leaving the breeding centre. dogs from the same area may have come into contact before entering the training kennel as they attended local puppy classes and crcov may well have been spread amongst these dogs at such occasions. however no records are available to determine which of the dogs attended these classes. the virus seems to have been rarely present at kennel b itself as only one of the negative dogs there became positive for crcov antibodies during its stay. generally the crcov antibody titres in the samples from seven dogs at kennel b were higher than those from seven dogs at kennel a. the samples analysed from kennel a were from dogs that had just seroconverted and may reflect a primary antibody response to crcov. the dogs from kennel b that were tested had been positive upon arrival at the kennel and had potentially encountered the virus repeatedly during their first year of life. in the majority of dogs from both kennels the antibody titres declined at least two fold in less than a year and two dogs became negative three months after seroconversion. this may indicate a poor immunogenicity of crcov; however as all dogs in this investigation became infected naturally, low and rapidly declining titres observed in some dogs may have simply been the result of a low infectious dose. crcov was only rarely detected by pcr and not at all by culture even in samples from dogs that subsequently seroconverted. the methods for rna extraction and rt-pcr had previously been used successfully for the detection of crcov in dogs [ ] , however in that study tissue samples were available. it is therefore possible that the detection of crcov by pcr failed due to an insufficient number of infected cells obtained by tonsillar swabs. the pcr primers were designed towards conserved regions of the spike genes of bovine coronavirus and human coronavirus oc strains. nevertheless these primers may have failed to detect variant strains of crcov. in addition crcov has proven to be difficult to isolate (unpublished results). alternatively crcov replication in the tonsils may be limited and nasal swabs might have given a better result. in contrast to crcov, seroconversions to chv in kennel a were detected throughout the year and did not appear to be seasonal. the constant presence of chv at kennel a could be due to long periods of viral shedding after infection or due to recurrent introduction of the virus by acutely infected dogs entering the kennel. dogs suffering from cird showed a subsequent seroconversion to chv more frequently although the difference was not statistically significant. chv may be able to cause or aggravate the disease but not all dogs with cird showed evidence of chv infection whereas some healthy dogs seroconverted. moreover chv can establish latent infections that may have been reactivated by stress or by respiratory disease caused by other microorganisms. this could also explain why seroconversions to chv were rare at kennel b where the prevalence of cird was very low. chv was not detected by pcr or virus culture despite studies showing that chv is present in the tonsils of infected dogs [ ] . in a recent study we were able to detect chv by pcr and culture in tracheal samples from kennelled dogs [ ] .as mentioned earlier the amount of cells obtained in the present study may not have been sufficient. this also means that the failure to detect canine parainfluenza virus and canine adenovirus by pcr does not rule out their presence in dogs in this investigation. however all dogs were vaccinated regularly against these viruses. only dogs housed at kennel b were vaccinated against bordetella bronchiseptica, which may have been in part responsible for the rare occurrence of cird at this kennel at the time of the study. however no conclusions can be drawn from this study on the efficacy of the vaccination program as this would have required groups of vaccinated and unvaccinated dogs to have been present at both kennels. in conclusion seroconversions indicated the presence of crcov and chv at a training kennel, which experienced frequent outbreaks of cird during the study period. however further studies will be necessary to determine the role of crcov and chv in the pathogenesis of the disease. moreover it will be important to increase our understanding of the interactions of viral and bacterial agents present simultaneously in the respiratory tract of dogs. canine infectious tracheobronchitis short review: kennel cough, st edn pathogenesis of canine herpesvirus in specific-pathogen-free dogs: -to -week-old pups studies of respiratory disease in random-source laboratory dogs: viral infections in unconditioned dogs detection of canine herpesvirus in a wide range of tissues using the polymerase chain reaction acute cooling of the body surface and the common cold detection of a group coronavirus in dogs with canine infectious respiratory disease longitudinal study of viruses associated with canine infectious respiratory disease avian infectious bronchitis virus cird and antibodies to crcov and chv epidemiology of coronavirus respiratory infections infectious bovine rhinotracheitis, parainfluenza- , and respiratory coronavirus experimental production of canine tracheobronchitis (kennel cough) with canine herpesvirus isolated from naturally infected dogs canine infectious tracheobronchitis: effects of an intranasal live canine parainfluenza-bordetella bronchiseptica vaccine on viral shedding and clinical tracheobronchitis (kennel cough) occurrence and frequency of coronavirus infections in humans as determined by enzyme-linked immunosorbent assay a serological study of canine herpes virus- infection in the english dog population prevalence of antibodies against canine herpesvirus in dogs in the netherlands in - seroprevalence of canine herpesvirus- in the belgian dog population in prevalence of feline chlamydia psittaci and feline herpesvirus in cats with upper respiratory tract disease equine herpesvirus and infections: an update author's address: dr. kerstin erles, department of pathology and infectious diseases this work was supported by the guide dogs for the blind association (united kingdom).we wish to thank the management and staff at the kennels participating in this investigation for their support and would also like to thank c. toomey and s. greaves for technical assistance. key: cord- -gon bzat authors: mondal, shankar; chang, yung-fu; balasuriya, udeni title: sequence analysis of infectious bronchitis virus isolates from the s in the united states date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: gon bzat to better understand the molecular epidemiology of infectious bronchitis virus (ibv) in the united states following the introduction of commercial ibv vaccines, we sequenced the s and n structural protein genes of thirteen ibv field isolates collected in the s. analysis of the s sequence showed that seven isolates were of the massachusetts (mass) genotype, five were se , and one was of the connecticut (conn) genotype, suggesting that these three ibvs were circulating in commercial poultry raised in different regions in the united states during the s. the s genes of mass-type isolates had high levels of sequence variation, representing . - . % nucleotide (nt) and . - . % amino acid (aa) identity when compared to those of the se -type isolates. in contrast, the n genes from the same isolates were less variable (> % nt and > % aa identity) when compared to those of the se -type isolates. phylogenetic analysis based on the s gene indicated that one isolate (l ) was more closely related to the mass type. in contrast, phylogenetic analysis based on the n gene showed that l was more closely related to the se type, indicating that there had been exchange of s genetic materials between mass- and se -like viruses. in addition, the mass-type isolates had high levels of sequence identity in the s gene compared with widely used modified live vaccines (mass , ma and h ) and modern field strains from the usa and other countries, suggesting a common ancestor. electronic supplementary material: the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. avian infectious bronchitis virus (ibv) is a ubiquitous, highly contagious respiratory pathogen of chickens that inflicts serious economic losses to the commercial poultry industry worldwide [ , ] . ibv, along with turkey coronavirus, belongs to the genus gammacoronavirus in the subfamily coronavirinae, family coronaviridae, order nidovirales [ , ] . ibv has a single-stranded, positivesense rna genome of approximately . kilobases. the genes (open reading frames [orfs]) encoding ibv structural proteins are located downstream of the viral replicase genes (orfs a and b) and in order from to are as follows: s (spike), e (envelope), m (membrane) and n (nucleocapsid). the s glycoprotein is posttranslationally cleaved into a transmembrane domain (s ) and an outer domain (s ), which expresses the serotype-specific epitopes of the virus [ ] . the n phosphoprotein, which forms the capsid of the virion, is involved in rna replication and carries group-specific antigenic determinants [ ] . both s and n proteins play critical roles in the induction of immune responses against ibv [ , ] . ibv was first isolated in the early s in the united states [ ] . the massachusetts (mass)-type viruses were believed to be the only serotype found in the usa until the mid- s, when a second ibv serotype connecticut (conn) was reported [ ] . immediately thereafter, the use of commercially produced mass-and conn-type modified live virus (mlv) vaccines began [ ] . a number of new ibv serotypes, antigenic variants and field strains were isolated in the subsequent years in the usa, and many ibv serotypes are currently known to exist worldwide [ , ] . mlv vaccines containing strains of ibv from different serotypes are routinely used to protect commercial chickens against infectious bronchitis (ib). mlv vaccine strains are selected to represent the antigenic spectrum of likely challenge viruses by incorporating the serotypes of viruses most commonly circulating in a particular country or region. in the united states, most ibv isolates causing ib belong to the mass, conn, and arkansas (ark) serotypes [ ] . disease outbreaks in vaccinated chickens may result when the flock is infected with ibvs that are antigenically unrelated to the vaccines used to immunize them. new variants of ibv can emerge from either wild-type or vaccine viruses by acquiring point mutations and/or genomic recombinations [ ] . one of our previous studies indicated that antigenic and genetic diversity existed during the s [ ] , well before the introduction of ibv vaccines. however, many ibv strains from the usa isolated following the introduction of commercial mlv vaccines have never been characterized by sequencing, and it is essential to sequence these isolates to understand the evolution of ibv geographically and to implement effective vaccination strategies to control modern ibv strains that are in circulation. the main objective of this study was to examine a number of archived ibv isolates from the s in order to determine ( ) which genotypes of ibv were prevalent in the united states during that time and ( ) whether genetic information obtained from these isolates would shed light on the evolution of the currently circulating serotypes. during the s, clinical signs of bronchitis with upper respiratory distress were frequently observed in broiler flocks, even in ibv-vaccinated birds, but rarely in layer flocks [ ] . through the cooperation of numerous hatcheries and laboratories, a number of field isolates of ibv were recovered in the mid- s from the respiratory tracts of naturally-infected chickens raised in different regions of the united states (winterfield and hitchner, unpublished data). following isolation at cornell university, the viruses were passed a minimum number of times (no more than three) in embryonated chicken eggs, and allantoic fluids from the embryos were lyophilized and stored at °c. for the present study, we used thirteen representative ibv field isolates from all regions of the us, generously provided by dr. benjamin lucio-martinez, college of veterinary medicine, cornell university, ithaca, ny ( table ). the lyophilized samples were reconstituted in ml of diethylpyrocarbonate (depc)-treated water and used directly for rna extraction without further passage in chicken embryos. ibv rna was extracted from ll of each sample using trizol ls reagent (gibco brl, grand island, ny) according to the manufacturer's instructions and resuspended in ll of rnase-free water. the reconstituted rna from each isolate was used as the template for rt-pcr amplification of the s and n protein genes. briefly, the rt reaction was performed using a superscript iii firststrand synthesis kit (invitrogen, inc., carlsbad, ca) with random hexamer primers according to the manufacturer's instructions. the rna template was removed by digestion with u of e. coli rnase h for min at °c. amplification of cdna with gene-specific primers (news oligo and degenerate [ ] for s , and nfor and nrev [ ] for n genes) was performed using an advantage polymerase mix kit containing high-fidelity dna polymerase (clontech laboratories, inc., mountain view, ca). the amplicons ( , bp [s ] and , bp [n]) were analyzed by gel electrophoresis using . % agarose with ethidium bromide ( . lg/ml). with the exception of isolate l , all other isolates resulted in , -bp n gene rt-pcr products, and therefore, only twelve n gene sequences were included in the analysis. rt-pcr products were gel-purified using a qiaquick gel extraction kit (qiagen inc., valencia, ca), and directly sequenced using a bigdye terminator cycle sequencing kit (perkin-elmer, branchburg, nj) and an applied biosystems model abi automated dna sequencing system. a combination of flanking and internal primers was used to sequence both strands of dna in their entirety. assembly of contiguous sequences and translation of nucleotide sequences into amino acid sequences were performed with vector nti advance software (invitrogen inc., carlsbad, ca). the blastn program (http:// www.ncbi.nlm.nih.gov/blast/) was used to search genbank for homologous ibv s and n gene sequences. selected sequences from genbank were included in the alignment. after aligning the s and n sequences, the geneious program (biomatters ltd, auckland, new zealand) was used to construct phylogenetic trees by the neighbor-joining method. subsequently, bootstrap analysis was performed with , replicates to determine the bestfitting tree for each gene. pairwise blast searches were also performed when there were no significant hits in the blastn search. all of the s and n gene sequences reported herein have been deposited in the ncbi genbank database, and the accession numbers are listed in table . the s and n gene sequences of other ibv strains were also obtained from genbank. the , -bp s gene sequences (nt , - , , numbered according to the beaudette strain, genbank accession number dq ) of thirteen s isolates were compared with those of other ibv strains (supplementary table ) from the usa and other countries. a phylogenetic tree based on the nt sequences of the complete s genes showed that the s ibv isolates were divided into three distinct lineages, mass, se and conn genotypes (fig. ) , which suggests that these three ibvs were circulating in commercial poultry raised in different regions of the usa in the s. seven ibv field isolates (l , l , l , l , l , l , l ) were clustered with mass-type reference strains (beaudette, mass ), vaccine strains (mass/bvial , mass/cvial , ma , h ) and field strains from the usa and other countries. these viruses had . - . % nt ( . - . % aa) sequence identity when compared to prototype mass . five ibv isolates (l , l , l , l , l ) were clustered with se virus, and they had . - . % nt ( . - . % aa) sequence identity when compared to prototype se . interestingly, only one ibv isolate (l ) clustered with the conn-type reference virus (conn ), the vaccine strain (conn/cvial ), and field strains from the usa and other countries. this virus had . % nt ( . % aa) sequence identity to the prototype conn strain. less-frequent isolation of the conn-type virus was probably due to the high level of conn-type immunity from widespread use of the vaccine in young flocks [ ] . the s sequences of ibvs closely related to the mass-type isolates were , bp ( aa) in length. the s sequences of all ibvs grouped with se were , bp ( aa) in length due to an additional -nt ( -aa) insertion at positions - and - . the s sequence of the conn-type isolate l was bp long ( aa), the same as the conn virus. the mass-type isolates were genetically distantly related to other s viruses and had only . - . % nt ( . - . % aa) sequence identity when compared to se -type isolates and . - . % nt ( - . % aa) sequence identity when compared to conn-type l isolate. the s gene of the s isolates were also distantly related to those of the gray, jmk, ark , n-m and n-m strains, showing . - . % nt ( . - . % aa) sequence identity. the deduced aa sequence of the s proteins had multiple substitutions that were distributed throughout the s protein (data not shown). the mass-type isolates had - aa differences when compared to mass reference (beaudette, mass ) or vaccine (ma , h ) strains. isolate l had a maximum differences, but only two (s r, l r) differed from the mass sequence. the se -type isolates had - differences compared to prototype se virus, and the conn-type l had three substitutions compared to conn virus. the majority of the differences in mass, se and conn-type viruses were concentrated between aa positions and , and and , which correspond to the two hypervariable regions (hvrs) [ ] that are known to carry serotype-specific antigenic determinants. the cleavage recognition sites between the s and s subunits of the s isolates were compared. all mass-type isolates had an rrfrr sequence, as in other mass viruses, and the se group had an hrsrr sequence like the gray and ark strains (data not shown). the s genes of the mass isolates were found to be genetically closely related to those of widely used mlv vaccine strains and modern field isolates from the usa and other countries (fig. ) , suggesting a possible common ancestry and emergence of virus strains that are essentially similar to the mlv vaccines that are used. previous studies have indicated that some strains of ibv isolated in different countries may have almost identical s genes [ , ] , reflecting some of the common features of ibv evolutionary direction. the spreading of a virus from one region or country to another could be due to its inadvertent introduction by the trading of birds or by the use of mlv vaccines. it is known that the mass strain of ibv is widely used as a mlv vaccine throughout the world [ ] . unlike the s genes, the n gene sequences of the s isolates were highly conserved, having [ % identity at fig. phylogenetic tree created from the nucleotide sequences of the s glycoprotein genes of infectious bronchitis virus (ibv) isolates. the neighbor-joining tree was constructed from the pairwise nucleotide differences for the s genes, which represented the relatedness between isolates from the s and heterologous ibv strains from the united states and other countries. the s isolates (*) clustered with massachusetts, connecticut and se -type reference (#) or vaccine (^) strains of ibv. the length of each pair of branches represents the distance between the sequence pairs. the scale at the bottom indicates the number of substitution events the nt ([ % at the aa) level as compared to the n genes of the other ibv strains (supplementary table ). the phylogenetic tree based on the n gene nt sequences showed that the s ibvs were also divided into three clades, representing the mass, se (unfortunately, the n gene sequence of prototype se was not available in genbank, and we did not have access to the isolate) and conn genotypes (fig. ) . of them, five (l , l , l , l , l ) were closely related to the mass strains (mass , h ), showing . % nt ( . % aa) sequence identity with mass , and they were even more closely related ([ %) to isolates ck/ch/lsd/ from china and ind/ tn/ / from india; six (l , l , l , l , l , l ) ibv isolates, those clustered with se (except for l which clustered with mass) based on s phylogeny, were closely related to turkey coronavirus (tcov) in the n gene, showing . % nt ( . % aa) sequence identity, and they had a similar (c %) genetic relationship to the ark , conn, and gray strains of ibv. the isolate l clustered with conn-type reference (conn ) and field strains, showing c % sequence identity. the n gene of all the s isolates was , bp ( aa) in length and differed from one another by zero to . % nt ( . % aa) (data not shown). our data is consistent with previous reports describing recombination between ibv and turkey coronaviruses [ ] . genetic variation was observed in the s and n genes of the isolate l in this study. phylogenetic analysis of the s gene showed that the mass-type isolates, including l , clustered together (fig. ) , as would be expected of viruses of the same serotype. interestingly, the l isolate had much different levels of sequence similarity to each other in the n gene, and it clustered randomly with other se -type isolates (fig. ) . the shift in sequence homology in the s and n genes indicated that exchange of genetic material took place subsequent to coinfection with a mass-like virus and an se -like virus for l , probably by homologous recombination. a similar recombination event has been observed in some japanese isolates [ ] , where the s protein was found to be closely related to australian isolates [ ] and the n protein was very similar to those of north american viruses [ ] . in addition, two us isolates (md and pp ) exhibiting % and % identity, respectively, to se in the s genes are known to be derived from se by recombination [ , ] . se was first reported by hopkins [ ] as a new serotype of ibv isolated in from a closed research flock of chickens with classic signs of ib. herein, we report five se isolates from samples collected in (l , l ) and (l , l , l ) from commercial chickens raised in different regions of the usa. unlike se , the conn and mass (mass , ma , h , h ) strains have been used simultaneously as mlv vaccines in commercial poultry in the usa [ ] . the use of se or its derivatives as an mlv vaccine is not known. because the chickens from which the s isolates were isolated most likely had been exposed to live mass vaccines [ ] , the vaccine might be implicated as a source for the mass-like sequences in the se s gene. the se source of genetic material was probably a strain naturally infecting these affected flocks, since they had not been exposed to se vaccine. although this study cannot prove or disprove that recombination has occurred in l , we can point to the evolutionary trends in the s gene as compared to the evolutionary trends in the n gene. this study demonstrates that the s gene of ibv tends to evolve more rapidly by mutation and genetic recombination when compared to the more conserved n gene [ ] . this suggests that these two genes of ibv are under very different selective pressures and that the exchange of genetic material may have occurred between vaccine strains and field isolates. based on these findings, we conclude that widespread vaccination with mlv vaccines against previously known dominant serotypes (e.g., mass , ark ), in association with the high recombination and mutation abilities of ibv, have led to the emergence and broad dissemination of the present-day novel serotypes. new strategies to control ibv infection effectively in chickens should be considered with the use of either inactivated or subunit vaccines. complete nucleotide analysis of the structural genome of the infectious bronchitis virus strain md reveals its mosaic nature veterinary microbiology coronavirus avian infectious bronchitis virus infectious bronchitis family coronaviridae infectious bronchitis virus types: incidence in the united states the evolution and emergence of rna viruses serologic and immunologic properties of a recent isolate of infectious bronchitis virus recombinational histories of avian infectious bronchitis virus and turkey coronavirus immune responses to structural proteins of avian infectious bronchitis virus genetic and antigenic diversity in avian infectious bronchitis virus isolates of the s immunological differences in strains of infectious bronchitis virus serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s ) gene molecular cloning and sequence comparison of the s glycoprotein of the gray and jmk strains of avian infectious bronchitis virus redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the de strain of infectious bronchitis virus s glycoprotein gene analysis of infectious bronchitis viruses isolated in korea phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan sequence analysis of the s glycoprotein of infectious bronchitis viruses: identification of a novel genotypic group in australia an apparently new respiratory disease of chicks complete nucleotide sequences of s and n genes of infectious bronchitis virus isolated in japan and taiwan infectious bronchitis virus variants: a review of the history, current situation and control measures evidence of natural recombination within the s gene of the infectious bronchitis virus comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses sequence analysis of s ibv isolates acknowledgements the authors would like to thank dr. benjamin lucio-martinez from cornell university, ithaca, ny, for providing the s ibv isolates used in this study. the authors would also like to thank dr. carol j. cardona from the university of minnesota, st. paul, mn, for reading the manuscript. key: cord- -oq zrnuv authors: taguchi, f.; matsuyama, s.; saeki, k. title: difference in bgp-independent fusion activity among mouse hepatitis viruses date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: oq zrnuv mouse hepatitis virus (mhv) utilizes a mouse biliary glycoprotein (bgp) as a receptor. co-cultivation of mhv-nonpermissive hamster bhk cells devoid of mouse bgp with mouse dbt cells infected with mhv-a or jhmv induces syncytia formation on bhk cells (bgp-independent fusion). this study shows the difference in bgp-independent fusion activity among various mhv strains. under a phase contrast microscopy, jhmv (cl- , sp- ) induced the bgp-independent syncytia on bhk cells similar to those observed on dbt cells, while such syncytia were not seen with the infection of other mhv strains (mhv- , mhv- , mhv-a , mhv-s, srr , srr and srr ). tiny syncytia detectable only by immunofluorescence were produced with the latter mhv strains except for srr which failed to produce syncytia. mhvs except for srr grew in bhk cells after bgp-independent infection. the bgp-independent fusion by jhmv was inhibited either by anti-s or anti-s antibodies. these results showed that the jhmv spike protein had a remarkably high bgp-independent fusion activity. (s n ) in which a conformational structure plays an important role [ , ] , while the s is not involved in this activity [ ] . several different regions and amino acid residues in the s have been reported to be important for fusion activity [ , , , ] , although it is unclear which of those are actually involved in the fusion event. biliary glycoprotein (bgp) , a member of carcinoembryonic antigen (cea) gene family, serves as a highly functional receptor for mhv [ ] . several different bgp isoforms are generated by an alternative splicing [ ] and expressed in the liver, brain and other organs. the bgp has two allelic forms. bgp a and bgp b , expressed in mhv-susceptible balb/c and resistant sjl mice, respectively [ , ] . both of these proteins retain the mhv receptor function, although the bgp a has much higher virus-binding activity than does the bgp b [ ] . such difference could result in the difference of mhv susceptibility in the whole animal level [ ] . two other species of protein, bgp [ ] and brain specific cea [ ] , both of which are cea members, have been reported to work as mhv receptor in mice. mhv- (jhmv) and mhv-a are known to infect cells lacking mhvspecific receptors, i.e., bgp-independent infection [ , ] . these viruses fail to directly infect bgp-negative bhk (bhk) cells, but infect them after cocultivation with the bgp-positive dbt cells infected with these viruses [ , ] . the s protein is involved in the bgp-independent infection and fusion, since the antibodies against the s protein prevented the infection [ ] and the expression of the s protein alone induced fusion on cells devoid of bgp [ , ] . in this paper, we compared the bgp-independent fusion activity of various mhv strains. mhv strains mhv- , mhv- , mhv-s, mhv-a [ , ] , jhmv cl- [ ] , sp- [ ] as well as different soluble receptor-resistant (srr) variants derived from cl- [ ] were used in this study. of these the amino acid sequences of a [ ] and jhmv cl- [ ] s proteins have been reported. sp- s has a -amino acid deletion in the hypervariable region of the cl- s [ ] . srr and srr have amino acid mutations at positions (leu→phe) and (cys→his) in the s , respectively. srr has a mutation at position of (leu→his) in the s . to see the bgp-independent fusion activity, dbt cells cultured in well-plate (corning) were infected with those viruses at an m.o.i. and incubated at • c for h. after times of washing with dulbecco's modified minimal essential medium (dmem, nissui, tokyo), infected cells were cultured in ml of dmem supplemented with % fetal calf serum (fcs, gibco) for to h. the cells were then trypsinized and cells were overlaid onto bhk cell monolayer cultured in well-plate ( × cells). the syncytia of bhk cells were examined by a phase contrast microscopy during h after overlay of mhv-infected dbt cells. viral antigen was also examined by immunofluorescence using anti-s mabs [ ] . only jhmv cl- and sp- induced the bgp-independent syncytia on bhk cells detectable by a phase contrast microscopy. the syncytia could be observed from about h after overlay. the syncytia formed by cl- were significantly larger than those by sp- ( fig. a and b ). this size difference observed on bhk cells between cl- and sp- was in good agreement with the size difference in anti-s mabs and anti-mouse igg labelled with fitc for immunofluorescence plaque produced on dbt cells [ ] . interestingly, no syncytia formation was demonstrated on bhk cells with other strains or srr mutants under the phase contrast microscopy, though all of them produced syncytia on bgp-positive dbt cells [ ] . mhv-a induced tiny syncytia on bhk cells comprising roughly to cells detectable only by immunofluorescence (fig. c) . other mhv strains, mhv- , mhv- and mhv-s produced tinier syncytia consisting of less than cells ( fig. d and e) . srr mutants have also failed to induce syncytia comparable to those produced by wild type cl- . srr caused syncytia including about to cells (fig. g ). srr showed very tiny syncytia consisting of less than cells. no obvious syncytia was observed on bhk cells overlaid with srr -infected dbt cells. single cells were mhv antigen-positive (fig. f ). these single cells presumably represented srr -infected dbt cells overlaid on bhk cell monolayer. we have examined whether these viruses multiply in bhk cells. dbt cells infected with jhmv-cl- , mhv-a and srr mutants were either overlaid onto bhk cell monolayer as described above or allowed to seed alone in the -well plate and virus growth in these cells was examined. as shown in fig. , all viruses except srr grew significantly higher in bhk cells co-cultured with mhv-infected dbt cells than in dbt cells alone, indicating that all viruses examined but srr multiplied in bhk cells. though cl- produced significantly larger syncytia on bhk cells in a bgp-independent manner as shown in fig. , it replicated to almost the same extent as other viruses. no significant difference in the growth on bhk cells between cl- and other mhv strains can not be accounted for at present. the cl- s protein could have a very strong bgp-independent fusion activity, but its replication in bhk cells could be less efficient than the other mhv strains. no replication of srr in bhk may be due to its failure to spread from infected dbt cells to bhk cells. next we have examined whether the anti-s and anti-s antibodies inhibit the bgp-independent fusion. we used neutralizing mabs , and recognizing the s n as well as mabs and recognizing other regions in the s [ ] . anti-s antibodies with neutralizing activity were produced using rabbits. rabbits were immunized with a synthetic peptide consisting of amino acid sequence nespllgcigstcaed which encompassed an immunodominant epitope in the s recognized by a neutralizing mab b . [ ] . the rabbit sera were then affinity-purified using the synthetic peptide. bhk cell monolayer overlaid with cl- -infected dbt cells that produced about syncytia was cultured in -fold serial dilutions of the anti-s and anti-s antibodies. cells were stained with giemsa solution at to h after infection and the syncytium number was counted under the light microscopy. the inhibition of syncytium formation was calculated in comparison with the syncytium number formed in the absence of neutralizing antibodies. standard neutralization test using cl- was also performed with these antibodies on dbt cells as described previously [ ] . as shown in fig. , all of these antibodies showed fusion inhibition (fi) activity, though the fi titers were variable; anti-s mabs showed to higher fi and viral neutralization activities compared with anti-s antibodies. the antibodies with higher neutralization titer exhibited the higher fi titer. this could imply that fig. . growth of mhv in bhk cells after bgp-independent infection. ten thousands of dbt cells infected with jhmv cl- , mhv-a , srr , srr or srr were overlaied on × bhk cells or allowed to seed alone in -well plate. at intervals after overlay, virus titers in the cultures were measured by plaque assay the attachment of the s protein to an unidentified receptor on bhk cells, which is conceibably prevented by the anti-si mabs ( , and ) recognizing the receptor-binding domain on the s, is an inevitable step for the bgp-independent fusion events or that the s is more profoundly involved in the bgp-independent fusion activity than the s . these results appear to suggest that both s and s are involved in the bgp-independent fusion activity. on dbt cells mhv-a and mhv- produce large plaques similar to those produced by cl- , while other mhvs used in this study produce smaller plaques similar in size to those produced by sp- . the plaque is composed of a single, large syncytium. this indicates that all these mhvs have a potential to induce fusion on bgp-positive cells. present study showed that jhmv (cl- and sp- ) could induce syncytia on bhk cells detectable by microscopy, whereas the other mhvs and srr mutants failed. these findings have indicated that jhmv, particularly cl- with a large s protein [ ] , has a strong bgp-independent fusion activity. the high fusogenicity of jhmv was also reported by gallagher [ ] who described that exogenously added jhmv viral particles had a strikingly high fusion activity on bgp-positive cells as compared with mhv-a . the srr mutants showed extremely reduced bgp-independent fusogenicity in spite of their high fusogenicity on bgp-positive dbt cells [ ] . this suggests that mutated amino acids in the srr mutants could play a critical role for the bgp-independent fusion. srr with a mutation at a position in the s has a low receptor-binding activity [ ] which also may affect on the fusion activity in bgp-negative cells. srr and srr have mutations in the amino acids at positions and in the s , respectively [ ]. gallagher et al. reported that both of these residues were involved in the fusion on bgp-positive cells [ , ] . their mutant virus which had mutations at a position of as well as two other positions showed an acid-ph dependent fusion activity on bgp-positive cells [ ] . this virus had a reduced fusion activity on bgp-negative cells [ ] . gallagher also reported the importance of cystein residue at a position of jhmv s protein for syncytia formation on bgp-positive cells using anti-cystein reagent [ ] . both amino acids at positions and are conceivably important for overall fusion activity, whether it is bgp-dependent or bgp-independent. nash and buchmeier reported that the mab to bgp prevented mhv- (jhmv) infection on bgp-positive cells, yet it failed to prevent syncytia formation in infectious center assay on mouse dbt cells [ ] . this implies that the bgp is not necessary for syncytia formation. in the present study, we showed all the viruses except for jhmv failed to produce microscopically-detectable syncytia on bhk cells, while they produced syncytia on dbt. these results could suggest that the bgp plays an important role in the syncytia formation by mhv strains but jhmv. expression of the s proteins of these mhvs in bgp-positive and bgp-negative cells will clarify the importance of bgp in syncytia formation. present study suggested that both s and s were critical for the bgp-independent fusion. the s could be important for the binding to the molecule on bhk cells which is an alternative receptor for mhv. the s protein-receptor interaction is an inevitable step prior to fusion events. such receptor is conceivably not so efficient as the bgp and therefore work as a receptor only when large amounts of s protein expressed on mhv-infected dbt cells are in contact with it [ ] . it is interesting to note that the recombinant mhv between jhmv and mhv-a arisen in a mixed culture of dbt and bhk cells as well as mutants derived from 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fragments analysis of the receptor binding site of murine coronavirus spike glycoprotein the s subunit of the murine coronavirus spike protein is not involved in receptor binding comparison of six different murine coronavirus jhm variants by monoclonal antibodies against the e glycoprotein molecular cloning and expression of a spike protein of neurovirulent murine coronavirus jhmv variant cl- characterization of a variant virus selected in rat brain after infection by coronavirus mouse hepatitis virus jhm the receptor for mouse hepatitis virus in the resistant mouse strain sjl is functional: implications for the requirement of a second factor for viral infection technical assistance by nozomu ishida and reading manuscript by noralyn dudt are greatly appreciated. this work was financially supported by grants from science and technology agency and the ministry of education, science and culture of japan. authors' address: dr. f. taguchi, national institute of neuroscience, ncnp, - - ogawahigashi, kodaira, tokyo , japan.received february , key: cord- -cx jz wf authors: dadar, maryam; fakhri, yadolah; bjørklund, geir; shahali, youcef title: the association between the incidence of covid- and the distance from the virus epicenter in iran date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: cx jz wf since the first official report of the spread of sars-cov- infections in the city of qom in mid-february, iran has become the country most affected by the covid- epidemic in the middle east. all iranian provinces are now affected, although to a different extent. the aim of the present study was to evaluate whether the distance from the epicenter of the infection (qom) or demographic factors such as population density and the ratio of the elderly population are associated with the incidence of covid- in different iranian provinces. for the purpose of determining whether the distance from the virus epicenter could be associated with the spread of infection, linear regression analysis was performed using stata . software. the association of the incidence of covid- with the population density and the ratio of the population over years old in iranian provinces was also evaluated. according to our results, a strong association was found between the incidence of covid- in iranian provinces and their respective distance from qom (p < . ; c = - . ). the incidence of covid- in iranian provinces was also positively associated with the ratio of the population over years old (p = . ; c = . ), while no significant association with population density was found (p = . ; c = . ). these results suggest that the implementation of travel restrictions from highly affected areas to other provinces could considerably reduce the rate of transmission of the disease throughout the country. also, provinces with a higher proportion of elderly people (over ) were identified as particularly at risk for the spread of sars-cov- infections. these results will contribute to better management of the covid- outbreak in iran, taking into account demographic and geographic characteristics of different provinces. with a population of about , , inhabitants, iran was the country most affected by the coronavirus disease (covid- ) epidemic in the middle east. located in southwestern asia and bounded by the caspian sea to the north and the persian gulf to the south, iran is at the crossroads between europe, the middle east, and asia. its central location in eurasia, spanning over an area of , , square kilometers, accounts for its strategic geographic importance. iran has a mean population density of inhabitants per square kilometer. however, the population density varies considerably throughout the country due to the presence of a vast unpopulated central desert plateau and massive mountain ranges, which occupy most of the land area. the northern provinces of tehran, alborz, gilan, mazandaran, and qom have the highest population density among the iranian provinces (table ) . iran announced its first cases of sars-cov- infection in the city of qom (the capital of qom province) on february [ , ] . despite the relatively rapid identification of the source and area of the infection, no city has been isolated or locked down, and the virus quickly spread throughout the country [ , ] . according to the latest assessment by the iranian health authorities, , cases have been confirmed, and , patients have died (http://behda sht.gov.ir). at least six neighboring countries, [ ] . however, this is only a partial picture of the situation in the region, as the number of cases of covid- was underreported in many countries due to inadequate screening of suspected cases and a shortage of diagnostic kits [ ] . according to official iranian data, the first reported cases of covid- were in qom [ , ] , a holy city near the capital tehran and a major destination of pilgrimages in the region [ ] . in the absence of a citywide lockdown, covid- spread very quickly to adjacent provinces such as tehran, isfahan, and markazi [ , ] . in late february, the iranian authorities decided on a set of measures aimed at limiting the spread of disease, such as the closure of educational institutions, major religious sites, and public monuments, as well as the cancellations of events giving rise to rallies. furthermore, substantive information and education programs have been carried out at both the local and national level to improve practices and people's knowledge regarding covid- [ , ] . faced with the growing covid- epidemic and the wave of departure throughout the country due to the nowruz holidays, travel between cities was prohibited starting march , a week after the iranian new year's vacation. however, according to the iranian red crescent, three million people had left the thirteen provinces most affected by covid- by road since march [ ] . in addition, about , afghans returned to afghanistan by land just before the implementation of travel restrictions [ ] . according to gis-based maps, the spread of covid- throughout iran mainly occurred from the north-central provinces of qom and tehran [ ] . through regression analysis, this study aimed to evaluate whether the distance of different iranian provinces from the epicenter of the infection (qom) was associated with the incidence of covid- at the early stages of the epidemic in iran. the association of the disease with the population density and the ratio of the population over years old was also evaluated and compared. with a population of about , , inhabitants and a total land area of , , km , iran became the epicenter of the covid- epidemic in the middle east and central asia. covid- has spread to all iranian provinces, and the city of tehran, the densely populated capital with over million people located km northeast of qom, leads the country in covid- cases ( table ) . the incidence rate for each province was calculated as the number of covid- cases diagnosed until march divided by the population of the province in and multiplied by , . the number of covid- cases diagnosed until march in each province was provided by the iranian ministry of health and medical education (available from: http://behda sht.gov.ir/). the population and demographic data of the iranian provinces were extracted from the census results and official projections provided by the statistical centre of iran (www.amar.org.ir). demographic data and covid- incidence rates for the iranian provinces are listed in table . the present study (id . ) was approved by the ethics committee of the hormozgan university of medical sciences. it did not involve human samples or participants. pearson's correlation coefficient (r) was measured in order to determine the linear association between variables. the assumption of normality of the data distribution was checked using the kolmogorov-smirnov (ks) test prior to analysis. the statistical significance at the % level was evaluated. wald p-values and the corresponding % confidence intervals are presented for the impact of independent linear and categorical items. one-way anova was used to determine the statistical significance of differential incidence rates of covid- reported in iranian provinces based on their relative distances from qom. figure was produced using r version . . all other analyses were performed using stata software, version . (stata corp, college station, tx, usa) [ ] . all iranian provinces were affected. however, they were impacted to varying degrees. tehran, the highly populated capital and the largest city in the country, reported the largest number of coronavirus cases. however, its incidence rate per , people is not the highest and was topped by the less densely populated provinces of semnan, qom, markazi, yazd, mazandaran, qazvin, guilan, alborz and isfahan (fig. , table ). the present study revealed a highly significant association between the incidence of covid- infections in iranian provinces and their respective distance from qom (p < . ; c = - . ) (fig. ) . the majority of covid- cases ( %) were centered in an area of km around qom, with an average incidence of per , people (table ) . one-way analysis of variance (anova) showed statistically significant differences in the covid- incidence between the iranian provinces (table ) located in an area of km around qom province and those at a distance of to km (p = . ) and more than km (p < . ). the incidence of covid- in the iranian provinces was positively associated with the ratio of the population over years old (p = . ; c = . ), while no significant association was shown with population density (p = . ; c = . ) (fig. ). after the first two months of the epidemic of covid- in iran, the country faces serious threats due to the rapidly growing epidemic and rising incidence of covid- in different iranian provinces, most of which are not as well equipped and prepared as the capital city, tehran, to cope with the increasing numbers involved. unlike many affected countries, strict containment and mass quarantine regulations have not been implemented in iran since the first official report of the spread of sars-cov- infections in the city of qom in mid-february. through linear regression analysis, the present study tried to determine the relationship between the incidence of covid- in iranian provinces and their respective distance from the virus epicenter (qom). although the incidence per , inhabitants was higher in densely populated areas ( table ; fig. ), this association was not significant (p = . ; c = . ). it seems, therefore, that the density of the population does not itself drive the spread of the disease, as indicated by the lower incidence of covid- in tehran when compared to the less densely populated provinces of semnan, qom, markazi, yazd, mazandaran, qazvin, guilan, alborz and isfahan (fig. , table ). thus, other demographic and environmental factors may explain the differences in vulnerability to the covid- epidemic among the iranian provinces. our statistical analyses showed that the association between the incidence of covid- in iranian provinces and their respective distance from qom is highly significant. this indicates that the implementation of strict travel restriction from highly affected areas to other provinces could considerably reduce the rate of transmission of the disease throughout the country. the chinese experience showed that the home-based quarantine of people living in hubei province (the epicenter of the epidemic) and strict travel policies, extended even long after the lunar new year holiday, have been particularly helpful in controlling of the transmission of the disease to the wider community and other provinces [ ] . iranian provinces with an aging population were identified as particularly at risk for the spread of covid- . age distribution has previously been shown to significantly influence the case-fatality rate due to the epidemic [ , ] . in this respect, it has been proposed that the older population is particularly at risk because of reduced immune function [ ] . in iran, older age and having comorbidities were strongly correlated with the risk of death among patients suffering from covid- [ ] . this highlights the need to implement proactive measures to protect the health of the elderly population from covid- with a special emphasis on physical distancing and hygienic measures for individuals exposed to higher risks [ , [ ] [ ] [ ] . this is a challenge that is coupled with a moral and social responsibility to take care of the health, morals, and safety of our elders in the view of the effectiveness and sustainability of the efforts in tackling the pandemic [ ] . to conclude, we found a negative linear association between the incidence of covid- in iranian provinces and their respective distance from qom. our regression analysis showed that the relationship between the variables is strong (p < . ; c = - . ). the incidence of covid- in iranian provinces was also positively associated with the ratio of the population over . these data stress the need for preventive and monitoring measures to counter the spread of the disease throughout the country, taking into account demographic and geographic characteristics of different provinces. covid- battle during the toughest sanctions against iran emerging novel coronavirus ( 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interpretation association of the incidence of covid- with the density of the population (a) and the proportion of the elderly population (b) in iranian provinces covid- control in china during mass population movements at new year case-fatality rate and characteristics of patients dying in relation to covid- in italy characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid- ) during the early outbreak period: a scoping review epidemiological characteristics of coronavirus disease (covid- ) patients in iran: a single center study responding to covid- : how to navigate a public health emergency legally and ethically covid- , an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics. hum vaccine immunother individual risk management strategy and potential therapeutic options for the covid- pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declared no competing interests. key: cord- -wet go authors: jia, w.; karaca, k.; parrish, c. r.; naqi, s. a. title: a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: wet go an antigenic variant of avian infectious bronchitis virus (ibv), a coronavirus, was isolated and characterized. this strain, cu-t , possesses a number of unusual features, which have not been previously observed in ibv. the s glycoprotein of cu-t carries virus-neutralizing and serotype-specific epitopes of two ibv serotypes, arkansas (ark) and massachusetts (mass). sequence analysis revealed that the virus, originally an ark serotype, has acquired the mass-specific epitope by mutation(s). this provides evidence that point mutations may lead to generation of ibv antigenic variants in the field. it was further observed that two independent recombination events involving three different ibv strains had occurred in the s glycoprotein gene and n protein gene of cu-t , indicating that genomic rna recombination in ibv may occur in multiple genes in nature. it was especially significant that a sequence of holland (a vaccine strain) had replaced half of the n gene of cu-t . this proves that recombination among vaccine strains is contributing to the generation of ibv variants in the field. based on these observations it is predicted that every ibv field isolate could have unique genetic nature. therefore, several recently reported diagnostic and serotyping methods of ibv which are based on dot-blot hybridization, restriction fragment length polymorphism (rflp), and polymerase chain reaction (pcr), may not reveal the true antigenic and/or genetic nature of ibv isolates, and may in fact yield misleading information. results in heavy economic losses to the commercial poultry industry, worldwide [ ] . ibv has a single-stranded, positive sense rna genome. it is . kilobases long, encodes three major structural proteins, and is organized as '-pol-s-gene -m-gene -n- ' (fig. ) [ , , ] . the three structural proteins of ibv are the spike glycoprotein (s protein), the membrane glycoprotein (m protein) and the nucleocapsid protein (n protein). the s protein of ibv is cleaved post-translationally into n-terminal s and c-terminal $ proteins [ ] . the s protein carries antigenic epitopes which induce virus neutralizing (vn) antibody, and also determine the virus serotype [ , ] . the n protein may induce cellular immune responses against ibv [ ] . ibv isolates have been grouped into serotypes based on vn test [ , ] . although more than twenty distinct ibv serotypes have been reported in the usa [ , ] , most isolates causing disease in the field belong to massachusetts (mass), connecticut (conn), or arkansas (ark) serotypes [ ] . live vaccines containing strains of those three serotypes have been routinely used in the field. however, more frequently than ever, outbreaks of the disease are being observed in vaccinated flocks. these vaccine failures, however, are not always due to infection by distinct serotypes, but could be caused by antigenic variants of ibv emerging from wild-type or vaccine viruses by point mutation(s) or genomic rna-rna recombination [ , ] . although point mutation(s) are believed to contribute to the generation of new antigenic variants of ibv, no direct evidence for that has been presented. rna-rna recombination has been shown to occur at a high frequency both in vivo and in vitro in mouse hepatitis virus (mhv), a coronavirus i- , , , [ ] [ ] [ ] ], yet, no isolation of recombinant mhv from natural disease in mice has been reported. in the case of ibv, one japanese and three european isolates have been suggested to be possible recombinants [ , ] . however, the origin of ~-~'~unknown-oriqin sequence[-------]hol - ike sequence ~ hvr fig. . schematic characterization of the cu-t genome. pol rna polymerase gene. s s protein gene; $ s protein gene; $ $ protein gene. n n protein gene. u tr ' end untranslated region. hvr hypervariabte region the recombination fragments in those isolates was unknown. more recently, two american field isolates were found to contain fragments of mass-like sequences in the s gene, which were % to % homologous to ibv strain mass [ ] . all the recombinations suggested in ibv so far have been in the s gene, and none of those reports have addressed whether the recombinations had altered the antigenic characteristics of the viruses. here we describe an ibv serotype and recombination variant, cu-t , which reacted with two monoclonal antibodies (mabs) specific to the vn epitopes of ibv serotypes mass and ark, respectively. we present evidence that the cu-t was originally an ark serotype which subsequently acquired a mass-specific epitope on the s protein through mutation(s). we also show that cu-t had undergone two independent genomic rna recombination events within the $ and n protein gene (n gene), respectively, involving three different ibv strains. furthermore, we provide evidence that cu-t had acquired a sequence of holland (ho ), a vaccine strain of mass serotype used in the field. implications of such genetic and antigenic changes in diagnosis and serotyping of ibv are discussed. four previously characterized ibv strains, ark , hol , corm , and mass [ , ] were obtained from our own laboratory stocks. cu-t was isolated from an adult commercial chicken flock in new york state, which was experiencing reduced egg production and poor egg shell quality associated with ibv infection. virus isolation from the flock was performed through the use of "sentinel" chickens. briefly, ten specificpathogen-free (spf) chickens were placed among the affected birds in separate cages for ten days. subsequently, the "sentinel" chickens were euthanized and the tracheas were collected for passage in spf chicken embryos. cu-t strain was recovered from the allantoic fluid of inoculated embryos, and was subsequently propagated and plaque purified in chicken kidney cell (ckc) culture monolayers [ ] . mabs , , and specific to sl-associated serotype-specific epitopes of ibv prototype ark, mass, and conn, respectively, have been described elsewhere [ ] . the protocols for both antigen-capture and competitive-binding elisas have also been reported previously [ , ] . the elisas were performed using whole virions, or purified s protein of cu-t according to the procedures used previously [ ] . viruses were propagated in chicken embryos and purified by sucrose gradient as described previously [ ] . viral genomic rnas were extracted with phenol-chloroform and used for cdna synthesis and rna sequencing. cdna synthesis, cionin and sequencin for cdna synthesis, an uni-zap ii kit (stratagene, la jolla, ca) was used in conjunction with a synthetic primer complementary to a sequence located downstream of the s genes of w. jia et al. several mass serotype strains [ ] ( '-gaactagtctcgaggaaggacgtgggact-ttg-y). the cdna library was screened by hybridization with a ' end-radiolabeled probe prepared from a styi fragment derived from a cdna clone (pbsm m) of s gene of mass obtained from solvay animal health, inc., mendota heights, mn. phagemids containing ibv genes were prepared by in vivo excision in escherichia coli (e. coli) using a protocol furnished by stratagene (la jolla, ca). dna sequencing was carried out by the dideoxy method, using a sequenase . dna sequencing kit (united states biological (usb), cleveland, oh). cdna clones park - pl, pbark - p , pbt -t , pbt -t , and pbt -t were used for sequencing. both strands of each cdna clone were sequenced. sequence data were obtained from cdna clones, except for the first bases of the s gene, the last bases of the n gene, the ' end non-coding region of cu-t , and the partial gene of ark and ho , which were obtained from direct sequencing of ibv genomic rna. direct sequencing of ibv genomic rna was also performed at least twice for those regions, using an rna sequencing kit (usb, cleveland, oh). sequence alignments and analysis were performed using the genetics computer group (gcg) program. the nucleotide sequences reported here have been deposited with the genbank. the accession numbers are as follows: s protein gene of ibv ark , l ; s protein gene of ibv cu -t , u ; n protein gene of ibv cu-t , u ; ' end non-coding region of ibv cu-t , u . tables and summarize results of the three types of elisas performed with the ark, conn, and mass serotype-specific mabs. in both the indirect and antigen-capture elisa, the ark-specific and mass-specific mabs reacted with both the whole virions and the purified s protein of cu t strain (table ) . in the competitive-binding elisa, unlabeled mass-and ark-specific mabs blocked reaction of the homologous labeled mabs, but not with the heterotogous mabs. in order to determine the genetic basis for the expression of the ark-and mass-specific epitopes on the $ protein ofcu t , the entire s gene of cu t was sequenced. the entire s gene of ark was also sequenced for comparison. all base positions indicated in this paper are calculated from the s gene start codon [ ] . amino acid positions indicated are of the s protein precursor, including the signal peptide. the gene of ark was found to be bases long, with a capacity to code amino acids. the s and $ genes were and bases long, respectively, and were predicted to encode and amino acids, respectively. comparison of the deduced amino acid sequences of the s proteins of ark and mass [ ] , revealed an . ~o homology ( / ). however, the s protein showed only . ~o homology ( / ), compared to . % ( / ) for the $ (data not shown). amino acid variations in the s proteins of the two viruses were more than times as frequent as for the $ protein. alignment of the $ gene of ark with that of mass and several european ibv isolates revealed that fifteen bases ( '-tggaagtgctacgcc- ') between bases and of the ark strain were unique to that virus (data not shown). a . ~ homology was observed between the first bases of the $ gene of ark and mass , while the last bases of the two genes were identical (fig. ) . the nucleotide sequences of s genes of cu-t and ark exhibited over ~o homology° the fifteen-base insertion in the s gene of ark described above was also observed in the cu-t $ gene, but its location was between bases t agggtcttaa tgactc-tt-atagaccttg aaa-a-t-tc aatactcaaa acttatatta agtggccttg gtatgtgtgg ttagccatag cttt-gccac t c a a a gc tc (~ gt c q a tattatcttc atc-taata-tagg-tgggt tttcttcatg actgg-tgtt gtggttgttg ttgtggatgc tttggcatta tgcctctaat gagtaagtgt t c t t and , as compared to bases and for ark . the difference in the position of the insertion was due to a three-base deletion (taa) between bases and of the s gene of cu-t compared to the s gene of ark . a single noncoding substitution was noted within the fifteen-base insertion of cu-t (t versus c at position ). a . % homology was observed between the amino acid sequences of the s proteins of cu-t and ark . seventeen amino acids were found to be different in the s protein of cu-t compared to those in ark , and those differences were distributed throughout the gene (data not shown). a . ~o homology was observed in the first bases of the $ genes of ark and cu-t , but, the following bases had only . /o homology (fig. ) . comparison with published sequences showed that the sequence of this -base region of cu t was more similar to a fragment in the ' end of the $ gene of a japanese isolate kb [ ] than to those of all other strains. although nucleotide substitutions were found in that fragment of the cu-t and kb , of those were synonymous substitutions (fig. ) . the downstream crossover site of this fragment was about to bases downstream of the stop codon of the $ gene of cu-t . through a substitution of t by g in the taa stop codon, the $ gene of cu-t was bases longer than that of ark , but of the same length as the $ gene of kb (fig. ) , indicating that a fragment of approximately bases was incorporated into the $ gene of cu-t by recombination. to further study the possible evolutionary origin of the cu-t strain, the first bases of gene , the n gene and the ' non-coding region of cu-t were sequenced subsequently. the same regions of gene of ark and ho were also sequenced for comparison. the first bases of the gene of cu-t matched more closely with those of ark gene ( . ~o homology), than of mass [ ] , kb [ ], or ho (table ) . the n gene of cu-t was t bases long, with a capacity to code amino acids. around bases at the ' end and bases at the ' end of i i g a g g a c t a g t g atggc~gcg gt~ggc~c tggaaagaca gacgccccag cgccagtcat caaactagga ggaccaaa-c cacctaaagt tggttcttct ggaaatgcat c t t c tt t a t t t cttggtttca agc~t#~ gcc~gaagc taaattcacc tccacct~g tttg~ggta gcggtgttcc tgat~tg~ ~tcttaaaa c~gccagca g t c ac t c a t t a g c t g t a t acatgg-tac tggagacgcc ~gc-aggtt t~gccaggt aaaggcggaa gaaaaccagt cccagatgct tggtacttct attatactgg aacaggacca t g c c gt c a t a gccgctgacc tg~ttgggg tgatagcc~ gatggtatag tgtgggttgc tgcaaagggt gctgatacta ~tctagatc t~ccagggt ac~gggacc t t tt g a c gaa at t t t atc~cagc-gcttcatcag cagcatctag tagagcaccg tcgcgtgatg gctcgcgtgg -cgtag~g-gg-gctg~g atgatcttat tgctcgtgca t a ~ ag t c c c t gc~j~agatca ttcaggatca gcag~g~g ggttctcgca ttactaa-gc t~ggc-gat gaaatggctc atcgccggta ttg- t t c c a -gttacagca atgctcaacc tagtccctag cagccatgct tgtctttttg gaagtagagt gacgcccaaa cttc~ccag atgggctgca cttga-attt cc t t a a t q t ag t t g~tttacta ctgtggtttc acgtgatgat ccgcagtttg at~ttatgt gaaaatttgt gatcagtgtg t- g c c a t a g c g g ta t t g~gcaggat gatgaagtag ataaagcatt gacctcagat gaggagagga acaatgcaca gctggaattt gatgatg~c cc~ggtgat t~-tggggg ac t a gt g a tt a tt a c ac t a gattcagc-- t-ggtgag~ tg~ctttga cut n gene were very similar to the comparable regions of ark . however, two crossover sites were observed around bases and (fig. ) . between those two sites only five bases were different ( . % homology) between the cu-t and the ho strains, and of those, only two were non-synonymous (fig. ) . within the same region, bases were different between cu-t and ark ( . % homology), and eight of those changes were non-synonymous (fig. ). there were many more differences in that region between cu-t and other ibv strains, including mass , gray, kb and beaudette (data not shown). the ' end untranslated region (utr) of the ibv genome was previously shown to contain an approximately -base hypervariable region (hvr) [ ] (fig. ) . that region of the cu-t sequence had a high homology with ark ( . ~), but lesser homology with kb ( . %), ho ( . %) and mass ( . ~) (data not shown). the results show that cu-t is a serotype and recombination variant which originated from an ark-like ibv. this is based on both the sequence data and mab studies of the virus. the nucleotide sequence homologies between the s gene, most of $ gene, gene and the ' utr of cu-t and ark all attest to the origin of cu-t from an ark-like virus. the fifteen-base insert observed in the $ genes of both ark and cu-t , which was absent in the mass serotype strains, further strengthens this conclusion (fig. ) . the seventeen amino acids in the s protein of cu-t which were different from those in ark were distributed throughout the gene, indicating that these amino acid changes were not due to recombination. therefore, it may be concluded that cu-t acquired the mass-specific serotype epitope on the s protein as a result of point mutation(s). the results of competitive-binding elisas indicated that the ark-and mass-specific epitopes on cu-t are independent. these facts provide direct evidence that point mutation(s) can result in the generation of antigenic variants during a natural infection. the results show that an approximately base-long fragment of unknown origin was incorporated into the ' end of cu t $ gene by recombination. interestingly, this recombinant fragment was similar to one previously observed in kb , a possible recombinant strain of ibv [ ] . recombination fragments in other two european isolates ( / and d ) were also found within the same regions [ ] , although they were of shorter length than those of cu-t and kb strains. in an in vitro transfection experiment it was observed that rna recombination in mhv occurred preferentially at certain selected sites (hot spots) [ ] . in ibv, a secondary structure most likely exists at the ' end of gene [ ] . we found that the downstream crossover sites of both cu-t and kb were located around that secondary structure (data not shown). the clustering of recombinations in ibv suggests that a recombination 'hot spot' most likely exists around the ' end of the s gene and the ' end of the gene , although the clustering may be a result of selection [ ] . an additional recombination which resulted in the replacement of half ( . ~) of the n gene was also observed, indicating that genomic rna recombination of ibv may involve more than two strains and may occur in multiple genes during natural infection. more interestingly, that recombination fragment in the cu-t is almost identical to a sequence ( . ~o homology) in the n gene of ho strain. since both ark and ho strains have been used simultaneously as live vaccines in the usa [ ] , this finding provides convincing evidence that recombination among vaccine strains is contributing to the emergence of variants in the field. the significance of recombination in the n gene must also be viewed in the light of a recent report that the n protein may induce cellular immune responses against ibv [ ] . the sequence of s portion of ark s gene obtained from a cloned pcr product has been published more recently [ ] . when the s portion of the entire s gene sequence of ark obtained in this study was compared with that sequence, nine bases were found to be different, which would result in four amino acid changes in the s protein at positions , , and . our data suggest that both inter-strain recombinations and mutation(s) are contributing to the generation of ibv variants in the field. we have shown here that genomic recombination between ibv strains may lead to replacements of large rna fragments in multiple genes. the replacement, however, may not result in change of antigenic nature of ibv. in contrast, mutation(s) on the s protein alone may generate new vn epitope(s). the data imply that every field isolate of ibv could be unique in rna sequence. therefore, recently reported diagnostic and serotyping methods of ibv, such as dot-blot hybridization [ ] , restriction fragment length polymorphism (rflp) and polymerase chain reaction (pcr) [ , ] , may not reflect the true antigenic and/or genetic nature of the virus. indeed, an exclusive use of such assays may yield misleading information on the antigenic and/or genetic characteristics of the virus. a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus random nature of coronavirus rna recombination in the absence of selection pressure establishing a genetic recombination map for murine coronavirus strain a complementation groups cloning and sequencing of the gene encoding the spike protein of the coronavirus ibv comparison of the spike precursor sequences of coronavirus ibv strains m and / with that of ibv beaudette induction of anti-viral immune responses by immunization with recombination-dna encoded avian coronavirus nucleocapsid protein sequencing of coronavirus ibv genomic rna: three open reading frames in the ' 'unique' region of mrna d sequence of the nucleocapsid genes from two strains of avian infectious bronchitis virus completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus sequence analysis of strains of avian infectious bronchitis coronavirus isolated during the s in the infectious bronchitis virus: evidence for recombination within the massachusetts serotype location of the amino acid differences in the s spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus coronavirus ibv: partial amino terminal sequencing of spike polypeptide $ identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor polypeptide of ibv strains beaudette and m coronavirus ibv: virus retaining spike glycopolypeptide $ but not s is unable to induce virus-neutralizing or haemagglutination inhibiting antibody, or induce chicken tracheal protection coronavirus ibv: removal of spike glycopolypeptide s by urea abolishes infectivity and haemagglutination but not attachment to cells serotyping of avian infectious bronchitis viruses by the virus-neutralization test variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens serological comparisons of strains of infectious bronchitis virus using plaque purified isolates the neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures immunologic differences in strains of infectious bronchitis production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes in vivo rna-rna recombination of coronavirus in mouse brain rna recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus a and fusion-negative mouse hepatitis virus avian infectious bronchitis in: calnek bw sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis rna recombination in animal and plant viruses recombination between nonsegmented rna genomes ofmurine coronaviruses rna recombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus high-frequency rna recombination of murine coronaviruses an overview of infectious bronchitis virus serotypes dot-blot hybridization using digoxigenin-labeled cdna probe complementary to the s gene of avian infectious bronchitis virus permits discrimination between virus strains a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes rapid detection and identification of avian infectious bronchitis virus we thank ms. beverley bauman, and ms. alice adriguetto for their technical assistance, and dr. bruce calnek and dr. randall renshaw for critical reading of the manuscript. the clone of mass s gene was kindly provided by dr. georges thiry, sotvay animal health, inc. this work was partially supported by a grant from sotvay animal health, inc.recombination among ibv strains key: cord- - tqnt l authors: aita, tsunehiko; kuwabara, masaki; murayama, kazunori; sasagawa, yuri; yabe, shizuka; higuchi, ryohei; tamura, tsutomu; miyazaki, ayako; tsunemitsu, hiroshi title: characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: tqnt l bovine torovirus (btov) is recognized as an enteric pathogen of calves, but its etiological role in diarrhea and epidemiological characterization in adult cows remain unclear. in - , three outbreaks of epidemic diarrhea occurred in adult cows at three dairy farms in niigata prefecture, japan. btov was the only enteric pathogen detected in these outbreaks, as determined by electron microscopy, reverse transcription-pcr, bacteria and parasite tests of fecal samples, and antibody tests with paired sera. the epidemiological features of the three outbreaks were similar to those of bovine coronavirus infection, except for the absence of bloody diarrhea, with diarrhea spreading among most adult cows, but not in calves, within several days and diarrhea lasting for - days with anorexia. decreased milk production and mild respiratory symptoms were also observed in two of the outbreaks. nucleotide sequence analysis of the btov nucleocapsid, spike, and hemagglutinin-esterase (he) genes revealed a close relatedness among the detected btov strains from each outbreak and those of japanese btov strain aichi/ . furthermore, we isolated a btov strain, designated niigata (tc), from a fecal sample using a human rectal tumor cell line. sequence analysis of this isolate and aichi/ indicated that both strains have truncated he genes with deletions in the ′ region that occurred through cell culture-adaptation. the short projections that are believed to be formed by the he protein on virus particles were not observed in these cultured strains by electron microscopy. taken together, these results suggest that btov causes epidemic diarrhea in adult cows and should be included in the differential diagnosis of diarrhea in adult cows. in addition, our findings indicate that the he protein of btov may not be necessary for viral replication. epidemic diarrhea in adult cattle is frequently observed worldwide, particularly among dairy cattle, and results in large economic losses from marked reductions in milk production. a major causative agent of adult cattle diarrhea is bovine coronavirus (bcv) [ , ] , which leads to the disease winter dysentery (wd). however, other viruses, including bovine torovirus (btov), are also suspected to be etiologic agents of diarrhea [ , , ] , although the epidemiological situation of infections caused by these viruses in adult cattle remains unclear. btov, a member of the genus torovirus within the family coronaviridae, is a spherical, oval, elongated, or kidney-shaped enveloped virus and displays a double fringe of peplomers on its surface [ ] . the virus has a single-stranded rna genome of positive polarity encoding rna polymerase and four structural proteins: the spike (s), membrane (m), hemagglutinin-esterase (he), and nucleocapsid (n) proteins [ ] . since the first isolation of btov in from a case of neonatal calf diarrhea in the united states [ ] , several reports have confirmed the association of btov with calf diarrhea in experimentally infected gnotobiotic calves and under field conditions [ , , , , ] . serological surveys have also revealed a high prevalence of antibodies to btov in cattle populations, ranging between % and % [ , , , , ] . recently, kuwabara et al. [ ] succeeded in isolating btov using a human rectal tumor cell line , which is expected to facilitate the diagnosis of btov infection by virus neutralization tests and virus isolation. btov has been detected in fecal samples from adult cattle with diarrhea [ , , ] . in addition, a few studies have reported that adult cows with wd-like diarrhea exhibited various degrees of seroconversion to btov [ , ] . these results suggest that btov may be a causative agent of epidemic diarrhea of adult cattle. however, the epidemiological data for btov infection in adult cattle under field conditions are limited, and the etiologic role of btov in epidemic diarrhea of adult cattle remains unclear. in the present study, we report the epidemiological features of three outbreaks of epidemic diarrhea in adult cows between may and february at three dairy farms in niigata prefecture, japan, in which btov was detected as the only pathogen. further, we describe the isolation of a cytopathogenic btov strain from diarrheic feces using hrt- cells, as well as the molecular characterization of the detected btovs. three epidemic outbreaks of adult cattle diarrhea occurred between may and february in niigata prefecture, japan. a total of fecal samples were collected from diarrheic adult cows aged to months: samples (niigata - to niigata - ) from outbreak (farm ), samples (niigata - to niigata - ) from outbreak (farm ), and samples (niigata - to niigata - ) from outbreak (farm ). nasal swabs were also collected at the time of fecal sampling from the cows involved in outbreak . paired serum samples were collected in the acute phase of diarrhea and to days later. fecal samples were diluted : in . m phosphate-buffered saline (pbs; ph . ), and nasal swabs were suspended in ml pbs. the resulting suspensions were clarified by low-speed centrifugation at , g for min and used for virus isolation and reverse transcription-polymerase chain reaction (rt-pcr). fecal samples were also tested for salmonella species using a standard technique, while coccidium species and cryptosporidium species were detected by a sucrose floatation method. in addition, fecal samples were examined for rotavirus double-stranded rna by polyacrylamide gel electrophoresis [ ] . virus neutralizing antibody titers against btov in paired sera from affected cows were measured using hrt- cells and the btov strain aichi/ [ ] . virus neutralization (vn) tests were also conducted for bovine viral diarrhea (bvdv) type , bovine adenovirus type (badv- ), bovine herpesvirus (bhv- ), and bovine respiratory syncytial virus (brsv) as described previously [ ] . antibody titers against bcv, adenovirus type (badv- ), and bovine parainfluenza virus type (bpiv- ) were determined by hemagglutination inhibition (hi) tests [ ] . seroconversion was defined as a fourfold or greater increase in paired serum antibody titers to the examined virus. fecal suspensions and the supernatants of infected cell cultures were partially purified by ultracentrifugation through a % (wt/wt) sucrose cushion, negatively stained with % sodium phosphotungstic acid (ph . ), and examined using an electron microscope (jem- s; jeol, ltd., tokyo, japan). two lines of human rectal tumor (hrt- ) and madin-darby bovine kidney (mdbk) cells were used for virus isolation. one line of hrt- cells (designated hrt- aichi cells), which had been maintained in aichi prefecture, japan, was used for the first isolation of btov in cell culture [ ] , and the second hrt- cell line (designated hrt- niigata cells) had been maintained in our laboratory. confluent monolayers of these cells in -well plates were washed with eagle's mem (emem) and then inoculated with . ml of the fecal suspensions diluted from : to : in emem with and without lg/ml trypsin (type i; sigma-aldrich, st. louis, mo, usa). after adsorption for min at °c, the cells were washed once with emem, . ml emem was added, and they were further incubated for or days at °c and examined for cytopathic effects (cpe). after incubation, the cells and culture supernatant were frozen and thawed once to harvest cell lysates, and subsequent passages were carried out in the same manner with . ml of cell lysate. after two passages, the viral isolate was cloned three times in hrt- cells by limiting dilution. the isolate was identified by indirect immunofluorescence and vn tests with gnotobiotic calf antisera against btov (kindly supplied by dr. linda saif, the ohio state university) [ ] and bcv as described previously [ , ] . for vn tests, btov strain aichi/ [ ] and bcv strain kakegawa [ ] were used as reference viruses. electron microscopy and rt-pcr were also performed for the identification of the isolate. viral rna was extracted from fecal suspensions, nasal swabs, and infected cell culture supernatants using trizol ls regent (invitrogen corp., carlsbad, ca, usa) according to the manufacturer's instructions. rt-pcr assays were performed using a onestep rt-pcr kit (qiagen, valencia, ca, usa) and primer pairs targeting fragments of the n-, s-, and he-specific genes of torovirus. the primers, which were identical to those described previously by hoet et al. [ ] and smits et al. [ ] , were as follows: ( rt-pcr was also performed for detection of the specific genes of bcv [ ] , group a rotavirus (gar) [ ] , group b rotavirus (gbr) [ ] , group c rotavirus (gcr) [ ] , and bvdv [ ] . sequence analysis pcr products of the n, s, and he genes of btov were sequenced directly by cycle sequencing with an automatic sequencer (abi prism ; applied biosystems, tokyo, japan). sequencing was also performed for the he gene of strain aichi/ [ ] , which was passaged nine times in hrt- aichi cells, and for the n, s, and he genes from the large-intestinal content of the original specimen from which aichi/ was isolated, designated as aichi/ (lic). sequence data were analyzed by the clustal w method [ ] using the megalign . program of lasergene software (dnastar, madison, wi, usa). phylogenetic trees were constructed using the mega program [ ] . the newly determined sequences have been deposited in the ddbj nucleotide sequence database and assigned the following accession numbers: niigata (n gene, ab ; s gene, ab ; he gene, ab ), niigata (n gene, ab ; s gene, ab ; he gene, ab ), niigata (n gene, ab ; s gene, ab ; he gene, ab ), niigata (tc) (he gene, ab ), aichi/ (lic) (he gene, ab ), and aichi/ (he gene, ab ). on may , , diarrhea and anorexia were observed in a few adult lactating cows on a dairy farm (farm ), with of adult cows on the farm displaying similar symptoms within several days. the diarrheal feces were watery and brownish. all affected cows recovered from diarrhea within - days, but milk production had decreased by up to % on the second day following the first finding of diarrhea and continued for one week. on november , , a few adult lactating cows on a dairy farm (farm ) showed pasty or watery diarrhea that was accompanied by nasal discharge. within days, of adult cows showed diarrhea and most of them had nasal discharge. milk production decreased by approximately % on the fourth day after the onset of diarrhea. all affected cows recovered from diarrhea within - days. the farm was located approximately km from farm . no epidemiological relationship was observed between farms and . on february , , symptoms of pasty or watery diarrhea, anorexia, and mild cough were observed in a few adult lactating cows on a dairy farm (farm ), located km away from farm . within one week, all adult cows on the farm showed similar clinical signs. all cows recovered from diarrhea after - days, and no clinical signs were observed on the tenth day after the first finding of diarrhea. milk production did not change during the outbreak. several common epidemiological features were observed among the three outbreaks. affected cows did not show bloody diarrhea or fever, and each cow recovered within - days after the onset of diarrhea without clinical treatment. notably, no clinical signs, including diarrhea, were observed in calves during these outbreaks of adult cow diarrhea. all fecal samples were rt-pcr negative for bcv, bvdv, gar, gbr, and gcr, and were also negative for species of salmonella, coccidium, and cryptosporidium. in addition, none of the samples were positive for rotavirus rnas by polyacrylamide gel electrophoresis. in contrast, all fecal samples except for niigta - were positive for n, s, and he genes of btov by rt-pcr. one nasal swab sample from outbreak was also positive for the btov n gene by rt-pcr, while we could not detect the s and he genes of btov in any nasal samples. examination of paired sera from of affected cows showed seroconversion (c -fold increase) to btov, and serum samples from the remaining two cows had antibody titers of c at the acute phase (table ) . no significant change in bcv antibody titers was observed in any of the outbreaks (table ) . additionally, antibody titers of the other viruses examined (bvdv type , badv- , badv- , bhv- , brsv, and bpiv- ) did not markedly change in any of the paired sera. after the second passage, cpe appeared in hrt- aichi cells inoculated with one fecal sample, niigata - , diluted : with emem containing trypsin. cpe, which was characterized by the enlargement of cells, was observed to days after inoculation, with the cells eventually detaching from the plastic surface. the isolate was cloned by limiting dilution three times, and the infective titers reached . tcid /ml by passage . indirect immunofluorescence microscopy of hrt- aichi cells that had been inoculated with the isolate and reacted with gnotobiotic calf antiserum against btov revealed cytoplasmic fluorescence, indicating the presence of btov antigens [ ] . in contrast, no fluorescent cells were observed when the infected cells were reacted with anti-bcv antiserum. in vn tests, antiserum to btov neutralized strain aichi/ and the isolate at titers of , and , , respectively. in contrast, antiserum to bcv failed to neutralize either strain (titer, \ ). in addition, the isolate was positive in rt-pcr targeting the n, s, and he genes of btov. electron microscopy and nucleotide sequencing of the rt-pcr products were also conducted for identification of the isolate. from these results, we concluded that the isolate from fecal sample niigata - was btov and designated the new isolate as strain niigata (tc). in contrast, no cytopathogenic viruses were isolated from any samples using hrt- niigata or mdbk cells. coronavirus-like particles were observed by electron microscopy in of fecal samples (niigata - , - , - ; niigata - , and niigata - ) from the three outbreaks. the particles were spherical, oval, elongated, or kidney-shaped, approximately - nm in diameter, and surrounded by club-shaped projections of - nm in length. the particles also had short projections of approximately nm in length (fig. a, b) . no other virus-like particles were observed in any of the fecal samples. in the culture supernatants of infected htr- aichi cells, coronavirus-like particles with long club-shaped projections similar to those detected in the fecal specimens were observed. however, short projections were not seen on these particles (fig. c, d) . the nucleotide sequences of the n ( nucleotides [nt]), s ( nt), and he genes ( nt) of btov from nine fecal samples obtained from the three farms and btov isolate niigata (tc) were compared with those of published toroviruses. the nine selected fecal samples were as follows: niigata - , - , and - from outbreak , niigata - and - from outbreak , and niigata - , - , - , and - from outbreak . in addition, the nucleotide sequences of the viral genes of aichi/ and aichi/ (lic) were compared (fig. ) . as the nucleotide sequences of btov genes from the same outbreak were identical, btov detected from the feces in each respective outbreak was designated as strains niigata , niigata , and niigata . for the n gene, the nucleotide sequences of niigata and niigata were identical to that of the b strain detected in the netherlands and showed % nucleotide identity to niigata . for the s gene, niigata and niigata showed % nucleotide identity, and they displayed %- % nucleotide identity to niigata and aichi/ . for the he gene, niigata , niigata , and niigata showed %- % nucleotide identity to each other and %- % nucleotide identity to aichi/ . in addition, the nucleotide sequences of both the n and s genes between niigata and niigata (tc) and between aichi/ (lic) and aichi/ showed % identity. phylogenetic analysis of the n, s, and he genes indicated that strains niigata , niigata , and niigata were grouped within the same cluster as other btov strains, including aichi/ and b (fig. ) . in the he gene, the detected open reading frames of strains niigata , nii-gata , niigata , and aichi/ (lic) encoded amino acids, which is identical to the number reported previously for other strains of btov. in contrast, both cell-cultureadapted aichi/ and niigata (tc) strains of btov had incomplete he open reading frames that only encoded and amino acids, respectively (fig. ) . btov has been suspected to be a causative agent of adult cattle diarrhea based on its detection in diarrheic feces and because various degrees of seroconversion to btov have been observed in outbreaks of wd-like diarrhea in adult cows [ , , , , ] . however, prior to the present study, no conclusive evidence for a link between btov and epidemic diarrhea in adult cattle had been reported. furthermore, epidemiological characterization of these diarrheal outbreaks remained unclear. here, we examined the etiological agents and conducted detailed serological tests with samples from three outbreaks of adult cattle diarrhea in niigata, japan, and found that btov was the only causative pathogen. in the three outbreaks examined, most affected cows developed watery diarrhea, anorexia, and decreased milk production, and additionally, some cows also showed slight respiratory signs. the observed clinical signs were similar to those encountered in wd and bcv infections. notably, none of the calves developed diarrhea during the outbreaks, a diagnostic characteristic that is also observed in wd and bcv infections in adult cows [ , ] . taken together, these results suggest that adult cattle diarrhea caused by btov may have been misdiagnosed as wd or bcv infection by clinical investigations. we isolated the cytopathogenic btov strain nii-gata (tc) using hrt- cells. of note, a clear difference in sensitivity to btov was observed between the two hrt- cell lines used in this study. namely, the nii-gata (tc) strain could be isolated and propagated in hrt- aichi cells, but not in hrt- niigata cells. similarly, strain aichi/ could also not be propagated in hrt- niigata cells, although both hrt- cell lines displayed high sensitivity to bcv (data not shown). these results indicate that the use of hrt- aichi cells is critical for the in vitro propagation of btov. although the reason for the different sensitivity between the two hrt- cell lines to btov is unknown, it is possible that the properties of the hrt- aichi cells may have changed during passaging to increase btov tropism. btovs have been classified into two serotypes on the basis of reactivity in the hemagglutination/hi test [ ] . recently, however, btovs have been proposed to consist of a single serotype that includes at least two subtypes [ ] . in the present study, rabbit antisera to strain aichi/ showed no significant differences in virus neutralizing antibody titers between the aichi/ and nii-gata (tc) strains, suggesting that these two strains belong to the same serotype. nucleotide sequence analysis indicated that the btov strains detected in the fecal samples from three outbreaks in niigata prefecture were closely related to each other and to strain aichi/ . this finding, together with the serological results, suggests that these japanese btov strains may have evolved from a recent common ancestor. our sequencing analyses revealed that both cell-cultureadapted btov strains, niigata (tc) and aichi/ , possessed truncated he genes with deletions in the region that occurred through cell culture-adaptation. electron microscopy photographs indicated that virus particles in fecal samples had long and short projections, whereas short projections were not seen on those in the culture supernatants of infected htr- cells. interestingly, in many laboratory strains of mouse hepatitis virus, the he genes are inactivated, which apparently represents an in vitro artifact resulting from adaptation to propagation in cultured cells [ ] . it is noteworthy that cell-culture-adapted equine torovirus berne virus also possesses a truncated he gene with deletions in the region [ ] . taken together with the data presented in this study, the he protein in btov may be unnecessary for viral replication. further examination of the biological properties of the he protein in fecal-and cell-culture-adapted strains of btov is needed to confirm this speculation. in conclusion, we presented the epidemiological characteristics of btov diarrhea in adult cattle based on three outbreaks in niigata, japan. we propose that examination for btov should be included in the routine diagnosis of adult cattle diarrhea. propagation of the kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters detection of breda virus antigen and antibody in humans and animals by enzyme immunoassay genus torovirus assigned to the coronaviridae detection of bovine group b rotaviruses in feces by polymerase chain reaction the complete sequence of the bovine torovirus genome detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens detection of bovine torovirus in neonatal calf diarrhoea in lower austria and styria (austria) enteric and nasal shedding of bovine torovirus (breda virus) in feedlot cattle detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle epidemiological analysis of bovine torovirus in japan genetic and antigenic characterization of newly isolated bovine toroviruses from japanese cattle detection of bovine torovirus in fecal specimens of calves with diarrhea in japan seroepidemiology of breda virus in cattle using elisa association of diarrhea in cattle with torovirus infections on farms first isolation of cytopathogenic bovine torovirus in cell culture from a calf with diarrhea a comparison of simian rotavirus sa preparations maintained in different laboratories cellular lesions in intestinal mucosa of gnotobiotic calves experimentally infected with a new unclassified bovine virus (breda virus) winter dysentery in dairy herds: electron microscopic and serological evidence for an association with coronavirus infection hemagglutination by calf diarrhea coronavirus evidence of torovirus infection in diarrhoeic cattle comparison of the genome organization of toro-and coronaviruses: evidence for two nonhomologous rna recombination events during berne virus evolution phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events epizootic diarrhoea of adult cattle associated with a coronavirus-like agent mega : molecular evolutionary genetics analysis (mega) software version . clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice winter dysentery diagnosed by farmers in dairy herds in central sweden: incidence, clinical signs and protective immunity isolation of bovine coronavirus from feces and nasal swabs of calves with diarrhea sequence comparison of the vp gene encoding the outer capsid glycoprotein among animal and human group c rotaviruses experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr a serologic investigation for coronavirus and breda virus antibody in winter dysentery of dairy cattle in the northeastern united states pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis antibodies to berne virus in horse and other animals studies with an unclassified virus isolated from diarrheic calves comparative studies on three isolates of breda virus of calves immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types and , infectious bovine rhinotracheitis virus, bovine parainfluenza- virus, and bovine respiratory syncytial virus when administered intranasally in young calves heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses acknowledgements we thank n. kawanishi, t. miyamoto, and n. hattori for technical assistance. key: cord- -br uorg authors: zhou, pei; fu, xinliang; yan, zhongshan; fang, bo; huang, san; fu, cheng; hong, malin; li, shoujun title: antiviral effect of lithium chloride on infection of cells by canine parvovirus date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: br uorg canine parvovirus type causes significant viral disease in dogs, with high morbidity, high infectivity, and high mortality. lithium chloride is a potential antiviral drug for viruses. we determined the antiviral effect of lithium chloride on canine parvovirus type in feline kidney cells. the viral dna and proteins of canine parvovirus were suppressed in a dose-dependent manner by lithium chloride. further investigation verified that viral entry into cells was inhibited in a dose-dependent manner by lithium chloride. these results indicated that lithium chloride could be a potential antiviral drug for curing dogs with canine parvovirus infection. the specific steps of canine parvovirus entry into cells that are affected by lithium chloride and its antiviral effect in vivo should be explored in future studies. canine parvovirus type (cpv- ), which was first identified and described in in both the united states and australia, is closely related to feline panleukopenia virus (fpv) [ , , , ] . this virus was named cpv- after an unrelated virus, canine parvovirus type (cpv- ), which causes neonatal death in puppies [ , ] . cpv- causes severe diarrhea and vomiting, and it has a predilection for young puppies, resulting in high mortality due to myocarditis and enteritis [ ] . cpv- has spread rapidly, becoming globally distributed only two years after it was first identified, and it has been demonstrated to be a contagious pathogen to all populations of canids [ , ] . furthermore, cpv- has been evolving, and genetic variants continue to be identified. during and , cpv- was completely replaced globally in canids by a new variant, cpv type a (cpv- a) [ ] [ ] [ ] . the virus underwent further antigenic drift, and a new variant, cpv type b (cpv- b), was observed [ ] . subsequently, in , another novel cpv variant (cpv- c) was detected in several countries [ ] . the mutations of variants have been mapped for vp , which is the most abundant structural protein (cpv- a: val- -ile, asp- -tyr, ala- -gly, ile- -thr and met- -leu; cpv- b: ile- -val reversion and asp- -asn; and cpv- c: asp- -glu). cpv- is highly infectious and can lead to high morbidity and mortality in dogs. currently, vaccination is the best measure for prophylaxis against cpv infection. nevertheless, regardless of the relatively high costs, there are some concerns about the effectiveness of the existing vaccines [ , , , ] and some concerns about the efficacy of existing clinical therapies, which, in addition to symptomatic treatment, include antiserum and interferon treatment. therefore, drug therapy for cpv- infection, as an alternative strategy, warrants more attention. lithium salts are significant therapeutic agents that are used to treat several non-infectious diseases [ , , , ] . in , the antiviral effect of lithium chloride (licl) on dna and rna viruses was investigated. licl inhibited replication of the dna virus herpes simplex but did not inhibit the replication of the rna viruses encephalomyocarditis virus and influenza virus [ ] . more recently, several reports have demonstrated the antiviral effect of licl on dna viruses, such as herpes simplex virus [ ] , pseudorabies herpesvirus [ ] , and porcine parvovirus [ ] . lately, the antiviral effect of licl on rna viruses, such as bronchitis coronavirus [ , ] , transmissible gastroenteritis virus [ ] , and type ii porcine reproductive and respiratory syndrome virus [ ] , has also been demonstrated. these reports indicate that licl might be a potential antiviral drug for other viruses. in this study, we investigated whether licl could inhibit the replication of cpv- in vitro and further explored the antiviral mechanism of licl. the newest strain cpv- c used in this study was maintained in our laboratory. the virus was isolated from a sick dog in guangdong province, china, in . vp was sequenced by the pcr method and found to contain the substitution asp- -glu. feline kidney cells (f ) were obtained from the american type culture collection (atcc). they were cultured in dulbecco's modified eagle medium (dmem) (gibco, usa) containing % fetal bovine serum (fbs) (hyclone, logan, ut) and % penicillin-streptomycin (gibco, ca) at °c and % co . licl (sigma, st. louis, mo, usa) was dissolved in dmem and sterilized by passage through a . -lm filter. cytotoxicity assays were performed according to the manufacturer's instructions for cck (donjindo, japan). for the assay, f cells were cultured in -well plates in serum-free dmem (to prevent cell replication) at °c and % co for hour to allow the cells to adhere to the plates. the cells were washed three times with pbs and then incubated with ll of licl at a series of concentrations ( , , , , , , , mm) in dmem with % fbs (five wells/dilution) for - h. as a control, five wells were mock treated. after washing with pbs, ll of dmem and ll of cck solution were added to each well, and the plate was incubated at °c for - h. the optical density (od) value was measured using a microplate reader (bio-rad, usa) at a wavelength of nm. the relative cell viability was calculated as (mean od drug)/(mean od control) %. the % cytotoxic concentration (cc ) was calculated using graphpad prism (graphpad software, san diego, usa). f cells ( cells) were cultured in -well plates, and nontoxic concentrations ( , , , and mm) of licl mixed with cpv( - tcid /cell) were added to the cells, which were then incubated at °c for h. as a control, cells infected with the same dose of cpv were not treated with licl. subsequently, the antiviral efficacy was evaluated by analysis of viral rna levels, protein expression level and cpe. in addition, interferon (inf-a and inf-b) expression levels were also determined. f cells ( cells) were cultured overnight in -well plates with % fbs dmem, and nontoxic concentrations ( , , , and mm) of licl mixed with cpv ( - tcid /cell) was inoculated into cells, which were then incubated for h at °c (maximal binding) [ ] . as a control, cells were infected with the same dose of cpv with no licl treatment. after removing the drugs and the unbound viruses by washing with cold dmem, the cell lysates were subjected to three freezethaw cycles in preparation for measuring viral loads. f cells ( cells) were cultured overnight in -well plates with % fbs dmem and infected with cpv ( - tcid /cell) at °c for h. after removing the unbound viruses with cold dmem, the cells were incubated with nontoxic concentrations ( , , , , , , and mm) of licl at °c for h. as a control, cells were infected with the same dose of cpv with no licl treatment. after washing with cold dmem, the cells were cultured in % fbs dmem for h. the cell lysates were subjected to three freeze-thaw cycles in preparation for measuring viral loads. f cells ( cells) cultured overnight in -well plates with % fbs dmem, and infected with cpv ( - tcid /cell) at °c for h to allow virus entry [ ] . after washing with cold dmem, the cells were treated with nontoxic concentrations ( , , , , , , and mm) of licl and cultured in % fbs dmem at °c for h. as a control, cells were infected with the same dose of cpv with no licl treatment. subsequently, the cell lysates were subjected to three freeze-thaw cycles in preparation for measurement of viral loads. total dna was extracted using a ra pure viral dna kit (magen, china) according to the instructions of the manufacturer. the pcr primers for the vp gene of cpv are listed in table . total mrna was extracted with trizol reagent (invitrogen, carlsbad, ca, usa) and converted to cdna using oligo d(t) primers and pcr primers for inf-a and inf-b, which are listed in table . real-time quantitative pcr was performed using a real-time pcr system (applied biosystems, usa) with a sybr Ò green pcr master mix kit (applied biosystems, usa), according to the instructions of the manufacturer. the -ddct method with normalization to gapdh was used to calculate the relative mrna expression levels [ ] . f cells cultured in -well plates were inoculated with the cell lysates that were serially diluted tenfold in dmem with five replicates. after cultivating at °c for h, cells were observed for cytopathic effect, and the tcid was calculated by the method of reed and muench [ ] . cells were washed with pbs, fixed with % paraformaldehyde for min, and permeabilized with . % triton x- for min. after washing three times with pbs, the cells were incubated with mouse anti-cpv antibody ( : ) (abcam, ab , britain) for h. subsequently, fitc-conjugated goat anti-mouse igg ( : ) (zhongshan, china) was used as the secondary antibody. as a reference protein, nuclear staining was done with , -diamidino- -phenylindole (dapi) according to instructions of the manufacturer (invitrogen, carlsbad, ca, usa). finally, fluorescence was observed under a leica dmi b microscope (leica, wetzlar, germany). all experiments were performed in triplicate, and the results are reported as the mean ± standard deviation (sd). the significance of differences between experimental groups was determined using an unpaired t-test and a oneway anova using prism . software (graphpad software). a p-value . was selected to indicate significance. the % cytotoxic concentration (cc ) of licl was . mm (fig. ) . licl caused serious cellular toxicity at high concentrations (e.g., mm). concentrations of , , , , and mm were above the % cytostatic concentration ( fig. ) but had no effect on cell morphology when compared with mock-treated cells (data not shown). therefore, - mm was used as the nontoxic concentration range of licl for antiviral tests. to investigate the antiviral activity of licl against cpv, licl was added in a series of concentrations ( , , , mm) prior to cpv infection. for real-time qpcr assays, the mean relative viral dna level of mock-treated cells and cells treated with , , , mm licl was . %, . %, . %, . %, and . % (with mock-treated cells set at %), respectively ( fig. a) . for virus titration ( % tissue culture infected dose, tcid ), the viral titers of mock-treated cells and those treated with , , , and mm licl were . , . , . , . , and . log tcid /ml, respectively (fig. b) . for indirect immunofluorescence assay (ifa), mock-treated f cells produced stronger fluorescent signals at hours after infection with cpv. the fluorescence signals declined after treatment with , , and mm licl (fig. c) . examination by microscopy showed that cpv infection results in the detachment of many cells, while mm treatment prevented most of cells from detaching (fig. ) . these results indicate that treatment of f cells with licl inhibits cpv infection and reduces the cytopathic effect in a dose-dependent manner. the relative mrna levels of inf-a and inf-b were also determined, but the levels in cells that had cpv, licl, and cpv?licl were not significantly different when compared to the mock-treated cells (fig. c ). viral attachment, entry, and replication assays were performed to determine which step in the viral life cycle is affected by licl treatment of f cells. no significant differences in the relative levels of viral vp gene dna or viral titers were observed between drug-treated and mocktreated cells, indicating that licl had no effect on cpv attachment and replication in f cells ( fig. a and b) . in viral entry tests, the relative levels of viral vp gene dna in mock-treated cells and those treated with , , , and mm licl were . %, . %, . %, . % and . %, respectively (fig. a) , and the viral titers of mock-treated cells and those treated with , , , and mm licl were . , . , . , . , and . log tcid /ml, respectively (fig. b) . these results indicate that cpv entry into cells is inhibited in a dosedependent manner by treatment with licl. licl has potential as an antiviral agent the purpose of this study was to determine whether licl could be used as a potential curative agent for cpv- . first, licl concentrations of - mm were determined to have no significant toxicity in f cells and no significant effects on cell morphology. second, after licl treatment, viral dna and viral protein levels decreased and cell morphology improved. these results indicated that licl inhibits cpv infection of f cells. however, ifn expression was not affected by licl treatment, which indicates that the innate immune system is not impacted by licl. thus, the potential antiviral effect of licl should be explored. the viral life cycle of cpv infection includes attachment to cells, entry into cells, and replication in the nucleus. these stages of the viral life cycle were evaluated after licl treatment in this study. cpv attaches to specific receptors on f cells in the initial step of the viral life cycle. the attachment step is significant for virus host tropism. we found that viral attachment was not affected by licl treatment. cpv replicates in the nucleus and requires that certain cellular factors are expressed during the s phase of the cell cycle. we also found that viral replication was not affected by licl treatment. cpv enters cells via an endocytic route that involves microtubule-dependent delivery of cpv to endosomes. this process is mediated by rapid removal of virus via clathrin-coated vesicles [ , ] . our results indicate that the cpv entry into f cells is inhibited in a dose-dependent manner by licl, which indicates that clathrin-coated vesicles might be affected by licl treatment. in conclusion, cpv infection was inhibited in a dosedependent manner by licl treatment of f cells. the antiviral effect of licl was at the step of cpv entry into cells, and this inhibition might involve clathrin-coated vesicles. further research is required to determine how cpv entry into cells is affected by licl and whether licl has an antiviral effect in vivo. fig. the viral load in f cells treated with different concentrations of licl as determined by real-time qpcr(a) and viral titrations (b) at different steps of the viral life cycle (attachment, entry and replication). ns, no significant difference; *, p \ . ; **, p \ . ; ***, p \ . status report: canine viral enteritis canine parvovirus vaccine infectious entry pathway for canine parvovirus recovery and characterization of a minute virus of canines minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing antiviral effect of lithium chloride on infection of cells by porcine parvovirus evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type c severe parvovirus in a -year-old dog that had been repeatedly vaccinated canine parvovirus-a review of epidemiological and diagnostic aspects, with emphasis on type c long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination canine parvovirus infection: which diagnostic test for virus? lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture canine parvovirus types c and b circulating in north american dogs in and an enteric disease of dogs resembling feline panleucopaenia comparative analysis of the effect of glycyrrhizin diammonium and lithium chloride on infectious bronchitis virus infection in vitro analysis of relative gene expression data using real-time quantitative pcr and the -ddct method inhibitory effects of licl on replication of type ii porcine reproductive and respiratory syndrome virus in vitro signal transduction pathways. molecular targets for lithium's actions lithium inhibits alzheimer's disease-like tau protein phosphorylation in neurons cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrinmediated endocytosis, followed by slower intracellular trafficking natural variation of canine parvovirus canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses the global spread and replacement of canine parvovirus strains mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones a simple method of estimating fifty per cent endpoints action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus characteristics and taxonomy of parvoviridae the effect of lithium chloride on the replication of herpes simplex virus antiviral effect of diammonium glycyrrhizinate and lithium chloride on cell infection by pseudorabies herpesvirus the three-dimensional structure of canine parvovirus and its functional implications intracellular route of canine parvovirus entry lithium chloride restores host protein synthesis in herpes simplex virus-infected endothelial cells acknowledgments this work was supported in part by the national natural science foundation of china ( ) and the special fund for agro-scientific research in the public interest ( ). conflict of interest the authors declare no competing financial interests. key: cord- - vam pt authors: li, hao; zhang, bin; yue, hua; tang, cheng title: first detection and genomic characteristics of bovine torovirus in dairy calves in china date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vam pt bovine torovirus (btov) is a diarrhea-causing pathogen. in this study, diarrheic fecal samples from five farms in four provinces in china were collected and tested for btov using a rt-pcr assay, and . % samples were found to be btov positive. moreover, two complete btov genome sequences (mn and mn ) were obtained from the clinical samples, which were , and , nucleotides in length, respectively. sequence analysis showed that the two isolates shared identical amino acid mutations in the s protein compared to the complete s sequences of btov available in the genbank database. in addition, seven consecutive amino acid mutations were found from aa , to , in the s protein of isolate mn . moreover, the two isolates shared one identical amino acid mutation in the receptor binding sites of the he protein. to the best of our knowledge, this is the first report on the epidemic and genomic characterization of btov in china, which is helpful for further understanding the genetic evolution of btov. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. toroviruses are members of the family coronaviridae, order nidovirales, and include bovine torovirus (btov) [ , ] , berne virus (etov) [ ] , porcine torovirus (ptov) [ ] , and human torovirus (htov) [ ] . btov mainly causes diarrhea in calves and adult cattle. the virus can not only be detected in feces but also in the respiratory tract, indicating that the virus has dual tissue tropism [ ] [ ] [ ] [ ] . btov has been detected in countries with a wide geographical distribution [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, a sequence reported to be from goat torovirus is present in the genbank database, and in , researchers detected the presence of ptov in pig herds by rt-pcr in china [ ] . presently, three btov genomic sequence (ay , lc , lc ) can be obtained from the genbank database. the btov genome encodes four structural proteins: spike glycoprotein (s), membrane glycoprotein (m), hemagglutinin esterase (he), and nucleocapsid glycoprotein (n) [ ] . the s protein is involved in viral infectivity and induces the production of neutralizing antibodies [ ] . the m protein plays a role in btov assembly and nucleocapsid recognition [ ] . the he has a putative f-g-d-s motif and has acetylesterase activity specific for n-acetyl- -oacetylneuraminic acid [ , ] . he contains three functional domains: a lectin domain (r), an esterase domain (e) and a membrane-proximal domain (mp) [ ] . the n protein is the only viral rna-binding polypeptide found in infected cells. it protects the genome and ensures its timely replication and reliable transmission, as well as playing a role in virus transcription and translation [ , ] . diarrhea is a common disease in dairy cows in china, which leads to serious economic losses. bovine coronavirus (bcov), bovine group a rotavirus (brva), bovine viral diarrhea virus (bvdv), bovine norovirus (bnov), and nebovirus (nev) had been identified as common diarrheacausing viruses in bovines in china [ ] [ ] [ ] [ ] [ ] . however, there is currently no information regarding btov in china. the goal of this study was to detect and the genome of characterize of btov in calves in china. from september to december , diarrheic fecal samples were collected from calves (aged < months) from five farms in four provinces of china. these included samples from sichuan province (one farm), from liaoning province (two farms), from shanxi province (one farm), and from henan province (one farm). all samples were stored at - °c in sterile -ml centrifuge tubes until further testing. total rna from μl of the fecal suspension was extracted using a qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. complementary dna (cdna) was synthesized using a primescript™ reverse transcription kit (takara, dalian, china) and stored at − °c. btovs were detected using a specific rt-pcr assay established in our laboratory. the specificity and reproducibility of the rt-pcr assay has been validated, and the detection limit is . × − pg/µl. briefly, a pair of primers to investigate coinfection with brva, bcov, bvdv, nev and bnov, all btov-positive samples were subjected to previously described specific rt-pcr assays for these viruses. [ ] [ ] [ ] ] the detection of brva, bcov, nev were following our previous reports, bvdv was following the previous report [], bnov was following the previous report []. to investigate the genomic characteristics of btov isolates from china, primer pairs were designed to amplify their genomes (table s ). the ' and ' ends of the viral genome were sequenced by rapid amplification of cdna ends using a smart race cdna amplification kit (takara). the pcr products were cloned into the pmd -t simple vector (takara) for further sequencing (sangon biotech). sequences were assembled using seqman software (version . ; dnastar, inc., wi, usa). putative orfs in the linear genomes were identified using the orf finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). nucleotide and deduced amino acid sequences were compared using the megalign program of lasergene software, version . (dnastar, madison, wi, usa). a multiple sequence alignment was performed, and a neighbor-joining phylogenetic tree was built using the interior branch test method with the aid of mega software. possible recombination events were identified using simplot software (version . . ) and the recombination detection program (rdp . ) by the rdp, chimaera, bootscan, seq, geneconv, max-chi, and siscan methods. among the diarrheic samples, . % ( / ) tested btov-positive by rt-pcr. the detection rate was . % a portion of the m gene of positive samples was sequenced, and all partial sequences of the m gene (gen-bank accession no. mn -mn ) were submitted to the genbank database. the nucleotide sequences were . % to % identical to one another and . % to . % identical to those of previously reported btov strains. a phylogenetic tree was constructed using the bp m fragment sequence, including the btov sequences from calves as well as other btov sequences from the genbank database. the btov sequences from this study were divided into two different groups (fig. s ). one of these groups included btov isolates from three different provinces, which formed a unique large branch, but the one remaining strain clustered with the turkish strains ht and ht . to obtain more-precise information about the evolutionary relationships of the btov isolates from calves, six representative btov isolates were chosen from four positive farms, and two btov isolates were chosen from each province. however, we were only able to successfully assemble two complete genomic sequences (sc- sichuan / and sc- sichuan / ), both of which were from the same farm in sichuan province. these sequences were submitted to the genbank database with accession numbers mn and mn . the length of the linear genome was , and , nt, respectively, and the g + c content was . %. the complete genome of the strains shared . - . % nt sequence identity with the genome sequences of mammalian tovs in the genbank database. analysis of the complete genome sequences revealed that the two chinese btovs shared . % nucleotide sequence identity with each other, . % to . % identity with the two genome sequences from japan, and shared . % nt identities with the prototype strain bread (fig. ). the complete s genes of strains sc- and sc- were both , bp in length and encoded a protein of , amino acids. the complete s genes shared . % nt sequence identity and . % aa sequence identity with each other and shared . - . % nt sequence identity ( . - . % aa sequence identity) with the btov s sequences available in the genbank database. a phylogenetic analysis based on the complete amino acid sequence of the s protein showed that the btovs could be separated into four groups (fig. ) , designated tentatively as group to group . the two btovs clustered with the btov strains b and ht belonging to group and were independently clustered in a small branch. further analysis showed that, compared to the available btov s sequences, strains sc- and sc- had amino acid mutations, of which two were located in the signal peptide, six were located in the putative s region, and two were located in the putative s region. in addition, sc- had a fig. phylogenetic tree based on the complete genomic nucleotide sequence of all mammalian toroviruses. sequence alignments were performed using clustalw in mega . software. the tree was constructed by the maximum-likelihood method with bootstrap values calculated for replicates. the scale bar indicates amino acid substitutions per site. bovine torovirus was found in calves and may be related to members of the genus coronavirus. the bovine torovirus strains btov/sc- /china and btov /sc- /china investigated in this study are indicated by black triangles fig. phylogenetic tree based on the deduced -aa sequence of the complete s gene. sequence alignments were performed using clustalw in mega . software. the tree was constructed by the maximum-likelihood method with bootstrap values calculated for , replicates. the bovine torovirus strains investigated in this study are indicated by by black triangles continuous -amino-acid mutation at the end of the putative s region, and sc- had a unique amino acid mutation in the putative s region (fig. ). the complete he gene of strains sc- and sc- are , bp in length and encode a protein of amino acids. f-g-d-s, the putative esterase active site in all he proteins, was located at aa positions - . this complete he gene shares . - . % nt sequence identity ( . - . % aa sequence identity) with all available btov he gene sequences. phylogenetic trees constructed using full-length he gene sequences showed that the two he sequences clustered into the same subgroup and belonged to genotype ii (fig. ) . furthermore, there was a unique amino acid variation (l f) in strains sc- and sc- compared to the available btov he sequences, and it is interesting that this mutation is located in the neutralizing epitope. in addition, the sc- strain had a unique amino acid variation (i y), and the sc- strain had three unique amino acid variations (k s, s q, w g) compared to the other available btov he sequences (fig. ). btov sc- and sc- were predicted to be recombination strains using the recombination detection program (rdp . ) software, and standard similarity plot analysis was performed using simplot . . (fig. ) . notably, both strains sc- and sc- had the same predicted breakpoints in the crossover region, at nucleotide positions , and , in the ' end of orf b and the ' end of the n coding region, respectively. in this study, . % ( / ) of the diarrheic stool samples collected from dairy cows were found to be btov positive. these samples were from four farms in three provinces, and the two more distant farms were separated by more than kilometers. the results indicate that btov has circulated in chinese cows with wide geographical (table ) , which is similar to the previous report from the usa [ ] . this may lead to an increase in clinical severity and increased difficulty in diagnosing and controlling calf diarrhea. in this research, we determined the obtained two complete genome sequences of two btov isolates from the same farm in sichuan province, increasing the number of btov genome sequences in the genbank database to five, thus contributing to a better understanding of the genome structure and genetic evolution of btov. phylogenetic analysis indicated that these two btov isolates had a close genetic relationship to strains from japan. strains sc- and sc- were predicted to be recombinants derived from btov strains breda and ptov, in agreement with a previous report [ ] . moreover, the two chinese strains shared identical unique amino acid changes in the s and he genes when compared to the other strains with sequences available in the genbank database, indicating the unique evolution in chinese btov strains. interestingly, although these two isolates came from the same farm, their s and he genes had their own unique amino acid variations. these results will be helpful for understanding the genetic evolution of btov. the s protein is involved in the induction of neutralizing antibody production during the infection process and plays an important role in the pathogenesis of btov, which is similar to that of coronaviruses [ , , ] . another member of the family coronaviridae, bcov, has a genomic structure similar to that of btov, and mutations in the s protein are associated with viral antigenicity, pathogenicity, tissue tropism, and host range change [ , ] . it has been reported that cleavage most likely occurs between amino acids , and , of the mature s protein in btov, resulting in the formation of the s and s proteins [ ] . the two chinese strains shared two identical amino acid mutations in the signal peptide that might affect its function. it is worth noting that these two strains have six identical amino acid mutations in the putative s region. since the s protein is responsible for receptor recognition and neutralizing antibody induction, mutations at amino acids and (t i, l p) might affect virus replication [ ] . therefore, the effects of these unique amino acid variations on the function of the proteins of strains sc- and sc- s is worth further study. based on the he gene sequences, btov can be divided into three genotypes [ ] . in this study, a phylogenetic tree constructed using all complete he gene sequences available in the genbank database showed that the two he fig. similarity plot analysis of the complete genome sequences of ptov shi (blue line), btov breda (red line), and ptov npl (pink line), with btov sc- and sc- as query sequences, using a sliding window of nt and a moving step size of nt sequences belonged to a genotype that is prevalent throughout the world [ , , , ] . the torovirus he protein contains three domains: mp, e and r, among which the r domain is involved in receptor recognition and plays an important role in the process of btov infection [ ] . the two chinese strains shared an identical unique amino acid change in the e region. in addition, strain sc- had another unique amino acid change in the e region. therefore, the ability of these mutations to affect viral receptor binding requires further investigation. in conclusion, this study is the first to confirm the existence and prevalence of btov in chinese calves with diarrhea, contributing to the diagnosis and control of calf diarrhea in china. moreover, two complete btov genome sequences were obtained from the clinical samples, and these two btov isolates had unique amino acid changes in the s and he proteins. these findings will enhance our understanding of the epidemic and the genetic evolution of btov. nidovirales: a new order comprising coronaviridae and arteriviridae detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms antibodies to berne virus in horses and other animals identification and characterization of a porcine torovirus du pasquier p ( ) an enveloped virus in stools of children and adults with gastroenteritis that resembles the breda virus of calves the complete sequence of the bovine torovirus genome enteric and nasal shedding of bovine torovirus (breda virus) in feedlot cattle detection and characterization of bovine torovirus from the respiratory tract in 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transcription-pcr assays for detection of bovine enteric caliciviruses (bec) and analysis of the genetic relationships among bec and human caliciviruses case-control study of microbiological etiology associated with calf diarrhea a single amino acid change within antigenic domain ii of the spike protein of bovine coronavirus confers resistance to virus neutralization coronavirus spike proteins in viral entry and pathogenesis crystal structure of bovine coronavirus spike protein lectin domain genetic and antigenic characterization of newly isolated bovine toroviruses from japanese cattle characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows hemagglutinin-esterase, a novel structural protein of torovirus acknowledgements this study was supported by funding from the key: cord- -gev xq a authors: zhu, liqian; yang, shen; tong, wu; zhu, jianping; yu, hai; zhou, yanjun; morrison, robert b.; tong, guangzhi title: control of the pi k/akt pathway by porcine reproductive and respiratory syndrome virus date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: gev xq a phosphatidylinositol- -kinase (pi k)/akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. in the present study, we compared the phosphorylation status of akt during infection of marc- cells and porcine alveolar macrophages (pams) with highly pathogenic prrsv (hp-prrsv) strain hun . we observed that biphasic activation of akt was induced in at both the early stage ( , and min postinfection) and the late stage ( and h postinfection) of hp-prrsv infection of marc- cells, while an early-phase activation of akt was found exclusively in virus-infected pams in vitro. analysis with the pi k-specific inhibitor ly confirmed that pi k acted as the upstream activator for the virus-induced activation of akt. uv-irradiation-inactivated virus still induced the early event in pams but not in marc- cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated akt within different cell cultures. we further demonstrated that foxo and bad, which serve as downstream targets of akt, were phosphorylated in virus-infected marc- cells. moreover, the suppression of phosphorylated akt with ly significantly inhibited the virus-induced cytopathic effect (cpe) on marc- cells, but it had a negligible effect on virus propagation. collectively, our data provide new evidence of a novel role for the pi k/akt pathway in prrsv infection of marc- cells. porcine reproductive and respiratory syndrome virus (prrsv) is a small, enveloped, positive-strand rna virus belonging to the family arteriviridae [ , ] . since it emerged in late s in europe and north america, it has caused significant economic losses to the pork industry worldwide [ , ] . in , an outbreak of hp-prrsv described as ''pig high fever disease'' occurred in china and caused disastrous loses to the farmers [ , ] . this disease is currently a major concern for the swine industry worldwide. phosphatidylinositol- -kinase (pi k)/akt is a key signaling transduction pathway in the regulation of cell survival, cell proliferation and differentiation, and cell apoptosis [ ] [ ] [ ] ] . the activation of pi k-akt pathway promotes cell survival by the phosphorylation of numerous substrates such as glycogen synthase kinase- (gsk- ), foxo , bad and mtor [ , , , , ] . many viruses are known to manipulate this pathway in favor of their replication, such as influenza a virus [ ] , hepatitis b virus [ ] , hepatitis c virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] , junín virus [ ] , human immunodeficiency virus type [ ] , bovine herpesvirus type [ ] and infectious bursal disease virus [ ] . previous studies have shown that during prrsv infection of porcine monocyte-derived dendritic cells (mo-dcs), the pi k/akt pathway is activated at min and h postinfection (h p.i.), and it is inhibited at h p.i. [ ] . in vivo, the virus shows a very narrow cell tropism and infects a specific subpopulation of porcine macrophages [ , ] . in vitro, efficient prrsv replication is only observed in primary pig macrophages (e.g., alveolar macrophages), differentiated monocytes [ ] or cells derived from african green monkey kidney, such as marc- [ ] . for easy manipulation, marc- cells provide an important tool for the study of prrsv replication. previously, it was reported that the pi k/akt pathway is regulated by prrsv in a complex manner in mo-dcs [ ] , but whether this is a universal phenomenon in all permissive cells is unknown. a number of studies have shown that the pi k/akt signaling pathway is functionally dependent on downstream substrates of akt such as foxo , bad and mtor for regulation of cell survival [ , , , ] . the downstream targets of akt controlled by prrsv have not been identified. the objective of this study was to address how the pi k/akt pathway is modulated in infection of marc- cells and pams with highly pathogenic prrsv. we demonstrated that the pi k/ akt pathway was activated by hp-prrsv in a cell-culturedependent manner and that the downstream targets foxo and bad were regulated through this pathway. in addition, we found that activated pi k was required for the development of cpe in marc- cells, but not for production of infectious progeny. the hp-prrsv strain hun used in the present study was isolated from a pig showing signs of ''high fever syndrome'' originating from a swine farm in the region of hunan province, china, in [ , ] . a stock of hun virus at passage was prepared in marc- cells, titrated, and stored at - °c until use. marc- cells were maintained in dulbecco's modified eagle's medium (dmem) (gibco brl) supplemented with % fetal bovine serum (gibco brl) in an incubator at °c containing % co . the pam cultures were obtained by broncho-alveolar lavage of -week-old domestic piglets from a prrsv-negative herd affiliated with shanghai agricultural science institute. all animal experiments were conducted according to the guidelines and approved protocol of the shanghai veterinary research institutional animal care committee. the pams were cultured with rpmi- medium supplemented with % fetal bovine serum. all of our experiments were conducted at veterinary laboratory biosafety level . rabbit monoclonal antibodies (mab) recognizing phospho-akt (ser ), phospho-bad (ser ), phospho-foxo (ser ) and phospho-mtor (ser ) were purchased from cell signaling technology inc. mouse monoclonal b-actin antibody was obtained from santa cruz biotechnology, inc., santa cruz, calif. horseradish peroxidase (hrp)-conjugated anti-rabbit igg and anti-mouse igg were purchased from zhongshan golden bridge biotechnology co., ltd. pi k-specific inhibitor ly was supplied by cell signaling technology, and a stock of this inhibitor was prepared with dmso. marc- cells in -mm dishes were grown to %- % confluence, whereupon they were subjected to serum starvation for h in serum-free dmem. subsequently, growth-arrested cells were infected at a multiplicity of infection (moi) of with hun virus or were mock infected with the same medium. one hour postinfection (p.i.), the cells were washed with phosphate-buffered saline (pbs, ph . ) and then cultured in fresh dmem for various times as indicated. for inhibitor experiments, marc- cells were pretreated with the pi k inhibitor ly for h. cells were then infected with the virus at an moi of for h, washed with pbs, placed in serum-free medium containing fresh inhibitor, and sustained for h. dmem containing . % dmso (v/v) was used for the mock treatment unless otherwise specified. growth-arrested pam cells cultured in -mm dishes were infected at an moi of with hun or were mock infected with medium and then incubated for various times as indicated. the cells were then collected for western blotting analysis. to inactivate hp-prrsv by uv irradiation, the virus stocks were dispersed in -cm tissue culture dishes and placed directly under a uv lamp ( w) for min. complete inactivation of the virus was confirmed by the titration on marc- cells. both marc- cells and pams were exposed to uv-irradiation-inactivated virus to analyze the variation of phosphorylated akt within h p.i. as indicated. after hp-prrsv infection for the lengths of time indicated, cells were washed with pbs and lysed with lysis buffer ( % triton x- , mm sodium chloride, mm edta, mm egta, mm sodium fluoride, mm sodium pyrophosphate, mm phenylmethylsulfonyl fluoride, . lg/ml leupeptin, mm benzamidine, and mm sodium orthovanadate in mm tris-hcl, ph . ). the cell lysates were cleared by centrifugation for min at , g prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) under reducing conditions. the separated proteins were transferred onto nitrocellulose membranes (millipore). the membranes were blocked for h at room temperature with skim milk solution ( % in tris-buffered saline containing . % tween ). blots were incubated overnight at °c with primary antibodies followed by incubation for h with the secondary antibody (conjugated horseradish peroxidase). after extensive washing, the immunoreactive bands were detected by enhanced chemiluminescence (ecl) (pierce) and subsequently reprobed for total protein loading using anti-b-actin antibody. to analyze whether inhibition of pi k by ly would affect virus replication, serum-starved marc- cells seeded in -well plates were pre-incubated with the inhibitors or mock pretreated with dmem containing . % dmso for h at °c and then infected with hun virus for h together with or without the inhibitor. after extensive washing with pbs, the cells were placed in dmem with or without fresh inhibitor for or h. after two rounds of freezing and thawing, the infectious progeny was titrated with marc- cells using a tcid assay. we first used the marc- cell model to examine the kinetics of akt phosphorylation at ser , which is required for akt activation, to determine whether hp-prrsv infection modulates the pi k/akt pathway. cells that were mock infected with dmem medium or infected with hp-prrsv strain hun were harvested at min, min, min, min, h, h, h, h, h and h postinfection for western blot analysis. it was found that akt was activated in a biphasic manner after infection of marc- cells with hp-prrsv (fig. a, top panel) . the first phase of akt activation occurred early ( and control of the pi k/akt pathway by prrsv min p.i.) in virus infection (fig. a) . at h p.i., the phosphorylation of akt had returned to the basal level, and after a certain interval, akt phosporylation increased again in the late stage of infection. the amount of phosphorylated akt was evidently increased at h p.i., and this level was sustained for the reminder of the investigation ( h p.i.) (fig. a, upper panel) . the changes in akt phosphorylation were not due to variation in the total amount of protein loaded (fig. a, bottom panel) . since akt can be activated by both pi k-dependent and pi k-independent pathways [ ] , the role of activated pi k in the virus-induced akt activation was subsequently investigated. here, the chemical ly , a potent and specific inhibitor of pi k, was used. as demonstrated by immunoblot assay, lm of ly could efficiently inhibit the phosporylation of akt induced by the virus at the late stage of infection (fig. b) . this indicated that the activated pi k accounted for the virus-triggered akt activation. taken together, the data indicate that the pi k/akt pathway was activated in infection of marc- cells by prrsv. since pams and monocytes are the major natural target cells during both acute and persistent prrsv infection [ ] , it deserved to be investigated whether the virus controls the pi k/akt pathway during infection of pams. here, the kinetics of phosphorylated akt was determined in hp-prrsv-infected pams in vitro. serum-starved pams that were mock infected with rpmi- medium or infected with hun at min, min, min, min, h, h, h, h, h and h postinfection were collected for western blotting. as illustrated in fig. , the amount of phosphorylated akt increased dramatically from to min p.i. however, this activation was transient and quickly returned to the basal level at min postinfection. in addition, the enhanced phosphorylation of akt was not observed at the later stage. this suggested that akt was only activated by hp-prrsv at the early phase in the infection of pams, which is different from what was observed in marc- cells. since increased phosphorylation of akt induced by hp-prrsv was observed at min and min postinfection in virus-infected marc- cells, and at , and min postinfection in pams, we hypothesized that the virus entry process accounted for this early-stage activation. thus the uv-irradiated hp-prrsv virus was employed to address this issue. under the conditions described above, the virus could be completely inactivated (data not shown). such inactivated virus fails to express viral proteins due to the formation of thymidine dimers, which prevent the transcription of viral genes but do not interfere with the capacity of the virus for receptor binding and entry into host cells by endocytosis [ ] . as showed in fig. , the exposure of cells to uv-irradiated virus led to phosphorylation of akt at and min postinfection in pams (fig. b ), but not in marc- cells (fig. a) . this indicates that virus-cell interactions during the binding or entry process are probably responsible for this event in pams, whereas in marc- cells, a different mechanism may account for this activation. by western blotting using rabbit monoclonal antibodies against phospho-bad (ser ), phospho-foxo (ser ) and phospho-mtor (ser ). compared to the mockinfected control, virus replication resulted in significantly increased phosphorylation of both foxo and bad, while no change in mtor was observed (fig. a) . phosphorylation with both foxo and bad were most evident at h p.i., which corresponds in time to the change in phosphorylated akt. to address whether the change in phosphorylated foxo and bad was induced by the virus through the pi k/akt pathway, the pi k-specific inhibitor ly was employed for further investigation. as shown in fig. b) (fig. a) . unexpectedly, the virus-induced activation of the pi k/akt pathway was not essential for production of infectious prrsv progeny virus. the cytopathic effect (cpe) in hun -infected marc- cells is characterized by cell congregation and contraction [ ] . interestingly, in infected marc- cells treated with ly , the cpe was significantly alleviated. under normal conditions, prrsv-infected marc- cells showed considerable cpe at h p.i. (fig. b, b) , but no cpe was observed in hun -infected marc- cells when lm of ly was used to treat the cells (fig. b, d) . in addition, the inhibitor at a concentration of lm could not induce cpe, and it was apparently not cytotoxic to marc- cells, (fig. b, c) , which was confirmed by mtt assay (data not shown). thus, these findings suggest that the pathway may be linked to hp-prrsv-induced cpe formation in marc- cells. many viruses interfere with signaling pathways in their infected host cells to favor productive infection, which can also impact on the physiology of the host cell as well as pathogenesis. therefore, the identification of cellular factors involved in virus replication is important for the design of novel antiviral strategies. in this regard, the manipulation of cellular pathways regulating cell apoptosis or survival affords great advantages to virus replication [ ] , and many viruses have been demonstrated to regulate cell survival via the regulation of the pi k/akt pathway [ , ] . a previous report has indicated that the pi k/akt pathway is activated in the early phase and subsequently inhibited at h p.i. during prrsv infection of mo-dcs [ ] . here, hp-prrsv strain hun was used to further investigate how and why this pathway is controlled by the virus. here, we report that the activation of the pi k/akt pathway was significantly enhanced by the hun virus at both the early and late stage of infection of marc- cells, while a distinct pattern was adopted during infection of pams in which the phosphorylation of akt occurred exclusively in the early stage, as early as min p.i., and then returned to the basal level at min p.i. (figs. a and ). when the cells were exposed to the uv-irradiationinactivated virus, significantly increased phosphorylation of akt was observed in pams, but not in marc- cells. entry of macrophages by prrsv occurs via a few similar but also different mechanisms compared to entry into marc- cells [ ] . it could be inferred that the virus entry process in pams was enough to activate the pi k/ akt pathway, while a different mechanism of activation was used in marc- cells. it is possible that a different entry mechanism results in this difference in the regulation of akt. together with previous data reported by zhang et al. [ ] , we generalize that prrsv regulates the pi k/ akt pathway by different mechanisms in mo-dcs, pams and marc- cells. various pro-apoptotic proteins have been identified as downstream targets of akt, including the bcl- family members bad, gsk- a/b, and mtor and the foxo transcription factor family members (fkhr) [ , , , , ] . they are normally regulated in a pi k/akt-dependent manner. for example, the phosphorylation of bad (ser- ) was decreased but the phosphorylation of mtor (ser ) and foxo (ser ) was increased by infection with varicella-zoster virus [ ] . similarly, it has been shown that the pi k/akt/mtor cascade contributes to the establishment of persistent hcv infection [ ] . in the present study, we demonstrated that both foxo and bad were regulated by prrsv through the control of the pi k/ akt pathway in the late stage of infection in marc- cells (fig. ) , and therefore the, pi k/akt/foxo and pi k/akt/bad cascades may be manipulated by prrsv to control host-cell survival. the pi k/akt pathway has been shown to play important roles in different steps of the life cycles of a variety of viruses [ ] . for example, this pathway has been shown to be involved in the regulation of ebola virus entry of host cells [ ] , to contribute to arenavirus budding [ ] , and to be involved in the control of synthesis of viral rna of nonsegmented negative-stranded rna viruses [ ] . unexpectedly, our investigation with the pi k-specific inhibitor ly showed that the pi k/akt pathway has a negligible effect on the titer of infectious prrsv progeny in marc- cells (p [ . ), as determined at both and h p.i. (fig. a ), but inhibition of this pathway significantly blocked virus-induced cpe formation. to our knowledge, similar results have not been documented for other viruses. therefore, it will be worthwhile in the future to investigate how the pi k/akt pathway regulates cpe formation during prrsv infection. in summary, our findings, together with a previous report of prrsv regulating akt in mo-dcs, suggest that pi k/akt signaling is modulated by prrsv in different ways in different cell types. foxo and bad were employed by the virus as downstream targets of the pi k/ akt pathway. unlike other viruses for which the control of this pathway contributes significantly to virus replication, for prrsv, it is not important for generation of infectious progeny, but this modulation changes the physiology of the host cell, as revealed by the significant alleviation of cpe. this finding may offer a new and important insight into the role of pi k/akt in virus infection, or even in viral pathogenesis. porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection secondary enterovirus infection in the murine model of myocarditis. pathologic and immunologic aspects akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor the torrid affairs of viruses: effects of mammalian dna viruses on the pi k-akt-mtor signalling pathway cell cycle and death control: long live forkheads phosphoinositide -kinase signalling pathways akt/pkb and other d phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation involvement of pi k/akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group the pivotal role of phosphatidylinositol -kinase-akt signal transduction in virus survival inhibition of glycogen synthase kinase- by insulin mediated by protein kinase b susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and cd effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) hijakt: the pi k/akt pathway in virus replication and pathogenesis apoptosis. a bad kinase makes good regulation of hepatitis b virus replication by the phosphatidylinositol -kinase-akt signal transduction pathway control of the pi k/akt pathway by prrsv alterations in microrna expression profile in hcv-infected hepatoma cells: involvement of mir- in regulation of hcv replication via the pi kinase/akt pathway enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line participation of the phosphatidylinositol -kinase/akt pathway in junin virus replication in vitro targeting the pi k/akt cell survival pathway to induce cell death of hiv- infected macrophages with alkylphospholipid compounds activation of the n-ras-pi k-akt-mtor pathway by hepatitis c virus: control of cell survival and viral replication lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav jnk and pi k/akt signaling pathways are required for establishing persistent sars-cov infection in vero e cells the forkhead transcription factor foxo regulates adipocyte differentiation mammalian target of rapamycin is a direct target for protein kinase b: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states varicella-zoster virus requires a functional pi k/akt/gsk- alpha/beta signaling cascade for efficient replication phosphoinositide- kinase-akt pathway controls cellular entry of ebola virus effect of the phosphatidylinositol -kinase/akt pathway on influenza a virus propagation akt plays a critical role in replication of nonsegmented negative-stranded rna viruses highly pathogenic porcine reproductive and respiratory syndrome the pi k/akt pathway contributes to arenavirus budding porcine reproductive and respiratory syndrome virus entry into the porcine macrophage infectious bursal disease virus activates the phosphatidylinositol -kinase (pi k)/akt signaling pathway by interaction of vp protein with the p alpha subunit of pi k activation of the pi k/akt signaling pathway during porcine circovirus type -infection facilitates cell survival and viral replication serine phosphorylation of death agonist bad in response to survival factor results in binding to - - not bcl-x(l) a dual effect of porcine reproductive and respiratory syndrome virus replication on the phosphatidylinositol- -kinase-dependent akt pathway cell biology. gsk- beta and microtubule assembly in axons highly virulent porcine reproductive and respiratory syndrome virus emerged in china biphasic activation of pi k/akt and mapk/erk / signaling pathways in bovine herpesvirus type infection of mdbk cells key: cord- - xi ee authors: evermann, j. f.; heeney, j. l.; roelke, m. e.; mckeirnan, a. j.; o'brien, s. j. title: biological and pathological consequences of feline infectious peritonitis virus infection in the cheetah date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: xi ee an epizootic of feline infectious peritonitis in a captive cheetah population during – served to focus attention on the susceptibility of the cheetah (acinoyx jubatus) to infectious disease. subsequent observations based upon seroepidemiological surveys and electron microscopy of fecal material verified that cheetahs were indeed capable of being infected by coronaviruses, which were antigenically related to coronaviruses affecting domestic cats, i.e. feline infectious peritonitis virus/feline enteric coronavirus. coincident with the apparent increased susceptibility of the cheetah to infectious diseases, were observations that the cheetah was genetically unusual insofar as large amounts of enzyme-encoding loci were monomorphic, and that unrelated cheetahs were capable of accepting allogenic skin grafts. these data provided the basis for a hypothesis that the cheetah, through intensive inbreeding, had become more susceptible to viral infections as a result of genetic homogeneity. research aimed toward understanding the pathogenesis of viral infections of endangered species constitutes a major component of the species survival management plan for the cheetah [ , ] . one of the primary reasons for this emphasis was the recent occurrence of a devastating epizootic of feline coronavirus infection (feline infectious peritonitis [fip] ) in captive cheetahs in - [ , , ] . since that time, there have been other reports on the apparent susceptibility of the cheetah to infectious diseases with emphasis on fip [- , , , , , ] . since there are currently no vaccines available for control of fip in either domestic or exotic cat populations it is essential that biologists, diagnosticians, and veterinarians be aware of the impact that this infection has upon the cheetah, so that appropriate management steps can be taken to minimize the chance for infection and thereby lessen the risk of fatal disease. the purpose of this review is to present desciptions of the various forms of coronaviral infections in the cheetah relying upon studies of both natural infections, as well as experimental infections in other species with coronaviruses, such as mouse hepatitis virus (mhv), canine coronavirus (ccv), transmissible gastroenteritis virus (tgev) of swine, and bovine coronavirus (bcv) of neonatal calves [ , , , , , , , , , ] . the biology of the feline coronaviruses has a short history since isolates were not available for laboratory studies until [see for review]. prior to that time, the disease was referred to as feline systemic proliferative and exudative vasculitis [ , ] , and later feline infectious peritonitis (fip) [ ] . recognition of the first coronavirus associated with fip in cats was determined by electron microscopy in [ ] . inasmuch as cell culture-adapted isolates of the fip virus (fipv) were not yet available, the disease condition was experimentally transmitted by the inoculation of liver homogenates from diseased cats into the peritoneal cavity of susceptible cats. during the period of experimental transmission of fip by tissue homogenates, serologic assays were developed which utilized indirect immunofluorescence on cryostat sections of liver obtained from cats with clinical fip [ ] . subsequent serologic studies relied upon cell culturegrown homologous virus, fipv, or heterologous cross-reacting coronavirus strains, such as ccv and tgev [ , , ~ , , ] . seroprevalence studies conducted during the s indicated that certain populations of cats had a high percentage of antibody, suggesting that either fipv was not % fatal as was generally believed, or that cats were also being [ , , , ] . since those initial reports on the divergent nature of feline coronaviruses and the designation of fipv and fecv strains, there have been additional reports on the detection and/or isolation of feline coronaviruses [ , ] . the spectrum of disease resulting from these various isolates is presented in table . although there are marked phenotypic differences in terms of in vivo virulence, efforts to distinguish fecv strains from fipv strains in vitro have not been successful [ , , , ] . it is conceivable that molecular technology will reveal differences at the genomic level, which reflect variation in strain virulence or pathology [ , , , ] . diagnostic molecular procedures would be useful in the detection of cats that were shedding fipv into the environment. however, if fipv is the progeny of a random mutation from fecv, or a recombinant of two enteric coronaviruses, then the application would vary according to the frequency of these mutational events [ , , , , , , ] . although the majority of research on the feline coronaviruses has been conducted in domestic cats, access to serologic tests has allowed investigators to test serum from other felidae, as well as from closely related members of the cat family, such as the cheetah [ , ] . the serologic results, as well as reports of sporadic cases of fip in cheetahs, indicated that they were susceptible to infection and in some cases succumbed to disease [ , , , , ] . the susceptibility of the cheetah to fip was of particular interest during an outbreak of the disease in a wildlife park in - [ , ] . in this incident, an infected cheetah was imported to the facility, which was a leading center for breeding cheetahs in north america. infectious agents were isolated in crandell feline kidney (crfk) cells from tissues submitted from cheetahs that died during the fip epizootic of - [ , ] . a common feature of the isolates was their reduced cytopathic effect (cpe) in crfk cells and cytoplasmic immunofluorescence when stained with antisera to a strain of moderate virulence, fipv ucd- ( fig. a, the cheetah virus has been maintained in cell culture as a persistent noncytolytic infection [ , ] . however, infected cells do express periods of cytopathogenicity referred to as "crisis periods", but following each crisis, cells emerge and the infection is maintained. assays for cell-free, virus have been hampered due to the low release of extracellular virus. ultrastructural studies conducted on the cells persistently infected by the cheetah coronavirus have revealed the presence of virus particles in cytoplasmic vacuoles, but minimal virus at the cell surface [ ] . this is in contrast to the high number of virus particles observed, both within virus-infected cells, as well as at the surface of cells with the cytopathogenic coronaviruses, fecv - and fipv - [ ] . the phenotypic parameters of the cheetah coronavirus in vitro indicate that the virus is a biological variant of a virulent coronavirus or is in a partially nonpermissive host cell [ ] . this is further suggested by its cell-associated nature and the lack of cell fusion capability, which would indicate incomplete expression of the essential e protein (fusion protein) [ , , , ] . this observation may be due to a partially defective virus [ ] or to a virus which fails to mature properly due to the absence of a host-cell protease [ , ] . the cheetah agent represents the least virulent in cell culture as compared with other feline coronaviruses (table ) except for fecv ucd, which has not yet been cultured in vitro, but must be maintained by passage through cats [ , . enzymatic enhancement of coronavirus replication and expression of cpe in vitro have been reported for a number of different coronaviruses including bcv and tgev [ , , , , ] . the addition of low amounts of trypsin ( ixg/ml) to serum-free culture media enhances the cpe of the feline corona- viruses fipv ucd- and fipv ucd- , and also appears to augment the expression of the cheetah coronavirus [ ] . studies on the effects of trypsin on various coronavirus strains, including fecv and fipv, have suggested that trypsin-sensitive and trypsin-resistant phenotypes occur in nature [ , ] . those strains that are highly resistant have been hypothesized to survive longer in the gastrointestinal tract and therefore be infectious longer, both for the infected host, as well as for other susceptible animals in the population which come in contact with the shedding host [ , ] . initially, definitive information on the natural spread of the feline coronaviruses was hampered due to the lack of cell-free virus; however, subsequent studies with cell-free the incubation periods for feline coronavirus infections are variable depending upon the dose and strain of virus and the age of and route of entry into the host. pedersen has conducted the most definitive studies to date on the pathogenicity of the viruses [ , ] . his results indicated an incubation period of - days for fecv-induced enteric infection, and days to months for the fipv strains. in some cases the outcome of fipv infection is dependent upon concurrent immunosuppression such as may occur with feline leukemia virus infection [ ] . the clinical features of the feline coronaviruses in domestic cats and cheetahs appear to be very similar ]- , , , , , ] . symptoms in domestic cats following fecv infection may range from subclinical to a mild diarrhea in uncomplicated cases [ ] . although not well documented in cats, multiple enteric infections with other viruses or bacteria should be considered in making a differential diagnosis. this has been reported to be the case in dogs with concurrent ccv and canine parvovirus infections [ ] . the clinical manifestations of fip include anorexia, icterus, and elevated serum proteins in fluids within the thoracic/abdominal cavities (wet form). although experimental studies of fip in cheetahs has not been conducted, information obtained from naturally occurring cases has indicated that the majority show early signs of liver dysfunction followed by periods of anorexia, dehydration and death [ , ] . it is important to recognize that although cheetahs appear to be very susceptible to fip-related disease, there are other factors which may contribute to a generalized liver dysfunction, such as dietary deficiencies [ , ] . pathologic studies of cheetahs diagnosed as having fip have many features in common with the lesions reported in domestic cats [ , , , , ] . a predominant feature in the fip lesions in cheetahs is multi-focal necrosis throughout many organs including the liver, kidneys, pancreas, spleen, lymph nodes, and thymus. the necrotic areas are characterized by karyorrhexis, karyolysis, cytolysis, and infiltration by lymphocytes and macrophages. fibronecrotic plaques are present on many organs in both the pleural and peritoneal cavities. necrotic foci containing lymphocytic infiltrates, macrophages, and neutrophils are also present in mesenteric fat and within the muscular layers of the small and large bowel. lymphoid aggregates in the spleen and lymph nodes were depleted. the fundamental histopathological lesions are a generalized vasculitis and perivasculitis [ , , , , , ] . although the course of the disease and distribution of lesions bear a close resemblance to fip in domestic cats, it is important that a complete necropsy and detailed histological examination be conducted in order to confirm the occurrence of fip in the cheetah [ ] . several other lesions have been noted in cheetahs in addition to changes attributed to fipv [ , ] . the gastric mucosa of four cheetahs contained superficial erosions of mucosal epithelium. in most cases, the changes were relatively mild with hyperemia of superficial mucosal vessels, mild focal hemorrhages and mild fibrosis of the interstitium. gastric ulcers have been observed in one cheetah. epithelium within the gastric pits was generally intact, although fibroblastic response bridged gastric pits in some areas. a moderate interstitial infiltrate of lymphocytes, plasma cells and macrophages was associated with areas of erosion. the immune response of the cat to the feline coronaviruses presents a critical factor in the pathogenesis of fipv infection. many of the lesions observed in cases of fip can be directly attributed to the immune response; i.e., an immunemediated vasculitis [ , , , , ] . these findings are supported by the reports of enhancement of fip by prior infusion of passively acquired antibody and the failure of conventional vaccine preparations to protect cats against subsequent challenge [see , for review] . currently, at least three distinct cellular lineages of the cat's immune system appear to be critical in determining the outcome of fipv infection [ , t, , , , ] . these include the macrophage, the b-cell and the t-cell populations. the macrophage appears to be one of the primary sites for fipv replication in vivo and was demonstrated to be a source of virus in vitro prior to the isolation of viruses in conventional cell cultures [ ] . although definitive studies have not been reported yet with fipv, it may be predicted that cells of the monocyte-macrophage lineage are very important in conferring resistance to infection, and in subsequent immune reactions of the processed fipv antigens with the b-and t-cell populations. coronaviral infection of the macrophage has been reported to be one of the major criteria for distinguishing resistance and susceptibility in mice with the murine coronavirus, mhv [ , , , , , , , , ] . fip-inducing strains apparently infect and replicate in feline macrophages while non-fip strains, e.g., the fecv strains, are primarily restricted to mucosal infections without replication in macrophages [see , for review]. in the majority of cases of fip, serum antibody has been measured by the indirect immunofluorescence assay (ifa), a group-specific test that does not distinguish between antibody to fipv and fecv [ , , , , ] . serum from cats with high ifa antibody titers has been demonstrated to enhance the pathogenesis of fipv when passively administered to cats six hours prior to virus inoculation [ ] . the role of antibody in the pathogenesis of fip has been controversial, but is not without precedent since there are several viral infections which have been documented to have an immune-mediated disease sequelae. these include dengue fever of humans [ , , ] , and yellow fever virus [ ] . the mechanism(s) of antibody-mediated enhancement may take the form of non-neutralizing antibodies, enhancing antibodies, or blocking antibodies (bind to/or block t-cell receptors) [see for review]. a likely explanation is that certain strains of feline coronavirus, i.e., fipv variants or mutants [ , , , ] , infect and replicate in macrphage cells, perhaps altering normal b-and t-cell interactions. the b-cell response is polyclonal and antibodies that are formed appear to detect all the major viral proteins as determined by western blot techniques [ ] . certain types of these antibodies may serve as enhancing antibodies in terms of increased viral uptake and replication within macrophages, which are subsequently spread throughout the body. the effectiveness of the t-cell response appears to play a critical role in controlling fipv infection, but may also play a part in the subsequent immunemediated pathogenesis. impairment of the t-cell response, such as may occur during concurrent viral infection with either feline panleukopenia virus or feline leukemia virus, appears to predispose cats to fip [ , , evermann, unpublished observation]. without t-cell surveillance of virus-infected macrophages, cell-associated viremia occurs and dispersal of virus occurs throughout the body [ ] . the actual mechanism(s) of fipv-induced tissue damage is being investigated, but may be a combination of inflammatory response with neutrophils imparting tissue damage alone or in combination with cytotoxic t cells attempting to rid tissues of virus-infected macrophages [ ] . the basic lesions in fip are a result of the cat's immune response producing large quantities of antibodies and the formation of immune complexes which are subsequently deposited within the vessels of the serosa [ , , , , ] . persistence of viral antigens in the serosal blood vessels provokes a hypersensitivity reaction with the migration and infiltration of mononuclear cells into vessel adventitia and media, resulting in severe vascular damage [ , ] . the outcome from the vessel damage usually includes a serous effusion and accumulation of fluid in the major body cavities. this type of immune response is characteristic of a type iii hypersensitivity reaction [ , , , ] . the genetic regulation of the immune response has been of interest in studying viral infections of animals, since it has been shown that resistance to certain viruses appears to be under genetic control [ , , , ] . more recently, the cheetah has been shown to be genetically unusual insofar as large amounts of enzyme-encoding loci are monomorphic in natural populations [ , , ] . this observation, coupled with the extraordinary finding that unrelated cheetahs would immunologically accept allogenic skin grafts, was interpreted in the context of a hypothesis that the cheetah had undergone intensive inbreeding in its recent natural history [ ] . such a situation would have significant effect upon the immune defenses against microbial pathogens. by analogy to other species, natural populations have multiple loci which are polymorphic for functions which influence the outcome of viral-induced disease outbreaks [- ] . notable amongst such genes are those forming the major histocompatibility complex, whose gene products function directly in monitoring immune response to viral infections [ , , , ] . the genetic homogeneity of the cheetah may be an important contributing factor in the increased susceptibility of the species to viral infections, such as fipv and feline herpesvirus [ , , ] . since the fatal outcome of the fip epizootic of - , it has been apparent that cheetahs are highly vulnerable to infection and disease from the feline coronaviruses [ , , , ] . in an effort to determine the prevalence of feline coronavirus infection in cheetahs, a serologic survey and an electron microscopic analysis were conducted on captive and wild-caught cheetahs [ ] . the serological results from captive cheetahs in zoologic parks in north america (table ) indicated that of ( %) captive cheetahs were seropositive to feline coronavirus by ifa (group-specific serology test). these results were higher than those of captive cheetahs located in southern africa and europe, but similar to captive cheetahs in eastern africa as well as to wild-caught cheetahs ( table ). the serologic results indicated that the cheetahs were being infected by a virus which was antigenically related to the feline coronavirus group, and that there was a comparable risk of acquiring infection whether efforts to control fip in captive cheetahs are structured around periodic serologic testing and the segregation of cheetahs that are seropositive to feline coronaviruses by ifa. the limitations of these recommendations are the possible lack of specificitly of ifa for identifying exposure to pathological variants, and the lack of complete understanding of the coronaviruses affecting cheetahs [ , , , ] . however, until further information is reported regarding coronaviral diseases of exotic cats, especially the cheetah, then the management should follow basic guidelines for control of an infectious disease (table ). the cheetah has assumed a prominent position in the zoologic and wildlife communities due to its endangered status and its apparent increased susceptibility to microbial infections [ , ] . these factors have emphasized the need for further research into the genetics and the diseases that may affect the cheetah in captivity, as well as in the wild [ ] . although coronaviral infections of the domestic cat have been recognized for over decades, the emergence of pathogenic variants in the cheetah populations has only recently been acknowledged as having a potentially severe impact on its survival [ , , ] . primary amongst the coronaviruses affecting domestic cats is fipv, which is regarded as being % fatal once clinical signs are manifested [ , ] . the occurrence of coronaviral infections of the cheetah have now been documented based on serology, electron microscopy of fecal contents, and the occurrence of fatal forms of infectious peritonitis compatible with the clinicopathologic signs observed in domestic cats with fip [ , , , , , , , ] . these observations may indicate that the cheetah has acquired a unique group of coronaviruses which have antigenic similarity to the domestic cat coronaviruses or that the cheetah is susceptible to cross-species transmission of domestic cat coronaviruses [ , , , ] . serological studies support the contention that both situations may be important in the epizootiology of fipv in cheetahs [ , , ] . although cheetahs in the wild have a comparable seroprevalence to the feline coronavirus group, their antibody titers are invariably lower than those titers detected in captive cheetahs during a disease outbreak [ , ] . the seroprevalence of feline coronavirus infection in cheetahs in captivity, both in north america and africa, indicates that the virus is transmitted with equal frequency. while evidence in support of cross-species transmission of feline coronaviruses to the cheetah is lacking, it is known that the cheetah cells are susceptible to some of the domestic cat coronaviruses in vitro [ ] . it is apparent that further analysis of cheetahs in captivity needs to be conducted in order to determine more about their microbial flora and their immune response [ ] . this information is essential in order to determine the risk of infection and disease from viruses such as fipv and feline herpesvirus [ , ] . until more is known regarding the pathogenesis of coronaviral infections of the cheetah, it would be prudent to establish surveillance programs which utilize a combination of serologic monitoring and quarantine procedures in order to insure the survival of the cheetah in captivity [ ]. feline infectious peritonitis. an immune-mediated coronaviral vasculitis transmissible gastroenteritis (tge) of swine: survivor selection of tge virus mutants in stomach juice of adult pigs genetics of resistance of animals to viruses. i. introduction and studies in mice feline coronaviral infections host age and genotypic effects on enterotropic mouse hepatitis virus infection response of 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infectious peritonitis: comparisons with dengue hemorrhagic fever pathogenesis of feline infectious peritonitis: nature and development of viremia proteases involved in the processing of viral polyproteins viral interference-dominance of mutant viruses over wild-type viruses in mixed infections feline infectious peritonitis: review of gross and histopathologic lesions lesions in the small intestine of newborn pigs inoculated with porcine, feline and canine coronaviruses feline coronavirus. in: appel mj (ed) virus infections of carnivores genetic variation of major histocompatibility complex class i genes in various species: the lack of polymorphism in the african cheetah the authors wish to express their appreciation to drs. niels pedersen key: cord- -j ye ed authors: loemba, h. d.; mounir, s.; mardassi, h.; archambault, d.; dea, s. title: kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: j ye ed the kinetics of appearance of antibodies directed to the major structural proteins n, m and e of porcine reproductive and respiratory syndrome virus (prrsv) was followed in pigs naturally- and experimentally-exposed to the virus. specific igm antibody titers were first detected by indirect immunofluorescence (iif) at the end of the first week of prrsv infection, peaked by day to post-inoculation (p.i.), then rapidly decreased to undetectable levels by day to p.i. on the other hand, specific igg antibody titers peaked by day to p.i. and remained unchanged to the end of the - or -week observation period; in addition, a persistent viremia was observed. virus neutralizing (vn) antibody titers > were not detected until to weeks p.i. taken together, the results obtained by western blotting analyses using purified virus ande. coli-expressed orfs to gene products, suggested that antibodies directed against the envelope e protein appear by day p.i., whereas antibodies directed against the nucleocapsid n and membrane m proteins can only be detected by the end of the second week p.i. no correlation could be demonstrated between vn and iif antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i. summary. the kinetics of appearance of antibodies directed to the major structural proteins n, m and e of porcine reproductive and respiratory syndrome virus (prrsv) was followed in pigs naturally-and experimentallyexposed to the virus. specific igm antibody titers were first detected by indirect immunofluorescence (iif) at the end of the first week of prrsv infection, peaked by day to post-inoculation (p.i.), then rapidly decreased to undetectable levels by day to p.i. on the other hand, specific igg antibody titers peaked by day to p.i. and remained unchanged to the end of the -or -week observation period; in addition, a persistent viremia was observed. virus neutralizing (vn) antibody titers > were not detected until to weeks p.i. taken together, the results obtained by western blotting analyses using purified virus and e. coli-expressed orfs to gene products, suggested that antibodies directed against the envelope e protein appear by day p.i., whereas antibodies directed against the nucleocapsid n and membrane m proteins can only be detected by the end of the second week p.i. no correlation could be demonstrated between vn and iif antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i. the porcine reproductive and respiratory syndrome virus (prrsv) is an enveloped rna virus which has been found to be responsible for reproductive failure in sows of any parities (late-term abortions, increased numbers of stillborn, mummified and weakborn pigs) and respiratory problems affecting pigs of all ages [ , ] . this new porcine virus has been provisionally classified h.d. loemba et al. within the genus arterivirus, according to its biochemical and morphological characteristics [ , ] . the viral genome is a positive single-stranded rna molecule approximately kb in length that contains eight open reading frames (orfs) similarly organized to that of the coronaviruses [ , ] . the virion contains three major structural proteins: a nucleocapsid protein n of kda, an unglycosylated membrane protein m (matrix protein) of - kda and a glycosylated membrane protein e (envelope associated glycoprotein) of - kda which are encoded by orfs, , and , respectively [ ] . clinical symptoms and production losses may vary widely among herds, but the vast majority of prrsv infections are subclinical [ , ] . presently some field and experimental evidence exists that indicates persistent lifelong viremia during prrsv infection [ , ] . an antibody-dependent enhancement mechanism has been reported to explain the persistent infection of porcine alveolar macrophages and circulating monocytes [ , ] . so far, little is known about the immunological status of prrsv-infected pigs. some investigators suggest that prrsv causes immunosuppression because of the recrudescence of secondary bacterial infections among the infected pigs [ ] . previous data indicated that antibodies to prrsv may be detected as soon as to weeks post-exposure using indirect immunofluorescence [ , ] and the immunoperoxidase monolayer assay [ ] . virus neutralizing antibodies were reported to appear later during the infection, usually when clinical signs are resolved [ , ] . the purpose of the present study was to evaluate the humoral immune response of prrsv-infected pigs. the kinetics of the appearance of specific igm and igg antibody titers during prrsv infection, in addition to viral structural protein specificities of the antibodies and duration of viremia have been investigated in pigs naturally-and experimentally-exposed to prrsv. the qu bec cytopathic strain iaf-klop of prrsv [ ] was propagated in marc- cells [ , a highly permissive cell line to prrsv kindly provided to us by j. kwang, u. s. meat animal research center, clay center, nebraska. serological identification of the cell culture-adapted iaf-klop strain was confirmed by indirect immunoftuorescence (iif) using monoclonal antibody (mab) sdowl [ ] , kindly provided by d. a. benfield and e. nelson, department of veterinary science, south dakota state university. this mab was found to be directed to an epitope of the n protein shared by both the north american and european isolates of prrsv [ , ] . homologous hyperimmune sera to the iaf-klop strain of prrsv were produced in pigs and rabbits, as previously described [ ] . in the first experiment, seven spf piglets were placed in a clinically prrsvinfected herd, whereas two similar piglets were introduced in a clinically-healthy neighboring barn [ ] . the spf pigs were selected from a farm free of atrophic rhinitis, mycoplasma hyopneumoniae, transmissible gastroenteritis virus, swine influenza virus, encephalomyocarditis virus, s. hyodysenteriae, sarcoptic mange, salmoneltosis, a. pleuropneumoniae and prrsv. in the second experiment, four five-week old spf piglets were intranasally inoculated with l s tcidso of the iaf-klop strain of prrsv. clinical signs of both the above piglets and a simultaneously mock-infected group of three pigs were monitored daily. blood samples were collected at days and p.i., then weekly during -day and day observation periods for sentinel piglets and experimentally-infected pigs, respectively. among the principal clinical signs observed in both naturally-and experimentally-infected pigs were intermittent raising of body temperature ( - °c), anorexia, lethargy, periocular oedema, conjunctivitis and blue discoloration of the ears. coughing and dyspnea (abdominal thumping) were usually noticed by the end of the first week of exposure [ , ] . no symptoms were observed in control piglets. for each group, one representative animal was necropsied at the end of the first week p.i. macroscopic and histopathological lesions of nonsuppurative interstitial pneumonitis were only demonstrated in the two infected pigs [ , ] . at the end of the -day observation period, no significant macroscopic and histologic lesions could be observed in pigs that had been infected experimentally. however, naturally-exposed sentinel pigs still manifested mild to moderate macroscopic and histologic lung lesions days p.i., as previously described [ ] . to attempt virus isolation, marc- cell monolayers were inoculated with sera samples collected from prrsv-infected pigs and monitored daily for the appearance of a cytopathic effect (cpe) or the presence of specific fluorescent foci [ ] . for all pigs tested (naturally-exposed and experimentally-infected) the virus could be isolated from sera samples collected by the end of the first week of exposure till the end of the observation periods (data not shown). at no time, could prrsv be recovered from serum samples collected from the control pigs. the iif test was carried out using acetone fixed prrsv-infected marc- cells, and fluorescein-conjugated anti-pig igm (kirkegaard & perry laboratories inc.) or anti-pig igg (sigma), as previously described [ ] . the iif antibody titers were expressed as the reciprocal of the highest serum dilution giving specific cytoplasmic fluorescence (table ). in both naturally-and experimentally-infected pigs, igm antibodies to prrsv were detected as early as days p.i. (iif titers of to ) and maximal titers were reached by day p.i. (iif titers of ). then, specific igm antibody titers drastically decreased until day or day p.i. specific igg antibodies were also detectable by the end of the first week of exposure, maximal titers being obtained around day to p.i. (iif titers of to ). thereafter, there was generally little change in igg antibody titers to prrsv of sera collected till the end of the -or -week observation period. the modified procedure described by yoon et al. [ ] was used for in vitro detection of virus neutralizing (vn) antibodies to prrsv. the vn antibody titers were expressed as the reciprocal of the highest dilution of sera that neutralized cpe induced in marc- cell monolayers by a constant dose of tcid o of virus. specific vn antibodies to prrsv could be detected in sera samples from only half of the infected piglets (data not shown). antibody titers > were not observed until - weeks p.i., with maximal titers ranging between ---. to study the reactivity of pig sera to prrsv specific proteins, immunoblotting experiments were performed using sucrose-gradient purified virus, as previously described [ ] . briefly, replicas of viral proteins, separated by sds-page and electrophoretically transferred onto nitrocellulose membranes, were incubated for h at °c in the presence of : dilution of the tested porcine sera. after washing in a . m tris-buffered saline (tbs) solution containing . % tween , the membranes were further incubated in the presence of : dilution of an alkaline phosphatase-conjugated anti-porcine igg (sigma). the immune complexes were revealed using a commercial alkaline phosphatase conjugate substrate kit (biorad, mississauga, ont.), containing nitroblue tetrazolium and -bromo- -chloro- -indolyl phosphate in tbs buffer. under reducing conditions, the three major prrsv structural proteins n ( kda), m ( kda) and e ( . kda) were consistently identified with the tissue cultureadapted iaf-klop strain of prrsv using homologous porcine hyperimmune serum [ ] . in the immunoblotting experiments with sera from both naturallyand experimentally-infected pigs (fig. ) , seroconversion to the m and n proteins of prrsv could not be demonstrated before the end of the first two weeks of infection. on the other hand, seroconversion to the e glycoprotein was generally obtained by the end of the third or fourth week p.i. viral protein specificity of the humoral immune response of infected pigs was further confirmed by western immunoblotting analyses using e.coli-expressed orfs to gene products. genomic rna was extracted from purified virus by the one-step guanidinium isothiocyanate-acid phenol method [ ] , then respective viral genes were amplified by rt-pcr, as previously described [ , ] . six oligonucleotide primers, containing ecori (sense primers) and bamhi (antisense primers) restriction sites at their '-end, were designed according to the sequence of the iaf-exp. strain of prrsv (embl/genebank accession number l ) [ ] . the pcr amplified products were purified using the geneclean ii as illustrated in fig. , with clinical sera samples no. and (naturallyinfected pigs), antibodies directed against the recombinant e protein could be detected by the end of the first week of infection, whereas antibodies directed against the recombinant n and m proteins could only be detected by the end of the second week of the prrsv infection. no reaction has been noticed with mbp protein (fig. c) and gst protein (data not shown) alone. in the present study, prrsv-infected piglets generally developed mild clinical signs of a respiratory disease within the first week post-exposure to the virus that lasted not more than to days. however, all the animals tested remained viremic till the end of the or -day observation period. our data are in agreement with previous observations by others who demonstrated that prrsv may persist for many weeks, even months in the infected pigs, despite their relatively high iif antibody titers [ , , ] . noteworthy, our results on the detection of igg antibodies to prrsv in infected pigs sera are also compatible with previous findings that specific igg antibodies can be detected by iif from - days p.i. up to several weeks (more than - months) after exposure to the virus [ , ] . in general, igg antibody titers detected by iif peaked by the end of the third or fourth week post-exposure. more recently, nelson et al. [ ] demonstrated that peaked igg antibody titers to prrsv (iif titers > ) may persist in experimentally-infected pigs for more than months, then decrease progressively to reach very low levels (iif titers < ) after more than days p.i. consequently, detection of igg antibodies in pig sera may indicate that the animals have been infected by prrsv in a recent or distant past. since no correlation could be demonstrated between the levels of igg antibody titers determined by iif and the viremic status of the animals, one cannot precisely pin point when the exposure to the virus had occurred or whether the pigs had been carrying the virus for a long time. interestingly, our results indicate that detection by iif of igm antibodies to prrsv may provide more precise information in the serological diagnosis of prrsv infection. indeed, the short-term persistence of circulating specific igm antibodies may be considered in the differentiation between acute and chronic prrsv infections in pigs. also in agreement with previous findings by other authors, antibodies with in vitro vn activity were relatively slow to appear and were not detected until - weeks p.i. [ , , ] . although the vn test has been reported to be less sensitive than the iif tests and immunoblotting [ ] , the long term persistence of prrsv in the experimentally-or naturally-infected animals despite high levels of antibodies to prrsv, raises the question as to what role the humoral immune response has in the protection to prrsv infection. also, it is possible for antibodies to enhance viral infection of fc-receptor-positive cells such as macrophages, by forming immune complexes that use the fc receptor to bind to the macrophages [ ] . thus, the putative protective role of neutralizing antibodies in prrsv infection needs to be further documented. western blotting analyses demonstrated that the immune response of experimentally-and naturally-infected pigs was directed to the three major viral structural proteins n, m and e, as previously reported in cases of pigs that have been experimentally-infected with the reference atcc-vr us strain [ ] . these preliminary observations were further confirmed by testing the reactivity of the porcine anti-prrsv sera against e. coli-expressed orfs to gene products. in light of our observations, it appears that antibody development to the various viral proteins in prrsv-infected pigs is chronological, initial detection of antibodies to the n, m and e proteins varied among pigs, ranging from to days p.i. these results are in agreement with previous findings by others [ , ] . however, it has been postulated that the stronger signal observed to the n and m proteins may reflect a higher molar ratio of these proteins in the virion relative to the e protein, or it may suggest a greater immunogenicity of these proteins in the pigs [ , ] . the results obtained using e. coil-expressed orfs to are in favor of the first hypothesis. indeed, antibodies to the recombinant mbp-e protein could be demonstrated as early as days pi, whereas antibodies to the n and m proteins could only be detected by the end of the second week p.i. this finding was not expected in case of the recombinant mbp-e protein since reactivity towards the viral envelope glycoprotein in immunoblotting experiments, using purified virus, could not be detected until the end of the third or fourth week p.i. this discrepancy may be due to the fact that when dealing with recombinant proteins, comparable amounts of the various proteins could be transferred onto the nitrocellulose membranes, whereas with purified viral preparations, the n and m proteins are expected to be present at a higher molar ratio comparative to the e glycoprotein [ , ] . indeed, in the case of equine arteritis virus it has been demonstrated that with the exception of gs (small envelope glycoprotein), the proteins are approximately equal in abundance, being present at a molecular ratio of (n): (m): (gi: large envelope glycoprotein) [ ] . the gs protein, which is expressed at a level similar to that of m in infected cells, is strikingly underrepresented in virus particles ( to %) [ ] . thus, it can be expected that for prrsv, the immunogenicity of the e glycoprotein in the pig is likely comparable to that of the n and m proteins. from results obtained with recombinant proteins, as well as with purified virus, there was no clear correlation between the appearance of circulating antibodies as detected by vn and iif tests, viremia and protein specificities of the circulating antibodies after various time p.i. the neutralization process may be the result of virus interaction with antibodies directed against epitopes located on different viral proteins. recently, we have been able to demonstrate that individually recombinant n, m and e proteins expressed by e. coli cells can induce the production of specific antibodies in rabbits and pigs; although iif antibody titers to iaf-klop strain ranged between to , the antisera are devoid of neutralization activity. expression of recombinant viral proteins in eucaryotic vectors should permit us to demonstrate if the conformation and glycosylation of viral envelope proteins are essential requirements for the expression of epitopes involved in virus neutralization. comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody 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 antibody-dependent enhancement of sirs virus replication single-step method of rna isolation by acid guanidium thiocyanate-phenol-chloroform extraction molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group structural proteins of equine arteritis virus porcine reproductive and respiratory syndrome enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line antigenic 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porcine reproductive and respiratory syndrome virus by monoclonal antibodies serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus antibody production and blastogenic response in pigs experimentally infected with prrs virus mystery swine disease in the netherlands: the isolation of lelystad virus morrison rb (t ) an indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera a modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera persistent and contact infection in nursery pigs experimentally infected with porcine reproductive and respiratory syndrome virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection we thank louise wilson, micheline ch nard, and nicole sawyer tbr their technical assistance. this work was supported in part by grants received from the national research council of canada (strategic grant # strgp- ), the minist re de t'agriculture, des p~cheries et de l'alimentation du qu bec, la f d ration des producteurs de porcs du quebec, and vetrepharm research inc., london, ontario, canada. received august , key: cord- -bfvdhai authors: hattermann, k.; müller, m. a.; nitsche, a.; wendt, s.; donoso mantke, o.; niedrig, m. title: susceptibility of different eukaryotic cell lines to sars-coronavirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: bfvdhai in order to define and characterize target cells of sars-coronavirus (sars-cov) we studied the susceptibility of different permanent and primary eukaryotic cell lines to sars-coronavirus. beneath vero e cells sars- coronavirus infection could also be demonstrated in two pig cell lines (poek, ps) and one human cell line (huh- ) using the indirect immunofluorescence assay and a newly established quantitative real-time pcr. in all susceptible cell lines mrna of the angiotensin-converting enzyme (ace ), the functional receptor for sars-cov infection, could be detected by rt-pcr. our results show that there is a correlation between the abundance of ace mrna and sars-cov susceptibility. markets and sometimes eaten in china [ ] . however, these species are not necessarily the natural hosts. in our study we investigated if sars-cov could infect and replicate in permanent cell lines and primary cells of different species in order to i) define and characterize potential target cells of sars-cov, ii) to understand the mechanism of virus transmission and the nature and range of target cells and organisms. we furthermore investigated sars-cov susceptible and not-susceptible cell lines for ace mrna. to study the kinetic of sars-cov infection, various cell lines were infected with sars-cov strain hong kong at multiplicities of infection (m.o.i.) of approximately . the production of sars-cov was determined in the supernatant and in the infected cells at definite time points post infection using a quantitative real-time pcr [ ] . in parallel, infected cells were investigated for the presence of viral protein using an indirect immunofluorescence assay (ifa) [ ] . for stock production, sars-cov (strain ) isolated from a hong kong patient (kindly provided by wilina lim, government virus unit hong kong) was added to vero e cells (american type culture collection, atcc, crl ). after h of incubation the supernatant and the infected cells were harvested, stringently centrifuged ( min at × g) and the supernatant was aliquoted. afterwards the virus titre was determined ( . * pfu/ml). all cell lines used (table ) were grown in the appropriate culture medium recommended by atcc or ecacc (european collection of cell cultures). porcine peripheral blood mononuclear cells (pbmc) were isolated from a healthy pig and grown in roswell park memorial institute medium (rpmi ) (gibco, paisley, uk) with % fcs and mm glutamine (icn, costa mesa) and µg/ml streptomycin and u/ml penicillin (biochrom, berlin, germany). chicken embryo fibroblasts were prepared from -day-old chicken embryos and cultivated in dulbeccos modified eagle medium (d-mem) (gibco, paisley, uk) supplemented with % fcs and µg/ml streptomycin and u/ml penicillin. one day before infection adherent cells were seeded onto sterile glass slides in -well plates while suspension cells were cultivated in -well plates. adherent cells were infected with µl and suspension cells with µl of infectious supernatant of sars-cov ( . * pfu/ml). vero e cells were used as a positive control while uninfected cells were used as negative controls. for quantitative real-time pcr rna from approximately . * cells was prepared using the rneasy protect mini kit (qiagen, hilden, germany). similarly, infected cell supernatant was centrifuged at . rpm in a heraeus varifuge to remove cells; rna was extracted from µl supernatant using the qiaamp viral rna mini kit (qiagen, hilden, germany). the amount of sars-cov rna was determined in triplicate by quantitative real-time pcr as described elsewhere [ ] . for rt-pcr analysis total rna was isolated using rneasy protect mini kit (qiagen) and was twice digested with dnase i (ambion, huntingdon, uk) as described in the manual instructions. for the detection of ace mrna, cdna was produced from total rna of all cell lines susceptible for sars-cov sars-cov infected cells were cultivated for a total of h. at , , , and h after infection i) cell morphology was assessed by inspection with a light microscope for cpe diagnosis, ii) supernatant and cells were investigated for viral rna load using the quantitative real time pcr, iii) cells were fixed and investigated for viral protein with the indirect immunofluorescence assay and analyzed by confocal laser scanning microscopy. furthermore sars-cov susceptible and not-susceptible human cell lines were analyzed for mrna of ace . analysis with a light microscope revealed cpe in vero e cells starting h after infection and in huh- cells starting h after infection. vero e cells formed syncytia or progressed from typical elongated morphology to round dead cells with cell debris in the supernatant. by h after infection almost all vero e cells were detached from their support, whereas huh- cells were still growing in monolayer and tended to syncytia formation. in contrast, no visible changes were observed in the porcine cells (data not shown). ifa revealed that h after infection viral protein could be detected in approximately % (data not shown) and h after infection in approximately % of the vero e cells (fig. /ii a, b) . in contrast, % of the huh- cells were positive for viral antigen not until h after infection ( the quantification of sars-cov rna by quantitative real-time pcr revealed a significant increase of intracellular viral rna in vero e , huh- , poek, (fig. /i a-c) and ps cells (data not shown). to determine whether extracellular virus particles had been produced by sars-cov infected human and porcine cells, cell-free supernatants were tested by quantitative real-time pcr at different times post infection. an increase of sars-cov rna was detected in the supernatant of infected vero e , huh- , poek ( fig. /i a-c) and ps cells (data not shown). as expected, the investigation of all sars-cov susceptible cell lines (vero e , huh- , poek and ps) for mrna of ace was positive in all cases though we failed to detect ace expression by ifa, western blot and facs analysis using commercially available monoclonal and polyclonal antibodies (alpha diagnostics, san antonio, usa) against human ace (data not shown). although same amounts of cdna were used and the experiments were repeated three times the signals of the porcine cell lines maintained much weaker ( fig. a lane and ) . after sequencing, blast analysis of the resultant pcr products of sars-cov susceptible vero e and huh- cells showed a % homology to the mrna of the humanace (genbank accession no. nm ), while porcine poek and ps cells showed % homology to the mrna of the human ace (genbank accession no. nm ). no mrna oface could be detected in the not-susceptible cell lines (fig. b ). all cdna samples used in the ace pcr have been tested positive in the control pcr (fig. c) . to examine whether the infection efficiency in porcine poek could be increased by viral adaptation cells were infected with sars-cov hong kong as mentioned above and cultivated for weeks. after this period of time the indirect immunofluorescence assay was carried out and sars-cov positive cells were counted. an adaptive effect resulting in a -fold higher infection rate could be observed in poek cells (fig. /ii g) . in our studies we could demonstrate that the green monkey cell line vero e and the human huh- cells show a high susceptibility to sars-cov resulting in high infection rates within hours. using the quantitative real-time pcr we could detect up to * rna copies in . * sars-cov infected vero e cells h after infection. these data could be confirmed with the ifa showing up to % infection of vero e cells and % infection of huh- cells h after infection. another finding in our studies was the demonstration that sars-cov could replicate in porcine ps and poek cells. these observations have also been made by others [ ] who demonstrated replication of sars-cov in the porcine cell line pk- . however, in our experiments infection rates of the porcine cells with sars-cov were clearly lower. the angiotensin-converting enzyme has been identified to play an important role in sars-cov entry [ ] . it can be expected that the sars-cov susceptible cells express a sars-cov specific receptor. we could identify mrna of ace in sars-cov susceptible vero e , huh- , porcine poek and ps cells but ace protein expression could not be verified by several methods suggesting that the expression level may be very low. recent studies have revealed that recombinant ace expressed on the cell surface does trigger viral permissiveness and that infection can be blocked by soluble ace [ , ] . the different infection rates in porcine poek and ps cells may be due to a lower expression rate of the ace which we showed on mrna level. moreover there could be the necessity of an accessory factor for virus adsorption and entry. this could be confirmed by chan et al. [ ] who found ace expression also in cells that were not susceptible for sars-cov. another possibility for lower infection rates in porcine cells may also be that the sequence homology of the human ace strongly deviates from the porcine ace . interestingly, infection efficiency in poek cells could be increased quickly by adaptation of the virus to poek cells. present studies will therefore clarify the question to what extent the adapted viruses differ from the original virus stock by growth comparisons and sequencing. persistent infection of sars coronavirus in colonic cells in vitro virus detectives seek source of sars in china's wild animals identification of a novel coronavirus in patients with severe acute respiratory syndrome retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-pcr-based search for a human circovirus susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme and infection can be blocked by soluble receptor severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts newly discovered coronavirus as the primary cause of severe acute respiratory syndrome angiotensin-converting enzyme is a functional receptor for the sars coronavirus retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme guideline to reference gene selection for quantitative real-time pcr cumulative number of reported probable cases of sars for the period this work was supported by the european union (spc. ).we thank anette teichmann and inga nehlmeier for excellent technical assistance. in addition we thank dr. stephen norley for critically reading the manuscript and any helpful discussions. key: cord- -i rxesrg authors: oh, jongsuk; lee, kyung-won; choi, hwan-won; lee, changhee title: immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: i rxesrg porcine epidemic diarrhea virus (pedv) is a highly contagious enteric pathogen of swine. acute pedv outbreaks have continually emerged in most swine-producing asian countries and, recently, in the united states, causing significant economic losses in the pig industry. the spike (s) protein of pedv is a type transmembrane envelope glycoprotein and consists of the s and s domains, which are responsible for virus binding and fusion, respectively. since the s domain is involved in a specific high-affinity interaction with the cellular receptor and induction of neutralizing antibody in the natural host, it is a primary target for the development of effective vaccines against pedv. in this study, a codon-optimized pedv s gene containing amino acid residues – was synthesized based on a multiple alignment of the s amino acid sequences of pedv field isolates and used to establish a stable porcine cell line constitutively expressing the pedv s protein. the purified recombinant s protein was found to mediate highly potent antibody responses in immunized rabbits. the antibodies strongly recognized the recombinant s protein from cell lysates and supernatants of s -expressing cells, whereas they bound weakly to the authentic s protein of pedv vaccine strain sm - . furthermore, a serum neutralization test revealed that the rabbit antisera completely inhibit infection of the pedv vaccine strain at a serum dilution of : . we then tested the ability of vaccination with the recombinant s protein to protect piglets against pedv. late-term pregnant sows were inoculated intramuscularly with the purified s protein, and the outcome was investigated in passively immunized suckling piglets after a virulent pedv challenge. the results showed that vaccination with s protein efficiently protected neonatal piglets against pedv. our data suggest that the recombinant s protein shows potential as an effective and safe subunit vaccine for ped prevention. porcine epidemic diarrhea (ped) is a devastating swine disease that is characterized by acute enteritis and lethal watery diarrhea followed by severe dehydration leading to death, with a high mortality rate in piglets [ , , ] . the disease was initially recognized in england in [ ] , but the causative agent of this disease, ped virus (pedv), was later identified in [ ] . ped epidemics were first reported in asia in , and since then, ped has continued to threaten swine health, causing substantial economic losses in the asian swine industry [ , , , ] . in , ped outbreaks suddenly occurred in the united states and have swept through the pork industry across the country, raising concerns about control measures for ped prevention [ , ] . in korea, pedv appeared in [ ] ; however, a retrospective study indicated that the virus had been present as early as [ ] . although periodic vaccination strategies have been implemented nationwide to control ped in korean swine herds, pedv has continually emerged, causing tremendous harm to the productivity of korean pig farms. pedv, a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales, is a large, enveloped virus possessing a single-stranded, positive-sense rna genome of approximately kb with a cap and a polyadenylated tail [ , ] . the pedv genome is composed of the untranslated region (utr), at least seven open reading frames (orf a, orf b, and orf through ), and the utr [ ] . the two large orfs a and b make up the two-thirds of the genome and encode the non-structural replicase genes. the remaining orfs in the terminal region code for four major structural proteins: the - -kda glycosylated spike (s) protein, the - -kda membrane (m) protein, the -kda envelope (e) protein, and the -kda nucleocapsid (n) protein [ , ] . the s protein of pedv is a type i membrane glycoprotein composed of , to , amino acids (aa), depending on the strain. it contains a putative signal peptide (aa - ), a large extracellular region, a single transmembrane domain (aa , - , ), and a short cytoplasmic tail. although pedv has an uncleaved s protein because it lacks a furin cleavage site, the s protein can be divided into s (aa - ) and s ( -the last aa) domains based on homology with s proteins of other coronaviruses [ , , , ] . like other coronavirus s proteins, the pedv s protein is known to play a pivotal role, interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host [ , ] . more precisely, previous studies have shown that the s domain includes the main neutralizing epitopes and the receptor-binding region [ , ] . furthermore, along with the full-length s gene, the s portion is known to be a suitable region for determining genetic relatedness among the different pedv isolates and for developing differential diagnostic assays [ , ] . considering these molecular and biological features of the s domain, it would be an appropriate target for developing effective vaccines against pedv. in the present study, therefore, we first synthesized a full-length, codon-optimized pedv s gene and then generated a stable porcine-origin cell line constitutively expressing the recombinant s protein. the protective efficiency of a recombinant-s -protein-based vaccine against pedv was then evaluated in the natural host. the recombinant s protein was capable of inducing an efficient antibody response in immunized rabbits. moreover, immunization with the pedv s protein was found to elicit specific neutralizing antibodies in sows and to protect passively immunized suckling piglets against pedv challenge. cells, virus, antibodies, and plasmid hek- t cells (crl- ) were purchased from the american type culture collection (atcc, manassas, va) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca) with % fetal bovine serum (fbs, invitrogen) and antibiotic-antimycotic solutions ( ; invitrogen). pk- cells were grown in rpmi medium (invitrogen) supplemented with % fbs and antibiotic-antimycotic solutions. vero cells were cultured in alpha minimum essential medium (a-mem, invitrogen) with % fbs and antibiotic-antimycotic solutions. the cells were maintained at °c in a humidified % co atmosphere. the pedv vaccine strain sm - was obtained from the korean animal and plant quarantine agency and propagated in vero cells as described previously [ ] . challenge pedv was prepared from the small intestine of a -day-old suckling piglet orally inoculated with small intestine homogenate containing the field virus. small intestine tissues were collected and homogenized in a % suspension with a-mem using a magna lyser (roche diagnostics, mannheim, germany) with three repetitions of s at a speed of , rpm, and suspensions were clarified by centrifugation at , g (hanil centrifuge fleta , incheon, korea). the clarified supernatant was filtered through a . -lm-pore-size syringe filter (millipore, billerica, ma), aliquoted, and stored at - °c until use as crude challenge virus. all of the horseradish peroxidase (hrp)-conjugated secondary antibodies were purchased from santa cruz biotechnology (santa cruz, ca). the pedv s-protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, korea). a plasmid encoding the s fragment of severe acute respiratory syndrome coronavirus (sars-cov), pcdm -sars-cov-s -ig, was kindly provided by hyeryun choe (harvard medical school, boston, ma). dna manipulation and cloning were performed according to standard procedures [ ] . the e. coli strain dh a (rbc bioscience, taiwan) was used as the host for general cloning. the plasmid encoding the full-length s gene of the pedv field strain knu- , pcdm -pedv-s -ig, was described previously [ ] . to construct the plasmids expressing s and its variants, the consensus sequence of pedv s was identified based on a multiple alignment of the s aa sequences of pedv field isolates [ ] and utilized to synthesize a full-length, codon-optimized pedv s gene (encoding aa - ) according to a method described previously [ ] . the codon-optimized pedv s gene was cloned into a modified expression vector, pcdm -fc, which contains the cd signal sequence and the fc domain of human igg [ ] , thereby producing a human fc-tagged fusion protein, rs -ig. all of the pedv s -truncated variants used in this study were generated using this template with the previously described primer sets [ ] . an fctagged pedv rs -ig fragment obtained from pcdm -fc-rs -ig was then subcloned into a pfb-neo retroviral vector (stratagene, la jolla, ca) using the sali and xhoi restriction sites to construct a pedv rs -ig gene expression plasmid, pfb-neo-pedv-rs -ig. all of the constructed plasmids were verified by nucleotide sequencing. generation of a stable pk- cell line expressing pedv rs -ig the retrovirus gene transfer system (stratagene) was applied to generate a cell line constitutively expressing the recombinant pedv rs -ig gene or an empty retroviral vector as described elsewhere [ , ] . antibiotic-resistant continuous cell clones were examined by rt-pcr to verify the presence of the full-length rs -ig gene, and the positive clones (pk-rs -ig) were then amplified for subsequent analyses. immunofluorescence assay (ifa) pk-rs -ig cells were grown on microscope coverslips placed in -well tissue culture plates. at h post-seeding, the cells were fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were subsequently blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with a goat anti-human igg antibody conjugated with fluorescein isothiocyanate (fitc) (santa cruz biotechnology). finally, the cells were counterstained with , -diamidino- -phenylindole (dapi; sigma, st. louis, mo), and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). fluorescence-activated cell sorting (facs) analysis rs expression in pk-rs -ig cells was analyzed by flow cytometry. briefly, cells were trypsinized at h postseeding and centrifuged at g (hanil centrifuge fleta ) for min. the cell pellet was washed with cold washing buffer ( % bsa and . % sodium azide in pbs) and cells were resuspended in % formaldehyde solution in cold wash buffer and fixed at °c in the dark for min, followed by incubation in . % triton x- in pbs for permeabilization at °c for min. following centrifugation, the cell pellet was resuspended in normal mouse igg antibody (santa cruz biotechnology) and incubated at °c for min. the cells were washed and reacted with fitc-conjugated anti-human or anti-mouse igg secondary antibody at °c for min in the dark. the stained cells were washed again and analyzed by facscan flow cytometry. western blot analysis hek t cells were transfected with each plasmid using lipofectamine (invitrogen) according to the manufacturer's protocol and solubilized in lysis buffer at h post-transfection as described previously [ ] . cell lysates were also prepared from vero cells infected with pedv sm - at a multiplicity of infection (moi) of . at the indicated time points using lysis buffer. the protein concentrations of the cell lysates were determined using a bca protein assay (pierce, rockford, il). pk-rs -ig cells were grown at cells/well in a -well tissue culture plate, and the protein-containing culture supernatants were harvested on days , , and . soluble proteins were immunoprecipitated with protein a sepharose cl- b beads (ge healthcare, piscataway, nj) in the presence of protease inhibitors at °c for h. the beads were collected by centrifugation at , g (eppendorf centrifuge r, hamburg, germany) for min at °c and washed three times with . m nacl in pbs. the cell lysates or the beads were mixed with nupage sample buffer (invitrogen) and boiled at °c for min. the proteins were separated on a nupage - % gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore). the membranes were then blocked with % powdered skim milk (bd biosciences, belford, ma) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at °c for h and reacted directly with the goat anti-human igg hrp-conjugated secondary antibody, the anti-s rabbit serum or the anti-pedv s mab, followed by the corresponding hrp-labeled secondary antibody at a dilution of : , for h at °c. finally, the proteins were visualized using enhanced chemiluminescence (ecl) reagents (ge healthcare) according to the manufacturer's instructions. protein purification pk-rs -ig cells were grown at cells/well in a -well tissue culture plate in serum-free medium (optipro sfm; invitrogen). at h post-seeding, the protein-containing culture supernatants were harvested and soluble proteins were immunoprecipitated with protein a sepharose cl- b beads in the presence of protease inhibitors at °c for h. the beads were collected and washed as described above. the samples were subsequently eluted with mm sodium citrate/ mm glycine (ph . ) and neutralized with m tris-hcl (ph . ). the purified proteins were concentrated with amicon ultra centrifugal filters k (millipore). protein concentration was measured using a bca protein assay (pierce, rockford, il) and the final products were analyzed by western blotting to confirm target protein purification. two new zealand white rabbits were immunized intradermally with lg of purified rs -ig resuspended in pbs in the presence of freund's complete adjuvant and boosted four times with a freshly prepared emulsion of lg immunogen and freund's incomplete adjuvant at -week intervals. pre-immune sera were collected before starting the immunization, and antisera were collected at each boost. the in vivo swine experiments described here were performed at the choongang vaccine laboratory animal facility under the guidelines established by its institutional animal care and use committee. a total of eight pregnant sows were obtained from a pig farm with no outbreaks or vaccination with pedv and randomly divided into four groups of two sows. all animals were determined to be free of antibodies to pedv. the design for the present immunogenicity study involving eight pregnant sows is outlined in table . the sows in group were immunized intramuscularly with attenuated pedv live vaccine and inactivated pedv vaccine obtained from choongang vaccine laboratory, in order at -week intervals prior to farrowing. the pigs in group were immunized intramuscularly with pedv live vaccine and lg of purified rs -ig resuspended in pbs in the presence of an oil-in-water adjuvant, in order at -week intervals before farrowing. both the pedv live and inactivated vaccines were administrated according to the manufacturers' manuals. the sows in group and group were inoculated intramuscularly three times at -week intervals with lg of purified rs -ig mixed with the oil adjuvant or with cell culture medium as a negative control. paired blood samples and colostrum were collected at -week intervals before farrowing and at farrowing, at delivery, respectively. one -to -day-old piglet ( piglets in total) was selected randomly from each farrowing sow in the immunized and control groups for challenge exposure with virulent pedv. piglets from all groups were challenged orally with ml of small intestine homogenate containing tcid /ml of pedv field virus, equivalent to . viral genome copies per ml, determined using real-time rt-pcr as described previously [ ] . clinical signs of diarrhea and death in challenged piglets were monitored daily throughout the study. stool samples from all groups were collected every day with -inch cotton-tipped swabs and subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, korea) according to the manufacturer's protocol. all piglets from the vaccinated and control groups were euthanized at days after challenge for post-mortem examination. small-intestinal tissue specimens collected from each piglet (\ mm thick) were fixed with % formalin for h at rt and embedded in paraffin according to standard laboratory procedures. the formalinfixed paraffin-embedded tissues were cut into -to -lmthick sections on a microtome, floated on a °c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for min and washed in decreasing concentrations of ethanol ( , , , , and %) for min each. the deparaffinized intestinal tissues sections were subjected to immunofluorescence assay using an n-specific mab and alexa fluor -conjugated goat anti-mouse antibody as described above. the presence of pedv-specific neutralizing antibodies in serum and colostrum samples collected from sows in all groups was determined using a serum neutralization (sn) test in -well microtiter plates using pedv vaccine strain sm - as described previously [ ] . briefly, individual virus stocks were diluted in serum-free a-mem to make pfu in a ll volume. the diluted virus was then mixed with ll of twofold dilutions of individual pedv s immunogen a pedv s immunogen pedv s immunogen placebo placebo placebo a placebo (cell culture medium) and protein (pedv s ) immunizations were given intramuscularly with the oil-in-water adjuvant b commercial pedv live and inactivated vaccines were administrated intramuscularly according to the manufacturers' instructions with the oil-in-water adjuvant inactivated sera in -well plates and incubated at °c for h. next, the mixture was incubated at °c for h, approximately vero cells in ll of a-mem were added to each well, and the mixture was maintained at °c in a % co incubator for to days. the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited the virus-specific cytopathic effect in both of the duplicate wells. results were also visualized by staining the wells with a crystal violet-formaldehyde staining reagent ( . % crystal violet, . % ethanol, and % formaldehyde in . m pbs) for h at rt. the student's t test was used for all statistical analyses, and p-values of less than . were considered statistically significant. generation of stable porcine-origin cell lines expressing the full-length, codon-optimized s protein we have previously constructed a series of expression plasmids encoding the full-length s -ig (s ). although expression of this gene could be detected in transiently transfected cells by western blot analysis, its expression level was relatively low and insufficient for further protein purification study (fig. , lane ) . thus, to enhance s protein expression, we first synthesized the full-length, codon-optimized s gene and constructed a panel of expression plasmids encoding the codon-optimized rs -ig (s ) and two c-terminally truncated s variants: rs - -ig and rs - -ig. these constructs were independently transfected into hek- t cells, and expression of each construct was robustly detectable by western blot analysis using an anti-human igg secondary antibody (fig. , lanes to ) , compared with a normal s protein (lane ). this result indicates that codon optimization greatly increases the expression level of s upon transient transfection. subsequently, to easily produce preparative amounts of the s protein, sublines of pk- cells were established to stably express the recombinant codon-optimized s under the control of a retroviral ltr promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blotting to identify s gene expression at the mrna and protein level, respectively (data not shown). based on the results of western blot analysis, one pk-rs -ig cell clone that consistently expressed the highest level of s was chosen for subsequent studies. to characterize pk-rs -ig cells, intracellular and extracellular expression levels of s were examined by immunofluorescence, facs analysis, and western blotting. as shown in fig. a , specific cell staining was clearly evident when pk-rs -ig cells were reacted with the antihuman igg antibody, confirming a consistent high expression level of the s protein. the majority of the cells consistently exhibited specific fluorescent signals, indicating a homogenous population of cells expressing s (fig. b) . time-course western blot analysis using culture supernatants revealed that the pk-rs -ig cells stably express and cumulatively secrete high levels of approximately -kda s (fig. c ). in addition, the overall growth kinetics of s -gene-expressing pk- cells were found to be similar to those of the parental pk- cells, indicating that s expression has no effect on cell growth (data not shown). further, the recombinant s protein tagged with the human igg fc domain expressed in the supernatants of stable pk-rs -ig cells was purified using protein a sepharose beads. the purified s protein was detectable at a high level by ponceau s staining, and this was confirmed by immunoblotting with anti-human igg antibody (fig. d ). using our purification and concentration procedures, we were able to obtain approximately lg of the s protein from ml culture supernatant of pk-rs -ig cells cultivated in one well of a -well plate for h. antibody response of the recombinant s immunogen in rabbits rabbit antisera were collected before immunization (preimmune) and at each boost at -week intervals. the serum samples at : , to : , dilutions were examined for binding to the recombinant fusion protein rs -ig or the authentic s protein by western blot analysis. the antiserum collected at the second boost reacted with the purified rs protein in a concentration-dependent manner (fig. a, top panel) , similar to the binding capacity of the anti-igg secondary antibody, which can directly recognize the protein fused to the fc region of human igg (middle panel). the antisera were found to retain nearly equivalent reactivity at the third and fourth boosts (data not shown). in contrast, the rabbit antiserum bound weakly to the authentic s protein prepared from cells infected with a pedv vaccine strain (top panel), whereas an mab raised against the vaccine strain reacted strongly with the authentic s protein (bottom panel). the rabbit antisera were further tested for their neutralizing activity against the pedv sm - vaccine strain, which can be grown in vero cells in the absence of trypsin. as shown in fig. b , the antisera collected at the final boost at : dilution fully protect vero cells from sm - infection (left panel), whereas pig hyperimmune sera raised against the vaccine virus were highly effective in inhibiting virus infection with neutralizing antibody titers of [ : (right panel). the rabbit sera at the second and third boosts possessed comparable neutralizing activities against the vaccine strain (data not shown). taken together, our data indicated that the recombinant s immunogen elicits potent antibody responses in immunized rabbits. however, the rabbit antisera strongly recognized the homologous s protein representing the s protein of field isolates but recognized the heterologous s protein of the pedv vaccine strain inefficiently, suggesting that there are antigenic differences between the vaccine strain and field pedvs. to determine the immunogenicity of the s -based subunit vaccine, pregnant sows assigned to four groups were immunized intramuscularly, as outlined in table . serum samples were collected at weeks and weeks prior to farrowing and at delivery and were subjected to a serum neutralization test against the pedv vaccine strain. nonimmunized sows showed only minimal neutralizing antibody titers, whereas immunized sows exhibited gradually increased neutralizing antibody titers, which increased considerably after the final vaccination (fig. ) . briefly, sows immunized with two doses of pedv live and inactivated vaccines at -week intervals (group ) had neutralizing titers of [ : at delivery. in contrast, sows immunized with two doses of pedv live and s protein vaccines (group ) or three doses of s protein vaccine (group ) at -week intervals produced relatively lower neutralizing antibody titers of : to : compared to those in group . in addition, colostrum samples from each group were found to have neutralizing antibody titers comparable to those in the corresponding serum samples (fig. b) . these results were coincident with the rabbit study described above, since the rabbit antisera generated by immunization with the recombinant s protein also contained low levels of neutralizing antibodies to the heterologous pedv vaccine strain. lastly, to assess the efficacy of immunization with the s protein, eight -to -day-old neonatal piglets from each sow were arbitrarily selected and challenged orally with wild-type pedv. clinical observations of death, diarrhea, and virus shedding in challenged piglets are summarized in table . in the control group, one piglet died and the other piglet experienced severe watery diarrhea post-challenge. although none of the piglets from any of the groups of immunized sows died during the challenge experiment, the number of piglets exhibiting diarrhea after challenge varied depending on the group. all piglets delivered from group sows showed mild-to-severe diarrhea lasting for the entire experiment at days post-challenge. in contrast, piglets from sows in groups and experienced only mild diarrhea for or days after challenge or throughout the challenge experiment. likewise, all piglets from immunized sows (groups , , and ) exhibited mild intestinal lesions, and viral antigens were detected only in their small intestines, whereas the majority of enterocytes over the entire villi in the control piglets (group ) were affected by pedv, showing moderate-to-severe villous atrophy (fig. ) . altogether, all immunization methods used in this study were capable of protecting passively immunized neonatal piglets against mortality and severe disease after challenge. however, immunizations involving at least one dose of pedv s protein vaccine (groups and ) were more efficacious than immunization of sows with two doses of pedv live and killed vaccines (group ) in reducing the overall degree of diarrhea, in terms of the duration and severity, in the suckling piglets. pedv continues to have a severe economic impact in swine-raising countries in asia and, more recently, in the united states. vaccination against pedv is an important and effective prevention measure. pedv entry into host cells is mediated by the s glycoprotein on the viral surface, which interacts with the cellular receptor and induces direct membrane fusion. the pedv s protein is also responsible for inducing neutralizing antibodies in the natural host and hence is a logical target in the development of effective vaccines. furthermore, the s domain is a key functional portion of the s protein, which is associated with viral binding to host-cell receptors and contains neutralizing epitopes [ , ] . therefore, the s protein of pedv is considered to be a potential candidate antigen for vaccination attempts. in the present study, the first aim was to stably express the full-length, codon-optimized s gene of pedv in porcine-origin cells and to evaluate the immunogenicity and efficacy of the recombinant s protein. our codon-optimization approach dramatically enhanced the expression level of the gene of interest upon transient transfection using a mammalian expression system. subsequently, we were able to successfully generate a stable pk cell line continuously producing large amounts of the codon-optimized s protein. following the purification and concentration processes, approximately - lg of the recombinant s protein could be consistently harvested from pk-rs -ig cells in each well of a -well plate. since the antibody response is a critical indicator to assess the effect of a vaccine, we immunized rabbits with the s -based immunogen prepared from culture supernatants of pk-rs -ig cells and determined whether or not they developed humoral immunity. pedv-specific antibodies were strongly detectable in rabbit sera collected from the second boost, even at the highest dilution ( : , ), when reacted with the recombinant pedv-s protein purified from s -expressing pk-rs -ig cells. in contrast, the binding capacity of the antiserum was dramatically reduced when the authentic s protein in wholecell lysates prepared from cells infected with sm - was used as the antigen for western blotting. moreover, the rabbit antisera could completely block infection of sm - only at a serum dilution of : , whereas the pig sera raised against sm - contained high levels of neutralizing antibody against the homogeneous virus ([ : ). since the pedv field isolate propagated in the current culture system is unavailable to us, we were unable to test the neutralizing capacity of those antisera against the field virus in this study. however, it is conceivable that rabbit antisera directed against the s protein may be more effective in neutralizing infection with the field virus than with sm - . on the other hand, the weak interaction and neutralizing activity of the anti-s rabbit serum against the sm - s protein may be attributed to genetic variations between the s proteins of the vaccine strain and field isolates. indeed, the korean field isolates, including the challenge strain used in this study, were found to display a high degree ([ %) of genetic heterogeneity, especially in the s domain, compared with the pedv vaccine strain sm - [ , ] . nevertheless, western blot and virus neutralizing assays showed that the recombinant s protein efficiently elicits humoral immune responses against pedv. in korea, several management strategies, including vaccination, have been employed to control pedv in pig farms. the most highly recommended immunization schedule involves two doses of attenuated live and inactivated killed vaccines in gilts at -to -week intervals before mating and in pregnant sows at and weeks of gestation. in the present study, we compared the efficacy of this common vaccination protocol and s protein-based vaccination. although all of the immunized sows developed neutralizing antibodies against sm - , only the sows immunized with live and killed vaccines (group ) had high neutralizing antibody titers, ranging from : to : . however, consistent with the rabbit immunization study, sows in groups and , immunized with at least one dose of the s -protein-based subunit vaccine, developed weak neutralizing responses to the vaccine virus, with titers ranging from : to : . successful protection against pedv is based on the presence of specific neutralizing antibodies in immune sows that are passively transferred to their piglets through colostrum and milk. our data showed that, regardless of vaccination group, all neonatal piglets from immunized sows survived after challenge with virulent pedv, suggesting that the s subunit vaccine provides effective lactogenic immunity to prevent mortality comparable to whole-virus-based vaccines. however, the s based vaccination strategies, which produced relatively low neutralizing antibody responses, exhibited more-efficient protection, with respective to the duration and severity of diarrhea, than the common live and killed vaccination procedure. it is therefore plausible that the actual levels of neutralizing antibodies raised against the s subunit vaccine are underestimated by the vaccine-virus-based neutralization test. thus, improved diagnostic tools are needed to differentiate vaccinal antibodies from those resulting from natural infection with the field virus. for this purpose, the recombinant s protein purified from pk-rs -ig cells could be further used as the diagnostic antigen in an enzyme-linked immunosorbent assay (elisa), and this aspect is currently under investigation. additionally, it is possible that the full-length s protein may induce stronger immune responses than s alone because it contains multiple functional domains and neutralizing epitopes. in fact, meng et al. [ ] have recently reported that the full-length pedv s gene induces a better immune response than the n-terminal half alone, using the recombinant dna plasmid in a mouse model. however, production of the full-length s protein may be unachievable in our system because its expression may cause cytotoxicity due to the presence of potential fusion activity in the c-terminal portion of the s protein. in conclusion, to the best of our knowledge, this is the first evaluation of the immunological and protective effects triggered by recombinant s protein in rabbit and pig models. the results presented here indicate that the recombinant s protein can elicit a specific antibody response and induce neutralizing antibodies, suggesting its excellent immunogenicity in the natural host. furthermore, challenge experiments revealed that the s protein-based vaccine protected passively immunized suckling piglets against field pedv. despite the nationwide use of commercial live and killed pedv vaccines, swine herds in korea have continued to experience repeated outbreaks, leading swine practitioners and researchers to question their protective efficacy. based on the present and previous studies, we hypothesize that antigenic and genetic variation between the vaccine virus and field pedvs responsible for periodic outbreaks in herds may be the cause of the low efficacy or failure of vaccination. accordingly, current pedv vaccines manufactured from cell-adapted viruses should be reassessed to determine their efficacy and improved if necessary. although further studies with a larger number of animals will be needed to better evaluate the efficacy of the s protein vaccine and to optimize immunization procedures, the recombinant s protein has potential for use in improving or developing effective and safe vaccines for ped prevention. amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the coronavirus spike protein is a 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multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus completion of the porcine epidemic diarrhea coronavirus (pedv) genome sequence isolation of porcine epidemic diarrhea virus (pedv) in korea mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea the n-terminal region of the porcine epidemic diarrhea virus spike protein is important for the receptor binding outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea new variants of porcine epidemic diarrhea virus, china evaluation on the efficacy and immunogenicity of recombinant dna plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus deadly pig virus slips through us borders contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection letter to the editor retrospective study of porcine epidemic diarrhea virus (pedv) in korea by in situ hybridization a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus coronaviruses molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic cleavage of peplomeric glycoprotein e of mhv yields two k subunits and activates cell fusion spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan acknowledgments we gratefully thank hyeryun choe from harvard medical school for providing reagents. this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology ( - ). key: cord- -bgm smuo authors: li, lan; zheng, qisheng; zhang, yuanpeng; li, pengcheng; fu, yanfeng; hou, jibo; xiao, xilong title: antiviral activity of recombinant porcine surfactant protein a against porcine reproductive and respiratory syndrome virus in vitro date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: bgm smuo porcine reproductive and respiratory syndrome virus (prrsv) has caused significant economic losses in the swine industry worldwide. however, there is not an ideal vaccine to provide complete protection against prrsv. thus, the need for new antiviral strategies to control prrsv still remains. surfactant protein a (sp-a) belongs to the family of c-type lectins, which can exert antiviral activities. in this present study, we assessed the antiviral properties of recombinant porcine sp-a (rpsp-a) on prrsv infection in marc cells and revealed its antiviral mechanism using a plaque assay, real-time qpcr, western blotting analysis and an attachment and penetration assay. our results showed that rpsp-a could inhibit the infectivity of prrsv in marc cells and could reduce the total rna and protein level. the attachment assay indicated that rpsp-a in the presence of ca( +) could largely inhibit marc cell attachment; however, in the penetration assay, it was relatively inactive. furthermore, our study suggested that virus progeny released from infected marc cells were blocked by rpsp-a from infecting other cells. we conclude that rpsp-a has antiviral activity against prrsv, most probably by blocking viral attachment and the cell-to-cell transmission pathway, and therefore, rpsp-a holds promise as a novel antiviral agent against prrsv. porcine reproductive and respiratory syndrome virus (prrsv) infection is common worldwide and has become one of the most economically important diseases in the swine industry [ ] . prrsv can cause reproductive failure in sows and is associated with a respiratory disease complex that can affect pigs of all ages [ ] [ ] [ ] . prrsv was discovered almost simultaneously in the late s on the european and north american continents and was divided into type prrsv (european genotype) and type prrsv (north american genotype) [ , ] . prrsv is an enveloped, single-stranded positive-sense rna virus. its genome is approximately . kb in size and has at least open reading frames (orfs) [ ] . orf a and orf b encode non-structural proteins, and the other genes encode structural proteins, including gp a, gp b, gp , gp , gp , gp a, matrix protein m and the nucleocapsid protein n [ ] [ ] [ ] [ ] [ ] [ ] . of these, four are glycoproteins: the minor proteins gp a, gp and gp and the major protein gp , which is thought to be important for virus assembly and entry into permissive cells. researchers have made great efforts towards preventing and controlling prrsv infection. currently, vaccination is the main method to control prrsv infection. although many commercially available modified live and inactivated prrsv vaccines have been developed, they fail to provide complete protection against the devastating disease caused by this virus [ , [ ] [ ] [ ] . furthermore, a large amount of literature has reported natural compounds that exhibit activity against prrsv infection, including flavaspidic acid ab [ ] , matrine [ ] , -deoxyphorbol -phenylacetate -acetate (dppa), ouabain, valinomycin and bufalin [ ] , and glycyrrhizin [ ] . additionally, other therapies designed to control prrsv infection have been reported, such as licl [ ] , polyinosinic-polycytidylic acid [ ] , type i and type iii interferons [ ] , double-stranded rna (dsrna) activated caspase oligomerizer (draco) [ ] , and micrornas [ ] . however, none of these aforementioned therapies can effectively treat or prevent prrsv infection, so there is an urgent need to develop new antiviral strategies. surfactant protein a (sp-a) belongs to a family of mammalian c-type lectins, and it is an important innate immune molecule that is involved in a range of immune responses. the primary structure of sp-a is composed of four regions, including an n-terminal region, a collagenlike region, a coiled-coil neck peptide, and a carbohydraterecognition domain (crd) [ ] . the crd can interact with sugar moieties that are present on the surface of many pathogens, including viruses, fungi and bacteria, resulting in inhibition of infectivity in a calcium-dependent manner. previous studies on the antiviral activity of sp-a have been focused on human medicine. in the late s, human sp-a was shown to inhibit respiratory syncytial virus by blocking viral entry and subsequent syncytium formation [ ] . research on porcine sp-a activity against prrsv was limited to one report indicating that recombinant porcine sp-a could exert anti-prrsv activity [ ] . unfortunately, no additional studies on the anti-prrsv potential and mechanism of sp-a were performed. however, studies on the antiviral activity of sp-d, which has a primary structure that resembles that of sp-a, have been reported more frequently. sp-d had an inhibitory effect on hiv infection through inhibition of viral entry by affecting gp -cd interactions [ ] . marine and colleagues demonstrated that recombinant porcine sp-d prevented the attachment of human seasonal h n and h n virus to receptors on epithelial cells of the upper respiratory tract [? ?] . notably, porcine ficolin is also a collagenous lectin that was established to reduce prrsv infection in marc- cells in neutralization assays and to inhibit the replication of infectious viral particles in a glcnac-dependent manner [ ] . in the present study, we report the antiviral properties and mechanism of action of recombinant porcine sp-a in marc- cells. our results indicate that rpsp-a affects virus attachment and blocks the cell-to-cell transmission pathway to suppress prrsv infection in marc- cells, which indicates that rpsp-a may be a useful antiviral agent for the treatment of prrsv infection. marc cells were grown in dulbecco's modified eagle's medium (dmem; gibco, grand island, ny, usa) supplemented with % newborn bovine serum (nbs) at °c and % co . spodoptera frugiperda cells (sf cells), purchased from invitrogen, were grown in grace's insect medium ( ; gibco, grand island, ny, usa) supplemented with % heat-inactivated fetal bovine serum (fbs; sciencell, ca, usa) for recombinant baculovirus production or were grown in sf ii sfm (gibco, grand island, ny, usa) for protein expression at °c. hp prrsv strain nj-a (type prrsv) was isolated from a pig on a farm with an atypical prrs outbreak in nanjing city, jiangsu province, china, in . it was identified and stored by the national research center of engineering and technology for veterinary biologicals. the nj-a strain was propagated and titrated by the plaque method in marc cells to prepare the viral stock used in this study. rpsp-a was obtained using a bac-to-bac baculovirus expression system (invitrogen, ca, usa). an n-terminal in-frame fusion of the psp-a (genbank accession no. nm_ ) gene with an -histidine tag was codon-optimized for baculovirus expression, synthesized, and cloned in the puc vector by genscript (piscataway, nj, usa). the recombinant psp-a gene was subcloned into a pfastbac vector under the control of the ph promoter. once generated in a pfastbac tm construct, the plasmid dna was used to transform max efficiency Ò dh bac tm competent e. coli for transposition into a bacmid. blue/ white selection was used to identify colonies that contained a recombinant bacmid. high-molecular-weight rpsp-a bacmid dna from dh bac tm e. coli was isolated using a bac/pac dna isolation kit (omega, usa). the concentration and purity of the rpsp-a bacmid were estimated using a thermo scientific nanodrop tm spectrophotometer, which was then used to transfect sf insect cells using cellfectin ii reagent. the recombinant baculovirus was harvested days post-transfection and was amplified twice to obtain higher titer virus stocks. recombinant p viral stocks were used to infect sf cells at an moi of . pfu/cell. at days postinfection, cells were harvested and washed twice in phosphate-buffered saline (pbs). cell pellets were immediately stored at - °c. the protein rpsp-a was extracted and purified using a -ml-size ni ? -sepharose histrap hp tm affinity column (ge healthcare, sweden) according to the instructions provided by the manufacturer. imidazole in the eluate, which contained rpsp-a, was removed on a hitrap tm desalting column ( ml) and then exchanged with storage buffer ( mm tris, ph . , mm nacl, . mm pmsf). then, the eluate was filter-sterilized through a . -lm membrane filter (millipore) and analyzed by western blotting using an anti-his antibody (sunshinebio, china). protein concentrations were determined using a bca protein assay reagent kit (pierce, rockford, il, usa). the cytotoxic effect of rpsp-a was examined using an mtt assay. briefly, marc cells in -well plate were exposed to various concentrations of rpsp-a at °c in a % co atmosphere for h. after incubation, the culture medium was replaced with fresh medium containing ll of mtt at a concentration of mg/ml, and the cells were incubated for an additional h. after removal of the supernatant, ll of dmso was added to each well to dissolve the crystals for min. absorbance at nm was recorded using an elisa microplate reader. antiviral activity was detected by plaque assay using marc cells in -well plates. a preliminary study was performed to determine the concentration of infectious virus particles using a - dilution of virus stock ( . pfu ml - ). subconfluent monolayers of marc cells in -well plates were treated with rpsp-a at the indicated concentrations and were simultaneously infected with prrsv. the cells continued to be cultured at °c for h. then, the cells were washed five times with pbs and covered with ml of medium containing a : mixture of dmem and % low-gelling-temperature agarose (sigma, usa). after h, the agarose was removed, and cells were stained with % crystal violet dissolved in % formaldehyde at °c for h. after washing gently with water, plaques were counted. antiviral activity was calculated using the following formula: inhibition rate (%) = À number of plaquestest number of plaquescontrol  % total rna was extracted from samples using a takara minibest universal rna extraction kit (takara, osaka, japan). rna was reverse transcribed into firststrand cdna using primescript tm reverse transcriptase with random primers in a -ll final reaction volume according to the manufacturer's instructions. first, ll of total rna was added to mixture i containing ll of random primer and ll of dntps ( . mm), and then the mixture was heated at °c for min and then kept at °c for min. second, a reaction mixture was prepared by adding the above mixture to mixture ii, containing ll of primescript buffer, ll of rnase inhibitor, ll of primescript reverse transcriptase and ll of rnase-free h o. the reaction was performed under the following conditions: °c for min, °c for min, °c for min and, finally, °c for min. real-time qpcr was performed in a lightcycler ii system (roche, basel, switzerland) using an evagreen x qpcr mastermix-no dye kit (abm, canada). reaction mixtures were prepared according to the manufacturer's instructions and incubated for min at °c, followed by cycles of s at °c and min at °c, followed by s at °c, min at °c, and s at °c. a portion of the orf gene was detected by real-time qpcr, and gapdh was used as a control. the data were analyzed using the -ddct method. customized primers are listed in table . total protein was extracted using cell lysis buffer ( mm tris, ph . , mm nacl, % triton x- , . mm pmsf) and quantified using a bca protein assay reagent kit (pierce, rockford, il, usa). then, lg total protein was subjected to % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to a polyvinyl difluoride (pvdf) membrane, which was and the attachment assay was carried out as described previously [ ] with some modifications. marc cells were seeded into -well plates. cells were washed with cold pbs followed by dmed and then were pre-chilled at °c for min. cells were incubated with prrsv at an moi of . and purified rpsp-a ( lg ml - ) at °c for h. cells were washed with cold pbs to remove unbound virus and rpsp-a, and then, fresh medium containing % fbs was added. the cells were then cultured at °c for h, after which the virus titer was measured. the penetration assay was performed as described previously with some modifications [ , ] . prrsv could be internalized from the surface of marc cells within h to h [ ] . thus, we examined the kinetics of activity against prrsv penetration using time-of-addition assays. marc cells were seeded into -well plates. cells were washed with cold pbs, followed by dmed, and were then pre-chilled at °c for min. cells were incubated with prrsv at °c for h. cells were washed with cold pbs. rpsp-a was added at h, h, and h and removed at h following a temperature shift. the time point when the cell culture was shifted to °c was designated as h. cells were washed with cold pbs to remove unbound virus and rpsp-a, and fresh medium containing % fbs was added. cells were incubated for h at °c, and the virus titer was measured. the data are presented as the mean ± standard deviation from three independent experiments. the data were analyzed using student's t-test with graphpad prism software. the differences between the mean values were considered statistically significant when the p-value was less than . . plaque assay results revealed that rpsp-a had potency against prrsv. as shown in fig. a , the number of visible plaques was significantly reduced in the presence of lg of rpsp-a per ml. furthermore, as shown in fig. b and c, treatment with lg of rpsp-a per ml reduced plaques by % from . pfu ml - to . pfu ml - (p \ . ), whereas treatment with lg of rpsp-a per ml reduced plaques by % from . pfu ml - to . pfu ml - (p \ . ). treatment with lg of rpsp-a per ml in the absence of ca ? was used as a negative control, and no inhibition was observed. to rule out the possibility that nonspecific toxicity caused by rpsp-a was affecting prrsv replication, we investigated its cytotoxicity in marc- cells at concentrations of , , , , , , lg/ml. as shown in fig. , hours after treatment, the cells cultured in medium containing rpsp-a at concentrations below lg ml - retained approximately % viability relative to the control. to elucidate the mechanism of the antiviral effects of rpsp-a, a virus entry assay was utilized. the data showed that rpsp-a could largely inhibit virus attachment; however, it was relatively inactive against virus penetration. in the virus attachment assay, marc cells were incubated with prrsv at an moi of . in the presence of rpsp-a at °c, a condition in which viruses attach to cells but do not penetrate. as shown in fig. , treatment with lg of rpsp-a per ml largely inhibited marc cell attachment. the mean viral titer was . pfu ml - in the absence of rpsp-a, but it was reduced to . pfu ml - in the presence of lg of rpsp-a per ml with the addition of ca ? (p \ . ). no significant difference was observed between and lg ml - of rpsp-a in the absence of ca ? (p [ . ). in the penetration assay, we studied the inhibition kinetics of rpsp-a against prrsv. the mean viral titer in the presence of lg of rpsp-a per ml with the addition of ca ? was . pfu ml - at h (p \ . ), . pfu ml - at h (p \ . ), and . pfu ml - at h (p [ . ) compared to . pfu ml - in the absence of rpsp-a (fig. ) , suggesting that the inhibitory effect of rpsp-a on virus penetration decreased over time, although there was no significant difference at h. additionally, lg of rpsp-a per ml in the absence of ca ? did not affect virus penetration. we next attempted to investigate the kinetics of the rpsp-a-mediated effect on the intracellular viral load. first, marc cells were incubated with prrsv at an moi of . for h, and the medium was replaced with fresh medium containing lg of rpsp-a per ml. then, the total rna in marc cells was extracted for prrsv rna genome detection by real-time rt-pcr after culture for h, h, h or h. as shown in fig. a , the addition of rpsp-a resulted in a significant reduction in the total rna level at h, h and h compared with the non-drug-treated cells (p \ . ), and this reduction reached a peak at h after addition of rpsp-a. however, there was no significant difference at h following rpsp-a addition compared with the non-drug-treated group (p [ . ). moreover, we also detected n protein of prrsv in marc cells by western blotting after treatment with rpsp-a for h. as shown in fig. b , western blotting analysis indicated that rpsp-a reduced the level of n protein in cells in a dose-dependent manner. surfactant protein a, an antimicrobial defense molecule of the innate immune system, has the potential to be exploited as an antiviral drug in the future. for this reason, we sought to develop it as a recombinant product in this study. here, rpsp-a was obtained using a bac-to-bac baculovirus expression system. we found that rpsp-a expressed by sf insect cells could block virus entry. viral entry represents an attractive target for chemotherapeutic intervention [ ] . however, drugs that inhibit virus entry prevent infection of cells by cell-free virus particles and also prevent virus transmission between virus-infected and uninfected cells [ , ] . the capacity to inhibit both modes of infection by a single drug is one of the reasons why sp-a may be superior to other microbicide drugs. thus, sp-a might be a candidate agent for preventing prrsv infection. so far, there have been limited data available regarding the anti-prrsv activity of porcine sp-a. in china, highly pathogenic prrsv appears to be widely prevalent, and therefore, the highly pathogenic prrsv strain nj-a, a type prrsv strain, was used in this study. to explore the anti-prrsv activity of porcine sp-a, further studies need to be performed on type prrsv. our data indicate that rpsp-a significantly suppresses prrsv infection in a dose-dependent manner without causing cytotoxicity in marc cells. we also found that rpsp-a exerts potent antiviral activity in a ca ? -dependent manner, indicating that rpsp-a may interact with prrsv via the carbohydrate-recognition domain, which is consistent with previous studies [ , ] . researchers have confirmed that lectins inhibit virus entry [ , , , ] , and each step of the viral entry pathway is a potential target for antiviral agents. we conducted virus attachment and penetration assays to assess whether rpsp-a also interfered with prrsv entry. the virus entry assay showed that rpsp-a strongly inhibits virus attachment, but its effect on virus penetration was, at most, modest, suggesting that rpsp-a most probably blocks virus attachment, like other lectins. it has been shown that hippeastrum hybrid agglutinin (hha), a mannose-specific lectin, probably interferes with sars coronavirus attachment. when attachment occurred at °c, hha was twice as active (ec = . lg ml - ) as it was in a situation where both attachment and penetration were allowed to occur at °c (ec = . lg ml - ) [ ] . in another study, recombinant porcine sp-d was shown to inhibit iav attachment using human trachea tissue, and it fully prevented binding of human seasonal h n and h n ia at higher dose [ ] . our data indicate that the suppression of prrsv penetration by rpsp-a gradually weakened and even disappeared as the time between infection and addition of rpsp-a increased, suggesting that rpsp-a might act on prrsv entry into marc cells only at the cell surface and that it has no effect once the virus has entered the cell. a better understanding of the underlying mechanisms by which rpsp-a influences viral entry might lead to new antiviral interventions. taking the previously reported data and our own findings together, we hypothesize that rpsp-a inhibits prrsv entry by binding to glycoprotein gp , gp , gp or gp on the surface of prrsv, thereby preventing it from interacting with its receptors on marc cells. the mechanism of the antiviral activity of lectins has been studied by several researchers. it has been shown that human sp-a can bind to the f (fusion) glycoprotein on the surface of respiratory syncytial virus and may neutralize rsv by directly inhibiting the fusion function of this protein, thereby inhibiting viral entry [ ] . in another study, marine et al. reported that the interaction between rpsp-d and the viral ha prevented attachment of the virus to target cells and contributed to the neutralizing effects of rpsp-d [ ] . in addition, other lectins such as sp-d and plant lectins have been shown to bind to glycoprotein gp of hiv- and block access of gp to the receptor protein cd [ , , , ] , thereby blocking viral entry to reduce hiv- infection. the nature of the interactions among prrsv, rpsp-a and cells is not yet known, so this model should be assessed in further studies. interestingly, our data also indicated that rpsp-a had no effect on the total rna level when the virus was allowed to undergo only a single cycle of replication but caused the total rna level to decrease greatly at h, h and h. a possible explanation for this is that virus progeny released from infected marc cells are blocked by rpsp-a and prevented from infecting other cells. since marc- cells are not of porcine origin but of monkey origin, the mechanism of infection of porcine alveolar macrophages (pams) is not identical to that in marc cells [ , ] ; furthermore, a previous study showed that some lectins had a stronger inhibitory effect on virus infection when pams were treated with lectins than when the virus was treated with lectins prior to infection, in contrast to what was observed with marc cells, suggesting potentially complex interactions among pams, lectins and prrsv [ ] . more studies will be needed to characterize the antiviral activity of rpsp-a in pams, which are the main target cells for prrsv in vivo. in conclusion, we show that rpsp-a exerts potent activity against hp-prrsv in marc cells. we clearly demonstrate that rpsp-a interacts at the level of virus attachment and blocks the cell-to-cell transmission pathway, which has never been observed before. rpsp-a most probably interacts with the glycoproteins of prrsv, thereby preventing it from binding to host-cell receptors and subsequently interfering with transmission. a better understanding of the mechanisms by which rpsp-a influences viral infection in porcine alveolar macrophages might lead to new antiviral interventions. porcine reproductive and respiratory syndrome virus vaccine does not fit in classical vaccinology porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis expression and antibody preparation of gp a gene of porcine reproductive and respiratory syndrome virus north american and european porcine reproductive and respiratory syndrome viruses differ in nonstructural 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(euphorbiaceae) large-molecular-weight carbohydrate-binding agents as hiv entry inhibitors targeting glycoprotein gp inhibition of hiv entry by carbohydratebinding proteins plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro entry of hepatitis c virus and human immunodeficiency virus is selectively inhibited by carbohydratebinding agents but not by polyanions effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptormediated endocytosis functional analysis of porcine reproductive and respiratory syndrome virus n-glycans in infection of permissive cells decreased surfactant protein a in patients with bacterial pneumonia ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- - pee i o authors: kang, hyeonjeong; lee, changhee title: sasa quelpaertensis nakai extract suppresses porcine reproductive and respiratory syndrome virus replication and modulates virus-induced cytokine production date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: pee i o although sasa quelpaertensis nakai, a dwarf bamboo, is known to exert a variety of beneficial effects on health, its antiviral effect remains to be elucidated. porcine reproductive and respiratory syndrome virus (prrsv) is one of the most devastating viral pathogens of swine and has a substantial economic impact on the global pork industry. therefore, the present study was conducted to determine whether sasa quelpaertensis nakai extract (sqe) inhibits prrsv infection in cultured porcine alveolar macrophages (pams). our results demonstrated that sqe treatment suppressed the replication of prrsv in a dose-dependent manner. the antiviral activity of sqe on prrsv replication was found to be primarily exerted at early times postinfection. treatment with sqe resulted in marked reduction of viral genomic and subgenomic rna synthesis, viral protein expression, and progeny virus production. notably, pro-inflammatory cytokine production in pam cells infected with prrsv was shown to be modulated in the presence of sqe. taken together, our data indicate that sqe has potential as a therapeutic agent against prrsv. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome virus (prrsv), a pathogenic macrophage-tropic arterivirus of pigs, is the etiological agent of acute respiratory illness in young piglets and reproductive failure in pregnant sows [ ] . prrsv is a small enveloped single-stranded positivesense rna virus that belongs to the family arteriviridae of the order nidovirales [ , , ] . the prrsv genome contains two large open reading frames (orfs), a and b, which comprise the two-thirds of the viral genome and encode non-structural proteins (nsps). the remaining orfs located in the -terminal region code for structural proteins [ , ] . the initial translation of orf a and orf b yields the a and lab replicase polyproteins, respectively, which are then proteolytically processed into at least functional nsps, including viral rna-dependent rna polymerase (rdrp) [ , , , ] . the rdrpcontaining replication complex mediates genomic rna replication and subgenomic (sg) mrna transcription, eventually generating a nested set of -coterminal sg mrnas that are individually translated to produce structural proteins [ , ] . prrsv preferentially replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months [ , , , ] . as a result, prrsv infection suppresses normal macrophage functions and immune responses, which may render pigs susceptible to secondary bacterial or viral infections, leading to more-severe disease than caused by either agent alone [ , , , ] . furthermore, a highly pathogenic prrsv infection, which is characterized by high fever and high mortality in pigs of all ages, has recently emerged in china and other asian countries and severely affected local pig husbandries [ ] . although both killed and modified live vaccines against prrsv have been developed to control the disease and are used in the global market, most commercial vaccines have problems related to their efficacy and safety. despite the availability of the vaccine, prrsv has still continued to plague most pork-producing nations, causing enormous financial losses for the swine industry worldwide [ , ] . thus, the lack of effective vaccines increases the need for new compounds against prrsv infection. sasa quelpaertensis nakai, known as jeju-joritdae, is a type of bamboo grass that is widely distributed on mt. halla on jeju island in south korea. its leaf extract possesses a number of health-promoting activities, including anti-ulcerogenic, anti-obesity, anti-inflammatory, and anticancer properties [ - , , ] . to date, there are still no reports on the effectiveness of sasa quelpaertensis nakai extract (sqe) against viral infections in human or veterinary subjects. in the present study, therefore, we tried to investigate the antiviral activity of sqe and its mechanism of action in target cells upon prrsv infection. treatment of target cells with sqe significantly impaired prrsv replication. further experiments revealed that sqe suppresses post-entry steps in the replication cycle of prrsv, including viral genomic and sg rna synthesis, viral protein expression, and virus production. the presence of sqe notably altered expression of cytokine genes in prrsv-infected pam cells, suggesting that sqe activity is involved in the modulation of inflammatory responses during viral infection. collectively, our results suggest that sqe may be an excellent therapeutic option for prrsv infection. cells, viruses, reagents, and antibodies pam-knu cells (immortalized pam cell line; [ ] ) were cultured in rpmi medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; invitrogen), antibiotic-antimycotic solution ( ; invitrogen), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ; invitrogen) in the presence of lg/ml g (invitrogen). the cells were maintained at °c in a humidified % co incubator. prrsv strain vr- was propagated in pam-knu cells as described previously [ ] . sqe was a kind gift from yong chool boo (kyungpook national university, daegu, south korea). it was prepared and dissolved in distilled water (dw) as described elsewhere [ , , ] . the yield of sqe from dried s. quelpaertensis leaves ( kg) was approximately %. p-coumaric acid was purchased from sigma (st. louis, mo) and dissolved in dimethyl sulfoxide (dmso). a monoclonal antibody (mab; sdow ) against the prrsv n protein was purchased from rural technologies (brookings, sd). anti-bactin antibody and horseradish peroxidase (hrp)-conjugated secondary antibody were purchased from santa cruz biotechnology (santa cruz, ca). the cytotoxic effects of reagents on pam-knu were analyzed by a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability. briefly, pam-knu cells were grown to a density of cells/well in a -well tissue culture plate with sqe or p-coumaric acid treatment for and h. after incubation, ll of mtt solution ( . mg/ml) was added to each well, and the samples were incubated for an additional h. the supernatant was then removed from each well, after which ll of dmso was added to dissolve the formazan crystals produced from the mtt. the absorbance of the solution was measured at nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-knu cells grown on microscope coverslips placed in -well tissue culture plates were pretreated with sqe or pcoumaric acid for h and mock infected or infected with prrsv at a multiplicity of infection (moi) of in the presence of each compound. the virus-infected cells were allowed to grow in the presence of sqe or p-coumaric acid until hpi, fixed with % paraformaldehyde for min at room temperature (rt), and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with an n-specific mab for h. after being washed five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca), followed by counterstaining with , -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on glass microscope slides in mounting buffer ( % glycerol and . % sodium azide in pbs), and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). pam-knu cells were grown in -well tissue culture plates for day and were mock infected or infected with prrsv at an moi of . at the indicated times, cells were harvested in ll of lysis buffer ( . % tritonx- , mm b-glycerophosphate, mm q-nitro phenyl phosphate, mm mops, mm, mgcl , mm nacl, mm egta [ph . ], mm sodium orthovanadate, lg/ml e , lg/ml aprotinin, lg/ml leupeptin, and mm pmsf) and sonicated on ice five times for s each. homogenates were lysed for min on ice and clarified by centrifugation at , g (eppendorf centrifuge r, hamburg, germany) for min at °c. the protein concentrations of the cell lysates were determined by a bca protein assay (pierce, rockford, il). the cell lysates were mixed with nupage sample buffer (invitrogen) and boiled at °c for min. the proteins were separated on a nupage - % gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immobilon-p membranes (millipore, billerica, ma). the membranes were subsequently blocked with % powdered skim milk (bd biosciences, belford, ma) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at °c for h and reacted at °c overnight with the primary antibody against prrsv n or b-actin. the blot was then incubated with the secondary horseradish peroxidase (hrp)-labeled antibody at a dilution of : , for h at °c. proteins were visualized by addition of enhanced chemiluminescence (ecl) reagent (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify viral protein production, the band densities of prrsv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/ facilities/wcif/imagej/) based on the density value relative to b-actin gene. pam-knu cells were infected with prrsv at an moi of as described above. at - , , , , , , , , , or hpi, sqe was added to reach the indicated final concentration for the remainder of the time course experiment. the virus-infected and sqe-treated cells were further maintained, and the infection was terminated at hpi by fixing the monolayers with % paraformaldehyde for min at rt. the fixed cells were subjected to ifa to assess the presence of prrsv infection as described above. an internalization assay was performed as described previously [ ] . briefly, pam-knu cells grown in -well culture plates were infected with prrsv at an moi of at °c for h in the presence of sqe. unbound viruses were then washed with pbs, and the cells were incubated at °c or °c in the presence of sqe for h, followed by treatment with proteinase k ( . mg/ml) at °c for min to remove the bound but non-internalized virus particles. the prrsv-infected cells were then serially diluted in rpmi and added to fresh pam-knu cell monolayers in -well tissue culture plates. at h post-incubation, internalized viruses were titrated by ifa as described above, and the % tissue culture infectious dose (tcid ) was calculated. quantitative real-time rt-pcr pam-knu cells were incubated with sqe for h prior to infection and then inoculated with prrsv at an moi of for h at °c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing sqe for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent (invitrogen) and treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentration of the extracted rna was measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets as described previously [ ] . the primers used in this study were described elsewhere [ , ] , and primer sequences are available on request. the rna levels of viral and cytokine genes were normalized to that of mrna for the porcine b-actin gene, and the relative quantities (rq) of mrna accumulation were determined using the -ddct method [ ] . to detect alteration of genomic rna and sg mrna levels in the presence of sqe during prrsv infection, the results obtained using sqe-treated samples were compared with those obtained with vehicle-treated samples. to assess the effect of sqe on transcriptional activation of pro-and anti-inflammatory cytokines in virusinfected cells, the relative fold change of each cytokine gene was calculated and compared between virus-infected and mock-infected pam cells and between vehicle-treated and sqe-treated virus-infected cells. pam-knu cells were treated with sqe ( mg/ml) for h prior to infection, followed by prrsv inoculation at an moi of for h at °c. the infected cells were maintained in fresh medium containing sqe or vehicle for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent and treated with dnase i as described above. northern blotting was conducted using a northernmax kit (ambion, austin, tx) according to the manufacturer's instructions. samples of total rna ( lg) were loaded onto a % agarose gel and separated by electrophoresis, using a denaturing buffer system. the separated total rna was then transferred to a brightstar-plus nylon membrane (ambion) for h and cross-linked by exposure to uv light for min. pre-hybridization was performed at °c for min, followed by detection using the genotype-specific probe for the utr ( -aatttcggccgcatggttttcgccaattaaatctt acccccacacggtcgc- ) or porcine s rrna ( -g cggttgcaccatttgggtgtcctg- ). the blot was hybridized to biotin-labeled oligonucleotide probes in ultrahyb reagent at °c overnight and washed twice with low-stringency blocking buffer and wash buffer. target viral rnas were detected using a brightstar biodetect kit (ambion) according to the manufacturer's protocols. the membrane was incubated with alkaline-phosphatase-conjugated streptavidin, followed by reaction with the chemiluminescent substrate cdp-star (ambion). the overlaid films were exposed in a dark cassette box in a dark room, and quantitation was performed by densitometry of the corresponding bands as described above. pam-knu cells were infected with prrsv along with sqe treatment as described above. the culture supernatant was collected at different time points ( , , , , and hpi) and stored at - °c. the titer of prrsv was measured by limiting dilution on pam-knu cells by ifa as described above and then quantified as the tcid per ml. student's t-test was used for all statistical analyses and pvalues of less than . were considered statistically significant. to investigate the antiviral activity of sqe, prrsv was chosen because it is one of the most important viral pathogens that have an economic impact on the global pork industry. based on the mtt assay, the highest concentration of sqe that was noncytotoxic was mg/ml in pam-knu cells. pam-knu cells were pretreated with sqe at concentrations of . to mg/ml or with dw as a vehicle control for h prior to infection, and sqe was present during the entire period of infection. virus production was initially measured by monitoring the cytopathic effect (cpe) after infection and confirmed by immunofluorescence at hpi using an n-protein-specific mab (fig. ) . in vehicle-treated control cells, visible cpe appeared at hpi and became prominent by hpi, and n-specific staining was clearly evident in many cell clusters, indicating infection and the spread of the virus to neighboring cells. in contrast, sqe had an obvious inhibitory effect on virus propagation. as shown in fig. a and b, sqe significantly decreased virus-induced cpe and viral gene expression at concentrations of * mg/ml. n protein staining revealed that the number of cells expressing viral antigen decreased during sqe treatment, resulting in a maximum of * % inhibition at a concentration of mg/ ml ( fig. a and b) . furthermore, the effective dose for inhibiting prrsv infection by % (ed ) was determined to be about . mg/ml. taken together, these results demonstrate that sqe efficiently suppresses the replication of prrsv. to determine the point at which sqe acted during prrsv infection, pam-knu cells were treated independently with sqe at various time points postinfection. at hpi, the level of prrsv replication was measured indirectly using ifa to determine the number of cells expressing the n protein (fig. ) . treatment of cells with mg/ml sqe at - , , and hpi resulted in a more than % decrease in prrsv-positive cells when compared to untreated controls. addition of sqe between and hpi led to a % to % decrease in the number of prrsv-infected cells. in contrast, no significant impairment of prrsv propagation was observed when sqe was added at hpi. these data indicate that the inhibitory effect of sqe was exerted mainly during the initial period of infection, suggesting that its action occurs at early time points after prrsv infection. to further assess the effect of sqe on prrsv replication, virus yield was determined during treatment with sqe. viral supernatants were collected at hpi and viral titers were measured. as shown in fig. a , treatment with sqe inhibited the release of viral progeny in a dose-dependent manner. the peak viral titer was determined to be . tcid /ml in the vehicle-treated control for prrsv, but the addition of mg/ml sqe decreased the titer of prrsv to . tcid /ml ([ log reduction compared with the control). a growth kinetics study further demonstrated that the overall process of prrsv replication was significantly delayed when cells were treated with sqe (fig. b) . consequently, these findings revealed that sqe inhibits the optimal release of progeny virus from the natural host cells. to evaluate which steps of the replication cycle of prrsv were targeted by sqe, we started focusing on the earliest step, virus entry. to address this issue, an internalization assay was performed as described previously [ ] . pam-knu cells were inoculated with prrsv at °c for h to allow virus attachment and further maintained either at °c or at °c to permit virus internalization in the presence of sqe. the samples were then treated with proteinase k to remove the remaining viruses from the cell surface. the serially diluted infected cells were subsequently subjected to an infectious center assay on uninfected pam-knu cell monolayers, and virus titers were measured days later by ifa. as shown in fig. c , the titer of prrsv was virtually the same in cells treated with sqe or without sqe at °c and was determined to be . tcid /ml, indicating no differences between those cells. in contrast, only minimal viral production of about tcid /ml was observed in prrsv-infected cells maintained at °c, which was likely due to inefficient removal of the bound virus. these results indicated that sqe has no inhibitory effect on the virus entry process. following the uncoating process, like all positive-sense rna viruses, the genome of prrsv is released into the cytoplasm and immediately serves as a template for viral translation by a cellular cap-dependent mechanism. early viral translation produces replicase polyproteins that are posttranslationally cleaved into nsps by viral or cellular proteinases. subsequently, the replicase proteins mediate de novo synthesis of viral rna, including genomic rna replication and sg mrna transcription. the structural proteins of prrsv are translated later from their respective sg mrna transcripts. thus, to investigate inhibitory effects of sqe on post-entry steps of the prrsv life cycle, we first investigated whether viral protein translation was affected by sqe. to accomplish this, pam-knu cells were treated with sqe for h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. the expression level of the prrsv n protein in the presence or absence of sqe was evaluated at hpi by western blot analysis. the production of the prrsv n protein was drastically diminished during sqe treatment (fig. ) . densitometric analysis of the western blot revealed that the intracellular expression of the viral protein was reduced by sqe treatment, with a maximum of about % inhibition at a concentration of mg/ml (fig. ) . this marked reduction was probably not the result of a nonspecific decrease in translation, since ponceau s staining indirectly indicated that the overall expression levels of cellular proteins remained similar following treatment (data not shown). taken together, these results demonstrated the inhibitory effects of sqe specifically against viral protein expression during prrsv replication. for positive-strand rna viruses, viral nonstructural proteins are required for viral rna replication and transcription. thus, it is conceivable that the impaired viral protein expression was caused by suppression of viral rna synthesis. since prrsv infection produces two rna entities, genomic and subgenomic, we investigated whether sqe specifically affects genome replication and sg mrna transcription. to answer this question, the relative levels of genomic rna and sg mrna were measured by quantitative real-time strand-specific rt-pcr in the presence or absence of sqe upon prrsv infection. as shown in fig. , sqe treatment resulted in a maximal reduction in the synthesis of prrsv genomic rna and sg mrna of % and %, respectively, at a concentration of mg/ ml, when compared with untreated infected cells. the decrease in viral rna levels after the addition of sqe was not due to nonspecific inhibition of transcription, as the mrna level of the internal b-actin control remained unchanged in all samples (fig. c ). the impairment of prrsv transcription in the presence of sqe was confirmed by northern blot analysis (fig. ). in the absence of sqe, prrsv rna synthesis was distinctly observed (lane ). a [ % reduction of rna-synthesizing activity occurred in the presence of mg of sqe per ml (lane ), while viral rna synthesis was almost completely abolished in the presence of mg of sqe per ml (lane ). altogether, these results indicated that sqe specifically suppresses the synthesis of prrsv genomic rna and sg mrna. since sqe has been reported to have anti-inflammatory properties, we examined whether sqe affects the transcriptional activation of immune-response genes upon prrsv infection to determine whether this contributes to b fig. reduction of viral progeny production by sqe treatment. (a) pam-knu cells were pretreated with sqe for h and were mock infected or infected with virus. sqe was present in the medium throughout the infection. at hpi, virus-containing supernatants were collected and virus titers were determined. (b) growth kinetics of prrsv on pam-knu cells treated with sqe, using the same experimental conditions as in panel a. at the indicated time points postinfection, culture supernatants were harvested and virus titers were measured. (c) a virus-internalization assay was conducted by infecting pam-knu cells with prrsv at an moi of at °c for h. after washing with cold pbs, infected cells were maintained in the presence or absence of sqe, either at °c or °c, for an additional hour. bound but non-internalized virus particles were removed by treatment with proteinase k. the infected cells were then serially diluted and plated onto pam-knu cells in -well tissue culture plates. at days post-incubation, internalized viruses were titrated, using ifa for detection, and the % tissue culture infectious dose (tcid ) was calculated. results are expressed as the mean values from triplicate wells, and error bars represent standard deviations. , p \ . fig. inhibition of viral protein translation by sqe treatment. sqetreated pam-knu cells were mock-infected or infected with prrsv for h and were further cultivated in the presence or absence of sqe. at and hpi, cell lysates were collected, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted using a prrsv-specific antibody that recognizes the prrsv n protein. the blot was also reacted with mouse mab against b-actin to verify equal protein loading. the amount of each viral protein was estimated by densitometry in terms of its band density relative to that of b-actin, and sqe-treated sample results were compared to vehicle-control results. values are representative of the mean from three independent experiments, and error bars denote standard deviations. , p \ . inhibition of prrsv by sasa quelpaertensis nakai extract its antiviral activity. we found that numerous immunerelated genes regulated by prrsv infection were significantly altered by treating the cells with sqe when compared with results obtained with vehicle-treated virusinfected cells (fig. ) . among these, upregulation of il- a, il- , il- , il- , tnf-a, and amcf- mrna levels by prrsv infection was dramatically reduced in the presence of sqe. however, chemokine genes that are modulated by prrsv, including mcp- and rantes, were synergistically elevated by sqe treatment. more interestingly, unchanged or slightly increased mrna levels of interferon regulatory factors (irfs), toll-like receptors (tlrs), and antiviral genes including mx and isg- were b fig. inhibition of viral rna transcription by sqe. pam-knu cells pretreated with sqe were mock infected or infected with prrsv for h and were incubated in the presence of sqe. total cellular rna was extracted at hpi (black bars) and hpi (white bars), and viral genomic rna and sg mrna were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna (a) and sg mrna (b) were normalized to mrna for porcine b-actin mrna (c), and relative quantities (rq) of mrna accumulation were evaluated. sqe-treated sample results were compared with untreated results. values are representative of the mean from three independent experiments, and error bars denote standard deviations. *, p = . to . ; , p \ . fig. suppression of viral rna synthesis by sqe treatment. pam-knu cells were treated with sqe, followed by prrsv inoculation, as indicated above the lanes. total rna was extracted from lysates of the infected cells at hpi and subjected to northern blot analysis using genotype-specific biotin-labeled oligonucleotide probes against the utr. porcine s rrna was used as a control to correct for variations in loading during viral rna quantification. the amount of viral sg mrnas was quantified by densitometry and normalized to that in prrsv-infected, untreated control cells ( %). the positions of the genomic rna and sg mrnas are indicated next to the gel significantly increased in prrsv-infected pam cells treated with sqe compared with vehicle-treated infected cells. these results indicated that sqe modulates the cytokine genes for inflammatory responses during prrsv infection. prrsv is not only a devastating viral pathogen in the pig population, but it can also be used as an animal virus model to study nidoviruses that are important in human and veterinary medicine. despite tremendous efforts to control disease caused by this virus, prrsv infection has continuously plagued pig-producing countries and has had a tremendous economic impact on the global swine industry. this is partially due to the absence of efficient vaccines capable of conferring full protection against heterologous viral infection as well as antiviral agents for treating prrsv. although sqe is widely known to possess several health-promoting properties [ , ] , its antiviral activity and mode of action on dna and rna viruses are currently unknown. the present study demonstrated that sqe exerts an adequate antiviral effect against prrsv in vitro, as determined by the ed for viral replication (antiviral activity) of approximately . mg/ml. treatment of cells with sqe resulted in significant inhibition of post-entry steps during the replication of prrsv, as demonstrated by reduced progeny production, diminished viral protein expression, and reduced synthesis of genomic rna and sg mrna. furthermore, a growth kinetics experiment indicated an approximately -log reduction in the titer of prrsv in the presence of sqe at h after infection, and the titer continued to remain at a similar level h after infection. taken together, our data indicate that sqe potently elicits antiviral activity against prrsv in natural target cells. compounds from medicinal plants or natural sources are of interest for their potential to inhibit a variety of viral infectious diseases and cancer. bamboo grasses have been used as traditional medicines throughout asia, and modern studies have reported their beneficial health effects. sasa quelpaertensis nakai is among these and is well known as jeju-joritdae in south korea. sqe, its leaf extract, has a number of biological properties, including anticancer and anti-inflammatory effects. since prrsv infection modulates inflammatory cytokine expression in pam cells, the antiviral activity of sqe may result predominantly from its anti-inflammatory effect. to elucidate potential mechanism(s) responsible for the antiviral effect of sqe on prrsv, we first tested whether sqe affects the induction of cytokine genes by prrsv in immortalized pam cells. the present study provided evidence that various cytokines induced in prrsv-infected cells are transcriptionally modulated in the presence of sqe. our results indicate that treatment with sqe robustly reduced infection-induced expression of pro-inflammatory cytokines, including il- a, il- , il- , il- , and tnf-a. in contrast, sqe resulted in a synergic elevation in gene expression levels of chemokines. notably, the induction of irfs, tlrs, and genes involved in the antiviral immune response was specifically enhanced in sqe-treated virus-infected cells. prrsv is known to possess the ability to escape host defense mechanisms by interfering with innate immune responses to favor its survival and spread in the natural host. thus, sqe appears to disrupt one of the pivotal viral mechanisms of immune evasion by modifying the expression of immune-related genes to facilitate virus infection, resulting in the suppression of prrsv replication. however, we were unable to identify which chemical constituent(s) in sqe is responsible for the regulation of immune-response gene expression that leads to its antiviral activity upon prrsv infection. although p-coumaric acid is known to be a major component of sqe ( . mg/g) and has diverse pharmacological properties, including antimicrobial, anticancer, and antiulcer activities [ , ] , this chemical was incapable of significantly impairing the replication of prrsv, even at the highest noncytotoxic concentration of lm (fig. a ) . it is conceivable that other minor constituents such as tricin, with a content in sqe of . mg/g, may possess the biological characteristics that give sqe its antiviral activity. indeed, recent studies have demonstrated inhibitory effects of synthesized tricin or its derivative on viral infections [ , ] . in addition to prrsv, our experiment was further extended to other nidoviruses to confirm the antiviral activity of sqe. porcine epidemic diarrhea virus (pedv) is one of pathogenic coronaviruses of pigs. like prrsv, it belongs to the order nidovirales, whose members all have a similar genome organization and replication strategy. thus, we attempted to assess whether sqe also affects pedv infection but found that it had no significant inhibitory effect on pedv infection in vitro (fig. a ). this observation might be explained by the tropism of each virus, since prrsv is a porcine macrophage-tropic arterivirus causing acute respiratory illness, whereas pedv is a pathogenic enterocytetropic coronavirus of swine causing acute enteritis and watery diarrhea. although sqe failed to block pedv infection, it still has potential as an antiviral agent against other coronaviruses of human and livestock, for which the modulation of immune-response cytokine gene production in the natural host is related to their replication. in conclusion, our findings described here demonstrate that sqe at subcytotoxic doses effectively prevents the replication of prrsv in natural host cells. additionally, sqe treatment significantly altered prrsv-induced fig. regulation of immune-related genes by sqe during prrsv infection. in the presence of vehicle or sqe ( mg/ml), pam-knu cells were mock infected or infected with prrsv at an moi of . total rna was extracted from lysates of the infected cells at hpi (left bar set) and hpi (right bar set). the mrna level of each cytokine gene was assessed by quantitative real-time rt-pcr and normalized to that of porcine b-actin. the relative fold difference between mock-infected and virus-infected cells (y-axis) was then calculated for each gene and compared between untreated and sqetreated virus-infected cells. data are representative of the mean values from three independent experiments carried out in duplicate, and error bars represent standard deviations. *, p = . to . ; , p \ . cytokine production in pam cells. therefore, we propose that one of the modes of action of sqe to elicit an antiviral effect against prrsv is to regulate the expression of immune-related genes that are associated with the ability of prrsv to escape the host innate immune responses. although further in vivo studies are needed to evaluate the efficacy and safety of sqe, the results presented here indicate that it is a good candidate as an antiviral agent against prrsv. inhibitory effects of tricin derivative from sasa albo-marginata on replication of human cytomegalovirus epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive 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interferon-b production by inhibiting irf activation in immortalized porcine alveolar macrophages human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus straus se (eds) fields virology family coronaviridae highly pathogenic porcine reproductive and respiratory syndrome effects of sasa health, extract of bamboo grass leaves, on spontaneous mammary tumourigenesis in shn mice 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 duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine acknowledgments we gratefully thank yong chool boo from kyungpook national university for providing sasa quelpaertensis nakai extract. key: cord- - dxzf ip authors: dongliu, yuan; guoliang, yang; haocheng, xu; shuaijia, qing; li, bing; yanglei, jia title: outbreak of acute febrile respiratory illness caused by human adenovirus b p h f in a military training camp in shandong china date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: dxzf ip this study reports an outbreak of acute febrile respiratory illness caused by human adenovirus b [p h f ] in a military training center in china between may and june . in total, military personnel were affected, and two patients were admitted into the intensive care unit of the military regional central hospital. a hadv-b [p h f ] virus was confirmed as the etiological pathogen of this acute outbreak of febrile respiratory illness based on clinical manifestations, epidemiological characteristics, specific molecular detection results, phylogenetic analysis, and serological assays. the virus was isolated by the rhabdomyosarcoma cell culture method, and the complete sequences of the e a, penton base, hexon, and fiber genes were determined and deposited in the genbank database. phylogenetic and sequence homology analyses indicated that the isolated strain is most closely related to some hadv- strains from mainland china. however, this strain appeared to be less virulent than former hadv- strains. according to the chest x-ray results of affected patients, there was no radiological evidence of pneumonia. the most frequent symptoms in these patients were sore throat ( . %, / ) and tonsillitis ( . %, / ). during the course of the outbreak, incorrect response measures and some potential risk factors, such as fire training and marching training, may have exacerbated the spread of the infection. this outbreak illustrates the urgent need to improve the epidemiological and etiological surveillance of hadv infections and to improve the ability of doctors and health officials in basic units of the chinese army to respond effectively to febrile respiratory illness. human adenoviruses (hadvs) are responsible for a broad spectrum of clinical diseases, including febrile respiratory illnesses (fri), gastroenteritis, pneumonia, and kidney infections [ ] [ ] [ ] [ ] . these ubiquitous viruses belong to the genus mastadenovirus in the family adenoviridae. so far, genotypes of hadv have been recognized and classified into seven species (a-g) based on their biological and genetic characteristics (http://hadvwg.gmu.edu/). among these genotypes, a re-emerged hadv-b [p h f ] virus has attracted increasing attention in china. this hadv-b [p h f ] virus is an intertypic recombinant of hadv-b and hadv-b . it has a hadv- genome chassis, including the hadv- penton gene and fiber gene, but a partial hadv- hexon gene, which encodes the antigenic epitopes of the virus [ ] . this virus not only could possess the virulence of hadv- but also could avoid the neutralizing antibody against hadv- , which exists much more widely in the population than that against this hadv-b [p h f ] virus [ ] . an hadv-b [p h f ] virus was first identified as an atypical hadv- strain in spain in [ ] . it then appeared in a turkish military camp in , where hundreds of military trainees fell ill and one man died in this y. dongliu, y. guoliang, x. haocheng and q. shuaijia contributed equally to this paper. outbreak. in recent years, this acute fri pathogen has reemerged several times, once in singapore in [ ] , and twice in china, in shannxi province in [ ] and in beijing in [ ] . in some outbreaks, it has caused severe respiratory disease and death in immunocompetent adults, including military recruits and high school students [ , ] . in a serological study, this respiratory tract pathogen was classified as ''hadv - intermediate'' [ ] . based on genome typing by restriction enzyme analysis, this virus was designated as hadv-b a [ , ] . when the computational genomic analysis method was used in by walsh et al. , the results showed that within the context of the molecular evolution of hadv, this pathogen is a novel adenoviral type and should be designated hadv-b [p h f ] [ ] . according to a previous study, hadv- has become a major causative agent of acute severe pneumonia in the chinese population [ ] . here, we report an outbreak of acute fri in a chinese military training center in . over days, from may to june, individuals were affected. molecular characterization, phylogenetic analysis, and serological assays revealed that hadv-b [p h f ] virus was the etiological agent of this outbreak, and the complete e a gene, penton base gene, hexon gene, fiber gene of this virus all belonged to the hadv- type. this is the first epidemiological and etiological report of a type- -like hadv-b [p h f ] virus associated with an fri outbreak in a chinese military camp. the clinical characteristics of this outbreak differ from those of previous hadv- outbreaks reported in chinese civilians and foreign armies. unlike other hadv- outbreaks, no case of pneumonia developed in any patient. responses to the outbreak according to the training plan, the training schedule included daytime mid-range marches (twice a week) and night fire training (twice a week) between april and may. in may , clustered cases of acute fri (febrile respiratory illnesses) appeared in this mtc, which was defined as an individual with a body temperature[ . °c and with at least one sign or symptom of acute respiratory tract infection, such as a cough or sore throat. in the early stage of the outbreak, - cases of fri were admitted to the mtc hospital every day. this situation did not draw the attention of the mtc managers, and the outbreak was mistaken for severe seasonal influenza by the doctors of the mtc hospital. however, after may, the incidence of disease suddenly increased to more than cases per day, and all patients showed similar symptoms. on may, in an effort to prevent an outbreak of the illness, about soldiers were gathered into several crowded classrooms to attend a health lecture. the mtc managers also appealed to the regional center for disease control and prevention (cdc) for help. on may, an epidemic survey was conducted by the regional cdc. at the same time, disease control and prevention measures were adopted to control the outbreak, including the cessation of training, isolation of affected individuals, environmental disinfection, and morning temperature screening. no new cases were reported after june. to investigate the etiological agent of this outbreak, throat swab specimens (n = , from the date of onset on may to june) were collected. sixty serum samples from fri patients affected after may were collected on may. on june, paired serum samples from patients in the convalescent phase were also collected. at the same time, another healthy soldiers were randomly selected from all of the training camps as the control group. their paired sera were collected on may (as the outbreak-phase samples) and june (as the after-outbreak-phase samples). the sera and swab specimens were transferred to the regional cdc laboratory at °c within h and stored at - °c and - °c, respectively, until further analysis. extraction and analysis of viral nucleic acids from throat swabs viral nucleic acids were extracted directly from the throat swab specimens using a minibest viral rna/dna extraction kit (takara, dalian, china). a one-step reverse transcription (rt)-pcr was then performed using an rv pcr detection kit (neuro hemin, hangzhou, china). this pcr kit can detect types of rna virus (including influenza type a viruses, influenza type b viruses, human respiratory syncytial viruses a and b, human metapneumovirus, human parainfluenza viruses , , and , human rhinovirus, human coronaviruses e and oc , and human enteroviruses) and two types of dna virus (human bocavirus , , , and human adenovirus). the primary identification of hadv in the positive specimen was performed by pcr using the adenovirusspecific primers described by allard et al. [ ] . the amplified target of this specific pcr was the partial hexon gene sequence ( bp) in the hadv genome. the amplicon was sequenced and analyzed using the blast program (national center for biotechnology information). rhabdomyosarcoma (rd) cells (atcc ccl- ) were cultured in dulbecco's modified eagle's medium (dmem, containing % fetal bovine serum, u/ml penicillin g, lg/ml streptomycin; gibco, carlsbad, california, usa) at °c in an closed system with % co incubation. to isolate the virus, rd cells were inoculated with the patients' throat swab specimens ( ll of each specimen) and cultured in dmem containing % fetal bovine serum, u/ml penicillin g, lg/ml streptomycin (gibco, carlsbad, california, usa) at °c in an closed system with % co incubation. the cultures were observed daily for a cytopathic effect (cpe). in - days, the cultures displaying an adenovirus-like cpe were passaged again to confirm the presence of the virus. the positive isolates were identified by adenovirus-specific pcr as described by allard et al. [ ] . viral dna was extracted from the hadv-positive cell cultures using a takara minibest viral rna/dna extraction kit. the complete e a, penton base, hexon, and fiber genes were amplified and sequenced using specific primer sets designed at the jinan junqu cdc (listed in table ). the pcr was performed using a ready-to-use pcr kit (pfu b ; sangon, shanghai, china). the -ll pcr reaction mixture contained ll of hifi pcr master, ll of sterile ddh o, ll of dna template, and . lm each primer. the pcr cycling parameters were initial denaturation at °c for min, followed by cycles of denaturation at °c for s, annealing at °c for s, and extension at °c for s, with a final extension step at °c for min. the amplification products were analyzed by . % agarose gel electrophoresis. the amplified products were sequenced in both directions by the dye terminator method (bigdye terminator v . cycle sequencing kit; applied biosystems) using an abi prism genetic analyzer (applied biosystems). the nucleotide and predicted amino acid sequences were analyzed using the bioedit program (http://www.mbio.ncsu. edu/bioedit/bioedit.html). nucleotide sequence homologies were inferred from the identity scores determined using the blastn program (national center for biotechnology information, bethesda, md, usa). sequence alignments were constructed with clustalx version . [ ] , and phylogenetic analyses were performed with the mega program [ ] . the reference sequences were retrieved from genbank (www.ncbi.nlm.nih.gov/genbank). the reliability of the phylogenetic trees was examined with bootstrap pseudoreplicates. hadv-specific antibody detection using enzymelinked immunosorbent assays (elisas) all serum specimens from patients (including specimens collected in the acute phase and specimens collected in the convalescent phase) in this outbreak and from healthy controls (including the paired specimens collected during the outbreak phase and after the outbreak phase from healthy soldiers) were examined using an elisa the clinical characteristics of the patients are summarized in table . among the hospitalized patients, all presented with fever, and most had a sore throat or tonsillitis. other symptoms reported by the patients were headache, fatigue, cough, sputum, nasal discharge, and diarrhea. two patients were admitted to the intensive care unit (icu) of the military regional central hospital because of the prolonged fever (fever more than days, peak temperature over than °c. patient was diagnosed with acute suppurative tonsillitis, with a peak temperature of . °c, elevated neutrophilic granulocytes (ne %, . %, reference value range, - %), and decreased lympholeukocytes (lym %, . %,) reference value range, - %). patient was diagnosed with upper respiratory tract infection, with a peak temperature of . °c, decreased leukocyte counts (wbc, . /l, reference value range . - . /l)). other patients were observed and treated at the mtc hospital. on average, the fri lasted for - days and all of the patients recovered well. x-ray examinations were conducted of patients (including the two patients admitted to the icu and patients in the mtc hospital), but no radiological evidence of pneumonia was confirmed. therefore, no case of pneumonia developed in this fri outbreak. a series of serological tests and routine blood cultures were performed to detect streptococcus, coxiella, rickettsia, mycoplasma, chlamydia, legionella, brucella, and typhoid fever, but all were negative. a diagnostic one-step rt-pcr was performed on all throat swab samples using an rv pcr detection kit (neuro hemin, hangzhou, china). of the patient swab specimens, specimens were positive for hadv dna, and all specimens were negative for other viruses. then, all specimens were re-tested by hadv-specific pcr with primers specific for a to isolate the virus, rd cells were inoculated with patient throat swab specimens (n = ). the characteristic adenovirus-like cpe was observed in six cultures from six throat swab specimens, and all these cultures were confirmed to be hadv positive by hadv-specific pcr. according to the blast analysis, the amplicon sequences (partial hexon gene fragment, bp) of the six hadv isolates were % identical to each other and to their sequencing results before virus isolation. one of the six hadv isolates (strain sd ) was selected for further genetic study. the complete penton base, hexon, fiber, and e a genes were amplified and sequenced using the specific primer sets listed in table . the sequence data were deposited in genbank. (genbank accession numbers: sd strain complete hexon gene sequence: kr ; penton base gene sequence: kt ; fiber gene sequence: kt ; e a gene sequence: kt ). to investigate the genetic relationships between isolate sd and other hadv strains, phylogenetic trees were constructed by the maximum-likelihood method with bootstrap pseudoreplicates using the mega program [ ] . the phylogenetic analyses were based on the complete hexon, penton base, fiber, and e a gene sequences of strain sd and their counterparts in other hadv strains representing hadv species a-g. in these analyses, hadv isolate sd clustered with the hadv- strains (fig. ) . in the phylogenetic analysis based on the complete hadv hexon gene sequence (fig. a) , the hexon gene sequence of sd clustered with those of the hadv- strains and hadv- strains. the strains most similar to sd were hadv- strain qs_dll_china and ak _argentina (blast score, ; identity, %), followed by singapore hadv- a strain sgn and taiwanese hadv- a strain (blast score, ; identity, %). on the phylogenetic tree based on the entire penton base gene sequence (fig. b) , isolate sd clustered with all the hadv- strains and two hadv- strains: de-wit and gz . the penton base sequence of almost every hadv- strain was % identical to that of sd , including that of singapore strain sgn , chinese strains qs-dll and cq- , egyptian strain ak , and argentinian strain ak (blast score, ; identity, %). on the fiber sequence phylogenetic tree (fig. c ), sd clustered with the hadv- strains and hadv- strains. according to the blastn analysis, the fiber gene sequence of sd was % identical to those of hadv-b strain _taiwan, strain qs-dll_china, and strain south-dakota_usa (blast score, ; identity, %), and % identical to hadv-b strains _spain, ak _argentina, sgn _singapore, and ak _egypt (blast score, ; identity, %). in the phylogenetic tree based on e a (fig. d) , the e a sequence of isolate sd clustered with those of the hadv- and hadv- strains. most of the hadv- strains were % identical to sd in the e a gene sequence. to determine the genetic relationships between sd and the other hadv-b strains, a phylogenetic tree was constructed based on the complete hexon gene sequences of isolate sd and reference strains of hadv subspecies b (fig. e) . on this tree, isolate sd clustered with the hadv- strains and hadv- strains. four main clades were formed in the hadv- ( a) cluster. the first clade was composed of argentinian strain ak , some chinese hadv- strains, and sd . the second clade contained taiwanese strains. the third clade included the hadv- a strains from egypt, to confirm the etiological agent of this outbreak, hadvspecific serological assays were performed. all serum specimens from patients and healthy controls were examined using a classic elisa hadv iga kit and igg kit (institute virion/serion gmbh). the detection results are summarized in table . the statistical analysis revealed that in the acute phase (within days of onset), the proportion of iga-positive samples was significantly higher in the patient group than in the control group, but the proportion of igg-positive samples did not differ significantly between the patient and control groups (table ). in the convalescent phase ([ days after onset), the proportion of iga-positive samples was significantly higher in the patient group than in the control group, and the proportion of igg-positive samples was also significantly higher in the patient group than in the control group. these results confirm that hadv was associated with this outbreak. on the other, in hand, the healthy group, there were significant differences in the proportions of iga-positive samples ( . % vs . %, p-iga \ . , v = . ) and in the proportions of igg-positive samples ( . % vs . %, p igg \ . , v = . ) between the outbreak phase and the after-outbreak phase (same period of acute phase and convalescent phase). this implies the presence of undetected infections in this military camp, which may have played an important role in the transmission of hadv. in conclusion, the regional cdc officials concluded that a type- -like human adenovirus b human/chn/sd / /[p h f ] virus was the etiological pathogen responsible for this acute fri outbreak based on the clinical manifestations in the patients, the epidemiological characteristics of the outbreak, the hadv-dna-specific pcr results, isolation of the virus, sequencing and alignment of the pcr amplicons, homology and phylogenetic analyses based on the complete e a, penton base, hexon, and fiber gene sequences, and serological assays specific for hadv iga/igg. this report describes an acute fri outbreak caused by a type- -like hadv-b [p h f ] virus. this virus shared the most sequence similarity with the hadv- in the complete e a gene, penton base gene, hexon gene, and fiber gene. hadv- infections are often associated with severe respiratory tract diseases and pneumonia, including recombination is an important feature of the genetic evolution of hadv. according to our analysis, the e a, penton base, and hexon genes of isolate sd are most closely related to those of chinese mainland strain qs-dll_ and argentinean strain ak _ . however, its fiber gene is % identical to those of usa strain south dakota/ _ , taiwanese strain _ , and chinese strain qs-dll_ but differs slightly from that of the argentinean strain. these characteristics may be the features of regional variants of the same genotype but these variations may have affected the infectivity or virulence of the new recombinant. further research, including complete genome sequencing and analysis, is required to identify any other relevant genetic variations that are responsible for the milder clinical manifestations of this hadv-b [p h f ] strain. many seroprevalence studies among military recruits have shown that lack of pre-existing immunity to some particular serotypes of pathogen is a critical factor determining susceptibility to infection. from an epidemiological perspective, outbreaks of respiratory illness in a military camp usually show higher infection rate among new recruits than among regular soldiers. however, in the present outbreak, there was no significant difference in the incidence rates of the new recruits and regular soldiers. this phenomenon suggests that the main pathogen of this outbreak is an unfamiliar pathogen to these troops, or even in this whole area. therefore, both the recruits and regular soldiers lacked pre-existing immunity to this pathogen. some earlier statistical analyses have shown that marching training and firing training are significantly associated with the onset of pneumonia and fri, especially in hadv outbreaks [ ] . during a respiratory illness outbreak in an mtc in the usa, hadv was detected on rifle surfaces [ ] . during the early period of the present outbreak, the main components of the training schedule were mid-range marches and night firing training, which may have contributed to the increased patient numbers or exacerbated their symptoms. based on this experience, appropriate measures must be identified and implemented to prevent outbreaks of respiratory illnesses in military training units, such as postponing firing training until after the peak stage of the epidemic and disinfecting the training rifles when they are transferred from one trainee to another. this fri outbreak also illustrates the pressing need to improve the fri epidemic response of the doctors and health officials in basic army units. in the acute fri outbreak reported here, about new cases appeared with the same symptoms in the days after may. however, this situation did not alert the mtc managers or health professionals. no correct diagnostic procedures were implemented, no epidemiological investigation was commenced, and the supervisory cdc received no information about the new cases. when a large wave of new cases (n = ) appeared between may and may, in an effort to control the outbreak, the mtc healthy managers gathered about soldiers into several crowded classrooms for health education, which may have contributed greatly to the transmission of the disease, and days later, the highest incidence peak of the entire outbreak was observed (fig. ) . had the health officials dealt with the outbreak correctly from the very beginning, the spread of this fri may have been restricted to a much smaller scale. because outbreaks of hadv- infections have been frequent in recent years, hadv- -associated fri pose a serious threat to the chinese army. therefore, improved epidemiological and virological surveillance of hadv and many other dangerous pathogens by the chinese military health system is urgently required. some simple and accurate diagnostic methods that distinguish hadv- infections from other similar diseases must be established. moreover, in view of the u.s. army's experience with an hadv vaccination program, the development and application of hadv vaccines may be an excellent way to protect the health of chinese soldiers. the present study has several limitations. first, although . % ( of ) of throat swab specimens were positive when using an rv kit and hadv-specific pcr and the amplicons from the pcr-positive samples were found to have high sequence similarities to the hadv-b strains, the remaining fri cases were still not detected by the specific pcr and sequencing analysis. therefore, it was still difficult to ensure this hadv-b -like virus was the only pathogenic agent responsible for this outbreak. it is possible that some other pathogens were involved in this outbreak. in some previously published descriptions of outbreaks of hadv infection among military trainees, the co-circulation of two or more types of hadv had already been reported. second, the members of the healthy control group were randomly selected from the asymptomatic soldiers in the same mtc on may. at the same time, the disease prevention and control measures had been implemented. none of the healthy controls showed any respiratory tract disease symptoms during the outbreak period. however, these healthy controls had not been screened by laboratory detection methods such as viral nucleic acid detection. considering the results of the iga and igg detection in the healthy control group, there may have been asymptomatic infection cases hiding in the healthy control group. many ''healthy control group'' members had already been infected by this hadv virus. in a future outbreak study, the healthy controls should be selected from a camp where the virus was not present and be screened using laboratory detection methods to differentiate the asymptomatic infection cases. worldwide epidemiology of human adenovirus infections adenovirus infections in immunocompetent and immunocompromised patients pring akerblom p ( ) rapid and quantitative detection of human adenovirus dna by real-time pcr epidemical features of hadv- and hadv- in pediatric pneumonia in respiratory disease caused by a species b adenovirus in a military camp in turkey genomic analyses of recombinant adenovirus type a in china occurrence of respiratory illness due to an atypical strain of adenovirus type during a large outbreak in spanish military recruits outbreak of febrile respiratory illness associated with adenovirus a infection in a singapore military training camp severe community-acquired pneumonia caused by adenovirus type in immunocompetent adults in beijing outbreak of acute respiratory disease in china caused by b species of adenovirus type antigenic characterization of intermediate adenovirus - strains associated with upper respiratory illness in a military camp genetic relationship between thirteen genome types of adenovirus , , and with different tropisms molecular epidemiology and brief history of emerging adenovirus -associated respiratory disease in the united states computational analysis identifies human adenovirus type as a re-emergent acute respiratory disease pathogen emergence of community-acquired adenovirus type as a cause of community-onset pneumonia polymerase chain reaction for detection of adenoviruses in stool samples clustal w and clustal x version . mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods outbreak of febrile respiratory illness caused by adenovirus at a south korean military training facility: clinical and radiological characteristics of adenovirus pneumonia transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting acknowledgments this study was sponsored by the china postdoctoral science foundation ( m ). the sponsors had no role in the study design, data collection, decision to publish, or preparation of the original manuscript. this study was approved by the review board of the jinan junqu center for disease control and prevention. informed consent was received from all participants before the study. the authors declare that they have no competing interests. key: cord- - d w xyd authors: jeon, ji hyun; lee, changhee title: cellular cholesterol is required for porcine nidovirus infection date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: d w xyd porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. our results showed that depletion of cellular cholesterol by treating cells with methyl-β-cyclodextrin (mβcd) dose-dependently suppressed the replication of both nidoviruses. conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. the addition of exogenous cholesterol to mβcd-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. the antiviral activity of mβcd on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral rna and protein biosynthesis and progeny virus production. taken together, our data indicate that cell membrane cholesterol is required for porcine nidovirus entry into cells, and pharmacological drugs that hamper cholesterol-dependent virus entry may have antiviral potential against porcine nidoviruses. nidovirales is a large order of enveloped positive-sense, single-stranded rna viruses that consists of the families arteriviridae, coronaviridae, roniviridae, and mesoniviridae, whose members infect a broad range of hosts including humans and other mammals, birds, fish, insects, and crustaceans [ , , , ] . although the genome sizes and virion morphologies of nidoviruses are strikingly different, the genome organization and replication strategy are comparable across the order. the nidovirus genome is composed of two large open reading frames (orfs), a and b, encompassing the ′-proximal two-thirds of the viral genome that encode non-structural proteins (nsps) and the remaining orfs located in the ′-proximal genome part that code for structural proteins [ , ] . the initial translation from replicase orf a and orf b yields large polyprotein (pp) precursors, pp a and pp ab, via a - ribosomal frameshift (rfs), which then undergo autoproteolysis by viral proteases to eventually produce functional nsps, including the viral rna-dependent rna polymerase (rdrp) [ , , ] . the membrane-bound rdrp-containing replication complex engages in viral genomic rna replication and subgenomic (sg) mrna transcription. the latter finally generates a ′ co-terminal nested set of sg mrnas that are used to express nidoviral structural proteins [ , , ] . porcine reproductive and respiratory syndrome virus (prrsv) is a pathogenic macrophage-tropic arterivirus of swine that results in reproductive failure in pregnant sows abstract porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. our results showed that depletion of cellular cholesterol by treating cells with methyl-β-cyclodextrin (mβcd) dosedependently suppressed the replication of both nidoviruses. conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. the addition of exogenous cholesterol to mβcd-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. the antiviral activity of mβcd on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral rna and protein biosynthesis and progeny virus production. taken together, and acute or chronic respiratory illnesses in pigs of all ages. prrsv primarily replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months. as a result, prrsv infection suppresses normal macrophage function and immune responses and is often associated with severe disease outcomes, including increased pre-weaning mortality in growing pigs resulting from secondary bacterial or viral infections, thereby affecting the swine production system [ , , ] . porcine epidemic diarrhea virus (pedv) is a pathogenic enterocyte-tropic swine coronavirus that causes acute enteritis with high mortality rates in neonatal piglets. pedv infection is characterized by severe villous atrophy in the small intestine that results in watery diarrhea followed by fatal dehydration and death in newborn piglets [ , ] . although pedv outbreaks have been reported in europe and asia, the most serious epizootics for nearly the past three decades have occurred in asia. however, since the virus first emerged in the united states in [ ] , pedv has become recognized globally as a highly contagious and deadly virus. these two viruses, prrsv and pedv, represent emerging and re-emerging porcine nidoviruses that continue to threaten pork-producing countries around world, leading to huge financial losses to the global swine industry [ , ] . lipid rafts, which are enriched in cholesterol, sphingolipids, and associated proteins, are unique liquidordered microenvironments in the plasma membrane and are involved in a variety of cellular processes as well as in multiple stages of the virus life cycle. cholesterol, a major constituent of lipid rafts, maintains the tight packaging of sphingolipids, and several proteins are partitioned into these microdomains. cholesterol depletion destroys this structural order, leading to disorganization of lipid raft microdomains and dissociation of bound proteins [ , ] . therefore, plasma membrane cholesterol plays important roles in the infection processes of various non-enveloped and enveloped viruses [ , , , ] . in particular, enveloped virus entry requires cholesterol in either the viral or cellular membrane or both [ , , , , , , , , ] . however, there are few reports on the potential relationship between cholesterol and the replication of porcine nidoviruses, although cellular membrane cholesterol has been shown to be a determinant of prrsv entry in african monkey kidney marc- cells [ , ] . in the present study, therefore, we investigated the requirement for cholesterol and its mechanism of action in porcine nidovirus infection. independent depletion of cholesterol from the plasma membrane of target cells by treatment with methylβ-cyclodextrin (mβcd) significantly impaired prrsv and pedv infection. these inhibitory effects on viral replication were partially reversible by replenishment with exogenous cholesterol. in contrast, porcine nidoviruses were shown to be resistant to pharmacological reduction of the cholesterol content of the viral envelope. our data indicate that cholesterol-enriched microdomains are essential for prrsv and pedv in the cellular membrane, but not in the viral membrane. further experiments revealed that pharmacological depletion of cellular cholesterol primarily interferes with virus binding and penetration and subsequently influences post-entry stages of the prrsv and pedv replication cycle, including viral genomic and sg rna synthesis, viral protein expression, and virus production. altogether, our results suggest that cholesterol in the cellular membrane is critical for porcine nidovirus entry and that disruption of the cholesterol-dependent entry process may be an excellent therapeutic option for nidovirus infection in human or veterinary subjects. pam-pcd cells [ ] were cultured in rpmi medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs, invitrogen), antibioticantimycotic solution ( ×, invitrogen), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ×, invitrogen) in the presence of μg of zeocin (invitrogen) per ml. vero cells were cultured in alpha minimum essential medium (α-mem, invitrogen) with % fbs and antibiotic-antimycotic solution. st-papn cells [ ] were cultured in α-mem with % fbs and antibiotic-antimycotic solution in the presence of μg of g (invitrogen) per ml. the cells were maintained at °c in a humidified % co incubator. prrsv strain vr- was propagated in pam-pcd cells as described previously [ ] . pedv strain sm - was kindly provided by the korean animal and plant quarantine agency and propagated in vero cells as described previously [ , ] . mβcd and water-soluble cholesterol were purchased from sigma (st. louis, mo) and dissolved in ethanol and phosphate-buffered saline (pbs), respectively. these compounds were diluted to the desired concentrations in maintenance medium. prrsv n and pedv n protein-specific monoclonal antibodies (mab) were obtained from choogang vaccine laboratory (cavac; daejeon, south korea). antibodies to porcine cd (pcd ) and β-actin were purchased from abd serotech (raleigh, na) and santa cruz biotechnology (santa cruz, ca), respectively. the polyclonal antibody recognizing porcine aminopeptidase n (papn) obtained from balb/c mice immunized with purified papn (sigma) was a gift from bang-hun hyun (animal and plant quarantine agency, gimcheon, south korea). the cytotoxic effects of reagents on pam-pcd and vero cells were analyzed using a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (sigma) that allows detection of cell viability. briefly, pam-pcd and vero cells were grown at × cells/well in -well tissue culture plates with mβcd or water-soluble cholesterol treatment for h. after days of incubation, μl of mtt solution ( . mg/ml) was added to each well and the samples were incubated for an additional h. the supernatant was then removed from each well, and μl of dmso was added to dissolve the formazan crystals produced by mtt. the absorbance of the solution was measured at nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd and vero cells grown on microscope coverslips placed in -well tissue culture plates were pretreated with mβcd or ethanol for h and mock infected or infected with prrsv and pedv, respectively, at a multiplicity of infection (moi) of . virus-infected cells were then grown in the presence of mβcd or vehicle for h, fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with n-specific mab for h. after washing five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca), followed by counterstaining with ′, -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on glass microscope slides in mounting buffer ( % glycerol and . % sodium azide in pbs), and cell staining was visualized using a leica dm il led fluorescence microscope (leica, wetzlar, germany). in addition, pam-pcd and st-papn cells stably expressing pcd and papn, respectively, were grown in the presence of mβcd or vehicle for h, fixed, and subsequently subjected to ifa with anti-pcd or anti-papn antibody as described above. cell staining was analyzed using a confocal laser scanning microscope (carl zeiss, gattingen, germany). quantification of virus-infected cells after treatment with mβcd was analyzed by flow cytometry. pam-pcd and vero cells were pretreated with mβcd, infected with virus, and maintained as described above. virus-infected cells were trypsinized at h postinfection (hpi) and centrifuged at × g (hanil centrifuge fleta ) for min. cell pellets were washed with cold washing buffer ( % bsa and . % sodium azide in pbs), and cells were resuspended in % formaldehyde solution in cold wash buffer for fixation at °c in the dark for min, followed by centrifugation and incubation of the pellets in . % triton x- in pbs at °c for min for permeabilization. after centrifugation, the cell pellets were resuspended in a solution of primary anti-n mab, and the mixture was incubated at °c for min. the cells were washed and allowed to react with an alexa fluor -conjugated anti-mouse igg secondary antibody at °c for min in the dark. the stained cells were washed again and analyzed on a facsaria iii flow cytometer (bd biosciences). expression of the viral receptor on the cell surface upon cholesterol depletion was also evaluated by facs analysis as described previously with some modifications [ ] . briefly, pam-pcd and st-papn cells were trypsinized at h post-seeding. the detached cells were fixed and then reacted with the primary antibody or normal mouse igg (santa cruz biotechnology), followed by incubation with secondary antibody as described above. the stained cells were either left untreated or were treated with mβcd at °c for h and analyzed using a flow cytometer. pam-pcd and vero cells were infected with prrsv or pedv and treated with mβcd or vehicle. the culture supernatants were collected at different time points ( , , , , and hpi) and stored at - °c. the prrsv titer was measured by limiting dilution on pam-pcd cells in duplicate by ifa as described above, and the % tissue culture infectious dose (tcid ) per ml was calculated using the spearman-kärber method [ ] . the pedv titer was determined by plaque assay using vero cells as described previously [ ] and was expressed as plaque-forming units (pfu) per ml. viral stocks were treated with mβcd at various concentrations at °c for h followed by ultracentrifugation to remove the mβcd. the mβcd-treated virus supernatants were purified through a % sucrose cushion (wt/vol) prepared in te buffer [ mm tris-hcl (ph . ), mm edta] by centrifugation at , rpm for h at °c in a p at rotor (model cp wx; hitachi, hitachinaka, japan). the virion cholesterol content was determined using fluorescence intensity analysis. briefly, -well plate wells were coated with ng of the purified viruses in mm sodium bicarbonate buffer (ph . ) and incubated at °c overnight. plates were washed three times with washing buffer ( . % tween- in pbs) and blocked with % powdered skim milk (bd biosciences, belford, ma) in pbs at °c for h. after washing, filipin iii (cayman chemical, ann arbor, mi) was added in triplicate for h in the dark. the plates were washed, and fluorescence intensity was measured with a spark m multimode microplate reader (tecan, männedorf, switzerland). in parallel, the purified samples were used to infect pam-pcd or vero cells for h, and the virus-infected cells were independently subjected to facs analysis and virus titration to determine prrsv or pedv infection as described above. pam-pcd and vero cells were first preincubated with vehicle or mβcd at various final concentrations for h and then supplemented with or without μg/ml or μg/ml exogenous cholesterol, respectively, and incubated for h. the cells were then inoculated with prrsv or pedv as described above. the virus inoculum was removed, and the infected cells were maintained in fresh medium containing mβcd and exogenous cholesterol. at h dpi, the virusinfected cells were harvested and subjected to facs analysis to assess the infectivity of prrsv and pedv as described above. in parallel, the cellular cholesterol content was determined using a cholesterol cell-based detection assay kit (cayman chemical) according to the manufacturer's instructions. briefly, virus-infected cells were cultivated in the presence of mβcd and exogenous cholesterol for h, fixed with cell-based assay fixative solution (cayman chemical) for min at rt, and then washed three times for min each with cell-based assay wash buffer (cayman chemical). the cells were incubated with filipin iii for h in the dark. after washing three times in wash buffer, the cells were counterstained with dapi, and filipin staining was visualized using a fluorescent leica dm il led microscope. pam-pcd and vero cells were infected with prrsv and pedv, respectively, at an moi of as described above. at - , , , , , , , , , or hpi, mβcd was added to maintain the indicated final concentration over the remainder of the time course experiment. the virus-infected and inhibitor-treated cells were further maintained and trypsinized at hpi, followed by centrifugation. the harvested cells were subjected to facs analysis to assess the presence of prrsv or pedv infection as described above. binding and internalization assays were performed as described previously with some modifications [ ] . pam-pcd and vero cells grown in -well culture plates were pretreated and infected with prrsv and pedv, respectively, at an moi of at °c for h in the presence of mβcd. unbound viruses were then removed by washing with pbs, and the cells were either incubated at °c (allowing virus binding only) or °c (permitting virus binding and internalization) in the presence of mβcd for h. in the latter case, the cells were further treated with proteinase k ( . mg/ml) at °c for min to remove bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi medium and inoculated onto fresh pam-pcd cell monolayers in -well tissue culture plates. at h post-incubation, bound or internalized viruses were titrated by ifa as described above, and the tcid was determined. for pedv, the serially diluted infected cells were inoculated onto uninfected vero cells, and, after h, viruses were titrated using plaque assay and quantified as pfu per ml. pam-pcd and vero cells were incubated with mβcd for h prior to infection and then inoculated with prrsv or pedv at an moi of for h at °c. the virus inoculum was subsequently removed, and the infected cells were maintained in fresh medium containing mβcd for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent (invitrogen) and then treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets as described previously [ ] . the rna levels of viral genes were normalized to that of mrna for the β-actin or glyceraldehyde- -phosphate dehydrogenase (gapdh) gene, and relative quantities (rq) of mrna accumulation were determined using the -ΔΔct method. to detect alterations in genomic rna and sg mrna levels in the presence of mβcd during porcine nidovirus infection, the results obtained from drug-treated cells were compared with those from vehicle-treated cells. pam-pcd and vero cells were grown in -well tissue culture plates for day and were mock infected or infected with prrsv and pedv, respectively, at an moi of in the presence of mβcd. at the indicated times, cells were harvested in μl of lysis buffer ( . % triton x- , mm β-glycerophosphate, mm ρ-nitrophenyl phosphate, mm mops, mm mgcl , mm nacl, mm egta [ph . ], mm sodium orthovanadate, μg of e per ml, μg of aprotinin per ml, μg of leupeptin per ml, and mm pmsf) and sonicated on ice five times for s each. homogenates were lysed for min on ice and clarified by centrifugation at , × g (eppendorf centrifuge r, hamburg, germany) for min at °c. the protein concentrations of the cell lysates were determined by bca protein assay (pierce, rockford, il). the cell lysates were mixed with × nupage sample buffer (invitrogen) and boiled at °c for min. the proteins were then separated on a nupage - % gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with % powdered skim milk in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at °c for h and reacted at °c overnight with primary antibodies against prrsv n, pedv n, or β-actin. the blots were then incubated with secondary horseradish peroxidase (hrp)-labeled antibody (santa cruz biotechnology) at a dilution of : , for h at °c. proteins were visualized using enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify the viral proteins produced, band densities of prrsv n and pedv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/), based on the density value relative to that of the β-actin gene. all statistical analyses were performed using student's t-test, and p-values less than . were considered statistically significant. to investigate whether cholesterol plays a role in viral infection, we used mβcd, which is the most common cholesterol-sequestering agent used for plasma membranes. to examine the effect of mβcd on porcine nidovirus replication, prrsv and pedv were selected because they are economically important viral pathogens in the pork industry. based on mtt assay, none of the doses of mβcd tested in the current study caused detectable levels of pam-pcd or vero cell death (fig. a) . pam-pcd and vero cells were pretreated with mβcd at concentrations of . to mm or with ethanol as a vehicle control for h prior to infection. mβcd or vehicle was present throughout infection. virus production was initially measured by monitoring cytopathic effect (cpe) after infection and then confirmed by immunofluorescence using the respective anti-n protein mab at hpi (fig. b) . in vehicle-treated control cells, visible cpe appeared at hpi (data not shown) and became predominant by hpi, and virus-specific staining was pronounced in many cell clusters, indicating infection and spread of the viruses to neighboring cells. in contrast, mβcd had an obvious inhibitory effect on porcine nidovirus propagation. as shown in fig. b , the cholesterol-sequestering compound dramatically diminished virus-induced cpe (first and fourth panels) and expression of prrsv and pedv genes in a dose-dependent manner. based on the quantification of n protein by flow cytometry, the proportion (%) of virus-infected cells was noticeably reduced after mβcd treatment. a maximum of ~ % inhibition of both viruses was observed in response to . mm mβcd ( fig. c and d) . in addition, the effective doses for inhibiting % (ed ) of the replication of prrsv and pedv were determined to be about μm and μm, respectively. taken together, these data show that cholesterol depletion of target cells efficiently suppresses the replication of porcine nidoviruses. we then investigated the effects of cholesterol depletion on the envelopes of porcine nidoviruses. each viral stock was treated with mβcd up to mm prior to inoculation of the respective target cells. the virion cholesterol content after mβcd treatment was measured using filipin iii as a fluorescent polyene antibiotic that binds to cholesterol. as shown in fig. a , viral cholesterol levels were significantly reduced in mβcd-treated viruses compared to those in vehicle-treated viruses. however, in contrast to depletion of cellular cholesterol, the removal of cholesterol from virions resulted in no significant reduction in the replication of prrsv and pedv, even at the highest concentration used (fig. b) . furthermore, the titers of both prrsv and pedv remained unchanged upon treatment of each virus with mβcd (fig. c) . our results indicate that the viral cholesterol content is irrelevant to prrsv and pedv infection in vitro. to verify the importance of cellular cholesterol in porcine nidovirus infection, we first examined whether replenishment of exogenous cholesterol restored mβcd-induced inhibition of porcine nidovirus infectivity. to accomplish this, cholesterol-depleted cells were treated with μg or μg of exogenous cholesterol per ml, which is the highest noncytotoxic concentration for pam-pcd or vero cells, respectively, before virus inoculation. both mβcd and exogenous cholesterol were supplied throughout the course of infection. the addition of exogenous cholesterol to mβcd-treated and virus-infected cells was found to significantly reverse the antiviral activity of mβcd through depletion of cellular cholesterol. incubation with mβcd alone greatly reduced prrsv production to % and % at . mm and mm, respectively, whereas supplementation with exogenous cholesterol enhanced virus production to % and % at the same concentrations of mβcd (fig. a) . likewise, although pedv infection declined to %, %, and % in the presence of mβcd alone at mm, . mm, and mm, respectively, virus production increased to %, %, and % at the same concentrations of mβcd when exogenous cholesterol was added (fig. b ). to verify these results, we also investigated alterations in cellular cholesterol content in cells treated with mβcd and exogenous cholesterol using a fluorescent filipin iii. cellular cholesterol levels specifically decreased in virus-infected and mβcd-treated cells compared to those in virus-infected and untreated cells. supplementing exogenous cholesterol distinctly elevated the cholesterol level in virus-infected and mβcd-treated cells (fig. ) . altogether, the data reveal that cellular cholesterol content plays a pivotal role in porcine nidovirus infection. to determine the point at which mβcd acts during porcine nidovirus infection, pam-pcd and vero cells were treated with mβcd at various time points postinfection. at hpi, the levels of prrsv or pedv replication were measured indirectly by quantifying the cells that expressed viral n protein using flow cytometry (fig. ). treating cells with mm mβcd at - and hpi resulted in an approximately % and % decrease in prrsv production, respectively, in comparison with control levels (vehicletreated cells). strikingly, the addition of mβcd at hpi and thereafter (post-entry periods) had no significant inhibitory effect on prrsv infectivity compared to the control levels. similarly, treatment of pedv-infected cells with mm mβcd up to hpi suppressed viral production by - %, whereas exposure to the compound at - hpi resulted in no reduction in pedv infectivity. these data showed that mβcd had to be present pre-infection or at an early stage of viral infection to exert its antiviral effect as a cellular cholesterol depletion reagent. therefore, there is an important effect of cholesterol in the pre-entry period during porcine nidovirus infection. next, we sought to pinpoint the step(s) in the replication cycle of porcine nidoviruses that were precisely targeted by pharmacological depletion of cellular cholesterol. to address this, the earliest steps, the two stages of virus entry (virus attachment and penetration), were examined using an internalization assay after treatment with mβcd. pam-pcd and vero cells were inoculated with prrsv and pedv, respectively, at °c for h to allow only virus attachment and were further maintained either at °c or °c to restrict or permit virus internalization, respectively, in the presence of mβcd. the samples incubated at °c were subsequently treated with proteinase k to remove remaining viral particles from the cell surface. serially diluted infected cells were then subjected to an infectious center assay on uninfected pam-pcd and vero cell monolayers, and virus titers were measured days later by ifa or plaque assay. as shown in fig. , the titers of prrsv and pedv were reduced in a dose-dependent manner in cells treated with mβcd maintained at °c to permit virus binding but prevent internalization, indicating that cholesterol depletion has an inhibitory effect on virus attachment to these cells. moreover, production of both viruses was diminished in mβcdtreated cells incubated at °c to allow virus entry to proceed, which suggested that cholesterol sequestration disturbs internalization of prrsv and pedv. in addition, we analyzed the amount of pcd or papn expressed on the cell surface after mβcd treatment and found that the surface expression level of the viral receptor in cells treated with mm mβcd was similar to that of on vehicle-treated cells (fig. ) . taken together, these results indicate that pharmacological depletion of cholesterol hinders virus attachment and its subsequent penetration event without altering viral receptor expression and that cellular membrane cholesterol is indispensable for the porcine nidoviral entry process. like other positive-sense rna viruses, following virus entry, the nidovirus genome is released into the cytoplasm and promptly serves as a template for translation of viral proteins by hijacking the host translational machinery. early nidoviral translation produces the replicase polyproteins that are proteolytically processed into nsps, which subsequently drive de novo synthesis of nidoviral rna. therefore, we focused on the post-entry phases of the viral life cycle to investigate the functional mechanisms of sequestration of cellular cholesterol in pedv infection. because nidoviral infection generates genomic and sg rna species, we first tested whether the removal of cellular cholesterol specifically affected genome replication and sg mrna transcription. for this purpose, relative levels of both genomic rna and sg mrna were assessed by quantitative real-time strand-specific rt-pcr in the presence or absence of mβcd following porcine nidovirus infection. as shown in fig. a , mβcd almost completely inhibited the synthesis of prrsv genomic rna and sg mrna at a concentration of mm when compared to untreated infected cells. furthermore, an analogous effect of mβcd on genome replication and sg mrna transcription of pedv was observed. little pedv genomic rna and sg mrna was detected in cells treated with . mm mβcd (fig. b) . the decreases in viral rna levels caused by mβcd did not reflect nonspecific inhibition of transcription because the internal control (β-actin or gapdh) mrna level remained unchanged in all samples (data not shown). taken together, these results indicated that treatment with mβcd subsequently suppressed synthesis of nidoviral genomic rna and sg mrna. since nidoviral structural proteins are translated from their respective sg mrna transcripts late in the infectious cycle, it is conceivable that suppression of viral protein expression is a consequence of cascade-like inhibition of viral rna synthesis. thus, we examined whether viral protein translation was affected by depleting cholesterol from cell plasma membranes. to accomplish this, pam-pcd and vero cells were exposed to mβcd for h prior to infection, and the compound was allowed to remain in the culture medium during infection and subsequent incubation. the expression levels of prrsv and pedv n proteins in the presence or absence of mβcd were evaluated at hpi by (fig. ). densitometric analysis of the western blots revealed that the intracellular expression of both n proteins was dramatically reduced by mβcd, with a maximum of more than % inhibition at the highest concentration (fig. ) . these data suggested that the sequestration effect of cellular cholesterol on viral protein expression is attributable to its specific preceding actions on viral rna biosynthesis during nidoviral replication. at hpi, cell lysates were prepared, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted using antibodies that recognize the prrsv n protein or pedv n protein. the blot was also reacted with mouse mab against β-actin to verify equal protein loading. viral protein expression was quantitatively estimated by densitometry and expressed as the density value relative to that of the β-actin gene, and mβcd-treated sample results were compared to those of the vehicle control. the values shown are the means from three independent experiments, and the error bars denote standard deviations. **, p < . in addition, virus yields were determined during pharmacological depletion of cellular cholesterol to investigate whether endogenous cholesterol is necessary for production of infectious viral progeny. after infection, viral supernatants were collected at hpi, and viral titers were measured. as illustrated in fig. a , the presence of mβcd suppressed the growth of viral progeny in a dose-dependent manner. the peak viral titer was determined to be . tcid /ml and . pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of mm mβcd reduced titers of prrsv and pedv to . tcid /ml and . pfu/ml, respectively (representing a more than -log reduction compared to control levels). examination of the growth kinetics also indicated that porcine nidovirus replication was markedly delayed when the cells were treated with mβcd at its optimal concentrations for each virus (fig. b) . these findings confirmed that cellular cholesterol content is integral in optimal progeny virus production from host cells. the order nidovirales is a monophyletic group of enveloped, positive-strand rna viruses with human and various animal hosts that produce a ′ co-terminal nested set of sg mrnas during infection. this order unites the four distantly related families arteriviridae, coronaviridae, roniviridae, and mesoniviridae, based on a number of common properties such as genome organization, predicted proteomes, and synthesis of genomic and sg viral rnas, and it also separates them into large-(coronaviruses, toroviruses, and roniviruses), intermediate-(mesoniviruses), and small-genome (arteriviruses) nidoviruses to stress the clear genome size differences [ , , , ] . research on porcine nidoviruses is necessary not only for developing strategies to control these viruses in pig populations but also for understanding the molecular biology of human or veterinary-important nidoviruses. despite extensive attention and research investment, two porcine nidoviruses, prrsv and pedv, continue to plague pig-producing countries, causing a significant economic impact on the swine industry worldwide. this is partially attributable to the lack of efficient vaccines that can confer full protection against nidoviral infections and the lack of antiviral agents to treat these infections. although cholesterol is required for optimal infectivity of diverse non-enveloped and enveloped viruses [ , ] , its contribution to and specific function in porcine nidovirus replication are currently unknown. the present study showed that pharmacological sequestration of cholesterol in the cellular membrane but not the viral envelope exerts an efficient antiviral effect against prrsv and pedv in vitro. this effect could be counteracted by the addition of exogenous cholesterol, indicating the importance of membrane cholesterol for porcine nidovirus infection. depletion of cellular cholesterol using the drug mβcd primarily affected the virus attachment and internalization stages, significantly affecting post-entry steps in the replication of porcine nidovirus. altogether, our data indicate that cellular membrane cholesterol plays a critical role in entry of prrsv and pedv into target cells. because viruses are obligate intracellular parasites, they have evolved elaborate relationships with their host cells and developed the ability to modulate lipid composition, lipid synthesis, and host cell signaling pathways [ ] . in particular, cholesterol-rich microdomains appear to be important in the entry of various viruses. indeed, many viruses have been shown to exploit cholesterol, which is present in either the viral envelope [ , ] , cellular membrane [ , ] , or both [ , ] , for maximal virus entry. furthermore, accumulating evidence indicates that cholesterol is an essential component in the life cycle of several nidoviruses. the depletion of cellular cholesterol inhibits the entry of coronaviruses, including mouse hepatitis virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] , human coronavirus e [ ] , and avian infectious bronchitis virus [ ] as well as arteriviruses, including equine arteritis virus [ ] and prrsv [ , ] . on the other hand, transmissible gastroenteritis virus (tgev) and canine coronavirus require cholesterol both in the target cell membrane and in the viral envelope [ , ] . the current study revealed that prrsv and pedv are sensitive to the depletion of cholesterol only in the cell membrane. although cholesterol depletion has been shown to inhibit prrsv entry into african green monkey kidney-derived marc- cells, whether plasma membrane cholesterol is involved in either virus attachment or penetration or both is unknown [ , ] . in the present study, we used a continuous prrsv-permissive pam cell line that is considered the primary cell target for prrsv in the natural host, making it a good system for studying virushost interactions [ ] . in a previous study, yin et al. [ ] suggested that cholesterol is critical for a post-adsorption step in the entry of tgev, another porcine alphacoronavirus. porcine cd and apn have been shown to confer permissiveness of non-susceptible cell lines to prrsv and pedv, respectively, and have been identified as key molecules in the entry of porcine nidoviruses [ , , ] . a previous report showed that cholesterol depletion did not alter cd expression in marc- cells [ ] . likewise, the present study indicated that pharmacological depletion of cellular cholesterol had no effect on the levels of pcd and papn expression in porcine cells. recent studies have indicated that apn is not a functional cellular receptor for pedv, suggesting that the presence of the authentic virus receptor is essential for viral entry [ , ] . therefore, it is still possible that cellular cholesterol is quantitatively related to a hitherto unidentified receptor for pedv. based on our results, nevertheless, we propose that cellular cholesterol is important in both the binding and internalization stages of prrsv and pedv entry into susceptible cell lines. these findings are striking in that these two viruses are known to use different cell entry mechanisms for the initiation of virus infection: prrsv enters pam cells via receptor-mediated endocytosis followed by ph-dependent fusion between viral and endosomal membranes [ ] , whereas pedv enters target cells via virus-receptor interactions, followed by direct ph-independent fusion of the viral and plasma membranes [ ] . because cholesterol is an essential lipid component of cell membranes, its depletion has the potential to inhibit virus entry via several mechanisms. cholesterol is a critical structural component of lipid rafts, together with sphingolipids. these lipids influence viral infection by regulating viral and/ or cellular membranes and thus can function by preferentially partitioning into specific membrane microdomains [ ] . cholesterol may affect virus entry by modifying interactions between virus particles and host cell membranes, and lipid recognition by certain viral constituents may be essential for virus entry [ ] . in the case of sars-cov, cholesterol in the plasma membrane plays an important role in interactions of the viral spike protein and cellular receptor angiotensinconverting enzyme for optimal infection [ ] . by analogy, it is feasible that cellular cholesterol depletion might disturb binding of prrsv and pedv to specific cellular receptors. secondly, the level of cholesterol is important for maintaining biological membrane fluidity, and its removal could reduce the potential for lateral diffusion in the membrane [ ] . this reduction in membrane fluidity could have an influence on the entry of prrsv and pedv. thirdly, the lipid environment, including the cholesterol level, is known to contribute to the charge of ion channels formed in cellular membranes [ ] . considering this issue, ion channel alterations in response to the lack of cellular cholesterol may affect the entry process of porcine nidoviruses. lastly, cholesterol removal has been shown to result in the inhibition of cellular signaling pathways [ ] . we previously found that prrsv and pedv activate specific intracellular signaling networks such as mitogen-activated protein kinase (mapk) cascade pathways to favor replication of these viruses, but these signaling pathways are irrelevant to virus internalization [ , [ ] [ ] [ ] . based on these previous data, cellular cholesterol does not appear to act through mapk signaling pathways. although our analysis did not provide clear evidence of the mechanism by which cholesterol promotes porcine nidovirus entry, we assume that the mechanism may differ among viruses and that cholesterol-dependent virus entry might be dependent on more than one of the aforementioned pathways simultaneously. in conclusion, our findings indicate that optimal infectivity of porcine nidoviruses requires cholesterol in the plasma membrane and that this is critical for the entry of prrsv and pedv. however, cholesterol depletion resulted in a reduction, but not abolishment, of virus infectivity, indicating that virus entry may still occur with lower levels of cholesterol but that increased cholesterol content makes this process more efficient. future work should address the question of whether cholesterol facilitates prrsv and pedv entry through interactions between viral attachment proteins and cellular receptors and/or by affecting membrane fluidity. the current study indicates that cellular cholesterol is a key player in the early stages of porcine nidovirus infection, including attachment and penetration. impeding porcine nidovirus entry is a viable antiviral strategy because it likely acts on extracellular targets, thereby limiting cell damage, and these viruses might be used as surrogate models for testing antiviral agents against human nidoviruses. although further studies based on in vivo assessments are needed to evaluate the efficacy and safety of the cholesterol-depleting agent mβcd, the results presented here indicate that molecules or drugs that interfere with cholesterol function in virus entry should be considered candidates for antiviral approaches to porcine nidoviral diseases. the fusion peptide of semliki forest virus associates with sterol-rich membrane domains lipid raft disruption by cholesterol depletion enhances influenza a virus budding from mdck cells functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses specific association of glycoprotein b with lipid rafts during herpes simplex virus entry lipids: a key for hepatitis c virus entry and a potential target for antiviral strategies regulation of receptor function by cholesterol suppression of coronavirus replication by inhibition of the mek signaling pathway 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cellular cholesterol role for influenza virus envelope cholesterol in virus entry and infection cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in marc- cells virus infection and lipid rafts differential effect of cholesterol on type i and ii feline coronavirus infection lipids as modulators of membrane fusion mediated by viral fusion proteins 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 porcine reproductive and respiratory syndrome virus entry into the porcine macrophage cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus critical role of cholesterol in bovine herpesvirus type infection of mdbk cells the authors declare that they have no conflict of interest.ethical approval this article does not contain any studies with animals performed by any of the authors. key: cord- -snrumw x authors: sugiyama, k.; amano, y. title: morphological and biological properties of a new coronavirus associated with diarrhea in infant mice date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: snrumw x biological and morphological properties of a virus, isolated from the intestine of infant mice with clinical signs of diarrhea and designated as diarrhea virus of infant mice (dvim), were examined. the first infective virus was detected on the cells hours post infection, followed by rapid release of the virus into the culture fluids. virus-induced syncytia in balb/c- t cell cultures caused hemadsorption at ° c and viral antigens were shown to be located in the cytoplasm of the syncytia by immunofluorescent techniques. by scanning electron-microscopy, budding virus-like particles were detected on the surface of virus-induced syncytia. morphologically the virus was shown to be enveloped and approximately nm in diameter. two types of projections were demonstrated, one type of projection was club-shaped, nm in length and the other type was small, granular, nm in length. the latter type of projection might be the basal part of the clubshaped type and related to the hemagglutinating activity. among the coronaviruses, there are strains that cause intestinal infections leading to enteritis and diarrhea in swine ( , ) , calves ( , , ) , rodents ( ) and possibly man ( ) . recently, a new viral agent (dvim) was isolated from an infant mouse with diarrhea, by the utilization of cultured cells. it has also been shown by complement fixation tests that this new isolate is antigenically related to known coronaviruses, avian infectious bronchitis virus (ibv) and mouse hepatitis virus strain- (mhv- ). the viral agent was propagated in balb/c- t cells with the formation of sync)~ia and caused hemadsorption and hemagglutination of mouse or rat erythrocytes ( ), " - / / / / $ . ( , ) . a virus isolated from t h e intestine of a n i n f a n t mouse w i t h clinical signs of diarrhea, d e s i g n a t e d dvim, was generously p r o v i d e d b y dr. k o z a b u r o sato (central l a b o r a t o r y of shionogi p h a r m . co., osaka, j a p a n ) . g r o w t h a n d purification of d v i m h a v e been previously described ( ) . h e m a d s o r p t i o n a n d h e m a g g l u t i n a t i o n (ha) assays were p e r f o r m e d as previously described ( ) cells were cultured on glass cover-slips ( × ram) infected with d v i m as described above. a t designated intervals cover-slips t r e a t e d or u n t r e a t e d w i t h erythrocytes were rinsed five t i m e s w i t h eold-pbs, fixed w i t h g l u t a r a l d e h y d e ( p e r cent in pbs), dried in a criticalpoint dryer a n d coated w i t h gold-palladium alloy in v a c u u m ( - tort) a n d e x a m i n e d b y sem. tubes were collected r a n d o m l y a n d e x a m i n e d for extracellular virus (ecv). the same tubes were w a s h e d five times with pbs, a n d t h e cells were d i s r u p t e d w i t h t ml of m a i n t e n a n c e m e d i u m b y two cycles of freezing a n d t h a w i n g . the s u p e r n a t e s of t h e d i s r u p t e d cells were collected after c e n t r i f u g a t i o n a t x g for m i n u t e s a n d t h e virus c o n t a i n e d in t h e s u p e r n a t e was designated cell-associated virus (cav). the i n f e c t i v i t y of e c v a n d cav was a s s a y e d as described above, a n d expressed as a n u m b e r of m e d i a n tissue culture infectious doses (tcids ) per . ml. hyperimmune serum h y p e r i m m u n e serum a g a i n s t purified d v i m was p r e p a r e d in a r a b b i t as follows: f o r t h e first a n d second inoculation, virus was injected i n t r a m u s c u l a r l y w i t h a n equal v o l u m e of complete f r e u n d ' s a d j u v a n t within a three-week interval, a n d a booster injection was given w i t h o u t a d j u v a n t t h r e e weeks a f t e r t h e second-injection. a n t ib o d y t i t e r for t h e i m m u n e serum was d e t e r m i n e d as , a n d , in h e m a g g l u t i n ation a n d n e u t r a l i z a t i o n test respectively. i n f e c t e d cell cultures on coverslips were fixed w i t h acetone at ° c for t m i n u t e s a n d tile indirect m e t h o d using r a b b i t a n t i -d v i m s e r u m a n d fluoresceine isothioc y a n a t e -e o n j u g a t e d g o a t a n t i -r a b b i t globulin was applied. virus samples were negatively stained with per cent potassium phosphotungstate (ph . ) and examined in jem- b electron microscope. the eytopathic effect of dvim was characterized by the formation of syncytia in balb/e- t cell cultures. when cells were infected at low multiplicities of infection (approximately, m o i = . ) , small syneytia were detectable at hours p.i. at to hours p.i., all areas of the cell sheet consisted of syncytia. subsequently, syneytial lysis began from the center of each syncytium and progressed toward the periphery. the relationships between virus production and formation of syncytium were examined by a comparison of ha titers of the culture fluid with the cell-fusion ratio during the replicative cycle oi the virus (fig. ) . as can be seen from fig. , ha activity was detected at hours p.i. but development of syncytium lagged behind. maximum syneytium development occurred between and p.i. ha titers reached a peak by hours p.i. and remained constant. the surface of uninfected cells were covered with microvilli (fig. a) , as previously shown for many celt lines, but following infection with dvim (t hours p.i.) the surface area of syney¢ia were shown to be covered with a large number of spherical buds (fig. b) . the buds were moderately pleomorphic in shape, approximately -- nm in diameter. particles of a similar size were not detected on the surface of uninfected cells. therefore we conclude those particles are budding virions. this conclusion is further substa.ntiated by the observation that mouse erythrocytes were exclusively adsorbed to syncy~ia. moreover, as shown in fig. a the size of the syneytia could be delineated by the location of adsorbed erythrocytes. at a high magnification, a large number of particles were etearly visible in the area of hemadsorption (fig. b ). fig. shows the growth curve of dvim in balb/c- t cells. the production of cav was detected within hours p.i., followed by a rapid exponential increase. at hours p.i., cav titer reached a maximum level ( . tcids / . ml). the production of ecv was later than that of cav, but the titer was . tcid / . ml at hours p.i. although syncytium formation was very rarely detected at hours p.i., approximately per cent of the cells were fused by hour and detachment from the glass surface followed. h a titers of cav increased rapidly from to hours p.i. corresponding to a rapid increase in virus production from °.s to .a tcids / .i ml, and ~he titer continued to increase until hours p.i. ha titers of ecv were delayed by approximately hours compared to those of cav. infectivity of cav and ecv decreased from hours p.i., corresponding to rapid degeneration of infected cells and possibly virions, while the ha titer of ecv remained constant until hours p.i. at ° c. the estimated time required for one step growth of dvim was approximately hours. an eleetron-mierograph of partially purified dvim is shown in fig. . the diameter of virions varied from to nm, excluding the surface projections. the projections formed a fringe radiating from the viral envelope and they appeared to be club-shaped, nm in length and t nm in width at the distal end of the projections. in addition, noticeable structures which consisted of apparent additional small granular projections were observed. these small projections were nm in length and firmly fixed on the viral envelope. purified virus partieles, in mcsg of the preparations, were only partially covered by club-shaped projections but always covered with small granular projections, even after a considerable period of sonieation. fig. shows the negatively stained dvim particles after sonication at l(hz for minutes. although club-shaped projections had completely disappeared, small projections were still present on the surface of partially disrupted virions. furthermore, the inner structure of the virion was clearly observed after sonieation suggesting penetration of the stain. the thickness of the envelope was measured as nm and a fine filamentous structure, irregularly tangled, was observed in broken virions. recently, some viral agents, which are responsible for diarrhea of new-borne mice, have been classified, geovirus is a major cause of routine diarrhea. ( ) . epizootic diarrhea of infant mice (edim) virus is now classified as a rotavirus by holmes st al. ( ) . mouse hepatitis virus (mhv), a member of coronaviruses, has been described as a common cause of enteric infection in baby mice ( , ) . on the other hand, lethal intestinal virus of infant mice (livim), first described by k~ast, is probably the most important viral agent of diarrhea in new-born mice ( , ) , but there was no viral agent detected. however, the epidemiological and pathological characterization of diarrhea of new-borne mice is consistent with the recorded pathology of livim, and suggests that a mhv could be the cause of this disease ( , , , ) . previous results reported by sato st al. ( ) indicated that the infectious agent (dvim) isolated from the intestine of mice with diarrhea possesses i~na and lipid. the agent passes through a nm filter but not a nm filter. serologically, dvim was shown to be related to ibv and mhv- by a complement fixation test but they were clearly distinguishable from each other by neutralization assays. these observations suggested that this new isolate, dvim, is a member of coronavirus. however, it was important to establish the morphological characteristics of purified dvim, since eoronavirus classification, is established mainly by means of characteristic morphology at present. the results presented in this report showed that diameter of virion was approximately nm, excluding surface projections. characteristic coronavirus projections attached to the virion were demonstrable. however morphologically dvim appears to be slightly different in that additional small granular projections were observed. a similar small projection on enteric bovine coronavirus has been reported recently ( ) . but no additional projection has been detected on mhv- , propagated and purified in the same manner as dvim (data not shown). the morphological relationships of the two projections, club-shaped and granular, are at present not clear. however, it is possible that the club-shaped projection might be attached to the granular projection. moreover, the ha activity of dvim was only slightly affected by sonication, which suggests that the ha activity of ])vim might be associated with the small granular projection. we showed by sen that a large number of particles were present on the dvim-induced syncytia but not on uninfected cells. the size of the particle was similar to that of negatively stained dvim, while hemadsorption was restricted to the cells which possess surface particles. the surface profile of dviminfected cells has a prominent resemblance to the cells infected with ortho-or para-myxoviruses ( , , ). as described previously ( , it is clear that the hemadsorption of dvim was not due to the "pseudohemadsorption" ( ). these observations suggest that dvim particles are assembled and bud through the plasma membrane of syncytium. in addition to these morphological properties, the existence of ha and receptor destroying activities as described previously ( ), have not been reported for any of the other routine coronaviruses. several studies of coronaviruses by the thin sections and transmissible electron microscopy show unequivocally that coronaviruses are formed by budding from the surface of intracellular cistelme ( , , , ) . the more detailed morphogenesis of dvim remains to be investigated in future. electron microscope study of hemadsorption in measles virus infection morphogenesis of avian infectious bronchitis virus and a related h u m a n virus (strain e) lethal intestinal virus infection of mice (livim). amer replication of an enteric bovine eoronavirus in intestinal organ cultures lethal enteritis in infant mice caused by mouse hepatitis virus intracellular development and mechanism of hemadsorption of h u m a n coronavirus lethal intestinal virus of infant mice is mouse hepatitis virus mouse hepatitis, reo- , and the theiler viruses coronavirus particles in faeces from patients with gastroenteritis morphogenesis of avian infectious bronchitis virus in primary chick kidney cells a transmissible gastroenteritis in pigs itemadsorption of m u m p s virus examined by light and eleetron microscopy growth and intracellular development of a new respiratory virus new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice infantile enteritis viruses. morphogenesis and morphology isolation of mouse hepatitis virus from infant mice with fetal diarrhea an apparently new lethal virus disease of infant mice coronavirus; a comparative review. c u r l top pathology of neonatal calf diarrhea induced by a coronavirus-like agent electron microscopic studies of coronavirus mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice some characteristics of corona-like virus isolated from infant mice with diarrhea and inf]arnentory submaxillary gland of rats characterization of a calf diarrhea coronavirus hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice morphology of transmissible gastro-enteritis virus of pigs. a possible member of coronaviruses adsorption-hemagglutination test for influenza virus in monkey kidney tissue cultures we wish to thank dr. kozaburo sato, central laboratory, shionogi pharma. co., osaka, for his useful advices during this work. key: cord- -tsxniu j authors: sha, jianping; li, yuan; chen, xiaowen; hu, yan; ren, yajin; geng, xingyi; zhang, zhiruo; liu, shelan title: fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: tsxniu j middle east respiratory syndrome coronavirus (mers-cov) was first isolated in . the largest known outbreak outside the middle east occurred in south korea in . as of june , laboratory-confirmed cases ( deaths; . % case fatality rate [cfr]) had been reported from countries, particularly in the middle east. however, the cfr for hospital outbreaks was higher than that of family clusters in the middle east and korea. here, we compared the mortality rates for nosocomial outbreaks in the middle east and one outbreak of mers-cov in south korea. our findings showed the cfr in the middle east was much higher than that in south korea ( . % [ / ] vs. . % [ / ], p = . ). infected individuals who died were, on average, older than those who survived in both the middle east ( years [ – ] vs. years [ – ], p = . ) and south korea ( years [ – ] vs. . years [ – ], p = . ). similarly, the co-morbidity rates for the fatal cases were statistically higher than for the nonfatal cases in both the middle east ( . % [ / ] vs. . % [ / ], p = . ) and south korea ( . % [ / ] vs. . % [ / ], p = . ). the median number of days from onset to confirmation of infection in the fatal cases was longer than that for survivors from the middle east ( days [ – ] vs. days [ – ], p = . ). thus, older age, pre-existing concurrent diseases, and delayed confirmation increase the odds of a fatal outcome in nosocomial mers-cov outbreaks in the middle east and south korea. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. the first report of middle east respiratory syndrome (mers) was identified in saudi arabia in june . the middle east respiratory syndrome coronavirus (mers-cov) isolated from this patient was similar to severe acute respiratory syndrome coronavirus (sars-cov), which caused an epidemic in - [ ] . both novel viruses are single-stranded rna viruses belonging to the genus betacoronavirus [ , ] , and the diseases they cause share common clinical characteristics, including fever, cough, diarrhea, and shortness of breath [ ] . jianping sha, yuan li, and xiaowen chen equally contributed to this work. as of june , the world health organization (who) had been notified of laboratory-confirmed cases with mers-cov (globally), including at least deaths; the case fatality rate (cfr) was . % ( / ) [ ] . a total of countries in the world have been affected, including countries in the middle east (egypt, iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates, yemen), africa (algeria, tunisia), europe (austria, france, germany, greece, italy, the netherlands, turkey, the united kingdom), asia (china, the republic of korea, malaysia, philippines, thailand) and north america (united states) (http://www.who.int/ emergencies/mers-cov/en/). so far, all cases of mers have been linked through travel to or residence in countries in or near the middle east. generally, the middle east is the primary region where mers-cov originates, circulates and is exported. in contrast, since the first report of sars-cov in china in , a total of sars cases, including deaths, have been reported to who. these have involved countries, predominantly in south east asia, with only one case identified in kuwait in , and no cases were found in the middle east since then (http:// www.who.int/csr/sars/country/table _ _ /en/). the fatality risk for mers-cov is much higher than that for sars-cov, which has a cfr of . % [ , ] . furthermore, the cfr for patients with co-morbidities is greater ( % in mers vs. % in sars) than those without preexisting diseases [ ] . generally, the cfr is attributed to both host factors and virus factors (e.g. virus replication and mutation) and local medical expertise [ , ] . one unique common epidemiological characteristic of these two diseases is that the spread of both mers-cov and sars-cov infection has been largely driven by human-to-human transmission in healthcare settings [ ] . failures in infection prevention and control in healthcare settings have occasionally resulted in large numbers of secondary cases in nosocomial outbreaks. the earliest identified nosocomial mers outbreak was traced back to march from clusters of severe respiratory illness among healthcare personnel (hcp) in jordan [ ] . since then, a series of nosocomial mers outbreaks in small numbers have been identified in the middle east (jordan in , saudi arabia in - ) [ , , , , ] in this study, we conducted a preliminary mortality risk factor analysis for nosocomial mers-cov outbreaks in south korea and the middle east. the findings from this study might help to reduce the severity and number of deaths from hospital-clustered cases by leading to the adoption of appropriate control measures. in , scientists in the republic of korea and china completed full-genome sequencing of coronaviruses from the mers outbreak in korea. the findings were analysed by a group of virologists convened by who, and preliminary results suggested that the mers cov viruses isolated in the republic of korea were similar to those isolated in the middle east (http://www.who.int/mediacentre/news/ mers/briefing-notes/update- - - /en/). mers-covs associated with the korean and middle east outbreak belong to lineage of mers-cov, which has been the predominant infectious agent in saudi arabian camels since november [ ] . the mers-cov variants associated with the recent outbreak of human infections in south korea (e.g., chinagd -v / and kor/knih/ - / ) show the highest similarity ( . - . %, full genome) to a camel virus (camel/riyadh/ry / ) sampled in march , followed by the latest strain (kt ) prevalent in saudi arabia ( . % identified) [ ] . however, the mers-covs in korea have the ability to cause large outbreaks in environments that are different from that of the middle east (http://www.who.int/emer gencies/mers-cov/en/). the national health and family planning commission of china determined that the collection of data from one human mers-cov infection imported from korea was part of the public health investigation of an outbreak and was exempt from institutional review board assessment. all other mers cases were obtained from publicly available data sources. all data were supplied and analysed without access to personal identifying information. information on all laboratory-confirmed mers cases was obtained from various publicly available sources, including who global alert and response updates, documents officially released by the local health bureau, news releases from middle eastern and south korean authorities, the weekly epidemiological record, promed posts, and literature published from april to june (http:// www.who.int/csr/don/archive/disease/coronavirus_infections/ en/). the latest cases that had not been officially announced by who were identified by searching promed posts, which confirmed announcements by individual countries' ministries of health. based on the available data, we initially established a database of a line list of human nosocomial mers outbreaks (supplementary tables s , s and s ). according to the who's july interim reporting definition (http://www.who.int/csr/disease/coronavirus_ infections/case_definition/en/), a person with mers has a laboratory-confirmed mers-cov infection, irrespective of clinical signs or symptoms. a case may be laboratoryconfirmed by detection of viral nucleic acid or serology. the presence of viral nucleic acid can be confirmed by either a positive reverse transcription polymerase chain reaction (rt-pcr) result on at least two specific genomic targets or a single positive target with sequencing of a second target. a case confirmed by serology requires demonstration of seroconversion in two samples, ideally taken at least days apart, by enzyme-linked immunosorbent assay (elisa), by indirect fluorescent antibody (ifa) screening, or by a neutralization assay [ , ] . a direct epidemiological link with a confirmed mers-cov patient may include ( ) healthcare-associated exposure, including providing direct care for mers-cov patients, working with healthcare workers infected with mers-cov, visiting patients or staying in the same close environment of individuals infected with mers-cov; ( ) working together in close proximity or sharing the same classroom environment with individuals infected with mers-cov; or ( ) travelling together with individuals infected with mers-cov in any kind of conveyance or living in the same household as individuals infected with mers-cov. in addition, the epidemiological link may have occurred within a -day period before or after the onset of illness in the case under consideration [ ] . we used a comparative epidemical analysis of the dates of onset of illness and the characteristics of the fatal and surviving cases. all statistical analysis was conducted using the statistical analysis system, version . (sas institute, cary, nc, usa). quantitative measurements are presented as the median and range of the observed values, and qualitative measurements are presented as relative and absolute frequencies. an analysis of variance (f test) was applied to the measurement data. v tests were used to compare the distribution of the different variables of qualitative measurements between fatalities and survivors. fisher's exact test was used in the analysis of contingency tables when the sample sizes were small (the expected values in any of the cells of a contingency table were below ; the number of total samples was no more than ; the data were very unequally distributed among the cells of the table). any p-values given were two-sided and considered statistically significant at . . as of march , we had identified human laboratory-confirmed clusters with mers-cov, involving cases, of which were fatal. all clusters had been reported to who or published by the local authority or in pubmed. these mers-clustered cases were distributed in nine countries: clusters from the kingdom of saudi arabia (ksa), six from the united arab emirates (uae), four from jordan, three from qatar, and one each from france, iran, italy, tunisia, and the united kingdom (uk). the numbers of clusters per year were as follows: three clusters including cases in , clusters including cases in , and clusters including cases in . for the control groups, we chose a total of sporadic cases of mers-cov, composed of fatal and nonfatal cases from the following countries: cases from the ksa, cases from the uae, cases from jordan, from qatar and from tunisia. the numbers of sporadic cases per year were as follows: cases in , cases in and cases in . fatality risks for nosocomial mers outbreaks the results showed that the percentages of hcp in mers clusters were much higher than those in sporadic cases ( . % [ / ] vs. . % [ / ], p = . ) ( table and table s ). however, the hcp-specific cfr was much lower than the overall cfr from mers clusters ( , p = . ). the median age in fatal cases in hcp was much lower than in fatal cases overall ( . years vs. years , p = . ) ( table ) . we stratified the age groups between the fatal and nonfatal cases groups. the results showed a statistical difference in the distribution of the - , - , - , - , and ? year-olds between the two groups (p = . ). males dominated both the fatal and nonfatal groups of the clustered and sporadic cases (p [ . ) ( table ) . a history of exposure to camels prior to onset of disease was not significantly correlated with survival ( . five time periods useful for public health surveillance were evaluated. the median time from onset to confirmation of infection in the fatal groups was much longer than that for survivors in mers clusters ( . days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] vs. days [ - ], p = . ) and in sporadic mers cases ( days vs. days [ - ], p = . ). however, there were no statistical differences in the median time from onset to hospital admission, onset to hospital discharge, and subsequent death or the number of hospitalized days between the fatal and nonfatal cases for the two groups (table ) . by march , we had obtained data on nosocomial outbreaks involved in confirmed cases (all nosocomial outbreaks were from the middle east; the above clusters were not included in these outbreaks), including iran (one cluster), ksa ( clusters), jordan (three clusters), france (one cluster) and uae (five clusters). we also had one nosocomial outbreak with confirmed cases with mers-cov in south korea (table and table s ). the observed average cluster size ( ) for mers from south korea was much greater than that for the middle east ( , range - ). human nosocomial outbreaks with mers-cov in the middle east occur throughout the year and peak in the spring, especially february to april. mers outbreaks in south korea were reported from march to june , concomitant with peaks in the reporting of mers nosocomial outbreaks in the middle east ( table ) . the average age in the fatal groups was much higher than that in the survival groups ( years old vs. table ) . we found no difference between the fatal and nonfatal cases with respect to exposure to camels and other animals (horses, sheep and goats). in contrast, the level of humanhuman transmission was much higher in the nonfatal cases in the middle east than in the fatal cases ( . table ) . the middle east group showed a statistical difference between fatal and nonfatal cases for the median days from ], p = . ). however, there were no differences in the percentage of total deaths between the index and secondary cases ( table ) . the mean age of the deaths was significantly higher than that of the survival cases for the index ( years vs. years [ - , p = . ) and secondary cases ( years vs. years , p = . ). patients in the age groups c and - years were the most common in the fatal and survival cases, respectively, for the index group, while the - and - -year age groups were the common groups in the fatal and nonfatal cases, respectively, for the secondary cases. there was no significant difference in gender distribution between the fatal and nonfatal cases in the index and secondary groups ( table ) . the ratio of co-morbidity was much higher in the fatal groups than in the non-fatal groups from the secondary cases ( . % [ / ] vs. . % [ / ], p = . ); however, there was no difference between the fatal and nonfatal groups from the index cases. similarly, a history of exposure prior to onset was common for the fatal and nonfatal groups from the index and secondary cases ( table ) . there were no differences between fatal and nonfatal cases in the median time from onset to hospitalization, onset to confirmation, onset to discharge or death or hospitalized days (table ) . however, the median time from onset to hospitalization was shorter in the secondary cases compared to the index cases ( days the secondary cases was slightly shorter than in the index cases ( days vs. days , p = . ); however, the median time from onset to hospital discharge for secondary survivors was days ( - ), which was significantly shorter than the days ( - ) for index survivors (p = . ). acute respiratory tract infections with mers-cov cause considerable morbidity and mortality and pose a threat of repeated outbreaks in healthcare facilities [ , , , [ ] [ ] [ ] ] . the resulting transmission among patients, visitors, and hcp has been a defining feature of mers-cov epidemiology since its emergence in [ ] . in this study, we compared the mortality risk factors in two different nosocomial outbreaks, based on nosocomial outbreaks of mers-cov infection in the middle east and one large outbreak identified in south korea. our findings showed the final cfr for the middle east ( . %) was significantly higher than that for south korea ( . %). both estimated cfrs were significantly lower than that for one hospital outbreak of mers (cfr % [ / ] ) in saudi arabia in and another nosocomial outbreak (cfr . % [ / ]) in saudi arabia [ , ] . the cfr of this latter outbreak was also much higher than that of one extended family cluster ( . % [ / ]) in saudi arabia in [ ] . these results demonstrate that the survival rate of clustered patients with mers-cov in korea was higher than in the middle east. there are several possible explanations for the observed differences between the cfrs in south korea and the middle east. first, there may be disparities in national surveillance and available expertise [ ] . second, the cfr for the middle east might have been overestimated because a large number of mild and asymptomatic cases are likely to go undetected there [ ] . third, it is possible that primary cases accounted for a higher percentage of the cluster patients in the middle east than in south korea [ ] . the findings on age were consistent in hospital outbreaks in the middle east and from south korea. our results showed that the median age in fatal cases was much higher than that in nonfatal cases. this is in agreement with a saudi arabian case series report that showed individuals older than years had a greater association with mortality. a multivariate logistic regression model estimated that for every -year increase in age, the odds of dying increased by % [ ] . in all, this indicates that older age is associated with death in cases of mers-cov infection [ , , ] . in particular, the median age in fatal hcp cases was also much higher than that in nonfatal hcp cases, but lower than the overall average. this is in agreement with the findings of liu et al. [ ] . the reasons for the higher fatality rates in older individuals are not understood but have been attributed to cultural practices that result in an increase in the exposure risk that older people are willing to take [ ] . in addition, older people may be more likely to smoke and to have underlying diseases and impaired immune functions, which may increase susceptibility and progression of infections and even increase the chance of death [ ] . the sex characteristics of mers outbreaks in the middle east are similar to those in south korea. the patients in mers outbreaks in both areas were predominantly male, and the proportion of males in the study populations did not differ [ ] . furthermore, there was no difference in the male-specific cfr between the mers clusters of the two groups, a finding that is similar to other reports [ , , , ] . our findings suggest that the gender distribution is not linked to a fatal risk factor in mers outbreaks. hcp are at high risk of acquiring emerging mers infections due to occupational exposure and are affected mostly by nosocomial outbreaks [ , , , , ] [ ] . in total, the fatality risk for hcp was significantly lower than the overall fatality risk in the middle east and south korea. these findings can be attributed to three facts: first, the majority of hcp developed asymptomatic or mild symptoms and moderate symptoms [ ] ; second, hcp were confirmed as secondary cases under medical investigation, which led to earlier confirmation and good outcomes [ ] ; third, epidemiological analysis showed that hcp were much younger and had fewer co-morbidities compared to total mers cases [ ] . in contrast with sars, about % of patients with mers had at least one additional illness, and patients who died were more likely to have an underlying condition ( % of patients who died vs. % of recovered or asymptomatic patients) [ , ] . similar to the middle east, this study showed that the odds of dying were four times higher for individuals with concurrent health conditions than for those without these conditions in south korea. the odds of fatality were much lower than those estimated by the logistic regression model (seven times) [ ] . this is in part due to higher viral loads in the respiratory tract and longer shedding in patients with underlying diseases compared to cases without co-mortalities [ , ] . human-to-human transmission of mers-cov has been confirmed by epidemiological and genomic studies of cases associated with hospital and household mers outbreaks [ ] . spread was assumed to occur largely via large droplets and contact [ ] . our study indicated that the percentage of human-to-human transmission in nonfatal cases was slightly higher ( . % vs. . %) than in fatal cases in mers clusters, and two reasons could explain this: first, the survivors in secondary cases were younger and had fewer co-morbidities [ , , , , ] ; second, most of the secondary cases were under medical investigation, and therefore, the infection could be confirmed early once symptoms were observed, making timely treatment possible [ , , , , , , ] . overall, human-to-human transmission seems to have had a positive effect on the outcome of the secondary cases from the mers nosocomial outbreaks in the middle east. rapid diagnosis and providing supportive care may be of marginal consequence in the mers clusters [ , ] . the progression of illness in fatal and nonfatal infections in nosocomial outbreaks with mers-cov in the middle east does not follow the typical pattern of south korea infections [ ] . in the middle east, the median time from onset to confirmation in fatal cases ( days) was clearly longer than in nonfatal cases ( days). in south korea, however, there was no difference in the median time between fatal and nonfatal cases. this is consistent with other retrospective studies of mers virus infections [ , , ] . furthermore, the time between suspected symptom onset and laboratory confirmation ( . days) in the fatal clusters was also slightly longer than the overall average [ ] . in particular, this finding indicated that delayed confirmation is a high-risk factor for human nosocomial outbreaks with mers-cov in the middle east. in conclusion, the overall cfr for nosocomial outbreaks in the middle east was much higher than in south korea. however, the mortality risk factors for mers infections in the middle east were similar to those identified for nosocomial outbreaks in south korea. older age, underlying diseases and delayed confirmation were the major risk factors for fatal outcome in human nosocomial outbreaks. in contrast, person-to-person transmission was associated with a good outcome for secondary cases during nosocomial outbreaks. interestingly, gender, exposure history and median days were not indicators of death with mers nosocomial outbreaks. the severity of nosocomial outbreaks and the risk of fatal infection in hcp were significantly lower than the overall rate in the middle east and south korea. nosocomial outbreaks of mers-cov infection are associated with knowledge deficits, unrecognized disease, insufficient infection control measures, poor compliance, and an overwhelming number of patient cases [ , , , , ] . therefore, early and rapid detection of suspected cases, especially in older people and hcp, along with appropriate infection control practices, education and timely preparedness, are important strategies to reduce nosocomial transmission and to improve the clinical outcome in health settings in the future [ , , , ] . hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus transmission in extended family epidemiological, demographic, and clinical 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corticosteroids critical role of nosocomial transmission in the toronto sars outbreak fatality risks for nosocomial mers outbreaks middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome acknowledgements we thank the reporting countries with the confirmed mers cases. we appreciate their assistance with field investigations, administration and data collection and sending the data to who. conflict of interest none declared. key: cord- -uadfehr authors: zhang, x. w.; yap, y. l.; danchin, a. title: testing the hypothesis of a recombinant origin of the sars-associated coronavirus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: uadfehr the origin of severe acute respiratory syndrome-associated corona-virus (sars-cov) is still a matter of speculation, although more than one year has passed since the onset of the sars outbreak. in this study, we implemented a -step strategy to test the intriguing hypothesis that sars-cov might have been derived from a recombinant virus. first, we blasted the whole sars-cov genome against a virus database to search viruses of interest. second, we employed recombination detection techniques well documented in successfully detecting recombination events to explore the presence of recombination in sars-cov genome. finally, we conducted phylogenetic analyses to further explore whether recombination has indeed occurred in the course of coronaviruses history predating the emergence of sars-cov. surprisingly, we found that putative recombination regions, located in replicase ab and spike protein, exist between sars-cov and other coronaviruses: porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), bovine coronavirus (bcov), human coronavirus e (hcov), murine hepatitis virus (mhv), and avian infectious bronchitis virus (ibv). thus, our analyses substantiate the presence of recombination events in history that led to the sars-cov genome. like the other coronaviruses used in the analysis, sars-cov is also a mosaic structure. sars, a new disease characterized by high fever, malaise, rigor, headache and non-productive cough, has spread to over countries with around % of mortality rate on average. sequence analysis of sars coronavirus (sars-cov) [ , ] showed that it is a novel coronavirus [ ] . anand et al. [ ] reported a three-dimensional model of sars-cov main proteinase and suggested that there are a number of methods and software packages that have been developed for detection of recombination events in dna sequences. the performance of these methods has been extensively evaluated and compared on simulated and real data [ , ] . in the present study we applied these methods to rna viruses. sars-cov and other coronavirus genomes (sars-cov, ibv, bcov, hcov, mhv, pedv, tgev) were first aligned using clustalw [ ] . sites with gaps were removed and a -nt alignment was generated. subsequently, seven methods were employed to detect the occurrence of recombination (see corresponding reference in parenthesis for details of each method): bootscan [ ] , geneconv [ ] , dss (difference of sums of squares) [ ] , hmm (hidden markov model) [ ] , maxchi (maximum chi-square method) [ ] , pdm (probabilistic divergence measures) [ ] , rdp (recombination detection program) [ ] . bootscan, maxchi and rdp are implemented in rdp software package, http://web.uct.ac.za/depts/microbiology/microdescription.htm. geneconv is implemented in the program, http://www.math.wustl.edu/∼sawyer/geneconv/. dss, hmm and pdm are implemented in topali software package, http://www.bioss.sari.ac.uk/software.html. basically default parameter settings were used in all the programs, except the following values: gscale = (geneconv), internal and external references (rdp), window size = and step = (dss, hmm and pdm). after potential recombination events were identified by at least methods above, separate neighbor joining trees were constructed for each putative recombination region to better evaluate the evidence for conflicting evolutionary histories of different sequence regions. all trees were produced with topali mentioned above. table summarizes the results of bootscan analysis with % bootstrap support and significant p-value (< . for uncorrected and mc corrected pvalue). two regions ( - and - , position in alignment) are identified as putative recombination regions and all coronaviruses are potential parents with sars-cov as potential daughter. geneconv detected putative recombination events occurred in a wide range of positions - (in alignment) at a significant level p < . for two p-values: simulated p-value (based on , permutations) and blastlike bc ka p-value (table ). all coronaviruses are potential parents with sars-cov as potential daughter. maxchi identified putative recombination events (table , possible misidentification events are not retained). most of the breakpoints are significant at about . level; the position located in alignment spans from to , but some beginning or ending breakpoints are not determined. similarly, coronaviruses are potential parents with sars-cov as potential daughter. rdp revealed that putative recombination events occur in the domain of alignment - (table ) , with the uncorrected and mc corrected pvalue at less than . and . respectively. in this case, coronaviruses (ibv, bcov, mhv and pedv) are potential parents with sars-cov as potential daughter. figure shows the dss profiles of putative breakpoints between sars-cov and other coronaviruses (dotted line indicates the percentile under the null hypothesis of no recombination): sars-cov, ibv, bcov and mhv (fig. a) , sars-cov, mhv, pedv and tgev (fig. b) , sars-cov, ibv, hcov and tgev (fig. c ). there are about different breakpoints (significant peaks): and (fig. a) , and (fig. b) , , and (fig. c) . hmm plots for sars-cov, ibv, bcov and hcov (fig. ) revealed that the putative breakpoints are at about position and . there is a clear transition from state (sars-cov grouped with ibv) (fig. a) into state (sars-cov grouped with hcov) (fig. c) . the region between and is noisy, and at this moment no information can be provided by hmm. figure shows the results of pdm analysis performed on sars-cov and other coronaviruses (dotted line indicates the % critical region for the null (fig. c, d) , , , , and (fig. e, f) . posada [ ] suggested that one should not rely too much on a single method for recombination detection. here we consider the regions identified by at least methods as putative recombination regions. the results are summarized in table . seven putative recombination regions span a range of positions in sars-cov phylogenetic trees constructed by using putative recombination regions and nonrecombination regions identified by above techniques are shown in figure . the left panels stand for non-recombination regions and the right panels for recombination regions. we compared each row of figures and found that the phylogenetic tree in the left panel (non-recombination region) had very different topology when compared to the phylogenetic tree in the right panel (recombination region), which indicates that recombination has occurred. for example, in fig. a , coronaviruses are divided into groups: group for tgev, hcov and pedv, group for bcov and mhv, group for ibv, and group for sars-cov, consistent with marra et al. [ ] ; while in fig. b, coronaviruses are divided and sars-cov, suggests that sars-cov is most closely related to bcov and mhv, which is consistent with a recent report [ ] . at the same time, sars-cov is also most closely related to tgev (fig. d) and ibv (fig. f) . thus, phylogenetic analysis substantiates the presence of recombination events in the history that led to the sars-cov genome. in this study, seven recombination detection methods and phylogenetic analyses were performed on sars-cov and the six coronaviruses identified by blast (ibv, bcov, hcov, mhv, pedv and tgev). these techniques successfully identified recombination events in bacteria and viruses [ , , , , , ] . our analysis concurred to suggest the occurrence of recombination events between ancestors of sars-cov and these coronaviruses. indeed, pairwise alignment showed that many segments of high homology with ibv, bcov, hcov, mhv, pedv and tgev do exist in sars-cov genome, table exhibits the segments with length > nt and identiy > %, and fig. shows the mosaic structure of the region - in sars-cov genome based on the segments with length > and identity > %. of course, the other coronaviruses used in the analysis are also mosaic structures, for more sequence similarities exist among them than with sars-cov. it is noted that all the sequence comparisons in this study are based on nucleotide sequences. while the protein sequences in sars-cov are largely different from those in the known three groups of coronavirus [ ] , such as, for s protein, the identity is: . % for sars-cov and bcov, . % for sars-cov and hcov, . % for sars-cov and ibv, . % for sars-cov and mhv, . % for sars-cov and pedv, . % for sars-cov and tgev. although sars-cov is close to bcov, mhv, tgev and ibv, the corresponding protein, replicase a, is still different: with identity . % for sars-cov and bcov, . % for sars-cov and ibv, . % for sars-cov and mhv, . % for sars-cov and tgev. naturally, we should take into account the role of convergent evolution, which would bear its mark on the viral genome. the recombination events that we witnessed in sars-cov are present in six different viruses, suggesting sequential horizontal transfers and progressive adaptation to new hosts cells or animals. indeed because viruses need both receptors to permeate host cells and resist the immune response of the host, their outer layer proteins are submitted to an extremely strong selection pressure that may restrict considerably the possible variations of the corresponding proteins (and accordingly of the corresponding genome pieces of sequences). it is nevertheless remarkable that, despite the inclusion of all possible types of viruses in our sample set (as well as shuffled genomes from the viruses we have identified as relevant) we find a more or less single category of viruses as similar to sars-cov. this suggests that even if the contribution of convergent evolution is important, this happened on a more or less common phylogenetic background, suggesting several steps of recombination followed by fine adaptation. in this context, we would like to suggest that ancestors of pedv, mhv or both are the most plausible origin of sars-cov. guan et al. [ ] based on phylogenetic techniques and bootscan recombination analysis stavrinides and guttman [ ] indicated that the replicase of sars-cov was a mammalian-like origin, the m and n proteins have an avian-like origin, and the s protein has a mammalian-avian mosaic origin. while in the present study we used phylogenetic analysis and recombination detection methods, including the powerful methods of maxchi and geneconv among methods studied (simplot (bootscan), geneconv, homoplasy test, pist, maxchi, chimaera, phypro, plato, rdp, recpars, reticulate, runs test, sneath test, triple) [ , ] , to conduct whole genomewide recombination analysis. we identified seven putative recombination regions, which encompass, in terms of proteins involved, replicase a, replicase b and the spike glycoprotein. stavrinides and guttman [ ] primarily inferred the occurrence of recombination qualitatively, but did not identify the precise recombination region in the protein involved (the s protein is an exception, they identified a recombination region in s protein, located between nucleotides and of the s protein, i.e. between nucleotides and of the sars-cov genome, basically covered by the last recombination region for s protein (table ) ). most importantly, each of our recombination regions is identified by at least methods, because one should not rely too much on a single method, as suggested in [ ] . in general, we believe two studies lead to the overall conclusion: the evolution of sars-cov has involved recombination. the recombination event in the replicase is related to the fact that the rna polymerase of coronaviruses utilize a discontinuous transcription mechanism to synthesize mrnas. the viral polymerase must jump between different rna templates regularly during positive-or negative-strand rna synthesis and depending on the rejoining sites, the resultant rna recombination will be either homologous or nonhomologous. this is the copy-choice model of recombination in rna viruses [ , , , ] . the recombination event in s protein is certainly important since this allows the virus to alter surface antigenicity and escape immunesurveillance in the animals, thus adapting to a human host. the existence of sars-cov-like viruses ( . % homology to human sars-cov) in several wild animals in a live animal market in guangdong [ ] indicated that interspecies transmission among the human and animal sars-cov-like viruses had occurred. the mutation analysis of sequence variations among these isolates will help identify the genetic signature of sars virus strains when a sufficient amount of sequence data is available. the very fact that several species of animals are affected does not allow one to trace directly the origin of the virus as endemic in one of these species, but, rather, might be indicative that animals and men might have been contaminated by a virus from a common origin, presumably located in animal food present in local markets in the guangdong province. investigating a wide variety of animal coronaviruses, especially in relation to rodents, birds, snakes and farm animals, would be interesting with regard to the origin of the sars-cov that caused disease in humans. finally, a challenging question arises. what is the molecular basis of recombination in sars-cov? many requirements are needed for recombination to occur: ( ) two coronaviruses can infect a host simultaneously and continue to replicate without interference with each other; ( ) sufficient nucleotide identity between these genomes is essential for genome-switching to occur during rna replication; ( ) the proteins arising from recombination must be functional; ( ) the recombinant virus must have some selective advantage for its survival. that is, the recombination that creates a successful "new" coronavirus is probably a rare event. so, we must stress that the potential recombination events in sars-cov, identified in the present study, are most likely "old" events, which may 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through sequence weighting, positions-specific gap penalties and weight matrix choice regulation of transcription of coronaviruses recombination in sars-cov flood of sequence data yields clues but few answers evidence of natural recombination within the s gene of infectious bronchitis virus evolutionary implications of genetic variations in the s gene of infectious bronchitis virus experimental confirmation of recombination upstream of the s hypervariable region of infectious bronchitis virus widespread intra-serotype recombination in natural populations of dengue virus author's address: dr we wish to thank the hong kong innovation and technology fund for supporting the present research. ending in length identity match percent source sars sars (%) mhv tgev hcov bcov ibv hcov hcov bcov ibv mhv hcov tgev pedv bcov ibv bcov ibv mhv pedv hcov tgev mhv hcov mhv ibv tgev pedv hcov pedv tgev hcov ibv hcov mhv ibv bcov mhv key: cord- -imx a authors: christianson, k. k.; ingersoll, j. d.; landon, r. m.; pfeiffer, n. e.; gerber, j. d. title: characterization of a temperature sensitive feline infectious peritonitis coronavirus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: imx a the characteristics of a temperature sensitive feline infectious peritonitis virus (ts-fipv) were examined. ts-fipv, unlike its parent strain, df wild type fipv (wt-fipv), propagated at °c (permissive temperature) but not at °c (nonpermissive temperature). this temperature preference of ts-fipv was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures ( – °c) prevail. viral structural proteins and rna were synthesized at °c but some undefined maturational defect prevented the formation of infectious ts-fipv at its nonpermissive temperature. ts-fipv was more thermolabile than wt-fipv which indicated alterations in the structural proteins of ts-fipv, and a difference in the envelope protein of the two viruses was revealed by western blot analysis. plaque assay characterization showed that ts-fipv produced small plaques in comparison to the large plaques of wt-fipv. these unique characteristics possessed by ts-fipv may account for its nonvirulent nature and ability to stimulate protective immune responses in cats. feline infectious peritonitis (fip) is a complex and fatal disease of cats caused by infection with feline infectious peritonitis virus (fipv). previous attempts to consistently protect cats by immunization with other antigenically related coronaviruses such as porcine transmissible gastroenteritis virus (tgev) [ ] , canine coronavirus (cev) [ ] , and human coronavirus [ ] have been unsuccessful. inconsistent protection was found when cats were given a sublethal dose of virulent fipv and cats vaccinated with an avirulent fipv were more easily infected than were nonvaccinated cats [ ] . recently, it has been demonstrated that an intranasally (in) administered temperature sensitive fip¥ (ts-fipv) vaccine is efficacious and safe upon fipv challenge [ ] . the pathogenesis of f i p is complicated and not fully understood. evidence indicates that fip is an immune-mediated disease [ ] . the virus has been shown to replicate initially in the upper respiratory tract and small intestine [ ] . macrophages, the primary f i p v target cell, may then cross the mucosal barrier and spread virus throughout the cat [ , , ] . furthermore, a correlation between fipv virulence in vivo and ability to infect macrophages in vitro has been observed [ ] . it has been suggested that a strong cell-mediated immune response to f i p v may be more important than humoral immunity in protecting cats from this disease [ , ] . however, the role of local i m m u n e responses in the upper respiratory tract and intestinal tract has not been carefully evaluated and may represent an important i m m u n e defense mechanism against fip. ts-fipv was developed by serial passages at a reduced temperature, followed by ultraviolet irradiation. the selected virus will propagate at its permissive temperature ( °c) but not at its nonpermissive temperature ( °c). temperature sensitivity was accompanied by the appearance of various characteristics that distinguish ts-fipv from its virulent parent strain. the purpose of this report is to present these distinguishing characteristics which include plaque size, temperature stability, ability to synthesize r n a , expression of structural proteins and temperature dependent replication in vitro and in vivo. norden laboratories feline kidney (nlfk) cells were used from passage to . cells were propagated in basal medium eagle (bme) supplemented with % fetal bovine serum (fbs) and ram hepes buffer. the df wild type fip virus (wt-fipv) was originally isolated from a cat liver explant. after several passages of tissue homogenates in specific pathogen free (spf) cats, the virus was adapted to nlfk cells by cocultivation with infected primary spleen cells. the df wt-fipv strain was grown on nlfk cells at °c for passages i through and then passed at °c up to passage . the virus collected at passage was ultraviolet irradiated ( cm distance with a westinghouse - lamp, v, cycles, . amps) for min. the virus was plaque purified and designated ts-fipv. passage of the plaque purified ts-fipv was used in the characterization studies. the plaque assay was done with confluent cell monolayers inoculated with . ml of a tenfold virus dilution. after adsorption for h at °c or °c, the cells were overlaid with ml of . % carboxymethylcellulose in medium. monolayers were fixed and stained with a solution of % formalin, % crystal violet and % ethanol h postinfection. viral growth curves were conducted to compare the growth of ts-fipv and wt-fipv at both °c and °c. ts-fipv and wt-fipv were inoculated onto confluent cell monolayers at a multiplicity of infection (m.o.i.) of approximately . . at predetermined times over h, viral fluids were aseptically removed, stored at - °c, and titrated for infectivity at the completion of the growth curve. the tcids assay for virus infectivity was done in -well microtiter plates by infection of wells each with gl of a tenfold virus dilution. ts-fipv was titered at °c and wt-fipv was titered at °c. wells were examined for cytopathic effects (cpe) on day and the method of reed and muench [ ] was used to calculate the tcids . thermolability studies were performed at °c with wt-fipv and ts-fipv. each virus was diluted : in serum-free bme. at intervals between and m in, samples were removed, immediately cooled in an ice bath and titrated for residual infectivity. nlfk cells were used to determine ts-fipv virus-specific rna synthesis. cell monolayers were infected at an m.o.i, of approximately for h at °c. after removal of the inoculum, ml of bme containing % dialyzed fbs, ~g/ml actinomycin d and txci [ - h]uridine was added to duplicate cultures. following incubation for h, the radiolabeled cellular rna was extracted according to miller et al. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the rna pellets were solubilized in readi-protein (beckman, fullerton, ca) and counted in a liquid scintillation counter (model ls , beckman instruments, fullerton, ca). a parallel set of cultures without radiolabel was used to monitor infectivity. the unlabeled cells and medium collected at h were titrated after one freeze-thaw cycle. spleen cells from balb/c mice immunized with either ccv or wt-fipv were fused with ns /ag mouse plasmacytoma cells (american type cell culture, rockville, md) using standard procedures [ ] . hybridoma culture fluids were screened for specific antibody production by elisa and western blot. selected hybridomas were cloned twice by limiting dilution and ascites was produced by intraperitoneal injection of × hybridoma cells into pristane-treated balb/c mice. the monoclonal antibodies chosen for use in this study were broadly cross-reactive against ccv, fipv, and feline enteric coronavirus (fecv). three antibodies designated mab-p, mab-n, and mab-e were selected based on their specificities for the peplomer protein (mab-p), nucleocapsid protein (mab-n) and envelope protein (mab-e). mab-p and mab-n were obtained from a fusion performed with a ccv-immunized mouse and mab-e originated from a mouse immunized with wt-fipv. all three antibodies were used as the primary antibody in the western blot and indirect immunofluorescence assays (ifa) (see below). the structural proteins of wt-fipv grown at °c and ts-fipv grown at °c were examined by sds-page and western blot. sds-page was performed by a modified laemmli system [ ] . after electrophoresis, the proteins were blotted to immobilon pvdf transfer membrane (millipore, bedford, ma) by the procedure of towbin et al. [ ] . following transfer, the blot was blocked with % nonfat dried milk in pbs, incubated with mab-p, mab-n, or mab-e and then reacted with alkaline phosphatase-conjugated goat anti-mouse igg (h + l) (kirkegaard and perry, gaithersburg, md). ts-fipv proteins in supernatants from cells infected at °c and °c were examined by western blot. nlfk monolayers were infected at an m.o.i, of approximately or for k . k . christianson et al. h at °c or °c. the inocula were removed, the cultures were washed twice with bme and finally ml of bme was added. at intervals between and h post-adsorption, the culture medium was removed and centrifuged (t , x g) for min. the supernatants were frozen at - °c until western blot analysis. wt-fipv and ts-fipv intracellular and surface antigens expressed at °c and °c were examined by an indirect ifa [ ] . cell monolayers were infected with wt-fipv and ts-fipv at an m.o.i, of approximately . . at the appropriate time point the cells were rinsed with pbs and then fixed in acetone for min at - °c. the slides were then exposed to mab-p, mab-n, or mab-e for rain at °c. following incubation, the slides were rinsed in pbs and then exposed to fluorescein-conjugated goat anti-mouse igg (h + l) (kirkegaard and perry, gaithersburg, md) for rain at °c. detection of viral surface antigens by indirect ifa was done as described for detection of intracetlular antigens except the monolayers were not fixed. the cells were observed and photographed with a zeiss axioskop photomicroscope equipped with a zeiss x objective. in vivo fate of the virus spf cats (liberty labs, liberty corner, nj), to months old, were used in this study. three cats were vaccinated in with ml of ts-fipv ( . tcids /ml ). at , , and days after inoculation one ts-fipv vaccinated cat was sacrificed. one cat was infected orally with wt-fipv ( . tcids /ml ) and sacrificed on day . after sacrifice, tissues were aseptically removed from the cats and frozen at - °c. tissues were then thawed and pulverized with a manually operated tissue grinder to make a % to % (w/v) suspension using bme with antibiotics as a diluent. the suspensions were clarified by centrifugation ( , x g) for min and . ml was then added to the cell monolayers for h at °c for ts-fipv and °c for wt-fipv. after incubation the tissue suspensions were removed and the monolayers were washed once with bme followed by the addition of ml bme. the plates were incubated at the optimal temperature for each virus and were observed for viral cpe daily for days following inoculation. a standard virus neutralization test [ ] was done with mab-p on the virus positive tissue homogenates to confirm the presence of coronavirus. cat tissues were examined by direct ifa for viral antigen. the effect o f incubation temperature on the in vitro growth o f t s -f i p v is shown in table . t s -f i p v h a d optimal infectivity when p r o p a g a t e d and titrated at °c. the cpe produced at °c was characterized by formation of syncytia. when grown at °c, ts-fipv showed infectivity initially when titrated at °c. however, after days at °c cpe was observed on less than ten percent of the monolayer and all plaques that formed were healed within days. when grown at °c, ts-fipv had no detectable infectivity when assayed at °c, but showed minimal infectivity when assayed at °c. the growth curves (fig. ) as can be seen in fig. , ts-fipv was rapidly inactivated at °c. ts-fipv infectivity decreased by five logs in min. wt-fipv was more stable at °c than ts-fipv since only two logs of infectious virus were inactivated within min. the synthesis of ts-fip virus-specific rna, as measured by h-uridine incorporation, was compared at permissive and nonpermissive temperatures. viral rna synthesis at °c and °c was similar h following infection, indicating that normal viral entry and initial synthesis occurred at the nonpermissive temperature. the h-uridine incorporation at °c was , cpm and at °c was , cpm. however, a difference in the number of mature, infectious virions was apparent by h post-infection. virus grown for h at the permissive temperature had a titer of . × tcids /ml, while virus grown at the nonpermissive temperature had a titer of only . × tcids /ml. thus, at the nonpermissive temperature, it appears that early viral rna synthesis occurred without a concomitant virus maturation process. the structural proteins of wt-fipv and ts-fipv grown at °c and °c, respectively, were compared by western blot using coronavirus-specific monoclonal antibodies. the western blots showed that wt-fipv and ts-fipv had structural protein profiles characteristic of that reported for other coronaviruses [ ] . the peplomer protein band had a molecular weight of kda and the apparent molecular weight of nucleocapsid was kda for both viruses (fig. ) . conspicuous differences appeared in the pattern of blotted envelope proteins for the wild type and temperature sensitive viruses. the wt-fipv envelope (fig. , e, a) , whereas the envelope protein of ts-fipv produced several protein bands from kda to kda (fig. , e, b) . the kda component of ts-fipv was present but not at the same intensity as it was for wt-fipv. the differences in molecular weight of the envelope protein of the two viruses were confirmed using immune cat sera. immune sera from both ts-fipv vaccinated cats and wt-fipv challenged cats showed the same differences in the envelope protein of the two viruses as did the monoclonal antibody (data not shown). thus, the differences in the molecular weights of the envelope polypeptides were not due to differences in reactivity to the monoclonal antibody. there was not a difference in the molecular weights of the envelope polypeptides from ts-fipv grown at °c or °c (data not shown). the specificity of the monoclonal antibodies used for antigen detection is demonstrated by the lack of reactivity with nlfk cell extracts (fig. , c) . the culture supernatant from ts-fipv infected cells was monitored for the appearance of structural proteins at both the permissive and nonpermissive temperatures. ts-fipv grown at °c for , , , or h had all three structural proteins detected in the culture supernatant (fig. a) . when ts-fipv was grown at °c for similar lengths of time, only nucleocapsid was found (fig. b) . nucleocapsid may be the only protein released at °c or the less abundant peplomer and envelope proteins were not detected. figure also shows that the nucleocapsid protein released at °c may be associated with infectious rna or a few viral particles were released at °c because these culture supernatants were infective at °c. however, at h the infectivity of intracellular synthesis of ts-fipv and wt-fipv proteins was compared at °c and °c. synthesis was monitored by ifa using coronavirus-specific monoclonal antibodies on acetone-fixed, infected cells. all three structural proteins of ts-fipv were detected in the cells by ifa at both the permissive and nonpermissive temperatures ( table ). the ts-fipv proteins appeared at both temperatures within h post-infection. similar results were observed with wt-fipv infected cells at °c. when incubated at °c, wt-fipv proteins appeared somewhat later. nucleocapsid was visible by h while peplomer and envelope proteins were apparent by h. examination of the surface of infected cells by immunofluorescence using a peplomer monoclonat antibody indicated that ts-fipv was present on the surface of living cells by h post-infection at °c but not °c (table ). small areas of envelope surface fluorescence were observed for ts-fipv by h at its permissive temperature only. after h at °c peplomer and envelope antigens were detected on the surface of wt-fipv infected cells, but at °c only slight peplomer fluorescence was found after h. nucleocapsid was not detected on the surface of cells infected with either virus. in order to determine if the temperature sensitive characteristics of ts-fipv observed in vitro were reflected by growth of the virus in vivo, cats were inoculated with either ts-fipv or wt-fipv. at predetermined times postinoculation, cats were sacrificed and tissues were examined for the presence of virus. evidence of ts-fipv replication in the upper respiratory tract was found by virus isolation and immunofluorescence (table ) . virus was isolated from the cervical lymph node, tonsil, trachea and turbinate at , , or days postvaccination. ts-fipv antigen was identified by direct ifa in the mandibular lymph node and the tonsil. in contrast, by days after oral infection with wt-fipv, the virus had disseminated throughout the cat. wt-fipv was isolated from four different lymph nodes (cervical, mandibular, mediastinal and ruesenteric), the oral/nasal/pharyngeal area, as well as from the thymus and spleen. all of these tissues except the thymus were positive for viral antigen by direct ifa. the differences in the characteristics of ts-fipv and wt-fipv may account for the protective immunity afforded by ts-fipv [ ] and the onset of disease by wt-fipv. ts-fipv and wt-fipv differ in temperature specificity and stability, plaque size, and structural protein expression. in addition, ts-fipv and wt-fipv disseminate differently in the cat. although the lesions responsible for the temperature sensitive defect have not been located, the characteristics of ts-fipv in relation to its parent strain suggest a maturation defect. ts-fipv growth is impaired at the optimal temperature ( °c) for wt-fipv but ts-fipv replicates normally at its permissive temperature ( °c). this temperature preference allows ts-fipv to grow in the upper respiratory tract of cats but retards systemic growth in cats where the temperature is °c. replication of ts-fipv in the upper respiratory tract may stimulate mucosal responses that may be required to protect cats against fipv challenge. mucosal immunity provided by ts-fipv may be important in stopping the primary infection of fipv since stoddart et al. [ ] has shown that fipv administered orally replicated initially in the tonsil and small intestine. also, the temperature preference of ts-fipv may prevent a vaccine-induced sensitization of the cat. this contrasts with a previous study by pedersen and black [ ] in which vaccination with a modified-live fipv not only failed to protect cats against disease, but appeared to sensitize cats so they were more susceptible to fipv challenge than were nonvaccinated cats. wt-fipv produced larger plaques in nlfk cells than did ts-fipv. tupper et al. demonstrated that two virulent fipv strains ( - and nor , a plaque purified df wt-fipv) produced larger plaques than the nonvirulent fecv strain - [ ] . mckeirnan and coworkers also documented this difference in plaquing profiles between these same feline coronavirus strains [ ] . it appears that avirulent feline coronaviruses produce small plaques, whereas virulent fipv produces large plaques. ts-fipv rna synthesis occurred normally at °c for approximately h, even though viral growth did not proceed normally. the absence of intact virion production in the presence of rna synthesis at °c suggests a defect in the maturation and assembly of the virion which has been shown in other temperature sensitive viruses [ , , ] . the detection of ts-fipv structural proteins by indirect ifa in nlfk cells at °c without concomitant virus production also indicates a maturation defect at °c. surface expression of ts-fipv and wt-fipv peplomer and envelope proteins but not nucleocapsid at the optimal temperature for each virus resembles the situation in fipv-infected macrophage-like cells [ ] . the expression of viral antigen on the cell surface may be important in the pathogenesis of fipv. the absence of ts-fipv surface antigen at °c may account for the lack of sensitization in ts-fipv vaccinated cats. a difference was evident in the molecular weights of the envelope polypep-tides of ts-fipv and wt-fipv as shown by western blotting. the low molecular weight ( kda) component was detected to a lesser degree for ts-fipv than wt-fipv. this same kind of difference was observed with two virulent strains of fipv; ucd did not have the low molecular weight component that was present in the dahlberg strain of fipv i- ]. these observed differences in the envelope protein may be due to differences in glycosylation. further investigation by two-dimensional gels is needed to clearly differentiate the envelope proteins of ts-fipv and wt-fipv. different characteristics are apparent between ts-fipv and its parent strain wt-fipv. the importance of these ts-fipv characteristics in relation to the protective response of the virus in cats is under investigation. experimental inoculation of cats with human coronavirus e and subsequent challenge with feline infectious peritonitis virus experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus isolation and characterization of conditionamethal mutants of sindbis virus physiological characterization of heatdefective (temperature-sensitive) poliovirus mutants: preliminary classification protection against feline infectious peritonitis (fip) by a temperature sensitive-fip virus vaccine inoculated intranasally virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus comparison of serologic assays for measurement of antibody response to coronavirus in cats expression of feline infectious peritonitis coronavirus antigens on the surface of feline macrpohage-like cells cleavage of structural proteins during the assembly of the head of bacteriophage t feline infectious peritonitis (fip)--the present state of knowledge comparative properties of feline coronaviruses in vitro temperature sensitive coronavirus characterization kinetics of accumulation and processing of simian virus rna in xenopus laevis oocytes injected with simian virus dna immunoglobulin-producing hybrid cell lines attempted immunization ofcats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd , and fipv-ucd a simple method for estimating fifty percent endpoints the biology of coronaviruses intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus the sites of early viral replication in feline infectious peritonitis semliki forest temperature-sensitive mutants: isolation and characterization electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose: procedure and some applications antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus pathogenesis of feline infectious peritonitis: nature and development of viremia pathogenesis of feline infectious peritonitis: pathogenic changes and immunofluorescence cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses the authors thank dr. b. huseman for performing the necropsies and b. suiter and dr. m. mellencamp for monoclonal antibody production. the critical review of this manuscript by dr. w. beckenhauer, s. christianson, dr. k. haffer and dr. a. torres was greatly appreciated. received july , key: cord- -cndq aqb authors: xue, chunyi; wang, wei; liu, qiliang; miao, zhongwei; liu, kang; shen, huifang; lv, lishan; li, xiaoming; chen, xiaochun; cao, yongchang title: chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus gp protein and the influenza virus ha and m proteins date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: cndq aqb both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. these diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. in this study, we have developed a chimeric virus-like particle (vlp) vaccine candidate for porcine reproductive and respiratory syndrome virus (prrsv) and h n influenza virus and investigated its immunogenicity in mice. the ha and m proteins from the h n influenza virus and the prrsv gp protein fused to the cytoplasmic and transmembrane domains of the na protein were both incorporated into the chimeric vlps. analysis of the immune responses showed that the chimeric vlps elicited serum antibodies specific for both prrsv gp and the h n ha protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. taken together, the results suggested that the chimeric vlp vaccine represents a potential strategy for the development of a safe and effective vaccine to control prrsv and h n influenza virus. influenza a virus is a segmented, negative-stranded rna virus belonging to the family orthomyxoviridae. among the large variety of species that influenza a viruses infect naturally, swine influenza virus (siv) causes an acute, highly contagious respiratory disease in swine. epithelial cells in the swine respiratory tract have receptors for both avian and mammalian influenza viruses [ ] . therefore, pigs might serve as ''mixing vessels'' for the generation of new reassortant strains with pandemic capacity. three predominant subtypes are prevalent in different countries: h n , h n , and h n . porcine reproductive and respiratory syndrome virus (prrsv), the causative agent of porcine reproductive and respiratory syndrome (prrs), is a member of the family arteriviridae in the order nidovirales [ ] . this virus causes one of the most economically important infectious diseases for the swine industry worldwide [ ] . prrs is predominantly characterized by reproductive failure in breeding swine, pre-weaning mortality, and respiratory disorders in pigs of all ages [ , , ] . vaccination has been an effective way to reduce the incidence of diseases resulting from influenza virus and prrsv infections. compared to conventional vaccines such as killed vaccine and attenuated vaccine, virus-like particles (vlps) have been demonstrated to be a promising alternative candidate [ , , ] ,. as a new form of vaccine candidate, the non-infectious nature of vlps and their lack of viral genomic material are attractive safety features that may be suitable for a variety of viruses [ , , , , , ] . both b cell-mediated antibody and specific t-cellmediated cellular responses were elicited by vlps. vlps not only mimic the overall structure of the virion, but they also present conformational epitopes of surface proteins, which can be readily recognized and processed by antigenpresenting cells [ , , , , ] . the protective effects of vlps have been demonstrated in clinical and preclinical trials [ , , , , ] . an important advance would be the development of new vlps with an enhanced breadth of immunity, which could potentially be used to prevent infection by prrsv and siv. in a previous study, we expressed the gp proteins of prrsv on the surface of chimeric vlps, which elicited a humoral and cellular immune response and a neutralization antibody response to prrsv [ , ] . based on this platform, we hope to generate chimeric vlps for protection against both prrs and influenza. in this study, we chose the h n influenza virus as the basis for vlp production. as expected, the fusion protein na/gp in combination with ha and m effectively formed chimeric vlps. next, we demonstrated that the chimeric vlps induced a potent immune response to both prrsv and h n siv in a balb/c mice model. hence, the results suggested that the chimeric vlp vaccine is a new vaccine candidate for protection against both prrs and h n influenza. spodoptera frugiperda sf cells were maintained in serumfree sf ii medium (gibco) at °c in spinner flasks at a speed of rpm. the prrsv strain gdkp/ / [ ] was propagated on marc- cells that were maintained in dulbecco's modified eagle's medium (dmem, gibco) supplemented with penicillin-streptomycin and % fetal calf serum at °c and % co . the h n strain of the influenza virus (a/swine/guangdong/ / (h n )) was propagated in mdck cells under the same conditions. t cells were maintained in dmem supplemented with penicillin-streptomycin and % fetal calf serum at °c and % co . the ha, na and m genes of the h n influenza virus (accession numbers fj . , fj . and fj . ), the gp gene of prrsv (accession number gq ), and the na/gp fusion gene were inserted into the pfast-bac-dual vector as described previously [ , ] . the na/gp and m genes were cloned into the same vector (pfast-bac-dual) under the control of different promoters. all of the plasmids were confirmed by dna sequencing to ensure that no additional changes were introduced during the pcr. the recombinant baculoviruses (rbvs) were derived from the transfer plasmids pfast-bac-dual-ha, pfast-bac-dual-gp and pfast-bac-dual-na/gp -m using the bac-to-bac baculovirus expression system. the viruses harvested from the supernatant were subjected to three rounds of plaque purification. sf cells were co-infected with rbvs expressing na/gp -m and ha at different ratios ( . , , , , , and ) and then incubated for h at °c. the sf cells showed a high degree of cytopathology. the culture supernatants were collected and centrifuged at g for min at °c and analyzed by western blot. the expressed influenza virus proteins ha and m were detected with mouse polyclonal sera against the h n influenza virus. the fusion protein na/gp was detected with mouse polyclonal sera against prrsv. horseradish peroxidase (hrp)conjugated goat anti-mouse igg polyclonal antibodies were used as the secondary antibody (ptglab, usa). to produce vlps containing ha, m and na/gp , sf cells were co-infected with rbvs expressing the ha and na/gp -m proteins at ratios of and . after incubation for h at °c, the vlps were harvested and purified using %- %- % (w/v) discontinuous sucrose step density gradient ultracentrifugation at , g for min at °c. the fractions were collected and analyzed for the presence of the ha, m , and na/gp proteins by western blot. the na/gp content of chimeric vlps was quantified by grayscale scanning of an sds-page gel. the hemagglutination activity of the vlps was determined using chicken red blood cells. for electron microscopy, the sucrose-gradient-purified vlps were applied to a carboncoated formvar grid for min. then, the grid was immediately stained with % phosphotungstic acid (ph . ) for s. the stained vlps were observed by transmission electron microscopy (jem- cx-ii, jeolltd, japan). four groups of six-week-old, female, inbred spf balb/c mice (n = ) were housed in microisolator units and allowed free access to food and water. the mice were immunized intramuscularly (i.m.) with lg of the chimeric vlps containing lg na/gp proteins with complete freund's adjuvant (cfa) for the primary immunization (weeks ) or incomplete freund's adjuvant (ifa) for subsequent boosts (weeks and ). the positive control groups were immunized i.m. with % formalininactivated prrsv in which the amount of gp was equal to that of the chimeric vlps or % formalin-inactivated h n influenza virus in which the amount of ha was equal to that of the chimeric vlps. pbs was injected as the negative control. blood samples were collected before immunization on the th, th, and nd day after primary immunization. the experiments were carried out in accordance with the ethical guidelines for animal protection in china and approved by the sun yat-sen university animal ethics committee. all procedures were performed under anesthesia, and all efforts were made to minimize suffering. briefly, -well plates were coated with ll of antigens at a concentration of lg/ml in coating buffer ( . m sodium carbonate, ph . ) at °c overnight. the antigens used as targets were extracts prepared from t cells transfected with pfast-bac-dual-ha (to express ha protein) or pfast-bac-dual-gp (gp protein) [ , ] . the plates were then blocked with pbs containing . % tween- and % bsa at °c for h and incubated with serial dilutions of each sample at °c for h. following thorough washing in pbs containing . % tween- , all of the samples were incubated with horseradish peroxidase (hrp)-labeled goat anti-mouse igg with a stock concentration of mg/ml, : , diluted in pbs containing . % tween- and . % bsa at °c for h. the unbound antibodies were removed, and the wells were thoroughly washed. the substrate , , , -tetramethylbenzidine (tmb, sangon, china) in citrate-phosphate buffer (ph . ) containing . % h o was used for color development. the reaction was terminated with . m h s , and the absorbance was determined at nm using a spectrophotometer (bio-tek elx uv, usa). lymphocytes were isolated from the spleens for cytokine elispot assays. briefly, spleens were carefully rinsed with sterile pbs and depleted of erythrocytes by treatment with ammonium chloride ( . m, ph . ). following thorough washing with pbs, cells were isolated from spleens using mouse lymphocyte separation medium (dakewe, china). the collected cells were centrifuged at g for min at room temperature and then resuspended in lympho-spot serum-free medium rodent (dakewe). cell viability was determined by staining with . % trypan blue (sigma). pre-coated anti-mifn-c or anti-mil- ( lg/ml in coating buffer, bd/pharmingen) -well plates (millipore) were incubated with ml lympho-spot serum-free medium rodent at °c for min and then were incubated with splenocytes isolated from vaccinated mice at /well. splenocytes were stimulated with gp protein or ha protein at a concentration of lg/ml. additional wells of cells were stimulated with pma ( ng/ml), and ionomycin ( ng/ml) or were mock stimulated. the plates were incubated for h at °c with % co . plates were thoroughly washed with pbs containing . % tween- and incubated with biotinylated anti-mifn-c or anti-mil- at °c for . h. then, the plates were washed and incubated with streptavidin conjugated to alkaline phosphatase at °c for . h. following extensive washing, antibody-cytokine-antibody complexes were incubated with stable bcip/nbt chromagen at °c for min. the plates were rinsed with ddh o and air dried at °c for h. the spots were counted using an immunospot elispot reader (bio-reader , bio-sys, germany). the sera were complement-inactivated at °c for min before testing, serially diluted twofold in dmem without serum, and then mixed with prrsv ( tcid ). the mixtures were added to prewashed marc- cells growing in -well tissue culture dishes and incubated at °c with % co for h. the cells were examined to observe the appearance of the cytopathic effects (cpe). the neutralization titers were expressed as the reciprocal of the highest serum dilution that neutralized tcid of prrsv in % of the wells. the hemagglutination inhibition (hi) assay was used to assess the ability of functional ha-specific antibodies to inhibit agglutination of chicken erythrocytes. briefly, sera were treated with receptor-destroying enzyme and serially diluted twofold in v-bottom -well microtiter plates. an equal volume ( ll) of four adjusted hemagglutination units of the inactivated h n strain of influenza virus was added to each well. the plates were covered and incubated at room temperature for min followed by the addition of ll of % red blood cells (rbcs) in pbs. the hi titer was determined as the reciprocal dilution of the last row that contained non-agglutinated rbcs. negative serum controls were included for each plate. the geometric mean hi titers and standard error were calculated within each group. all parameters were recorded for individual mice within all groups. statistical comparisons of the data between groups were carried out using an analysis of variance test (anova). statistical analyses were performed using student's two-tailed test with equal variance. p-values less than . (p \ . ) were considered statistically significant. cells were coinfected with recombinant baculoviruses expressing na/gp -m and ha proteins at different ratios. the culture supernatant was harvested and analyzed by western blot (fig. a and b ). different ratios led to different levels of protein expression. the results suggested that infection with rbvs expressing ha and na/gp -m proteins at a ratio of : was optimal for chimeric vlp production. chimeric vlps were produced and released into the culture supernatant of insect cells coinfected with rbvs expressing ha, m , and na/gp as described. the vlps were purified by sucrose gradient ultracentrifugation and characterized by western blot. the fractions containing vlps could be observed at a sucrose density between - %, and the incorporation of ha, m , and na/gp protein into the vlps was confirmed ( fig. a and b) . electron microscopic examination of negatively stained samples revealed the presence of vlps with a diameter of approximately nm (fig. c) . the ha titer was . the immunogenicity of the chimeric vlps was evaluated in a mouse immunization trial in the presence of freund's adjuvants. ha protein was used as an antigen for h n influenza virus antibody detection. mice vaccinated with the vlps and inactivated virus vaccine formulations elicited serum anti-ha protein antibody responses following the first vaccination that were boosted by the second vaccination (fig. b) . the group in which vlps were infected intramuscularly and the positive control group showed considerable igg titers that were significantly higher than those of the negative control group (p \ . , fig. b ). the differences in igg titers between the vlp group and the positive control was not statistically significant (p [ . ). importantly, the chimeric vlps induced significant igg titers against the prrsv gp protein compared with the negative control group (p \ . , fig. a) , and the difference in igg titers between the vlp group and the positive control group was not statistically significant (p [ . ) which was the same as the immune response to the ha protein. these data show that the fusion of the ectodomain of na did not hinder the function of gp as an antigen. meanwhile, the production of an immune response to the ha protein indicated that these chimeric vlps administered via the intramuscular route could effectively induce a response to two different antigens that was comparable to that induced by the positive control vaccine. the numbers of ifn-c and il- cytokine-secreting cells were determined using specific and sensitive elispot assays. spleens were harvested from vaccinated mice, and lymphocytes were isolated after the last immunization. the cells were stimulated with either gp protein or ha protein. our data showed that significant numbers of ifn-cand il- -secreting cells were induced in the chimeric vlp group or the positive control group, but not in the negative control mice (p \ . ), whether gp protein (fig. a and b) or ha protein (fig. c and d) was used as the stimulus. it is notable that the mice immunized with the chimeric vlps elicited both th -and th -type cellular immune responses. this might be helpful to suppress viral replication and reduce viral infection for both the prrsv and h n influenza virus. because neutralizing activities play an important role in the first line of defense against prrsv infection and the clearance of prrsv, most likely conferring protective immunity, we performed neutralization assays. as shown in fig. , prrsv-specific neutralizing antibodies were detected on the th day after primary immunization, they were elevated on the th day, and they increased to the highest level in the nd day. furthermore, no neutralizing antibodies against prrsv were detectable in the pbs group. the hi results demonstrated that a single intramuscular immunization with the vlp vaccine in the presence of adjuvant elicited a relatively modest hi titer against the inactivated h n influenza virus strain, and the two booster immunizations induced a high hi titer, indicating that the vlps were strongly immunogenic (fig. ) . prrsv and siv coinfect pigs under certain conditions. these two agents associated with mycoplasm hyopneumoniae (mhyo), pasturella multocida, and porcine circovirus type (pcv ) have been reported to constitute the porcine respiratory disease complex (prdc) [ ] . a previous study indicated that siv and prrsv are the primary etiological agents associated with respiratory disease in pigs [ ] . the presence of prrsv between vaccinations reduced the efficacy of the vaccine, but it did not negatively impact either the systemic or local antibody response to either siv vaccination or challenge [ ] . previously, we prepared chimeric vlps composed of the m protein from h n influenza virus and a fusion protein, denoted as na/ gp , containing the cytoplasmic and transmembrane domains of the h n virus na protein and the prrsv gp protein. we demonstrated that immunization of balb/c mice with these chimeric vlps could stimulate a robust immune response [ , ] . based on this result, an important advance would be the development of new vaccines, which could be used to prevent coinfection by prrsv and siv. fig. . serum samples were collected on the th, th, and nd day after primary immunization to determine the neutralization antibody titers. the data represent the mean s.d. for eight mice. the differences in the neutralization antibody titers between the experimental groups were statistically significant (**p \ . ) fig. hemagglutination-inhibition (hi) titers. serum samples were collected on the th, th, and nd day after primary immunization of mice ( per group) vaccinated with chimeric vlps, inactivated h n virus, or pbs. the hi responses were assessed compared with the h n viruses. the differences in hi titers between the experimental groups were statistically significant (**p \ . ) the influenza virus virion is surrounded by a lipid membrane containing two major glycoproteins, ha and na. the ha protein is the most abundant glycoprotein and is responsible for the attachment of the virus to terminal sialic acid residues on host cell receptors and mediating fusion between viral and cellular membranes [ ] . influenza vlps could be obtained by the expression of four viral proteins (ha, na, m , m ) [ ] , three viral proteins (ha, m , na) [ , , , , ] , two viral proteins (ha, m ) [ , , ] , or even ha alone [ ] . recently, it has been demonstrated that influenza vlps can be made from m fusion proteins [ ] . therefore, influenza vlps were chosen as a platform on which prrsv proteins (gp ) can be expressed. a previous report showed that, based on the assembly of newcastle disease (nd) vlps [ ] , it was possible to develop chimeric vlps containing respiratory syncytial virus (rsv) g proteins using nd vlps as a platform [ ] , as well as nd vlps containing rsv g and f proteins [ ] . in our study, we showed that the ha protein could assemble with na/gp and m to form chimeric vlps. compared to our previous results, ha had no influence on the formation of chimeric vlps, but the size and morphology of the chimeric vlps were more similar to those of the natural influenza virus. the ha titer of the chimeric vlps indicated that ha was structurally and functionally incorporated into the particles. the results suggested that it might be feasible to generate chimeric influenza vlps in this way. the immunogenicity of chimeric vlps in mice demonstrated that they were effective for inducing immunity. our data showed that both the inactivated virus and the chimeric vlp vaccine generated comparable amounts of prrsvspecific or h n -influenza-virus-specific antibodies, as measured by elisa. splenocytes proliferated vigorously and produced both ifn-cand il- in response to stimulation with gp or ha antigen. following three immunizations, the vlp vaccine induced hi antibodies against the homologous influenza strain in mice. our findings provide evidence that chimeric vlps can induce protective immunity against h n influenza virus similarly to the killed virus. meanwhile, when we measured the neutralizing antibody titers, significantly elevated neutralizing antibodies to prrsv were achieved in the vlp group as well as with inactivated prrsv (p \ . ). this result demonstrated that the prrsv gp protein co-incorporated with influenza h n na and formed chimeric vlps with ha and m , which could be identified and transferred efficiently as a prrsv antigen. it was reported recently that vlps of the hepatitis b virus core protein containing five mimotopes of infectious bursal disease virus (ibdv) protected chickens against ibdv [ , ] . the use of both genetically engineered norovirus vlps incorporating relevant epitopes from multiple strains and multivalent vaccine formulations increases the breadth of the immune response to diverse variants within a genotype [ ] . in summary, we found that the chimeric vlps induced antibodies against two diseases at the same time, in contrast to the chimeric vlps in our previous study. the data presented in this study showed that intramuscular immunization of mice with these chimeric vlps induced systemic immune responses, including both humoral and cellular immune components. similar to our research, chimeric sars-cov s glycoprotein and influenza m efficiently form vlps that protect mice against challenge with sars-cov [ ] . experimental triple-ha vlps containing ha proteins derived from the h n , h n , and h n viruses were immunogenic and protected ferrets from challenge with all three potentially pandemic viruses. additionally, vlps containing ha subtypes derived from the seasonal h n , h n , and type b influenza viruses protected ferrets from three seasonal influenza viruses [ ] . therefore, it is clear that the chimeric vlps were as effective as the killed viruses. the results showed that the immune responses to these chimeric vlps were similar to those to the vlps composed of na/ gp and m proteins. the less an animal needs to be handled to deliver vaccines, the better. the presence of multiple virus targets in a single vaccine will be a new advantage of the chimeric vlp vaccine. such vaccines could also reduce the burden of vaccine production and administration in comparison with regular vaccination. after this basic step, studies of the protective efficacy of chimeric vlps in a swine model need to be carried out. in summary, in this study, we provide a proof of concept for a chimeric vlp vaccine generating an anti-prrsv and anti-h n -influenza-virus immune response. these chimeric vlps could serve as a promising vaccination strategy to control prrs and h n influenza in swine. although the response to immunization with the chimeric vlps was not more effective than the response to inactivated viruses, the fact that chimeric vlps are not infectious and lack viral genomic material means that they could provide a safer method to prevent viral infection. furthermore, the production of the chimeric vlps is faster and presents a lower risk relative to the traditional method. thus, this approach has greater potential for vaccine development. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design chimeric influenza-virus-like particles containing prrsv gp cross-clade protective immune responses to influenza viruses with h n ha and na elicited by an influenza virus-like particle influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin nidovirales: a new order comprising coronaviridae and arteriviridae using plant cells as influenza vaccine substrates retrospective analysis of etiologic agents associated with respiratory diseases in pigs elicitation of protective immune 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norovirus consensus gii. virus-like particles in monovalent and bivalent vaccine formulations clinical signs and economic losses caused by porcine reproductive and respiratory syndrome virus in a large breeding farm intranasal vaccination with influenza virus-like particles protects mice and ferrets from lethal and h n influenza virus challenge chinese-like strain of porcine epidemic diarrhea virus recombinant h n virus-like particle vaccine elicits protective immunity in ferrets against the pandemic h n influenza virus influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes influenza virus-like particles comprised of the ha, na, and m proteins of h n influenza virus induce protective immune responses in balb/c mice virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus a bivalent influenza vlp vaccine confers complete inhibition of virus replication in lungs a trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets porcine reproductive and respiratory syndrome immunology of the porcine respiratory disease complex production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus gp protein virus-like particles of hepatitis b virus core protein containing five mimotopes of infectious bursal disease virus (ibdv) protect chickens against ibdv fabrication of influenza virus-like particles using m fusion proteins for imaging single viruses and designing vaccines vaccination with coxsackievirus b virus-like particles elicits humoral immune response and protects mice against myocarditis key: cord- -sf lu f authors: hirano, n.; ono, k.; sada, y.; inoue, a.; murakami, t.; takamaru, h. title: replication of rat coronavirus in a rat cell line, lbc date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: sf lu f rat coronavirus readily propagated and induced marked cytopathic effect in a rat cell line, lbc cell culture, which provided a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus. pa~ker et al. ( ) have isolated an agent with characteristic morphology of eoronavirus ( ) from the lung of rats. this rat colony was positive for complement fixing antibody against mouse hepatitis virus (mhv). the agent, designated as rat eoronavirus (rcv), was shown to share common antigen(s) with mhv. by intranasm inoculation with i~cv, a fatal pneumonitis was produced in newborn rats. the growth of rcv has been reported on a primary rat kidney cell culture but not on any established cell lines. because of the lack of the susceptible cell lines for igcv, consistent work with i~cv still remain difficult. this brief communication recommends a rat cell line, lbc, providing propagation of rcv and useful tool for infectivity assay. rcv, strain , was kindly supplied by dr. j. c. parker, microbiological associates, bethesda, maryland, u.s.a. the lbc cell line was established in by dr. k. kai, institute of medical science, university of tokyo, from a spontaneous mammary tumor occurring in a lewis rat by con-ventionm methods. further dermis of this cell line are still under investigation. the lbc cells were grown at °c in eagle's minimum essential medium (mem) containing per cent fetal calf serum (fcs) and kanamyein ( . mg/ml). the fcs concentration was reduced to per cent for maintaining the cells or harvesting the virus. cells grown in ml culture bottles were washed once with dulbeceo's phosphate buffered saline ph . (pbs), and inoculated with . ml of virus material. after virus adsorption at ° c for minutes, the inoculated cultures were given maintenance medium and incubated at ° c. cytopathic effect (cpe) was first detected at hours postinoculation (p. i.). at hours p.i. rounding of cells and formation of syncytia developed in the whole cultures, as observed in a primary rat kidney cell culture by parker et al. ( ) . passages of rcv in the lbc cell monolayers were readily carried out at intervals of days with undiluted culture fluid. after a few passages, cpe became complete within hours p.i. (fig. ) . the culture fluid sampled at ¢ hours p.i. was assayed for infectivity by inoculating into lbc cells prepared in × mm test tubes, showing an infectivity titer of i . per cent tissue culture infective doses (tcids )/ . ml. indirect immunofluorescence and neutralization test were made using anti-rcv rat serum, which was kindly supplied by dr. j. c. t)arker. coverslip cultures of lbc cells were inoculated with the virus, fixed at hours p.i. with cold acetone and subjected to immunofluorescence. the samples were first treated with a : dilution of the rat antiserum in pbs and then with a : dilution of fluorescein isothiocyanate-conjugated anti-rat igg rabbit serum (miles bioehemic~ls, u.s.a.) in pbs, respectively, at ° c for minutes. the virus specific antigen was found abundantly in the cytoplasm of mono-as well as multi-nucleated cells, as shown in fig. . serial -fold dilution of the antiserum in mem were m~xed with an equal volume of virus material ( tcidso/ . ml) and incubated at ° c for minutes. then, . ml of the mixtures were assayed for cpe by inoculation into lbc cells prepared in test tubes. the antiserum was able to neutralize the lbc-passaged virus showing an antibody titer of : . as control, the titer of rat serum derived from rcv-free colony was within : . the supernatant of infected culture fluid was observed by electron microscopy (hitachi h- a) after negative staining with per cent phosphotungstic acid. numerous spherical particles t to nm in diameter with characteristic peptomers were observed as shown in fig. . the lbc cells can be readily grown in vitro without risk of contamination with latent rat viruses from primary cultures, and are very sensitive to i~cv, yielding higher titered viruses compared with the previously described cell system ( ) . the lbc cell culture might be a much more useful tool for rcv propagation and assay. the dbt cell line yielding high-titered mhv ( , ) is not able to support growth of t~cv. the lbc cells can also support growth of simodaeryoadenitis virus of rat, strain ( ), but not of mhv strains (unpublished observation). chaxaeterization of tile virus of sialodaeryoadenitis of rats: a member of the coronavirus group replication and plaque formation of mouse hepatitis virus (mi-iv- ) in mouse cell line dbt culture utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats the authors thank dr. k. kai for kindly supplying the lbc cells. this study was supported by grants-in-aid for scientific research ( ) from ministry of education, science and culture of japan and :naito foundation. key: cord- -vlszl i authors: chen, si; liu, dafei; tian, jin; kang, hongtao; guo, dongchun; jiang, qian; liu, jiasen; li, zhijie; hu, xiaoliang; qu, liandong title: molecular characterization of hlj- , a recombinant canine coronavirus strain from china with an orf abc deletion date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vlszl i canine enteric coronaviruses (ccovs) are important enteric pathogens of dogs. ccovs with different variations are typically pantropic and pathogenic in dogs. in this study, we isolated a ccov, designated hlj- , from a dead -week-old male pekingese with gross lesions and diarrhea. interestingly, sequence analysis suggested that hlj- contained a -nt deletion in orf abc compared with reference ccov isolates, resulting in the loss of portions of orf a and orf c and the complete loss of orf b. phylogenetic analysis based on the s gene showed that hlj- was more closely related to members of the fcov ii cluster than to members of the ccov i or ccov ii cluster. furthermore, recombination analysis suggested that hlj- originated from the recombination of fcov - and ccov a , which were both isolated in the united states. cell tropism experiments suggested that hlj- could effectively replicate in canine macrophages/monocytes and human thp- cells. this is the first report of the isolation of strain hlj- in china, and this virus has biological characteristics that are different from those of other reported ccovs. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. canine coronavirus (ccov) is a member of virus family coronaviridae, order nidovirales. ccov is single-stranded positive-sense rna viruses with a genome that is between and kb in length [ ] . based on antigenic and genetic relationships, ccov consists of two distinct genotypes, ccov-i and ccov-ii [ , ] . ccov was first recognized as an enteric pathogen of dogs in , when it was observed to cause clinical signs including anorexia, lethargy, vomiting, and mild-to-severe diarrhea [ ] . the clinical signs usually lasted - weeks and were followed by recovery, or occasionally by dehydration that led to death, mainly in puppies [ ] . in recent years, researchers have reported an increasing number of cases of virulent ccov infection in dogs and the emergence of ccov variants with recombinant s genes. recombination occurs when different ccov types infect the same host simultaneously [ , , , ] . therefore, ccov has become a cause of concern as an emerging pathogen in dogs. in the present study, we report the emergence and molecular characterization of an, fcov-like recombinant ccov-hlj- , which was isolated from a fecal sample from a dead dog that exhibited enteritis. during the spring of , a dead dog, a privately owned -week-old male pekingese, was submitted for laboratory investigation. the clinical symptoms were lethargy, inappetence, fluid diarrhea, vomiting and dehydration with death after days. necropsy of the dog showed hemorrhagic enteritis and severe lesions in the liver, spleen and lung. hemorrhages of the liver and spleen were observed on their surfaces. multiple areas of congestion were found in the lung. a fecal sample was collected and subjected to virological investigations. rapid kits were employed to identify common canine viral pathogens, including canine distemper virus (cdv), canine parvovirus (cpv), canine adenovirus- (cav- ), cav- and ccov (bionote, hwaseong-si, gyeonggi-do, south korea). the primers p-f and p-r (table s ) were employed to confirm the above results [ ] . the crfk cell line was used for virus isolation. after three rounds of purification by plaque assay [ ] , the virus was propagated and titrated and then harvested by one cycle of freezing and thawing. viral rna was extracted from the original fecal sample and from crfk cells infected with ccov using trizol reagent (invitrogen, carlsbad, ca, usa). fifteen primer pairs were designed based on the conserved regions of ccov strain tn- (table s ). the rt-pcr assay was performed as described previously [ ] . the protocol for electron microscopy was described previously [ ] . an indirect immunofluorescence assay (ifa) was conducted using a standard procedure. an anti-ccov antibody (vmrd, wa, usa) and fitc-conjugated rabbit anti-dog igg (sigma, ca, usa) were used as primary and secondary antibodies, respectively. a ccov-n polyclonal antibody was prepared as follows [ ] : briefly, the complete n gene was amplified using a forward primer ( ' ttt gga tcc atg gcc aac cag gga caa cgc ') and a reverse primer ( ' ttt gcg gcc gctta gtt cgt tac ctc atc aat '). then, the products were cloned into the vector pgex p- . purified gst-n recombinant protein was used as an antigen to inject female balb/c mice. after three immunizations, serum was collected and stored at - °c. rna was extracted from fecal and organ samples using a previously established taqman-based real-time rt-pcr assay for rapid detection and quantification of ccov rna [ ] . ??the primers?? df f and orf cr (table s ) [ ] were used to detect sg mrnas from the orf abc region by rt-pcr, using a one-step rt-pcr kit (abm, richmond, bc, canada). a phylogenetic tree based on the sequences of the structural proteins was constructed using the neighbor-joining (nj) method of molecular evolutionary genetics analysis (mega) software (version . ). bootstrap values were estimated for , replicates. simplot . . was used for nucleotide sequence comparison of hlj- to the reference fcov strains and ccov strains. the hlj- sequence obtained in this study was assembled and submitted to the genbank database under accession number ky . canine blood monocytes originating from specific-pathogen-free (spf) dogs were isolated [ ] . phorbol -myristate -acetate (pma) was purchased from sigma-aldrich (st. louis, usa). the human monocytic cell line thp- was provided by the national institute for communicable disease control and prevention, chinese centers for disease control and prevention (beijing, china). thp- cells were induced to differentiate into a mature-macrophage-like state by stimulation with ng of pma per ml for h before treatment. we isolated a canine coronavirus with a deletion in the orf abc region from a dead dog in china. real-time rt-pcr assays were carried out to determine the viral load in the faeces, intestine, lung, liver, spleen, kidney and heart of the dead dog. all of the organs except the kidney were found to be positive for ccov (table s ), indicating that this strain is a pantropic ccov strain. next, after three rounds of plaque assay screening, a novel isolate, hlj- , was successfully isolated and purified. the presence of virus particles was confirmed by electron microscopy of negatively stained samples (fig. s ) . sequence analysis suggested that there was a -nt deletion in orf abc when compared to isolates - , tn- and bgf- . this deletion resulted in truncations in orf a ( nt) and orf c ( nt) and the complete loss of orf b ( fig. a and b) . to investigate the stability of the orf abc region in the hlj- genomic rna, the virus was propagated in crfk cells for nine generations. total rna was extracted from hlj- -infected crfk cells and from the original faecal sample. analysis by pcr confirmed that the orf abc deletion was present not only in the purified virus but also in the original faecal sample (fig. s ) , indicating that the deletion was not due to viral adaptation during cell culture passage. examination of subgenomic orf abc mrnas showed that the genome of hlj- contained a truncated orf abc (fig. c) . previous studies have demonstrated that this region is variable, and a natural cb/ strain that causes fatal infections and long-lasting lymphopenia has also been shown to contain a deletion in orf b [ , , ] . strains / and na/ are closely related to the prototype strain cb/ , which causes diarrhea and acute lymphopenia [ , ] . furthermore, strain bgf also has a deletion in the orf abc region [ ] . these results demonstrate that the orf abc region may be prone to being lost during the evolution of the virus. recombination plays a vital role in the evolution of ccov, allowing the emergence of new strains with altered virulence and immunogenicity. in recent years, ccovs with different genotypes and subgenotypes have been detected. ccov-ii is the oldest genotype, which has been known since [ ] . more recently, recombination between ccov and tgev has been detected, which has led to the emergence of ccov-iib isolates in which the n-terminus of the s gene is very similar to that of tgev, while the rest of the genome resembles that of the reference ccov-ii isolate [ ] . a ccov strain (ucd- ) that had potentially recombined with tgev was identified in the late s [ ] . genetic analysis of several ccovs circulating in italy first revealed a new canine genetic cluster in which recombination events within the s gene had increased the similarity of ccovs to the feline homolog [ ] . on the basis of their genetic relationship to fcov-i, the new genotype was initially designated "fcov-like ccovs". recently, an additional orf, named orf , located between the end of the s gene and the orf a gene, was also detected [ ] . in this study, phylogenetic analysis based on the s protein revealed that hlj- is closely related to members of the fcov-ii cluster and is distinct from ccovs ( fig. a) . analysis of the s (receptor-binding) domain showed that it clustered closely with fcov-ii - (fig. b) , while the s (fusion) domain clustered with ccov-ii, fcov-ii and tgev (fig. e) . the coronavirus s protein consists of two independent subdomains, the n-terminal domain (ntd) and the c-terminal domain (c-domain) [ ] . the ntd clustered closely with cb/ and - (fig. c) , and the c-domain clustered with - (fig. d) . in contrast, the m gene clustered with a and had a -amino-acid deletion compared with a , cb/ , / (fig. h) , and the e gene cluster with tgev and did not clustered with ccov and fcov (fig. g) . these data indicate that hlj- is probably a recombinant of tgev, fcov-ii and ccov-i/ ii, with recombination sites located between the s, e and m genes. simplot analysis was carried out to identify possible recombination events and breakpoints in the complete nucleotide sequence of the hlj- genome. the analysis the orf abc region of feline coronavirus has been shown to affect viral cell tropism. truncated orf abc of fipv df- has been shown to effectively replicate in macrophages/monocytes, while infection with fcovs with the complete orf abc region was confined to the intestinal tract [ ] . in the present study, we found that hlj- and fipv df- could also effectively replicate in canine macrophages/ monocytes (fig. a) . however, hlj- could replicate in thp- cells, while df- could not (fig. b) . these results suggest that hlj- has the ability to alter its tissue tropism from the intestinal tract to systemic infection and to alter its viral cell tropism from dogs to humans, which should be regarded as a potential threat to domestic dogs. accordingly, epidemiologic studies are required to determine whether domestic ccov isolates have similar characteristics. we report for the first time that ccov hlj- , with an orf abc deletion, has been isolated in china. hlj- represents a recombinant coronavirus with distinct host cell tropism. acknowledgements this work was supported by the state key laboratory of veterinary biotechnology foundation (sklvbp ). conflict of interest the authors declare that they have no competing interests. virus taxonomy, th report of the international committee on taxonomy of viruses molecular characterization of feline infectious peritonitis virus strain df- and studies of the role of orf abc in viral cell tropism recovery and characterization of a coronavirus from military dogs with diarrhea canine coronavirus highly pathogenic for dogs canine coronavirus highly pathogenic for dogs new enteric viruses in the dog an update on canine coronaviruses: viral evolution and pathobiology immunity after natural exposure to enteric canine coronavirus does not provide complete protection against infection with the new pantropic cb/ strain recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr a pantropic canine coronavirus genetically related to the prototype isolate cb/ molecular characterisation of the virulent canine coronavirus cb/ strain molecular characterization and phylogenetic analysis of transmissible gastroenteritis virus hx strain isolated from china feline and canine coronaviruses: common genetic and pathobiological features preparation and characterization of mouse polyclonal antibody against conserved region of human foxo . xi bao yu fen zi mian yi xue za zhi canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins molecular characterization of a canine coronavirus na/ strain detected in a dog's organs crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy characterization of a recombinant canine coronavirus with a distinct receptor-binding (s ) domain molecular characterization of a virulent canine coronavirus bgf strain cocirculation of canine coronavirus i and iia/b with high prevalence and genetic diversity in heilongjiang province plaque assay for canine coronavirus in crfk cells 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 key: cord- -apfcql m authors: paraguison-alili, rubigilda; domingo, clarissa yvonne j. title: phylogenetic tracking of current porcine epidemic diarrhea virus (pedv) strains in the philippines date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: apfcql m to trace the possible route of introduction of porcine epidemic diarrhea virus (pedv), the phylogenetic relationships of pedv strains in the regions of epidemicity in the philippines to pedv strains that are endemic in other countries were investigated. partial nucleotide sequences of the s spike gene was determined from the pedv-positive samples and compared with s sequences from other countries. phylogenetic analysis indicated that pedv strains in the philippines segregate into two groups. members of group are related to strains from the usa, taiwan, japan and canada, while those in group are related to strains from china and vietnam. porcine epidemic diarrhea (ped) is an infectious enteric disease that poses a threat to the swine industry and is characterized by acute enteritis and diarrhea, with the infection causing more-severe disease in piglets. the causative agent of ped is porcine epidemic diarrhea virus (pedv), which belongs to group a of the genus coronavirus, family coronaviridae, order nidovirales. pedv is an enveloped rna virus with a single-stranded positivesense rna genome approximately kb in size that contains six genes: replicase (rep), spike (s), orf , envelope (e), membrane (m), and nucleoprotein (n). the full-length s gene is about . kb and consists of the s and s domains of approximately . and . kb in length, respectively. since the receptor-binding sites and majority of the neutralization epitopes are located in the s portion, this region has been subjected to sequencing and molecular analysis to determine the genetic relatedness of different pedv viruses [ ] [ ] [ ] . the current study characterizes the spike s portion of the virus. the s protein has a crucial role in viral and cell fusion activity and in inducing a host immune response [ ] [ ] [ ] . the spike protein makes up the surface projections of the virus and plays a major function in interaction with cell receptors [ ] . the sequence of the full-length s gene, the s portion, or the s portion reflects the genetic diversity of the virus. the s gene, particularly the s portion, is a highly variable region and is appropriate for sequencing to study the genetic relatedness and molecular epidemiology of pedv [ ] . ped may occur year round, with peak incidence during the cold season, and it occurs in herds that have been exposed to the virus and have developed partial immunity. pigs from week of age are usually affected, and those that survive ped can be carriers for up to days. infected sows may be febrile, with occasional abortion, but they usually recover within - days. in the philippines, the first case was reported in and was identified as a tge-like virus on a farm of sows in pampanga. recorded outbreaks occurred in january in pampanga, tarlac, pangasinan, batangas, cavite, laguna, quezon and negros occidental. sporadic and isolated outbreaks were recorded every year thereafter. through , outbreaks have been reported particularly in the provinces of bulacan, rizal, pampanga, tarlac, pangasinan, batangas, cavite, laguna, bacolod and general santos, with severe cases in adult animals. the mortality rates due to ped were as follows: birth to days old, % to %; to days old, % to %; to days old, % to %; days or older, rare. the mortality decreases as age increases. morbidity is high in growers and finishers, but mortality is low, and affected pigs may be stunted. in may to august , an outbreak of ped occurred in the province of batangas, killing % ( , head) of , sick pigs, resulting in huge economic loss [ ] . linkages with swine farms in the provinces of pampanga, tarlac, batangas and agusan were established. using data from swine farms in these provinces and diagnosis data from the college of veterinary science and medicine in central luzon state university, the incidence is % in pampanga, % in tarlac, % in batangas, and to % in agusan. the overall prevalence of ped is %, % on smallholding farms and . % on commercial farms. during the wet season of , from july to october, swine farms from the provinces of agusan, batangas, pampanga and tarlac, which are known to experience ped outbreaks, were requested to provide clinical samples such as intestines, fecal samples, fecal swabs and environment swabs whenever there were reported cases. selection of commercial and small swine farms was done in coordination with provincial veterinarians. reports on cases of gastrointestinal infections with symptoms of diarrhea were obtained from veterinary offices. a list of affected farms was obtained and used in constructing the sampling frame. rna was isolated using trizol reagent (invitrogen usa) according to manufacturer's recommendations. reverse transcription pcr was performed using a onestep rt-pcr kit (qiagen germany). a pair of primers was designed to specifically amplify a portion of the s region: fs m (gtc atg gca ctg acg atg atg ttt c) and peds r (cag atg tgt aat aaa cac ctg cca a). thermocyling conditions were as follows: °c for minutes and °c for minutes for the rt reaction, followed by cycles of amplification at °c for seconds, °c for seconds, and °c for minute, with a final extension at °c for minutes. good-quality rt-pcr products from samples that were positive for pedv were selected and processed for dna sequencing: batangas, sample; tarlac, ; pampanga, and agusan, . a total of pedv-positive samples were processed for dna sequencing of the -bp portion of the s gene. genbank accession numbers for pedv strains or clones from other countries are listed in table . the partial s gene sequences of pedv were aligned with those of strains from other countries, using clustalw. a phylogenetic tree for the -bp gene segment was constructed using the maximum-likelihood method with the general time-reversible nucleotide substitution model. the confidence level for each branch was tested by the bootstrap method with , replicates. phylogenetic and molecular evolutionary analysis was conducted using mega version . . software [ ] . this is the first report of pedv s gene sequences from the provincial pedv hotspots of the philippines, which include batangas, pampanga, tarlac and agusan. a phylogenetic tree was created by the maximum-likelihood method with , bootstrap replicates, using the mega . . software [ ] . interestingly, sequence analysis of the s gene from current pedv strains from the philippines revealed that these current strains cluster into two groups. viruses in group share common phylogenetic roots with strains from the usa, taiwan, japan and canada, while those in group were more closely related to strains from china and vietnam. pedv identified on swine farms in pampanga, tarlac (central luzon), batangas (southern luzon), and agusan (mindanao) were clustered within group . interestingly, other samples from central luzon, particularly pampanga and tarlac, clustered separately within group (fig. ) . it has been reported that all us pedv strains form a tight cluster and are most closely related to a strain isolated in in china (ah ) [ ] , while philippine strains are closely related to vietnamese pedvs in an orf genetic tree [ ] . in another study, comparative analysis of fulllength sequences of the whole genome revealed that isolates from germany show very high nucleotide sequence similarity to strain oh , which was found in the united states in [ ] . phylogenetic analysis of the complete pedv genome also indicated that japanese strains, table reference sequences from ncbi genbank, described by strain or clone names and accession numbers including two novel pedv variants, were closely related to strains that were widespread in the united states and korea in - [ ] . moreover, sequencing and phylogenetic analysis of the spike gene and orf of pedv revealed that the prevailing pedv isolates in japan had the greatest genetic similarity to us isolates [ ] . in taiwan, phylogenetic analysis of the s gene showed that all pedv strains from taiwan were closely related to the non-s indel strains from the usa, canada and china [ ] . taken together, recent reports are consistent with our findings that the philippine pedv strains share common ancestors with those from the usa, taiwan, japan and canada (group ) as well as those from china and vietnam (group ). pedv is present in many countries in asia and has been present in europe since the s. it was first discovered in europe, but it has become increasingly problematic in asian countries such as korea, china, japan, the philippines, and thailand. it has also spread to north america and was discovered in in indiana, usa [ ] , and in canada in the winter of . in january , a new variant strain of pedv with three deletions, one insertion, and several mutations in the spike region was identified in ohio by the animal disease diagnostic lab of the ohio department of agriculture [ ] . findings presented in this paper provide important information regarding the route of introduction of pedv in the philippines and provide linkages to other countries. the emergence of different pedv strains can occur via introduction of the virus from overseas, since this virus is versatile and can be easily spread. our study also detected pedv contaminants from inanimate objects such as swabs of floor pens and farrowing crates, fan blades of tunnel vent farms, raw feed ingredients, and inner surfaces of empty feed sacks. hence, proactive disease control measures should include the identification of the source of imported goods for pigs, which may require thorough biosecurity procedures to limit the spread of the agent and the outbreak. acknowledgments this project was funded by the philippine council for agriculture, aquatic, and natural resources research and development (pcaarrd). grateful acknowledgments to our colleagues at the college of veterinary science and medicine, central luzon state university for helpful advice. we thank the veterinarians for submitting the clinical samples from the different provinces for pedv screening. we also thank the division of bioresources hokkaido university for dna sequencing of the rt-pcr products. above all, to god for the wisdom and knowledge. conflict of interest the authors whose names are listed below certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria, educational grants, participation in speakers' bureaus, membership, phylogeny was based on partial spike s gene sequences. evolutionary history was inferred using the maximumlikelihood method based on the kimura -parameter model [ ] . the initial tree for the heuristic search was generated automatically by applying neighbor-join and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach. evolutionary analysis was conducted using mega [ ] employment, consultancies, stock ownership, or other equity interest, and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. we have no conflict of interest. statement of human rights all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. statement on the welfare of animals all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. informed consent informed consent was obtained from all individual participants included in the study. pathogenesis comparison between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains in conventional neonatal piglets veterinary virology: the third edition characterization of the structural proteins of porcine epizootic diarrhea virus, strain cv the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus lipa city veterinary office mega : molecular evolutionary genetics analysis version . for bigger datasets phylogenetic analysis of the spike (s) gene of the new variants of porcine epidemic diarrhoea virus in taiwan molecular characterization of the spike and orf genes of porcine epidemic diarrhea virus in the philippines comparison of porcine epidemic diarrhea viruses from germany and the united states molecular characterization of pig epidemic diarrhoea viruses isolated in japan from us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states american association of swine veterinarians presentation to the pork management conference a simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences key: cord- -fe ezt authors: traavik, t.; mehl, r.; kjeldsberg, elisabeth title: “runde“ virus, a coronavirus-like agent associated with seabirds and ticks date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: fe ezt from i. uriae collected in the seabird colonies at runde, norway, two identical virus strains demonstrating no antigenic relationships to major arbovirus groups were isolated. the new strains demonstrated a corona-virus like morphology, haemagglutinated chicken red cells and were sensitive to sodium desoxycholate. multiplication with cpe was demonstrated in bhk /c and bsc- cells, and without cpe in vero and gmk cell cultures. the mouse pathogenicity was relatively low. in gel precipitation three to five specific lines were seen. precipitating antibodies have been found in seabird species commonly infested byi. uriae. the ecological circumstances of the isolates indicate an earlier unrecognized arbovirus circulating between seabirds andi. uriae. this corona-like virus has been tentatively termed runde virus. in investigations were undertaken to evaluate the extent, distribution and ecological circumstances of arbovirus loci in norway. until then., no arbovirus isolates had been reported from this country, although ecological and cli-nicai/epidemiological considerations ( , , ) and a limited serological survey on bovine sera ( ) indicated the existence of central-european tick-borne encephalitis virus fool. the early phase of the project concerns the study of the importance of ticks as vectors; later, mosquito-borne agents will be investigated. in norway five ixodes species are abundant: _[. ricinus ( ) , i. tria.nguticeps ( ) , i. hexagonus ( ) , i. tividus ( ) and . uriae ( , ) . the latter species is present at great numbers in the vast norwegian seabird colonies. its ability to attack man and mammms has been questionable, but this has been recently documented ( ) . this paper describes studies with ixodes uriae as a possible vector. these studies were undertaken since: a) the tick is part of rather confined ecosystem which might be suitable for studies on virus-vector-host interrelationships. b) virus-transmission by this tick might have mainly zoonoticm but also some public health importance. c) reports of arbovirus isolates from seabird colonies have been presented earlier ( , , , , ) . the seabird colonies at runde were chosen from a great number of possibilities due to relative ease of access, and because an unusually high and unexplainable chick mortality had recently been reported. from i. uriae ticks collected at runde in late september , three virus strains have been isolated. one of these isolates belongs to the uukuniemi group ( , ) . the additional two strains are serologically closely related or identical. some characteristics and ecological circumstances of the latter viruses are reported in the present article. description o/ the biotope the island runde is situated at ° 'n ' ° 'e, approx. kin from the city alesund (fig. ) . the total area is about . km and the circumference kin. there are two small settlements on the island, l~unde in southeast and goksoyr in northeast : most of the island consists of an uneven mountain platea.u, and has altitudes varying between and metres above sea level. in the southwest part, the plateau is terminated by cliffs bordering the sea. most seabird colonies are situated in this area. approaching sea-level there is naked stone with niches and clefts inhabited mainly by rissa tridactyla, sula bassana, alca torda and uria aalge. further up towards the plateau there are rockfalls covered by thin layers of soil supporting various gramnivor plants. these parts are inhabited by the common puffin (fratercula arctica). we visited the island between september -- , , and collected ixodes uriae in the puffin rockfalls. according to local ornithologists, the puffins had migrated south about weeks earlier. we found the ticks under and between the rocks. signs of engorgement were never seen, and they seemed to have entered into diapause. most of the ticks were to varying degrees surrounded by desiccated skinflaps indicating that they had newly emerged to their present stage. some had not completed their development from nymph to adult, and were totally surrounded by nymph cutis. the ticks were kept on dram-vials with moist plaster of paris, and transported alive to the laboratory. we collected a totm of i. uriae, of which were processed for virus-isolation (table ) . isolation procedures the ticks were divided according to stage and sex, pools of imagos consisting of and nymph pools of individuals. after rinsing in saline, the ticks were ground in a mortar in . per cent bovine albumin in pbs pi-i . (apbs) with mycostatin, streptomycin and penicillin. suspensions were clarified by centrifugation at × g and inoculated i.c. into baby mouse litters, born: nmri (spe), aged one to three days. suspensions not inoculated the same day as processed were kept at -- °c (l~evco ultrmow). mice were observed during weeks for signs of illness. diseased mice were killed, brains removed aseptically and homogenated in apbs with antibiotics, centrifuged and passed into new baby mice. litters which remained healthy were killed after weeks, brains processed and passed as described. two blind passages were performed routinely. calculations of per cent end point, titers, lds baby mice (bmlda ) were performed according to ir,~d and mu~nci ( ) , based on ten-fold titrations in mice. virus-multiplication has been tested in bhk /c t , itela bristol, bsc- , rk- and vero celt lines and also in primary gmk cells. bi-ik /c i cells were grown in the medium described by maephe~asos" and sto~e~ (it). all other cultures were grown in eagle's medium with per cent inactivated calf serum. tubes were seeded with cells in ml medium. cultures were used when confluent monolayers were present. cells were washed with saline, virus was diluted ~enfold from - to t - in the medium, a volume of . ml of each dilution was inoculated into three tubes and allowed to adsorb for hour at room temperature before washing with saline and addition, of new medium, culture tubes were incubated for days at ° c and inspected daily for a cytopathie effect (cpe). after days culture fluids were inoculated i.c. in baby mice litters, . t ml per mouse. cell controls were treated in parallel. bhk /e cultures in roux bottles were established by seeding × a cells/ml. efforts to plaque the isolates were performed in bitk /c cells under carboxymethylcellulose overlay by a method recently described ( ) . one to three hundred times concentration of cell culture fluids were performed by precipitation with polyethylene glycol (peg) and nac as described by mcs~arry and benzingee ( ) . in some instances the precipitates were sonicated at w for × seconds in a branson b sonifier, sensitivity to sodium-desoxycholate (si)c) was determined in baby mouse litters by the method of t~ei~a ( ) . for haemagglutination (ha), immunoelectroosmophoresis (ieop) and closed hexagon immundiffusion (chi) experiments, the following antigen preparations have been employed : a) crude suckling mouse brain (smb) preparations: a per cent infected suspension in apbs which has been centrifuged for minutes at , rpm. b) sucrose-aceton (sa) extracted infected mouse brains: prepared according to c~ar~:e and casals ( ), but omitting the final lyophilization step. c) cell culture antigens: culture fluids from infected bhk /c roux bottle cultures were precipitated with per cent peg (macrogolum, norsk medisinaldepot) and . per cent naci ( ) . the -- times concentrated virus-precipitates were sonieated. antibodies were produced in adult, white mice inoculated with infectious suckling mouse brain preparations. initially each mouse received a virus dose of approximately . x - bmld~ in . ml brain suspension mixed thoroughly with . mi freund's complete adjuvant (difco). subsequently weekly injections with approximately × - bmld~ in . ml suspension were given. the last injection, identical to the initial one, was performed one week thereafter. another -- days later paraeentesis and bleeding from the retro-orbital sinus were performed. arltibody preparations for tahyna virus were produced in the same way. a fowl antiserum to avian infectious bronchitis virus (aib) with a ni of . , was kindly provided by the institute of veterinary medicine, oslo. reference mouse antibody preparations to tbe, tribec, eee, wee and uukuniemi (s ) were supplied by the yale arbovirus research urlit. an ornithological expedition to tternyken, rost (approx. ° °n, °e (fig. ) , collected seabird sera in early may . the composition of this material is cited in table . serological methods tiaemagglutination (ha) and haemagglutination inhibition (hai) tests were performed according to clarke and casals ( ), modified for microtitration equipment (cooke eng. co.). ha activity was tested within the ph range . -- . at °, ° and ° c. erythrocytes from a variety of species have been investigated. these results will be reported separately, and all ha and hai titers in the present paper refer to the employment of . per cent chicken erythrocytes. (ieop) twelve ml gel was poured onto precoated × cm lantern slides. for antigen detection i per cent agarose (l'industr. biol. franc.) and for antibody detection a mixture of . per cent agarose and . ~ per cent difco baeto agar was used ( ) . three rows of paired wells with mm diameter were punched out. the well interdistance was ram. in the i-lepascreen eleetrophoresis apparatus (spectra biologicals, oxnard, calif., ) slides were run simultaneously for i-- hours. in screenings, this allows testing of unknown samples in one run. gel precipitation this was performed by a sensitive modification of the micro ouehterlony technique termed closed hexagon immunodiffusion (chi) ( ) , and also with varying patterns on lantern slides. to obtain the maximum number and intensity of the precipitation lines, the antigens were applieated several times ( -- ) during the hours prior to antiserum application. due to the lower diffusion rate of antigens, an interval of -- hours between the last antigen and the antiserum application was adopted. the first lines then could be seen -- hours after antiserum application. the gels were incubated at room temperature overnight, and then several days at -~ ° c before staining and final reading. ieop and chi gels were stained by per cent tannic acid as previously described ( ) . negative contrast eiv[ infected culture fluids from bgk /cl cells grown in roux bottles were concentrated times by peg /nac precipitation ( ) , resuspended in borate saline pit or pbs pti . and sonicated. then . ml suspension was diluted / in pbs and centrifuged for hour in a sorvm rc -b with rotor -- at , rpm. the pellet was suspended in a few drops of distilled water and negatively stained with per cent phosphotungstie acid pi-i . or . per cent uranyloxalate pi-i . . one drop virus-stain mixture was placed on a formvar-earbon coated grid and excess fluid was withdrawn with filterpaper. the grid was examined in a jem b electron miscroseope at a magnification of , ×. thin section em infected and control bhk /c cultures were harvested days p.i. by means of a rubber policeman. the ceils were fixed in per cent glutaraldehyde for hour at -~ ° c, washed three times in eacodylate buffer, postfixed in per cent osmiumtetroxyde for hour at room temperature and centrifuged for minutes at rpm. the cell pellet was resuspended in a few drops of caeodylate buffer and centrifuged in micro capillary tubes in a hematocrit eentrifuge for minutes at , rpm. the pellets were removed from the capillary tubes, dehydrated in acetone and embedded in spurt by a rapid procedure ( ) . ultrathin sections (silver) were cut on a u -reichert ultramierotome and doubly stained by saturated uranylacetate in per cent ethanol and reynolds lead citrate ( ) . electron microscopy was carried out in a jem b at a magnification of , ×. two pools, both processed from unengorged female i. urine, termed e and e were inoculated into mouse litters and days old respectively. the mice seemed unaffected during the weeks observation period. however, in the first passage (m ) the mice were moribund after . days for e and days for e . in the second passage (m ), incubation periods were reduced to i and days respectively and in the subsequent passages to -- days for both strains. the viruses were reisolated twice from the original tick pools during the following year. these results are summarized in table . the original tick pools were diluted / in bhk-medimn and inoculated on five bhk /c tube cultures. the tubes were harvested after five days. no cpe was recorded. the culture fluids were incubated into three baby mouse litters each. the mice were moribund after -- days. the viruses were shown to be serologically identical to the strains isolated in mice by hai, chi and ieop. the ru e t tick pool was diluted / in bhk-medium, and three bhk cultures in roux bottles were infected. tile cultures were harvested after four days. infected culture fluid was inoculated i.c. into baby mouse litters, which were paralysed at day p.i. the rest of the culture fluids were concentrated times by peg /naci, and used as antigen. the isolates were shown to be serologiealty identical, and also identical to the mouse-passaged strains by hai, chi and ieop. a per cent brain suspension from the third suckling mouse brain passage (m ) titered -s bmlds /ml in day old mice. the virus suspension was titrated in parallel in and . days old mice. as shown in table , week old mice were nearly as sensitive as the newborn, while there was a drop in infectivity of . logs and a prolongation of average survival time from . to . days for week old mice. during production of antibody preparations it was demon-strafed that adult mice were refractary to intraperitoneally injections with both e and e . in hela bristol and rk- cultures no signs of virus multiplication were detected during primary inoculation and consecutive blind passages. no cpe was present, and culture media concentrated -- times by peg /nac precipitation did not affect -- days old suckling mice. as mentioned before, virus multiplication but no cpe was recorded after infection of bhk /c cultures with the original tick pools. plaquing in bhk /e cells was not successful by the method used. both i~unde es and e virus demonstrated a very marked sensitivity to treatment with sodiumdesoxycholate. the titers were reduced from . to . in the case of ru e and . to . in the case of l~u e . l~unde virus produces antigens in suckling mouse brains as well as in bhk l c . the antigens demonstrate specific reactions with mouse antisera and immune aseitie fluids in hai, chi and ieop. the antigenic preparations have been kept at, -- ° c for months without loss in reactivity, itaemagglutinating (ha) antigens can be produced ~om infected smb by the sa extraction method of cl~r~:e and casals ( ), hut mso suspensions of crude infected smb and peg /nac precipitated cell culture fluids contain haemagglutinating activity. the ability to agglutinate chicken erythrocytes is relatively independent of pit and temperature. the highest titers and most clearcut endpoints have however been attained by pit . -- . at ° c. in hai no serologic differences between rue and e could be demonstrated. the mouse antisera and immune ascitie fluids were inhibitory to i-ia units of antigen to dilutions of / -- . normal mouse sera were not inhibitory, neither was a fowl antiserum to aib virus or antisera to tbe, tribec, eee, wee and tahyna. precipitating antigens, as revealed by chi and i e o p , also were present in the same preparations as cited above. i n citi -- specific lines have been noted (fig. ) , while in i e o p double lines were seen. no antigenic difference between the two strains was detected. virus from mouse brains and cell culture demonstrated total i d e n t i t y b y these methods. no reaction was observed with a n antiserum aga, inst avian infectious bronchitis virus. the seabird sera were screened by i e o p , and the specificity secured by chi. foul" out of birds had precipitating antibodies to r u e t . the composition of the material and the results are sho~nt in table . negative contrast em a series of electron micrographs of negatively stained preparations are presented in figs. , . fig. shows corona-virus-like structures in a preparation from l~u e infected cell culture supernate. similar structures were never seen in uninfected controls. most of the particles have an approximate total diameter of nm, but in other preparations particles with diameters varying from l l to n m have been seen. fig. a demonstrates an intuct particle at a greater magnification. i t seems to contain an inner structure nm in diameter. fig. b and c demonstrate partly and completely disrupted particles with loss of knobs and release of inner helical material. staining with . per cent uranyloxalate (fig. d ) produces a sort of -dimensional picture of the virus-like particle. thin section em thin sections of r u es infected b h k /e cultures days p.i., demonstrate virus-like particles in the cytoplasm and extraeellularly between broken and intact cells (fig. ) . the particles have a diameter of -- nm. some of them look empty, while others are penetrated by stain and expose inner structures. the particles seem to have a double outer membrane, but no knobs are visible by thin sections and positive staining. when the runde virus strairts were first isolated, it had to be kept in mind that they might originate from the staff handling the tick pools, or from the baby mice used for isolation. the reisolations from the original tick suspensions (table ) , a~d the properties differing from known human, rodent and also avian eoronaviruses ( , ) , could not entirely rule out these possibilities. but the fact that antibodies to the virus were demonstrated irt seabirds and the isolation of virus also in cell cultures seem to settle this question. circumstances of tickcollection strongly support that runde virus is an arbovirus in the ecological sense, and not a mechanical pick-up : . i t is highly unlike that a relatively labile virus should survive for weeks in i. uriae without active multiplication. . the lack of engorgement or pregnancy of the female i. uriae, and the fact that they were in diapause and surrounded by desiccated skinlaps, strongly indicate that t~unde virus m a y have passed interstadially in the virus-carrying ticks. still, the final inclusion of t~unde virus amortg the arboviruses must await the demonstratiorl of true biological transmission by .i. uriae. although the morphology of gunde virus strikingly resembles that of the eoronavirus group, some characteristics of the virus differ considerably from * those of known coronaviruses. this holds true for the pathogenicity to mice ( , ) , the ability to grow in cell cultures ( , , ) and the haemagglutinating properties ( , ) . besides, the size of the virion seems larger than for other members of the coronavirus group ( ) . the discrepancy in size between negative contrast and thin sections may be partly due to shrinkage during fixation and dehydration prior to embedding and thin sectioning, and partly to collapse of the particles by negative staining adding to their actual size. the most reliable staining procedure for size calculations probably is negative staining by uranyloxalate which also stabilizes the structure of the particles. detailed taxonomic considerations for runde virus must await further characterization of biological, serological and molecular properties. but runde virus is probably a "new" coronavirus on the grounds of: its morphology, the lack of cross reaction with avian infectious bronchitis virus and the lack of serological reactions with control mouse sera probably eliminating the possibility of mi-iv isolates. also, our department of experimental animals have no indications of mouse hepatitis in the mouse colony which have been used in these studies. the demonstration of this hitherto unrecognized virus associated with seabirds and ticks also necessitates further efforts to clarify-the ecological and possible epizootologieal implications. as alre~ody mentioned, an unusually high chick mortality ill the seabird colonies at runde has been reported during the ias~ few years. bacteriological and toxocological research has not been able to reveal any definite etiological factor, although pesticide residues have been demonstrated to some extent. this must of course bring viruses into consideration. a synergistic action between pesticides and virus seems one possible working hypothesis in this connection. since norwegian seabird colonies in general are rather frequently visited by scientists, students, holiday travellers and the local public, the recent documentation of the ability of i. uriae to attack man ( ) raises the question of public health implications. isolations from i. urine of uukuniemi viruses ( , , ) orbiviruses ( , ) and flaviviruses ( , ) ill addition to runde virus further stresses this point. at present nothing is known concerning the distribution and general infection rates of runde virus. some indications that it might be widespread in norwegian seabird eolonies can be given, however. firstly, the isolation of two virus strains from only i. urine collected at runde indicates a rather high infection rate of the vectors in this location. secondly, the demonstration of a high rate of seropositive birds at tlernyken, rest, proves that virus-circulation is not restricted to runde. further field work and experiments which may throw more light upon th(~ ecological interrelationships of runde virus as well as upon the characteristics of the virion are in progress. we are indebted to eva mood petterson, einar brunvold and hatlgrim sog£rd for their skilfui technical assistance, and to tone lea for her valuable help with the preparation of figures. the propagation of "eoronaviruses" in tissue-culture coronaviruses of mart meningoeneephalomyeloradieulitis in sunnhordland. tidsskr. for den norske l~egeforening techniques for haemagglutination inhibition with arthropod-borne viruses replication of avian infectious bronchitis virus in african green monkey kidney cell line veto two-hour embedding procedure for intracellular detection of viruses by electron microscopy some characteristics of haemagglutination of certain strains of "ibv-like" virus a serological tool for the diagnostics of virus b hepatitis. aeta path. microbiol, scand tuleniy virus--a new group b arbovirus isolated from ixodes (ceratixodes) putus pick.--camb. collected on tuleniy island sakhalin v i r u s --a new arbovirus isolated from ixodes (ceratixodes) put~ts p i c k . --c a m b . collected on tuleniy island, sea of khotsk polyoma transformation of hamster cell c l o n e s --a n investigation of genetic factors affecting cell competence great island and bauline : two new kemorovo group orbiviruses from ixodes uriae in eastern canada growth in suckling mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease concentration and purification of vesicular stomatitis virus by polyethylen glycol "precipitation lopper og lundelus p£ sjofugl p& t~ost . fauna (oslo) records of eetoparasitic insects and mites on birds and mammals in norway lopper, f~tt og midd p~ piggsvin i norge. fauna (oslo) ultrastructure of animm viruses and bacteriophages: an atlas a simple method of estimating fifty percent endpoints the use of lead citrate at high p i t as an electron opaque stain in electron:microscopy arboviruses in finland. ii. isolation and characterization of uukuniemi virus, a virus associated with ticks and birds notes on norwegian ticks ixodes ricinus som mulig vektor for sykdommer hos mennesker i norge action of sodium desoxycholate on arthropod-borne viruses arbovirusencephalitt overfort av ixodes ricinus a sensitive modification of the ouehterlony technique. detection of hepatitis associated antigen by immunodiffusion in a closed hexagonal system. acta path. mierobiol, seand serological investigations indicating the existence of tick-borne encephalitis virus loci along the norwegian coast. aeta path. microbiol, seand development of a modified immunoelecgroosmophoresis method for uukmfiemi and i~unde virus serology tickborne viruses in norway uukuniemi group viruses isolated ill norway tickborne viruses in western north america. ii. yaquina head, a new arbovirus of the kemorovo group isolated from ixoedes uriae tick-borne viruses from seabird nesting sites in oregon and alaska authors' address: dr. t. t~aavik, institute of medical biology, university of tromso, tromso, norway.received august , key: cord- -h sg shp authors: kochan, g.; gonzalez, d.; rodriguez, j. f. title: characterization of the rna-binding activity of vp , a major structural protein of infectious bursal disease virus date: journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: h sg shp infectious bursal disease virus (ibdv) is the agent of an immune-depressive disease affecting the poultry industry worldwide. infection of ibdv leads to expression of five mature virus-encoded proteins. proteolytic processing of the virus-encoded polyprotein generates vp which coats the inner surface of the ibdv capsid. in this report, we describe the characterization of the rna-binding activity of vp . for these studies, the vp coding region was fused to a histidine tag and expressed in insect cells using a recombinant baculovirus. the histidine-tagged vp was affinity-purified and used to study its ability to bind rna molecules using three complementary methods: (i) northwestern blotting; (ii) binding of vp protein-rna complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. the results demonstrated that vp efficiently bound ssrna and dsrna. under the experimental conditions used in this study, the formation of vp -rna complexes did not depend upon the presence of specific rna sequences. a series of histidine-tagged vp deletion mutants spanning the whole vp coding region were generated. the use of these mutants revealed that the vp rna-binding domain layed in a highly conserved aa stretch close to the n-terminus of the protein. avibirnavirus of the family birnaviridae [ ] . infection of young chickens with ibdv leads to infectious bursal disease (ibd), characterized by the destruction of the bursa of fabricious that results in a severe immunosuppression. ibd causes major economical losses to the poultry industry [ , ] and it might also affect several species of wild birds [ , , ] . ibdv vp coding sequence, corresponding to polyprotein aa positions to , was subcloned from plasmid pvote /poly [ ] into the pfastbac htc vector (gibco brl). briefly, pvote /poly cdna was digested with bamhi and partially with ncoi, and the resulting dna fragment containing the vp gene was ligated to plasmid pfastbac htc restricted with the same enzymes, generating the plasmid pfastbac-vp -his. this plasmid contained the vp gene fused to a histidine tag under the control of the polyhedrin promoter. vp deletion mutants were generated by overlap extension pcr [ ] using synthetic primers containing the desired deletions and using the pcdna -poly plasmid [ ] as a template. pcr products were obtained either with primer a ( -gcgcgagctccgtttccctc acaatccacgcgac- ) with the corresponding reverse-sense primer (table ) or with primer b ( -gcgcggatcctcatccaaggtcctcatc agagaca- ) with the corresponding reverse-sense primer ( table ). both products were recombined by pcr using primers a and b. pcr products were digested with bamhi and xhoi and cloned into the pfastbac-vp -his plasmid restricted with the same enzymes. in this way, a series of vp mutants containing small deletions (≈ bp) spanning the whole coding region of the protein was generated ( table ). the carboxy-terminal deletion mutants vp - (numbers represent positions of aminoacids in the polyprotein, flanking the deletions) and vp - presenting deletions of and residues, respectively, were obtained by cloning the vp fragment resulting from digestion with xbai and noti from plasmids pcdna -vp - and pcdna -vp - (maraver et al., submitted), respectively, into the pfastbact-htc vector. all constructs were sequenced to confirm the mutations. to study vp rna-binding activity, different [∝ - p]-labeled single-stranded rna (ssrna) probes, containing viral and non-viral sequences, were generated using the ribo-max kit (promega). briefly, transcription reactions were carried out by the t rna polymerase for h at • c. radioactive labeling was introduced including µci[∝ - p]atp ( ci/mmol) in transcription reactions ( µl final volume). synthesized rnas were purified using microspin columns s- hr (amersham pharmacia biotech). two ibdv virus-derived ssrna probes were produced, probes a and b. rna probe a ( nt) was synthesized from ibdv orf a by transcription from the plasmid pcdna-poly [ ] linearized with smai. the sequence of ssrna probe a corresponded to ibdv polyprotein coding region. rna probe b ( nt) contained the first nt fused to the nt non-coding regions (utrs) from segment b. this probe was produced using the plasmid tv - (maraver et al., submitted for publication) linearized with smai. two non-viral ssrna probes, gfp and pl, were also produced as described above. rna probe gfp ( nt) was obtained from green fluorescent protein (gfp) mrna fused to the encephalomyocarditis virus (emcv) leader sequence. this probe was produced by in vitro transcription using as a template the pvote -gfp plasmid restricted with psti and klenow-treated. plasmid pvote -gfp was obtained by blunt-end ligation of plasmid pvote- [ ] restricted with drai and smai, and the gfp gene excised by noti digestion from the green lantern plasmid (gibco brl) followed by treatment with klenow. rna probe pl ( nt), was obtained by run off transcription of the polylinker region of the pcdna plasmid (promega) linearized with xbai. the purity of all synthesized ssrna probes was confirmed by polyacrylamide gel electrophoresis under denaturing conditions and detection by autoradiography. the concentration of rna preparations was estimated by either electrophoresis in polyacrylamide or agarose gels and ethidium bromide staining or g. kochan et al. for binding studies to double-stranded ibdv rnas (dsrnas), virus dsrna was purified from bsc cells infected with ibdv using the ultraspect rna extraction reagent kit (biotecx laboratories, inc.) following the instructions provided by the manufacturer. subsequently, ibdv dsrna segments a and b were separated in % agarose gels, and segment a was purified using rnaid kit (q. biogene). plasmid dna pcdna linearized with smai was used as double-stranded dna (dsdna) competitor probe. single-stranded dna from phix phage virus (biolabs) was used as ssdna competitor probe. wt and mutant vp proteins were expressed using a baculovirus-based expression system. briefly, a mm diameter culture plate of confluent h insect cells were infected with rbvs at a multiplicity of infection (moi) of . infected cells were harvested at hpi and lysed on ice with a dounce homogenizer in tnn buffer ( mm tris, mm nacl, and . % np- , ph . ) in the presence of protease inhibitors (complete mini, roche). cell lysates were clarified by centrifugation at x g for min at • c. recombinant proteins were purified from the resulting supernatants using a ni-his trap affinity column (pharmacia lkb) following the instructions provided by the manufacturers. the expressed proteins were eluted from the column with a continuous imidazol gradient from mm to mm. his-tagged wt vp protein was eluted at an imidazol concentration of mm. for elution of each vp deletion mutant, imidazol concentrations were optimized. the purity of eluted proteins was assessed by sds-page analysis and coomassie staining. the concentration of purified proteins was estimated using the bca system (pierce). to confirm the identity of baculovirus-expressed vp proteins, western blot analyses were carried out from infected cell lysates as described [ ] . briefly, after separation of the cellular proteins by sds-page and transfer onto nitrocellulose membranes, filters were saturated with % non-fat dried milk in pbs for h at room temperature (rt), followed by incubation with specific anti-vp monoclonal (mab) or polyclonal antibodies [ ] diluted : in blocking solution. excess of unbound antibodies was removed by washing three times in pbs buffer. after washing, horseradish peroxidase-conjugated secondary goat-anti rabbit mab or rabbit anti-mouse mab (cappel) were added in blocking solution ( : dilution) and incubated for h at rt. after removing the unbound conjugates, membranes were developed in pbs containing . % -chloro- -naphtol and . % hydrogen peroxide. after developing, membranes were washed in distilled water and dried. northwestern blot assays were carried out with cellular extracts from cells infected with wt or rbvs as described [ ] . infected cells were collected at hpi and pelleted by centrifugation at x g for min at • c. cell pellets were washed with pbs and lysed in tnn buffer by incubation on ice for min with frequent and intensive vortexing. after cell lysis, cellular extracts were mixed ( : ) with loading buffer ( % glycerol, . % sds, mm dtt, . % bromophenol blue, mm tris, ph . in pbs). samples were incubated at • c for min with gentle agitation and centrifuged at x g for min at rt to remove cell debris. in all cases µg of cellular extracts from cells infected either with wt bv or rbv expressing vp (vp -bac) was separated in % sds-page gels and transferred onto nitrocellulose filters in tris-glycine buffer ( mm tris, mm glycine, ph . ). filters were incubated h at • c in renaturing buffer ( mm nacl, mm edta, . % ficoll, . % bovine serum albumin (bsa), . % polyvinylopirolidone, . % triton x- , mm tris, ph . ). afterwards, membranes were incubated in ml of renaturing buffer containing cpm of p-labeled probe b ( . ng/ml) in the presence of an excess ( ng/ml) of yeast whole rna over labeled probe. after incubation for h, membranes were washed four times with renaturing buffer for an additional hour to remove unbound probe. after drying, membranes were analyzed with a phosphorimager (storm , molecular dynamics). the same membranes were processed for western blot using specific anti-vp antibodies. experiments were repeated three times with independent preparations of cell extracts. to characterize the vp ssrna-binding activity, a filter-binding assay was performed as described [ ] . the affinity of vp protein to ssrna was estimated from three independent experiments using p-labeled virus ssrna probes. briefly, increasing amounts (from × − mm to × − mm) of purified vp or bsa, used as a control for non-specific interaction, were mixed with cpm of p-labeled ssrna probe b ( × − mm) in binding reaction buffer ( mm tris, mm kcl, mm nacl % glycerol, , % triton x ), in µl final volume, so the vp /rna molar ratio in these experiments varied from to . binding reaction mixtures were incubated at rt for h and filtered through nitrocellulose membranes using a dot-blot apparatus. in order to always use the same protein/rna ratio, when other probes were used the amount of the probes were corrected according to their size. the proportion of the retained probe in the filters was quantified by densitometry using a phosphorimager apparatus. a vp -rna binding curve was drawn by plotting the percentage of retained radioactivity as a function of vp protein concentration. assuming that the plateau reached represented the complete binding of the rna, the apparent kd corresponded to the concentration of vp protein required to reach half saturation [ ] . kd estimation was performed assuming a simple molecule-to-molecule binding equilibrium reaction. to transform the non-linear curve observed for vp -rna binding to linear, a hill plot was obtained as described [ , ] , by plotting log [retained radioactivity/( -retained radioactivity)] as a function of log (vp concentration). for the hill plot analysis, means of retained radioactivity from three independent experiments were used. a linear regression was performed by the least squares procedure ( degrees of freedom) and the associated probability to the correlation coefficient (r) was calculated obtaining a t value for a t-student test, as described [ ] . the hill coefficient (n h ) was obtained as the slope of the regression line and the k . value (amount of vp concentration to give half saturation, apparent kd) was calculated [ ] . competition assays based on nitrocellulose binding were carried out using virus-derived radiolabeled ssrna probes together with increasing concentrations of non-radioactive competitor rna and dna probes. briefly, vp -rna binding reactions were carried out as described above using radiolabeled ssrna probes b and a. binding reactions were performed in the presence of increasing concentrations, molar ratios of : , : , : and : , of competitor unlabeled rna or dna probes. reaction mixtures were filtered through nitrocellulose membranes, and the retained radioactivity was quantified as described above. competition experiments were also repeated three times to obtain representative means. the interaction of vp with ssrna was analyzed by emsa as described [ , ] with minor modifications. briefly,vp -ssrna complexes were allowed to form using increasing amounts of purified vp as described above. after incubation, aliquots of the reaction mixtures were loaded onto % polyacrylamide gel in tbe buffer ( mm tris-borate, mm edta, ph . ), and gels were run in tbe buffer at • c. gels were dried and exposed using a phophorimager apparatus. excess of bsa (from to mm) was used as a non-specific protein in rnabinding reactions. for supershift assays, after a min incubation at rt, the reaction mixtures were supplemented with µg of either vp -specific monoclonal antibodies (mabs) or nonspecific antibodies (preimmune mouse igg) and maintained for h at rt. thereafter, reaction mixtures were resolved in polyacrylamide gels and vp -ssrna complexes were revealed as described above. experiments were repeated three times. to find the level of sequence conservation of the vp ssrna-binding domain among different birnaviruses, known vp sequences from representative members of the family were aligned and compared using the clustal algorithm [ ] . for alignment conditions, the table of residue weights used was pam with a gap penalty of [ ] . for this purpose, the sequences of vp proteins from different species belonging to the avibirnavirus, aquabirnavirus, and entomobirnavirus genera as well as to unclassified birnaviruses were retrived from genebank. for genus avibirnavirus, ibdv virus serotype , strain soroa (af . ), strain oh (u . ), strain stc (d ), cu- wt virus (af . ), and serotype (af . ) were used. for aquabirnavirus genus, vp sequences from ipnv strains drt (d . ), jasper (nc ), sp (af . ), and n (d ) were used. in the case of genus entomobirnavirus, the vp protein sequence from drosophila x virus (u ) was used. for unclassified birnaviruses, the species marine birnavirus (ab ) and yellowtale ascites virus (ab . ) were used. previous studies performed with the aquabirnavirus ipnv had shown the presence of vp in ribonucleoprotein complexes isolated from virus particles [ ] . this observation along with the fact that vp coats the inner surface of the virus particle [ , ] suggested the possibility that vp might establish direct interactions with rna. to study this possibility, a recombinant histidine-tagged version of the ibdv vp was produced and purified. purified vp was then used to perform a number of assays to unambiguously determine its ability to interact with different rnas in the absence of other ibdv-encoded proteins. since preliminary results indicated that vp bound ssrna as well as dsrna, ssrna probes were used for subsequent rna-binding analyses. in order to obtain sufficient purified vp to perform a series of biochemical experiments described below, vp -bac, a rbv, was generated. this virus expressed a n-terminal histidine-tagged version of vp under the control of the polyhedrin promoter. the ability of vp to bind ssrna was initially tested by northwestern blotting using total cell extracts from either wt baculovirus or vp -bac-infected cells. the assays were carried out using p-labeled ssrna probe b. this probe (fig. ) , vp was expressed at high levels in cells infected with vp -bac. a significant binding with ssrna probe b was observed by the expressed vp in vp -bac infected cell extracts (fig. ) . a weak but significant band of bound probe was observed also in wt-infected cells, at the same position as ibdv vp . this band accounted for a % of total bound rna probe when compared to the cell lysate containing vp , as determined by densitometric analysis. western blot analysis with a vp specific mab confirmed that the rna-binding protein expressed after infection with vp -bac was in fact vp protein (fig. ) . the rna-binding protein present in cells infected with wt baculovirus did not react with vp -specific antibodies, suggesting that this protein was either cellular or viral, but not vp . these results also showed that monomers of vp fixed to nitrocellulose filters bound the ssrna probe b. to further characterize the binding properties of purified vp to ssrna, emsa assays were performed with p-labeled probe b. purified vp was used for these studies instead of total cellular extracts due to the existence of an rna-binding protein in control cell extracts that might interfere with the results (fig. ) . a series of shifted bands of rna-protein complexes were visualized after incubation of purified vp with radioactive probe b (fig. ) . the intensity of these bands was directly dependent upon the amount of vp used in the binding reaction mixtures ( fig. a) , reaching a plateau above a vp concentration of × − mm. results were reproducibly observed in all experiments. no shifted bands were observed when relatively high amounts of bsa were used in the binding mixtures, as expected (fig. ) . these results further showed that baculovirus-expressed vp bound ssrna probe b. since pure vp was used for the studies, these results also showed that vp did not require the presence of other virus or cellular proteins to bind ssrna. to confirm that the shifted bands corresponded to ribonucleoprotein complexes containing vp and not to aggregated rna, supershift assays were performed using purified vp protein-specific mabs (fig. b) . when bsa was used as a control at high amounts, no shifted bands were observed (fig. b) . in constrast, vp alone or in the presence of a non-specific igg formed at least three ribonucleoprotein complexes of different electrophoretic mobilities (fig. b ). the complex of higher electrophoretic mobility probably corresponded to monomer vp molecules binding to the rna probe, since when low amounts of vp protein were used in binding reactions this was the only shifted band ( fig. a) . addition of vp protein-specific mabs caused the supershift of all the complexes (fig. b) . these results unambiguously confirmed that the shifted bands corresponded to ribonucleoprotein complexes containing vp . after confirming that vp bound ssrna probe b, vp -rna complex dissociation constant was estimated by binding ribonucleoprotein complexes to nitrocellulose filters, as described in materials and methods. the percentage of the retained radioactivity in nitrocellulose filters was represented as a function of vp concentration (fig. a) . a plateau of vp -rna binding was observed at vp concentrations above − mm. the vp -rna dissociation constant was derived from binding curves, and it was estimated from independent experiments to be of ± . × − mm (n = ; spearman's coefficient of variation, cv = %). similar results were obtained with different ssrna probes (data not shown). a hill plot for the binding of vp to ssrna was derived (fig. b) , calculating a regression line with a correlation coefficient (r = . ; degrees of freedom; t = . ) -associated probability (p) of . × − , showing a very significant correlation of data to a linear relationship. the hill coefficient (n h ) derived from the regression line was . (fig. b) , slightly lower than , showing that vp bound ssrna in a non-cooperative manner. the k . was calculated from the regression line, giving a result of . × − mm, close to the estimated apparent kd (fig. b) . the experiments described above were performed using probe b that contained the and utrs from ibdv genome segment b. competition experiments were performed to find out whether the vp -rna interaction was sequence-specific. for this, increasing concentrations of different unlabeled rna or dna competitor probes were included in the vp /rna reaction mixtures. after incubation, samples were filtered though nitrocellulose membranes, and the amount of retained radioactivity estimated. as expected, binding of vp to labeled ssrna probe b was efficiently competed by the addition of unlabeled probe b (fig. a) . a similar result was obtained when ssrna probe a was used (fig. b) . nonviral ssrna fig. . inhibition of vp binding to labeled ssrna probes by unlabeled competitor rnas. a graphs represent the percentage of retained radiactivity in nitrocellulose membranes from complexes formed by vp and labeled ssrna probe b, as a function of the ratios of unlabeled ssrna probes b (left graph) or pl (right graph) to labeled probe b. data is represented as the mean of three independent experiments together with error bars. b graphs represent the percentage of retained radiactivity in nitrocellulose membranes from complexes formed by vp and labeled ssrna probe a, as a function of the ratio of unlabeled ssrna probes a (left graph) or gfp (right graph) to labeled probe a probes pl and gfp also competed with probes b and a, respectively ( fig. a and b), indicating that vp binding to ssrna was sequence-independent. ibdv contains a dsrna genome probably in close contact with vp molecules in ibdv virions [ , ] . to find out whether vp presented a binding activity to ibdv dsrna, competition experiments using purified ibdv dsrna were performed. as shown in fig. , ibdv purified dsrna segment a efficiently competed the binding of vp to labeled ssrna probe a. this also suggested that vp probably bound ssrna and dsrna through the same interaction domain. interestingly, dsdna did not significantly compete the binding (fig. ) . single-stranded dna efficiently competed with the binding of vp to probe a, showing that vp also bound ssdna (fig. ) . in order to identify the vp protein region involved in ssrna-binding, a set of mutants with deletions of about aa covering the whole protein was generated (fig. a ). vp deletion mutants were expressed and purified as described in materials and methods. after purification, proteins were analyzed and quantified by sds-page and coomassie staining (fig. b) . interestingly, except for the vp - deletion mutant, the samples contained two vp electrophoretic forms, resulting in doublets in sds-page gels (fig. b) . bands corresponding to higher molecular mass species migrated according to their expected molecular masses. estimation of the molecular masses of the lower protein bands indicated a consistent reduction of about . kda compared to the expected size (fig. b) . northwestern analyses showed that both wild-type-derived vp forms interacted with labeled ssrna probes (data not shown). the affinity of each mutant protein to ssrna was determined by membrane filter binding assays, using probe b, as described above. the amounts of the purified deletion mutant proteins used in these assays were normalized according to their respective molecular masses. identical molar amounts of each mutant protein were used in the assays. the ssrna-binding activity of wt vp was estimated to have an apparent kd of ( ± . ) × − mm (n = , cv = %) (figs. and ) . deletion mutants vp - and vp - presented a slightly reduced affinity compared to that of wt vp . carboxy-terminal deletion mutants showed an affinity similar to that of the complete vp protein, indicating that the ssrna-binding domain was not affected by these deletions (fig. ) . in contrast, mutants presenting deletions at the region between residues and showed significantly lower affinities to ssrna, up to -fold reduction in the case of vp - deletion mutant. these results indicated that the vp ssrna-binding domain was located within a region comprising from residues to (fig. b) . to investigate whether the identified ssrna-binding domain presented highly conserved sequence features, a comparison was performed using the polyprotein sequences of representative species from the family birnaviridae. the alignment and comparison of vp protein sequences revealed that in the region implicated in rna-binding, strains from the genus avibirnavirus contained a aa sequence absent in their counterparts from the aquabirnavirus genus (fig. ). in addition, sequence comparisons showed two absolutely conserved residues, arg and gly , within this region. a highly hydrophobic region, including the conserved gly (g-fa-p-w-a-n) was also found between residues and (fig. ) . interestingly, when this region was deleted from vp (vp - ), the ssrna-binding activity was most severely impaired ( fig. ) , suggesting that this region might play a critical role for ssrna-binding. other highly conserved short sequences were also found, but rna or dna binding motifs previously described in the literature such as zinc fingers, arginine-rich regions or rg boxes, were not found within vp [ ] . whether this region contains a previously unpublished rna-binding motif, or if it comprises a double-stranded rna-binding motif [ ] is currently under investigation. a detailed mutagenesis study is being carried out to identify which of the conserved regions in the rna-binding domain is directly involved in rna-binding. capsids of dsrna viruses having a life cycle including an extracellular phase contain two or more protein layers [ , ] . strikingly, capsids of members of the birnaviridae family are much simpler. they contain a single protein shell [ , ] . this unique feature makes especially interesting the study of both the molecular architecture and the morphogenesis of birnaviruses. the inner surface of the ibdv particle is coated by y-shaped vp trimers in close contact with the continuous shell formed by vp [ , ] . although the precise capsid location of the putative rdrp, vp , has not been established yet, it has been shown that it directly interacts with vp [ , ] . therefore, vp appears to play a crucial structural role. it has been suggested that vp might interact with the dsrna genome segments [ , , ] . indeed, its presence in ribonucleoprotein complexes isolated from disrupted ipnv virions [ ] , makes it a good candidate for playing a role in genome packaging and/or stabilization. it was therefore interesting to analyze the ability of vp to interact with rna. we decided to undertake this analysis using a histidine-tagged version of the protein produced in insect cells infected with rbvs. the protein obtained under these conditions was highly pure and soluble, thus enabling us to carry out a number of highly specific assays. rbv-expressed vp exhibited a ssrna-binding activity as determined by northwestern and emsa assays. however, extracts from cells infected with wildtype baculovirus also contained an rna-binding protein. the presence of this either cellular or viral protein prevented further studies on the vp rna-binding properties, especially for kd estimations. for this reason further studies were carried out with affinity-purified vp . vp presented a ssrna-binding activity with an approximate kd of ( ± . ) × − mm as directly determined from vp -rna binding curves. vp -rna binding curves were also represented as a hill plot, in order to calculate kd and the hill coefficient. the calculated n h ( . ) for vp -ssrna binding was slightly lower than . however, it really cannot be considered as negative cooperativity, but rather as non-cooperative binding. an alternative derivation of kd by calculating k . in the hill plot also agreed with the estimated kd for vp -ssrna binding. this kd value falls within the range of other viral and cellular nucleic acid-binding proteins [ , , ] . interestingly, the emsa assays revealed at least three vp -ssrna complexes with different electrophoretic mobilities. two possibilities could explain the presence of the multiple shifted bands observed: i) vp could contain more than one rna-binding domain; and ii) vp could form oligomers that bind rna. the most abundant complex corresponded to the faster migrating band, even when extremely low concentrations of vp were used in the assays. this observation suggested that most likely the faster migrating band was formed by vp monomers. moreover, the binding of vp to ssrna in northwestern assays showed that vp monomers generated under denaturing conditions bound ssrna. the ability of vp to form oligomers has been assessed using a two-hybrid system approach [ ] , and the structural analyses of ibdv particles and vlps have revealed that vp molecules form trimers in the internal surface of the capsid [ ] . therefore, it is likely that vp might also form trimers under the conditions used for rna-binding studies, explaining the presence of lower mobility shift bands when vp concentration was increased. however, the ability of recombinant vp to form trimers has not been tested in this work. competition experiments revealed that vp bound ssrna in a sequenceindependent manner. the lack of any binding specificity to ssrna probes used in this study might indicate that, as it is the case with other virus-encoded nucleoproteins [ , , , ] , other factor(s) could be necessary to confer specificity. vp , the putative ibdv rdrp, is the most likely candidate for this role. interestingly, vp bound dsrna and ssdna, apparently through the same ssrna-binding domain, but not dsdna. these binding characteristics are usually found in rna-binding proteins, especially in dsrna-binding proteins [ ] . the ability of vp to interact with ssrna and dsrna could reflect the possibility that genome replication and transcription, assumed to be carried out by vp , could be assisted by vp binding both to ibdv ssrna and dsrna. in order to identify the vp protein domain involved in rna-binding, a series of deletion mutants were expressed and purified. interestingly, two protein forms were observed for each mutant, except for vp - . this is consistent with a proteolytic processing at the vp carboxy terminus, since vp - deletion mutant lacking the c-terminal residues only presented a single band of the expected molecular mass (fig. ). vp presents two electrophoretic forms in ibdv-infected cells. vp doublets have been described in previous reports. however the nature and function of these forms remain unknown [ ] . interestingly, only the full length vp protein is incorporated in ibdv virions, suggesting that the shorter form might have additional, non-structural, functions. it could also be possible that cellular proteases such as caspases [ ] could be involved in vp processing. alternatively, vp could exhibit an autoproteolytic activity not previously reported. the kd for each vp deletion mutant was determined, and it was found that deletions affecting to the vp region between residues to abrogated vp ssrna-binding activity. the loss of ssrna-binding activity exhibited by these vp deletion mutants was not due to loss of protein solubility, since in all experiments vp mutants were soluble. although deletions could affect the vp structure, this is unlikely. small deletions were introduced to avoid dramatic changes in protein structure. additionally, only deletions between residues and did have an effect on ssrna-binding activity, while the rest of the mutants efficiently bound ssrna. interestingly, ssrna-binding was severely impaired by the deletion of a short, highly conserved sequence between residues - . whether this region is directly involved in binding to ssrna and dsrna, is a subject of investigation. to our knowledge, this is the first time that an rna-binding activity has been demonstrated for a birnavirus vp protein. vp protein bound ssrna and dsrna, exhibiting no specificity for virus-derived ssrna probes. the identification of critical residues for rna-binding activity within vp rna-binding domain is currently under investigation. the implications of vp rna-binding activity are also being investigated in the context of the virus replication and transcription. nuclear factor i family members regulate the transcription of surfactant protein-c adding the third dimension to virus life cycles: three-dimensional reconstructions of icosaedral viruses from cryoelectron micrographs relationships between the magnitude of hill plot slopes, apparent binding constants and factorability of bindings polynomials and their hessians a non-canonical lon proteinase lacking the atpase domain employs the ser-lys catalytic dyad to exercise broad control over the life cycle of a double-stranded rna virus 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and virulence seroprevalence of infectious bursal disease virus in free-living wild birds in japan in vitro recombination and mutagenesis by overlap extension pcr linear correlation a major structural protein of ibdv sequence-specific interaction between hiv- matrix protein and viral genomic rna revealed by in vitro genetic selection infectious bursal disease and hemorrhagic enteritis proteolytic processing in infectious bursal disease virus: identification of the polyprotein cleavage sites by site-directed mutagenesis bioinformatics: a practical guide to the analysis of genes and proteins quantitative analysis of the binding of turnip crinkle virus coat protein to rna fails to demonstrate binding specificity but reveals a highly cooperative assembly interaction interactions in vivo between the proteins of infectious bursal disease virus: capsid protein vp interacts with the rna-dependent rna polymerase, vp stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells infectious bursal disease in western australia induction of apoptosis in vitro by the -kda nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis we are grateful to j. castón and a. maraver for critical reading of the manuscript; to s. gonzalez and p. gastaminza for interesting and helpful discussion; to h. müller for anti-vp monoclonal antibodies; a. maraver for plasmid tv - ; to e. camafeita for protein analyses.this work was supported by grants bio- - from the comisión interministerial de ciencia y tecnología, b/ / from the subdirección general de investigación of the comunidad autónoma de madrid. key: cord- -ddcz zck authors: yang, jin; fang, mei-xin; li, jie; lou, guo-qiang; lu, hang-jun; wu, nan-ping title: detection of hepatitis c virus by an improved loop-mediated isothermal amplification assay date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ddcz zck an improved, sensitive, specific, and rapid one-step reverse transcription loop-mediated isothermal amplification (lamp) assay targeting the ′ untranslated region (utr) was developed to detect hepatitis c virus (hcv) infection. based on an accelerating primer (ap), the present assay, named ap-lamp, has the advantages of rapidity and sensitivity over the routine lamp method. the possible ap-based amplification pathway during the reaction was revealed by restriction enzyme digestion and eletrophoresis. the detection limit of the ap-lamp assay was approximately iu/ml, and no cross-detection was observed. the assay was evaluated further with clinical specimens, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for detection of hcv rna. the hepatitis c virus (hcv) pandemic has become a major public health concern, with such increasing prevalence that nearly million individuals are infected worldwide [ ] . the majority of acute hcv infections present an asymptomatic course. many infected individuals are therefore not seen in a medical setting [ ] . nearly % of infected people develop persistent infection and are at risk of longterm complications, ranging from mild liver damage to severe chronic hepatitis that can develop into cirrhosis, end-stage liver disease, or hepatocellular carcinomas [ ] . therefore, a rapid and accurate diagnosis of hcv is important for the prevention of viral transmission and management of disease progression. screening of antibodies against hcv, however, is not a reliable method of diagnosing acute hcv infection, since the appearance of antibodies against hcv can be delayed in up to % of patients at the onset of symptoms [ ] . moreover, the window period can be even longer in immunocompromised patients, who need to be screened routinely for hcv viremia [ ] . nucleic-acid-based detection techniques are currently the most reliable methods for detecting hcv infection. a variety of molecular diagnostic assays, such as reverse transcriptase pcr [ ] , nucleic-acid-sequence-based amplification [ ] , transcription-mediated amplification [ ] , branched-chain dna assay [ ] , and in-house realtime pcr [ ] , have been developed for the detection of hcv rna. these assays, whether qualitative or quantitative, are relatively time-consuming, labor-intensive, and dependent on specialized equipment. in resource-limited or point-of-care settings, the cost and technology requirement limit their universal application. since most hcv-infected individuals are asymptomatic, there are clear advantages to targeted screening for hcv in those who are at high risk. earlier detection of infection results in earlier treatment and thus earlier recovery [ ] . for this reason, there is still a great need for a tool to simplify the detection of hcv rna with acceptable sensitivity and specificity, short turnaround time, and cost-effectiveness. loop-mediated isothermal amplification (lamp) is a novel rapid, accurate, and economical nucleic acid test [ ] . the method is characterized by employing a dna polymerase with strand-displacement activity, along with two inner primers (fip, bip) and two outer primers (f , b ) to form auto-cycling immediates. loop primers (lf, lb), first described by nagamine et al, could accelerate and enhance the sensitivity of the lamp assay [ ] . onestep lamp assays have been successfully applied to the rapid detection of a number of rna viruses, such as influenza virus [ ] , mumps virus [ ] , west nile virus [ ] , severe acute respiratory syndrome corona virus [ ] , and hiv- [ ] . lamp assays have also been developed to detect hepatitis viruses, such as hepatitis b virus [ ] , hepatitis a virus, and hepatitis e virus [ ] . at present, however, no hcv detection assay using this method has been reported. the attributes of the hcv target may interfere in complex ways with the lamp method. for instance, although the viral untranslated region ( -utr) is thought to be the most conserved portion of the hcv genome and is targeted by almost all of the commercial and in-house tests [ ] , one of the most important issues may be the existence of complex secondary structure across all of this region (fig. b) . also, the -utr is generally considered to be variable enough to distinguish all of the major types and many subtypes of hcv [ ] . in the study described here, we have developed a modified lamp method for rapid and economical detection of hcv rna. in contrast to the routine lamp (pre-lamp) method, we designed an accelerating primer (ap) and tested the performance of the ap-based lamp assay (ap-lamp). the new assay was further evaluated using clinical samples. clinical specimens and standard twenty-five blood samples collected from patients with confirmed chronic hcv infection and specimens obtained from patients suspected of having viral hepatitis who were admitted to hangzhou th and infectious hospital were used for evaluation in this study. confirmed cases of hcv infection were verified by a positive result in an enzyme-linked immunosorbent assay (kehua bio-engineering, shanghai, china) for antibodies against hcv or a quantitative real-time pcr for hcv rna. a panel of samples collected from healthy blood donors was also included as negative controls. in addition, five each of anti-hiv-antibody-, anti-hav-antibody-and hbv-dna-positive samples obtained from the corresponding patients were also tested. informed consent was obtained from all patients, and the study was approved by the local ethical committee, as per the declaration of helsinki ( ). five milliliters of blood was collected from each subject in a tube containing ll of . % edta. plasma was immediately separated after centrifugation at rpm for the in-house hcv rna standard was obtained by extracting rna from a hcv-rna-positive specimen with an hcv titer of iu/ml. this in-house standard was anti-hcv antibody positive and calibrated in triplicate in parallel with the national hcv rna reference material (gbw , . iu/ml - . iu/ml, genotype ) using several commercial real-time pcr tests (kehua bio-engineering, shanghai, china; daan biotech, guangzhou, china). the gbw panel was calibrated using the who hcv international standard (nibsc / ) [ ] . hcv genotyping was performed using sequencing of the ns b region of the hcv genome as described previously [ ] . sequence analysis indicated that the hcv genotype of the in-house standard was b. serial dilutions of the standard sample for experimental analysis were prepared in normal human plasma and stored at - °c until testing. viral rna was extracted from ll of plasma using a tianamp virus rna kit (tiangen biotech, beijing, china) as per manufacturer's instructions. this extraction protocol used a fast spin-column procedure. rna was eluted in ll of rnase-free water. the whole extraction procedure was done within an hour. to design an assay that can detect most of the genotypes of prevalent hcv strains, individual sequences of the utr region of hcv strains from the hcv database (http://hcv.lanl.gov/) were retrieved. through alignment analysis, the conserved fragments of the utr were used to design the primer set. the hcv genotype b sequence (genbank accession number ay ), chosen as a representative strain, was used as a reference for generating the set of primers. all of the primers, including two outer (f and b ), two inner (fip and bip) and two loop primers (lf and lb), were designed according to the guideline provided by primerexplorer v (http://loopamp.eiken.co.jp/). in this set, fip consisted of f c, complementary to the f sequence, and f sequence, and bip consisted of the b sequence and b c, complementary to the b sequence. f and b were located outside f and b , while the loop primers recognized the region between f and f , or b and b . to strengthen the power of lamp, an additional accelerating primer (ap), located between f and b , was added to the primer set. a schematic representation of the locations of the primers along with a representative alignment of the main hcv strains is shown in fig. . the details of the oligonucleotide primers used for the amplification are given in table . all primers were synthesized by invitrogen (invitrogen, shanghai, china). the routine one-step lamp (pre-lamp) reactions were carried out in a final volume of ll containing pmol each of the fip and bip inner primers, pmol each of the lf and lb loop primers, pmol each of the f and b outer primers, thermopol buffer (new england biolabs, beverly, ma), mm mgcl , m betaine (sigma aldrich, usa), . mm each deoxynucleotide triphosphate, u of bst dna polymerase (new england biolabs, beverly, ma), . u of amv reverse transcriptase (takara, dalian, china), and ll of template. the mixture was incubated at °c for min and then heated to °c for min to terminate the reaction. for the ap-lamp assay, the reaction mixture and the conditions were the same as those described for pre-lamp, except that pmol of ap was added to the reaction mixture. a negative control was included for each lamp run. serial dilutions of the standard were used as templates for the lamp assay to evaluate its sensitivity. the specificity of the assay was tested with samples from hav-, hbv-, and hiv-infected patients and healthy donors. after amplification, the amplified dna products were analyzed by electrophoresis in a . % agarose gel stained with ethidium bromide and visualized using a bio-rad transilluminator. the restriction enzymes nhei and smai (new england biolabs, beverly, ma) were used to digest amplified products to confirm amplification specificity. digested products were analyzed by gel electrophoresis on a . % agarose gel. for naked-eye visualization, one microlitre of diluted sybr green i (invitrogen, carlsbad, ca) was added to the reaction tube after amplification, and the reaction was observed visually. for a positive reaction, a change in the color of the reaction solution from orange to fluorescent green could be recognized. for real-time monitoring of the lamp reaction, the reaction was performed on an abi prism ht sequence detection system, with sybr-green i (invitrogen, carlsbad, ca) added to the reaction mixtures to provide the fluorescent signal. the run was set up as follows: cycles of min at °c ( cycle corresponding to min of reaction), with fluorescence reading at the end of each of these cycles. a commercial hcv rna real-time pcr detection kit (kehua bio-engineering, shanghai, china) was used according to the manufacturer's instructions. as the template, . ll of rna extract was used in a -ll reaction. the clinical sensitivity of this quantitation kit was iu/ml [ ] . to design a lamp primer set covering the main genotypes of hcv isolates, a multiple sequence alignment, including genotypes - , was generated by retrieving and aligning the sequences stored in the hcv databases (http://www.hcv. lanl.gov). the primers were designed to maintain maximum conservation for annealing to the target regions. mismatches at the ' or ' ends of fip/bip were substituted by degenerate bases (table ) . a database search using blast from ncbi showed that all of the primers were specific for the hcv genome. the routine lamp format (pre-lamp) for the hcvspecific assay was performed by using rna templates extracted from standard samples. the amplified dna products were subjected to electrophoresis on . % agarose gels and visualized under uv light after ethidium bromide staining. as a result, a typical lamp laddering pattern was observed, indicating the different replication intermediates of the stem-loop amplification process, while no bands were obtained from the no-template control. eletrophoresis-based monitoring of the pre-lamp product showed a sensitivity of iu/ml ( fig. a) . to test the specificity of the test, the amplified product was digested with the enzyme nhei, resulting in the detection of strong bands (fig. b, lanes ) . furthermore, the possibility of crossreactivity with other viruses known to cause similar clinical signs was also investigated. no amplification of any viral rna (or dna) extracted from hbv-, hav-, or hivpositive samples was detected (fig. e) . to improve the efficiency of detection, an accelerating primer (ap) was designed to form an additional synthesisstarting site. different from the loop primer, which binds the single strand in the f -f or b -b region in the classical lamp [ , ] , the ap designed here is complementary to one of the double-stranded regions between f and b (fig. a) . adding ap to the pre-lamp reaction, the assay, named ap-lamp, was carried out to evaluate its performance. serial dilutions of the templates were amplified by the ap-lamp assay. as shown in fig. , the one-step ap-lamp assay had a detection limit of a iu/ml of rna template. specificity tests, including restriction enzyme analysis and the use of negative samples, were also conducted, and these showed no crossreaction. since visualization of amplification products from lamp reactions without special equipment would make the assay widely applicable and available, sybr green i was added to the reaction mixtures, resulting in a color change from orange to green. the amplified products yielded a green color in positive ap-lamp reactions, demonstrating that the sensitivity of this assay is equal to that of eletrophoresis (fig. b ). given that the sensitivity of the ap-lamp assay for detecting hcv is higher than that of the pre-lamp method, the pathway of ap-based amplification was investigated. a dumbbell-like intermediate is the initial auto-cycling product for the subsequent amplification steps in the lamp assay. apart from the classical lamp pathway that is followed when using fip and bip, as described elsewhere [ ] , the ap pathway involves the synthesis via ap priming to promote elongation followed by fip self-priming. the characteristic feature of the products of the ap pathway is that the end products are partly derived from the concatenation of ap-fip fragments (fig. ). more importantly, if the ap pathway is followed in the assay, it is logical to speculate that the amplification would still occur without the outer primer (named ap-b lamp in this study), which is strictly required in the classical lamp [ ] . three lamp formats are shown in fig. a . to test this hypothesis, we compared the pre-lamp, ap-lamp, and ap-b lamp assays using the same templates. a panel of serial log dilutions of hcv rna of known concentration was tested using the ap-lamp, pre-lamp, and ap-b lamp assays on the real-time ht platform. the results for each dilution tested in batches of three replicates in two separate runs are given in table . for the ap-lamp assay, the average threshold time (tt) required to detect a positive signal ranged from . ± . min (mean ± sd) when iu rna was present to . ± . min when iu rna was present, compared to . ± . - . ± . min in the pre-lamp assay and . ± . - . ± . min in the ap-b lamp assay. the tt value was defined as the reaction time necessary to achieve a positive signal above the baseline [ ] . these results demonstrated that the ap-lamp assay was faster by - min than the pre-lamp reaction and was much faster than real-time pcr. furthermore, using the assays to analyze low-concentration standards ranging from to iu, no amplification was obtained when the template concentration was less than iu/ml in the pre-lamp assay. the real-time ap-lamp assay consistently detected hcv rna at levels of iu/ml, while only three of six ap-b lamp assays detected hcv rna. by probit regression analysis, the ap-lamp method detected iu/ml with [ % probability of a positive result, as compared to iu/ml and iu/ml for the pre-lamp and ap-b lamp assay, respectively. therefore, the sensitivity of the ap-lamp assay for detecting hcv is about two times higher than that of the ap-b lamp assay and nine times higher than that of the pre-lamp assay. when comparing the electrophoresis bands of the products of the ap-lamp, ap-b lamp and pre-lamp assays, the first two showed a similar pattern. the main difference between the ap-lamp and pre-lamp assays is that there were ladder-like bands between and bp. the bands in this region (in rectangles with a broken line, fig. ) are likely to correspond to the self-primed amplification product bounded by the ends of the ap and fip stemloop structure (* bp in size), which match the expected size of the ap-pathway product. moreover, the result of the ap-b lamp assay provided evidence that the ap pathway is used during amplification. several reports have indicated that the lamp assay strictly requires the strand displacement function of the outer primers [ ] . no lamp amplification occurs when fip, bip, f or b is absent [ ] . by using the ap in the assay, this study using real-time monitoring or agarose gel electrophoresis confirmed that the outer primer . the extracted rna template was prepared from the standard plasma and was subjected to analysis by the pre-lamp and ap-b lamp methods. a pre-lamp assay using serial dilutions from iu/ml to iu/ml. b ap-b lamp assay using template concentrations from iu to iu/ml. c lowconcentration standards ranging from to iu/ml, detected by the pre-lamp assay. d the same standards ranging from to iu/ml, detected by the ap-b lamp reaction. the dashed box indicates the band pattern with the most significant difference between the two assays is not required. the ap-b lamp assay has higher sensitivity than the pre-lamp assay, but it is less sensitive than ap-lamp. this assay type also showed lower stability than ap-lamp, especially when the samples were at low concentration. in addition, taking into account the similar band patterns between the ap-lamp and ap-b lamp assay, these results indicate that ap-priming-based amplification is inferior to the classical pathway in the lamp process. the amplified products were digested with two restriction endonucleases to confirm the specificity and structure of the amplified products from three different lamp assays. the restriction enzyme smai recognizes the sequence between f and b , while nhei cuts between b and b . if the products were amplified specifically and formed the structures shown in fig. a , nhei digestion would yield fragments of , , , and bp, and smai digestion would yield fragments of , and bp. since the amplified product is a concatenation of dna fragments of different sizes, the amplification kinetics probably affect the actual end products of the lamp reaction. due to cutting in the region between b and b , nhei-digestion bands were observed at approximately - ( ? ) bp in the pre-lamp product, in contrast to - bp in the ap-lamp and ap-b lamp products. while cutting the region between f and b , the sizes of the fragments generated by smai digestion were approximately and bp (fig. d) , which is in good agreement with the predicted sizes. the feasibility of the ap-lamp assay for detecting hcv in clinical material was assessed by using both positive and negative plasma specimens. the real-time pcr assays were performed simultaneously, and the results of both (table ) . of the samples collected from healthy volunteers, all tested negative in both ap-lamp and real-time pcr. a total of samples were obtained from chronic hcv patients (genotype b, n = ; genotype a, n = ; all anti-hcv positive), with the viral load ranging from . to . iu/ml. none of the samples were missed by the ap-lamp assay. of acute-phase samples collected from patients suspected of having viral hepatitis, three were anti-hcv positive, and these were also detected by ap-lamp. of the remaining anti-hcv-negative cases, only two were positive for hcv rna by ap-lamp. these two patients later seroconverted after and months, respectively ( table ). the ap-lamp gave a total of ( . %) positive results, and the same result was obtained by real-time pcr. in total, the ap-lamp method demonstrated % agreement with real-time pcr when used for analysis of clinical samples. these preliminary results suggest that the ap-lamp assay described here can be applied for the diagnosis of hcv infection in a clinical setting. among the nucleic acid amplification tests available to date, lamp method has many characteristics that make it suitable for the rapid, sensitive, and simple detection of pathogens [ ] . the adaptation of the lamp technology for hcv detection in point-of-care or resource-limited setting has many potential advantages. for examples, the reaction occurs under isothermal conditions and thus does not require special equipment. the powerful amplification efficiency of the lamp assay makes it extremely rapid, and it exhibits high analytical sensitivity. furthermore, the end product can be observed immediately by visual observation, through turbidity or dye staining [ ] . up to now, however, no hcv nucleic acid test has been available outside of the laboratory setting, possibly due to time, cost and technology limitations. the use of multiple primer combinations is one of the key features of the lamp method, but this can affect the primer selection for a given template, such as the hcv 'utr, which has a complex ordered secondary structure and genotypic variation sites simultaneously. we devised an accelerating primer to improve the efficiency of amplification. just like the loop primer in lamp, the ap provides an additional starting site for dna synthesis (fig. ) during the amplification, thereby reducing the overall reaction time and increasing the sensitivity. it should be mentioned, however, that in the lamp reaction, the loop region is always in a single-stranded state during the process. in contrast, the ap is located in the doublestranded region and is complementary to one of its strands. when comparing the performance of the ap-lamp and pre-lamp assays, higher amplification efficiency was found in the former. a possible explanation for this is that apart from the classical amplification process in lamp assay, there is an ap-based amplification pathway in the ap-lamp method. this notion is supported by the following evidence: first, by comparing the band patterns of ap-lamp and pre-lamp, the products of the ap pathway (ap-fip) were observed in the former. next, the ap-b lamp assay still amplified the target in the absence of outer primer b . in the classical lamp format, the outer primer is important for strand displacement to form an auto-cycling intermediate product. no amplification occurs without this primer [ ] . finally, by the digesting the end products with restriction enzymes that recognize different sites in the target, the most favorable structure of the amplified products was found by length polymorphism analysis, as shown in fig. . it is well known that primer design in lamp is more complex and difficult than that in pcr [ ] . since lamp reaction efficiency and sensitivity strongly depend on primer selection, a more flexible way for lamp primer design could promote the use of this method. summing up the above points, the ap strategy could be applied in lamp design to meet special demands under certain conditions. for instance, because both ends of the fip/bip secondary structure play a key role in amplification cycling in the lamp-based assay, using an ap could reduce the problem of selecting fip/bip. adding ap to the principle of ap in lamp amplification. a the three lamp formats in this study. the primers commonly used in the lamp assay (pre-lamp) include inner primers fip and bip, outer primers f and b , and loop primers lf and lb. the loop primer anneals the partially single-stranded portion. the ap devised in this study is the additional accelerating primer located between the f and b fragments. the ap-b lamp assay does not use the outer primer b . b the cyclic amplification step for the ap pathway is illustrated: the dsdna reaches a dynamic equilibrium at °c, and thus the ap can bind the partly free ' end of the template to initiate strand extension. complementation of the hairpin structure (f -f c) induces self-primed dna synthesis pre-optimized lamp assay could avoid the need to design different primer sets for optimization. the performance of ap-lamp was investigated in this study. running the ap-lamp assay in a real-time pcr machine consistently achieved a lower limit of detection of iu/ml by probit analysis. this sensitivity is comparable to a series of in-house tests published recently [ - , , ] . although this method is essentially qualitative at the outset, the sensitivity limits represent good performance, since the hcv plasma viremia in acute infections is generally higher than copies/ml [ ] . as expected, a specificity test using seronegative samples and other nontargeted virus samples demonstrated % exclusivity of the assay. by applying the ap-lamp assay to clinical specimens, there was % agreement between ap-lamp and the real-time pcr test. the ap-lamp method consistent detected hcv-infected samples with a broad range of viral loads. since the samples comprised the hcv genotypes b, a, which are prevalent in china [ ] , this ap-lamp assay is expected to work for the majority of hcv-infected individuals in the local region. because of the high mutation rate of the hcv 'utr, it is not easy to generate a single lamp primer set to detect every viral strain of an individual subtype. degenerate design is most the common way to address the issue of genotype inclusivity, but this may lower the diagnostic sensitivity due to a hybridization effect. for this reason, the ap strategy developed here would potentially be applicable in a situation like this. future evaluation of detection efficiency using an extensive collection of hcv genotypes and larger samples would be desired to validate the performance of the assay. in addition to the high sensitivity and specificity of the ap-lamp assay, its other major advantages are its rapidity and the flexibility of its detection method. the ap-lamp assay itself could be carried out in less than min. only . h (including the extraction step) was needed to perform the lamp assay, compared to . - h for the real-time pcr assay. amplification in the lamp assay can be detected with the naked eye in the form of visual fluorescence, e.g., the original orange color of the dye changes to green under natural light in the case of a positive amplification reaction [ ] , thus eliminating the need for gel electrophoresis or real-time monitoring. our results, as well as the results of previous studies using a fluorescent reagent to detect the lamp product visually [ , ] , showed a similar detection efficiency when compared to real-time pcr or electrophoresis. therefore, this hcvspecific lamp assay may be applicable under clinical or field conditions. in conclusion, as demonstrated using hcv, we have developed a lamp test using a novel principle based on an accelerating primer and have provided an alternative way to design a lamp assay for a complex target. this study presents a sensitive and specific lamp method for screening for or confirming infection with hcv in a simple, rapid, and cost-effective manner. loop-mediated isothermal amplification assay for hcv national institutes of health consensus development conference statement: management of hepatitis c real-time quantitative lamp (loop-mediated isothermal amplification of dna) as a simple method for monitoring ammonia-oxidizing bacteria natural history of chronic hepatitis c virus infection an in-house method for the detection and quantification of hcv in serum samples using a taqman assay real time pcr approach taqman amplification system with an internal positive control for hcv rna quantitation hiv- and hcv detection in dried blood spots by sybr green multiplex real time rt-pcr sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of hiv- quantitation of hepatitis c virus using an in-house real-time reverse transcriptase polymerase chain reaction in plasma samples a novel diagnostic target in the hepatitis c virus genome loop-mediated isothermal amplification integrated on microfluidic chips for pointof-care quantitative detection of pathogens dynamics of viremia in early hepatitis c virus infection design of an antisense reverse-transcriptasepolymerase chain reaction primer efficient for all hepatitis c virus genotypes: comparison of its performance vs a commercial primer evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents sequence analysis of the ' untranslated region in isolates of at least four genotypes of hepatitis c virus in the netherlands reverse transcription-loop-mediated isothermal amplification assay for rapid detection of hepatitis e virus detection and quantification of serum or plasma hcv rna: mini review of commercially available assays hepatitis c virus genotype distribution in china: predominance of closely related subtype b isolates and existence of new genotype variants a novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis c viral rna with armored rna as internal control loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases use of sequence analysis of the ns b region for routine genotyping of hepatitis c virus with reference to c/e and ' untranslated region sequences accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna acute hepatitis c: prevention and treatment realtime reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases comparison of conventional pcr with real-time pcr and branched dna-based assays for hepatitis c virus rna quantification and clinical significance for genotypes to loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products hcv screening to enable early treatment of hepatitis c: a mathematical model to analyse costs and outcomes in two populations rnase-resistant virus-like particles containing long chimeric rna sequences produced by two-plasmid coexpression system real-time quantitative assay of hcv rna using the duplex scorpion primer mumps virus reinfection is not a rare event confirmed by reverse transcription loop-mediated isothermal amplification the authors declare that they have no conflict of interest. key: cord- -txryqil authors: kulka, m.; chen, a.; ngo, d.; bhattacharya, s. s.; cebula, t. a.; goswami, b. b. title: the cytopathic f strain of hepatitis a virus induces rna degradation in frhk cells date: journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: txryqil the mechanism responsible for the induction of apoptosis by the rapidly replicating hm / f strain of hepatitis a virus (hav) was investigated. full length hav rna and viral capsid protein vp were detected in f infected cells at earlier times post-infection than in hm /clone infected cells. analysis of total cellular rna from hm / f infected frhk cells by denaturing agarose gel electrophoresis and northern blot hybridization revealed extensive degradation of both the s and s ribosomal rna (rrna) molecules. similar degradation was observed when these cells were infected with human coxsackievirus b , a fast replicating enterovirus. in contrast, the parental strain of f, hm /clone did not induce rna degradation. inhibition of rna degradation correlated with inhibition of virus replication. the pattern of rrna degradation resembled degradation of rrnas by rnase l, an enzyme activated in interferon-treated cells following infection with certain viruses. ribosomal rna degradation was accompanied by the reduction in the levels of several cellular rnas including those for β-actin and glyceraldehyde- -phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. interferon mrnas could not be detected in either infected or mock-infected control cells, and stat , a key regulator of interferon action was not phosphorylated following virus infection. these results reveal a heretofore-undescribed pathway that involves the regulation of rna degradation and apoptosis following hav/ f replication in frhk cells. hepatitis a virus (hav), the only member of the genus hepatovirus within the picornaviridae family, is a major cause of infectious hepatitis worldwide [ ] . the virus is characterized by its resistance to heat, acid ph, polar solvents, and common disinfectants [ ] . it is primarily transmitted by the fecal-oral route and is a principal agent of localized food-borne outbreaks of infectious hepatitis in developed countries [ ] . the virus contains a single (+) stranded rna genome that is approximately . kb long and contains a poly (a) tail. the end is uncapped but covalently linked to the viral coded protein vpg [ ] . the viral genome has been cloned and sequenced from a number of strains. it codes for a single polyprotein, which is subsequently cleaved by the viral protease c and possibly cellular proteases into mature viral proteins [ , , ] . strains of hav can be grouped into three categories based on their distinctive growth characteristics in permissive cells: i) wild-type (wt) strains, defined by a long incubation period between infection and detection of replication and a cytopathic effect (cpe) is not observed; ii) tissue culture adapted (cc) strains, defined by shorter incubation periods than for wt strains and cpe is not observed; and iii) tissue culture adapted (cp) strains, defined by shorter incubation periods than for cc strains and cpe is observed. the cp strains arise during repeated passage of cc strains in permissive cells [ , , , , , , ] . although the molecular basis for the development of the cp phenotype is poorly understood, it is well documented that both viral genome replication and viral protein expression can be detected much earlier in permissive cells infected with cp strains such as hm / f, hm / a and hm /hb . , than in cells infected with the parental cc strains from which they were derived [ , , , , ] . the rapid replication and viral gene expression observed for cp strains has been attributed to a more efficient initiation of translation from the ires (internal ribosomal entry site) of cp strains due to mutations in this region [ , , ] , though some have disputed this interpretation [ , ] . apoptosis is observed following infection by a number of picornaviruses, especially when virus replication is restricted [ , , , ] . while cell killing by apoptosis may aid in spreading progeny virus or escape cellular defense mechanisms [ ] , premature induction of apoptosis may limit virus growth and spread [ , , ] . the cytopathic effect that is observed in cells following infection with cp strains of hav has been attributed to the induction of apoptosis [ , ] . apart from the demonstration of a link between rapid replication and cytopathogenicity of the cp strains of hav, the actual molecular events leading to apoptosis following infection with cp strains are unknown. expression of p region proteins b and c from either cp or cc strains in frhk cells result in the accumulation of a tubularvesicular network [ , ] . expression of the p region protein ab has been shown to result in membrane alterations in bacterial cells with resultant changes in membrane permeability [ ] . the lack of correlation between apoptosis and expression of either p or p region proteins prompted us to investigate alternative mechanisms of apoptosis induction by cp strains of hav. in this study, we report that infection of frhk cells with the hav cp strain hm / f results in the degradation of ribosomal rna (rrna) and the reduction of several cellular mrnas including β-actin and gapdh. ribosomal rna degradation was specific for the f strain and the fast replicating enterovirus coxsackievirus b (cbv ), while the parental cc strain of f did not induce rrna cleavage. the effect of f or cbv infection on rrna integrity was observed in the absence of interferon (ifn) induction. ribosomal rna degradation was detected early after virus infection and prior to induction of cpe. this degradation was blocked by inhibitors of virus replication. the pattern of rrna degradation suggests the involvement of the dsrna activated rnase l; the regulation of this enzyme could provide an alternative mechanism by which f infection induces apoptosis. frhk monkey kidney cell line was a kind gift of dr. g. kaplan (fda, center for biologics evaluation and research, bethesda, md). the cells were grown in eagles minimal essential medium (emem, life technologies, gaithersburg, md) containing % heat inactivated fetal bovine serum (fbs), mem non-essential amino acids and sodium pyruvate (all from life technologies), with routine weekly sub-culturing. under these conditions the cells increase in number about -fold in to days. the cell line is contact inhibited and can be kept in growth medium or maintenance medium ( % fbs) for several weeks without degeneration of the monolayer. hav strains hm / f and hm /clone [ , ] , and human coxsackievirus b (cbv ) were obtained from atcc. all three viruses were grown in frhk cells. virus stocks were prepared essentially as described [ , ] . f and cbv were titered by plaque assay, while clone virus was titered by an enzyme immunoassay (eia) in well plates as described previously [ ] using the havab eia diagnostic kit (abbott laboratories, abbott park, il). confluent cultures of frhk cells in cm flasks ( × cells) were infected with - pfu/cell of f or cbv, or tcid of clone in . ml of mem containing % heat inactivated fbs or mock infected. after h of adsorption, . ml of the same medium was added to each flask and incubation continued until the indicated times of cell harvest. cells were harvested by scraping, centrifuged to remove medium, and the pellets washed once with pbs (ca ++ and mg ++ free) and either stored frozen at − • c or used immediately. for rna isolation, cell pellets were dissolved in trizol (life technologies) containing mm β-mercaptoethanol (sigma), following the protocol suggested by the manufacturer. all rnas were routinely digested with rnase-free dnase (promega, madison, wi) in the presence of ribonuclease inhibitor (rnasin, promega) for min at • c, followed by phenol/chloroform extraction and alcohol precipitation. rnas were quantitated by uv absorption at nm. infected or mock infected cells were treated with µg/ml cycloheximide, . mm guanidine hydrochloride, or µg/ml of brefeldin a in mem containing % fbs immediately following the virus absorption period. for measurement of virus replication, well plates containing approximately . × cells were used. cells were fixed in methanol/pbs at hpi and viral antigens detected by eia [ ] . for rna isolation, cm flasks, containing approximately × cells at the time of infection were harvested at or hpi, and rnas isolated as described above. a cm flask was seeded with frhk cells and infected at approximately % confluence with clone virus as described above. the virus was allowed to replicate for days and thereafter the cells were trypsinized and seeded into a cm flask. subsequently, the flask was sub-cultured at weekly intervals by trypsinization and seeding a new flask with one tenth of the cell suspension. to monitor virus replication, periodically cells were seeded into well plates and cultured for to h until the plates were confluent. cells were fixed in % methanol and viral antigens were measured by the havab eia procedure [ ] . uninfected cells were used as a negative control. samples of rna ( µg unless otherwise indicated) were denatured by heating at • c for min in µl of buffer containing × mops buffer, % formamide and . % formaldehyde. samples were separated in a % agarose gel containing × mops buffer, . m formaldehyde, and µg/ml etbr [ ] . the gel was photographed under uv light and transferred overnight to nytran n membranes (schleicher & schuell) in × ssc. the membrane was washed briefly in × ssc, baked under vacuum and stored at • c prior to hybridization. probes for β-actin and gapdh were labeled by random priming of decatemplate tm -βactin-mouse and decatemplate tm -gapdh-mouse respectively (ambion, austin, tx), using α- p datp ( ci/mmol, icn, costa mesa, ca). probe for s rrna was decatemplate tm - s-mouse, also obtained from ambion and was similarly labeled. the anti-sense rna probe for the s rrna (ptri rna s, ambion) was labeled using sp polymerase and α- p utp. the probe for hav genomic rna was obtained by xba digestion of the plasmid phav/ (kindly provided by dr. s. emerson nih, bethesda, md). following gel purification, the large xba fragment, representing almost all of the genomic rna (except the ires), was labeled by nick translation using α- p datp. blots were prehybridized at • c in hyb- (gentra) supplemented with denatured salmon sperm dna or yeast trna (for the rna probe) for - h with continuous agitation and hybridized with probe ( - × cpm/ml) overnight. filters were washed once in × ssc/ % sds for min and then times in . × ssc/ . % sds for min each time at • c, prior to exposure to film or phosphorimager screen (molecular dynamics). anti-vp antiserum was obtained from rabbits immunized with a synthetic peptide cvpetf pelkpgesrht, corresponding to amino acids - of hav viral capsid protein vp , covalently linked to klh. synthetic peptide, free and klh-conjugated, was purchased from new england peptide, inc (fitchburg, ma). pre-immune serum was collected from new zealand white rabbits - days prior to immunization and stored at − • c. rabbits were immunized with klh-conjugated peptide emulsified in freund's complete adjuvant and challenged at regular intervals, over a to week period, with klh-conjugated peptide emulsified in incomplete freund's adjuvant. the animals were hyperimmunized using unconjugated peptide resuspended in sterile pbs. the serum was collected and stored at − • c. all animal studies were conducted in compliance with the guide for the care and use of laboratory animals under an approved iacuc protocol. culture media was removed from mock or virus infected cell cultures and the monolayer were scraped into ca ++ and mg ++ free phosphate-buffered saline (pbs). cells were pelleted ( × g, • c) and washed twice with cold pbs. to obtain a total cell lysate, the cell pellet (while on ice) was resuspended in cold ripa buffer ( mm tris-cl, ph . , mm nacl, % np- , . % sodium deoxycholate, . % sds) containing mm pmsf, and : dilution of protease inhibitor cocktail (sigma st. louis, mo) and phosphatase inhibitor cocktails i and ii (sigma). additional pmsf was added ( mm) to the lysate prior to its passage through a gauge needle ( - times). following min of incubation on ice, the sample was centrifuged at , × g for min • c to pellet insoluble material. the extracts were stored as aliquots at − • c. protein concentrations of lysates were estimated using the bca- protein assay (pierce chemical co., rockford, il) according to the manufacturer's instructions. sixty five µg of each protein extract were adjusted to equivalent volumes in denaturing sample buffer and heated at • c for min and subjected to electrophoresis in sds- % polyacrylamide gels followed by electrophoretic transfer onto supported nitrocellulose membranes (optitran, ba-s , schleicher and schuell inc.) in transfer buffer ( mm tris base, mm glycine) containing % methanol. the membranes were blocked with tbs-t (tris buffered saline/ . % tween ) containing % nonfat dried milk (nfdm) for min at room temperature and washed four times with tbs-t. using a mini-blotter manifold, membranes were incubated either overnight at • c with anti-stat and anti-p-stat antibodies (cell signaling technologies, beverly, ma) diluted : in tbs-t containing % bsa or h at room temperature with anti-actin antibody (sigma) and anti-vp antibody diluted : in tbs-t containing % nfdm. after four washes in tbs-t, the blots were incubated with goat anti-rabbit igg-hrp antibody conjugate (cell signaling) diluted : , in tbs-t containing % nfdm. following four washes at room temperature in tbs-t, the supersignal west pico chemiluminescent substrate kit (pierce chemical co. rockford, il) was used according to manufacturer's instructions followed by exposure to x-omat-xar (kodak, inc.) for the detection of bound antibody-hrp conjugates. cells were grown in cm flasks and infected when confluent with f as above and labeled at hpi for h with µci/ml of a mixture of s-methionine and s cysteine (icn) in methionine and cysteine-free medium supplemented with % dialyzed fbs (icn) following a min pre-incubation in methionine and cysteine-free medium. cbv infected cells and clone persistently infected cells were labeled similarly at h or days post-infection (pi), respectively. cells were collected by scraping, washed once with pbs and the pellets lysed in µl ripa buffer containing protease inhibitors cocktail (sigma). tca precipitable incorporation was measured using µl of clarified lysate and protein concentration was measured with µl lysate. labeled proteins were separated in a - % novex pre-cast gel (invitrogen, carlsbad, ca), after heating the samples at • c for min in × sample buffer containing dithiothreitol. following electrophoresis, the gel was fixed and stained with brilliant blue g (sigma) in % methanol, % acetic acid and destained in % methanol, % acetic acid. the gel was then treated with amplify (amersham biosciences, piscataway, nj) for min, dried and exposed to a phosphorimager screen, which was scanned in a typhoon scanner (molecular dynamics). total rna preparations from mock infected, f infected ( hpi), cbv infected ( hpi), and hm infected ( dpi) frhk cells were labeled with α- p datp using the cds primers for human . array (clontech, palo alto, ca), clontech enzyme and reagents provided with the array. the labeled cdna preparations were purified using the nucleospin extraction kit (clontech). four identical arrays were pre-hybridized at • c in hybridization bottles in a hybaid oven (national labnet, woodbridge, nj) in ml of expresshyb (clontech) containing µg/ml denatured sheared salmon sperm dna (eppendorf scientific, westbury, ny). hybridization was performed overnight at • c by the addition of denatured probe ( . × cpm/ml) and µg human cot- dna (clontech) directly to the pre-hybridization solution. the membranes were washed twice ( min each) with × ssc- % sds and twice ( min each) with . × ssc− . % sds at • c, followed by a final wash at room temperature with × ssc for min. the membranes were wrapped in saran wrap and exposed to phosphorimager screens at room temperature for to h. the screens were scanned in a molecular dynamics typhoon scanner at -micron resolution and analyzed using the atlasimage . software (clontech). µgrna samples were reverse transcribed in µl reactions using oligo (dt) as the primer and amv reverse transcriptase and µl of each cdna pool were pcr amplified using primers bg and bg as described [ ] . ten µl samples were withdrawn after and cycles and analyzed by agarose gel electrophoresis. bands were quantitated by the imagequant program from molecular dynamics following scanning of the etbr stained gel by a typhoon phosphorimager. at a moi of - pfu/cell, the rapidly replicating cp strain hm / f of hav ( f) induced cell rounding in frhk cells within h post-infection (pi) in approximately to % cells. the percentage of cells exhibiting cpe increased for the next h, so that by h, to % of the cells were rounded and detached from the surface of the culture flask. thus, even with the f virus, infection is characterized by a slow and asynchronous recruitment of cells into supporting viral replication. similar effects have been reported for other cp strains of hav, namely hm / a, and hb . [ , ] . in agreement with these results, we confirmed the induction of apoptosis in f infected frhk cells by a colorimetric tunel assay (data not shown). the parental hm /clone virus (clone ), also used in the present study, produced no changes in cell morphology after several months of continuous culture of infected cells, although viral antigens could be detected by eia after four weeks of weekly subculture following the initial infection. the coxsackie virus strain b (cbv ) used in this study replicated very rapidly in frhk cells, producing marked cpe within hpi and all cells were floating by to hpi. we observed the appearance of rna bands in the region between the s and s species, as well as below the s rrna band, upon denaturing agarose gel electrophoresis of total rna preparations isolated from f-infected cells (fig. ) . the apparent degradation products were easily detectable after ethidium bromide (etbr) staining of formaldehyde-agarose gels (fig. a) . rna degradation was observed only in f infected cells at and hpi (lanes and ), while no degradation was observed at hpi in clone infected cells (lane ) or mockinfected cells (lane ). degradation of rrna has not been reported previously in hav infected cells, therefore, the rrna origin of the observed degradation the probe for s rna was a . kb fragment of the mouse s rna gene (nucleotides to ), labeled by random priming with α- p datp. the probe for hav genomic rna was the large xba i fragment from a genomic clone phav/ , labeled by nick translation with α- p datp. an rna ladder ( . to . kb) was included in the analysis and the position of the . to . kb fragments identified in panel a. an arrow is used to identify the putative s rrna degradation product in a and the position of the s rna and its degradation products in b. full length hav rna is . kb (c) products was confirmed by northern blot analysis. duplicate rna samples were electrophoresed and transferred to nytran n membranes. one half of the blot was hybridized to a probe for s rna (fig. b) , and the other half to a probe for hav rna (fig. c) . the s probe hybridized to intact s rna and to two additional species of rna (indicated by arrows) from f infected cells, while these species were not detected in rna preparations from either mock or clone infected cells. apparently full length hav rna ( . kb) was detected in f infected cells at both and hpi, while no viral rna was detected in clone infected cells at hpi, confirming that the f virus indeed was a faster replicating strain. to further investigate whether rna degradation was specific for f infection, and to determine the temporal relationship between rna degradation and onset of cpe, we isolated total rna from cells infected with cbv , hm /clone , and hm / f at different times, and analyzed them by formaldehyde-agarose gel electrophoresis (fig. ) . in cbv infected cells, degradation of both species of rrna was observed after cbv infection at - hpi, when almost all the cells in the monolayer were rounded but still attached to the surface ( fig (fig. b, lane ) , though small amounts of degradation products may be detected at days following infection (fig. b, lane ) . the levels of viral rna and protein in clone infected cells at and days pi were comparable to those observed in f infected cells, as measured by rt-pcr and western immunoblotting (fig. ) and eia (data not shown). we have continuously cultured the persistently clone infected cells for over five months without significant changes in cell morphology or rrna integrity. when rna was obtained from f-infected monolayers following separation of the rounded cells from cells with a normal morphology by vigorous agitation, rrna degradation was apparent in both populations of cells (fig. b, lanes and ) . in lane , only µg of rna was applied, since the percentage of floating cells at hpi was low and the yield of rna much less compared to the cells that remained attached (fig. b, lane ) . in lane , no intact s or s bands were visible, indicating that almost complete degradation of the rrna species has occurred in these cells. it should be noted that cbv infection proceeds much faster and synchronously in frhk cells. since we did not examine rrnas from cbv infected cells between and hpi, it is quite possible that rna degradation occurs in cbv infection prior to the onset of morphological changes associated with cpe. to confirm that s rrna degradation was occurring after infection with f or cbv , a northern blot of the rna gel shown in fig. (panel a) was hybridized to a probe specific for s rrna (fig. ) . the short probe ( bases from the end of the s rna) hybridized strongly to a fragment migrating faster than the . kb marker, as well as to intact s rrna, but did not hybridize to apparent fig. a were subjected to denaturing agarose gel electrophoresis. the gel was northern blotted and hybridized to a labeled anti-sense rna probe specific for s rrna (nucleotides to ). the probe was synthesized from ptrirna- s by transcription with the sp polymerase. the arrow indicates the position of a s rna degradation product. b an identical northern blot was hybridized to a nick translated dna probe for hav as described in fig. . arrows indicate position of full-length hav rna ( . kb) and putative degradation products degradation products seen in etbr stained gels (figs. and ) that migrated to the region between the s and s species. it is likely that these latter products were derived from the end of the s rrna and could not be detected by a probe targeted to the end. nevertheless, it is clear that both the large and small rrna molecules were degraded in cbv and f infected cells, and that this degradation coincided with the appearance of cpe in cbv infected cells but preceded the appearance of cpe in f-infected cells. in contrast, infection of permissive cells with mouse hepatitis virus (a coronavirus) resulted in the degradation of only the s species [ ] . apparently intact hav rna was detected in f-infected cells at hpi and hpi, and, albeit at lower levels in clone infected cells at dpi (fig. b ). this blot was exposed longer to reveal the existence of hav rna in clone infected cells. while overexposure of the blot showed that some degradation of viral rna might also be taking place, the bulk of the viral rna remains intact, possibly protected from degradation by its association with capsid proteins. on the other hand, the smaller virus specific rna molecules could have resulted from premature termination of transcription [ ] . to investigate whether the degradation of rrnas observed in f infected cells required replication of the virus, we studied the effects of several inhibitors of virus replication on rna degradation. cyclohexamide (ch) is a strong inhibitor of protein synthesis in many cultured cell lines and produces a marked cpe in treated cells that is attributed to induction of apoptosis [ , ] . in agreement with the results of brack et al. [ ] , we observed f-like cpe after treatment of frhk cells with ch at concentrations ranging from to µg/ml. despite the induction of cpe/apoptosis, ch treatment failed to elicit rna degradation in uninfected frhk cells (fig. b, lane ) , and inhibited both virus replication (fig. a) , and degradation of rna in f infected cells (fig. b, lane ). the inhibition of virus replication was not unexpected since ch inhibits picornavirus genome replication by preventing translation of input genome to produce viral rna dependent rna polymerase [ ] . brefeldin a (bfa) has been shown to induce apoptosis in other cell lines through the activation of caspase and disruption of golgi complex function, and to inhibit replication of hav and some picornaviruses, as well as viral rna synthesis [ ] . treatment of frhk cells with µg/ml bfa produced marked apoptosis within h while suppressing f replication and f induced rna degradation (fig. , panels a and b respectively). thus, rna degradation in f-infected cells was prevented by both ch and bfa. ch and bfa induced apoptosis in frhk cells was synchronous and was complete by h as evidenced by cell rounding and eventual detachment from the growth surface. however, these changes were not accompanied by rna degradation following treatment with either ch (fig. b) or brefeldin a (data not shown). treatment of f-infected cells with . mm guanidine hydrochloride (guhcl), on the other hand, had a marginal effect on f replication at high moi, and a somewhat larger effect at low moi (fig. a) . however, guhcl treatment did not prevent rna degradation in f-infected cells (fig. b ). it has been reported that guhcl, an inhibitor of poliovirus replication, is ineffective against hav due to a mutation in the genome [ ] , but guhcl was found to be a strong inhibitor of hm /p virus [ ] . these experiments clearly show that in frhk cells, replication of the f virus is required for rna degradation, and that degradation of rna is not a consequence of apoptosis. degradation of rrna is a feature of virus infection in interferon (ifn) treated cells and is believed to be due to the availability of double stranded rna (dsrna) during replication or transcription of the viral genome, resulting in the activation of the rnase l pathway [ , ] . however, in the absence of ifn pretreatment, dsrna can activate the rnase l pathway by activating pre-existing - a synthetase, which ultimately results in cell death [ , , ] . since viral rna replication and expression occurs much earlier post infection in f-infected cells compared to clone infected cells, the degradation of rrnas could result from an early dsrna dependent activation of rnase l in f-infected cells. we further investigated whether the lack of rna degradation and cpe in clone infected cells observed at later times pi may correlate with diminished levels of viral rna or protein. frhk cells were infected with hm /clone , and the cells were serially passaged at weekly intervals until viral antigens were detectable by eia (data not shown). after days, the level of viral antigens was comparable to that observed in f infected cells after an acute infection. total cellular rna, and proteins were isolated from the persistently infected cells. the level of viral rna in the persistently infected cells was measured at and days of continuous culture by rt-pcr (fig. a) . the level of viral rna was somewhat lower in clone infected cells after days, but at days the levels were almost the same as in f infected cells at hpi. pcr amplification was carried out for and cycles to ensure that the signal strengths of the pcr products were obtained before the reactions reached saturation ( cycles) . western blot analysis with anti-vp antibody also showed lesser but easily detectable accumulation of the viral capsid protein vp in persistently clone -infected cells (fig. b) . however, the cells did not show any discernable morphological change after more than days of weekly subculture and produced viral antigens and rna as determined by eia and rt-pcr. the data indicate that, unlike f infection, clone replication and expression does not induce rrna degradation. a reduction in cellular rna and protein levels during virus infection may aid in the replication of viruses possessing a cp phenotype (e.g., hav hm / f), or a rapid growth/cpe characteristic (e.g., cbv) in cell lines permissive for virus growth. for example, translation of hav rna in infected cells requires interaction of trans-acting cellular protein factors with cis-acting elements present at the [ , ] . indeed, a competition between gapdh and polypyrimidine tract-binding protein (ptb) for stem-loop iiia of hav ires has been reported by yi et al. [ ] . while ptb functions as a positive effector of hav rna translation, the binding of gapdh to stem-loop iiia of hav ires has been shown to destabilize rna secondary structure and inhibit translation [ , ] . these results prompted us to investigate the levels of gapdh mrna in f-infected cells since decreased levels of this mrna may indicate a functional relationship between f replication and regulation of effector proteins such as gapdh. as a control, we also investigated whether virus infection affects the level of another cellular housekeeping gene β-actin. northern blot hybridizations to β-actin and gapdh specific probes were used to assess the levels of these two cellular mrnas (fig. ) . the levels of both mrnas were reduced in a time dependent manner in cbv and f infected cells while there was little effect of clone infection on these molecules. however, unlike the rrnas, we did not find any degradation products of these rnas. two reasons for this could be that these mrnas are transcriptionally repressed and/or the degradation products are too small to detect by this analysis. the degradation of rrna into discrete products is a hallmark of the antiviral effect of interferon. since the cell line used for these experiments was not treated with interferon prior to virus infection, it was important to determine whether the frhk cell line is a natural producer of interferon, or if interferon is induced as a result of virus infection. to investigate if interferon mrnas were present in frhk cells prior to or following virus infection, nylon arrays, each containing human genes including those for interferons α, β, and γ were hybridized to cdna probes synthesized using total rna from uninfected and virus infected cells as templates. surprisingly, we did not find significant hybridization signals table . induced and down-regulated genes during f infection of frhk cells. array hybridizations were carried out in duplicate with cdna probes prepared using two independent rna preparations each from mock and virus infected frhk cells. independent phosphorimager scans for each duplicate sample (control or infected) were averaged using atlasimage program to produce composite arrays for control and infected cells. the composite arrays were then compared to detect differences in signal levels using atlasimage software. gene signal values that were less than two-fold over background values were considered insignificant. ratios of less than one represent down-regulation in the infected cells. ratios of higher than one represent up-regulation in the infected cells. a less than two-fold difference of signal intensity between control and infected samples are considered insignificant. nd means the pixel value for that gene was equivalent to background corresponding to the interferon genes in either uninfected or hav infected cells (table ) . these results are supported by the data from pietiäinen et al. [ ] suggesting that ifn mrnas are neither present nor induced during pv , ev and cbv infection of hos and hela cells. however, low levels of ifn rnas, and any comparative differences between uninfected and infected cells, may not be detectable by array hybridization. rt-pcr analysis of total rna from virus infected frhk cells did not show increased level of β-ifn mrna compared to uninfected cells. however, treatment of these cells with dsrna resulted in β-ifn mrna induction (data not shown). we investigated the possibility that low levels of ifn may be present in control or infected frhk cells by looking at the phosphorylation status of stat by western immunoblotting (fig. ) . signal transducer and activator of transcription (stat) and are regulators of ifninduced gene expression. in cells treated with ifn, both stat and stat are phosphorylated by janus kinase (jak), which is a prerequisite for dimerization and translocation to the nucleus [ , ] . the antibody to stat readily detected the two forms of this protein in both control and virus infected cells, as well volumes of extracts containing equal number of cpm or protein were heated in sds sample buffer containing reducing agent prior to loading on a - % polyacrylamide gel that was run in mops-sds buffer along with molecular weight markers. the gel was stained, destained and, and treated with amplify (amersham) prior to drying according to standard protocol and exposed to phosphorimager screen, which was scanned by a typhoon scanner as in control and interferon treated hela cell extracts. both stat proteins in hela migrate as slightly larger molecules. using an antibody specific for the phosphorylated stat , stat phosphorylation was not detected in either control or f-infected frhk cells (fig. ) , confirming the results obtained by array hybridization. in addition, stat phosphorylation was not observed in cbv infected frhk cells while phosphorylated stat was readily detected in ifn treated hela cells (fig. , panel a) , and frhk cells (fig. , panel b) . these results are in agreement with those obtained by array hybridization, indicating that ifn is not expressed and, therefore, is not mediating rna degradation in f-infected cells. unlike most enteroviruses, even cp strains of hav lack the ability to shut off host protein synthesis. this inability to cause host shut-off is most likely due to a non-functional a protease, and is probably a major reason for the slow growth phenotype of hav [ ] . while it would contradict published reports, it seemed possible that f induced rna degradation could result in host shut-off triggered by events peculiar to frhk cells. therefore, we investigated the effect of f infection on overall protein synthesis by pulse labeling of cells for h and analysis of the products by sds-polyacrylamide gel electrophoresis (sds-page). as shown in fig. , cbv infection resulted in a dramatic reduction of cellular protein synthesis at hpi, and viral proteins could be clearly identified. in contrast, the effect of f infection was much less pronounced, and comparable to the effect of the parental cc (clone ) strain. thus no positive identification of hav specific proteins could be made from such an analysis. it should be noted that the clone infected cells used for protein labeling in this experiment have been routinely sub-cultured for more than two months, and were actively producing viral antigens as detected by a colorimetric eia procedure (data not shown) as well as viral rna and vp capsid protein (fig. ) . however, rna degradation was not evident in these cells. in conclusion, rrna degradation alone is not sufficient for host shut-off. cytopathic (cp) strains of hav have been isolated in several laboratories during the past few years. the cp strains arise during repeated passage of cc strains in permissive cells and the genetic basis for the development of the cp phenotype is poorly understood [ , , , , , , ] . there is no evidence to indicate conclusively which nucleotide changes arising in the cp genotype are responsible for both the rapid growth and the cp phenotype [ , , , , , ] . these characteristics may be the result of different mutational events that have occurred during cc strain replication. the data presented here identify a previously unreported event that occurs during infection with a cp strain of hav, namely the degradation of rrnas and the reduction in the level of cellular rnas. similar effects on rrna, and β-actin and gapdh mrnas were also observed after cbv infection. however, the non-cpe inducing parent strain of f, hm /clone had only a marginal effect on rrna integrity during long-term persistent infection. similar to the effects of enteroviruses, such as pv , cbv and ev , on the expression of certain cellular genes involved in apoptotic pathways, c-myc and c-jun levels were moderately increased in f-infected cells, while c-jun level was markedly reduced in clone infected cells (table ) . full length viral rna was detectable much earlier in f-infected cells compared to cells acutely infected with the clone virus (figs. and ) . significantly, viral rna and viral capsid protein vp were present at comparable levels following extended culture of clone -infected cells in the absence of significant rna degradation (fig. ) . degradation of rrna following infection of interferon treated cells has been reported for a number of dna and rna viruses [ , ] . moreover, dsrna, a likely intermediate of rna virus replication or transcription, has been shown to promote rrna degradation without interferon treatment in some cell lines [ , , , , , , , , ] . the function of dsrna as an effector of the interferon response is dependent on both the cell type and the nature of the virus [ , ] . for example, vaccinia virus is known to evade the effects of ifn treatment, but in suspension cultures of mouse l cells treated with ifn, rapid degradation of rrna was observed [ ] . activation of the - a/rnase l pathway has been reported during estrogen withdrawal from chick oviducts [ ] . the enzyme responsible for the synthesis of - a, - -oligoadenylate synthetase can apparently be also induced by hepatitis c virus coat protein [ ] . while the role of a viral protein in the activation of - a/rnase l pathway in f or cbv infected frhk cells cannot be ruled out, previous reports with emc virus and the studies involving dsrna clearly suggests a similar mechanism of rrna degradation reported here. it remains unclear why rrna degradation and apoptosis are absent in clone infected cells despite the presence of viral rna and protein. perhaps cellular inhibitors of rnase l play a role in blocking rna degradation in clone infected cells [ ] . it is also possible that a mechanism, analogous to the adenovirus vai rna mediated inhibition of pkr activity is used by the clone virus to disarm the rnase l pathway [ , ] . clearly, accumulation of sufficient viral rna or proteins cannot account for either rrna degradation or the cp phenotype, since cc strains accumulate enough viral rna and proteins after an initial lag (fig. ) . it is interesting to note that while the precursor of mature vp protein vp - a is present in equal amounts in both f and clone infected cells, vp itself accumulates to higher levels in f infected cells, suggesting a slower processing of the precursor in clone infection. surprisingly, we did not see a general inhibition of protein synthesis in frhk cells infected with the cp ( f) strain of hav despite massive rna degradation, although an almost total shutoff of host protein synthesis was observed in cbv infected frhk cells (fig. ) . this is in contrast to the general inhibition of protein synthesis observed upon activation of the rnase l pathway in some cell lines [ , , , , , , ] . the effect of cbv infection on protein synthesis is most likely a combination of rrna cleavage (this study) and cleavage of eif g, poly (a)-binding protein (pbp), and other translation factors mediated by an active a protease [ , , ] . it has been shown that both caspase activation and apoptosis induction can occur when - a synthetase is activated and rrna degradation is induced [ , , , , ] . while we have not directly assessed the level of - a following virus infection, no other rnase currently known produces the characteristic degradation pattern of rrna that we have observed. it is apparent from our data, however, that rna degradation is dependent upon cp virus strain replication, since compounds, such as cycloheximide and brefeldin a, that inhibited f replication also abolished rrna degradation in f infected cells (figs. and ) . treatment in the absence of infection did not induce rna degradation. additionally, the treatment of uninfected cells with these compounds induced apoptosis in the absence of rna degradation (figs. and ) . in view of the temporal relationship between onset of rrna degradation and induction of cpe/apoptosis, these results suggest that rna degradation following f (or cbv ) infection is the cause, rather than the effect, of apoptosis. on the other hand, an apoptotic reaction may also be induced by the preferential synthesis of apoptosis inducing proteins such as c-jun [ ] or c-myc [ ] . as shown in table , several gene transcripts linked to apoptosis are present in higher levels in f-infected cells compared to clone infected cells. particularly intriguing are lack of induction of c-myc and downregulation of c-jun in clone infected cells. we cannot rule out the possibility, therefore, that apoptosis in f-infected cells may be induced via regulation of relevant proteins such as c-jun or c-myc. another interesting feature of the cp strains of hav is that all apoptosis inducing cp strains are derived from persistently infected monkey kidney cells and induction of apoptosis (or cpe) is restricted to monkey kidney cells. therefore, the nature of the host cell is clearly as important as the genetic makeup of the virus. it is interesting to note that a base insertion is present in the ires of cp strains hm / f as well as hm / a. it would be tempting to speculate that conformational changes brought about by this or other mutations result in a better inducer of - a synthetase, a possibility that is currently under investigation. activation of rnase l resulting in rrna cleavage is usually accompanied by inhibition of protein synthesis [ , , , , ] and picornavirus growth [ , , ] . we believe that the apparent lack of inhibition of host protein synthesis in f-infected cells is a result of asynchronous rna degradation, which masks the effect of rna degradation on protein synthesis. the other possibility is the absence of activation of dsrna stimulated pkr, which may be lacking in these cells. however, several different mechanisms exist, through which viruses can disarm the pkr response [ , ] . further studies are required to assess the level of activity of this enzyme in virus-infected cells. recent studies using dna arrays have revealed that genes involved in the interferon pathway (interferon response genes, isgs) are the major targets of a diverse group of viruses [ ] . a direct effect of interferon in inducing - a synthetase during f or cbv infection is unlikely because we failed to detect ifn mrna or stat phosphorylation in either uninfected or virus infected cells. these results are supported by previous findings that cell culture adapted hav does not induce ifn in cultured cells [ ] . moreover, recent studies also have shown that a non-cytopathic cc strain of hav down regulates dsrna-induced transcription of β-ifn [ ] . however, there are now several studies to show that isgs can be regulated in the absence of ifn by viral dsrna or viral proteins [ , , ] . while many viruses have evolved mechanisms to disarm various facets of the ifn inducible antiviral response, the usurpation of the ifn response by the cp strain f to facilitate it's spread is unique. the fact that only a few cell lines of primate kidney origin are susceptible to apoptosis in response to rapidly replicating hav variants suggests that both cellular processes and genotypic changes in the viral genome are responsible for the establishment of a cp phenotype. in order to define the contributions of these two processes, it will be necessary to examine strain differences at the genetic level as they relate to substantive changes in viral protein expression/function, and to investigate the connection between viral and cellular events at the level of cellular protein expression. in conclusion, rrna degradation in combination with lower levels of cellular mrnas such as β-actin, gapdh and others could confer on the f virus a replicative advantage over the parental cc strain. clearly, reduction in the levels of cellular mrnas is advantageous to the virus, even if it leads to a small reduction rather than a dramatic shut-off of host protein synthesis. competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle cytopathology, plaque assay, and heat inactivation of hepatitis a virus strain hm rnase-l-independent specific s rrna cleavage in murine coronavirus-infected cells poliovirus protease c pro kills cells by apoptosis infection of polarized cultures of human intestinal epithelial cells with hepatitis a virus: vectoral release of progeny virions through apical cellular membranes differential kinetics of polypeptide expression and different biological activities in the human fibroblast response to dsrna or interferon treatment a cytopathogenic, apoptosis inducing variant of hepatitis a virus hepatitis a virus inhibits cellular antiviral defense mechanisms induced by double stranded rna in vitro characterization of an internal ribosomal entry site (ires) present within the nontranslated region of the hepatitis a virus rna: comparison with the ires of encephalomyocarditis virus caspase activation and specific cleavage of substrates after 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vitro prevention and control of hepatitis a hepatitis a viruses with deletions in the a gene are infectious in cultured cells and marmosets activation of p mitogen-activated protein kinase and cjun nh -terminal kinase by double-stranded rna and encephalomyocarditis virus: involvement of rnase l, protein kinase r, and alternative pathways activation of nf-κb by double-stranded rna (dsrna) in the absence of protein kinase r and rnase l demonstrates the existence of two separate dsrna triggered antiviral programs membrane permeability induced by hepatitis a virus proteins b and bc and proteolytic processing of hav bc replication of hepatitis a viruses with chimeric nontranslated regions cleavage of poly (a)-binding protein by coxsackievirus a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? rna-protein interaction at the end of the hepatitis a virus rna cellular factors in the transcription and replication of viral genomes: a parallel to dna-dependent rna transcription antigenic and genetic variation in cytopathic hepatitis a virus variants arising during persistent infection:evidence for genetic recombination maturation of the hepatitis a virus capsid protein vp is not dependent on processing by the c pro proteinase the rnase l inhibitor (rli) is induced by double-stranded rna direct induction of interferongamma-and interferon-alpha/beta inducible genes by double-stranded rna activation of the interferon inducible - -oligoadenylate synthetase gene by hepatitis c virus coat protein production of cytopathology in frhk cells by bs-c- passaged hepatitis a virus detection of defective genomes in hepatitis a virus particles present in clinical specimens effects of echovirus infection on cellular gene expression recombinant expression of hepatitis a virus protein a: interaction with membranes viruses and apoptosis caspase-dependent apoptosis by - -oligoadenylate activation of rnase l is enhanced by ifn-β constitutive expression of a , -oligoadenylate synthetase cdna results in increased antiviral activity and growth suppression proteinase c of hepatitis a virus (hav) cleaves the hav polyprotein p -p at all sites including vp / a and a/ b specific interaction of glyceraldehydes -phosphate dehydrogenase with the nontranslated rna of hepatitis a virus the structures of picornaviral proteinases stability of hepatitis a virus viruses and interferons how cells respond to interferons apoptosis-inducing and apoptosis-preventing functions of poliovirus apoptosis in acute and chronic central nervous system disease induced by theiler's murine encephalomyelitis virus isolation and molecular cloning of a fast growing strain of human hepatitis a virus from its double stranded replicative form hepatitis a virus infection and the interferon system hav f induces rna degradation regulation of two interferon inducible human genes by interferon, poly(ri):poly(rc) and viruses detection of a genome-linked protein (vpg) of hepatitis a virus and its comparison with other picornaviral vpgs low efficiency of the nontranslated region of hepatitis a rna in directing cap-independent translation in permissive monkey kidney cells functional significance of the interaction of hepatitis a virus rna with glyceraldehydes -phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function an infectious cdna clone of a cytopathic hepatitis a virus: genomic regions associated with rapid replication and cytopathic effect impact of rnase l overexpression on viral and cellular growth and death division of molecular biology we thank dr. darcy hanes for help in preparing the anti-vp antiserum, dr. joseph e. leclerc and dr. dan levy for critically reading the manuscript, dr. c.d. atreya and dr.stephen feinstone for helpful discussions, and marcia meltzer for help with the graphics. key: cord- - vv a authors: kuhn, jens h.; bao, yiming; bavari, sina; becker, stephan; bradfute, steven; brister, j. rodney; bukreyev, alexander a.; chandran, kartik; davey, robert a.; dolnik, olga; dye, john m.; enterlein, sven; hensley, lisa e.; honko, anna n.; jahrling, peter b.; johnson, karl m.; kobinger, gary; leroy, eric m.; lever, mark s.; mühlberger, elke; netesov, sergey v.; olinger, gene g.; palacios, gustavo; patterson, jean l.; paweska, janusz t.; pitt, louise; radoshitzky, sheli r.; saphire, erica ollmann; smither, sophie j.; swanepoel, robert; towner, jonathan s.; van der groen, guido; volchkov, viktor e.; wahl-jensen, victoria; warren, travis k.; weidmann, manfred; nichol, stuart t. title: virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family filoviridae date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: vv a the task of international expert groups is to recommend the classification and naming of viruses. the international committee on taxonomy of viruses filoviridae study group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. here, further guidelines are suggested to include their natural genetic variants. first, this term is defined. second, a template for full-length virus names (such as “ebola virus h.sapiens-tc/cod/ /kikwit- ”) is proposed. these names contain information on the identity of the virus (e.g., ebola virus), isolation host (e.g., members of the species homo sapiens), sampling location (e.g., democratic republic of the congo (cod)), sampling year, genetic variant (e.g., kikwit), and isolate (e.g., ). suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). we suggest that these comprehensive names are to be used specifically in the methods section of publications. suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. the proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. if applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the national center for biotechnology information (ncbi). furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling. abstract the task of international expert groups is to recommend the classification and naming of viruses. the international committee on taxonomy of viruses filoviridae study group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. here, further guidelines are suggested to include their natural genetic variants. first, this term is defined. second, a template for full-length virus names (such as ''ebola virus h.sapiens-tc/cod/ /kikwit- '') is proposed. these names contain information on the identity of the virus (e.g., ebola virus), isolation host (e.g., members of the species homo sapiens), sampling location (e.g., democratic republic of the congo (cod)), sampling year, genetic variant (e.g., kikwit), and isolate (e.g., ). suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). we suggest that these comprehensive names are to be used specifically in the methods section of publications. suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. the proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. if applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the national center for the international committee on taxonomy of viruses (ictv, http://www.ictvonline.org), the body tasked by the international union of microbiological societies (iums) to make decisions on matters of virus classification and nomenclature, is responsible for the assignment of viruses to taxa (orders, families, subfamilies, genera, and species). this task is supported by the intellectual input from specialized groups, the ictv study groups. study groups serve as advisory committees and connect the ictv taxonomists with laboratory virologists, who investigate viruses scientifically. ictv study groups and other expert groups, but not the ictv itself, propose unique virus names and abbreviations. fauquet et al. discussed in that ''[i]t is de facto accepted by the virologists that there is no homogeneity in the demarcation criteria, nomenclature and classification below the species level, and each specialty group is establishing an appropriate system for its respective family'' [ ] . unfortunately, most ictv study groups or other expert groups have not provided clear guidelines in the past, accepting strain and genetic variant names as they were suggested by different researchers in their publications rather than creating consistent nomenclature schemes that apply at least to all viruses of one family. the status quo is, therefore, that variants of particular viruses are often named according to different standards. for instance, one may be assigned a number only, whereas another may be referred to by a name, whereas yet another one may have been designated with the year of isolation. such variety is not necessarily cause for grave concern when the number of virus variants is very limited and experts in the field are aware of them. however, their number, and in particular the number of sequences deposited in databases, has increased considerably in recent years. it is therefore becoming difficult for researchers to be aware of them, and in particular, to know their specific characteristics. decreasing sequencing costs and ongoing improvements in sequencing technology have led to increased submission of genomic consensus sequences of viruses to databases without associated peerreviewed descriptive publications and without fulfilling (yet undefined) minimum standards for sequencing and metadata. in practice, this means that crucial information about these sequences and the associated viruses is lost when, for instance, the date of isolation or the location of isolation, i.e., the ''biological context'' in the form of metadata, are not deposited along with the sequences. in a recent report from a national center for biotechnology information (ncbi) workshop on virus genome annotation, the authors of the report concluded that ''…only when information…[is] included does a viral genome sequence become something more: a sample isolated in evolutionary time and environmental context, which can be compared to others, allowing inferences between sequence, host, chronology, and geography'' [ ] . the amount of genomic information for members of the family filoviridae is unlikely to become overwhelming in the short term. however, their importance in regard to biodefense measures, and recent calls for genetic filovirus variant standardization to expedite countermeasure development [ ] , make them suitable candidates for name standardization trends started by influenzavirus, coronavirus, and rotavirus experts. here, we propose guidelines for the establishment of a standardized nomenclature for natural genetic variants of filoviruses, and how to build designations for them from metadata in genbank records and publications. review of existing systems for nomenclature below the species level several nomenclature schemes have been brought forward for individual virus groups. the most commonly accepted one is the nomenclature for influenzaviruses (family orthomyxoviridae, genera influenzavirus a/b/c), which was published in by the world health organization [ ] and which has since been updated several times [ ] [ ] [ ] the influenzavirus nomenclature has proven very useful as it allows searching for and identifying particular influenzavirus isolates from the more than , deposited sequences. it is generally accepted within the influenzavirus research community and has the advantage that the isolate designation is mostly self-explanatory, allowing non-influenzavirus specialists to comprehend it quickly. its disadvantages are that (a) it has become partially redundant because the three ''antigenic types'' of ''influenza virus'' have been reclassified as three different viruses belonging to three different species (influenza a/b/c virus, species influenza a/b/c virus); (b) host designation permits the use of non-standardized animal names that lack specificity (such as ''duck'' ? which kind of duck?); and (c) it does not distinguish between strains and variants. in , the ictv caliciviridae study group proposed a nomenclature for caliciviruses [ ] : reminiscent of the influenzavirus nomenclature, this system is easily comprehensible in its unabbreviated form. however, its implementation is complicated by the requirement that virus species names be listed when in practice virus names are (and should be) used instead [ , , , [ ] [ ] [ ] and the problem that host designation permits the use of non-standardized host names that lack specificity (such as ''hot pepper'' ? which kind of hot pepper?). the abbreviated names are more difficult to grasp immediately and raise the question how countries, cities, and hosts should be abbreviated consistently. the most recent comprehensive virus nomenclature was proposed in by the rotavirus classification working group in conjunction with the development of an ncbi database for rotavirus genome sequences [ ] . this nomenclature is again similar to that used for influenzaviruses: the system envisions the use of particular denominators to point out missing information (''xxx''). suffixes in the \species of origin[ field point out whether a viral genome has been directly sequenced from a clinical specimen (''-wt'' for ''wild-type''), from tissue/cell culture-derived viruses (''-tc''), from viruses passaged through homologous hosts (''-hhp'') or from laboratory-generated or laboratoryengineered viruses that resemble viruses from nature (''-lab'') or cannot be found in nature (''-labstr''). contrary to other nomenclatures described here, the developers of the rotavirus nomenclature did address the need for a standardization of country abbreviations and suggested to use the unique -letter (''alpha- '') country codes used by the representation of names of countries (iso ) as prepared by the international organization for standardization (see http://www.iso.org/iso/country_codes.htm or https://www.cia.gov/library/publications/the-world-factbook/ appendix/appendix-d.html). a minor problem of this nomenclature is that one denominator asks for ''species of origin,'' yet the provided examples list actual animals (''human'' instead of ''homo sapiens'' for instance). furthermore, as in other nomenclatures described above, it is unclear which vernacular name of a given animal ought to be used (for instance: should ''cougar'' be used, or should ''puma,'' ''mountain lion'' or ''mountain cat'' be chosen?). instead, the authors provide simple examples for the ''species of origin'' denominator, such as ''mouse'' or ''bat,'' which could be more descriptive (which kind of ''mouse'' or ''bat''?), given that there are over a thousand different animals assigned to a roughly equal number of different mouse and bat species. the ictv filoviridae study group and other experts have recently proposed an updated taxonomy for filoviruses [ ] , which has been accepted by the ictv [ , ] . this taxonomy, summarized in table , also delineates the proper use of vernacular filovirus names. it is an attempt to ameliorate the confusion that exists generally among virologists regarding the difference between virus species and viruses [ , , , [ ] [ ] [ ] by introducing virus names distinct from species names [ ] as previously requested by some experts [ , ] . lastly, it accommodates recent developments in filovirology, such as the discovery of two novel filoviruses that require(d) the establishment of two new species and one new genus [ , , ] . whereas the demarcation of individual filoviruses (ebola virus vs. sudan virus vs. marburg virus, etc.) is now clearly defined [ ] , it is unclear how to distinguish their individual subclasses (strains, genetic variants, genotypes, mutants, etc.), mainly because of a lack of definitions for these terms and the absence of generally applicable guidelines for assigning viruses to them. the result is not only inconsistency and the ensuing difficulties for non-specialists to understand manuscripts, seminars, or figures [ ] , but also a broad range of different designations referring to the same filovirus, as well as typographical and transliterational errors [ ] . in recent years, filovirus disease outbreaks have been observed more frequently, and an ever-increasing number of isolates and genomic consensus sequences are becoming available. technological breakthroughs in sequencing also have allowed the identification of a novel filovirus (lloviu virus, llov) in the absence of replicating isolates [ ] . [ ] , and restv was isolated from apparently healthy domestic pigs (sus scrofa scrofa linnaeus, ) [ ] . llov was detected in schreibers's long-fingered bats (miniopterus schreibersii kuhl, ) [ ] . fragmented marv genomes were detected in greater long-fingered bats (miniopterus inflatus thomas, ) and eloquent horseshoe bats (rhinolophus eloquens andersen, ) [ ] . fragmented ebov genome consensus sequences were detected in tissues from deceased western lowland gorillas (gorilla gorilla gorilla savage, ) and central chimpanzees (pan troglodytes troglodytes blumenbach, ) [ ] , as well as from hammerheaded fruit bats (hypsignathus monstrosus allen, ), franquet's epauletted fruit bats (epomops franqueti tomes, ), and little collared fruit bats (myonycteris (myonycteris) torquata dobson, ) [ ] . conversely, several filoviruses are not available for research anymore and/or sequence information was lost, but awareness of them remains important for the understanding of historical reports [ ] . it is foreseeable that the discovery or creation of novel filoviruses will accelerate in the near future. it is therefore of the utmost importance to retrospectively and prospectively establish a consistent, easily comprehensible nomenclature for filoviruses that not only provides crucial information such as isolation host and place and date of isolation, but also describes whether the entity in question is natural in origin or artificial, and extant or extinct/ destroyed. in this article, a standardized nomenclature and guidelines for its further development are being proposed for natural filoviruses, i.e., filoviruses occurring in nature. follow-up articles will clarify the nomenclature for laboratory and artificial/synthetic filoviruses and provide datasets on all filoviruses known with name designations following the scheme proposed here. unfortunately, there is no universally accepted definition for the terms ''strain'', ''variant'', and ''isolate'' in the virology community, and most virologists simply copy the usage of terms from others. here, we propose not to add to the existing confusion by constructing radically novel definitions, but rather to employ or extrapolate from the few existing definitions that have been brought forward. it is important to point out here that no matter at which classification level one examines ''a virus'', one always deals with a varied population. a virus-infected cell will, after only one round or replication, already contain a population of genomes, and virions derived from these genomes will vary slightly from each other (quasispecies [ ] ). likewise, a sample taken from a virus culture or an infected animal will contain numerous virions, many of which vary slightly (quasispecies [ ] ). while single-virion analysis is theoretically possible, it certainly is not done routinely right now (it would be meaningless as far as naming is concerned), and even if it were, one would still have to work with virion populations to infect animalsand of course virions are not equal to viruses [ ] . consequently, ''a virus'', ''a strain'', ''a variant'', or ''an isolate'' always refers to populations and not to single physical entities, and their descriptions are therefore based on average properties. for instance, ''the sequence'' of ''an isolate'' is a consensus sequence of the population of genomes present in the analyzed sample. [ ] . these two definitions are also reflected in the words for ''strain'' in other languages, such as german (stamm), which back-translate to ''trunk'' rather than ''branch'', i.e., the word implies something fundamentally different from a reference entity despite it being directly related to it, possibly with little genomic sequence variation. a strain is therefore a genetically stable virus variant that differs from a natural reference virus (type variant) in that it causes a significantly different, observable, phenotype of infection (different kind of disease, infecting a different kind of host, being transmitted by different means etc.). ''genetically stable'' means that the genomic changes associated with the phenotypic change are largely preserved over time through natural selection. the extent of genomic sequence variation is irrelevant for the classification of a variant as a strain since a distinct phenotype sometimes arises from few mutations. ''observable phenotype'' means, for instance, that within a comparative animal experiment, it would be possible for the researcher to distinguish between the reference control virus-infected animal and the animal infected with the alleged new strain, without knowing which animal received which virus and without having any information about the differences between the two viruses. the designation of a virus variant as a virus strain would be the responsibility of international expert groups. thus far, despite the abundant indiscriminate use of the word ''strain'' in the filovirus literature, natural filovirus strains according to this definition have not been reported. all described genetic variants of ebov, for instance, cause a similar hemorrhagic fever in humans and even experimental animals and are transmitted similarly. none of the known ebov genetic variants can be distinguished from others on clinical grounds alone. in fact, their variety seems to be limited to subtle differences in growth kinetics and plaque formation in vitro or subtle changes in the duration of disease in experimental animals, and ultimately derives from limited, but often stable, differences in genomic sequence [ ] . this also holds true for the different genetic variants of marv, ravv, bdbv, restv, and sudv (currently, there is only one isolate of tafv and none of llov). we therefore recommend abstaining from using the word ''strain'' in context of any natural filovirus until either a particular genetic filovirus variant is discovered that causes a difference in disease phenotype and/or until expert groups establish a clear-cut definition of what ''phenotype'' means and to which extent phenotypes must differ to establish a filovirus as a strain. van regenmortel defined a virus variant as an isolate or a set of isolates whose genomic (consensus) sequence(s) differ(s) from that of a reference virus [ ] , i.e., the term ''variant'' is often equivalent to ''mutant''. according to fauquet et al., a virus ''variant is something that differs slightly from the norm…[i.e.,] it means a slightly different genome, symptom, or mode of transmission'' [emphasis added by the authors] [ ] . according to van regenmortel's definition, which we adopt, multiple genetic filovirus variants have been described during the last four and a half decades. a natural genetic filovirus variant is a natural filovirus that differs in its genomic consensus sequence from that of a reference filovirus (the type virus of a particular filovirus species) by b % but is not identical to the reference filovirus and does not cause an observable different phenotype of disease [ ] (filovirus strains would be genetic filovirus variants, but most genetic filovirus variants would not be filovirus strains if a strain definition would be brought forward). fauquet and stanley defined a virus isolate as ''a sam-ple…that has been cultured for study'' [ ] . van regenmortel has come to a similar conclusion and defined a virus isolate as ''simply an instance of a particular virus'' [ ] . we suggest adopting the latter definition for filoviruses, as advancement in sequencing now allows for the partial characterization of an instance of a virus variant in the absence of culturing. a natural filovirus isolate is an instance of a particular natural filovirus or of a particular genetic variant. isolates can be identical or slightly different in consensus or individual sequence from each other. it is important to point out that the designation of a filovirus as a ''genetic variant'' could change with time given the accumulation of new data that justify such a change. a novel isolate of a virus may at first be grouped with a particular genetic filovirus variant based on sequence information but later reclassified as a strain after experimental infections reveal it to behave phenotypically differently from a reference variant (for instance, if the filovirus did not cause viral hemorrhagic fever in a laboratory nonhuman primate but rather caused encephalitis). it would be the decision of international expert groups to change the designation under such circumstances. it is also worth mentioning that historically the term ''virus isolate'' was used to designate a particular virus detected in a certain biological organism at a certain time, but not for a virus batch prepared in a laboratory from a seed culture that ultimately is derived from that detected virus. we explicitly do not recommend labelling every instance of a filovirus culture in the laboratory as a separate isolate. ideally, filovirus taxonomy below the species level would follow an existing general scheme. unfortunately, as described above, such a global nomenclature does not exist-but the influenzavirus, avian coronavirus, and rotavirus nomenclatures are sufficiently similar to serve as examples. we suggest following the rotavirus proposal, and propose the following general template for filoviruses, to be used in the materials and methods sections of manuscripts: if an isolation host can only be identified to a taxon level higher than species, then the entire name of the lowest known taxon should be used. for instance ''hipposiderus'' (member of the genus hipposiderus). naming isolation hosts in this way is preferable to/more concise than using vernacular names, as these vary from language to language and one particular organism is often referred to by multiple vernacular names. see below for suffixes example for the full-length designation of an isolate in the methods section of a manuscript: ''ebola virus h.sapiens-tc/cod/ /kikwit- '' furthermore, we propose following the suggestions of fauquet et al. [ ] to define shorter versions of names for the convenience of writers and presenters. we suggest using the following medium-length designation for virus names in figures, such as phylograms, sequence alignments or diagrams: genbank records are indexed with regard to taxonomy, and each record must be associated with the ''organism'' field. following the approach of rotavirus and adenovirus experts, we suggest the species name be used as the ''organism'' name on genbank records. the virus name described above and the rest of the full-length designation described above (i. it is important to remember that ''…the rationale behind any specific naming scheme may not stand the test of time. yet, the metadata is [sic] a constant, and as long as the relevant metadata is [sic] included in every genome record, any naming format will be supported'' [ ] . this means that future developments in filovirology may result in the demand for a modified or different nomenclature, which, however, should not be difficult to create based on the metadata collected and archived for the current system. a logical consequence of this system is therefore also the development of a database that contains the information suggested here in conjunction with metadata available from publications, other databases, or research records. in fact, as many data as possible should be added to genbank records, and metadata ought to be updated on a constant basis. filovirus genetic variant/isolate designations are by no means planned to replace record metadata within genbank records, but rather are designed to be ''built'' from them. nomenclature for filoviruses characterized from passage material in , maniloff convincingly argued that isolates of viruses do not necessarily have to be available for their classification in existing taxonomical schemes, and that ''no special taxon need to be considered for uncultured viruses'' (such as the taxon candidatus used in bacteriology) as long as the relationship of the uncultured virus to existing ones can be inferred unequivocally [ ] . maniloff therefore extended to virology what has long been held in bacteriology, namely that the majority of infectious entities in nature most likely cannot be propagated in the laboratory due to their special adaptation to particular cell types and replication conditions. furthermore, sequence information from uncultured viruses (even if they could be cultured) is strongly desired because the virus in question could quickly adapt to culture conditions and therefore mutate rapidly. we agree with maniloff's proposition but argue that the sequence of at least a near-complete genome (only incomplete at its extreme and termini) of an uncultured filovirus has to be available before classification. llov is the only uncultured filovirus known at the time of writing for which near-complete genomic data are available, and it is also one of only two filoviruses we know of that have been sequenced before passaging in tissue/cell culture [ ] . to differentiate uncultured or passage filoviruses for which near-complete genomic data are available from those that exist in culture, we propose to follow the suggestions of the rotavirus classification working group and to add the suffix ''-wt'' (for ''wild-type'') to their genetic variant names as outlined above. nomenclature for filoviruses characterized from passage x material filoviruses that have undergone tissue/cell culture passaging should receive names supplemented with the suffix (''-tc''). the exact passaging history should be provided in genbank metadata fields and also in the methods section of manuscripts next to the virus designation. nomenclature for potential filoviruses only known from genome fragments in , voevodin and marx introduced the term ''fragvirus'' for presumptive viruses known only from fragmented genomic sequence data [ ] . we agree that the amplification of short stretches of filovirus genomes and their phylogenetic placement using adequate homologous sequences derived from existing filoviruses is not sufficient to recognize truly novel viruses. for instance, amplified sequences could be experimental artifacts or result from rna/dna cross-contamination of samples. near-fulllength genomic sequencing and/or isolation of a replicating filovirus is essential to prevent misinterpretations of filovirus endemicity and diversity. to distinguish presumptive filoviruses known primarily from genomic sequence fragments, we propose to add the suffix ''-frag'' (for ''fragment'') to their genetic variant names. as genetic variant assignment could change upon further accumulation of data, the genetic variant names ought to be placed in quotation marks to denote the fact that they are considered temporary. for instance, marburg virus r.aegyptiacus-frag/ken/ /''ke '' would be the designation for the virus hypothesized to exist based only on the availability of an np gene fragment sequence (genbank accession # gq ).''-frag'' viruses may be reclassified as ''-wt'' or ''-tc'' viruses upon the detection and description of near-complete genomic data or virus isolation, upon which the genetic variant designation could become official (quotation marks dropped: ''ke '' ? ke ) or be changed. storage of viruses is not always optimal, thereby resulting in their inactivation over time. furthermore, virus-infected samples, such as formalized, paraffin-embedded, or frozen tissues are often discarded when storage space is limited. it is therefore no surprise that once-isolated viruses have been inadvertently or deliberately destroyed. in particular, many marv isolates obtained during the first recognized marburg virus disease outbreaks in west germany and yugoslavia in , such as isolates ''flak'', ''hilberger'' ''lüdicke'' or ''kliebe'', may have been lost forever. others, such as ''popp'' or ''hartz'', are still available in the form of guinea-pig-adapted or guinea-pig-passaged versions. a considerable percentage of the early filovirus literature reports experiments performed with these virus isolates. their natural history often allows their closest still-available relatives to be inferred, thereby allowing for extrapolation of scientific data to virus isolates used today. we would like to emphasize the importance of studies done with now unavailable viruses while urging that it should be made clear to readers that viruses used for said studies are not available anymore and that results of experiments done at present with a closely related isolate may therefore not necessarily fit historical results. to distinguish unavailable filoviruses, we propose to add the suffix ''-hist'' (for ''historical'') to their genetic variant names. mediumlength designations are not necessary for ''-hist'' viruses, as genomic sequences are not available and therefore the construction of phylograms and alignments is impossible. we recommend that full-length isolate designations always be used in the materials and methods section of filoviruses: natural strains, genetic variants, isolates manuscripts next to the taxonomic placement of the isolate (order, family, species), genbank accession number if available, and its passaging history, if known, or to the extent that it is known: ''hela cells in -well plates were infected for h with ebola virus h.sapiens-tc/cod/ /kikwit- (order mononegavirales, family filoviridae, species zaire ebolavirus; genbank accession no. ay ) at an moi of . , , or . virus was obtained from the centers for disease control and prevention, atlanta, georgia, usa, and had been passaged twice through grivets (species chlorocebus aethiops) and twice through grivet kidney epithelial (vero e ) cells before use''. if the article does not address taxonomy, taxon (italicized) names should not be used elsewhere in the manuscript, and only the virus abbreviation should be used in the remainder of the manuscript text (in the example above: ''ebov'') after proper introduction as long as no other genetic variant or isolate of the same virus has to be addressed. we recommend using abbreviated designations in cases where several genetic variants or isolates of one filovirus are addressed: ''here, we demonstrate that infection of rhesus monkeys with ebov/may protects from subsequent infection with ebov/kik''. we propose to limit the use of medium-length designations to phylograms and sequence alignments (and to replace them with abbreviations if space is limited). upon discovery of a novel filovirus, it is, ideally, up to the discoverer to create an appropriate isolate designation according to the scheme proposed here. we strongly recommend (i) discontinuing the usage of patient names or patient name abbreviations for any part of the designation, as such practice is ethically problematic; (ii) avoiding the use of country names, as this has caused diplomatic problems in the past; (iii) avoiding the use of any ''unusual'' characters, such as those with diacritical marks, but to stick to the standard -letter latin alphabet for the sake of database input and handling; and (iv) choosing designations that can be pronounced easily in place of designations that solely consist of numbers as these are difficult to memorize. we further encourage all scientists to contact and seek the advice of the ictv filoviridae study group (http://ictvonline.org/subcommittee.asp?committee= &se=) before publication of a novel isolate name. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses discovery of swine (sic) as a host for the reston ebolavirus (sic) towards viral genome annotation standards, report from the ncbi annotation workshop. viruses taxonomy: get it right or leave it alone should all other biologists follow the lead of virologists and stop italicizing the names of living organisms? a nomenclature for avian coronavirus isolates and the question of species status improved clarity of meaning from the use of both formal species names and common (vernacular) virus names in virological literature revising the way we conceive and name viruses below the species level: a review of geminivirus taxonomy calls for new standardized isolate descriptors geminivirus strain demarcation and nomenclature family filoviridae taxonomy of the caliciviruses virus taxonomy-ninth report of the international committee on taxonomy of viruses filoviruses-a compendium of years of epidemiological, clinical, and laboratory studies proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations clarification and guidance on the proper usage of virus and virus species names being obsessive-compulsive about terminology and nomenclature is not a vice, but a virtue family filoviridae evaluation of perceived threat differences posed by filovirus variants fruit bats as reservoirs of ebola virus identification and classification of viruses that have not been propagated uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) discovery of an ebola virus-like filovirus in europe desk encyclopedia of general virology studies of reservoir hosts for marburg virus newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda isolation of genetically diverse marburg viruses from egyptian fruit bats a proposal to change existing virus species names to non-latinized binomials viruses are real, virus species are man-made taxonomic constructions virologists, taxonomy and the demands of logic virus species and virus identification: past and current controversies genetics and evolution of infectious diseases frag-virus: a new term to distinguish presumptive viruses known primarily from sequence data isolates of zaire ebola virus (sic) from wild apes reveal genetic lineage and recombinants world health organization ( ) a revised system of nomenclature for influenza viruses world health organization ( ) a revised system of influenza virus nomenclature-a report of the who study group on classification world health organization ( ) réexamen de la nomenclature des virus grippaux a: mémorandum oms filoviruses: natural strains, genetic variants acknowledgments we are indebted to philippe le mercier (swiss institute of bioinformatics, geneva, switzerland) and colleagues for providing unpublished thoughts on virus nomenclature below the species level. we also thank thomas s. postler (new england primate research center, southborough, ma, usa) and philip j. kranzusch (harvard medical school, boston, ma, usa) for their very useful editorial comments and suggestions. key: cord- -s a m authors: black, w. d.; hartley, c. a.; ficorilli, n. p.; studdert, m. j. title: reverse transcriptase-polymerase chain reaction for the detection equine rhinitis b viruses and cell culture isolation of the virus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: s a m equine rhinitis b virus (erbv), genus erbovirus, family picornaviridae occurs as two serotypes, erbv and erbv . an erbv-specific nested reverse transcriptase-polymerase chain reaction (rt-pcr) that amplified a product within the d(pol) and ′ non-translated region of the viral genome was developed. the rt-pcr detected all available erbv isolates and one available erbv isolate. the limit of detection for the prototype strain erbv . / was . % tissue culture infectious doses. the rt-pcr was used to detect viral rna in six of nasopharyngeal swab samples from horses that had clinical signs of acute febrile respiratory disease but from which erbv was not initially isolated in cell culture. the sequences of these six erbv rt-pcr positive samples had – % nucleotide identity with six other partially sequenced erbv isolates and one erbv . erbv was isolated from one of the six samples at fourth cell culture passage when it was shown that the addition of mg/ml mgcl( ) to the cell culture medium enhanced the growth of the virus. this isolated virus was antigenically similar to erbv . / . determination of the nucleotide sequence of the p region of the genome also indicated that the isolate was erbv , and it was therefore designated erbv . / . this is the first reported isolation of erbv in australia. the study highlights the utility of pcr for the identification of viruses in clinical samples that may initially be considered negative by conventional cell culture isolation. equine rhinitis b virus (erbv), formerly equine rhinovirus , was first isolated in switzerland [ ] , and the genomic sequence of the prototype isolate, p / , was determined [ ] , resulting in its classification in a new genus, erbovirus [ ] . viruses used to develop and validate the rt-pcr are listed in table . a stock of each virus was prepared by inoculation of monolayer cell cultures of rabbit kidney (rk ) cells as previously described [ ] . other cells used for virus culture, including virus isolation attempts, were vero cells (passage - ), rk cells (passage - ) and equine foetal kidney (efk) cells (passage ). cells were cultured in either eagle's minimal essential medium (mem, sigma-aldrich) with mm nahco and µg/ml ampicillin (sigma-aldrich) or, when it was shown that mgcl enhanced erbv growth in cell culture, in leibowitz' l- medium (l- , sigma-aldrich) with µg/ml ampicillin and mg/ml mgcl . each of the media included . % foetal bovine serum (fbs, jrh biosciences). viral rna was extracted using a qiaamp viral rna extraction kit (qiagen) according to the manufacturer's directions. reverse transcription was performed using µl of rna template and µl of µm oligo-(dt) reverse primer heated to • c for min, centrifuged briefly and cooled quickly on ice. samples were then incubated at • c before the addition of . µl of superscript ii-rnase h reverse transcriptase (invitrogen), . µl rnaguard (amersham biosciences), µl mm dntp, µl mm dtt, and µl × rt buffer. after incubation at • c for min, reactions were inactivated at • c for min and either used undiluted, or diluted : with water. if stored for later use, samples were heated to • c and vortexed immediately prior to use. pcr reactions were prepared in -µl pcr tubes (clp biological), with µl final reaction volume with nm of each primer, units taq polymerase (promega), taq buffer, . mm mgcl , µm of each dntp [ ] . primers used in the first round were: nm of primers erbv.outer.f (ttttgatgcttca cattctcc) and erbv.outer.r (cgctgtaccctcggtcctactc), to which µl of the undiluted rt mixture was added as template. one drop of mineral oil was added to the reactions. aerosol-resistant micropipette tips (clp biological) were used throughout. pcr cycle conditions were: min at • c, then cycles of sec at • c, sec at • c, and sec at • c. µl of the resulting first-round pcr mixture was diluted in ml of tris-hcl buffer (ph . ), and µl was then added to the second-round pcr reactions as template. second-round pcr reactions were performed as for the first-round pcr, but using primers erbv.inner.f (cttactaygaatgtgarggggc) and erbv.inner.r (gcctcggcgagtgaagag). cultured erbv isolates were diluted : in transport medium prior to viral rna extraction to avoid overloading the nested rt-pcr. amplicons were analysed in % agarose gels as previously described [ ] . purification of amplified viral cdna for sequencing were also as previously described [ ] . typically, for sequencing pmol of primer, µl of bdt . (applied biosystems) and - ng of template dna was used per sequencing reaction. both strands of at least three separate clones were sequenced. dilutions of erbv . / were used to determine the sensitivity of the specific rt-pcr. the virus titre of the erbv . / sample was determined, and the sample was then diluted to obtain , , , . , . , . and . tcid per µl, i.e., , , , − , − , − and − log tcid per µl. these samples were then each subjected to rna extraction and nested rt-pcr. the nested rt-pcr minimally detected . tcid of erbv . / in both rounds of the pcr. while there was no absolute difference in the detection limit of the first-and second-round pcr, the amount of amplified dna appeared much greater in the second round of the rt-pcr than the first round under the conditions described (data not shown). nasopharyngeal swabs samples from horses that had acute febrile respiratory disease that were obtained as part of another study (unpublished) were examined using the rt-pcr. nasopharyngeal swabs were obtained using a modification of a method previously described [ ] . briefly, dacron 'nu gauze' compress swabs (johnson & johnson) were used for mucous collection. following collection, swabs were placed in viral transport medium (mem supplemented with non-essential amino acids, % fbs, mm nahco , µg/ml ampicillin, µg/ml gentamicin, and µg/ml fungizone (sigma-aldrich)). the samples were transported on ice, then filtered through a sterile . µm membrane filter (millipore) and stored at − • c. serum samples were also obtained at the time of the nasopharyngeal swab, and a second sample was obtained approximately weeks later. the samples were tested for the presence of viruses by inoculation onto rk , vero and efk cells. when the erbv-specific nested rt-pcr was satisfactorily validated, the nasopharyngeal swab samples were re-tested specifically for the presence of erbv. for virus isolation, nearly confluent vero, rk and efk cell monolayers cultured in cm flasks were used. after the cell culture growth medium was removed, ml of filtered nasopharyngeal swab sample was inoculated onto the cells and incubated at • c for h. ml of maintenance medium (mem at ph . with . % fbs, µg/ml ampicillin and mm nahco ) was then added to each flask, and the flasks were incubated at • c for days with daily examination for cpe using an inverted microscope. the samples were passaged three times, using ml of the cell culture supernatant from the previous passage as inoculum, then stored at − • c. assays for sn antibody were performed in sterile, -well, flat-bottom microtiter trays as previously described [ ] . an erbv . / rabbit antiserum used in sn and immunofluorescence assays was prepared as previously described [ ] . immunofluorescence assay staining of rk cells infected with erbv . / and erbv . were performed using rabbit erbv . / antiserum [ ] , biotinylated swine anti-rabbit immunoglobulin (dako), and streptavidin-fluorescein isothiocyanate (dako) as previously described [ ] . nucleotide sequences determined in this study for the d pol - utr rt-pcr products from the six nasal swabs are genbank accession nos. ay -ay . the nucleotide sequences for the same region of four of the erbv isolates listed in table are genbank accession nos. ay -ay . the p region sequence for the isolated virus, erbv . / is genbank accession no. ay . the rt-pcr amplified a product from all of erbv isolates and one erbv isolate (erbv . / ) as listed in table (fig. ) . the products of both the first and second rounds of the pcr were of the expected size. the rt-pcr did not amplify any product from either of two tested erav isolates, nor was any product amplified from uninfected rk , vero or efk cells, or media control. products of the expected length were amplified from six of nasopharyngeal swab samples ( / , / , / , / , / and / ) using the erbv-specific nested rt-pcr ( fig. ) , and there was no evidence that the primers amplified products from any other viruses that may have been present in the swab samples. there was a high background in the first-round rt-pcr (data not shown) such that detection and nucleotide sequencing was based on the second-round product. these samples were previously considered erbv negative based on primary isolation attempts in cell cultures. the nucleotide sequences were then aligned with the published sequences of erbv . / and erbv . / (data not shown), and the percentage nucleotide identity between these sequences was determined. there was - % nucleotide sequence identity between the nucleotide sequences of all six erbv isolates and the six dna products amplified from nasopharyngeal swabs. the d pol and ntr regions were highly conserved amongst the erbv sequences table and of sequences from / , / , / , / , / , / that were obtained directly from the relevant nasopharyngeal swab samples from horses with acute respiratory disease. sequences for erav.perv/ and emcv are included for comparison. indicated are gaps (−) due to alignment incompatibility (erav and emcv only) or due to lack of sequence data (erbv n-and c-termini only). conserved amino acids are also indicated (.) in general in that no correlation could be discerned between the individual nucleotide sequences and serotype (erbv or erbv ), known phenotypes such as acid stability, or geographic location or year of isolation. there were several nucleotide differences between each of the sequences of the erbv isolates and the nasopharyngeal swab samples, however, suggesting that they were each from different samples rather than due to the cross-contamination of laboratory samples. to facilitate comparison with other more distantly related picornaviruses and to allow for the redundancy of codons, the erbv and swab nucleotide sequences were translated into amino acids and were aligned with erav and emcv amino acid sequences of the d pol gene (fig. ) . the amino acid alignment was then used to determine percentage amino acid identities and similarities between each sequence. the deduced amino acid sequences obtained from the nasopharyngeal swab samples were very similar to each other ( . % amino acid identity; . - % amino acid similarity), and were very similar to erbv sequences ( . - % amino acid identity; . - % amino acid similarity). in comparison to other known picornavirus sequences, the sequences from the swab samples were most similar to the sequence of emcv ( . - . % amino acid identity; . - . % amino acid similarity), and were relatively dissimilar to the sequence of erav ( . - . % amino acid identity; . - . % amino acid similarity). the nasopharyngeal swab samples were therefore concluded to be positive for erbv of unknown serotype and acid stability phenotype. subsequently it was shown that the isolated virus ( / ) was erbv (see below) and acid labile. each of the six erbv-positive samples came from horses that were seropositive for either erbv or erbv , or both viruses. there was, however, no clear association between erbv rt-pcr-positive horses and seroconversion to erbv or erbv , although some rises in sn antibody titres were observed between acute and convalescent serum samples. virus isolation in cell culture had been attempted for all nasopharyngeal swab samples, but no cpe was visible during three passages on rk , vero and efk cells. the cell culture supernatants from each passage and each cell type were then stored at − • c. the erbv-specific rt-pcr was used retrospectively on the cell culture supernatant samples from four of the six original nasopharyngeal swab samples that were erbv positive by rt-pcr, to determine if erbv was present in cell culture without visible cpe. only one nasopharyngeal swab sample, / da/pc, was putatively erbv positive by rt-pcr in the second and third passage in vero and rk cells, but not efk cells (data not shown). this indicated that an erbv had been isolated from the nasopharyngeal swab sample and had been cultured in vero and rk cells despite the absence of visible cpe. furthermore, this indicated that not all cell types e.g., efk, supported the growth of this virus under the conditions used. despite many attempts to isolate virus from the other rt-pcr-positive samples in all three cell types and using the modified culture conditions, only the one isolate was obtained. immunofluorescence was performed in attempts to visualize cells infected with erbv. / . rk monolayer cell cultures inoculated with either erbv . / or erbv. / showed similar clusters of rounded cells with strong cytoplasmic fluorescence when probed with rabbit antiserum to erbv . / , but not with preimmune rabbit serum (data not shown). after the fourth passage in cell culture the new isolate erbv. / produced complete cpe in established monolayers of rk cells in mem after [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] h. when cultures were inoculated with less than tcid , erbv. / in dmem formed small plaques in rk cells (approximately to µm diameter, as estimated by comparison with an hemocytometer grid), after which the plaques stopped increasing in size. the plaques were not visible days after incubation, and this was possibly related to the growth of surrounding cells into the previously clear plaque areas. this confounded the analysis of the erbv isolate / by virus titration and sn assays. this was also observed in the culture of erbv . / in rk and vero cells under similar conditions. to improve the visibility of the erbv plaques, virus titration assays were performed using various culture conditions including varying temperature, media, and supplementation with different solutes, conducted in -well replicates and repeated three times. the titre of the erbv isolate / was higher at • c in rk cells in l- medium ( . log tcid /ml) compared to dmem medium ( . log tcid /ml). the titre was higher in l- medium at • c ( . log tcid /ml), relative to • c ( . log tcid /ml), but the virus plaques remained small in each. all subsequent assays were performed at • c. the titre was higher in l- at • c with mm mgcl ( . log tcid /ml), or . mm zncl ( . log tcid /ml) relative to the same conditions without mgcl supplementation ( . log tcid /ml). in the presence of mgcl and zncl , the plaques were very large and visible (approximately - µm), but the uninfected cells also had some slight cytopathology presumably due to the toxicity of the solutes. zncl was cytotoxic at mm, causing complete destruction of the rk cell monolayer. other solutes such as cuso , nh so , mncl , glutathione and deae, were assayed, but were either cytotoxic or had little effect on the virus titre at the concentrations used. therefore, rk cells at • c in l- medium with mm mgcl supplement were used as the standard conditions for analyzing the erbv isolate / in titration and sn assays. erbv. / was neutralized similarly to erbv . / in an sn assay using monospecific erbv and erbv antisera from naturally infected horses. an erbv . / rabbit antiserum did not neutralize / , and rat antisera prepared against erbv . / neutralised the isolate with a pattern similar to erbv . / (data not shown). the isolate was therefore designated erbv . / . during the design of erbv-specific primers for an rt-pcr, several different primer sets were tested for suitability. these included primers based within the a and d pol regions, which amplified various erbv isolates including erbv . / , allowing the sequencing of this virus and its subsequent identification as an erbovirus [ ] . the non-translated region ( ntr) and the d pol sequences were highly conserved between all erbv sequences. the ntr contains a -base-long stem-and-loop structure, s m, [ ] , that is highly conserved between several viruses including human, sheep, cat, avian, turkey, and pig astroviruses, as well as avian infectious bronchitis and the human severe acute respiratory syndrome (sars) coronaviruses (genbank ay ), but not in picornaviruses, except erboviruses. the rt-pcr also amplified the expected product from three isolates of a distinct serotype of acid-stable equine picornavirus that has been proposed as an erbovirus (unpublished). the erbv-specific nested rt-pcr detected erbv . / at . tcid per µl of sample. this corresponded to a limit of detection of . tcid /ml. the detection of erbv in six of swabs tested, and the observation that erbv and erbv were isolated from the saliva of horses at titres from approximately - tcid /ml [ ] suggests that the erbv-specific nested rt-pcr is sensitive enough to detect erbv in clinical samples, assuming that nasal swabs and saliva samples are similar. while the first and second rounds of the nested rt-pcr were similarly sensitive in detecting cell-cultured erbv . / , the first round of the nested rt-pcr did not clearly detect erbv in clinical samples later found to be positive in the second round. this was presumed to be due rt-pcr inhibitors in the nasal swab samples, often observed to have suspended contaminants prior to filtration, and also mis-priming equine and other nucleic acids, which can reduce specific amplification. only after successful virus isolation from one of four nasopharyngeal swab samples that were erbv positive by nested rt-pcr was it determined that the virus growth in cell culture is significantly enhanced by modifications to the growth medium, particularly the inclusion of mm mgcl , which was not provided on initial culture of these swab samples. efforts were then made to improve the culture conditions for erbv in general and for erbv . / in particular. plaques produced by human rhinoviruses were reported to be larger in the presence of mm mgcl and also when low bicarbonate medium was used [ ] , while zinc ions may inhibit the growth and or cytopathogenicity of rhinoviruses [ ] . cardiovirus growth in cell culture is enhanced by mm magnesium ions [ ] . similarly, we found that the plaques formed by erbv . / and erbv . / were larger and more visible in l- medium supplemented with mm mgcl . the apparent enhancement of erbv . / titre in medium supplemented with mm mgcl was possibly due to the effect on the cells, rather than on the virus per se, given the similarity of effects using zncl . erbv . / was, however, stabilized at • c by m mgcl [ ] . furthermore, the culture of erbv was improved by the use of rapidly dividing cells [ ] . the development of an erbv-specific fully nested rt-pcr allowed the identification and first isolation of erbv in australia, demonstrating conclusively that erbv is present inaustralian horses. the erbv-specific rt-pcr would facilitate detection of erbv in nasopharyngeal swab samples by reducing the dependence on cell culture conditions and virus viability in samples. rt-pcr is advantageous because of high sensitivity and specificity, and the short time required to produce a result relative to established techniques such as virus isolation by cell culture, or the observation of seroconversion using paired serum samples and testing by elisa or sn antibody assay [ , ] . seroconversion of convalescent horses to erbv or erbv may not be a reliable means of diagnosis given that approximately % of horses sampled had detectable sn antibody to either virus, albeit at low sn antibody titre, and that most seropositive horses had only mild (i.e., < -fold) subsequent rises in sn antibody titre. erbv has been isolated from horses without detectable sn antibody, and conversely, some horses were observed to seroconvert without isolation of erbv [ ] , strengthening the need for a fast, reliable and sensitive method of detection, such as rt-pcr, that does not rely on a serum antibody response or virus isolation. the conclusive determination of erbv as the cause of equine respiratory disease by detection of virus in nasopharyngeal samples by nested rt-pcr is, however, also made difficult by the long-term persistent infection of horses with these viruses without causing apparent disease, as reported after the repeated isolation of erbv over years after two yearling colts were experimentally infected [ ] . conclusive demonstration of the association between erbv and clinical disease in future studies would be greatly facilitated by the use of the nested rt-pcr described above. sequence variation divides equine rhinitis b viruses into three distinct phylogenetic groups that correlate with serotype and acid stability respiratory disease in thoroughbred horses in training: the relationships between disease and viruses, bacteria and environment observations of picornavirus, adenovirus, and equine herpesvirus infections in the pirbright pony herd proc, twenty-fourth annual convention of the american association of equine practitioners. american association of equine practitioners infectious agents in acute respiratory disease in horses in ontario specificity, efficiency and fidelity of pcr. introduction to pcr prevalence of antibodies to equine viruses in the netherlands enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium equine picornavirus: isolation of virus from the oral cavity of healthy horses polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material equine rhinovirus infection -serologic evidence of infection in selected united states horse populations proc, fourth international conference on equine infectious diseases equine rhinitis b virus: a new serotype a common rna motif in the end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus beitrag zur kollektiven behandlung pharmakologischer reiihenversuche family picornaviridae prevalence of neutralizing antibodies to equine rhinitis a and b virus in horses and man equine rhinovirus is more closely related to foot-and-mouth disease virus than to other picornaviruses identification of noncytopathic equine rhinovirus as a cause of acute febrile respiratory disease in horses studies on the seroprevalence and frequency of equine rhinovirus-i and -ii infection in normal horse urine studies on picornaviruses isolated from the respiratory tract of horses physicochemical characterization of two serologically unrelated equine rhinoviruses an equine respiratory virus with enterovirus properties duration of symptoms and plasma cytokine levels in patients with the common cold treated with zinc acetate. a randomized, double-blind, placebo-controlled trial virus infections of horses at newmarket, and preparation and characterization of encephalomyocarditis (emc) virus equine rhinoviruses: new serotypes equine herpesviruses. i. isolation and characterisation of equine rhinopneumonitis virus and other equine herpesviruses from horses isolation and characterisation of an equine rhinovirus serological survey of some equine infectious diseases in the united arab emirates the effects of equine rhinovirus, influenza virus and herpesvirus infection on tracheal clearance rate in horses equine rhinovirus serotypes and : relationship to each other and to aphthoviruses and cardioviruses author we thank cynthia brown for technical assistance and rebbecca wilcox for providing data from a seroprevalence study from which the erbv equine antiserum was selected. we especially thank marianne weiss, university of berne, p. j. timoney and w. h. mccollum, university of kentucky, and dorothy holmes and ed dubovi, cornell university, for providing virus isolates and culture history. w.d.b. was the recipient of an australian postgraduate award (industry) with racing victoria the industry partner for the award. other financial support was from racing victoria and the cev special virology fund. key: cord- - f uqe authors: verbeek, a.; dea, s.; tijssen, p. title: genomic relationship between turkey and bovine enteric coronaviruses identified by hybridization with bcv or tcv specific cdna probes date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: f uqe genomic relationships between turkey and bovine coronavirus (tcv and bcv), which are currently placed in distinct antigenic groups, were demonstrated by hybridization using specific cdna probes. bcv-specific recombinant plasmid probes p , p , and p , holding inserts derived from (probably nonstructural) genes, and plasmids pn and pn holding the n and m gene, respectively, permitted the detection of isolates of both bcv and tcv with similar sensitivities. similarly, probing supernatants of cell cultures infected with several isolates of tcv, using probes pn and pm , respectively holding the n gene of bcv and tcv, resulted in equally intense detection signals. only a slight detection of mhv- , which is antigenically related to bcv, was observed, whereas the probes did not allow the detection of ibv, tgev, and hcv- e, which are placed in antigenic groups separate from those of bcv and tcv. detection of tcv was improved by hybridization with bcv-specific single-stranded (ss) probes holding sequences of the n and m genes and synthesized by the polymerase chain reaction. diagnosis of tcv in clinical samples by hybridization was better with pcr-produced ss bcv-specific probes than with ds pcr-produced probes or a combination of six recombinant plasmid probes holding non-overlapping bcv-specific cdna sequences. detection signals were absent when probing clinical samples with( )p-labelled puc-dna. coronaviral enteritis (bluecomb disease) causes significant economic losses to the turkey industry in the united states and canada [ , , ] . turkey coronavirus (tcv) infects and destroys the major absorbtive cells of the small and large intestines, leading to severe diarrhea in young poults but mild symptoms in adults' [ , , ] . initially, the disease was diagnosed by oral infection of susceptible poults with filtered intestinal contents. subsequently, detection of the virus was attempted by direct electron microscopy of clarified fecal samples [ ] or detection of viral antigens by immunofluorescence in the cytoplasm of mucosal cells in frozen intestinal sections [ , , ] . these techniques, however, are timeconsuming when large numbers of samples have to be screened and lack sufficient sensitivity. recently, an indirect elisa has been employed for detection of tcv in clinical samples [ ] . rabbit hyperimmune serum produced against purified egg-adapted and tissue culture-adapted minnesota strain of tcv crossreacted strongly with bovine coronavirus (bcv) and murine hepatitis virus type (mhv- ) although bcv and tcv are classified in different antigenic groups [ ] . subsequently, the existence of a strong antigenic relationship between bcv and tcv was confirmed by monoclonal antibodies produced against either virus [ i. in the current study, we investigated the genomic relationship between bcv and tcv by hybridization. several recombinant plasmids, containing bcvspecific cdna sequences [ , ] , were radioactively labelled by nick translation and used none or in combination for the detection of tcv. polymerase chain reaction (pcr)-produced probes which were highly efficient in detection of bcv [ ] , were also used to detect purified tcv or virus in supernatants of infected cell cultures. the use of one recombinant plasmid, either bcv or tcv specific, was usually unsufficient for the detection of tcv in clinical samples from diarrheic pours. therefore, a combination of bcv-specific probes or inherently more sensitive pcr-produced probes were used to detect virus in clinical samples to confirm the positive detection signals obtained with tissue culture-propagated virus. these results emphasized a genomic relatedness between the two viruses and ruled out the possibility of the activation in vitro of a latent bcv homologous coronavirus. the prototype egg-adapted minnesota strain of tcv [ ] , kindly supplied by dr. b. s. pomeroy (college of veterinary medicine, st. paul, mn, u.s.a.), was initially propagated by inoculation into the amniotic cavity of -to -days old embryonating turkey eggs and serially propagated in hrt- cells, a continuous cell line derived from a human rectum adenocarcinoma [ , ] . the origin of the tissue culture adapted tcv quebec isolate has been described previously [ ] . the ncdc (mebus) strain of bcv was obtained from the american type culture collection (atcc, vr ). the mebus strain and quebec bcv isolates, obtained from diarrheic calves, were also propagated in hrt- cells [ ] . turkey and bovine coronaviruses were purified from the supernatant fluids of infected cell cultures by differential and isopycnic ultracentrifugation on sucrose gradients as described previously [ , ] . the purdue strain of transmissible gastroenteritis virus (tgev, atcc, vr ) and serotype of murine hepatitis virus (mhv- , atcc, vr ) were propagated, respectively, in swine testicle and mouse fibroblast l cells [ ] . the beaudette, hollande, and massachusetts strains of infectious bronchitis virus (ibv) were propagated by inoculation into embryonating chicken eggs [ ] . purified human coronavirus hcv- e was a gift from dr. p. talbot (virology research center, institut armand-frappier). intestinal contents from -to -week-old turkey pours with mild to severe diarrhea were obtained from commerical flocks from different locations in quebec, canada. specimens from to l poxflts frnm the ~nme flock were pooled and used to make ten percent homogenates in . m tris-hc , ph . . the homogenates were clarified by centrifugation in an eppendorf centrifuge for min at room temperature. supernatants were stored at - °c until further use. virus from clinical specimens of diarrheic turkey poults was propagated by one to three passages on hrt- cells as described previously [ ] . the preparation of bcv-specific clones (covering about / of the genome and dispersed over its total length) has been described elsewhere [ , ] . to prepare tcv clones, the tissue culture-adapted minnesota strain of tcv [ ] was purified on sucrose gradients ( to %) and pelleted by ultracentrifugation for h at , rpm in a beckman sw . rotor. genomic rna was extracted from concentrated virus and copied into edna for cloning as described for bcv [ ] . briefly, purified rna, denatured by methyl mercuric hydroxyide treatment was used as template for first-strand edna synthesis using molony murine leukemia virus reverse transcriptase (pharmacia) in the presence of either oligod(t) or random primers (brl) [ , , ] . dna polymerase i and rnase h were used for second-strand synthesis [ ] . homopolymer tailing of the edna molecules [ ] , using dctp precursors, was followed by insertion into pst i linearized (dg)-tailed puc- (pharmacia) and transformation of jm cells [ ] . about tcv-clones from the edna library were screened for the presence of the genomic '-end and the n gene by colony filter hybridization [ ] using bcv-specific radiolabelled recombinant plasmids, containing sequences of the n gene [ , ] . one tcv-specific recombinant plasmid (pm ) with an insert of . kbp containing the entire sequence of the tcvn gene ( fig. ) (manuscript submitted), was selected as a probe to detect tcv. bcv-specific recombinant plasmids p , p , and p , which served previously for the optimization of hybridization conditions for bcv-rna detection [ ] , and probes pn and pn , holding respectively the bcv n and m gene ( fig. ) , were used in the detection of several coronaviruses in order to establish the presence of potential genomic homologies. the edna sequences of p and p of bp and bp, respectively, were determined (unpubl. data) and compared with sequence data available for all structural genes of bcv [- , , ] , using the ibi pustell sequence analysis programs. the translated sequence of the inserts revealed that they are parts of open reading frames. although both probes were found to be specific for bcv [- ] , no sequence homologies were found between these inserts and the structural genes of bcv, suggesting that these inserts represent fragments of non-structural genes. recombinant plasmid p , obtained by random priming of edna and holding an insert of . kbp was selected for its capacity to strongly detect bcv-rna in colony filter hybridization assays. the insert of p could be detected by probe p in southern blot analysis (not shown) and might represent at least a part of a non-structural gene. clinical diagnosis of tcv was assayed with a pool of six p-labelled bcv specific recombinant plasmids (pbcv-pool), holding non-overlapping edna sequences, in order to amplify the detection signal. this pbcv-pool was superior in clinical diagnosis of bcv when compared with em and elisa [ ] . recombinant plasmids were isolated from bacteria by alkaline lysis, purified on csc gradients [ ] , and labelled by nick translation in the presence of [ t p]dctp ( ci/ mmol; ) to specific activities of to x cpm/gg. recombinant plasmid pn was linearized in the vector polylinker with sali ( fig. ) and served as a template for single-stranded (ss) probe synthesis [ ] . primer piorf , ' tta cac cag agg tag ggg ttc, complementary to the sequence located to nucleotides from the '-end of the viral genome, was used in repeated primer extension using the pcr thermal cycler to synthesize probe molecules of approximately bases, corresponding to the internal open reading frame inside the n gene. double-stranded probes to the same sequence were synthesized by pcr using pn as template and piorf and piorf , ' atg gca tcc tta agt ggg ccg, to nucleotides from the '-end, as primers. plasmid pn , containing the bcv m gene (fig. ) , was restricted in the polylinker with hind iii for repeated primer extension in pcr, using the bcv-specific primer pxbav, ' gaa cat ttc tag att ggt cgg act g, to nucleotides from the '-end, resulting in probe molecules of approximately bases. pcr mixtures for ss probe synthesis consisted of ng template dna, ram tris-hct, ph . , mm kci, . mm mgci , mm dtt, i-tm of datp, ttp, and dgtp, pmol primer, gl [~ zp]dctp ( ci/mmol, . gm)and . u taq dna polymerase (cetus) in a final reaction volume of ~tl. about ng template was used in case of ds probe synthesis, whereas the final dctp concentration was adjusted with "cold" dctp to ~m. the reaction mixtures were overlaid with gl paraffin oil and subjected genomic relationship between turkey and bovine enteric coronaviruses to amplification cycles. each cycle included a denaturation for rain at °c, an annealing for min at °c, and a primer extension period for rain at °c. the last cycle included a rain incubation at °c, whereafter the products were separated from non-incorporated radionucleotides by spin-column chromatography, using sephadex g- . ss and ds probes were labelled to specific activities of about and t cpm/gg, respectively. clarified clinical samples from diarrheic turkey poults were extracted once with freon ( , , -trichloro trifluoroethane) before application to nitrocellulose membranes (pore size . gin), previously equilibrated in a x ssc solution ( x ssc = . g nac and . g sodium citrate dihydrate per liter). samples were spotted using a minifold ii slotblot apparatus (schleicher and schuell, inc.) followed by baking of the blots at °c for to h under vacuum. membranes were then rehydrated in a x ssc solution and prehybridized at °c for h or overnight in a standard hybridization solution consisting of: % formamide (or as indicated in the individual experiment), x denhardt's components, x sspe salts, . % sds, and gg/ml of denatured, sheared calf thymus dna [ ] . p-labelled recombinant plasmids and ds pcr-produced probes were heat-denatured and added to the prehybridization solution to a final concentration of ng probe/ml. pcr-produced ss probes were added without previous heat-denaturation at concentrations indicated in individual experiments. hybridization was for about h at °c, followed by blot-washing according to standard procedures [ ] . the double antibody-sandwich elisa was used for the detection of tcv in clinical specimens from diarrheic turkey poults as described elsewhere [ ] . suspensions of purified virus of several coronavirus strains were adjusted to approximately x virus particles per ml as determined by em by admixture with known amounts of latex spheres. gl of each suspension was spotted on nitrocellulose membranes and hybridized separately in triplicate under relatively stringent conditions (i,e., % of formamide) with p-labelled bcv specific recombinant plasmids p , p , and p . the three probes allowed individually the detection of the tissue culture-adapted mebus strain of bcv as well as two bcv quebec isolates. in addition, strong hybridization detection signals were obtained with the tissue culture-adapted minnesota strain and quebec tcv isolates (fig. ) . similar results were obtained when probing identical blots with pn and pn , respectively, holding sequences of the bcv n and m genes (data not shown). these bcv-specific probes allowed also individually the detection of twenty other quebec isolates of tcv (data not shown). only slight detection signals were seen with the antigenically related mhv- , whereas no detection signals were obtained with several strains of ibv and the human coronavirus hcv- e (fig. ) in antigenic groups distinct from the bcv and tcv groups. detection signals were absent when testing spotted supernatant fluids of non-infected hrt- cells (fig. ) and when probing identical blots with p-labelled puc-dna (not shown), confirming the specificity of the hybridization signals obtained. aliquots of serially diluted suspensions of purified tissue culture-adapted minnesota strain of tcv were spotted on nitrocellulose membranes. figure , lane , shows the detection signals obtained with the piorf -primed probe, synthesized on linearized plasmid pn t , whereas lane represents the detection signals obtained with the pxbav-primed probe, synthesized on pn . tcv detection, using a combination of both probes, resulted in an about -fold increase in sensitivity, as determined by the slope of the dose-response curve [ ] , whereas detectability was increased about -fold (fig. , lane ) . a minimum corresponding to about x viral genomes (as estimated via protein content), could be detected by combining both probes. as expected, detection of spotted puc- -dna was significantly reduced with the pcr-produced probes (fig. ) when compared to nick-translated recombinant plasmid probes (fig. ) , therefore increasing the specificty of detection. the efficacity of tcv-and bcv-specific probes in detecting tcv was established using seventeen clinical isolates, propagated by three passages in hrt- here, about gl of each diluted reaction mixture was added to the prehybridization solution of the individual blot. the virus quantity was calibrated by the estimation of protein content rather than rna due to the unstability of the latter. the amount of viral protein refers to the quantity of proteins measured according to bradford [ ] . an amount of pg viral proteins is estimated to correspond to about . x virus particles or about . pg genomic rna. ng puc- dna was spotted as a control on the hybridization itself. exposure was overnight at - °c cells. hybridization of virus-containing supernatants with probes p n and p m , respectively, holding the n gene sequences of bcv and t c v resulted in similar detection signals (fig. , lanes and ) , confirming the genomic relatedness between bcv and t c v in this gene as suggested by serological evidence. three cultured isolates (i.e., , , and ), identified as weak-positive by elisa [ ] were only slightly positive or negative by hybridization with either of the two nick-translated recombinant plasmid probes. hybridization with a combination of the pxbav-and p i o r f -primed p c r -p r o d u c e d ss probes showed a strongly increased detection sensitivity and resulted also in the identification of all samples as positive (fig. , lane ) . ten clinical samples, which were tcv-positive by elisa only after virus cultivation in hrt- cell monolayers, were also positive in hybridization assays using one bcv-specific probe (p ) on the supernatant fluid from the first passage (not shown). tcv in the original clinical specimens could hardly be detected with probe p alone (not shown). clinical diagnosis was attempted with either the pbcv-pool of recombinant plasmids to amplify the detection signal or with probes, synthesized by pcr as a result of labelling to specific activities about times higher than nick-translated probes. a total of clinical samples from birds with diarrhea were extracted with freon to eliminate binding competition between the virus and macromotecules present in the samples for sites on the nitrocellulose. treated samples were then spotted on nitrocellulose membranes for hybridization with bcv-specific probes (fig. ). among these, samples were identified as positive with ss pcrproduced probes, whereas of these were positive by hybridization with ds pcr-synthesized probes. only positive hybridization signals were obtained when the samples were probed with the pbcv-pool of recombinant plasmids. three of these samples were not identified as positive with either of the two pcr-produced probes and may have been false-positives. however, similar blots, incubated with radioactive-labelled puc- -dna to examine possible background hybridization with vector d n a did not reveal any significant hybridization detection signals, supporting the specificity of the signals obtained. an optimized elisa [ ] on all clinical specimens performed rather poorly (only samples were identified as positive; data not shown), may be as a result of repeated freezing and thawing of the samples. in the present study, genomic relatedness between bcv and tcv was demonstrated as bcv-specific probes to both structural and nonstructural genes could be used to detect isolates of tcv under relative stringent hybridization conditions. these results are in agreement with and extend previous findings on the serological relatedness between structural proteins of these two enteric coronaviruses as established by elisa and western immunoblotting [ ] . our bcv-specific probes could not detect the bcv-antigenically unrelated coronaviruses ibv and tgev, whereas only slight detection signals were seen with mhv- (fig. ) , which is placed in the same antigenic group as bcv [ ] . shockley et al. [ ] also reported a weak detection with another strain of mhv (mhv-a ), using a p-labelled insert probe corresponding to the bcv n and m genes [ , ] . their bcv-specific probe could in addition detect the serologically-related porcine hemagglutinating encephalitis virus and human coronavirus strain oc . clinical diagnosis of tcv with a bcv specific p-labelted probe (p ) was initially hampered by both the low concentration of virus in samples and by macromolecules, which clog the wells of the slot-blot apparatus and compete with the virus for binding sites on the nitrocellulose. in a previous report on the clinical diagnosis of bcv [ ] , we investigated several options for the elimination of these macromolecutes in bovine diarrheic samples and for the amplification of detection signals. similarly, tcv in clinical specimens, that were treated with freon before spotting, could be detected when using the pbcv-pool of recombinant plasmid probes (fig. ) . although not observed in this study, plasmid probes may in some cases cause difficulties in clinical diagnosis due to vector homology to plasmids in the sample [ , ] . therefore, pcr [ , , ] was applied to synthesize probe molecules from inserts in linearized recombinant plasmids to reduce labelling of vector sequences. pcrproduced ss probes were more efficient than nick-translated recombinant plasmid probes in detection of purified tcv and extracellular virions present in tcv-infected cell cfilture supernatant fluids (cf. figs. and ) . in combination, the pcr probes allowed the detection of a minimum amount of about viral genomes (fig. ) . hybridization signals obtained with spotted puc-dna (fig. ) were likely caused by molecules synthesized by polymerase-readthrough into vector sequences of incompletely digested templates. detection of spotted puc-dna was, however,, significantly reduced with pcr-probes when compared to recombinant plasmid probes (figs. and ) , improving the specificity of the detection assay. pcr-produced ss probes were superior to other probes used in clinical diagnosis, probably as a result of incorporation of label to high specific activities ( cpm/gg; [ ] ) and the absence of self-aamealing among the probes. double-stranded probes, synthesized in pcr with / of the dctp being radiolabelled were efficient in bcv detection but resulted in strong background after only h of autoradiography [ ] . in our studies on detection of tcv, the background was reduced by addition of more "cold" dctp for ds probe synthesis; in turn, however, fewer numbers of specimens were identified as positive. pcr-produced probes to the bcvn and m genes were, as in the detection of purified bcv [ ] and tcv (fig. ) , also superior in clinical diagnosis of tcv (fig. ). in conclusion, bcv or tcv probes may be valuable in molecular hybridization for routine clinical diagnosis of either virus but may be problematic when these two viruses have to be distinguished. studies are currently in progress to establish the nucleotide sequence of the tcv structural genes in order to determine the exact degree of genomic relatedness between the two viruses. isolation of transmissible enteritis agent of turkeys in avian embryos the vector homology problem in diagnostic nucleic acid hybridization in clinical specimens the use of a random priming procedure to generate cdna libraries of infectious bronchitis virus, a large rna virus nucleotide sequence of the glycoprotein sgene of bovine enteric coronavirus and comparison with the s proteins of two mouse hepatitis virus strains a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding coronaviruses associated with outbreaks of transmissible enteritis (bluecomb) of turkeys in quebec: hemagglutination properties and cell cultivation detection of turkey enteric coronavirus by enzyme-linked immunosorbent assay and differentiation from other coronaviruses isolation and trypsin-enhanced propagation of turkey entric (bluecomb) coronavirus in a continuous human rectal tumour (hrt- ) cell line antigenic and genomic relationships among turkey and bovine coronaviruses cossart ye (t ) false-positive results with hepatitis b virus dna not-hybridization in hepatitis b surface antigennegative specimens a simple and very efficient method for generating cdna libraries generation of single-stranded dna by the polymerase chain reaction and its application to direct sequencing of the hla-dqa locus studies on transformation of eschericia coli with plasmids une lign e cellulaire particuli~rement sensible fi le replication du coronavirus ent tique bovin: les cellules hrt- t ) sequence analysis of the bovine coronavirus nucleocapsid and matrix protein molecular cloning: a laboratory manual isolation and characterization of viruses associated with transmissible enteritis (bluecomb) of turkeys cloning and in vitro expression of the gene for the e haemagglutinin glycoprotein of bovine coronavirus fluorescent antibody test for rapid diagnosis of coronaviral enteritis of turkeys (bluecomb) antigenic relationships of the feline infectious peritonitis virus to coronaviruses of other species indirect fluorescent antibody test for the diagnosis of coronaviral enteritis of turkeys (bluecomb) bluecomb disease of turkeys combined immunofluorescence and transmission electron microscopic studies of sequential intestinal samples from turkey embryos and poults infected with turkey enteritis virus coronaviral enteritis of turkeys labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with dna polymerase i electron microscopy of coronavirus-like particles characteristic of turkey bluecomb disease terminal transferase-catalysed addition of nucleotides to the ' termini of dna enteric viruses in diarrheic poults primer-directed enzymatic amplification of dna with a thermostabte dna polymerase the generation of radiolabeled dna and rna probes with polymerase chain reaction diagnosis of porcine and bovine enteric coronavirus infections using cloned cdna probes tijssen p (t ) practice and theory of enzyme immunoassays biotinylated and radioactive cdna probes in the detection by hybridization of bovine enteric coronavirus genomic relationship between turkey and bovine enteric coronaviruses detection of bovine enteric coronavirus in clinical specimens by hybridization with cdna probes polymerase chain reaction for probe synthesis and for direct amplification in detection of bovine coronavirus this research was supported by the conseil des recherches et services agricoles du qu bec. av acknowledges the support received from the world university service of canada. this report was taken in part from a dissertation submitted by av to the department of virology, institut armand-frappier, universit du qu bec, in partial fulfillment of the requirements for the ph.d. degree. authors' address: p. tijssen, centre de recherche en virologie, institut armand-frappier, , boulevard des prairies, laval-des-rapides, qu bec h v b , canada.received october , key: cord- - f xe zc authors: rowland, r. r. r.; robinson, b.; stefanick, j.; kim, t. s.; guanghua, l.; lawson, s. r.; benfield, d. a. title: inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with -aminopurine date: journal: arch virol doi: . /s sha: doc_id: cord_uid: f xe zc porcine reproductive and respiratory syndrome virus (prrsv) belongs to a group of rna viruses that establish persistent infections. a proposed strategy for evading immunity during persistent prrsv infection is by preventing the induction of ifn activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. ifn-γ mrna expression was observed in the lymph nodes and lungs of pigs infected with wild-type prrsv strain sdsu- . pretreatment of marc- cells with ifn-γ inhibited wild-type (sdsu- p ) and culture-adapted (sdsu- p ) prrs viruses in a dose-dependent manner and at relatively low concentrations. the effect of ifn-γ on virus replication included reductions in the number of infected cells, virus yield, and rna content in single cells. virus replication was partially restored by the addition of -aminopurine ( -ap), an inhibitor of dsrna inducible protein kinase (pkr). the addition of -ap also restored the viral rna content per cell to near normal levels, suggesting that inhibition of viral rna synthesis was through pkr. the principal difference between p and p isolates was the recovery of p replication with lower concentrations of -ap. immunostaining with anti-pkr antibody showed a redistribution of pkr from the cytoplasm into nucleoli of infected cells. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus placed in the family arteriviridae, order nidovirales [ , , , ] . other members in this family include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv). the arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -coterminal set of subgenomic mrnas possessing a common leader and a poly-a tail [ ] . the outcome following prrsv infection depends on the age and reproductive status of the pig [ ] . infection of adult pigs generally produces a nonfatal disease characterized by flu-like symptoms, including mild interstitial pneumonia, elevated temperature, and inappetance. in sharp contrast, the infection of pregnant gilts and sows results in abortion and the birth of dead and weak-born piglets. surviving piglets exhibit the severest form of respiratory distress with mortality often approaching % within weeks after farrowing. surviving pigs continue to suffer the negative effect of prrsv by exhibiting increased susceptibility to secondary infection. during acute infection, prrsv replication is found in all organs and tissues, which is consistent with the macrophage as the principal cell population supporting virus replication [ , ] . non-macrophage cells, including type ii pneumocytes [ ] , bronchiolar epithelial cells and arteriolar endothelial cells [ ] may provide additional sources of virus in infected pigs. in culture, prrsv replicates in porcine alveolar macrophages, blood monocytes, monocyte-derived macrophages and monkey kidney lines derived from ma- cells [ , , , ] . pigs surviving acute prrs support a long-term asymptomatic infection, which has contributed to the worldwide spread of the disease [ , , , , ] . even though prrsv-specific humoral and cell-mediated immune responses appear early [ , ] , persistently infected pigs continue to shed virus for several months [ , ] . 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 [ ] or restrict the localization of virus replication within "immunoprivileged" anatomical sites, such as testes [ , , ] . the amino acid variability among prrsv field isolates within the ectodomain of the orf envelope protein suggests another strategy; antigenic drift [ ] . persistent viruses frequently incorporate evasion strategies that block the production or inhibit the activation of proteins up-regulated by interferon (ifn; [ , , , , ] ). studies suggest that prrsv may avoid ifn by preventing its induction in macrophages. both type-i and type-ii ifns efficiently inhibit prrsv replication in macrophages; however, small amounts of ifn are found in prrsv-infected pigs and infected macrophages in culture [ , , ] . since prrsv-specific cell-mediated immunity is present during infection, it is assumed that ifn-␥, a product of activated t cells and nk cells, is present in infected pigs [ , ] . however, nothing has been reported regarding the induction of ifn-␥ in prrsv-infected pigs or the mechanism of ifn-␥-inhibition of virus replication. the purpose of this study was to evaluate the production of ifn-␥ in pigs following infection with prrsv and to characterize the mechanism of ifn-␥ action following infection of marc- cells with wild-type and culture adapted prrsv isolates. the results show that ifn-␥ efficiently inhibits prrsv replication in marc- cells, and at least part of the antiviral activity may be through pkr. the north american macrophage-tropic strain, sdsu- -p , is a low-passage isolate possessing attributes similar to other north american field strains. the marc- celladapted strain, sdsu- -p , was obtained by passaging p an additional times on marc- cells. adaptation was performed by infecting marc- cells on -well plates with serial : dilutions of virus. medium from the last well showing cpe was retained, diluted : , and ml added to a new t- flask of marc- cells. after incubation at • c for hr, the monolayer was washed with pbs, and fresh medium added. after days the flask was freeze/thawed at − • c, and medium was again end-point diluted on a new well plate. this process repeated until the total number of passages reached . marc- cells [ ] were maintained in mem supplemented with % heat-inactivated fetal bovine serum (fbs) and antibiotics at • c in % co . the indiana strain of vesicular stomatitis virus (vsv) was obtained from christopher chase at south dakota state university. unless otherwise indicated, confluent marc- cells were infected at an moi of approximately . tcid /cell. increasing the moi of sdsu- beyond . does not produce a further increase in the number of cells infected during the first round of infection or the virus yield (personal observation). recombinant human ifn-␥ (boehringer mannheim) and ifn-␣ (prepo) were stored at − • c. a mm solution of the nitrate salt of -aminopurine ( -ap; sigma) was prepared in mem and ph adjusted by adding m tris until the cell culture medium changed to pink. ifn-␥ and -ap were added at approximately h and h, respectively, prior to infection with virus. after h the medium was removed and cells washed twice with pbs. incubation was continued in fresh medium, containing freshly prepared ifn-␥ and -ap. cytopathic effect (cpe) was observed by phase contrast microscopy of live cells and after staining acetone-fixed monolayers with % crystal violet. cytokine mrna expression in lung and lymph nodes was evaluated in pigs that were exposed to prrsv in utero. this model is based on observations that infected neonates exhibit the severest form of disease [ , ] . seronegative sows were infected at days gestation with wild-type p or attenuated p . sows were allowed to farrow and live-born piglets sacrificed at days after birth. samples of lung and lymph node tissues were immediately frozen in liquid nitrogen and stored at − • c. total rna was extracted from tissues according to christopher-hennings et al. [ ] and stored at − • c. rt-pcr of porcine cytokine and ␤ -microglobulin mrnas was performed according to reddy et al. [ ] . cdna was prepared from ug of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase and random hexamers as primers. pcr amplification of ifn-␥, il- and ␤ -microglobulin cdnas was performed using the primers described in table . pcr amplification consisted of cycles ( sec at • c, sec at • c, and sec at • c) and dna products electrophoresed on a . % agarose gel and visualized using ethidium bromide. the identity of each amplified product was confirmed by cloning and sequencing the gel-purified fragment. [ ] . serial -fold dilutions of virus were placed on -well tissue culture plates containing marc- cells. after days, plates were fixed in % acetone then stained with sdow- . results were reported as tcid /ml. the intracellular localization of pkr was determined by staining with anti-pkr mab (anti-p from transduction laboratories). acetone-fixed cells were incubated for h at room temp with a : dilution of anti-pkr antibody, followed by a h incubation with biotinylated anti-mouse igg (sigma), diluted : . a final one hr incubation included texas redstrepavidin (molecular probes), diluted : and fitc-labeled sdow- , diluted : . antibodies and stepavidin were diluted in pbs-fbs and slides washed twice for min in pbs between the addition of each reagent. fitc-labeled sdow- and texas red-labeled anti-pkr antibodies were visualized under a fluorescence microscope using nm and nm (rhodamine) excitation filters, respectively. for northern blots total rna was extracted from cells at h after infection using the acid phenol guanidinium technique of chomcynski and sachi [ ] . rna was electrophoresed on % agarose following denaturation with glyoxal and dimethyl sulfoxide [ ] , then transferred to a nylon membrane. the amount of rna added to each well was adjusted to reflect the viral rna from the same number of infected cells (determined by the percentage of sdow- -postive cells in a small sample of cells removed from culture prior to rna extraction). the [ p] dctp-labeled prrsv-specific probe, designed to detect genomic and subgenomic rnas, was prepared by random-primed labeling a bp cdna template derived from rt-pcr amplification of the orf region [ ] . nylon membranes were hybridized with the probe overnight at • c, using a hybridization buffer prepared according to church and gilbert [ ] . nylon membranes were washed in × ssc at • c followed by a high stringency wash in . × ssc at • c for min, then exposed to x-ray film. the relative viral rna content in single cells was determined using a modification of the in situ hybridization technique described by peng et al. [ ] . approximately cells in ul of tissue culture medium were spotted onto denhardt's-treated slides. fixation, pre-treatment, hybridization, and post-hybridization procedures were performed according to lawson et al., [ ] using a modification of the original method described by anderson et al. [ ] . the [ s] dctp-labeled random-primed probe was prepared from the same bp orf template used in northern blot hybridization. after the last post-hybridization wash, slides were dipped in nbt- autoradiography emulsion (kodak), exposed for h in the dark, developed, then h and e stained with mayer's hematoxylin and % eosin y in ethanol. prrsv-infected cells were identified as having a silver grain content above that of mock-infected cells. histogram distributions were constructed by counting the number of silver grains over the cytoplasmic and nuclear regions of infected cells. statistical analysis was performed using a two-tailed student's t test. previous studies [ , ] identified the reduced ability of pigs and pig macrophages to produce ifn-␣. the purpose of this experiment was to characterize ifn-␥ mrna expression in acutely infected pigs. sows, at days gestation, were infected with wild-type p or attenuated p isolates. all p -infected piglets (n = ) were positive for prrsv at the time of necropsy ( days after birth) and exhibited typical congenital prrsv pathology, including microscopic lesions in lungs and lymph nodes. the piglets born to the p -infected sow (n = ) showed no signs of prrs and were negative for virus at the time of necropsy, confirming that this virus was rapidly cleared from the mother and did not cross the placenta. three pigs from each of the p and p groups were randomly chosen for analysis of cytokine mrna expression in lungs and mandibular lymph nodes. ifn-␥ mrna expression was detected in the lymph nodes from two of the pigs infected with the wild-type p virus (fig. ) . a third p pig showed no ifn-␥ mrna in lymph node; however, ifn-␥ mrna was detected in the lungs. there was no evidence of ifn-␥ expression in the lymph nodes or lungs of the p detected by rt-pcr amplification of total rna from three infected (p virus) and three control pigs (p virus). infection of pregant sows with sdsu- wildtype p and attenuated p isolates is described in materials and methods. n lymph node; l lung pigs. ␤ microgloblin mrna was amplified to nearly equal levels in all tissues. il- mrna was also amplified from all tissues, a cytokine mrna we frequently find it in lymphocytes and lymphoid tissues of un-infected pigs. the effect of ifn-␥ on the growth of prrsv in marc- was studied by incubating -well plates of confluent marc- cells with human ifn-␥ overnight followed by infection with prrsv p or p isolates. the results of a representative experiment, presented in fig. a , showed loss of crystal violet staining in p and p cultures not treated with ifn-␥. the effect of ifn-␥ on cpe was apparent at concentrations as low as u/ml ifn-␥. intact cell monolayers were observed in wells incubated with or , u/ml of ifn-␥. phase-contrast microscopy, performed prior to crystal violet staining, confirmed the absence of cpe. for the purpose of comparison, ifn-␥-treated marc- cells were also infected with vsv, a virus sensitive to interferon [ , ] . the experimental conditions were the same as those used for prrsv infection. intact cell monolayers were only observed in vsv-infected cultures treated with , u/ml ifn-␥ ( fig. a) . the effect of human ifn-␣ on prrsv replication was also investigated. the experimental conditions were the same as described for the ifn-␥ experiments. representative results, presented in fig. b , showed ifn-␣ inhibition of p and p isolates. similar to ifn-␥, ifn-␣ activity was apparent at concentrations as low as u/ml. unlike ifn-␥, the p isolate was more sensitive to ifn-␣ than p (compare wells treated with and ug/ml ifn-␣). this difference was confirmed in subsequent experiments. further assessments of ifn-␥ inhibition of virus replication were made by counting the number of infected cells and by measuring virus yields in cultures incubated with ifn-␥ overnight. ifn-␥ treatment produced dramatic decreases in the percent-infected cells and virus yield for both p and p viruses (fig. ) . similar to the cpe data the effect was evident at concentrations as low u/ml. ifn-␥, reduced virus yields at all time points and consistently reduced peak yields of p and p by greater than to logtcid /ml. ifn-␥ also reduced the number of infected cells. the growth curve experiments also revealed differences between p ad p isolates. improved adaptation of p to marc- cells was evident by increased virus yields and percent infected cells. another difference was the timing of peak virus yield for p , which was observed at h after infection versus viruses, respectively. ifn-␥ treatment, infection, and measurement of virus yields were performed in the same manner as described in fig. h for p . the experiment presented in fig. also revealed what appeared to be an increased sensitivity of p to ifn-␥. however, due to the different growth characteristics of p and p isolates, a differential effect of ifn-␥ was difficult to assess. there are several approaches that can be used to study the activation of pkr. a role for activated pkr in the ifn-inhibition of prrsv replication was studied by following the recovery of virus replication after the addition of -ap, an inhibitor of pkr phosphorylation [ ] . even though we did not evaluate the phosphorylation state of pkr, we assumed that reversal of ifn inhibition of virus replication following addition of -ap could only occur by preventing pkr phosphorylation. in these experiments -ap was added h prior to infection and after overnight incubation with u/ml ifn-␥. inhibition of ifn-␥ activity by -ap was dosedependent and evident at concentrations as low as mm for p (fig. ) . the optimal concentration of -ap required for maximum virus recovery of p and p infection were and mm, respectively. following several experiments, it was noted that -ap never recovered virus replication to the same levels obtained in infected cultures not treated with ifn-␥ ( fig. ; compare treatments and for p and treatments and for p ). and as shown in fig. further increasing -ap to mm in p -infected cultures actually inhibited virus yield. a similar effect was observed for the p virus incubated mm -ap (data not shown). pkr is typically found in both cytoplasmic and nuclear compartments [ ] . in uninfected marc- cells pkr was distributed throughout the cytoplasm with increased anti-pkr fluorescence in the perinuclear region (fig. d ). we did not observe a significant increase or redistribution in pkr immunofluorescence in cells following overnight incubation with u/ml ifn-␥. however, the intracellular distribution of pkr was dramatically different in prrsv-infected cells. in a representative experiment (fig. e ) pkr immunofluorescence was still present in the cytoplasm, but with increased fluorescence around the perinuclear region and within the nucleoli of p -infected cells (fig. e ). similar results were observed in cells infected with p . incubation with mm -ap prior to infection with p did not alter the pattern of pkr staining, suggesting that pkr activation was not required for nucleolar localization. interestingly, the intracellular distributions of pkr and prrsv nucleocapsid (n) proteins were almost identical (compare fig. b and e) . to determine a possible association between pkr and n protein in the absence of infection, we performed a similar experiment that expressed the n protein fused to enhanced green fluorescent protein (egfp). a representative result, presented in fig. c and f, showed n-egfp fluorescence in the nucleolus and cytoplasm; whereas, pkr remained in the cytoplasm. since the n-egfp fusion construct may not be representative the native n protein, we repeated the experiment using a construct that expressed only the n protein, which was detected using fitc-sdow- . under these conditions the n and pkr proteins did not co-localize to the nucleolus (data not shown). the inability of -ap to completely recover virus replication following ifn-␥treatment suggested that other anti-viral pathways, up-regulated by ifn-␥, were activated during prrsv infection. for example, the , oligoadenylate synthetase ( - a synthetase) pathway, activated by dsrna, inhibits translation by degrading mrna [ ] . northern blots were used to evaluate the integrity of p subgenomic mrnas. the results of a single experiment, presented in fig. a , showed decreased expression of all prrsv rnas following treatment with u/ml ifn-␥. we did not observe degraded rna in the ifn-treated cultures. viral rna expression was also reduced in the presence of mm -ap alone; however, the addition of -ap to ifn-␥ treated cells increased the level of viral rna expression above the ifn-␥-treated culture. to further confirm that ifn-␥ treatment decreased viral rna expression, relative changes in viral rna content in single cells were measured using a modified in situ hybridization technique [ ] . histograms showing the number of p -infected cells versus number of silver grains/cell in infected cells at hr r are presented in fig. b . the mean silver grain content in infected cultures was . grains/cell (fig. b, panel ) . pre-treatment with u/ml of ifn-␥ shifted the distribution towards cells containing significantly less viral rna (mean = . silver grains/cell, p = . , fig. b panel vs panel ) . the addition of -ap restored the mean rna content per cell to near normal levels (fig. b, panel vs panel ). this study confirms previous reports describing ifn inhibition of prrsv [ , , ] . furthermore, we extend these observations by demonstrating that ifn inhibits the replication of both wild-type and tissue culture-adapted isolates derived from a north american prrsv isolate. inhibition of prrsv was observed following pre-incubation with either type-i or type-ii ifns. furthermore, the sensitivity of prrsv to ifn-␥ was greater than vsv ( fig. a) , a virus frequently used for the detection of ifn activity [ ] . consistent with other reports describing the presence of cell-mediated immunity during prrsv infection [ , ] , we observed ifn-␥ mrna expression in lymph nodes and lungs of prrsv-infected pigs (fig. ) . whether or not this amount of expression represents a biologically relevant quantity of ifn-␥ remains to be determined. even though we demonstrated the effectiveness of ifn in inhibiting prrsv replication in marc- cells, little is known regarding the properties of the macrophages supporting prrsv replication in vivo. in infected pigs, prrsv replication is initially found in all organs and tissues [ ] . during the long asymptomatic stage of infection, virus replication is restricted to a subpopulation of cells located in tonsil and lymph nodes (personal observation), suggesting that these cells may be more resistant to ifn-␥, even when exposed to local elevations of cytokines from activated t cells. this observation is similar to studies of ldv infection of mice. ldv normally replicates in a subpopulation of macrophages, but in some strains of mice infects motor neurons causing paralysis. injection of mice with ifn-␥ prior to infection inhibits virus replication in motor neurons without significantly affecting the overall level of viremia [ ] . we propose a model in which the induction of ifn-␥ production during prrsv infection is sufficient to suppress virus replication in permissive cells in non-lymphoid organs, but does not affect replication in a small subpopulation of permissive cells in lymph nodes and tonsil. the identity of this prrsv-permissive population remains to be determined. anti-viral proteins, including pkr, - a synthetase and mx are up-regulated in response to ifn [ , , ] . pkr, after binding viral rna, is autophosphorylated on serine and threonine residues and inhibits translation by phosphorylating the alpha subunit of eif . phosphorylation of pkr is inhibited by -ap, an atp analog. the addition of -ap to cells pre-incubated with u/ml ifn-␥ partially recovered prrsv replication in marc- cells (fig. ) . these results demonstrate that at least, in part, the interferon-induced anti-prrsv activity may occur through the pkr pathway. the ability of -ap to partially restore viral rna synthesis in ifn-treated cells supports the work of deboon et al. [ ] who concluded that ongoing protein synthesis is required for the production of arterivirus rnas. one explanation for the failure of -ap to completely restore virus replication is the activation of other antiviral pathways not regulated by pkr. for example the activation of the - a synthetase pathway can be identified by the presence of degraded forms of both viral and ribosomal rnas [ ] . degraded rna was not detected in northern blots (see fig. a ) in total rna from prrsv infected cultures (personal observation). recently, zhang et al. [ ] reported increased expression of mx in cultured macrophages infected with prrsv. a role for mx in the inhibition of prrsv replication has not been demonstrated. another explanation for the failure of -ap to restore prrsv replication is the inhibition of -ap sensitive phosphorylation event required for virus replication. for example, mm -ap prevents the phosphorylation of the pe /e envelope glycoprotein, which is required for the production of infectious progeny [ ] . pkr is normally distributed between the cytoplasm and nucleus, which is consistent with its roles in regulation of translation and transcription. in marc- cells, we found pkr primarily concentrated in the cytoplasm and perinuclear regions (fig. d ). an unexpected observation was the localization of pkr to the nucleoli of prrsv-infected cells (fig. e ). nucleolar localization was not affected by -ap, suggesting that pkr activation is not necessary for nucleolar localization. this observation confirms the result of besse et al. [ ] who identified a non-phosphorylated form of pkr in the nuclei of hela cells. furthermore, pkr nucleolar localization during prrsv replication is not the result of a physical association with the prrsv nucleocapsid protein ( fig. c and f). from these results we can conclude that nucleolar localization of pkr in marc- cells is dependent on prrsv infection, but is independent of activation or an association with the nucleocapsid protein. prrsv infection of pigs continues to represent a worldwide disease problem. this study, combined with the results of others, clearly demonstrates an important role for ifn-␥ in the control of prrsv infection and suggests that the induction of ifn activity should be considered in the design of new vaccines. however, the mechanism for evasion of host defenses during persistent infection remains to be determined. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview interferon-alpha response to swine arterivirus (poav), the 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pigs porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence neonatal infection of mice with lactate dehydrogenase-elevating virus results in suppression of humoral antiviral immune response but does not alter the course of viraemia or the polyclonal activation of b cells and immune complex formation the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- convenient assay for interferons molecular cloning: a laboratory manual antiviral actions of interferon: interferon-regulated proteins and their surprisingly selective antiviral activities rrna cleavage as an index of ppp(a 'p)na activity in interferon-treated encephalomyocarditis virusinfected cells in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection detection of intracellular porcine reproductive and respiratory syndrome virus nucleocapsid protein in porcine macrophages by flow cytometry antiviral defense in mice lacking both alpha/beta and gamma interferon receptors differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity porcine reproductive and respiratory syndrome virus: a persistent infection characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection key: cord- -d f a ds authors: pensaert, m. b.; de bouck, p. title: a new coronavirus-like particle associated with diarrhea in swine date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: d f a ds coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv , which was found to be distinct from the known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv , which was found to be distinct from the known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. in , do yr.e and hutc~i~gs ( ) described a viral diarrhea, in swine and called it transmissible gastroenteritis. until recently, transmissible gastroenteritis virus was the only virus known to be specifically associated with diarrhea in swine of all ages. in , following the discovery of rotaviruses in different, animal species, a porcine rotavirus was detected in the feces of pigs with diarrhea ( ) . diarrhea could be reproduced experimentally in piglets with this virus. in a search for rotaviruses on belgian swine breeding farms with diarrheal problems, a new coronavirus-like particle was detected b y electron microscopic examination of intestinal or fecal samples from sick pigs. the present report describes the morphology of this coronavirus-like particle, and shows that it is distinct from the known porcine coronaviruses and causes diarrhea. up to now, the only coronaviruses isolated from swine have been transmissible gastroenteritis virus {tgev) and hemagglutinating encephalomyelitis virus (hev). tgev has been described as a cause of diarrhea in swine in countries all over the world ( ) . numerous studies have been performed on the v i r u s --a n i m a l interactions of tgev, which is usually detected either by its isolation from fecal these studies were supported by the institute for the encouragement of l~esearch in industry and agriculture (iwonl), brussels, belgium. material in cell cultures or by immunoflu orescence in the smm intestinal epithelium of infected pigs ( , ) . tgev infections can also be diagnosed serologically. j:iev was first described in canada in as a cause of centrm nervous disorders in pigs ( ). the same virus was later associated with a disease syndrome called vomiting and wasting disease in several european countries ( , ) . the virus can easily be detected by cultivation in several porcine cell cultures ( ). both tgev and hev have been classified as coronaviruses mainly on the basis of their specific morphology ( ). in , a sudden outbreak of diarrhea was observed in swine of all ages on belgian swine breeding farms. the morbidity in sows was very variable and the animals recovered after a diarrhea which lasted to days. all the pigs showed a watery diarrhea. death occurred up to the age of days and the overali mortality rate in these piglets was approximately per cent ( ) . it decreased with increasing age. tgev was suspected as the cause of this diarrhea. however, the direct immunofluorescence test for the diagnosis of tgev, which is routinely applied on cryostat sections of the small intestine of sick pigs, was negative for these pigs. the absence of seroneutralizing antibodies to tgev in the blood of sows collected to weeks after the outbreak confirmed that tgev was not involved. in an attempt to arrive at an etiologie diagnosis, fecal material and intestinal contents from pigs of each farm were subsequently processed for examination in an electron microscope by negative staining. they were diluted t to (v/v) in phosphate-buffered saline, ph . and clarified at × g, at ° c, for minutes. the supernatant was layered on top of a per cent sucrose solution and centrifuged at , x g, at ° c, for minutes. the resulting pellet was resuspended in a few drops of distilled water, placed on mesh formvar coated grids, and stained with per cent k-phosphotungstate, ph . . grids were examined using a zeiss em s- electron microscope at an acceleration voltage of kv. micrographs used for particle size measurement were taken at an instrumental magnification of , x, which were then photographically-enlarged to , x or , ×. gotavirus particles were not detected. however, eoronavirus-like particles were observed in specimens of pigs from each of the breeding farms. one of the fecal samples containing these coronavirus-like particles was designated cv and was used for further studies. the etiologic relationship between the corona virus-like particles, cv , and the occurrence of diarrhea was established by oral inoculation of a per cent suspension of the fecal material contmning cv into a one day old colostrumdeprived pig. the experimental pig was killed hours later, at the height of diarrhea, and a virus stock was prepared from an homogenate of its small intestine and contents. a bacteria free filtrate of the supernatant of a per cent suspension of this material was used for inoculation of colostrum-deprived-hysterectomy-derived piglets, kept i n isolation. seven control pigs were used. the pigs were inoculated at the age of to days. all the inoculated pigs developed a watery diarrhea within to hours after inoculation whereas the control animals remained normal. coronavirus-like particles were detected by electron microscopic examination in the watery feces or intestinal contents of each of the experimentally inoculated pigs. such particles were not found in the feces of the same pigs prior to inoculation or in the fecal samples of the control animals. the particles, shown in figure , had typicm coronavirus morphology. they were pleomorphic with a range in diameter of to nm, including the projections, which were approximately nm in length. most particles were between t and t nm in diameter. the projections formed a single fringe radiating from the core. they appeared to be club-shaped. only the dilated distal ends of the projections were seen on the micrographs. the negative stain also appeared to settle on the surface of some particles and an electron opaque central area covered by surface projections was often seen (fig. a --a r r o w s and lc) . no internal structure was observed. it was impossible to distinguish these coronavirus-like particles morphologically from tgev or h e v particles from similar preparations. other particles, different from these coronavirus-like particles, were also observed in the majority of the fecal samples. as seen in figure , they were pleomorphic and very variable in size, ranging in diameter from to nm with an average diameter of to rim. they carried numerous short projections, of approximate length nm, on their surfaces. similar particles of unkno~m identity have been described in human and animal fecal samples ( , , ) . in the present studies such particles have also been found in the solid fecal samples of the control pigs. they appeared, therefore, not to be associated with diarrhea. rotaviruses and other recognizable virus particles were not seen in control or experimentally inoculated pigs. as already mentioned, tgev was eliminated as the cause of the diarrhea on the origina farms. additionmly out of the experimentally inoculated pigs, killed at, the heigth of diarrhea, were negative for tge viral antigens in their small intestinal epithelium by the direct immunofluorescenee test. furthermore, the remaining pigs, inoculated with cv at the age of days, were allowed to recover after a diarrhea which lasted -- days. a serum sample, collected from these pigs weeks later, did not contain neutralizing antibodies against the cell culture adapted purdue strain of tgev. fig. . one eoronavirus-like particle cv (arrow) together with pleomorphie particles of unknown identity. bar represents nm the possibility that the cv particles consisted of h e v was less likely since the latter virus does not cause diarrhea in pigs. cryostat sections of the sma~ intestine of experimentmly inoculated pigs were negative for fluorescence by the direct test using a conjugate directed against the vw isolate of h e v ( ) . furthermore, the pigs that had been allowed to recover did not possess hemagglutination-inhibiting or seroneutralizing antibodies against this t t e v isolate. preliminary attempts were made to cultivate the coronavirus-like particle, cv , in primary pig kidney cell cultures and in secondary porcine thyroid cells. four weekly blind passages were made. the cells were examined for cytopathic effect and hemadsorption with chicken red blood cells, and the cell culture fluids were examined for hemagglutination. no evidence of viral replication in the cell cultures was obtained. it is kno~n that the tiev can easily be isolated in primary pig kidney cell cultures using the same criteria ( ) . the present data suggest that, as well as tgev and hev, another previously unrecognized coronavirus-like virus is prevalent in swine. the results indicate that diarrhea can be reproduced in experimental pigs with this virus and that it is associated with certain outbreaks of epizootic diarrhea on belgian swine breeding farms. more details on the clinical disease in the field and on the results of the experim e n t a l infections will be reported later. vomiting and wasting disease of piglets a transmissible gastroenteritis in pigs the hunt for viruses in infections of the alimentary s y s t e m : a n immuno etectronmicroscopicat approach a hemagglutinating virus producing encephalomyelitis in baby pigs pleomorphic virus-like particles in human faeces virus-like particles in calves' faeces diagnosis of transmissible gastroenteritis in pigs by immunofluorescence characteristics of a coronavirus causing vomition and wasting in pigs a virus isolated from an apparently new epizootic diarrhea in swine. proe. th world int the size and morphology of transmissible gastroenteritis and vomiting and wasting disease viruses of pigs titration of two porcine respiratory viruses in mammalian cell cultures by direct fluorescent antibody staining isolation of the virus of transmissible gastroenteritis (tge) from naturally infected piglets in cell culture transmissible gastroenteritis of swine the isolation of reovirus-like agents (rotaviruses) from acute gastroenteritis of piglets key: cord- -kjgah ts authors: lee, do hyun; jeon, young-soo; park, choi-kyu; kim, seungjoon; lee, du sik; lee, changhee title: immunoprophylactic effect of chicken egg yolk antibody (igy) against a recombinant s domain of the porcine epidemic diarrhea virus spike protein in piglets date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: kjgah ts porcine epidemic diarrhea virus (pedv) is a highly contagious enteric pathogen of swine causing high mortality rates in piglets. pedv outbreaks have occurred continuously in most swine-producing asian countries and have recently emerged in the united states, leading to large economic losses for both the asian and us pig industries. the spike (s) protein of pedv consists of the s and s domains, responsible for virus binding and fusion, respectively. the involvement of the s domain in specific high-affinity interactions with the cellular receptor and induction of neutralizing antibodies in the natural host makes it a logical target for the development of effective vaccines and therapeutics against pedv. passive immunization by oral administration of egg yolk antibodies (igy) obtained from immunized chickens provides an alternative source of specific antibodies for the prevention and treatment of pedv in newborn piglets. in this study, we produced an igy against the pedv s protein and investigated its immunoprophylactic effect in neonatal piglets. a codon-optimized pedv s gene consisting of amino acid residues – was synthesized and used to establish a stable porcine cell line constitutively expressing a recombinant pedv s protein containing the chicken immunoglobulin fc fragment at its c-terminus. the purified recombinant s protein was found to mediate potent immune responses in immunized hens. we next tested the ability of oral passive immunization with anti-pedv s igy to protect piglets against pedv. specific chicken igy against the s protein was orally administered to neonatal piglets, and their responses subsequent to a virulent pedv challenge were monitored. the results showed that oral administration of anti-pedv s igy efficiently protects neonatal piglets against pedv, suggesting its potential as a prophylactic or therapeutic agent against acute pedv infection. porcine epidemic diarrhea (ped) is characterized by acute enteritis and watery diarrhea followed by severe dehydration, leading to high mortality rates in neonatal piglets [ , , ] . the disease was initially recognized in england in [ ] , and the causative agent, ped virus (pedv), was not identified until [ ] . ped epidemics were first reported in asia in , and ped has since continued to threaten swine health, causing substantial economic losses for the asian swine industry [ , , , , ] . in early , sudden ped outbreaks occurred in the united states sweeping through the pork industry across the country [ , ] . subsequently, starting in late , large-scale outbreaks of ped rapidly recurred in korea, taiwan, and japan; a us-strain-like pedv was found to be responsible for the recent outbreaks in those countries, raising global concerns regarding control measures for ped prevention [ ] [ ] [ ] ] . pedv is a large enveloped virus possessing a singlestranded, positive-sense rna genome of approximately kb with a cap and a polyadenylated tail, belonging to the genus alphacoronavirus within the family coronaviridae of the order nidovirales [ , ] . the spike (s) protein of pedv is a type i membrane glycoprotein consisting of , to , amino acids (aa), depending on the strain, that can be divided into the s (aa - ) and s ( -end) domains based on homology to the s proteins of other coronaviruses [ , , , ] . similar to other coronavirus s proteins, the pedv s protein plays a critical role by interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host [ , ] . more precisely, the s domain has been reported to contain the receptor-binding region and the main neutralizing epitopes [ , ] . furthermore, the s region has been established as a suitable target for determining genetic relatedness among pedv isolates and for developing differential diagnostic assays and effective vaccines [ , , , ] . in korea, both modified live and killed vaccines against pedv have been developed for disease control and use in the domestic market. however, the continued occurrence of ped nationwide has caused enormous financial losses for the korean pork industry, raising questions regarding the efficacy of these commercial vaccines. this phenomenon appears to be due to genetic and antigenic differences between the vaccine strain and the strains prevalent in the field [ , ] . thus, the lack of effective vaccines increases the need for next-generation field-virus-based vaccines or control measures against pedv infection. passive immunization by oral administration of specific antibodies represents an attractive approach against gastrointestinal pathogens in both humans and animals [ ] . egg yolk immunoglobulin (igy) from immunized chickens has been demonstrated to be a convenient large-scale source for specific antibodies; it has also been shown to be safe and effective against pedv in newborn piglets [ ] . the aim of the present study was to produce an igy against the pedv s protein and assess its immunoprophylactic properties in neonatal piglets. a codon-optimized pedv s gene was used to establish a stable porcine cell line constitutively expressing a recombinant s protein. the recombinant s protein was capable of inducing efficient cytokine and antibody responses in immunized hens. moreover, oral passive immunization using chicken igy raised against the pedv s protein was found to control and prevent ped post-challenge in suckling piglets. cells, virus, and antibodies hek- t cells (crl- ) were purchased from the american type culture collection (atcc; manassas, va, usa) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solution ( ; invitrogen). pk- cells were grown in rpmi medium (invitrogen) containing % fbs and antibiotic-antimycotic solution. vero cells were cultured in alpha minimum essential medium (a-mem; invitrogen) with % fbs and antibiotic-antimycotic solution. the cells were maintained at °c in a humidified % co atmosphere. the sm - pedv vaccine strain was obtained from the korean animal and plant quarantine agency and propagated in vero cells as described previously [ ] . challenge pedv was prepared from intestinal contents obtained from a field case that was found to be free of other common etiologic agents of neonatal porcine diarrheal diseases, as described previously [ ] . briefly, a -day-old suckling piglet was inoculated orally with a small-intestine homogenate containing the field virus. small-intestine tissues were collected and homogenized in a % suspension with a-mem using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of s at , rpm, and tissue suspensions were clarified by centrifugation for min at , g (hanil centrifuge fleta , incheon, south korea). the clarified supernatant was filtered through a . -lm-pore-size syringe filter (millipore, billerica, ma, usa), aliquoted, and stored at - °c until use as the crude challenge virus. horseradish peroxidase (hrp) or fluorescein isothiocyanate (fitc)-conjugated secondary antibodies were purchased from jackson immunoresearch laboratories (west grove, pa, usa), and alexa fluor -conjugated secondary antibody was obtained from molecular probes (carlsbad, ca, usa). the pedv s-protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, south korea), and the nucleocapsid (n)-protein-specific mab was obtained from choongang vaccine laboratory (cavac; daejeon, south korea). dna manipulation and cloning were performed according to standard procedures [ ] . escherichia coli strain dh a (rbc bioscience, taiwan) was used as the host for general cloning. the pfb-neo-pedv-rs -ig plasmid encoding a full-length, codon-optimized pedv s gene (aa - ) was described previously [ ] . the pcdna . /chicken fc plasmid encoding the fc domain of chicken igg was a kind gift from hyung-kwan jang (chonbuk national university, jeonju, south korea). the chicken fc (cfc) fragment from pcdna . /chicken fc was subcloned into pfb-neo-pedv-rs -ig by replacing the fc domain of human igg to construct the gene expression plasmid, pfb-neo-pedv-rs -cfc, which produces a chicken fc-tagged fusion protein, rs -cfc. the constructed plasmid was verified by nucleotide sequencing. generation of a stable pk- cell line expressing pedv rs -cfc the retrovirus gene transfer system (agilent technologies, santa clara, ca, usa) was used to generate a cell line constitutively expressing the recombinant pedv rs -cfc gene or an empty retroviral vector as described elsewhere [ , ] . antibiotic-resistant continuous cell clones were examined by rt-pcr to verify the presence of the fulllength rs -cfc gene, and the positive clones (pk-rs -cfc) were then amplified for subsequent analysis. pk-rs -cfc cells were grown on microscope coverslips placed in -well tissue culture plates. at h post-seeding, the cells were fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in phosphate-buffered saline (pbs) at rt for min. the cells were subsequently blocked with % bovine serum albumin (amresco, solon, oh, usa) in pbs for min at rt and then incubated with a goat antichicken igg antibody conjugated to fitc. finally, the cells were counterstained with , -diamidino- -phenylindole (dapi; sigma, st. louis, mo, usa), and cell staining was visualized using a leica dm il led fluorescence microscope (leica, wetzlar, germany). pk-rs -cfc cells were grown in -mm-diameter tissue culture dishes to % confluency in serum-free medium (optipro sfm; invitrogen). at h post-seeding, the protein-containing cell culture supernatants were harvested, and soluble proteins were immunoprecipitated with chicken igy precipitating resin (genscript, piscataway, nj, usa) according to the manufacturer's protocol in the presence of protease inhibitors at °c for h. the beads were collected by centrifugation at , g (eppendorf centrifuge r, hamburg, germany) for min at °c and washed three times with . m nacl in pbs. the samples were subsequently eluted with mm sodium citrate/ mm glycine (ph . ) and neutralized with m tris-hcl (ph . ). the purified proteins were concentrated with amicon ultracentrifugal filters k (millipore). protein concentration was measured using a pierce bca protein assay (thermo scientific, rockford, il, usa), and the final products were analyzed by western blotting to confirm target protein purification. the protein-containing cell culture supernatants or purified proteins as described above were mixed with nupage lds sample buffer (invitrogen) and boiled at °c for min. the proteins were separated on a nupage - % gradient bis-tris gel (invitrogen) under reducing conditions and stained using simplyblue safestain (invitrogen) according to the manufacturer's instructions or electrotransferred onto immobilon-p polyvinylidene fluoride (pvdf) membranes (millipore). the membranes were then blocked with % powdered skim milk (bd biosciences, belford, ma, usa) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at °c for h and then reacted directly with the goat anti-chicken igg hrp-conjugated secondary antibody or with anti-pedv s mab followed by the corresponding hrp-labeled secondary antibody at a : , dilution for h at °c. finally, the proteins were visualized using enhanced chemiluminescence reagents (ge healthcare, piscataway, nj, usa) according to the manufacturer's protocol. twenty -week-old white leghorn hens were randomly allocated into three groups and immunized by intramuscular injection into the breast muscle with either the binary ethylenimine (bei)-inactivated sm - pedv vaccine strain, lg of the purified pedv rs -cfc protein resuspended in pbs, or pbs with a mineral oil-based adjuvant (montanide isa vg; seppic, puteaux, france) ( table ). the hens were then boosted three times with the same immunogens emulsified with adjuvant at -week intervals. blood samples were collected prior to immunization, at each boost, and three weeks subsequent to the eggs collected from immunized hens were washed with diluted sodium hypochlorite solution (yuhanclorox, seoul, south korea), disinfected with % ethanol, and used for egg yolk fractionation. the separation of the yolk from the albumen and chalaza was performed as described previously [ ] . the egg yolk material was mixed with distilled water (dw) at a : (v/v) ratio immediately followed by the addition of strong acid electrolytic water (ph \ . ) at . % of the total volume. samples were thoroughly whisked using a hand blender for approximately min. the egg yolk mixture was kept at °c for h, and supernatants were then separated and clarified by centrifugation for min at , g (hanil centrifuge fleta ) to thoroughly remove lipids. the supernatants containing igy was collected, lyophilized using a spray dryer and stored at °c for further experiments. peripheral blood mononuclear cells (pbmcs) were isolated from whole blood using a standard gradient centrifugation purification protocol with histopaque (sigma) according to the manufacturer's instructions. total rna was extracted from the pbmcs of hens using trizol reagent (invitrogen) and treated with recombinant dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of the extracted rna were measured with a nanovue spectrophotometer (ge healthcare). quantitative real-time rt-pcr was done using a thermal cycler dice real time system (takara) with a one step sybr primescript rt-pcr kit (takara) and the following gene-specific primer sets as described previously [ , , ] : chicken ifn-a forward, -atcctgct gctcacgctccttct- ; chicken ifn-a reverse, -gg tgttgctggtgtccaggatg- ; chicken ifn-b forward, -gcctccagctccttcagaatacg- ; chicken ifn-b reverse, -ctggatctggttgaggaggctgt- ; chicken ifn-c forward, -agctgacggtggacc-tattatt- ; chicken ifn-c reverse, -ggctttgcgc tggattc- ; chicken il- forward, -caaggtgac ggaggaggac- ; chicken il- reverse, -tggcgag gagggatttct- ; chicken il- forward, -ccaag-cacacctctcttcca- ; chicken il- reverse, -gcaaggtaggacgctggtaa- ; chicken tnf-a forward, -gaagcagcgtttgggagt- ; chicken tnf-a reverse, -gttgtgggacagggtagg- ; chicken glyceraldehyde- -phosphate dehydrogenase (gapdh) forward, -ggtggtgctaagcgtgttat- ; chicken gapdh reverse, -acctctgtcatctctccaca- . the steady-state mrna levels of each cytokine gene were normalized against the level of chicken gapdh mrna, and the relative quantity (rq) of mrna accumulation was evaluated using the -ddct method. the relative fold change in the expression of each gene was then calculated by comparing pre-immune and immunized sera. pedv-specific neutralizing antibodies in the serum and igy samples collected from hens in all groups were determined using a serum neutralization test in -well microtiter plates with the sm - pedv vaccine strain as previously described [ ] . briefly, individual virus stocks were diluted in serum-free a-mem to a concentration of plaque-forming units in a volume of ll. the diluted virus was then mixed with ll of a twofold serial dilution of individual inactivated sera or igy solution dissolved in dw ( mg/ml) in -well plates, and the mixture was incubated at °c for h. next, approximately vero cells in ll of a-mem with % fbs were added to each well, and the mixture was further maintained at °c in a % co incubator for to days. the neutralization titer was calculated as the reciprocal of the highest serum dilution that inhibited virus-specific cytopathic effects in all duplicate wells. the in vivo swine experiments described here were performed at the improah animal facility under the guidelines established by the institutional animal care and use committee. a total of newborn piglets were obtained from seronegative pregnant sows at a commercial pig farm with no known prior ped outbreak or vaccination with pedv. all animals were determined to be free of antibodies to pedv, as well as to transmissible gastroenteritis virus and porcine reproductive and respiratory syndrome virus. no other animals aside from those included in the study were housed at the facility for the duration of the experiment. the design for the present pig experiment is outlined in table . piglets were randomly divided into four groups: group , administration of igy against pedv; group , administration of igy against pedv rs -cfc; group , administration of igy against placebo; group , control. the groups were housed in separate rooms, and no physical contact was allowed between the groups. all neonatal piglets except for animals in the control group were inoculated orally with ml of the small-intestine homogenate containing tcid /ml pedv field virus determined using real-time rt-pcr as described previously [ ] . following challenge exposure, piglets were administered orally with ml of the corresponding igy solution dissolved in dw ( mg/ml) at and days post-inoculation (dpi). clinical signs of diarrhea and the mortality in challenged piglets were monitored daily throughout the study. stool samples from all groups were collected daily with -inch, cotton-tipped swabs and subjected to rt-pcr using an i-tgev/pedv detection kit (intron biotechnology, seongnam, south korea) to detect the presence of pedv. fecal shedding of pedv was measured by quantitative real-time rt-pcr as described above, and the results were analyzed using the system software as described previously [ ] . all piglets from the challenged and control groups were euthanized at dpi for post-mortem examination. smallintestinal tissue specimens collected from each piglet (\ mm thick) were fixed with % formalin for h at rt and then embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut into -to -lm-thick sections on a microtome, floated in a °c water bath containing dw, and transferred onto glass slides. the tissues were then deparaffinized in xylene for min and washed in decreasing concentrations of ethanol ( %, %, %, %, and %) for min each. the deparaffinized intestinal tissue sections were stained with hematoxylin and eosin (h&e; sigma) to observe histopathological lesions of pedv infection and subjected to ifa using the pedv n-specific mab and goat anti-mouse igg secondary antibody conjugated to alexa fluor as described above. the student's t-test was used for all statistical analyses and p-values of less than . were considered statistically significant. generation of stable porcine-origin cell lines expressing the full-length, codon-optimized s protein previously, we synthesized a full-length, codon-optimized s gene and confirmed that codon optimization greatly enhanced the expression level of s upon transient transfection [ ] . in this study, the codon-optimized s gene was used for stable transfection to produce preparative amounts of the s protein. to accomplish this, sublines of pk- cells were established that stably expressed the recombinant codon-optimized s under the control of a retroviral ltr promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blot analysis to examine s expression at the mrna and protein level, respectively (data not shown). based on the results of the western blot analysis, one pk-rs -cfc cell clone that consistently expressed the highest level of s was chosen for subsequent studies. to characterize the pk-rs -cfc cells, intracellular and extracellular expression levels of s were examined by immunofluorescence and western blotting. as shown in fig. a , specific cell staining was clearly evident when pk-rs -cfc cells were reacted with the anti-chicken igg antibody, confirming a consistent high expression level of the s protein. western blot analysis of cell culture supernatants revealed that the pk-rs -cfc cells stably expressed and cumulatively secreted high levels of the approximately -kda s . the recombinant s protein expressed in the supernatants of stable pk-rs -cfc cells was purified using chicken igy precipitation resin beads. the purified s protein was detectable at a high level by simplyblue staining and was confirmed by immunoblotting with antichicken igg antibody (fig. b) . using our purification and concentration procedures, we were able to purify an average yield of lg of s protein per ml of pk-rsi-cfc cell culture supernatant cultivated in a -mm tissue culture dish for h. in addition, the overall growth kinetics of s gene-expressing pk- cells were found to be similar to those of the parental pk- cells, indicating that s expression has no effect on cell growth (data not shown). to produce igy, hens assigned to the three groups were immunized intramuscularly, as outlined in table . blood samples were collected before immunization (pre-immune), at each boost, and weeks after the final boost. pbmcs were prepared from blood samples obtained during the first ifn-a, ifn-b , ifn-c, il- , and il- , was significantly altered by immu-nization of inactivated pedv or s -cfc compared to levels in the placebo-inoculated group (fig. ) . these data indicate that the immunogens used in this study efficiently stimulated immune responses in chickens. serum samples collected at each collection time were subjected to a serum neutralization test against the pedv vaccine strain. non-immunized hens showed only minimal neutralizing antibody titers, whereas immunized hens exhibited gradually increasing neutralizing antibody titers (fig. a) . furthermore, hens immunized with inactivated pedv (group ) and s -cfc protein antigen (group ) at -week intervals produced similar neutralizing antibody titers ranging from : to : subsequent to the final immunization. in addition, egg yolk samples from each group were found to contain neutralizing antibody titers comparable to those in the corresponding serum samples, indicating the presence of neutralizing igy antibodies (fig. b) . to evaluate the immunoprophylactic efficacy of anti-pedv s igy, neonatal -to -day-old pedv-seronegative piglets were divided into four groups and challenged orally with wild-type pedv followed by the respective antibody treatment at days and post-challenge. animals in group were not challenged with pedv and given igy orally from non-immunized chickens at the same time points as the other groups. clinical observations of mortality and diarrhea in challenged piglets are summarized in table . none of the piglets from group died or developed any clinical signs of diarrhea. additionally, fecal shedding of pedv was also detected in the rectal swabs of these animals for the duration of the study (fig. ) . in contrast, all piglets from the challenged groups ( - ), regardless of igy administration, exhibited diarrhea that began at or days post-challenge and lasted throughout the challenge experiment. in group , one piglet died; the remaining piglets experienced severe watery diarrhea and shed pedv in their feces, with a mean cycle threshold (ct) value of . (range . - . ) during the study (fig. ) . although none of the piglets from groups and treated with igy died during the challenge experiment, the number of piglets exhibiting diarrhea post-challenge varied depending on the group. all piglets from these groups showed mild-to-severe diarrhea lasting for the entire experiment but recovered from the diarrhea by the end of the study. furthermore, fecal shedding of pedv from these piglets was significantly lower than in group piglets; pedv rna was detected in the fecal swab samples of all pigs in groups and , with mean ct values of . (range fig. constitutive expression of the recombinant s protein in pk-rs -cfc cells. (a) immunofluorescence assay for the rs protein. pk- or pk-rs -cfc cells grown in a -well tissue culture plate were fixed with % formaldehyde at h post-seeding and incubated with anti-chicken igg antibody (top panels). the cells were then counterstained with dapi (bottom panels) and examined using a fluorescent microscope at magnification. (b) purification of the rs protein. the recombinant s protein was purified from serum-free medium of pk-rs -cfc cells grown in a -mm tissue culture dish. the cell culture supernatant and the purified rs protein were resolved on a - % gradient bis-tris gel and stained with simplyblue solution (left panel) or electrotransferred onto a pvdf membrane (right panel). the membrane was blotted with a chicken igg-specific antibody . - . ) and . (range . - . ), respectively (fig. ) . however, despite the similar levels of diarrhea, the group piglets shed slightly higher amounts of pedv in their feces compared to those in group . gross intestinal lesions consistent with viral enteritis, including thin-walled and fluid-content-dilated small intestines, were typically observed in all group piglets; less-severe lesions were observed in piglets given either igy (data not shown). likewise, the majority of enterocytes over the entire villi in the control piglets with normal igy treatment (group ) were affected by pedv, showing moderate-to-severe villous atrophy and destruction, while piglets from groups and exhibited mild intestinal lesions comparable to those in the non-challenged group, and viral antigens were only detected in their small intestines (figs. and ). however, anti-pedv s igy treatment (group ) was more efficacious than treatment with igy raised against the whole virus (group ) in reducing the overall severity of macroscopic and microscopic intestinal lesions in the piglets. collectively, all immunoprophylactic methods used in this study were capable of protecting neonatal piglets against mortality and disease severity after challenge with a virulent pedv. vaccination against pedv is one of the most important and effective prevention measures by passively transferring specific neutralizing antibodies present in vaccinated sows to their litters through colostrum and milk. despite the availability of commercial attenuated and inactivated vaccines in korea, pedv continues to plague the domestic pork industry, raising issues regarding their protective efficacy. recently, severe outbreaks of pedv have reemerged in korea and have been estimated to affect over % of pig farms, causing serious economic impact on producers and customers [ , ] . our previous studies suggested that antigenic and genetic variations between the vaccine virus and field pedvs may be the cause of the incomplete efficacy or failure of vaccination, which appears to be responsible for the periodic outbreaks in domestic herds [ , ] . furthermore, a comparison of the amino acid sequence of the s domain of the pedv s protein, which is associated with viral binding to host cell receptors and contains neutralizing epitopes [ , ] , has shown a difference of over % between the vaccine strain and field isolates, suggesting the need for next- effect of igy against the pedv s protein generation vaccine development [ , ] . in addition to vaccination, artificial passive immunization using igy has been used commercially as an alternative method for controlling ped by providing supportive immunity to neonatal piglets exposed to acute infection in korea. however, opinions differ among swine practitioners and producers regarding the efficacy of this immunoprophylactic strategy. a similar debate regarding the commercial use of igy may arise since the vaccine strain is also used to immunize chickens for the production of anti-pedv igy in korea. we have previously demonstrated that the s protein of pedv could be considered a potential candidate antigen for vaccination [ ] . in the present study, the s protein of the field pedv was used as an immunogen to inoculate hens, thereby producing anti-pedv s igy. we first aimed to stably express the full-length, codon-optimized s gene of pedv in porcine-origin cells and to evaluate the immunogenicity and efficacy of igy against the recombinant s protein. subsequently, we were able to successfully generate a stable pk cell line continuously producing large amounts of the codon-optimized s protein tagged with cfc. following the purification and concentration processes, approximately - lg of the recombinant s -cfc protein could be consistently harvested from the culture supernatants of pk-rs -cfc cells grown in a -mm culture dish. since humoral immunity is an important indicator for evaluating the effect of the s -based immunogen used in this study, we immunized chickens with the s -cfc antigen prepared from the cell culture supernatants of pk-rs -cfc cells and investigated whether they developed cytokine and antibody responses. all of the proinflammatory cytokines tested, except for tnf-a, were stimulated in chickens immunized with either the whole virus or the s -cfc antigen. of these, the expression levels of il- and il- genes were distinctly enhanced in the s -cfc-inoculated group compared with the inactivated pedv-inoculated group. furthermore, the chicken sera raised against s -cfc contained higher levels of neutralizing antibody than those raised against the sm - vaccine virus. however, the final levels of anti-pedv igy were lower in the eggs of the s -cfc-immunized chickens compared to those of the sm - -inoculated group. these inconsistent results may be attributed to the use of the heterogeneous sm - virus in the serum neutralization assay, which possesses a high degree of genetic variation in field isolates. nevertheless, our data indicated that the recombinant s protein efficiently elicits immune responses in chickens. subsequently, we tested the prophylactic efficacy of each igy in suckling piglets post-challenge-exposure. our data showed that, regardless of the igy administered, all neonatal piglets treated with igy survived after challenge with virulent pedv, suggesting that the anti-pedv s igy provides effective passive immunity to prevent mortality comparable to whole-virus-based igy. despite the lower levels of virus shedding in the feces of animals treated with anti-pedv igy, the anti-s igy-based strategy resulted in more-efficient protection than the anti-pedv igy procedure, as determined by the duration and severity of diarrhea and histopathological lesions. these results indicated that the administration of anti-s igy could significantly reduce the mortality and clinical signs of piglets, suggesting that tissue specimens were collected from the small intestines of piglets from each group at the time of necropsy. the formalin-fixed and paraffin-embedded tissue sections were deparaffinized and stained with hematoxylin and eosin. the sections were examined using a light microscope at magnification. the inset images are enlarged versions of parts of the picture effect of igy against the pedv s protein application of anti-s igy is capable of partially blocking the virus from invading the small intestine. a more promising approach would be to use the entire field virus or its full-length s protein to produce igy instead of using only the s domain, since the s protein contains multiple functional domains and neutralizing epitopes to efficiently stimulate non-susceptible hosts. for this purpose, the production and application of igy against a korean field isolate are currently under investigation. in conclusion, to the best of our knowledge, this is the first study to evaluate the immunoprophylactic effect of igy against the recombinant s protein of the field virus in a pig model. the results presented here indicate that the recombinant s protein can elicit cytokine and antibody responses and induce neutralizing antibodies in chickens. furthermore, challenge experiments revealed that administration of anti-s igy efficiently protected suckling piglets against field pedv by providing passive immunity. pedv infection causes high mortality rates ( - % in neonatal piglets under days of age), and epidemiological observations indicate the rapid spread of the disease. in this circumstance, the preliminary results of the present study showed the potential of anti-pedv s igy application as a considerable measure against pedv. further experiments to optimize production procedures will be required to achieve higher titers of igy; field studies on farms will be needed to better evaluate the efficacy of anti-s igy. these studies will provide additional practical information for the future use of this alternative igy method as a supplement to passive immunity against ped and other economically important viral diseases. fig. detection of pedv in small intestine tissues of piglets. tissue specimens were prepared from small intestine of piglets from each group at the time of necropsy. the formalin-fixed and paraffinembedded tissue sections were deparaffinized and subjected to immunofluorescence staining with an anti-pedv n antibody. the sections were then counterstained with dapi and examined using a fluorescence microscope at magnification the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states 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genes of porcine epidemic diarrhea viruses isolated in korea the n-terminal region of the porcine epidemic diarrhea virus spike protein is important for the receptor binding outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea reemergence of porcine epidemic diarrhea virus on jeju island full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in south korea cytokine production in immortalized porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus new variants of porcine epidemic diarrhea virus, china us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review deadly pig virus slips through us borders contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein letter to the editor a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-b production by inhibiting irf activation in immortalized porcine alveolar macrophages human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus diseases of swine molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic cleavage of peplomeric glycoprotein e of mhv yields two k subunits and activates cell fusion spike protein region (aa ) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan acknowledgments this research was supported by technology development program for bio-industry, ministry for agriculture, food and rural affairs, republic of korea ( - - -hd ). conflict of interest the authors declare that they have no conflict of interest.ethical approval all procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. key: cord- -hw aijwf authors: banyard, ashley c.; johnson, n.; voller, k.; hicks, d.; nunez, a.; hartley, m.; fooks, a. r. title: repeated detection of european bat lyssavirus type in dead bats found at a single roost site in the uk date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: hw aijwf in august , european bat lyssavirus type (eblv- ) was isolated from a daubenton’s bat found at stokesay castle. in september , another bat from the same vicinity of stokesay castle also tested positive for eblv- . this is the first occurrence of repeated detection of eblv- from a single site. here, we report the detection of low levels of viral rna in various bat organs by qrt-pcr and detection of viral antigen by immunohistochemistry. we also report sequence data from both cases and compare data with those derived from other eblv- isolations in the uk. cases of european bat lyssaviruses type- and - (eblv) continue to occur across europe. eblv- seems to be restricted to the infection of serotine bats (eptesicus serotinus) [ ] , although it has been reported in 'spill-over' events into incidental hosts [ , , ] . in comparison to eblv- , eblv- has been reported on fewer occasions, having been isolated from both daubenton's bats (myotis daubentonii) and pond bats (myotis dasycneme) [ ] . the discovery of eblv- in a daubenton's bat in june in newhaven, east sussex, prompted concerns that bat rabies may be present within the uk and, furthermore, that the threat of rabies entering the uk via migratory bats was realistic [ ] . indeed, bat rabies cases in european bats in the mid s indicated a possible spread of the virus, especially in denmark and the netherlands. as well as having been isolated in the uk, eblv- isolates have also been reported in switzerland, holland and finland [ , ] . eblv- infection has also been observed in daubenton's bats in germany [ , ] . since , eblv- has been identified from several locations across the uk, suggesting that eblv- is endemic at a low level in british bats (table ) , and in august , a further uk isolate was identified in an adult female daubenton's bat found by a member of the public at stokesay castle, shropshire [ ] . the bat carcass was submitted to the veterinary laboratories agency (vla) for laboratory testing to confirm the presence of eblv- and was shown to be positive by the fluorescent antibody test (fat). confirmatory diagnosis was achieved by pcr using a hemi-nested rt-pcr that detected viral rna in brain, salivary gland and tongue samples (fig. a) . quantitative rt-pcr (qrt-pcr) was undertaken using previously described methods [ ] to determine levels of viral rna present within different organs of the infected animal ( fig. b) . high levels of viral rna were detected in the brain, with lower levels in the salivary glands, stomach and tongue, with rna also being detected in the intestine and heart (fig. c) . immunohistochemical analyses were restricted to sections of the spinal cord and detected virus antigen in dorsal root ganglia (fig. a) . the original observation of an eblv- positive bat at this site prompted measures to be taken to minimise the risk to members of the public encountering other bats from the roost. twice-daily checks were implemented to ensure that no injured, sick or dead bats were present in areas of the castle open to public access. importantly, control measures were implemented, including the wearing of protective gloves so that staff could handle a bat with minimal risk during the regular checks before members of the public entered the tower of the castle [ ] . signs were also erected at the entrance of the tower warning members of the public not to handle bats. in september , a dead bat was discovered on the top floor of the south tower of stokesay castle during one such check. the bat was a juvenile male daubenton's bat and was submitted to the vla for routine testing. this bat was also shown to be positive for infection with eblv- by rt-pcr, a -base-pair (bp) fragment of the nucleocapsid (n) gene being successfully amplified. unfortunately, the standard fat could not be undertaken, as the brain material had decomposed during storage and transit. the remainder of the carcass of this bat was, however, submitted for histopathological examination. several sections were taken, and despite advanced autolysis, specific labelling was found throughout the spinal cord in neurons, dorsal root ganglia (fig. b ) and peripheral nerves. the n-gene pcr product was sequenced in its entirety and found to be % identical to the isolate across the -bp region analysed. a phylogenetic analysis of the eblv- isolates across the uk to date was generated using the neighbour-joining method, using mega software (fig. d) . unfortunately, the carcass of this bat was severely decomposed, and further molecular tests, such as comparative qrt-pcr on a range of organs, could not be performed. the identification of eblv- positive bats from the same site more than year apart raises several questions regarding the basic transmission and biology of this virus within bat roosts. recent attempts to undertake scientific studies with bat species regarding the transmission and maintenance of these viruses between bats have resulted in limited conclusions as to how the virus is maintained within colonies. bite transmission seems the most likely route of transmission, although no direct evidence for this in captive bats infected with eblvs has been observed [ , , ] . the detection of high levels of virus antigen in the brain of infected bats is typical for these viruses, and generally, where eblv- has been detected in british bats, live virus has been isolated from brain material where samples have not decomposed. here, we have reported the detection of virus in other tissue types. presence of virus in these regions is likely to be linked to the degree of innervation, principally by the autonomic nervous system, although quantitation of neuronal involvement within different organs and tissue types to establish a basis for this hypothesis has not been undertaken [ , ] . however, studies with eblv- infection in the natural host, eptesicus serotinus, showed no substantial pattern of virus distribution in different non-neuronal organs in bats that developed disease [ ] . mechanisms of virus transmission within roosts remain an enigma. for genotype lyssaviruses it has been established that infection of the salivary glands may lead to the secretion of virus in the saliva for several days before the onset of clinical signs. whilst this is widely documented for larger species, low levels of viable virus or viral rna detected in saliva swabs tested during experimental studies with different bat lyssaviruses highlight the difficulty in determining the importance of this route of transmission for virus dissemination within a roost [ , , ] . aerosol spread of virus within a roost would seem feasible, as bats live in very close proximity. however, to date, transmission studies have only been reported with genotype rabies viruses, experimental attempts to transmit eblv- via this route proving unsuccessful [ ] . for the stokesay castle case, the detection of eblv- rna in tongue lends support to the transmission through bite or grooming, although again, this has not been conclusively shown. in , advanced autolysis prevented thorough histological examination or molecular assessment of the tongue. granular immunolabelling was, however, seen in nerves at the base of the tongue and along the jaw. unfortunately, no taste buds or epithelial tissue was available for further testing. the mechanisms of maintenance of eblvs within bat roosts and transmission between individuals remains unknown, although the 'small vector hypothesis' remains plausible [ ] . the restriction of eblv infection to certain species of bat as well as mechanisms by which individuals are able to survive infection, or at least exposure to the virus, are key questions that remain [ ] . viral load, bite/ exposure site and immunological competence of the exposed animal may all affect the outcome of infection. factors such as seasonal variation, pregnancy, nutritional status and immune status must also play an important part in dictating whether or not bats succumb to infection [ ] , but currently, our understanding of bat biology and immunology is low. the detection of a large number of viruses within different bat species has highlighted this lack of knowledge [ ] . isolation of a number of zoonotic viruses in bat species, including coronaviruses, astroviruses, henipaviruses and other lyssaviruses, will surely drive further scientific investigation [ , , , ] . clearly, (d) fig. a hemi-nested second-round pcr results for bat / . firstround pcr (primers jw and ) produced a negative result for all three bat samples tested. however, the second-round pcr (primers jw and ) produced a positive result for the brain and salivary gland [ ] ( brain, salivary gland, tongue, negative control mouse brain, rt positive control, rt negative control, pcr positive control, pcr negative control, pcr negative control for second-round reaction. with eblv- having now been identified on two separate occasions from the same roost, virus is being maintained and transmitted from bat-to-bat by some as yet undefined mechanism. the status of daubenton's bats, and indeed all bats across the uk, as protected species makes it difficult to undertake investigative studies at such sites. however, serosurveillance of bats are planned at the stokesay castle site that will allow determination of the level of seroconversion within this roost and highlight possible transmission mechanisms that will help understand the transmission biology of these elusive viruses and may identify those parameters needed to enhance strategies to combat neuroinvasion and subsequent disease development. bats: important reservoir hosts of emerging viruses european bat lyssavirus type : human exposure in england european bat lyssaviruses: an emerging zoonosis detection of high levels of european bat lyssavirus type- viral rna in the thyroid gland of experimentally infected eptesicus fuscus bats susceptibility of north american big brown bats (eptesicus fuscus) to infection with european bat lyssavirus type first isolation of eblv- in germany experimental infection of serotine bats (eptesicus serotinus) with european bat lyssavirus type a (eblv- a) isolation of european bat lyssavirus type from a daubenton's bat (myotis daubentonii) in shropshire antibodies against lagos bat virus in megachiroptera from west africa heminested pcr assay for detection of six genotypes of rabies and rabies-related viruses experimental infection of big brown bats (eptesicus fuscus) with eurasian bat lyssaviruses aravan, khujand, and irkut virus isolation of a european bat lyssavirus type from a daubenton's bat in the united kingdom airbourne transmission of lyssaviruses experimental study of european bat lyssavirus type- infection in daubenton's bats (myotis daubentonii) experimental infection of big brown bats (eptesicus fuscus) with west caucasian bat virus (wcbv) human rabies of bat origin in europe emerging pattern of rabies deaths and increased viral infectivity spill-over of european bat lyssavirus type into a stone marten (martes foina) in germany epidemiology of bat rabies in germany bat lyssaviruses in europe studies on the pathogenesis of rabies in insectivorous bats. iii. influence of the gravid state rabies in individual countries-denmark prevalence and genetic diversity of coronaviruses in bats from china natural and experimental infection of sheep with european bat lyssavirus type- of danish bat origin european bat lyssaviruses european bat lyssaviruses-an ecological enigma development of a real-time, differential rt-pcr taqman assay for lyssavirus genotypes , and first isolation of a rabies-related virus from a daubenton's bat in the united kingdom detection of diverse astroviruses from bats in china acknowledgments we wish to acknowledge denise marston for technical assistance. this work was supported by defra grants se and sev . key: cord- - d c g authors: seger, waleed; ghalyanchilangeroudi, arash; karimi, vahid; madadgar, omid; marandi, mehdi vasfi; hashemzadeh, masoud title: genotyping of infectious bronchitis viruses from broiler farms in iraq during - date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: d c g infectious bronchitis virus (ibv) is one of the most critical pathogens in the poultry industry, causing serious economic losses in all countries including iraq. ibv has many genotypes that do not confer any cross-protection. this virus has been genotyped by sequence analysis of the s glycoprotein gene. a total of tracheal and kidney tissue specimens from different commercial broiler flocks in the middle and south of iraq were collected from september to september . thirty-two ibv-positive samples were selected from among the total and were further characterized by nested pcr. phylogenetic analysis revealed that isolates belong to four groups (group i, variant [is/ -like]; group ii, /b-like; group iii, qx-like; group iv, dy - -like). sequence analysis revealed nucleotide sequence identities within groups i, ii, and iii of . %- %, . %- %, and . %- %, respectively. group i (variant ) was the dominant ibv genotype. one chinese-like recombinant virus (dy - -like) that had not been reported in the middle east was detected. in addition, the presence of qx on broiler chicken farms in the area studied was confirmed. this is the first comprehensive study on the genotyping of ibv in iraq with useful information regarding the molecular epidemiology of ibv. the phylogenetic relationship of the strains with respect to different time sequences and geographical regions displayed complexity and diversity. further studies are needed and should include the isolation and full-length molecular characterization of ibv in this region. coronaviruses are an important group of viruses that cause highly contagious respiratory and enteric diseases in animals and humans [ ] . avian corona viruses can infect a wide range of avian species, particularly those reared in close proximity to domesticated poultry, such as fowl, partridge, geese, pigeons, guinea fowl, teal, ducks, and pea fowl [ ] . infectious bronchitis virus (ibv) causes enormous problems, causing a highly contagious disease in chickens. respiratory, reproductive, digestive, and renal infections are the primary clinically important types of ibv infections in domestic chickens [ ] . ibv belongs to the genus gammacoronavirus, along with other avian coronaviruses. it is an enveloped, positive-stranded rna virus with a genome of about kb containing ' and ' untranslated regions (utrs) and a poly(a) tail [ ] . a major part of the genome is composed of two overlapping open reading frames (orfs), a and b, which are translated into large polyproteins a and ab, respectively, through a ribosomal frameshift mechanism. the primary structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) are encoded in the remaining part of the rna genome [ , ] . the glycoprotein s consists of two subunits (s and s ) and contains a wide variety of antigenic determinants that may induce the production of specific neutralizing antibodies. mutations within this genomic region may result in the emergence of new viral variants [ ] . in recent years, genotyping has been performed using s gene sequencing [ ] , which has become the procedure of choice for differentiating between vaccine and field viruses. more than ibv serotypes have been recognized worldwide [ ] . ib still causes serious problems in the iraqi poultry industry due to the inability of vaccines to provide cross-protection between different genotypes. due to the limited network of poultry diagnostic laboratories in iraq, differential diagnosis is can only be done based on clinical signs and gross lesions. the characterization of ibv has raised additional problems in terms of both epidemiology and control. although ibv on the poultry farms in iraq (with h and / strains) is presently controlled by inactivated and live attenuated vaccines, outbreaks of ib have nevertheless been observed on broiler farms [ , ] . thus, the spread of viral respiratory diseases has become the most commonly reported condition in commercial broiler flocks in iraq; however, there have been no studies on the detection and genotyping of these viruses using molecular techniques and sequencing. in this study, genotyping of ibv isolates from broiler farms in the south and middle of iraq was carried out based on partial s sequencing. samples were collected from broiler chicken farms in the south and middle (five governorates) of iraq (al-kut, al-najaf, thi-qar, al-muthanna and al-basrah; farms per governorate; fig. ) from september to december . the samples (trachea and kidney) were taken from chickens that showed clinical signs suggesting ib (respiratory problems such as gasping, sneezing and bronchial rales, and nephritis lesions such as enlargement, and congestion in kidneys). the samples were collected with aseptic technique and frozen at - °c. the details of positive samples are shown in table . rna was extracted from tissue samples using cinnapure rna extraction kit (sinaclone, iran). for cdna synthesis, ll ( . lg) of random hexamer primer (sinaclon, iran) was added to ll of extracted rna, and the mixture was heated at °c for minutes. fourteen ll of cdna master mix containing . ll of depc-treated water (sinaclon, iran), ll of dntp mix (sinaclon, iran), . ll of ribolock rnase inhibitor (thermo fisher scientific, usa), . ll of revert aid reverse transcriptase (thermo fisher scientific, usa), and ll of x rt reaction buffer was added to each tube, resulting in a final volume of ll. then, the mixture was incubated at °c for min, °c for min, °c for min, and °c for min. the cdna was stored at - °c until use. sampling, rna extraction, and cdna synthesis were conducted in iraq, and the remainder of the work was carried out in iran. real-time pcr for ibv detection based on the utr was chosen for this study. the amplification was performed by using an amplification kit (bioneer, south korea) with the forward primer gcttttgagcctagcgtt , reverse primer gccatgttgtcactgtctattg and taqman Ò dual-labeled probe fam-caccaccagaac ctgtcacctc-bhq as described by callison et al. [ ] . nested pcr was performed using spike gene primers that were designed to amplify a * -bp fragment of the gene [ ] . first-round amplification ( bp) was performed in a final volume of ll containing ll of distilled water, ll of sinaclon pcr master mix (sinaclon, iran), ll of sx ( cacctagaggtttgytwgcatg ) and sx ( tccacctctataaacaccyttac ) primers ( lm), and ll of cdna . the amplification was performed with a -cycle thermal profile ( °c for min, °c for s, °c for s, °c for s, °c for min). in the second round of the nested pcr, sx ( taatactggyaatttttcagatgg ) and sx ( aatacagattgcttacaaccacc ) primers were used. the second round of amplification was performed in a volume of ll ( . ll of distilled water, ll of sinaclon pcr master mix (sinaclon, iran), ll of sx and sx primers ( lm), and . ll of the first-round pcr product). the reaction was carried out under the same cycling conditions. the pcr product was analyzed by electrophoresis on a . % agarose gel and visualized under uv light. an accuprep Ò pcr purification kit (bioneer co., korea) was used for the purification of the pcr products. sequencing was performed with the primers (both directions) that were used in the second step of nested pcr (bioneer co., korea). chromatograms were evaluated with cromaspro (cromaspro version . ). a phylogenetic tree was constructed by the neighbor-joining method, using mega . software, and each tree was produced using a consensus of bootstrap replicates [ ] . the nucleotide sequences of a partial segment of the s gene were compared with several s sequences from genbank, including h ( table ). the s gene sequences of the ibvs were submitted to the ncbi genbank database with the accession numbers ku -ku . real-time pcr for ibv detection (ibv incidence rate) real-time pcr for ibv detection based on the utr was used in this study. of the samples tested, were positive for ibv by real-time pcr. the number of positive samples from each governorate is presented together with the sample size from each governorate. the total incidence rate for the middle and south iraqi governorates was %, where the highest incidence rate of ibv in broiler chickens was detected in al-najaf governorate / ( %), while the lowest rate / ( %) was detected in al-muthana governorate ( table ) . to carry out ibv genotyping, all positive samples were subjected to amplification of a portion of the s gene for sequencing. all samples that were positive in real-time pcr were positive in nested pcr. four genotypes were detected after sequencing. the percentage of is/ , /b, qx, and dy - genotypes in five iraqi governorates was . %, . %, . % and . %, respectively ( table and fig. ). the qx genotype was detected in al-kut and al-najaf governorates, while the dy - genotype was only detected in al-najaf governorate. is/ and /b genotypes were detected in all governorates ( table ) . the nucleotide sequences of the s gene from iraqi ibv isolates obtained in this study were aligned and compared with those of previously identified isolates from iraq, neighboring countries, and worldwide reference ibv strains, as shown in table . the analysis revealed that all sequences obtained in this study were genetically different. phylogenetic analysis of the strains (fig. ) revealed that iraqi ibv strains could be classified into four genetic groups or genotypes: group i, variant is/ -like viruses, including field isolates ( . %); group ii, /blike viruses, including field strains ( . %); group iii, qx-like viruses, including three field strains ( . ); and caused by bacteria and mycoplasmas have already been detected on broiler farms located in the central and southern parts of iraq. however, the molecular detection studies (infection rate and genotyping) of respiratory viral diseases such as ib in broilers in iraq are limited (personal communication with veterinary organizations). we conducted the first study to determine the infection rate and ibv genotypes on broiler farms, using molecular and phylogenetic techniques. the approved vaccine strains used in iraq are based on serotypes ma , h , and /b [ ] . ib still causes serious problems in the iraqi poultry industry due to the inability of the vaccines to provide cross-protection between different genotypes [ ] . the genotyping of ibv is necessary not only for understanding virus evolution but also for effective modification of the vaccination programs [ ] . in this study, the infection rate of ibv was %, which indicated widespread distribution of ibv in the southern and middle parts of iraq. the highest infection rate of ibv was observed in al-najaf ( %) governorate, while the lowest ( %) was observed in al-muthana (fig. ) . [ ] . the infection rate was lower than the rate previously reported on commercial farms within neighboring countries: . % and . % in jordan and iran, respectively [ ] . even where there were no poultry farms in iraq, the infection rate was as high as %. this may be attributed to poor biosecurity on the farms, resulting in the spread of infection between and within the flocks [ ] . phylogenetic analysis showed that the iraqi isolates clustered into four genetic groups (group i, variant (is/ )-like; group ii, /b-like; group iii, qx-like; and group iv, dy - -like). variant (is/ -like) viruses were the dominant ibv genotype infecting broiler chickens, with an overall prevalence of . % in ibvpositive samples. they shared high nucleotide sequence identity with ibv isolates from iran, israel, egypt, turkey, and kurdistan. the isolation of is/ / (eu ) ibv, one of two israeli variant strains, was first reported by meier and mahar in israel [ ] . ibv variants have been recognized in iraq since (iraqi veterinary directorate/ministry of agriculture). the present work is similar to another study in which three ibv genotypes were found in slemani-kurdistan, iraq, including group a (very similar to iranian isolates), group b (closely related to chinese isolates) and group c (similar to is/ and egypt/beni-seuf/ isolates) [ ] . these findings are in agreement with those of kanan et al., who reported that is/ / ibv was the most common genotype in egypt ( ), lebanon ( - ), jordan ( - ), kuwait ( ), and oman ( ) [ ] . the current study is similar to another study on an is/ strain in turkey [ ] . the second group consisted of /b-like viruses that were detected in different iraqi governorates at the high prevalence of . %. the present study revealed the presence of /b on the broiler farms. the ibv strains of the /b serotype were first identified in france in , followed by great britain in . subsequently, these strains spread to other countries in europe, asia (particularly to iran, turkey), and north america and are some of the most common ibv serotypes in some countries [ ] . our result were in line with those of mahmood et al., who conducted the first study of identification and genotyping of ibv isolates and indicated that the / -like virus is circulating on vaccinated broiler farms of the kurdistan region of iraq [ ] . the detection of /b in commercial flocks was previously reported in iran, jordan, and israel [ , , ] . however, because all flocks have been vaccinated, we concluded that the detected viruses probably belonged to the vaccine strains. therefore, the full-length s gene of the isolates should be determined to differentiate field and vaccine isolates. in this study, another important ibv variant (qx-like) known to cause respiratory, renal, and reproductive problems [ ] was detected with a prevalence of . %. in , qx was first described and identified in china, after which the prevalence of the so-called qx-like ibv genotype was reported, and it became one of the most dominant genotypes in many countries [ ] . the genotype, designated as qx ibv, has spread from asia, where it was described for the first time in in china, and spread to europe and recently to the southern part of the african continent [ ] . the qx-like genotype was also previously detected in central iraq by the iraqi veterinary directorate (official report). this finding was in line with another study in the kurdistan region of iraq, which showed that the phylogenetic aspects of the kurdistan viruses were closely related ( . %) to the qx strain reported in china from to [ ] . qx-type virus was isolated in israel in [ ] . the pcr lab/ / (jx ) strain was c fig. phylogenetic tree of iraqi isolates and reference strains of infectious bronchitis virus based on the nucleotide sequences of the s gene. the phylogenetic tree was constructed, using mega version , by the neighbor-joining method with bootstrap replicates (bootstrap values are shown on the tree). the isolates from this study are indicated by black circles. white dots indicate iranian reference strains, and black triangles indicate iraqi reference strains isolated in iran, which is genetically ( . %- . %) related to strains of the present study [ ] . this finding revealed that the qx strain has been widely transferred from other countries (probably iran) to iraq. although no information is currently available about the introduction of the qx strain from china into other countries, it has been hypothesized that wild birds may be the source of introduction based on the evidence that ibv may replicate in members of the order anseriformes [ ] . it is of interest that that dyi - -like ibv genotype was detected in the present study. this is the first report of this genotype in iraq, and it has not yet been reported in other countries in the middle east. the virus appeared following the recombination of ck/ch/gd/lz and ta ibv in china and is currently a circulating ibv genotype in china [ ] . it is also fair to say that the origin of this virus and the means by which it was introduced to iraq are not clear. based on the persistence of the dy - like genotype in china, it could be predicted that this genotype might spread in iraq and the middle east in the future. in this survey, some ibv genotypes, such as is/ , massachusetts, d , and q , were not detected. these ibv genotypes were detected previously and reported in iraq, iran, the united arab emirates and saudi arabia [ , , , ] . new serotypes or variant strains may emerge because of only a few changes in the amino acid sequence of the s protein. these changes could be due to immunological pressure caused by the extensive use of vaccines, recombination as the outcome of mixed infections, or a decrease in the prevalence of dominant serotypes as a result of vaccination, allowing other field strains to emerge [ ] . not much is known about the mode of spread of ibv between the countries in the middle east. however, crossborder movements of poultry and poultry-related products are likely to be important factors [ ] . in summary, our study demonstrated a high rate of infection with ibv ( %) in central and southern iraq. this is the first report indicating the presence of an ibv dy - like strain in the middle east. it is an updated and comprehensive study of genotyping of ibv in iraq and completes the ibv puzzle in the region. phylogenetic analysis showed that the detected strains are closely related to other ibv strains infecting broilers, pullets, and layers in the region. finally, it could be suggested that ) work be done on whole-genome sequencing, ) cross-protection studies be conducted for designing the best vaccination program in the iraqi poultry industry, ) molecular surveillance in other governorates in iraq be continued, and ) the pathogenesis of different iraqi ibv isolates be studied. molecular detection of infectious bronchitis virus and it is relation with avian influenza virus (h ) 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isolated in southern china during genotypes of infectious bronchitis viruses circulating in the middle east between turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review phylogenetic study of iranian infectious bronchitis virus isolates during - using glycoprotein s gene review of infectious bronchitis virus around the world efficacy of live infectious bronchitis vaccines against a novel european genotype, italy development and validation of rt-pcr tests for the detection and s genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples presence of is/ / genotype-related infectious bronchitis virus in breeder and broiler flocks in turkey evolutionary and bioinformatics analysis of the spike glycoprotein gene of h vaccine strain protectotype of infectious bronchitis virus from india fenner's veterinary virology isolation and molecular characterization of sul/ / avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east infectious bronchitis virus: a major cause of respiratory disease outbreaks in chickens in ghana identification of a novel nephropathogenic infectious bronchitis virus in israel complete genome sequences of two chinese virulent avian coronavirus infectious bronchitis virus variants detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in thailand investigation and molecular characterization of avian infectious bronchitis virus in suspected broiler farms in slemani governorate. council of college of veterinary medicine university of sulaimani in partial fulfillment of the requirements for the degree of master in veterinary medicine/clinical pathology by hana sherzad rauf bvm &s molecular subtype of infectious bronchitis virus in broiler flocks in jordan the pathogenesis of a new variant genotype and qx-like infectious bronchitis virus isolated from chickens in thailand a survey of the prevalence of infectious bronchitis virus type / in iran infectious bronchitis virus variants: a review of the history, current situation and control measures mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods molecular survey and phylogenic analysis of infectious bronchitis virus (ibv) circulating among chicken flocks in riyadh province, saudi arabia acknowledgments the authors gratefully acknowledge dr. hamideh najafi, dr. iraj ashrafi, and mr. behrooz asadi for their extensive technical support. funding financial support for this study was provided by the ministry of higher education and scientific research, iraq, university of basrah ( . . ), and the research council of the university of tehran under grant no. - - . ethical approval this article does not contain any studies of animals performed by any of the authors. key: cord- - nyew i authors: chen, yi-ning; loa, chien chang; ababneh, mustafa mohammed-khair; wu, ching ching; lin, tsang long title: genotyping of turkey coronavirus field isolates from various geographic locations in the unites states based on the spike gene date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: nyew i turkey flocks have experienced turkey coronaviral enteritis sporadically in the united states since the s. twenty-four field isolates of turkey coronavirus (tcov) from multiple states in the united states were recovered from to to determine the genetic relationships among them. the entire spike (s) gene of each tcov isolate was amplified and sequenced. pairwise comparisons were performed using the clustal w program, revealing . % to . % sequence identity in the full-length s protein, . % to . % in the amino terminus of the s subunit (containing one hypervariable region in s a), and . % to . % in the s subunit at the deduced amino acid sequence level. the conserved motifs, including two cleavage recognition sequences of the s protein, two heptad repeats, the transmembrane domain, and the golgi retention signal were identified in all tcov isolates. phylogenetic analysis based on the full-length s gene was used to distinguish north american tcov isolates from french tcov isolates. among the north american tcov isolates, three distinct genetic groups with % bootstrap support were observed. north carolina isolates formed group i, texas isolates formed group ii, and minnesota isolates formed group iii. the s genes of tcov isolates from the united states remained conserved because they contained predominantly synonymous substitutions. the findings of the present study suggest endemic circulation of distinct tcov genotypes in different geographic locations. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. turkey coronaviral enteritis of varying severity, caused by turkey coronavirus (tcov), has been reported in turkey flocks from multiple states in the united states since the s. the major clinical signs of tcov infection include depression, ruffled feathers, diarrhea, decreased body weight, and uneven flock growth. the most apparent gross lesions are markedly distended intestines with gaseous and watery content, particularly in the ileum and ceca. salient histopathologic findings include shortening of the intestinal villi, an increase in crypt depth, and widening of intervillous spaces [ ] . when turkeys are infected with tcov and other infectious agents such as astrovirus, small round virus, and escherichia coli (e. coli), they can develop poult enteritis-mortality syndrome (pems), which causes high mortality [ , ] . subsequent experimental studies of the tcov isolates vr- and tcov/on/mg / from canada have shown that tcov can cause symptoms electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. & tsang long lin tllin@purdue.edu similar to those caused by pems [ , ] . therefore, tcov has been suggested to be the major causative pathogen for turkey enteritis, and secondary infections caused by other opportunistic microorganisms enhance the severity of tcov enteritis and contribute to the development of pems. turkey enteritis associated with tcov infection has caused substantial economic losses in indiana, north carolina, arkansas, and other states in the united states [ , ] , as well as in canada [ ] , europe [ , ] , and brazil [ ] . currently, there are no vaccines to prevent the disease, and treatment of infected turkeys is often unsuccessful. a member of the species avian coronavirus (cov) in the genus gammacoronavirus and family coronaviridae, tcov has a positive single-stranded rna genome that is approximately kb in size. the major structural proteins of tcov include the spike (s), envelope (e), matrix (m), and nucleocapsid (n) proteins. comparisons of '-end coding regions [ , ] as well as the full genomes [ ] of tcov isolates and infectious bronchitis virus (ibv) have suggested that tcov arises through recombination in the s gene, because pairwise comparisons of s gene sequences have revealed only a % similarity between tcov isolates and ibv strains, whereas gene , m gene, gene , and n gene sequences have over % similarity [ , , ] . the s gene sequences of different tcov isolates ( %- . %) are more conserved than those of various ibv strains ( . %- . %), which could explain the close antigenic relationship of tcov isolates compared with the distant antigenicity of different ibv serotypes [ , , , ] . investigations during tcov outbreaks and genomic analyses of tcov isolates have revealed that distinct tcov isolates tend to circulate endemically, and their respective sequences group phylogenetically according to their state of origin [ , , ] . however, these observations may have been biased because of the small number of tcov sequences that were analyzed. in the present study, tcov isolates were recovered from clinical cases submitted to the indiana animal disease diagnostic laboratory at purdue university by turkey farms in minnesota, indiana, north carolina, missouri, arkansas, texas, south carolina, and pennsylvania between and . the objective of the present study was to elucidate the relationship between the genotypes and geographic distribution of tcov isolates from turkey farms in multiple states in the united states by using sequence analysis and comparing the full-length s gene. twenty-four field isolates of tcov were recovered from clinical cases submitted to the animal disease diagnostic laboratory at purdue university by turkey farms in minnesota, indiana, north carolina, missouri, arkansas, texas, south carolina, and pennsylvania between and (table ). field cases of tcov were confirmed by clinical signs, gross lesions, histopathologic findings, immunofluorescence antibody (ifa) assay with antiserum against tcov/in/ / , electron microscopy, and reverse transcription polymerase chain reaction (rt-pcr). all tcov isolates were propagated five times in embryonated turkey eggs as described previously [ ] . in brief, intestines from tcov-infected turkeys were homogenized as % suspensions in chilled sterile phosphate-buffered saline and clarified by centrifugation at rpm for minutes at °c. the supernatant was filtered through a . -lm membrane filter (millipore, bedford, ma, usa). the filtrate was inoculated into the amniotic cavity of -day-old embryonated turkey eggs. the embryo intestines were harvested after days of incubation for virus purification. the harvested intestines were homogenized and clarified at rpm for minutes at °c. the supernatant was layered on top of % and % sucrose and clarified using ultracentrifugation in an sw rotor at , rpm for hours at °c in an optima xl- k ultracentrifuge (beckman coulter, fullerton, ca, usa). the interface between % and % sucrose was collected and placed on top of a continuous %- % sucrose gradient and clarified by ultracentrifugation at , rpm for hours at °c. a band of buoyant density . - . g/ml (containing tcov) was collected and saved at - °c as the viral stock. the viral rna was extracted from the purified virus using rnapure tm reagent (genhunter, nashville, tn, usa) and chloroform, followed by precipitation using cold isopropyl alcohol and ethanol. the extracted rna was reverse transcribed to cdna using superscript tm iii reverse transcriptase (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the first reaction was minutes of incubation with the rna, random hexamer primer ( ng/ll), and mm dntps at °c, followed by minute on ice. the second reaction was minutes of incubation with a mixture of x firststrand buffer, . m of dithiothreitol (dtt), u of superscript tm iii reverse transcriptase, and u of rnaseout tm (invitrogen, carlsbad, ca, usa) at °c, followed by hour of incubation at °c and minutes of inactivation at °c. the full-length s gene (approximately . kb) was amplified by pcr using the cdna of each tcov isolate with the primers sup and sdown (online resource ). the mixture ( : , v:v) of taq (promega corp., madison, wi, usa) and pfu dna polymerases (stratagene, la jolla, ca, usa) with proofreading ability was used in a -well thermal cycler (geneamp, perkin-elmer cetus corp., norwalk, ct, usa) to maintain the fidelity of the pcr [ ] . the pcr products were electrophoresed on % agarose gels and purified using a zymoclean tm gel dna recovery kit twenty-four tcov isolates collected from eight states in the united states from to were purified and sequenced in our laboratory. tcov/in/ / was analyzed in a previous study [ ] , and the full-length s gene sequences of the other tcov isolates were published for the first time in the present study ( table ). the putative peptide cleavage site separating the amino-terminus of the s subunit from the carboxyl terminus of the s subunits as well as a second possible peptide cleavage site in the s subunit were detected using the prop server (http://www. cbs.dtu.uk/services/prop/). the s subunit of tcov was further designated as s a at the amino-terminus ( - in tcov/in/ / ) and s b at the carboxyl-terminus ( - in tcov/in/ / ) [ ] . figure shows a diagram of the s protein of tcov. the nucleotide and deduced amino acid sequence similarities of the s genes of all tcov isolates were analyzed using the clustal w alignment method in mega [ ] . a phylogenetic tree based on fulllength nucleotide sequences of the s gene was constructed using the maximum-likelihood method and the kimura -parameter model. a phylogenetic tree based on deduced amino acid sequences of the s a subunit, containing a hypervariable region (hvr), was constructed using the neighbor-joining method and the jones-taylor-thornton model. a codon-based z-test of positive selection for s gene sequences of various tcov isolates was conducted to analyze the differences in the number of nonsynonymous (dn) and synonymous (ds) substitutions per site by using the nei-gojobori method [ ] in mega . the variance of both trees and codon-based z-test were validated using bootstrap replicates. the s sequences of tcov isolates reported in the present study were submitted to the genbank databse, and their accession numbers ranged from kf to kf . the accession numbers of other covs used for phylogenetic analysis are also listed in table . the sizes of the s genes of tcov isolates reported in the present study ranged from to nucleotides. all tcov isolates exhibited similar s protein sequences (fig. ) . the consensus transcription-regulating sequence (trs), ctgaacaa, was identified nucleotides upstream of the start codon of the s protein. several conserved motifs and one hvr were found in all tcov isolates, and their sequences are listed for comparison in table . the consensus motif rxrr/x (x is any amino critical arginine was mutated to glycine and the second probable protein cleavage site was lost. rather than nqgr/s, french tcov isolates had the pqgr/s sequence as the conserved cleavage motif in the s subunit. only one hvr, spanning amino acid positions to (tcov/in/ / ) was found in the tcov isolates, rather than the three hvrs identified in ibv [ ] . two -aminoacid insertions in heptad repeats (hr and hr ) , the consensus motif (yikwpwyvwl) in the transmembrane domain, and the late golgi retention signal (yyttf) for s protein were also observed in all tcov isolates. among the amino acid residues of the neutralizing-epitopecontaining s fragment in the s subunit identified in a previous study [ ] , consensus residues were observed among the tcov isolates (online resource ). a pairwise comparison of the deduced amino acid sequences of the tcov isolates showed that the sequence identity ranged from . % to . % for the full-length s protein, . % to . % for the s a subunit containing hvr, and . % to . % for the s subunit phylogenetic trees based on the full-length s nucleotide sequences ( fig. a) and s a amino acid sequences containing hvrs (fig. b ) of different covs of the genus gammacoronavirus were generated. as shown in figures a and b, the ibv strains were separated from tcov isolates, and north american tcov isolates were separated from french tcov isolates. three genetic groups, referred to as groups i, ii, and iii, were observed in north american tcov isolates ( fig. a) because of the high degree of variation, most phylogenetic groupings based on the s a deduced amino acid sequences did not have a bootstrap value over % (fig. b) . nevertheless, the texas tcov isolates of group ii and all three tcov isolates of group iii shown in the phylogenetic tree based on the full-length s nucleotide sequences still clustered according to their s a amino acid sequences containing their hvr. turkey coronavirus isolates from different geographic areas in the united states have been shown to be antigenically related to one another [ ] . in the present study, an antiserum against the isolate tcov/in/ / reacted with all tcov isolates from tcov-infected turkeys and embryos by ifa assay (data not shown). the close antigenicity among tcov isolates was associated with the high similarity of the s gene sequences, which ranged from . % to . %. the s genes of tcov isolates were conserved compared with the diverse s genes among various ibv strains, which range from . % to %, resulting in the existence of many serotypes of ibv [ ] . the emergence of new ibv serotypes has been postulated to involve the recombination of s genes of vaccine strains of ibv in the field [ , ] . in a previous study, different serotypes of tcov/va/ / , tx/ / , and in/ / were identified by using a neutralization test in conjunction with real-time rt-pcr despite the high level of amino acid sequence identity ( % to %) among these tcov isolates [ ] . additional studies are necessary to clarify the antigenic relationships among the various tcov isolates and serotypes. in the present study, similar to previous findings with ibv strains, most of the variations in the s protein sequences among tcov isolates were observed in the amino-terminal half. along the alignment of these s protein sequences, the region of sequence with the most variation was between residues and of tcov/in/ / from the start codon of the s protein. various deletions occurred in this region in different tcov isolates. this region is in the vicinity of hvr ii (residues to ) of the ibv s protein. hvr i (residues to ), ii, and iii (residues to ) of the ibv s protein are associated with three neutralizing epitopes. the sequences of these regions could be used for differential diagnosis of ibv serotypes [ , ] . by contrast, similar regions of high variation corresponding to hvr i or iii of ibv were not detected among the tcov isolates examined. these differences illustrate why the s proteins of ibv strains are more diverse than those of tcov isolates. similar phylogenetic trees were constructed using the full-length s and s a proteins. thus, genotyping tcov field isolates based on the s a sequence containing hvr rather than the whole s gene or full-length s gene is more practical. the observation that tcov isolates originating from the same state were closely clustered together in the phylogenetic tree suggested endemic circulation of distinct tcov genotypes in various geographic locations. endemic circulation of distinct tcov genotypes in france and north american are recognized because french tcov isolates share only % amino acid sequence identity in the s protein with north american tcov isolates [ ] . distinct sources of recombination promoting the emergence of tcov in north america and europe has been suggested [ , ] . the groupings of north carolina, texas, and minnesota tcov isolates also support the theory of endemic tcov genotypes. the tcov isolates from the outbreaks in arkansas and north carolina in also clustered geographically [ ] and could be placed phylogenetically in group i in the present study. the . % amino acid sequence identity of the s proteins of two tcov isolates recovered years apart in minnesota (tcov/mn/atcc/ and tcov/mn/ / ) implied that the tcov isolate mn/atcc/ remained endemic and that no substantial genetic changes occurred over two decades. conservation of tcov isolates is also shown in the result that no positive selection of the s protein was found among the tcov isolates. because indiana isolates from and clustered in group i with most north carolina isolates and indiana isolates from and clustered in group ii with texas isolates, it is most likely that the turkey sources were the same for the turkey in conclusion, the relationship between tcov genotypes and the geographic distribution of tcov presented in the present study provides crucial information for the monitoring and control of diseases associated with tcov infection in the united states. characterization of turkey coronavirus from turkey poults with acute enteritis detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in canada antigenic relationship of turkey coronavirus isolates from different geographic locations in the united states investigating turkey enteric coronavirus circulating in the southeastern united states and arkansas during first full-length sequences of the s gene of european isolates reveal further diversity among turkey coronaviruses detection of turkey coronavirus in commercial turkey poults in brazil complete sequences of ' end coding region for structural protein genes of turkey coronavirus comparison of '-end encoding regions of turkey coronavirus isolates from indiana, north carolina, and minnesota with chicken infectious bronchitis coronavirus strains emergence of a group coronavirus through recombination recombinational histories of avian infectious bronchitis virus and turkey coronavirus relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus genetic and antigenic diversity in avian infectious bronchitis virus isolates of the s complete nucleotide sequence of polyprotein protein and genome organization of turkey coronavirus mega : molecular evolutionary genetics analysis version . simple methods for estimating the numbers of synonymous nucleotide substitutions acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells identification and characterization of a neutralizing-epitope-containing spike protein fragment in turkey coronavirus genotypic and phenotypic characterization of the california (cal ) variant of infectious bronchitis virus comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different arkansas delmarva poultry industry -derived infectious bronchitis virus vaccines typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene reverse genetics system for the avian coronavirus infectious bronchitis virus changes in nonstructural protein are associated with attenuation in avian coronavirus infectious bronchitis virus novel avian coronavirus and fulminating disease in guinea fowl identification of a novel coronavirus from a beluga whale by using a panviral microarray key: cord- -k z v vx authors: rong, q.; alexander, t. s.; koski, g. k.; rosenthal, k. s. title: multiple mechanisms for hsv- induction of interferon α production by peripheral blood mononuclear cells date: journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: k z v vx uv-inactivated, infectious, and other forms of herpes simplex virus (hsv- ) induced interferon (ifn) production by different routes in myeloid origin mononuclear cells (momc) (consisting predominantly of monocytes). gm-csf activated the momc (g-momc) to produce greater amounts of interferon while differentiation to dc, by the addition of granulocyte macrophage colony stimulating factor (gm-csf) and calcium ionophore (ga-momc), reduced the levels of interferon production upon challenge with some hsv strains. uv-inactivated virus induced more interferon than infectious virus. l-fucose, an antagonist of the mannose receptor, inhibited the induction of ifn-α by uv-inactivated virus and gb(−) virus (defective in penetration) in momc and ga-momc but not g-momc. l-fucose had little effect on interferon induction by infectious hsv- . the insensitivity of the g-momc to fucose inhibition distinguishes these interferon producing cells from the pdc cells previously described as natural interferon producing cells. the mannose receptor appears to be involved in the response to non-infectious forms of hsv but infectious virus appears to use a different pathway. these studies suggest that non-infectious virions and hsv infected cell debris effectively stimulate monocytes and pre-dendritic cells to produce ifn-α to initiate host protection against hsv infection. interferon alpha (ifn α) production is one of the earliest responses to virus infection, acting locally and systemically [ ] to inhibit virus replication and induce protective immune responses. ifn α can inhibit infection and the spread of hsv from neurons to epidermal cells [ ] in vitro, and application of plasmid dna expressing ifn α is sufficient to prevent disease upon vaginal [ ] or corneal [ ] infection in animal models. the most potent inducer of ifn-α is double stranded rna (dsrna), formed as the replicative intermediate of rna viruses or as a result of complementary rnas of a dna virus [ ] . complementary transcripts, which may provide an inducer, have been detected in vaccinia virus [ ] , adenovirus [ ] , herpes simplex virus (hsv) [ ] , and sv [ ] . ifn-α production can also occur upon inhibition of protein synthesis which can block the synthesis of repressors of ifn-α mrna synthesis [ ] . certain enveloped viruses, including sendai virus [ ] , human immunodeficiency virus (hiv) [ , , , ] , and hsv [ , , , - , , , , , , , , , , , ] promote the production of large amounts of ifn-α upon interaction with a population(s) of cells in freshly isolated peripheral blood. human monocytes produce ifn-α in response to sendai virus or hiv [ , ] and may also produce interferon in response to hsv. hsv induces high levels of ifn α in a minor cell component which have been termed natural interferon producing cells (nipc). the ifn α producing activity of these cells is lost upon overnight in vitro culture of the cell population [ ] . a candidate for the nipc has been identified as the pdc (dendritic cell precursor) [ ] . these cells lack myeloid or monocytic markers including cd (myeloid origin marker), cd , cd b, cd (markers expressed when precursor cells differentiate into myeloid lineage mononuclear cells), cd b and cd (strongly expressed on granulocytes), cd (monocyte marker) antigens, and also lack b-cell and t-cell antigens. other types of hsv responsive interferon-producing cells may also be present in peripheral blood or the peripheral tissue. for example, in the mouse, the marginal metallophilic macrophages and marginal zone macrophages of the spleen are the major interferon producers in response to iv challenge with hsv and murine dc lines can produce interferon in response to bacteria and viruses, including hsv [ ] . several forms of hsv can induce the ifn α response including infectious and uv inactivated virus [ , ] indicating that replication of the virus is not required. internalization of the virus is also not required since hsv fixed to glutaraldehyde cross-linked cells [ , ] and genetically engineered hsv- glycoprotein d (gd) obtained from mosquito cells are sufficient for induction of ifn-alpha [ ] . in this study, we evaluated an alternative source of cells to study the nature of the interferon response to hsv- . large numbers of non-lymphocytic mononuclear cells were obtained by leukophoresis and countercurrent centrifugal elutriation [ ] . these cells are predominantly of myeloid origin (momc) with a small percentage of immature dendritic cells. following treatment with granulocyte macrophage colony stimulating factor (gm-csf) and a (calcium ionophore, ionomycin, 'a') in serum-free cell culture, the monocytes and immature dendritic cells (idc) undergo a rapid and consistent change to become activated dendritic cells (dc) [ , ] . the differentiation to dc includes down-regulation of cd expression, acquisition of dendritic cell morphological properties, upregulation of mhc class ii and co-stimulatory molecule expression, and enhanced capacity for t cell sensitization [ ] . we demonstrate that the extent of interferon induction is different upon challenge with different strains and forms of hsv and that monocytes, gm-csf treated monocytes, and the mature dendritic cell populations respond differently to these challenges. the response of the momc to some strains of hsv- is enhanced by gm-csf to levels similar to that reported for nipc. comparison of the activities of different strains of infectious hsv, uv-inactivated hsv, and a mutant hsv incapable of penetration (k t) [ ] and results of treatment with a mannose receptor antagonist indicate that there are more than one mechanism for hsv induction of interferon in the momc origin cell populations. mononuclear myeloid cells (momc) isolated from different healthy donors on different occasions by leukopheresis and countercurrent centrifugal elutriation [ ] were frozen and thawed for each experiment. the momc preparations consist predominately of cd + cells (> %) (a myeloid cell surface marker) with approximately % monocytes (cd + cd + ) and - % idc (cd − cd + ) [ ] . after thawing, momc were washed once in macrophage-sfm medium (gibco) containing % penicillin-streptomycin (cellgro) and grown in -well ( × /well) cluster plates (costar) in ml serum free macrophage-sfm medium (gibco) with % penicillin-streptomycin in an atmosphere of % co at • c. recombinant human granulocyte-monocyte colony stimulation factor (gm-csf, ng/ml) (immunex) was added to specific wells of the momc (gm-csf treated momc, g-momc) directly after the plating. calcium ionophore (a) (a , ionomycin; sigma chemical co, ng/ml) was added to a set of gm-csf treated momc at h after thawing to induce differentiation of the cells (gm-csf plus a treated momc, ga-momc) [ , , ] . cells for each experiment were analyzed by flow cytometry to determine their immunophenotype [ ] . cells were incubated with mg/ml human igg (sigma chemical co.) for min to block fc receptors and double stained with fluorescein isothiocyanate (fitc)-conjugated mouse anti-human cd and phycoerythrin (pe)-conjugated mouse anti-human cd (b . ) or cd (b . ) (pharmingen) for min at • c. virus stocks were prepared by freeze-thaw and sonication of infected vero cell lysates and culture media. vero (african green monkey kidney) cells were grown in medium (gibco brl) supplemented with % fetal calf serum (hyclone) and % penicillin-streptomycin (cellgro) at • c. virus stock was quantitated in a standard plaque assay on vero cell monolayers. kos , a lab attenuated virus, was kindly provided by tom holland, wayne st. u. school of medicine, detroit, mi. sp and slp were generated for other studies in our lab [ ] . sp is a clinical strain obtained post mortem from the brain of a -day-old infant with disseminated hsv- disease. slp was generated by serial passage ( times) of plaque purified sp virus in vero cells at low moi (< . ). ang and ang-path virus [ ] , two related syncytial virus strains, were kindly provided by bradley m. mitchell (baylor college of medicine, houston, tx). ang-path was derived from ang by passage in mice. stocks of virus were prepared and quantitated as mentioned above. uv-inactivated virus was prepared by exposure of virus to uv light for min and then tested by plaque assay for residual infectivity. the quantity of uv-virus is referred to as pfu or moi-equivalents, which are based on the amount of infectious virus prior to uv inactivation. k t is a mutant of hsv- kos with a gene deletion in the transmembrane region of the glycoprotein b (gb) to prevent implantation into membranes and the virion envelope [ ] . k t was kindly provided by john docherty (northeastern ohio universities college of medicine, rootstown, oh). stocks of k t containing gb (k t-gb) were made by infecting d cells, a vero cell line expressing gb, with k t-gb. quantitation of k t-gb was done in a standard plaque assay on d cells. plaque assay was done on vero cells to detect potential revertants to the parental kos virus. k t virus was quantitated by two methods which gave comparable results. k t viral dna concentration was compared with that of a kos virus stock for which the titer was already known. viral dna was purified for both viruses using wizard genomic dna purification kit (promega, madison, wi). the dna concentration was quantitated by absorbence at nm and k t pfu-equivalents were calculated by comparison with the dna concentration of kos stock. in addition, k t strain was quantitated using a plaque assay in which polyethylene glycol (peg) was used to promote viral penetration and plaque formation (adapted from sarmiento et al., [ ] ). after a h adsorption period, the monolayer was washed once with pbs and the cells were exposed to a solution ( ml per well) that contained g of melted peg (sigma, st. louis, mo) and . ml of m without serum. the peg solution was removed by washing with solutions of : peg: serum-free m , : peg: serum-free m , and three washes with m containing % fetal calf serum. the cultures were incubated with m containing % fetal calf serum for h at • c to allow the cells to recover. the medium was then removed and m containing . % methylcellulose (kodak, rochester, ny) and % fetal calf serum was added. the cultures were incubated for two or three days at • c until plaques formed. cells treated with uv inactivated viruses were incubated for h at • c. for infectious virus, cells were incubated with hsv- for h at • c, the medium was replaced with sfm medium and incubated for h at • c. to evaluate the effect of fucose on interferon induction, different concentrations of fucose were added min before the addition of virus. after the h incubation, ml aliquots were removed and frozen at − • c. the cells and remaining medium were frozen at − • c and thawed for quantitation of virus production. ifn-α concentration was determined by elisa (biosource, camarillo, ca). the antibodies in the elisa kit can recognize the most common subtypes of ifn-α. myeloid origin mononuclear cell populations (momc) obtained by leukopheresis and countercurrent elutriation were used as an alternative source of nonlymphocytic mononuclear cells to study hsv induction of ifn α. momc are predominantly (> %) monocytes, contain a minor population of immature dendritic cells (idc) ( - %) and have minimal contamination by lymphocytes and neutrophils [ ] . momc treated with gm-csf (g-momc) appeared similar to the untreated momc but maintained their viability to a greater extent over the -day-course of the experiment. both the momc and g-momc expressed high levels of cd , the lps receptor, although g-momc expressed lower levels than momc with low or absent cd (b . ) or cd (b . ) but a sub-population expressed higher levels of cd expression. g-momc maintained in serum free medium and treated with the calcium ionophore a (ga-momc) readily and reproducibly converted to mature dc [ , ] . the ga-momc cells could be distinguished from the momc and g-momc by the lack of cd expression and upregulation of both cd and cd expression, characteristics of mature dendritic cells [ ] . the loss of cd expression and up-regulation of cd and cd was seen in each experiment. maintenance of these cells in serum free medium and separation from lymphocytes and granulocytes prevents the effects of bovine serum factors from affecting their development. the conversion was reproducible in cells from different donors. herpes simplex virus production by momc, g-momc and ga-momc was evaluated by quantitating the amount of virus released to the media by plaque assay on vero cells. all three cell populations were poor virus producing cells with an average yield of one virus per cell (data not shown). this was less than the input (moi = ) infectious virus. the low permissivity of myeloid cells for hsv replication has been reported by others [ ] . infection with higher multiplicities of infection (moi = ) caused considerable cytopathological effect and cell death. initial studies were performed to determine whether momc, g-momc and ga-momc have the ability to produce ifn α in response to uv-inactivated hsv- . the cells were challenged with different moi equivalents of uv-inactivated kos, a highly passaged, attenuated hsv- strain and extracellular medium was obtained after h and ifn α analyzed by elisa. ifn-α production, by each of the cell populations, increased upon challenge with increasing moi equivalents of uv-inactivated hsv- kos (fig. ) . the g-momc cell population produced the greatest amount, ga-momc produced an intermediate amount and the momc produced the least amount of ifn-α in response to uv inactivated kos. there was some variation in the amounts of interferon produced by cells obtained from different donors and for different dates of donation but the g-momc treated cells always produced the most ifn α. cells that were mock infected or treated with uninfected vero cell extract produced no ifn α (data not shown). the response to uv inactivated hsv confirms other studies [ , ] that show that complete virus replication is not necessary for ifn α induction. also, gm-csf activates the cells to produce more interferon upon hsv induction. the magnitude, range and trends for ifn α responses were different for uv inactivated and infectious virus and for different strains of hsv- (fig. ) . the difference in response to infectious and uv-inactivated virus is clearly shown for hsv- kos and uv-inactivated kos. the strain dependent difference in response was evaluated for hsv- strains that differ in their passage history, tissue culture behavior and their ability to cause lethal neuroinvasive disease in the mouse footpad and other models of hsv infection [ , , ] . a moi of or the equivalent amount of uv-inactivated virus was chosen for the virus challenge because the cytopathological effect of infectious hsv on the different types of momc cells was minimal at this dose. cells from different individuals were used for some of the experiments (different panels of fig. ). the first set of viruses to be tested included sp , a low passage neuroinvasive virus and slp , an attenuated virus derived from sp by passage in vero cells ( fig. a, b) . the response to uv-sp was greater than for uv-slp or uv-kos (data not shown). the response to infectious virus was much less than for the moi, or moi-equivalents for all experiments was uv-inactivated virus. the g-momc response was greater than for momc or ga-momc but sp was the poorest inducer of interferon in all three cell populations. these trends were observed for different individuals and on different occasions. a different trend in cellular response was observed for the ang and ang-path set of viruses. ang-path was derived from ang, a clinical virus, by passage in mouse brains to select for a more neuroinvasive virus [ ] . both ang and angpath cause syncytia formation in all three cell populations. ang-path induced interferon, but unlike the previously described viruses, the ga-momc produced more interferon upon induction by ang-path than did g-momc (fig. c) . ang was a poor inducer of interferon but induced a small, but measurably greater amount of ifn α in ga-momc. this strain dependent difference in the trend may indicate a different type of interaction of ang-path and ang with the momcorigin cells. in order to address the question of whether virus entry is required for the induction of ifn-α in momc origin cell populations, an hsv mutant defective in penetration was evaluated for its abilty to induce interferon production. the k t mutant was developed from kos by deletion of a -base-pair bsteii fragment in the gb-coding region, corresponding to amino acids of the transmembrane region. this virus yields normal virions lacking the glycoprotein b. the k t mutants bind efficiently, but cannot enter cells [ ] . stocks of infectious virus (k t-gb) were prepared by growth in a gb expressing vero cell line (d cells) and stocks of virus lacking gb (k t) were obtained upon infection of the non-complementing vero cells. an equivalent titer (with respect to kos) of k t virus was quantitated by two methods.aliquots of k t virus were allowed to bind to d cells and fusion of the cell-bound virus was promoted with peg treatment [ ] . this allowed plaque formation to occur in the complementing d cells. in addition, the dna concentration of aliquots of k t was compared to similar aliquots of kos, for which the titer was known. these assays indicated that the equivalent-titer of the k t virus stock was approximately × /ml. interferon induction by k t was compared to kos at moi equivalents of (data not shown) and . the results are corrected for levels of interferon produced by equivalent numbers of wild-type virus to the infectious virus that may be present in k t due to genetic reversion to the parental kos (approximately . kos virus per , k t virus) or the small amounts of k t viruses which would acquire or retain gb on their envelope (k t-gb) ( k t-gb virus per , k t virus). figure d shows that k t induced lower levels of ifn-α production than the parental kos virus in momc and g-momc cells. the ga-momc produced a larger amount of ifn α than momc or g-momc in response to k t and this response was greater than for kos. studies by other investigators implicated the mannose receptor as an important mediator of hsv-induced ifn-α production and fucose as an effective inhibitor of this interaction [ ] . initial studies demonstrated a concentration dependent inhibition of uv-inactivated hsv- kos induction of interferon in momc, g-momc and ga-momc. cells pretreated with fucose for min were incubated with uv-kos (moi = ) for h in the presence of fucose and then aliquots were removed and tested for ifn-α production. ifn-α production in the absence of fucose treatment was set as the % control. fucose treatment caused a biphasic concentration dependent inhibition of ifn α production by momc and ga-momc in response to uv-kos (fig. ) . although the % inhibitory dose was mm, the extent of inhibition (slope) was less at higher concentrations of fucose ( - mm) . interestingly, the effect of fucose treatment of g-momc was different from that of momc and ga-momc. ifn α induction in g-momc was inhibited by only % at mm fucose and this was the maximum level of inhibition (fig. ) . these results indicate that there are different mechanisms for ifn α induction between these different cell populations with fucose sensitive, fucose less sensitive and fucose insensitive routes of interferon induction. in addition, the primary route of interferon induction for uv-hsv in g-momc cells is a fucose insensitive route. subsequent studies evaluated the ability of fucose ( mm) to block interferon induction by uv-inactivated ang, infectious ang, infectious kos, and k t. as shown in fig. , fucose inhibited ifn α induction by uv inactivated kos and uv-inactivated ang in momc and ga-momc but not the g-momc cell population, consistent with the results shown in fig. . interestingly, fucose did not inhibit ifn-α production in response to infectious kos or ang virus in any of the three cell types. unexpectedly, fucose treatment appeared to enhance the ifn-alpha production in the g-momc cells. interferon induction by k t virus was very sensitive to fucose treatment. ifn α production was reduced by % in momc and ga-momc, and by % in g-momc. the difference in q. rong et al. different forms of hsv, including infectious virus, uv-inactivated-non-infectious virus, fixed hsv coated onto glutaraldehyde fixed cells [ ] and purified glycoprotein d [ , ] can induce an ifn α response. the nature of the responses to these different forms of virus has been assumed to be similar and the descriptions of the responses have been used interchangeably in many studies. the results from our study indicate that various forms and different strains of hsv induce ifn α to different extents and likely, by divergent pathways. in addition, cell types other than the pdc cell are likely to produce ifn α in response to hsv and that response is different for different forms or strains of virus. also, gm-csf potentiates the interferon response to some forms and strains of virus. these studies were made possible by the use of myeloid origin mononuclear cells (momc), a large population of cells which are predominantly monocytes, with small numbers of dendritic cell precursors and minimal contamination from lymphocytes and neutrophils [ ] . unlike cells used in other studies, the momc can be frozen and thawed and maintained in tissue culture for greater than h. use of serum free medium for these cells minimizes the interference from unknown cytokines and the serum free conditions may be more representative of the extra-vascular environments where pdc or monocytes may encounter an hsv infection [ ] . gm-csf appeared to prime or activate the interferon response to uvinactivated and infectious forms of kos and sp viruses but not for all the viruses. the amount of ifn-α produced by g-momc in response to uv inactivated hsv was in the same range as suggested in the literature for the nipc [ , ] , although direct comparisons are difficult due to differences in the means of analysis (elisa vs bioassay), the virus strain, and individual donor variation. the gm-csf also enhanced interferon production by peripheral blood mononuclear cells following stimulation by hsv bound to glutaraldehyde fixed wish cells [ ] . in vivo, gm-csf is an early response to infection and is produced by activated t cells, macrophages, endothelial cells or fibroblasts [ ] . treatment with recombinant gm-csf [ ] is sufficient to elicit protection against hsv- encephalitis in a rat model. our studies would suggest that an important component of the gm-csf induced protection is the potentiation of the interferon response to hsv- . differentiation of the g-momc into mature dc-like cells by treatment with a (ga-momc) was accompanied by a reduction in the production of ifn α in response to kos, sp and slp strains of hsv. decreased response upon differentiation is consistent with the loss (reduction) of interferon induction observed by others upon overnight incubation of peripheral blood mononuclear cells under normal cell culture conditions [ ] . the ga-momc dendritic cells were more responsive to challenge with ang-path and k t viruses than g-momc or momc. the difference in interferon response may reflect differences in the interaction of the virus with the interferon producing cells sinceang andang-path cause syncytia formation, k t binds, but is incapable of entering the cell, and all three viruses have mutations in or lack the glycoprotein b. other studies support our findings that dc can make ifn α in response to hsv and also hiv [ , ] . the different fucose inhibition patterns for the varied forms and strains of virus and for the different cell types suggests that there are different routes of hsv induction of interferon. the fucose sensitive cell surface route probably uses the mannose receptor and is activated by uv-kos, uv-ang and k t in both momc and ga-momc cell populations. this may also be the route used by uv-inactivated-non-infectious virus, fixed hsv coated onto glutaraldehyde fixed cells [ ] , and purified glycoprotein d [ , ] . a route that is less sensitive to fucose, as distinguished at high concentrations of fucose, may also be used by these activators. the mannose receptor does not seem to be extensively involved in induction of ifn α by infectious virus or by any of the forms of virus in g-momc. the insensitivity of the g-momc to fucose inhibition distinguishes the interferon producing cells in this population from the pdc cells that have been called nipc, which are sensitive to fucose inhibition [ ] . the large enhancement of interferon induction by uv-inactivation of infectious hsv observed herein and by linnavuori and hovi [ ] suggests that infectious virus may have the ability to limit ifn α production in momc related cells. hsv strains appear to differ in their ability to utilize this mechanism to evade host protection as indicated by comparison of the trend for interferon induction for the uv-inactivated viruses (sp > slp ) and infectious viruses (slp sp ). for the limited numbers of virus strains tested herein, the virus strains with a history of more extensive passage in non-human hosts (slp , kos, ang-path) appeared to induce more ifn-α production than the low-passage viruses (sp , ang). this suggests that the suppression of interferon production may be selected during human infection as a means to escape host defenses but this property may be lost upon infection of cells or animals of other species. other human-specific hsv mechanisms for escaping host protective responses include the hsv- ul protein block of mhci expression by blocking the tap [ ] and glycoprotein e binding to the fc portion of igg [ ] . the results of this study open up the possibility that myeloid origin monocytes and pre-dendritic cells are an important source of ifn α for host protection against hsv infection. the mechanism of induction for these cells may be different from the pdc cells (based on the fucose blocking studies) described by others [ ] . the greater response to uv-inactivated virus suggests that non-infectious virions and possibly hsv infected cell debris are the more potent activators of ifn α production and that replicating virus can limit the induction of interferon production as a means of escaping host protection. locally produced gm-csf would activate monocytes or pdc to enhance production of ifn α and protective responses. extensive symmetrical transcription of simian virus dna in virusyielding cells interferon induction by hiv glycoprotein : role of the v loop induction of interferon-alpha by glycoprotein d of herpes simplex virus: a possible role of chemokine receptors dendritic cells and the control of immunity granulocyte-macrophage colony-stimulating factor, interleukin- , hsv- induction of ifn alpha in myeloid cells interleukin- synergize with calcium ionophore to enhance dendritic cell function role of early cytokines, including alpha and beta interferons (ifnalpha/beta), in innate and adaptive immune responses to viral infections intrastrain variants of herpes simplex virus type isolated from a neonate with fatal disseminated infection differ in the icp . gene, glycoprotein processing, and neuroinvasiveness engagement of the cellular receptor for glycoprotein b of human cytomegalovirus activates the interferon-responsive pathway linker-insertion nonsense and restriction-site deletion mutations of the gb glycoprotein gene of herpes simplex virus type recombinant glycoprotein of human immunodeficiency virus is a potent interferon inducer infrequent but efficient interferon-alpha-producing human mononuclear leukocytes induced by herpes simplex virus in vitro studied by immunoplaque and limiting dilution assays interferons and the colony-stimulating factors il- and gm-csf enhance the ifn-alpha response in human blood leucocytes induced by herpes simplex virus glycosylation is required for coronavirus tgev to induce an efficient production of ifn-alpha by blood mononuclear cells il- switches the differentiation of monocytes from dendritic cells to macrophages calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells splenic marginal metallophilic macrophages and marginal zone macrophages are the major interferon-alpha/beta producers in mice upon intravenous challenge with herpes simplex virus calcium signaling inhibits interleukin- production and activates cd (+) dendritic cells that induce th cell development cd + blood dendritic cells 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dna encoding ifn-alpha postinfection enhances cumulative survival of herpes simplex virus type vaginally infected mice herpes simplex virus turns off the tap to evade host immunity the effect of granulocytemacrophage colony-stimulating factor on myeloid cells and its clinical applications when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna (review) rna synthesis in cells infected with herpes simplex virus x. properties of viral symmetric transcripts and of double-stranded rna prepared from them cd + monocytes as dendritic cell precursors: diverse maturation-inducing pathways lead to common activation of nf-kappab/relb single amino acid changes in the viral glycoprotein m affect induction of alpha-interferon by the coronavirus transmissible gastoenteritis virus inhibition of herpes simplex virus type -induced interferon synthesis by monoclonal antibodies against viral glycoprotein d and by lysosomotropic drugs herpes simplex virus as an inducer of interferon in human monocyte cultures bacterial lipopolysaccharide, tnf-alpha, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in cd + monocytes through distinct pathways that activate nf-kappa b alpha and gamma interferons inhibit herpes simplex virus type infection and spread in epidermal cells after axonal transmission hsv- induction of ifn alpha in myeloid cells the mannose receptor mediates induction of ifn-alpha in peripheral blood dendritic cells by enveloped rna and dna viruses neuroinvasive properties of herpes simplex virus type glycoprotein variants are controlled by the immune response poxviridae and their replication in vivo immune evasion mediated by the herpes simplex virus type immunoglobulin g fc receptor plasmid dna encoding ifn-alpha antagonizes herpes simplex virus type ocular infection through cd + and cd + t lymphocytes a leukocyte subset bearing hla-dr antigens is responsible for in vitro alpha interferon production in response to viruses human peripheral null lymphocytes ii producers of type- interferon upon stimulation with tumor cells, herpes simplex virus and corynebacterium parvum synthesis of complementary rna sequences during productive adenovirus infection properties of human natural interferonproducing cells stimulated by tumor cell lines determination of herpes simplex virus-induced alpha interferon-secreting human blood leukocytes by a filter immunoplaque assay herpes simplex viruses and their replication monocytes is the main producer of human leukocyte alpha interferons following sendai virus induction flow cytometric analysis of natural interferon-alpha producing cells membrane proteins specified by herpes simplex viruses iii. role of glycoproteins vp (b ) in virion infectivity the interferon system -a bird's eye view of its biochemistry the nature of the principal type-interferon-producing cells in human blood myeloid dendritic cells the cell surface phenotype of human natural interferon-alpha producing cells as determined by flow cytometry herpes simplex virus type icp inhibits human tap but not mouse tap hsv- induction of ifn alpha in myeloid cells antiviral activity induced by culturing lymphocytes with tumor derived or virus-transformed cells, identification of the antiviral activity as interferon and characterization of the human effector lymphocyte subpopulation spontaneous cell-mediated cytotoxicity in humans: role of interferon and immunoglobulins granulocyte-macrophage colony-stimulating factor expressed in t cells mediates immunity against herpes simplex virus type encephalitis herpes simplex virus regulation of herpes simplex virus type gene expression in nonpermissive murine resident peritoneal macrophages northeastern ohio universities college of medicine, sr this research was supported in part by public health service research grant r ns - from the ninds to ksr. key: cord- -f ffvkr authors: horváth, trén; mocsári, e. title: ultrastructural changes in the small intestinal epithelium of suckling pigs affected with a transmissible gastroenteritis (tge)-like disease date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: f ffvkr the small intestine of piglets collected during a sudden outbreak of diarrhoeal disease resembling transmissible gastroenteritis (tge) was examined by light and electron microscopy. the principal histopathological changes were moderate infiltration by mononuclear cells in the lamina propria of the villi and cytoplasmic vacuolation. these were most pronounced in the epithelial cells covering the villous tips. by scanning electron microscopy, the intestinal villi were swollen and the transverse furrows disappeared. microvilli were reduced in number leaving denuded areas on the brush border of the villous epithelial cells. the ultrastructural changes were restricted to the cytoplasm of affected villous epithelial cells. the cell organelles were missing in rounded areas leaving cleared areas in the cytoplasm. parallel fascicles and bundles were seen in these areas. viral particles with an average diameter of nm were found within the dilated apical tubulo-vesicular system, free in the cytoplasm, among the microvilli or lying free in the intestinal lumen. viral particles surrounded a non-membrane bound viroplasm in some cases. the negatively stained particles showed a typical coronavirus morphology. these particles were found to be distinct from the known coronaviruses of swine, tge virus and hemagglutinating encephalomyelitis virus by immune electron microscopy. transmissible gastroenteritis (tge) is a highly contagious enteric disease of swine of all ages causing important economic losses, especially in dense swine populations. the disease was first described by doyl]~ and hutchings ( ) in the united states, in . in hungary, szent-ivxn¥i et a l . ( ) reported the occurrence of tge in . following the elaboration of methods for the routine - / / / /$ . i ir]~n i-iorvith and e. m o c s~i : laboratory diagnosis of the disease ( ), cso~tos a n d benye;)a ( ) t u r n e d their a t t e n t i o n in to a new tge-like epizootic diarrhoea of swine in which the causative role of t g e virus was excluded on the basis of virological examinations. clinical observations and the experimental reproduction of diarrhoea in colostrumdeprived pigs with bacteria-free intestinal material have also supported the viral etiology of this diarrhoeal disease ( , ) . i n the past few years, an increased frequency of newborn pig diarrhoea caused b y rotavirus was reported ( , , , , , , ) . however, in a n u m b e r of instances, the significance of rotavirus could not be established ( ) . finally, in belgium pe~sae~t and de bouck ( ) and in e n g l a n d chasey and cart-wright ( ) detected coronavirus-like particles in the feces and intestinal epithelium of pigs affected with a tge-like epizootic diarrhoea. the present report describes the ultrastructural changes in the small intestinal epithelium and the detection of a virus morphologically similar to the coronaviruses in the intestinm content a n d epithelium of pigs affected with a tge-like disease, which has occurred periodically in h u n g a r y since . three to seven days old piglets were collected from large hungarian swinebreeding farms during sudden outbreaks of diarrhoeal disease affecting animals of all ages. all the pigs showed a watery diarrhoea. the etiological rote of tge coronavirus was excluded by the negative result of direct irnmunofluorescence tests carried out on serapings of the small intestinal mucosa, by unsuccessful viras isolation attempts on secondary swine thyroid cell cultures and by the absence of tge virus-neutralizing antibodies in the acute~ and convalescent-phase sera ( ) . direct immunofluorescence ( ) , counter-immunoelectrophoresis ( ) , electron microscopic ( t ) and immune electron microscopic examinations ( ) for rotavirus were also negative and no rotavirus antibodies were found by counter-immunoelectrophoresis ( ) in acute-and convalescent-phase sera from the recovered animals. intestinal segments were removed from six different regions of the small intestine of sacrificed piglets and fixed in buffered formalin. the sections were stained with hemotoxylin-eosin (he). disc-shaped portions of intestinal wall, approximately . em in diameter were stretched on a dental wax sheet. they were fixed in . per cent glutaraldehyde at ph . and postfixed in per cent phosphate-buffered osmium tetroxide, ph . . they were dehydrated in graded acetone and vacuum-dried. finally, tissue blocks were vapor coated with gold in an edwards' vacuum-evaporator. the preparations were examined under a jeol sm scanning electron microscope using an acceleration potential of kv. small segments were removed from six sites of the small intestine, adjacent to those taken for histopathology. the segments were fixed in karnowsky's paraformaldehyde or in . per cent glutaraldehyde at ph . and post-fixed in per cent phosphate-buffered osmium tetroxide at ph . . tissue blocks were dehydrated in graded ethanol and embedded in durcupan acm. the sections were cut with a reichert om u ultramierotome. semi-thin sections were stained with toluidine blue and thin sections with uranyl acetate and lead citrate. i n t e s t i n a l c o n t e n t s were diluted to (v/v) in p h o s p h a t e -b u f f e r e d saline (pbs), p h . a n d clarified b y c e n t r i f u g a t i o n at × g for m i n u t e s at ° c. the supern a t a n t was filtered t h r o u g h a n m millipore filter a n d centrifuged a t , × g for m i n u t e s a t °c in a n m s e ss ultracentrifuge. t h e resulting pellet was r e s u s p e n d e d in a few drops of distilled water, placed on mesh, f o r m v a r -c o a t e d grids a n d s t a i n e d w i t h per cent p h o s p h o t u n g s t i e acid. grids were e x a m i n e d u n d e r a philips cs electron microscope o p e r a t i n g at a n acceleration p o t e n t i a l of kv. porcine a n t i -t g e serum was p r e p a r e d in all s p f piglet orally exposed to t h e v i r u l e n t mitler- strmn of t g e virus ( ) . porcine a n t i -h e m a g g l u t i n a t i n g encephalomyelitis virus (hev) serum was o b t a i n e d from dr. m. b. pe~saert, u n i v e r s i t y of g h e n t , belgium. t h e sera were h e a t -i n a c t i v a t e d a n d stored frozen u n t i l used. t h e n e u t r a l i z i n g a n t i b o d y titre of t g e s e r u m was : . i t s o p t i m a l dilution for i e m was : . t h e n e u t r a l i z i n g a n t i b o d y titre of i-iev a n t i s e r u m was : , a n d its o p t i m a l dilution for i e m was : . t h e optimal a n t i b o d y dilution for i e m was considered to be t h a t which produced the largest v i r u s -a n t i b o d y aggregates. toluidine blue stain. × . c sem of villi from a non-exposed pig. these are long a n d slender w i t h p r o m i n e n t t r a n s v e r s e furrows (arrow). × t . d sem of t h e j e j u n u m from a pig affected with tge-like disease. the villi are swollen a n d t h e t r a n s v e r s e furrows are missing. × t oration were present in some of them (fig. b) . these alterations were most pronounced in the epithelial cells covering the tips of the villi but they were missing in those lining the crypts of lieberkiihn. examination of small intestines of normal pigs revealed long, finger-like villi of various lengths and configurations. the surfaces of most villi were interrupted by transverse furrows of various depths (fig. c) . in the small intestines of the affected pigs, villi were swollen and the transverse furrows disappeared. mierovilli were reduced in number leaving denuded areas on the brush border of the epithelial cells (fig. d) . principal ultrastructural changes were found in the cytoplasm of the villous epithelial cells of the small intestine, the ceil surface and the nucleus were generally intact (fig. ) . in some of the affected cells, the cell organelles were missing in rounded areas leaving cleared areas in the cytoplasm (fig. ) . viral particles were present along the border adjacent to the intact areas of cytoplasm (fig. ) . sometimes, viral particles surrounded an electron-dense, coarsely grannlated viral precursor material or viroplasm (fig. . the virions consisted of a central electron-opaque core surrounded by an intermediary electron translucent zone and by an electron-opaque outer coat (fig. ) . their average diameter was nm. viral particles were also present in the dilated apical tubule-vesicular system, free in the cytoplasm (fig. ) , among the microvilli (fig. ) , or lying free witt~in the dilated cisternae of rough endoplasmic reticulum (fig. ) . most viral particles were seen in the epithelial cells covering the tips of villi in the jejunum. their number decreased gradually approaching the inlets of the crypts of lieberkiihn and no viral particles were found in the epithelial cells lining the crypts. likewise, viral particles were not found within the nucleus. sporadically, parallel bundles and fascicles were seen in the cleared areas of cytoplasm (figs. and ) , disruption el microvilli of the brush border and the separation of infected epithelial ce!ls were also demonstrated occasionally. coronavirus-like particles were found in negatively stained preparations of the intestinal contents of all piglets. the particles were pleomorphie with short surface projections. the projections formed a single fringe radiating from the electron-opaque central core. the diameter of the particles ranged between and nm. the length of the surface projections was approximately nm (fig. ) . the direct iem for tge virus and hev was negative. previous observations and experimental results have assumed the viral etiology of this tge-like disease. experimental reproduction of diarrhoea in col ostrum-deprived pigs with bacteria-free fecal samples of sick pigs ( , ) and the explosive spread of diarrhoea within all age groups have eqr ally led to this assumption. the results of the present study proved the viral e~iclogy of this tge-like disease. virions with an average diameter of nm ~ ere found in the intestinal fig. . dilated tubule-vesicular system characteristic for suckling pigs t, h a t --besides maeromoleeules-can also promote the intake of infectious agents. viral particles (arrow) locating intra-and extraeellular. mieiovilli of the brush border are disrupted, × , villous epithelial cells of naturally infected piglets or free in the intestinal lumen, in the infected epithelial cells, viral particles were located mainly within the dilated eisternae of the rough endoplasmie retieulum. in addition, viral part, ides were found free in the cytoplasm or surrounded by a unit membrane. the location of viral particles closely parallels that of tge virus. the negatively stained particles showed a typical coronavirus morphology. these coronavirus-like particles were morphologically indistinguishable from tge virus and hev particles in similar preparations. however, by direct iem, they were distinct from these two eoronaviruses of swine. the size and morphology of the particles were similar to those of the recently recognized eoronaviruses reported by p~scsaert and de bouck ( ) and by chas~sy and caatummht ( ) in epidemic diarrhoea of pigs. as mentioned above, tge virus was excluded as the etiological cause of diarrhoea on the original farms. the histopathology of the intestine and the ultrastruetural changes in villous epithelial cells also differed from those caused by tge virus. in the case of tge, the marked shortening of villi of the small intestines that hoopea and haelteama~ ( ) referred to as villous atrophy proved to be a highly significant lesion. these morphological changes affect the entirety of the villi; they are not restricted to the absorptive cells. furthermore, simultaneously with the regressive alterations, a marked cell proliferation can be demonstrated in the crypts of lieberkiihn presumably replacing the destroyed and sloughed epithelial cells. in the case of tge-like diarrhoea, the morphological changes are restricted to the cytoplasm of the differentiated villous epithelial cells. in most cases, the cell surface was intact, even if the cytoplasm showed marked pathological changes. in addition, parallel bundles and fascicles were seen in vacuoles in the cytoplasm that were observed neither in normal (figs. a and ) nor in tge virus-infected pigs. all attempts to isolate and propagate the virus in cell cultures have so fat" been unsuccessful. tge-szcrfi megbeteged s el fordul~sa sert s~llomanyainkban antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus rotavirus as a cause of diarrhea in pigs virus-like particles associated with porcine epidemic diarrhoea detection of antibody to rotavirus by counterimmunoelectrophoresis in human serum, eolostrmn and milk j~rv~nytani s diagnosztika.i vizsg£ atok a malaeok viruses hasmendsdr isolation of the porcine rotavirus in belgium a transmissible gastroenteritis in pigs concepts of pathogenesis and passive immunity in transmissible gastroenteritis of swine comparative studies on transmissible gastroenteritis (tge) and tgedike disease of swine with special respect to the ultrastruetural changes thesis submitted for the specialist of veterinary virology degree beovirus-like agent associated with fatal diarrhea in neonatal pigs a reovirus-like agent (rotavirus) associated with diarrhoea in neonatal pigs vizsg~latok a sert s viruses hasmen se (tge) laborat riumi kdr-jelzgs nek t k etesit s re a new coronavirus-like particle associated with diarrhea in swine demonstration of reovirus-like particles in intestinal contents of piglets with diarrhoea immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine comparison of eounterimmunoeleetrophoresis and electron microscopy for laboratory diagnosis of human roevirus-like agent associated infantile gastroenteritis test for reovirus-iike agent a rnalacok virusos has-men se (a ,transmissible gastroeneritis" meg~tllapit~sa haz£nkban). magyar hla-torvosok lapja pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotie pigs diarrhoea in piglets inoculated with rotavirus an apparently new syndrome of porcine epidemic diarrhoea the isolation of reovirus-like agents (rotaviruscs) from acute gastrocnteritis of piglets the authors thank professor t. szent-[v~nyi, department of epizootiology, university of veterinary science, budapest, for his valuable suggestions and comments in the preparation of this manuscript. they also acknowledge the assistance of p. h tzl, ministry of agriculture and food, plant protection and agrieultm'al chemistry centre, budapest, in the scanning electron microscopic studies. authors' address : dr. i r~-horv~-ti~, central veterinary institute, t~bornok u. , h-t budapest xiv, hungary.received august , key: cord- -fqxjzavm authors: anindita, paulina duhita; sasaki, michihito; setiyono, agus; handharyani, ekowati; orba, yasuko; kobayashi, shintaro; rahmadani, ibnu; taha, siswatiana; adiani, sri; subangkit, mawar; nakamura, ichiro; sawa, hirofumi; kimura, takashi title: detection of coronavirus genomes in moluccan naked-backed fruit bats in indonesia date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: fqxjzavm bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (sars-cov). in the present study, we report the discovery of bat cov genes in indonesian moluccan naked-backed fruit bats (dobsonia moluccensis). a partial rna-dependent rna polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the rdrp and helicase genes could also be amplified from fecal samples. phylogenetic analysis suggested that these bat covs are related to members of the genus betacoronavirus. deltacoronavirus [ ] . members of the genera alphacoronavirus and betacoronavirus are known to cause human disease, whereas those of the genera gammacoronavirus and deltacoronavirus are causative agents of animal disease [ ] . bats are known to be reservoirs for many emerging infectious diseases that have zoonotic potential [ ] and are believed to be the original source of sars-cov [ ] and mers-cov [ ] . in southeast asia, bats harboring coronaviruses have been discovered in a number of countries, including the philippines [ , ] and thailand [ ] . however, there have been no studies focused on covs in indonesia. here, we report the discovery of bat covs in moluccan naked-backed fruit bats, dobsonia moluccensis, in indonesia. we university. fecal, tracheal swabs, and tissues samples including brain, lung, liver, spleen, and kidney from each bat were collected and placed into rnalater (life technologies, carlsbad, ca). all samples were exported to japan with the permission of the directorate general of livestock and animal health services, the ministry of agriculture, republic of indonesia. morphological features and nucleotide sequence analysis of both mitochondrial s rrna and cytochrome b were conducted as described previously [ ] to identify the species of each fruit bat. feces and tissue samples were subjected to rna extraction using trizol reagent (life technologies) according to the manufacturer's instructions. the rnas were then screened for genomes of covs using a nested rt-pcr with primer sets that specifically amplify the conserved region of rdrp, as described previously [ ] (region a in fig. ). the helicase region was amplified using degenerate primers specific for members of the genus betacoronavirus [ ] (region b in fig. ). pcr products of both the rdrp and helicase regions were gel-purified, and nucleotide sequencing was performed using a bigdye terminator v. . / . cycle sequencing kit (life technologies) and analyzed using an abi prism genetic analyzer (life technologies). one-step rt-pcr was performed using a new primer set and a primescript ii high fidelity onestep rt-pcr kit (takara, tokyo, japan) to amplify the region not covered in the first and second sets of pcr experiments (region c in fig. ). based on alignment of the sequences obtained in this study, the forward primer -cacgcaacttgttgtaatgcgtcagaga- and the reverse primer -cacgtgcttttgcaggcactat acgac- , corresponding to the rdrp gene at nucleotide position - and to the helicase gene at nucleotide position - , respectively, of bat coronavirus (batcov) hku (nc ) were used. the fragments obtained, covering part of the rdrp and helicase genes (region d in fig. ), were sequenced. pcr-amplified nucleotide sequences were aligned and compared to the sequences of known covs using the mega program. nucleotide sequence identity values were calculated using genetyx software ver. (genetyx, tokyo, japan). phylogenetic trees were constructed based on nucleotide sequences, using the maximum-likelihood method with the tamura-nei model and bootstrap replicates [ , ] . virus isolation was attempted by inoculation of african green monkey kidney (vero e ), yaeyama flying fox kidney (fbkt), and leschenault's rousette kidney (dem-kt ) cells. vero e cells were obtained from prof. ayato takada (research center for zoonosis control, hokkaido university, hokkaido, japan) [ ] , and fbkt and dem-kt cells were obtained from prof. ken maeda (yamaguchi university, yamaguchi, japan) [ ] . fecal and tissue homogenates that were positive for cov rna were used for inoculation of cells after clearance by centrifugation at g for min. cells were incubated at °c for min with fecal samples and for h with tissue homogenates. inocula were replaced by fresh culture medium supplemented with % fbs, % antibioticantimycotic solution containing penicillin ( , u/ml), streptomycin ( lg/ml) and amphotericin b ( lg/ml) (sigma, st. louis, mo), and gentamycin ( lg/ml). the supernatants of cultured cells were transferred to fresh cell monolayers each week. rnas were extracted from culture supernatants and subjected to nested rt-pcr targeting the rdrp gene up until the fifth passage. the presence of cytopathic effect (cpe) was also checked daily until the end of the experiment. the species of the captured bats were shown to be dobsonia moluccensis, acerodon celebensis, pteropus sp. (genetically close to pteropus hypomelanus), and pteropus vampyrus (table ) . nested rt-pcr with three out of a total of fecal samples from dobsonia species produced a fragment of bp in length after removal of the primer region, which was expected size of the rdrp fragment (region a in fig. and table ). the samples were ifb - f, ifb - f, and ifb - f. the rdrp gene is the one most commonly used for pcr amplification in cov surveillance, since it contains the most regions that are conserved in all covs [ ] . sequence analysis demonstrated that all samples contained the sequence of the rdrpgene-conserved motifs a (dypkcd) and c (xsdd), which form the polymerase catalytic active site [ ] . the partial rdrp sequences of ifb - f and ifb - f were identical. there were two nucleotide substitutions when the rdrp sequences of ifb - f and ifb - f were compared with the rdrp sequence of ifb - f. in addition to the partial rdrp gene, a portion of the helicase gene was also amplified, producing a fragment of bp (region b in fig. ), from the three samples that were positive for pcr targeting the rdrp region. in the helicase gene, no nucleotide differences were found between ifb - f and ifb - f. however, two nucleotide substitutions were observed between ifb - f and ifb - f or ifb - f. after sequencing the partial rdrp and partial helicase regions, we designed new primers to amplify the sequence between these regions (region c in fig. ) . a -bp fragment was detected in two samples (ifb - f and ifb - f). the sequence of a fragment (region d in fig. ), referred to as ''partial rdrp-helicase'', was then constructed by incorporating overlapping sequences of partial rdrp, partial helicase, and the region in between. the nucleotide sequence comparison of partial rdrp-helicase from ifb - f and ifb - f showed nucleotide substitutions. blast searches showed that ifb - f and ifb - f had the highest nucleotide sequence identity to batcov hku - - and batcov ky , at % and %, respectively. following removal of primer sequences from the partial sequence of the rdrp-helicase gene at the and end, the length of the sequence was , bp, and a phylogenetic tree comparing the sequence of the samples with known covs, including sars-cov and mers-cov, was constructed (fig. ) . the phylogenetic tree showed that ifb - f and ifb - f formed a distinct branch that was closely related to batcov hku (nc ), hku - (ef ), hku - - (hm ), and hku - - (hm ) from china [ ] as well as batcov ky from kenya (hq ) [ ] . all of these covs belong to the genus betacoronavirus. nucleotide sequences of partial rdrp-helicase genes derived from fecal samples ifb - f and ifb - f were deposited under accession numbers ab and ab , respectively, in the dna data bank of japan (ddbj, tokyo) nucleotide database. various tissues and tracheal swab samples collected from the bats where the feces yielded a positive result were also subjected to nested rt-pcr for the partial rdrp region. spleen tissue of ifb - f, brain tissue of ifb - f, and lung tissue of ifb - f showed positive signals for rdrp gene amplification. sequence analysis of tissue samples showed sequences identical to the partial rdrp sequence obtained from fecal samples (ifb - f and ifb - f). the respiratory epithelium is known to be a target for covs [ ] , and therefore, detection of bat cov genome fragments in lung samples might be expected. however, the detection of cov genome fragments in spleen and brain samples suggested that bat cov infection in these species may not be restricted to the respiratory or alimentary tract. attempts at virus isolation using vero e , fbkt, and demkt cells were unsuccessful. no cpe was observed in cell cultures, and viral rna was not detected by rt-pcr from the culture supernatants after the fifth passage. nucleotide sequencing and phylogenetic analysis of partial rdrp-helicase regions of the samples in the present study and these of known covs suggest that the cov genome fragments obtained in this study are related to [ ] . therefore, the covs detected in this study could not be defined at the species level, since only certain regions of conserved replicase domains were determined. however, the number of nucleotide substitutions over the partial rdrp-helicase sequence between ifb - f and ifb - f and phylogenetic analysis of partial rdrp-helicase suggested that the fruit bats harbor variants of a single coronavirus strain. in this study, cov genomes containing regions resembling those of members of the genus betacoronavirus were detected in dobsonia moluccensis, known as moluccan naked-backed fruit bat, in indonesia. dobsonia moluccensis belongs to family pteropodidae, which is known to harbor a number of viruses, including covs [ , ] , paramyxoviruses [ , ] and rhabdoviruses [ , ] . nipah and hendra viruses have also been detected in dobsonia moluccensis [ , ] . fig. phylogenetic tree of partial rdrp-helicase using a , -bp nucleotide sequence following removal of primer sequences at the and end of the partial rdrp-helicase gene obtained from fruit bats samples (ifb - f and ifb - f) and various coronaviruses derived from genbank. the tree was constructed by the maximum-likelihood method using mega software. bootstrap values were calculated for replicates and are shown next to the branches. bat coronaviruses detected in this study are indicated by black squares. sars-cov, severe acute respiratory syndrome coronavirus; avian ibv, avian infectious bronchitis virus; cov, coronavirus this report highlights the fact that indonesian fruit bats are a natural reservoir for various viruses, which have the potential to be zoonotically transmitted, since fruit bats are also consumed in certain parts of indonesia. what we are watching-five top global infectious disease threats, : a perspective from cdc's global disease detection operations center detection of coronaviruses in bats of various species in italy isolation and characterization of viruses related to the 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philippines sars-coronavirus ancestor's foot-prints in south-east asian bat colonies and the refuge theory molecular detection of a novel paramyxovirus in fruit bats from indonesia generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray evolutionary trees from dna sequences: a maximum likelihood approach mega : molecular evolutionary genetics analysis version . characterization of the envelope glycoprotein of a novel filovirus, lloviu virus isolation of novel adenovirus from fruit bat molecular model of sars coronavirus polymerase: implications for biochemical functions and drug design coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku belonging to a novel betacoronavirus subgroup detection of novel sars-like and other coronaviruses in bats from kenya differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses coronavirus genomes in fruit bats in indonesia bats: important reservoir hosts of emerging viruses bats host major mammalian paramyxoviruses bat lyssaviruses, northern vietnam nipah virus and bats a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? acknowledgements we thank prof. ayato takada for providing the vero e cell line, and prof. ken maeda for providing the fbkt and demkt cell lines for the current study. this study was partly supported by a grant from the japanese initiative of global research network on infectious diseases (j-grid) and a grant of the ministry of education, culture, sports, and technology (mext), japan. key: cord- -klv jdm authors: smith, abigail l.; barthold, s. w.; de souza, m. s.; bottomly, kim title: the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: klv jdm infection of balb/c mice with mouse hepatitis virus, strain jhm (mhv-jhm), at any of several intervals relative to ovalbumin (ova) administration resulted in elevated ova-specific igg a titers. since gamma interferon (ifn) has been implicated as an up-regulator of igg a production, attempts were made to determine whether levels of this cytokine were modified in sera of infected mice. serum ifn-γ was not detected, but treatment of mhv-jhm-infected mice with monoclonal anti-ifn-γ antibody resulted in high mortality with decreased survival times, enhanced virus titers in liver and spleen, and more severe virus-associated pathology, compared to mock-treated, infected mice. immunotherapy with recombinant ifn-γ ameliorated disease as reflected by mortality rates and virus titers in target organs. mice inoculated with soluble protein antigens preferentially produce igg and igg specific to that antigen [ , ] . in contrast, infection of mice with any of several viruses enhances the igg a response both to the infecting virus and to immunizing soluble protein antigens [ ] [ ] [ ] . gamma interferon (ifn), a cytokine preferentially produced by the th subset of cd + t cells [ ] , stimulates production of igg a by b lymphocytes activated in vitro and in vivo and inhibits igg secretion [ , , ] . thus, it has been postulated that the immune response triggered by viruses mainly activates the th subset of helper t cells [ -] . mouse hepatitis virus (mhv) is a singular name for a group ofcoronaviruses, family coronaviridae, infecting laboratory mice worldwide at high prevalence [ , , ] . mice infected with mhv can exhibit aberrant immune responses that interfere with their usefulness in biomedical research [ , , , [ ] [ ] [ ] . coutelier and co-workers [ ] showed altered protein-specific isotype responses of mice infected with the a strain of mhv one day prior to antigen administration. the initial goal of the currently reported experiments was to determine whether mhv-jhm infection yielded similar results and whether timing of virus exposure was critical to observing the effect, as has been reported for other experimental systems altered by mhv infection [ , ] . mhv-jhm infection of the balb/c mouse was exploited as representative of a combination of moderately virulent, pantropic virus in a commonly used permissive genotype. based on the observation of elevated ovalbumin (ova)-specific igg a levels in sera of mice infected with mhv-jhm at any of several intervals relative to antigen exposure, attempts were made to detect ifn- in sera of infected mice and to reverse the igg a elevation by administration of ifn-y-specific antibody. the effect of this antibody and of immunotherapy with exogenous recombinant ifn- on virus-associated mortality and pathology and on virus replication in target organs was examined. female balb/cbyj mice (the jackson laboratory, bar harbor, me) were five to seven weeks old at the time of virus exposure. young adult balb/c nu/nu (athymic) and nu/+ (euthymic) mice were obtained from life sciences, inc. (st. petersburg, fl). cr:orl sencar dams with litters were obtained from the animal genetics and production branch, nci (bethesda, md). randomly selected mice were free of antibody to common murine viruses on arrival, all mice were housed in micro-isolator cages (lab products, maywood, nj) and were given food and water ad libitum. manipulations and husbandry were performed in a class ii biological safety cabinet, and infected mice were housed in a facility separate from that used for control (uninfected) mice. an open-cage sentinel mouse seromonitoring program was in place during the course of the reported studies. seroconversion to none of common murine viruses or mycoplasma pulmonis was detected, except among mhv-inoculated, immunocompetent mice which seroconverted to the infecting virus. mhv-jhm (american type culture collection, rockville, md) was used in the form of an infected infant mouse brain homogenate. mice immunized with ova or given antibody to ifn- were exposed orally to infant mouse iclds of virus. mice treated with recombinant ifn- were exposed intranasally to the same dose of virus. intranasal exposure of balb/c mice generally results in nearly % mortality, whereas mortality rates after oral inoculation are less than % (unpubl. obs.). groups of mice were infected per os with mhv-jhm on days - , - , - , , + , or + relative to intraperitoneal immunization with ova ( ~tg/mouse) in freund's complete adjuvant (fca). additional groups of uninfected mice received ova plus fca or fca only. mice were killed with co gas and exsanguinated on days or post-ova or fca administration. data from three replicate experiments were pooled. the numbers of mice per group are shown in table . mice were monitored for seroconversion as evidence of infection by indirect immunofluorescent staining using individual sera diluted : and a bivalent antigen consisting of mhv-jhm and mhv-s, as previously described [ ] . results are expressed as geometric mean titers [ ] for sera with significant a values at a dilution of : or greater. selected sera from mice tested for igg a were eliminated from the analysis due to unacceptably high background readings. ten percent tissue homogenates were clarified by centrifugation, and the supernates were screened for infectivity by intracerebral inoculation of litters of two day old sencar mice. virus was quantified by inoculating serial log dilutions of tissue homogenates. mortality during a one week period was recorded, and virus titers were calculated by the method of reed and muench [ ] . monoclonal antibody to mouse gamma ifn, designated xmg . [ ] , was precipitated with saturated ammonium sulfate and extensively dialyzed against pbs, ph . . the final product contained x neutralizing units per ml of antibody when tested with units of recombinant gamma ifn in the wehi b cell lymphoma bioassay [ ] . mice were given x l neutralizing units daily by the intraperitoneal route, beginning one day prior to virus exposure through one day prior to necropsy. recombinant mouse ifn- (lot no. ; specific activity ~> x units/mg; purity > % according to manufacturer) was obtained from amgen biologicals (thousand oaks, ca). mice were given x units intraperitoneally and × units intranasally once daily on days (- ) through (+ ) relative to virus exposure. liver and spleen were fixed in % neutral buffered formalin, paraffin embedded, sectioned at ~tm and stained with hematoxylin and eosin. chi square analysis was used to test for differences in proportions, and student's unpaired t test was used to analyze differences in mean virus titers. sera from mice infected with mhv-jhm one day prior to ova administration contained substantially elevated levels of antigen-specific igg a on day (table ) with geometric mean titers that were times control (ova only) values at that interval. there was a suggestion of a delayed igg a response among mice infected with mhv-jhm three or five days after ova administration, since sera from one of mice tested on day contained ova-specific igg a, whereas four of eleven ova-immunized, uninfected mice had seroconverted at that interval (% = . ; p < . ). sera from higher proportions of mice in groups that received mhv-jhm on days - ( %), - ( %) or ( %) relative to ova contained ova-specific igg a on day than the proportion that were ova-immunized, but uninfected ( %); however, these differences were not statistically significant. mice infected four days prior to ova administration had ova-specific igg titers that were sevenfold lower than those of ova-immunized, uninfected mice (table ) , and the proportion of igg -positive sera ( %) was reduced compared to uninfected control sera ( %). results are shown as number positive at a dilution of : or greater/number tested, and geometric mean enzyme-linked immunosorbent assay titer ± sd for positive sera sera collected from mice at days after ova immunization yielded a more clear-cut pattern. igg a titers were elevated in sera from mice that received virus any time from seven days prior to ova through one day after ova ( table ) . as had been seen among mice bled seven days after administration of ova, the most marked increase in ova-specific igg a ( -fold increase over controls) occurred among mice infected one day prior to immunization. sera from all infected mice except those exposed to virus seven days prior to immunization contained reduced levels of ova-specific igg (table ) . however, the greatest reduction (mice infected three days after ova immunization) was three-fold. mice infected seven days prior to immunization yielded igg titers equivalent to uninfected controls. isotype distribution of ovalbumin-specific antibody was also analyzed by determining igg :igg a ratios for sera from mice bled on day (fig. ) . although the two tests may have inherently different sensitivities, this parameter would be expected to remain relatively constant, since the two assays were always run in parallel, and the same reagents were used throughout the study. this analysis revealed that igg levels in sera from uninfected, ova-immunized mice were -fold higher than igg a levels. in contrast, the ratio was less than one for sera from mice infected one day prior to immunization and measured on day . the elevated igg a ova-specific response of most groups of mhv-infected mice and the preferential production of this isotype by mice infected one day prior to antigen administration suggested that these mice were producing ifny. however, using the wehi b cell lymphoma bioassay [ ] , attempts to detect the cytokine in sera of infected mice failed, as had earlier attempts to ] . it was reasoned that, ififn-y was being produced below the level of assay detection and was responsible for the induction of an elevated igg a response, treatment of ova-immunized, mhv-infected mice with anti-ifn- should restore, at least partially, the igg ova-specific response. in two experiments, % of mice inoculated orally with mhv-jhm and treated with xmg . died with survival times substantially shorter than those of mock-treated, infected mice ( . - - . days vs . ± . days). this suggested that ifn-y was produced during mhv infection and was influencing the course of disease. in an attempt to delineate the role of ifn-y in the pathogenesis of mhv-jhm infection, groups of athymic or euthymic balb/c mice were treated with monoclonal anti-ifn-y (xmg . ) or were mock-treated with dialysis buffer (pbs) and exposed orally to virus. athymic mice were included because they produce ifn-y by virtue of fully competent natural killer cells [- ]. four mice per genotype and treatment group were necropsied daily on post-infection days one through five. liver and spleen sections from all mice were examined histologically, and virus in the same organs of mice necropsied on days four and five post-infection was quantified. on day , mock-treated athymic and euthymic mice and xmg . -treated euthymic mice had rare scattered foci of necrotizing hepatitis, with necrosis of hepatocytes and infiltration of neutrophilic leukocytes. xmg . -treated athymic mice had hepatitis that was similar in quality to that of their mock-treated counterparts, but necrotic foci were more numerous. on day , differences between treatment groups were more striking. xmg . treated athymic mice had numerous large, coalescing foci of hepatocellular necrosis, with only modest leukocytic response (fig. a) . in contrast, mocktreated athymic mice had fewer, smaller foci of hepatitis with less necrosis and infiltration with more leukocytes (fig. b) . xmg . -treated euthymic mice had more foci of hepatitis than either their mock-treated counterparts or mock treated athymic mice. lesions among the latter three groups were qualitatively similar, with infiltration of both neutrophilic and mononuclear leukocytes. microscopic differences between treatment groups were not seen in spleens of mice necropsied on day , but were evident on day . xmg . -treated athymic mice, and to a lesser extent xmg . -treated euthymic mice, had severe necrosis of the periarteriolar regions of the white pulp. necrosis was present in the same regions of spleens from mock-treated mice, but was less severe. virus titers in livers and spleens of xmg . -treated mice necropsied on days and post-inoculation, whether athymic or euthymic, were higher than those in the corresponding tissues of mock-treated mice ( table ) . for athymic mice, these differences were statistically significant for both tissues on both days. among euthymic mice, statistically significant differences in virus titers occurred in the spleen, but not the liver. since treatment of mhv-jhm-infected balb/cbyj mice with antibody to ifn-y resulted in decreased survival time and increased virus titers in two target (table ) . virus titers in liver, spleen and brain of ifn- treated mice at five days post-inoculation were lower than those in the corresponding organs of mock-treated mice (table ) , but only the differences in liver titers were statistically significant. there have been a few reports detailing the responses of mhv-infected mice to nonreplicating antigens or other viruses. mice infected intraperitoneally with mhv- two or four days prior to sheep red blood cell immunization yielded depressed igm and igg plaque-forming cell responses [ ] , whereas mice in-fected at the time of or one day after red cell administration yielded elevated plaque-forming cell responses. this could be correlated with the timing of ifna/ responses relative to antigen administration. more recently, intranasal administration of mhv-a one day prior to tetanus toxoid immunization resulted in elevated antigen-specific igg with a four-fold decrease in the percent of igg that was igg and a like increase in the proportion that was igg a [ ] . our results with mhv-jhm and a different immunizing antigen confirm and extend that finding by showing elevated igg a ova-specific responses over a wide range of infection intervals. timing is crucial, however, to the extent that igg a titers exceeded igg titers only among mice infected one day prior to antigen administration. mhv infection reportedly increases resistance to a variety of secondary viral infections. pre-infection of mice with mhv increased resistance to lethal sendai virus infection in a normally susceptible genotype and interfered with replication of pneumonia virus of mice in respiratory tract tissues [ ] . subclinical mhv infection delayed increased plasma lactic dehydrogenase (ldh) levels resulting from ldh-elevating virus [ ] . natural, inapparent mhv infection also increased resistance to encephalomyocarditis virus and reduced the protective effect of exogenous ifn-a or - [ ] . since mhv infection induces ifn-(z/ production [ , , ] , it was postulated that increased resistance to these secondary infections was due to endogenous ifn-a/ levels and that the effects of exogenous ifn were masked by the endogenous production [ ] . there are now a limited number of reports showing that ifn- ~ modulates the pathogenesis of virus infections. treatment of mice with a different rat antimouse ifn-~f monoclonal antibody or with a sheep-derived antibody against recombinant ifn- resulted in reduced clearance or enhanced replication of lymphocytic choriomeningitis virus (lcmv) in mice [ , ] . neutralization of ifn- in lcmv-infected athymic c bl/ resulted in enhanced virus replication [ as seen in the studies reported here. more recently, a rat antimouse ifn- monoclonal antibody prevented il -induced recovery of athymic mice infected with recombinant vaccinia virus encoding murine il [ ] , suggesting that il -induced ifn- production was responsible for rapid clearance of this construct. virus replication and associated mortality were unaffected among anti-ifn- -treated athymic mice infected with a control virus construct encoding hsv tk and a/pr/ / ha [ . however, administration of recombinant ifn- to mice infected with the control construct significantly increased their survival time, although all treated mice eventually died of disseminated infection [ ] . resistance of intraperitoneally inoculated a/j mice to mhv- also correlates with the ability of this genotype to produce ifn- after infection [ ] . the accumulated data strongly suggest that ifn- , whether induced during the course of infection or administered therapeutically, limits viral replication in vivo. in the case of vaccinia virus encoding il , ifn- apparently mediates recovery of athymic mice. our data with mice exposed by presumed natural routes to mhv-jhm show that endogenous ifn- modulates the pathogenesis of infection in both athymic and euthymic mice. in earlier studies with lcmv [ ] and the current experiments with mhv, ifn- was either present in very low concentration or undetected in sera of infected mice, despite the fact that anti-ifn-y treatment resulted in elevated virus titers. these observations may stem from insensitivity of the assays used to detect ifn- . the vesicular stomatitis virus cytopathic effect reduction assay was used in the lcmv studies, and the wehi cell bioassay was used in the current experiments. the wehi cell bioassay is as sensitive as the ia induction assay for detection of ifn- [ ] , but sensitivity comparisons with other methods have not been reported. use of the sandwich enzyme-linked immunosorbent assay, as reported by karupiah and co-workers [ ] , would likely reveal the presence of ifn- in sera or tissue homogenates from infected mice. based on both histologic evaluation and virus quantification, the effect of administration of anti-ifn- antibody was more marked in athymic than in euthymic mice. this may be due to higher endogenous concentrations of ifn- in euthymic mice by virtue of functional t cells as well as natural killer cells. neither natural killer cells nor endogenous levels of ifn- are sufficient to afford protection, however, since athymic mice do die after natural or experimental infection with mhv [- , ] . mhv-jhm also kills virtually all balb/ c mice exposed by the intranasal route. the lethal effect of infection could be partially overcome by administration of recombinant ifn- , with a concomitant and significant decrease in virus concentration in the liver. others have postulated that virus infections preferentially activate the ifny producing t h subset of cd + t cells [ ] , based on the fact that most infections result in enhanced igg a production. using ldh-elevating virus or the fl strain of mouse adenovirus, attempts were made to inhibit virusassociated igg a production by injection of several different ifn-y-specific monoclonal antibodies [ ] . these efforts failed, although it was not clear whether treatment simply did not affect isotype distribution or whether the mice succumbed to infection. in the current study, neutralization of ifn- in vivo resulted in high mortality with reduced survival times, thereby inhibiting our efforts to show a direct link between virus-induced ifn- production and enhanced igg a responses. among mouse strains susceptible to mhv after exposure by natural routes, lesions and mortality occur at or near the infectious dose level, and the severity of lesions is independent of virus dose [ , ] . in the case of mhv-jhm inoculation of the balb/c mouse by presumed natural routes, the median infectious and lethal doses are essentially identical (unpubl. data). therefore, subsequent studies designed to reveal whether neutralization of ifn- reverses preferential igg a production will require the use of mouse genotypes more resistant to the lethal effects of mhv-jhm infection [ ] or of a less virulent mhv strain. our studies with mhv-jhm in the balb/c mouse have shown, however, that endogenous ifn-y modulates coronavirusassociated mortality, virus replication and tissue damage in a susceptible host. mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm the polyclonal and antigen-specific ige and igg subclass response of mice injected with ovalbumin in alum or complete freund's adjuvant alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus two types of mouse helper t cell clone. iii. further differences in lymphokine synthesis between th and th clones revealed by rna hybridization, functionally monospecific bioassays, and monoclonal antibodies in vivo polyclonal b-lymphocyte 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recovery of athymic nude mice from recombinant vaccinia virus/interleukin infection gamma interferon in murine coronavirus infection cytotoxic t lymphocyte control of acute lymphocytic choriomeningitis virus infection: interferon gamma, but not turnout necrosis factor alpha, displays antiviral activity in vivo diagnosis of murine infections in relation to test methods employed enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice new a (ed) viral and mycoplasmat infections of laboratory rodents. effects on biomedical research a major role of macrophage activation by interferongamma during mouse hepatitis virus type infection. i. genetically dependent resistance prevalence of natural virus infections in laboratory mice and rats used in canada a simple method of estimating fifty percent endpoints inhibition of b lymphocyte activation by interferon gamma activationofnaturalkillercells and induction of interferon after injection of mouse hepatitis virus type in mice an immunofluorescence test for detection of serum antibody to rodent coronaviruses altered splenic t cell function of balb/ cbyj mice infected with mouse hepatitis virus or sendai virus antigenic characterization of tettnang virus: complications caused by passage of the virus in mice from a colony enzootically infected with mouse hepatitis virus in vitro splenic tcell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain jhm interferon-gamma and b cell stimulatory factor- reciprocally regulate ig isotype production ifn-gamma stimulates igg a secretion by murine b cells stimulated with bacterial lipopolysaccharide persistent infection with mouse hepatitis virus of low virulence in nude mice the role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (mhv- ) statistical methods in serum surveys this research was supported by nih grants rr and rr . the authors thank deborah f. winograd, deborah s. beck, and jane dunn for excellent technical assistance. received january , key: cord- -ftzjdvfj authors: bhatt, p. n.; jacoby, r. o. title: experimental infection of adult axenic rats with parker's rat coronavirus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ftzjdvfj the pathogenesis of parker's rat coronavirus (prcv) was studied in axenic cd rats. three to four to week old rats were euthanized daily for eight days after intranasal inoculation. rats remained free of clinical disease. virus was recovered from the nasopharynx and trachea after twenty-four hours and from the lung by day three but was not detected in respiratory tract after seven days. viral antigen was detected by indirect immunofluorescence in the mucosal epithelium of upper respiratory tract and in pulmonary alveolar septae from day two to six postinoculation. acute rhinitis developed by day two and was associated with mild focal necrosis of respiratory mucosal epithelium. mild nonsuppurative tracheitis and multifocal interstitial pneumonia appeared by day five and persisted through day eight. dacryoadenitis did not occur, sialoadenitis was detected in only three rats and virus was recovered from only one submaxillary salivary gland. this experiment indicates that prcv can be a primary pathogen for the respiratory system of adult rats. in contrast to sialodacryoadenitis (sda) virus the tropism of prcv for salivary and lacrimal glands is low. coronavirus infection is common in laboratory rats and two antigenically related viruses, sialodacryoadenitis virus (sdav) and parker's rat coronavirus (prcv), have been isolated from naturally-infected rats ( , ) . sdav causes severe self-limiting sialodacryoadenitis in naturally-infected or experimentallyinoculated adult rats ( , ) and recent evidence indicates that sdav is associated with naturally-occurring keratoeonjunetivitis in rats ( ) . prcv was originally isolated from the lungs of rats with a high incidence of complement-fixing (cf) : p.n. bha~'t and r. . jacob¥: antibody to mouse hepatitis virus (mhv) ( ) . in contrast to sdav, natural infection with prcv is reportedly asymptomatic in adults, but experimentallyinoculated neonates developed lethal interstitial pneumonia ( ) . this report described experimental prcv infection of adult cd rats. results indicate that prcv is pathogenic for the respiratory system. fifteen male and fifteen female, to i week old, axenic caesarian-derived (cd) rats (charles river breeding laboratories, wilmington, mass.), - g in weight, were housed in sterile isolators and were fed sterile rat food and water ad libitum. isolators were monitored for bacterial and fungal contamination by repeated sampling of feces, bedding and water bottles. they remained sterile throughout the experiment. seed prcv was obtained froln dr. john c. parker, microbiological associates, det:hesda, maryland. virus stock was prepared by one passage in primary rat kidney (pri~) cultures prepared from a germfree cd rat as previously described ( ) . aliquots of infected tissue culture fluid were stored at -- °c with equal volumes of fetal bovine serum (fbs). unanesthetized rats were inoculated intranasally with .i ml of culture fluid containing . log tcids of prcv. culture fluid was removed from isolators immediately after inoculation and was retitered for infectious prcv in prk monolayers as previously described ( ) . on each of eight consecutive days after inoculation three to four rats, selected at random, were anesthetized with pentobarbital sodium and exsanguinated. serum was stored at -- ° c. sections of trachea, lung, cervical lymph node, submaxillary and parotid salivary glands and exorbital and iiarderian lacrimal glands were harvested aseptically and were frozen at -- o c. nasopharyngeal cavities were irrigated with sterile minimum essential medium in hank's base containing per cent fbs (memh-fbs) and washings were also frozen at -- ° c. tissues were homogenized (i per cent w/v) in memii-fbs and were clarified by lowspeed eentrifugation. supernatants were serially diluted and inoculated into pi~k cultures. observations for cytopathic effects were carried out as previously reported ( ) and endpoints were calculated as described by reed and mvenci~ ( ) . virus in lungs, tracheas, nasal washings, salivary glands and lacrimal glands was quantitated for individual rats. lymph nodes were pooled by collection day before being titrated. antlpi~cv immune serum and prcv cf antigen were donated by drs. john parker and michael collins of microbiological associates, bethesda. serum neutralizing (sn) and cf antibody titers to sdav and prcv were determined as previously described ( ) . sections of nasal turbinate, trachea, cervical lymph node, submaxillary and parotid salivary gland and exorbital and ii[arderian lacrimal glands were snap frozen in dry icealcohol baths. lungs were inflated with . per cent gelatin and placed at ° c for one hour to gel, sliced and frozen on chucks at -- ° c. rat anti-sdav igg was prepared and atiquots were conjugated to fluoreseein isothiocyanate ( ). preliminary tests indicated that anti-sdav and anti-prcv sera gave equally good results for indirect immunofluoreseent staining of ptlcv-infeeted tissues. goat anti-mouse globulinfluoreseein isothioeyanate (lot aa , antibodies, incorporated, davis, california) was used with mouse anti-sdav immune ascitie-fluid for indirect staining. sections of snap frozen tissues mm thick were fixed overnight in acetone at -- ° c and were stained by the direct, and/or indirect, fluorescent antibody technic. they were examined with a zeiss microscope fitted with an i{bo osram amp, a bg- ultraviolet exciter filter and numbers : and barrier filters. coronal sections of nasopharynx at three levels and sections of trachea, lung (inflated with fixative via the trachea), cervical lymph node, smivary glands, lacrimal glands, heart, thymus, liver, spleen, kidney, pancreas, adrenal gland, gonad, eye and brain were fixed in i per eent neutral buffered formalin, sectioned at ~m, stained with hematoxylin-eosin and examined by light microscopy. rats were free of detectable clinical disease during the eight day experiment. prcv was recovered from nasal washes and trachea by day one, from cervical lymph nodes by day two and from lung by day three (fig. ) . titers were usually higher in trachea than in lung. virus was rarely detected in salivary or lacrimal glands and was not found in serum. virus was not detected in nasopharynx, trachea and lung a~ter day six and in other tissues after day seven except in ~he submaxillary salivary gland of one rat. intracytoplasmic viral antigen was de~ected by immunofluorescence in epithelial cells of nasal mucosa by day two. fluorescence was multifocal and persisted until day six when onty several small loci of antigen remained. tracheal fluores- cence was sparse; a few mucosal epithelial cells contained viral antigen on days two through four. fluorescence in lung was also sparse and was observed only on days six and seven. antigen was not detected in parotid, submaxillary, exorbital and itarderian glands or in cervical lymph nodes. gross lesions were confined to lung, were seen on days six and seven and consisted of several small (less than mm) red-brown to gray loci which were randomly dispersed over all lobes. a few lungs had patchy areas of pale red to gray discoloration on individual lobes, but the lobes were not firm. i-iistologieally, lesions occurred primarily in the respiratory system and were first seen on day two as mild rhinitis. there was multifocal or segmental necrosis of respiratory epithelium covering nasal turbinates. the lamina propria was mildly edematous and contained lymphoc es and a few neutrophils which occasionally infiltrated interstitial tissnes of underlying glands. some meatuses contained neutrophils and cell debris. similar lesions were fonnd through day four (figs. , ) although there was a relative increase in the proportion of neutrophils in the lamina propria and several necrotic aeini were seen among submueosal glands. hyperplasia of paraseptal lymphoid tissue was noticed by day four, but germinal centers were not present. by day five acute rhinitis was accompanied by mild nonsuppurative traeheitis. tracheal lamina propria had infiltrates of lymphoid cells and some neutrophils. neutrophils were also found in mucosal epithelium but epithelial necrosis was generally sparse. occasionally inflammation was more severe and ineluded substantial necrosis of epithelium and tracheal glands. lung lesions began by day five as focal peribronehial lymphoid cell hyperplasia and mild focal interstitial pneumonia. alveolar septae contained mononuclear cells and neutrophils and there were some inflammatory cells in adjacent (figs. , ) . by day six pneumocytes, foamy macrophages and edema fluid partially filled some alveoli, but lesions remained mild and focal. there were also traces of nonsuppurative perivasculitis in several lungs. necrosis of bronchial epithelium was not observed. nasopharyngeal and tracheal inflammation subsided by day seven and only traces of rhinitis remained by day eight. pulmonary lesions were not detected on day eight. salivary gland lesions were rare. parotitis was found in two rats on day seven and submaxillary sialoadenitis was found in one rat on day eight. lesions were alveolar spaces contain a few macrophages identical to those caused by sdav ( , ) and were characterized by necrosis of salivary ducts with periductular and interstitial i n f l a m m a t o r y edema. there were no lesions in lacrimal glands, eye, liver, spleen, heart, kidney, thymus, cervical l y m p h node, adrenal gland, gonad, or brain. anti-pi~cv a n d anti-sdav neutralizing a n t i b o d y were detected by day six and seven, respectively (table ) . cf a n t i b o d y to p g c v and sdav was not detected through day eight. parker and eoworkers previously demonstrated that prcv caused lethal interstitial pneumonia in experimentally infected suckling rats and they suggested that prcv could play a role in chronic respiratory disease of adult rats ( ) . we extended their hypothesis to include sdav by showing it was also a primary pathogen for the respiratory system of adu]t rats ( ) . experiments reported here show diree~ty that prcv infection of adult rats, although asymptomatie, causes enough inflammation of the respiratory tract to warrant continued scrutiny as an initiator or eopathogen in clinically severe respiratory-syndromes. it is clear that prcv and sdav are closely related viruses. their antigenic similarity has been well documented, but in cross neutralization tests titers to the homologous virus were persistently higher than to the heterologous virus ( ) . similar results were obtained in the present experiment where prcv-infeeted rats had sn antibody to prcv by day six and slightly lower sn titers to sdav by day seven. it is of interest in this regard that prcv and sdav were originally isolated from lungs of fisher rats and salivary glands of cd sprague dawley rats, respectively. these rats were raised in different rooms of the same animal facility. strains of mouse hepatitis virus (mhv) (coronavirus) differ in their pathogenicity and tissue tropism although they are closely related serologieally ( ). our findings indicate that the relationship between sdav and prcv is similar to that among mitv strains. therefore, we propose that sdav and prcv be considered different strains of rat eoronavirus. when the pathobiology of sdav ( ) and pi~cv infection in vivo is compared, additional differences emerge (table ) . first, clinical signs of rhinitis and sialo- up to eight days post inoculation cf antibody can develop if rats are tested at later times dacryoadenitis were observed frequently during experimental sdav infection whereas prcv infection in rats of the same strain and of the same age and source was asymptomatie. second, the tissue tropism of pi~cv differed from sdav. both viruses replicated in the respiratory tract and caused rhinotraeheitis but prcv also caused mild pneumonia whereas sdav did not. conversely, prcv replicated poorly in salivary and lacrimal glands and only rarely produced lesions, whereas sdav was severely pathogenic for these glands. nevertheless, sialoadenitis produced by prcv, although mild when it occurred, was morphologically compatible with sdav-induced lesions. the potential for pl~cv-induced sialoadenitis may be mitigated by factors such as strain, age and sex of the host. additional experiments should examine the effects these variables ha,~e on eoronavirus infections of rats. this study also underscores the usefulness of documenting and correlating virological and morphological data to assess the pathogenetie significance of infections which are clinically silent and which are usually detected only by serological monitoring. mouse hepatitis virus (mhv), viruses of vertebrates characterization of the virus of sialodacryoadenitis of rats : a member of the coronavirus group chromatographic purification of tetramethylrhodamine immune globulin conjugates and their use in the cellular localization of rabbit gammaglobulin peptide chains pathogenesis of sialodacryoa.denitis in gnotobiotic rats sialodaeryoadenitis in the rat (a light and electron miserocopic study) keratoeonjunctivitis associated with sialodaeryoadenitis in rats rat coronavirus (rcv): a prevalent., naturally occurring pneumotropie virus of rats a simple method of estimating fifty percent and endpoints supported by phs grants rr , rr and fr . the authors wish to acknowledge excellent technical assistance of miss m. nettleton, miss b. collett and miss d. davis. key: cord- - t fjld authors: khromykh, a. a.; westaway, e. g. title: rna binding properties of core protein of the flavivirus kunjin date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: t fjld kunjin virus (kun) c is a typical flavivirus core protein which is truncated in vivo to a mature form of residues enriched in lysine and arginine. in order to study the possible association of kun c with rna in vitro, we prepared several recombinant c proteins with specific deletions, each fused at the amino-terminus to glutathione-s-transferase (gst) and expressed ine. coli. they were reacted with kun rna probes transcribed in vitro from cdna representing the ′ untranslated region ( ′ utr, of nucleotides), the ′ utr ( nucleotides), and the ′ utr plus most of the c coding region (t′ core, nucleotides). fusion protein c (incorporating mature c) bound strongly to all kun rna probes with apparent specificity, being completely resistant to inhibition by mm nacl, and to competition by a large excess of trna. in reactions with labelled kun rna probes putative binding sites were identified in the isolated amino-terminal ( residues) and carboxyterminal ( residues) basic amino acid domains; this binding was strongly competed by unlabelled kun utr probes but weakly or not at all by trna. these small domains probably acted co-operatively in binding of mature c to kun rna probes. the kun rna-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral rnas. there are relatively few reports of in vitro binding of coat or core proteins to genomic sequences of positive-stranded rna viruses; a common but not universal feature is the high proportion of lysine/arginine residues associated with these binding reactions. these residues impart a positive charge to the core protein facilitating its binding to (negatively charged) rna during the process of virus assembly. both specific and nonspecific binding reactions have been reported. examples include alphaviruses [ , ] , mouse hepatitis virus [ , ] and several plant viruses [ , , ] . stem loop structures in the binding region of rna also have been reported. a predicted binding site of the nucleocapsid protein of mouse hepatitis coronavirus corresponds to sequences within a potential hairpin loop involving nucleotides through (in the leader region) of genomic rna; it was proposed that such binding may play a role in regulation of transcription [ ] . turnip crinkle virus coat protein binds to two sites in genomic rna able to associate in a stem loop structure surrounding the termination codon; it was proposed that this structure is involved in rna replication [ ] . a stem loop structure between nucleotides and from the ' end of alfalfa mosaic virus rna binds viral coat protein, thus possibly contributing to initiation of infection and to assembly of virus particles [ ] . the association of the flavivirus core protein with virion rna in intracellular and extracellular virus particles is well documented (for review see [ ] ). however, how and where this association occurs in cells is not understood. nucleocapsids have not been isolated from flavivirus-infected cells [ ] , and corelike particles have been observed by electron microscopy only in mosquito cells infected with the pr- strain of dengue- virus (but not with the ngc strain or with other flavivirus species; reviewed by hase etal. . the flavivirus genomic rna of about kilobases is flanked by about to t nucleotides in the ' untranslated region (utr) and to nucleotides in the ' utr (see [ , ] ). as noted above, binding of coat or capsid proteins to stem loops in the utrs of some positive-stranded rna viruses is possibly involved in encapsidation and/or rna synthesis; for flaviviruses similar interactions with core protein may involve the folded stem loop structures proposed in approximately the first nucleotides of the ' utr and the last nucleotides of the ' utr [ , , , , , ] . before any sequence data were available, a rote was proposed for flavivirus c in control of the switch of rna plus strand as a template for transcription to translation by attachment of c to the ' end, hence blocking polymerase attachment for initiation of copying from plus strand templates [ ] . flavivirus core protein c is relatively small (about kilodaltons) and about % of the amino acids are lysine or arginine (see [ , ] ). the carboxy terminal sequence of amino acid residues of kun c protein provides the hydrophobic signal sequence preceding prm that is cleaved posttranslationally, reducing c to its mature form of residues [ t]. the purpose of the present work was to investigate associations of c with rna which could be observed in vitro, as part of a continuing study of events during flavivirus replication. initially, we have analysed the binding of kun c representing the mature form (i.e. with the carboxy-terminal amino acids deleted), and with additional deletions in regions of basic amino acids, to kun rna probes representing the ' and ' utrs and to nonspecific rna. the utr regions were chosen as rna probes in this initial study because of their possible involvement in replication or assembly, and because of reports noted above of binding of coat or capsid proteins to ' utr regions of several plant viruses [ , , ] or to the ' leader sequence of a coronavirus [ , ] . for this study we exploited the use of glutathione-s-transferase (gst)-core fusion proteins attached to an insoluble matrix (glutathione sepharose b) for rapid rnaprotein binding assays, and extended some of these results by mobility shift assays [ ] which were possible because of the relatively small size of the rna probes, kun utr probes bound strongly to fusion proteins incorporating full length or mature c, and to the isolated amino-terminal or carboxy-terminal domains of mature c. the base pair fragment representing the sequence coding for the first amino acids of kun c protein, plus the complete ' utr and an upstream sp promoter, were pcr amplified (using appropriate primers) from cdna synthesized from kun virion rna ligated ( ' end to ' end) as described previously [- ] . the pcr fragment was then cloned into pbluescript ii sk vector (stratagene); from the resulting plasmid named pbsskcore, a cdna fragment containing the sequence coding for the first amino acids of c protein and the preceding nucleotides of the ' utr was excised by digestion with bgiii and bamhi, and subcloned into bamhi digested pgex- x expression vector for expression from a single open reading frame of c protein fused at its amino terminus to gst (fig. la, c ). constructs expressing gst fused to the first or amino acids of c protein (c or c respectively), were obtained by similar methodology. constructs expressing gst fused to the carboxy-terminal region of c protein, such as c - (amino acids - ) and c -t (amino acids - ), or to the middle region of c protein, such as c - (amino acids - ) and c - (amino acids - ), were obtained by direct cloning of digested pcr fragments into pgex- x vector. the construct c expressing the amino-terminal amino acids of c protein was obtained by digestion of plasmid construct c with restriction endonuclease sinai at the smai site adjacent to the codon for amino acid and the sinai site in the multiple cloning site of the vector, and subsequent religation of the digested plasmid. each of the described constructs ( fig. la) was expressed using ml cultures of e. coli dh c¢, induced by adding . mm isopropyl-b-d-thiogalactoside (iptg; progene). h later cells were pelleted and ruptured by sonication in ml of ripa buffer ( mm tris-hc ph . , mm nac , % np , . % sodium deoxycholate, . % sds) supplemented with protease inhibitors. cell debris was removed by centrifugation and fusion proteins were purified from the resulting supernatant by affinity chromatography with gl of glutathione sepharose b slurry using a batch method essentially as described in the manual (gst gene fusion system, phannacia p-l biochemicals inc., ). fusion proteins were eluted off the glutathione sepharose beads in ~ of elution buffer containing mm tris-hc ph . , mm glutathione, and mm nac . preparation of rna probes ' utr and ' core rnas, representing the first nt or first nt of kun rna sequence, respectively (fig. lb) , were obtained by sp rna polymerase transcription of plasmid dna pbsskcore digested with bg/ii (for ' utr) or bamhi (for ' core rna). to obtain the ' utr rna the sequence corresponding to the last nt of kun rna was pcr amplified from the cdna synthesized from ligated kun virion rna as described previously [ ] , cloned in pbluescript ii ks (pbsiiks) vector and then used for rna transcription with t rna polymerase. to obtain radiolabetled rna probes, [ s s] utp was included in these in vitro transcription reactions. all synthesized rnas were treated with dnase and purified by phenol-chloroform extraction and two ethanol precipitations with m ammonium acetate. digoxigenin (dig)-labelled ' core and ' utr rnas were synthesized in similar in vitro to obtain p-labelled total cell rna, a confluent monolayer of vero cells in a t- flask (nunc) was first incubated for h at °c with phosphate-free minimum essential medium (mem; gibco brl) plus . % bovine serum albumin and then for h with the same medium containing mci of [ p]orthophosphate. cells were collected and lysed at °c in lal of lysis buffer ( mm tris-hc ph . , . m nac , mm mgci;, . % np ) supplemented with units of rrnasin (promega). the lysate was kept at °c on ice for - min, and spun in a microfuge for rain at maximum speed at °c; the supernatant was adjusted to . % sds and digested with lag per ml of proteinase k for rain at °c. rna from the treated supernatant was isolated by phenol-chloroform and ethanol precipitation with . m sodium acetate. the quantifies of protein and trna used in the binding reactions were selected as optimal for detection after using a range of concentrations in titration assays. the binding reactions were thus performed with - gg (~ pmol) of fusion proteins attached to the glutathione sepharose beads ( - gl of bead slurry) and - gg (~ pmol) of in vitro synthesized untabelled or dig-labelled rnas, or - ng (~ . pmol) of s-labelled rnas, or cpm of p-labelled total veto cell rna, in gl of binding buffer (bb; ram tris-hc ph . , . % np , mm nac , mm [ -mercaptoethanol, . mm atp, and u of rrnasin) at °c for h. in competition experiments appropriate amounts of yeast trna (boehringer mannheim) or unlabelled in vitro synthesized rnas were added to reaction mixtures simultaneously with rna probes. after the reaction the beads were washed times with . ml bb to remove residual unbound rna. rna bound to the fusion protein on beads were recovered by digestion of the protein with proteinase k ( . mg per ml) in the buffer containing % sds, ram tris-hc ph . , ram nac , ram edta. recovered unlabelled rna was identified by ethidium bromide staining after electrophoretic separation in a nondenaturing agarose gel in tae buffer ( mm tris-acetate, mm edta). radiolabelled recovered rna was identified by counting of a sample in a [ -counter. rna-protein binding reactions using the northwestern blotting technique were performed with dig-labelled kun ' core and ' utr rnas and zp-labelled cell rna essentially as described by stohtman et al. [ ] . briefly, purified proteins were removed from the beads by boiling in gel loading buffer, separated in a % laemmli gel as described previously [ ] and transferred electrophoretically to a nitrocellulose membrane. transferred proteins were allowed to renature on the membrane at room temperature overnight in ml of sbb buffer ( mm tris-hc ph . ; mm nac ; mm edta; . % bovine serum albumin; . % ficoll; . % polyvinyl pyrrolidone). the membranes were then transferred into ml capped tubes, overlaid with ml of sbb buffer containing either gg of dig-labelled rna or , cpm of p-labelled total vero cell rna and incubated for - h at room temperature with constant rotation. after incubation the membranes were washed times for rain with sbb buffer and were either dried and exposed to x-ray film (for detection of p-labelled rna) or processed for colorimetric detection of dig-labelled rna as described in the manual (the dig system user's guide for filter hybridization, boehringer mannheim, gmbh, bioehemica, ). c was expressed in e. coli and purified by retention on glutathione sepharose beads as described in materials and methods. the three viral rna probes ' utr, ' core and ' utr ( fig. lb) bound to c on the beads in the presence of ram nac (fig. a) . most of added s-labelled ' core rna ( ng) was retained when bound to lag c on the beads ( % and % in two experiments) whereas a smaller proportion ( % and % in two experiments) of s-labelled ' utr ( ng added) was retained. larger amounts ( - gg) of unlabelled or dig-labelled rnas were used in subsequent experiments, hence an excess of rna was always added for binding to c fusion protein. a measure of the specificity of rna-protein binding is its resistance to inhibition . we therefore included mm nac in the binding reactions which were thus shown to be unaffected by high salt (fig. a) . reactions of c with the viral probes ' utr and ' core in mm nac were repeated in competition experiments with yeast trna. although trna bound strongly to c in the presence of both probes (using , , and gg trna), no clear evidence of competition with the kun rna probes was observed (fig. b, c) . thus the ' and ' utr regions of kun rna bound to full length core protein c in a relatively specific manner. in order to analyse possible binding of cellular rna to c, we prepared p-labelled vero cell rna (see materials and methods). radiolabelled veto cell rna bound strongly to c in northwestern blots (results not shown). in binding experiments on glutathione sepharose beads, we found by polyacrylamide gel analysis of the bound rna and autoradiography of dried gels that the major binding component of vero cell rna was trna (fig. d) , which was competed out by an excess of yeast trna (results not shown). we therefore continued to use yeast trna in competitive rna binding assays. a minor amount of p-labelled s and s rna also bound to c- on the beads. no labelled rna was bound to gst alone. in attempts to define which regions of the mature form of c were binding to rna, three regions enriched in basic amino acids were chosen arbitrarily for all these deleted fusion proteins bound to ' utr and ' core probes on beads as efficiently as c (fig. a) . similar binding results were obtained with the same reagents using northwestern blots as the assay system (fig. b) . in both binding assays gst alone did not bind to either kun probe. all the core fusion protein constructs migrated on sds-polyacrylamide gels according to their predicted molecular weights except for c which migrated faster than anticipated (fig. b) . the nucleotide sequence from which c mrna was transcribed in e. coti was checked and found to be correct. either c migrated anomalously in gels, or the carboxy terminus was degraded, possibly by e. coli proteases, reducing the size of c protein moiety by approximately %. ifc was truncated in this manner, seven to nine basic amino acid residues of the amino-terminal region would still remain (fig. la) . these results did not identify location of the binding regions in c, but showed that the presence of gst protein fused to the amino terminus of the core protein did not compete with or appear to interfere with binding of the rna probes. the northwestern blots established that the rna probes were binding only to the purified core fusion proteins, and not to any other proteins which could possibly be attached to the glutathione sepharose beads either directly from e. coli lysates or coprecipitated with gst-core fusion proteins during purification. binding of btg trna to the deleted fusion proteins c - and c was quantitatively similar to binding of lag trna to c , but apparently had reached saturation binding because little or no increase in binding of trna was observed when the larger amounts to lag trna were added to the assay. gg trna competed weakly with binding of the ' utr and ' core kun rna probes to c - and c . furthermore, this binding was not blocked by mm nac (results not shown). with c , trna bound weakly; a large excess ( lag) was required for detection of about lag trna bound. however, binding of both kun rna probes to c was as efficient as that to c , c - and c , but in contrast was blocked by mm nac and competed out by lag trna (results not shown). thus although c bound kun rna probes more readily than trna, its binding to the kun rna probes was weaker than that of c - and c . these results showed that binding of kun ' core and ' utr probes to c - and c was very similar to that observed with c , and trna competed weakly with binding of these kun rna probes to the deleted c polypeptides only when used at high concentrations ( lag or about -fold molar excess). the location of the possible binding domain(s) in c remained undefined. because no clear picture had emerged showing which if any basic amino acid region in c was the major contributor to the apparent specificity of utr binding to c , shorter fusion protein constructs were prepared each containing only a single arbitrary domain enriched in basic amino acids (c , c - and c - ; fig. la ). all of these fusion proteins containing such short sequences of c migrated similarly in sds-polyacrytamide gels as expected based on their predicted molecular weights (fig. a) . c - did not detectably bind ' core rna, but c and c - did (fig. b) . despite their small size (containing only or amino acids of c), c and c - bound similar amounts of kun utrs as c did previously under the same conditions. for example, c - bound about % ( % and % in two experiments) and about % ( % and % in two experiments) of s-labelled ' core and ' utr probes, respectively. c bound about % and %, respectively, of the same labelled probes. these results appeared to exclude the central region of c, incorporating the arbitrary domain in c - , as a kun utr binding region. competition binding experiments were extended with c and c - using s_labelled ' core and ' utr rna probes and excess of the same probes but unlabelled as competitors. homologous utr probes competed (excess of unlabelled versus s_labelled utrs) as did the kun heterologous utr probes (excess of unlabelled ' core versus ~s_labelled ' utr, and excess of unlabelled ' utr versus s-labelled 'core; results not shown). lag trna bound weakly to c and c - and did not apparently compete with the ' core rna probe fig. . sds-page analysis of purified kun core-fusion proteins (see fig. la ) and their use in competition rna binding experiments between ' core rna and trna. in a, gst-fusion proteins expressed in e. coli were purified with glutathione sepharose b beads, eluted with gtutathione as described in materials and methods, and identified by sds-page in a % gel stained with coomassie blue. gst is gst protein expressed and purified as for the others; m represents low molecular weight range of prestained proteins (bio-rad) as used in fig. b . in b, ' core rna ( ~tg) was mixed with , , or gg trna ( to molar excess) and used in rna competition binding experiments with c , c - and c - (see fig. la ) attached to gtutathione sepharose beads. rna bound to the beads was recovered and analysed in nondenaturing agarose gels (see materials and methods). m indicates marker lanes containing +x rf dna digested with hae iii (fig. b) . however, gg trna ( molar excess) successfully competed out the binding. in salt block experiments, binding of both ' core and ' utr probes to c and c - occurred at mm nac but inhibition was observed at mm naci (results not shown). thus although the short peptides in fusion proteins c and c - bound the kun utr probes, their strength of binding was reduced relative to that of the longer peptides in c - and c . as a further test for binding capacity of c and c - , we repeated the binding experiments using homologous rna and trna as competitors in gel shift assays. we also prepared a new construct c - (fig. la) in lieu ofc - to ascertain whether the addition of two basic amino acids (total content of ) would produce binding of utrs unattainable with c - (total content of basic amino acid residues) in the binding assays on glutathione sepharose beads. c - migrated similarly to c - (fig. a) . c , c - , and c - were purified from e. coli lysates on glutathione sepharose beads and eluted off the beads in buffer containing mm glutathione (see materials and methods). conditions for binding of the fusion proteins to the s_labelled rna probes were selected after titration experiments using gel shift assays. increase in concentration of fusion proteins (from . to . gg) resulted in increases in mobility shift, and . to . gg was selected for further assays. both c - and c caused obvious gel shifts after incubation with ss-labelled ' core and ' utr probes, which were readily competed with to -fold molar excess of unlabelled homologous probes (fig. a, b) . in contrast, trna in -fold molar excess failed to completely eliminate the gel shifts observed in reactions of c - with both s-labelled ' core and ' utr probes, or in reactions ofc with the ' utr probe. a -fold molar excess of trna competed, albeit weakly, with gel shift of s-labelled ' core rna and c , and more than a -fold molar excess was required to eliminate this gel shift completely. thus these two non-overlapping regions of kun c enriched in basic ~min acids (c or amino-terminal, and c - or carboxy-terminat) individually bound kun ' core and ' utr which were competed by excess of unlabelled homologous probes but only partially by a large excess of trna, in both gel shift assays and binding reactions on sepharose beads. when gel shift assays with c - were attempted under the same conditions used for c and c - , the migration of the ' utr and ' core probes was unaffected (results not shown), agreeing with the lack of binding observed previously in the bead assay using c - (fig. b) . thus the addition of two more basic amino acids (an increase of content from % in c - to % in c - ) was insufficient to induce rna-protein binding, and the result therefore apparently excludes this region of c in rna binding activity. therefore it can be concluded that the binding observed with c , c and c .was associated with domain , and that the stronger binding observed with c - and c - was assodated with domain (see fig. la ). these two binding domains were thus localized to peptides of amino acid residues (in c ) and residues (in c - ). this is the first report of rna binding of the genus flavivirus core protein in vitro. fusion protein c bound strongly to the kun utr riboprobes in a relatively specific matter, being resistant to competition by a large excess of trna and to binding inhibition by . m nac . c contains the virtual equivalent of kun mature c of amino acids which is the detectable form of c found in virions and in infected cells [ , ] . within the core protein sequence, putative kun utr binding domains were shown to reside in the aminoterminal and carboxy-terminal sequences, exemplified by the binding reactions reporter ( nucleotides) or vector ( nucleotides) sequences [ ] . however, bmv cp bound specifically to full length rna and nonspecifically to vector rna, based on the observation that only the specific complex was resistant to dissociation by . to . m nac [ ] . specific binding to cp by the ' utr of rna- and rna- of alfalfa mosaic virus (both containing conserved stem loop structures) was reduced but not eliminated by addition of excess nonhomologous competitor rna in gel shift assays [ ] . binding to the nucleocapsid protein of puumala hantavirus by the small negative-stranded genomic s rna segment was strongly inhibited also in gel shift assays by to mass excess of trna [ ] , even though the viral rna and nucleocapsid protein bind specifically during virus assembly. rna binding domains within capsid proteins and involving enrichment in basic amino acids have been analysed for several positive-stranded rna viruses, including the bmv cp discussed above [ ] . the amino-terminal region (amino acids - , % enriched in lysine and arginine) of hepatitis c virus core protein bound to hcv genomic rna and hepatitis b virus rna in northwestern blots, and to ribsomal subunits during centrifugation [ ] . masters [ ] described binding of the lysine-arginine enriched central region of mouse hepatitis virus (mhv) nucleocapsid protein n to cellular rna during in vitro translation of n, detected by gel shift analyses. however, stohlman's group [ , ] also identified an rna-binding domain in the central region of mhv native n protein (between amino acids and ) by northwestern blotting, using p-labelled riboprobe containing the mhv ' utr or leader rna sequence which could not be competed by a large excess of total cellular rna. sindbis virus capsid protein c ( amino acids) bound to cellular rna during in vitro translation; subsequently this cellular rna which had bound to c was successfully competed in a gel migration assay by a nucleotide sindbis rna probe containing the nucleotide packaging signal in ongoing studies on encapsidation by s. schlesinger and colleagues [ ] . despite acknowledged considerable variation between experiments, the authors established by deletion of amino-terminal and carboxy-terminal sequences in sindbis c that the regions between amino acids - ( % enriched in lysine and arginine) were the most important for binding of a amino acid peptide a to the nucleotide packaging signal. similar results with another alphavirus (semliki forest) were reported by forsell et at. [ ] who showed by deletions in genomic rna transcripts that most of the basic amino acids ( % enriched) in the amino-terminal residues of the capsid protein were required for capsidation in vivo and infectivity, and concluded that most of the region is involved in both specific and nonspecific interactions with encapsidated rna. interestingly, it was shown that the sindbis fusion protein gst-a or fi'ee a caused an (incomplete) gel shift of a "highly structured" s-labelled nucleotide viral rna fragment (containing four stem loops predicted by computer folding) with the capsid binding activity [ ] . bacterial rna and trna at about -fold mass excess were effective competitors ( to % inhibition) only when the sindbis -mer rna probe was reduced to a -mer still able to bind to the active amino acid sequence in gst-a . thus nonspecific binding became more apparent when the -mer sindbis rna probe was reduced to nucleotides. in similar experiments, the short kun ' utr riboprobe of nucleotides was still relatively specific; it bound c and resisted inhibition by . m nac (fig. a) . in most cases trna appeared to be able to bind independently of the presence of the kun riboprobes, and vero cell trna was retained on c in greater proportion than s and s ribosomal rna, both many times larger than trna (fig. d) . clearly our results are analogous to those obtained with other virus coat or capsid proteins involving both specific and nonspecific rna binding. all flavivirus c proteins have a similar distribution of basic amino acids (see [ ] [ ] [ ] [ ] [ ] ) which may be arbitrarily grouped for kun virus as in fig. : domain comprising amino acids - ( basic residues, % enrichment), domain comprising amino acids - [ , %] and domain comprising amino acids - ( , %). c and all the shorter fusion proteins except c - and c - bound the kun utr riboprobes, thus excluding the arbitrary domain from further consideration. binding of the kun riboprobes to the isolated aminoterminal domain (in c ) and to the isolated carboxy-terminal domain (in c - ) was inhibited by high salt, but was strongly or partially resistant to competition by large excess of trna (figs. b, ). thus our results show that rna binding to kun core fusion proteins is easily demonstrable, and that relatively specific binding appears to occur between c , the virtual equivalent of mature core protein c, and the ' and ' utr regions of kun rna. the binding specificity appears to reside in domains and , probably acting in concert. while the criteria for binding of viral coat proteins to rna including viral utrs cannot as yet be precisely defined, several general principles are emerging. enrichment in basic amino acids obviously facilitates binding and helps localize the binding domains experimentally - , , , , , , ] . the binding domains may be relatively short if the arginine/lysine content is in the vicinity of % or greater e.g. the a peptide ( residues) of sindbis c [ ] , in the capsid protein of semliki forest virus [ ] , and the and residue peptides in c - and c , respectively, of kun c. not surprisingly, binding was stronger for longer peptides and in some cases for longer rna sequences ( [ , ] , the kun binding data). secondary structures within viral rna may also enhance specificity [ ] , especially the stem loop structures in utrs of flaviviruses - , , , , , ] and in utrs of several other positive-stranded viruses for which binding has been reported (see introduction). as noted above, the ordered structure of trna bound more strongly than the longer ribosomal rna to the kun core fusion protein c (fig. d) , but was able to partially compete in binding of the kun ' and ' utr probes with the short core fusion proteins c and c - only when in large excess (figs. b, ) . a further criterion for binding is resistance to inhibition by high salt, as observed in the present study and in other reports [ , , ] . it seems reasonable that some specific association must occur in infected cells similar to that observed in vitro with c and the kun utrs and that the stem loop structures in the kun ' and y utrs acting together in virion rna may contribute to the strength of binding to mature c. it is not possible from this initial study to conclude what role the observed binding reactions play in vivo. some additional binding may occur with longer (coding) regions of kun rna, but such reactions were beyond the scope of the present study, and will be the subject of future work. whether nonspecific rna interactions also play a role in flavivirus replication is uncertain. there have been suggestions that a lack of rna specificity may aid in virion assembly [ , , ] . further work on the role or extent of rna-protein binding in replication of flaviviruses is needed because of the dearth of information in this area. sequence and secondary structure analysis of the y-terminal region of flavivirus genome rna the '-nucteotides of flavivirus genomic rna from a conserved secondary structure flavivirus genome organization, expression, and replication the p movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein nucleotide and complete amino acid sequences of kunjin virus: definitive gene order and characteristics of the virus-specified proteins identification of domains in brome mosaic virus rna- and coat protein necessary for specific interaction and encapsidation structure-function of the nh -terminal domain of the semliki forest virus capsid protein deletion annysis of the cap sid protein of sindbis virus: identification of the binding region rna binding of recombinant nucleocapsid proteins of hantaviruses conserved elements in the ' untranslated region of flavivirus rnas and potential cyclization sequences morphogenesis of flaviviruses. in: harris jr (ed) subcellular biochemistry completion of kunjin virus rna sequence and recovery of an infectious rna transcribed from stably cloned full-length cdna localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus kunjin core protein-rna binding detection of stable secondary structure at the ' terminus of dengue virus type rna localization of the rna-binding domain of mouse hepatitis virus nucleocapsid protein analysis of the terminal sequences of west nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis cellular proteins bind to the ' end of sindbis virus minus-strand rna the '-untranslated region of alfalfa mosaic virus rna contains at least two independent binding sites for viral coat protein nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution biosynthesis and biochemical properties of the hepatitis c virus core protein carboxy-terminal analysis of nine proteins specified by the flavivirus kunjin: evidence that only the intracellular core protein is truncated specific interactions between coronavirus leader rna and nucleocapsid protein interactions between viral coat protein and a specific binding region on turnip crinkle virus rna characterization of an internal element in turnip crinkle virus rna involved in both coat protein binding and replication interactions between sindbis virus rnas and a amino acid derivative of the viral capsid protein further defines the capsid binding site analysis of structural properties which possibly are characteristic for the '-terminal sequence of the genome rna of flaviviruses replication of flaviviruses flavivirus replication strategy this work was supported by a grant from the national health and medical research council of australia. key: cord- -tu g q authors: cheung, andrew k.; ng, terry f.; lager, kelly m.; bayles, darrell o.; alt, david p.; delwart, eric l.; pogranichniy, roman m.; kehrli, marcus e. title: a divergent clade of circular single-stranded dna viruses from pig feces date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: tu g q using metagenomics and molecular cloning methods, we characterized five novel small, circular viral genomes from pig feces that are distantly related to chimpanzee and porcine stool-associated circular viruses, (chiscv and poscv ). phylogenetic analysis placed these viruses into a highly divergent clade of this rapidly growing new viral family. this new clade of viruses, provisionally named porcine stool-associated circular virus and (poscv and poscv ), encodes a stem–loop structure (presumably the origin of dna replication) in the small intergenic region and a replication initiator protein commonly found in other biological systems that replicate their genomes via the rolling–circle mechanism. furthermore, these viruses also exhibit three additional overlapping open reading frames in the large intergenic region between the capsid and replication initiator protein genes. coronaviruses and enteroviruses were detected in these samples that were subsequently submitted to national animal disease center for additional study. fecal samples from six pigs were pooled and processed to prepare a viral nucleic acid library. briefly, viral particles were first purified using size filtration and nucleases [ ] . the extracted viral nucleic acids were amplified by random pcr with a specific nucleotide sequence tag for identification. several libraries, each prepared with a different sequence tag for identification, were combined and subjected to pyrosequencing and analyzed as described previously [ ] . sequencing was performed on a roche flx sequencer using titanium chemistry (roche, branford, ct). for comparative purposes, the best blastx results were used to categorize the sequences (contigs and singletons) into virus family and genus. of the , , total keypass reads generated, , reads contained sequence tags belonging to the six pooled fecal samples. positive sequence reads for a taxonomic group were identified based on deduced protein sequence similarity using a stringent expectation value, best blastx expectation scores of b - , as the cutoff. sequence reads at this cutoff level exhibited highly significant protein sequence similarities with known viruses in the database. viral sequences (coronavirus, enterovirus, rotavirus) corresponding to the viruses identified by the indiana animal disease diagnostic laboratory (west lafayette, in) were detected. other viral sequences belonging to the rna virus families (astrovirus, picobirnavirus, teschovirus, torovirus and sapelovirus) and dna virus families (anellovirus, circovirus, and parvovirus) were also observed. several sequences encoding amino acid sequences related to rep of chiscv and poscv were identified. the chiscv-and poscv -related nucleotide sequences detected by deep sequencing (designated tp and tp ) were used to design primers for pcr. dna amplification employing converging primers (conventional pcr) was used to confirm the presence of contig sequences in the sample, and diverging primers (inverse pcr) were used to amplify and clone the complete circular viral genomes. nucleic acids were extracted directly from fecal samples using a qiaamp minelutevirus vacuum kit (qiagen, valencia, ca) and subjected to rolling-circle amplification to amplify circular dna molecules (illustra genomiphi v dna amplification kit, ge healthcare biosciences, piscataway, nj). the amplified dna was used as a template for pcr using converging or diverging primers based on pyrosequencing results. the amplicons were resolved and excised from agarose gels, cloned into plasmid topo-clx and introduced into eschericheria coli top (invitrogen, carlsbad, ca) by transformation. multiple clones were picked and used for sequence determination using sanger methods. from the tp pcr product, three clones were analyzed, and they all yielded identical sequences. this viral genome was designated porcine stool-associated circular virus (poscv ; gen-bank accession number kc ). from the tp pcr product, four variant genomes were obtained, and the individual genomes were designated poscv - l , - l , -lt and - l with genbank accession numbers kc , kc , kc and kc , respectively. similar to the genome organization of other scvs, the tp clones (poscv and all four poscv clones) were about . kb in length (fig. a) . the viral genomes can be divided into four regions: two large orfs with deduced amino acid sequences exhibiting homology to the rep and cap of chiscv, a lir that encodes multiple overlapping orfs, and an sir that contains a palindromic sequence capable of forming a stem-loop structure. the rep orf and cap orf are transcribed divergently from the lir and converge at the sir. in contrast to the lir of poscv , which encodes two small orfs (orf and orf ) in the same orientation as the cap gene, the lirs of poscv and poscv also contain an additional orf (orf ) in the reverse orientation as the cap gene. the four poscv genomes were aligned, and a schematic representation is shown in fig. a . the lir, cap region and portion of the rep region exhibited few to no nucleotide differences. genetic differences were concentrated around the stem-loop structure in the sir and the portion of the rep orf. the four genome regions (sir, rep-orf, cap-orf and lir) are described individually in greater detail below. sir: the sir sequences of poscv and poscv are shown in fig. b . whereas the rep orf of poscv - l overlaps the stem-loop structure, the other four poscv genomes do not. all five genomes contain a palindromic sequence in the sir that is capable of forming a stem-loop structure whose nucleotide sequence is well conserved. this stem-loop structure may be part of the origin of dna replication. among the poscv genomes, the sir sequences on the cap-gene side are more conserved, while sequences on the rep-gene side exhibit the greatest differences. rep orf: phylogenetic and pairwise identity analyses were conducted to determine the relationship of tp clones to other viruses. a phylogram was created based on the deduced amino acid sequences encoded by the rep gene (fig. c) . the amino acid sequences were aligned using mafft . [ ] with the e-ins-i alignment strategy and previously described parameters [ , ] . a maximum-likelihood tree was created using raxml based on the mafft alignment with previously described parameters [ , ] . the resulting tree was midpoint rooted using mega [ ] . pairwise identity analysis of the poscv genes and orfs was also performed using mega [ ] . the results showed that the tp clones were most closely related to chiscv or poscv , and they clustered into a distinct clade with poscv and poscv , separated into two different sub-groups. there is limited amino acid sequence identity ( - %) between the tp clones and bovine scv (boscv) [ ] or pigscv. the amino acid sequence identities between tp:pos-cv and tp:chiscv were approximately % and %, respectively (table a) . the nucleotide or amino acid sequence identity between poscv and poscv was approximately %, and the sequence identity among the poscv variants was - %. in addition, rolling-circle replication (rcr) amino acid sequence motifs (rcr-i, rcr-ii, rcr-iii, walker a and walker b) commonly found among the rep proteins involved in rcr were detected [ ] (fig. b) . these motifs were conserved among members of this new clade. cap orf: the deduced cap protein sequences of selected scv were compared (table b) . there is limited amino acid sequence homology ( - %) between tp clones and boscv, chiscv, pigscv or poscv . the nucleotide sequence identity of the cap gene ( - %) was lower than the amino acid sequence identity ( - %) between poscv and poscv . in general, the rep gene is more conserved than the cap gene across the ssdna viruses. therefore, it was unusual to find that the nucleotide and amino acid sequence identities among the poscv cap genes ( - %) were higher than those of the rep genes ( - %). lir: the lir nucleotide sequence identity between poscv and poscv was . %, and the sequences of credence to the speculation that either orf may code for an important functional domain or protein. the lirs of poscv and poscv also code for an additional orf that is transcribed in the opposite orientation to the cap gene and overlaps orf and orf . the amino acid sequence identity between poscv and poscv was approximately %, and the sequence identity among the poscv variants was - % (table c) . thus, the amino acid sequence identity of orf between poscv and poscv was almost as high as that of the capsid protein identity of %. in this work, we report a clade of novel viruses that includes poscv and poscv , which encode a rep-like protein and a palindromic sequence capable of forming a stem-loop structure (in the sir), suggesting that their genomes may replicate via a common rcr mechanism. interestingly, this clade of viruses encodes three overlapping ''conserved'' orfs (orf , orf and orf ) in the lir. whereas the amino acid sequence identities between poscv and poscv for these orfs range from . % to . %, the amino acid sequence identities among the capsid proteins range from . % to . %. whether these additional orfs code for functionally important proteins is not known. likewise, the role of these viruses in any disease is unknown. the growing diversity of scv-related genomes currently reported in the stool of chimpanzees, cows, and pigs likely portend further identification in other mammalian species. however, it remains to be seen whether these stool-associated viruses replicate in the host or that they are pass-through viruses present in the diet. confirmation of their host and organ tropisms will require detection of scv-specific antibodies or finding virions in animal tissues. a high level of co-infections involving numerous known viruses (coronavirus, orf tp l l l orf tp l l l orf tp l l l tp ----tp ----tp --- enterovirus, rotavirus, astrovirus, picobirnavirus, teschovirus, torovirus, sapelovirus, anellovirus, circovirus and parvovirus) was detected in just six animals from this study. this report, and the work of others, demonstrates the growing complexity of the pig virome and the challenge to understand the biology, interactions and significance of these newly discovered viruses. novel circular dna viruses in stool samples of wild-living chimpanzees mafft version : improvement in accuracy of multiple sequence alignment identification of a novel single-stranded, circular dna virus from bovine stool diversity of viruses detected by deep sequencing in pigs from a common background metagenomic identification of a novel anellovirus in pacific harbor seal (phoca vitulina richardsii) lung samples and its detection in samples from multiple years high variety of known and new rna and dna viruses of diverse origins in untreated sewage simultaneous identification of dna and rna viruses present in pig faeces using process-controlled deep sequencing the fecal virome of pigs on a high-density farm discovery of a novel circular single-stranded dna virus from porcine faeces a rapid bootstrap algorithm for the ra ml web servers mega : molecular evolutionary genetics analysis (mega) software version . acknowledgments the authors thank n. otis, l. hobbs, d. michael and m. woodruff for technical assistance and s. ohlendorf for manuscript preparation. t.f.n. and e.l.d. were supported by r hl . key: cord- - guojyay authors: park, seong-jun; kim, hye-kwon; song, dae-sub; moon, hyoung-joon; park, bong-kyun title: molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: guojyay porcine epidemic diarrhea virus (pedv) has caused enteric disease with devastating impact since the first identification of pedv in in korea. in this study, we investigated molecular epidemiology, showed genetic diversity, and analyzed phylogenetic relationships of korean pedv field isolates with other pedv reference strains. genetic analysis of the complete m and orf genes showed that each pedv group had several unique characteristics, and this indicated that specific groups of pedvs may be differentiated from the other pedvs by specific nucleotide differences. especially, orf gene analysis can be used for discrimination between vaccine and wild-type pedvs. sequence and phylogenetic analysis showed that recent, prevalent korean pedv field isolates have close relationships to chinese field strains and differ genetically from european strains and vaccine strains used in korea. these results raise questions as to whether a new type of pedv vaccine may be necessary for preventing pedv infection more effectively in korea. porcine epidemic diarrhea virus (pedv), a member of the family coronaviridae, subfamily coronavirinae, genus alphacoronavirus, is an enveloped, single-stranded rna virus. pedv was first reported in belgium and the united kingdom in [ ] . since the first identification of pedv, outbreaks of pedv infections have been reported in many swine-producing countries, notably in europe and asia [ ] . porcine epidemic diarrhea (ped), caused by pedv, is an acute, highly contagious, and devastating enteric disease that is characterized by severe diarrhea, dehydration and significant mortality in swine, resulting in severe economic losses in the european and asian swine industry [ ] . coronaviruses have a genome organization with a common set of five genes arranged in a conserved order [ ] . the polymerase gene, occupying % of the genome, encodes the replicase polyproteins. the genes for structural proteins s, e, m, and n are located downstream of the polymerase gene [ ] . in addition, a variety of genes encoding accessory proteins whose number and sequence vary among different coronaviruses are studded between the structural genes, and are called accessory genes [ ] . one of the four structural proteins, the m protein, a structural membrane glycoprotein and the most abundant envelope component, is a triple-spanning membrane protein with a short amino-terminal domain on the exterior of the virion and a long carboxy-terminal domain on the inside [ ] . in addition to playing an important role in the viral assembly process [ , ] , the m protein induces antibodies that neutralize virus in the presence of complement [ , ] . the m protein has also been proposed to play a role in a-interferon (a-ifn) induction [ ] . it has been demonstrated that coexpression of m and e proteins allows the formation of pseudoparticles, which exhibit an interferogenic activity similar to that of complete virions [ ] . unlike the structural proteins, in most cases, little is known about the functions of the accessory proteins, and in general they are not required for virus replication in cultured cells [ , , , ] . quite the opposite, their expression might lead to a decrease in viral fitness in vitro, and mutants with inactivated accessory genes are easily selected during serial passage in cell cultures [ , , , ] . in field strains, however, accessory genes are generally maintained [ , ] , and their loss mainly results in attenuation in the natural host [ , , ] . especially, in the case of pedv, the orf gene is the only accessory gene, and it has been suggested to be an important determinant for virulence of this virus. virulence of pedv can be reduced by altering the orf gene through cell culture adaptation [ ] in a manner similar to tgev [ ] , and differentiation of orf genes between the highly cell-adapted viruses and field viruses could be a marker of adaptation to cell culture and attenuation of virus [ , ] . in addition, differentiation of the orf gene could be a valuable tool for molecular epidemiology studies of pedv [ , , ] . in korea, pedv was first isolated in [ ] . it has been detected frequently in many provinces and has become one of the most important viral enteric diseases. in spite of using the vaccine strategy at present, damage caused by pedv infection is continuous and serious in korea. to better control and prevent pedv infection, it is necessary for us to investigate the molecular epidemiology of pedv field isolates in korea. in this study, therefore, we investigate the molecular epidemiology and genetic diversity of korean pedv field isolates and analyze phylogenetic relationships of the korean pedv field isolates to other pedv reference strains reported previously. the present study focused on the m and orf genes because of their characteristics described above. porcine intestinal and fecal samples were collected between october and june from swine farms in six provinces of korea. all pigs from the farms showed signs of watery diarrhea and dehydration at the time of sample collection. fresh samples were collected from individual pigs, placed into a sterile specimen container, and submitted to the department of veterinary medicine virology laboratory, college of veterinary medicine, seoul national university. these intestinal and fecal samples were confirmed to be positive for pedv by reverse transcription-polymerase chain reaction (rt-pcr) [ ] . the attenuated dr strain and its parent strain were obtained from our laboratory [ ] . the attenuated dr strain was derived from the parent strain by serial passages in vero cells and was used for manufacture of the korean ped oral vaccine by the green cross veterinary product co., ltd. (yongin, korea). the kped- strain, used for manufacture of the korean live pedv vaccine, was kindly provided by the green cross veterinary product co., ltd. (yongin, korea), and the p- v strain for manufacture of the japanese live pedv vaccine was provided by the nisseiken regional distributor in korea. pedv-positive fecal samples were prepared as % (v/v) fecal suspensions in phosphate-buffered saline (pbs; . m, ph . ), and positive intestinal contents were also prepared as % (v/v) intestinal suspensions in pbs by homogenization. the sample suspensions were vortexed and centrifuged for min at x g. rna was extracted from a -ll starting volume of the centrifuged % sample suspensions using trizol ls (invitrogen corp., carlsbad, ca, usa) according to the manufacturer's instructions. viral rnas were extracted from attenuated dr , kped- and p- v strains as described above. reverse transcription (rt) was carried out using random hexamer primers (takara bio inc., otsu, japan), and the cdna was immediately used for amplification or stored at - °c. primers were designed based on the published sequences of the e and n genes to cover the complete m gene of pedv. the primers were pedm (forward), '-gtcttacatg cgaattgacc- ', and pedm (reverse), '-ggcata gagagataatggca- '. the size of the amplified product was predicted to be bp. pcr was carried out using a commercial amplification system (perkin-elmer, applied biosystems, foster city, ca) as described previously [ ] with simple modifications. the pcr was performed at °c for min, followed by cycles of °c s, °c s, °c s, and a final extension at °c for min, and the sample was then held at °c. the primer pair orf - /orf - [ ] , targeting the s/e regions (corresponding to nt , - , of cv ; bp) of pedv, was used for generating the complete orf gene of pedv, and pcr was performed using the protocol described previously [ ] . the rt-pcr products for each m and orf gene were analyzed by . % agarose gel electrophoresis and visualized by ultraviolet illumination after ethidium bromide staining. bands of the correct size were excised and purified using a qiaquick gel extraction kit (qiagen gmbh, hiden, germany) according to the manufacturer's instructions. the purified rt-pcr products corresponding to the full-length m and orf genes of pedv were cloned into pdrive cloning vector (qiagen gmbh, hiden, germany) as described previously [ ] , and the cloned plasmids were purified using a qiaprep Ò spin miniprep kit (qiagen gmbh, hiden, germany) before sequencing. sequencing of plasmid dna was carried out at least twice in both directions at the genotech institute (genotech co., ltd., korea) using t and sp primers and an automated dna sequencer (abi system , applied biosystems inc., foster city, usa). the nucleotide sequences of the full-length m and orf genes of korean pedv field isolates were aligned using clustalx version . [ ] , edited using bioedit version . . (http://www.mbio.ncsu.edu/bioedit/bioedit.html), and compared with those of pedv reference strains in the genbank database as well as in previous papers. sequence similarity analysis was performed for the aligned nucleotide and amino acid sequences using megalign software (dnastar inc., madison, wi, usa). phylogenetic analysis of the korean pedv field isolates with other pedv reference strains based on the nucleotide alignments was done by the neighbor-joining method and the minimum-evolution method of molecular evolutionary genetics analysis (mega version . ) using pairwise distances [ ] . the korean pedv field isolates and the other pedv reference strains used for sequence alignment, sequence analysis and phylogenetic analysis are indicated in the figure legends. phylogenetic analysis based on a complete m gene fragment of korean pedv field isolates, together with other pedv reference strains, confirmed that all pedvs, including korean field isolates, fell into three groups, and all pedvs isolated from korea belonged to group ( fig. ). more precisely, as shown in fig they did not have nucleotide deletions or insertions but did have point mutations. these isolates had a conserved ataaac sequence at nucleotides upstream of the initiator atg, as previously recognized in br / [ ] . sequence analysis of the complete m genes showed that all pedvs, including the korean field isolates, fell into three groups, and group had four subgroups ( - , - , - , and - ), as can be seen in the phylogenetic tree ( fig. ) . each group had unique differences in its sequences. group had seven specific nucleotide changes (g?c at , t?c at , c?t at , a?g at , a?c at , g?a at , and a?c at ) that were not found in the other groups, and two of the seven changes lead to amino acid changes (v?l at and g?s at ). group had one specific nucleotide change (c?t at ). the specific differences between group and other groups are as follows: subgroup - exhibited one specific nucleotide change (t?a at ). in particular, in the case of subgroups - , - and - , which include all of the korean field isolates, subgroups - and - shared one specific nucleotide change (c?t at ), and subgroups - and - shared two specific nucleotide changes (t?c at , and t?a at ). subgroup - had one specific nucleotide change (c?a at ). these results indicated that specific groups of pedvs may be differentiated from all other pedvs, including korean field isolates, by specific nucleotide differences, although more pedvs need to be analyzed for more accurate analysis. sequence homology analysis of the m gene sequence homology results based on the complete m gene of all pedvs, including the korean pedv field isolates, phylogenetic analysis of the orf gene phylogenetic analysis based on a complete orf gene fragment of korean pedv field isolates, together with other pedv reference strains, confirmed that all pedvs, including korean field isolates, fell into three groups (fig. ) . one group comprised the cv , br / , and lzc strains. the second group consisted of vaccine strains (the attenuated strains dr , kped- , p- v and cv vs), the ch/gsjiii/ strain, and the dbi korean field isolate. the third group was made up of korean field isolates, chinju , the parent strain dr , and chinese strains. the third group had two subgroups ( - and - ). nine korean field isolates from and ten chinese strains formed one subgroup ( - ), and korean field isolates from - formed a second subgroup together with chinese strain ch/s, korean strain chinju , and the parent strain dr . sequences of complete orf genes of korean pedv field isolates were determined and compared to those of other pedv reference strains. all korean pedv field isolates except dbi had a single orf of nucleotides encoding a protein of amino acids, with a predicted mr of . - . kda. on the other hand, dbi had a single orf of nucleotides encoding a protein of amino acids, with predicted mr of . kda because of a -nucleotide deletion at position - . all korean pedv field isolates, including dbi , had a conserved sequence (ctagac) at nucleotides upstream of the initiator atg, similar to that described above for the m gene. sequence analysis of the complete orf genes showed that all pedvs, including the korean field isolates, fell into three groups, and group had two subgroups ( - and - ), as can be seen in the phylogenetic tree (fig. ) . each group had differences in its sequences. group had eight specific nucleotide changes (c?t at , a?g at , a?g at , t?c at , a?g at , c?t at , t?c at and g?a at ) that were not found in the other groups, and four of the eight changes led to amino acid changes (a?v at , i?v at , t?a at and s?n at ). group showed one specific nucleotide change (c?a at ) and had two classes of large nucleotide deletions at position - , which are predicted to produce truncated proteins. the specific differences between group (containing all korean field isolates except dbi ) and the other groups are as follows: group had three specific nucleotide changes (t?g at , c?t at and c?t at ), and two of three changes led to amino acid changes (f?v at and l?f at ). in particular, subgroup - , which includes nine korean field isolates from and ten chinese strains, was differentiated from subgroup - by one specific nucleotide change (c?t at ). these results showed that these specific nucleotide differences may be used to differentiated specific groups of pedvs from other pedvs, including korean field isolates. however, more pedvs need to be analyzed. in addition, complete orf gene analysis can be used for differentiation between vaccine-and wild-type pedvs because all vaccine-type pedvs had a large nucleotide deletion in the orf gene. sequence homology analysis of the orf gene sequence homology results based on the complete orf gene of all pedvs, including korean pedv field isolates, in this study, the complete m and orf genes of korean pedv field isolates were amplified by rt-pcr, cloned and sequenced to investigate their molecular and epidemiological characteristics and their genetic diversity. in addition, phylogenetic relationships between korean pedv field isolates and other pedv previously reported reference strains were also analyzed. all korean pedv field isolates (excluding dbi ) have only point mutations in the m and orf genes, and all of them, including dbi , have ataaac and ctagac sequences at and nucleotides upstream of the initiator atg of the m and orf genes, as recognized previously [ ] . these sequences are hexameric motifs that are common to coronaviruses and are similar to the hexameric motifs xua(a/g)ac found adjacent to other pedv orfs. these hexameric motifs have been proposed to be a start site for the transcription of subgenomic mrnas [ ] . the dbi isolate has a -nucleotide deletion at position - of the orf gene, resulting in a truncated protein of amino acids in size. this size of this deletion is exactly the same as the one present in the vaccine strain cv and the chinese field strain ch/gsjiii/ [ ] . genetic analysis based on complete m and orf genes showed that each pedv group had several unique characteristics, and these results indicated that specific groups of pedvs may be differentiated from other pedvs, including korean field isolates, by specific nucleotide differences, but more pedvs need to be analyzed for more accurate analysis. especially, complete orf gene analysis can be used for discrimination between vaccine and wild-type pedvs because only vaccine-type pedvs had large deletions in the orf gene. according to sequence and phylogenetic analysis using the complete m gene, all pedvs isolated in korea are ped vaccines are commonly used in korea as losses caused by pedv infection increase. however, most of the korean pedv field isolates analyzed in this study differ from members of the group that includes the vaccine strains, and only a few isolates belonged to that group. these results may be attributable to immune pressure due to widespread vaccine use in korea. in addition, this reflects the existence of genetic diversity among the korean pedv field isolates and raises questions as to whether a new type of pedv vaccine may be necessary for more effective prevention of pedv infection. the dbi isolate has high sequence identity and a close phylogenetic relationship to the vaccine strains according to genetic and phylogenetic analysis of the complete orf gene, and this result implies that it might be derived from a vaccine strain. although the mechanism by which the dbi isolate emerged is unclear, two possibilities are suggested: ( ) the dbi isolate might have evolved from a vaccine strain; ( ) dbi isolate might have been produced in nature through recombination between a pedv field isolate and a vaccine strain. of these two possibilities, the latter is more likely because the partial s and orf genes of the dbi isolate are closely related to those of g - (which is genetically different from the vaccine strains) [ ] and group (which are genetically similar to the vaccine strains). this is similar to what was shown for other korean field isolates used in this study. in the case of the e and m isolates, partial s [ ] and orf genes were different from those of the vaccine strains, and the m genes were similar to those of the vaccine strains. the partial s gene of the e isolate differs from that of the vaccine strains [ ] , and the m gene is closely related to those of the vaccine strains. however, despite trying several times to amplify the complete m gene of the dbi isolate, we did not obtain any amplicons from this isolate, and for that reason, further studies, such as full-length sequencing as well as complete m gene sequencing of the dbi isolate, are needed to determine how the dbi isolate originated. the present study allows a better understanding of the molecular epidemiology, genetic diversity, and phylogenetic relationships of korean pedv field isolates with other pedv reference strains. we expect that the results of this study will help to prevent and control pedv infection more effectively. coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes molecular epidemiology of porcine epidemic diarrhea virus in china table nucleotide and deduced amino acid sequence similarity based on the full-length orf genes of the korean pedv field isolates and those upper and lower triangles indicate percent nucleotide (nt) and amino acid (aa) sequence similarity, respectively a european strains (cv and br / ) and chinese lzc strain b vaccine strains (attenuated strains dr , kped- d chinese strains ch/s, korean strains (chinju and parent dr ) and korean field isolates (bi , bi , bi , m , e , m , m , m , v , mf , bif , vf , cpf , cpf and pff genetic characteristics of porcine epidemic diarrhea virus isolated in korea heterologous gene expression from transmissible gastroenteritis virus replicon particles coronavirus particle assembly: primary structure requirements of the membrane protein the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses human coronavirus e encodes a single orf protein between the spike and the envelope genes sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf live, attenuated coronavirus vaccines through the directed deletion of groupspecific genes provide protection against feline infectious peritonitis the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes isolation of porcine epidemic diarrhea virus (pedv) in korea coronavirus: organization, replication and expression of gene single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus luxury at a cost? recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro coronavirus accessory proteins protein interactions during coronavirus assembly transmissible gastroenteritis coronavirus gene is not essential but influences in vivo virus replication and virulence cloning and further sequence analysis of the orf gene of wild-and attenuated-type porcine epidemic diarrhea viruses sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea the coronavirus membrane protein coronavirus immunogens murine coronavirus nonstructural protein ns is not essential for virus replication in transformed cells differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf mega : molecular evolutionary genetics analysis (mega) software version . the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools identification of proteins specified by porcine epidemic diarrhoea virus efficacy of a transmissible gastroenteritis coronavirus with an altered orf- gene in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the a open reading frame is not essential for replication severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice acknowledgments this work was supported by a grant (code # ) from biogreen program, rural development administration, republic of korea. key: cord- - lq r authors: hsu, tien-huan; liu, hao-ping; chin, chieh-yu; wang, chinling; zhu, wan-zhen; wu, bing-lin; chang, yu-chung title: detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in taiwan date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: lq r porcine deltacoronavirus (pdcov) was initially documented in hong kong and later in the united states, south korea, and thailand. to investigate if pdcov is also present in taiwan, three swine coronaviruses—pdcov, porcine epidemic diarrhea virus (pedv), and transmissible gastroenteritis coronavirus (tgev)—were tested using real-time reverse transcription polymerase chain reaction (rrt-pcr) in rectal swab samples from piglets exhibiting diarrhea between january and may on pig farms in taiwan. the rrt-pcr results were positive for pdcov ( / , . %), pedv ( / , . %), tgev ( / , . %), and coinfections ( / , . %). after cloning and sequencing, pdcov nucleocapsid genes were analyzed. phylogeny results indicated that the nucleotide sequences of all isolates were like those reported in other countries. to further trace pdcov in the period of to , an enzyme-linked immunosorbent assay (elisa) was used to detect antibodies against pdcov. the results showed that of , ( . %) sera were positive for the pdcov nucleocapsid protein, implying that pdcov might have existed in taiwan before . porcine deltacoronavirus (pdcov) is an enveloped, positive-sense single-stranded rna virus that belongs to the family coronaviridae. pdcov was first discovered in hong kong, china in [ ] . it was subsequently reported in the united states [ ] [ ] [ ] and south korea [ ] in , followed by thailand and mainland china in [ , ] . currently, there are at least three members of the family coronaviridae that can cause diarrhea in pigs: transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine deltacoronavirus (pdcov) [ ] . both tgev and pedv belong to the genus alphacoronavirus, whereas pdcov belongs to the new genus deltacoronavirus. pedv is an important enteric pathogen that causes piglet diarrhea worldwide, and it has caused significant economic losses in the swine industry in taiwan from to [ ] . however, pdcov and pedv share a similar clinical manifestation, and many studies have shown that coinfection with pedv and pdcov is common in piglets [ ] [ ] [ ] . thus, the purpose of this study was to identify the viruses responsible for causing diarrhea in piglets and to specifically investigate the prevalence of pdcov infection in taiwan. in this study, rectal swabs of piglets that suffered from diarrhea from pig farms located in central and southern taiwan were collected between january and may . all rectal swabs were transported in phosphate-buffered saline with % glycerol. the total nucleic acid content was then extracted from the rectal swabs using a labprep™ dna/rna mini kit (taigen biotechnology, taiwan). the isolated nucleic acid samples were tested for the presence of swine enteric coronaviruses-tgev, pedv, and pdcovusing the idexx™ realpcr ® test kits. pdcov-positive samples were further examined by traditional reverse transcription polymerase chain reaction (rt-pcr) with primers pdcov-n ( '-acc atc gct cca agt cat tcttg- ') and pdcov-n ( '-gag tgg agt tgg gtg ggt tta reverse-transcription reaction was performed at °c for minutes, and then a standard polymerase chain reaction was performed: cycles of °c ( seconds), °c ( seconds), and °c ( minute). after electrophoresis and gel elution, amplified products were cloned and sequenced. the nucleotide sequence data were analyzed using chromas lite, and the deduced amino acid sequences of the open reading frames were compared to other pdcov sequences using blast. the sequences of three complete pdcov nucleocapsid (pdcov-n) genes, taiwan , taiwan , and taiwan , were obtained and deposited in genbank (accession numbers ky to ky ). multiple sequence alignment and phylogenetic tree construction were then performed using the mega program [ ] . a pdcov-n clone (taiwan ) was further subcloned into the protein expression vector pet- a(+) using primers pdcov-n-f ( '-acc gga tcc atg gct gca cca gta gtccc- ') and pdcov-n-r ( '-cac aag ctt cta cgc tgc tga ttc ctgct- '). the pdcov-n protein was then expressed by adding isopropyl-β-d-thiogalactoside (iptg) (final concentration . mm) to a bacterial culture and was purified using immobilized metal affinity chromatography for a subsequent enzyme-linked immunosorbent assay (elisa). a total of , serum samples collected from pig farms in taiwan during to were tested, and specific-pathogen-free (spf) pig sera were obtained from the animal technology institute taiwan to be used negative controls. the elisa was conducted following the modified procedures described in a previous study [ ] . in brief, -well microtiter plates were coated with purified his-tagged pdcov-n ( ng/well) in mm carbonate buffer (ph . ) at °c for - hours and blocked with blocking buffer ( % bovine serum albumin in tris-buffered saline (tbs): mm tris-cl, ph . , mm nacl) at room temperature for hour. after washing three times with tbst (tbs containing . % tween- ), μl of the tested sera ( : dilution in blocking buffer) were added to the wells and incubated at room temperature for hour. after incubation, the plate was washed three times with μl of tbst and incubated at room temperature for hour with μl of goat α-porcine ig antibody conjugated with horseradish peroxidase at a concentration of : dilution in blocking buffer. after washing, μl of , ', , '-tetramethylbenzidine (tmb) substrate solution was added, and the plate was incubated at room temperature for minutes. the development reaction was stopped by adding μl of m h so , and the absorbance at nm wavelength was measured. all tests included a blank coated with antigen only, a second antibody control, and six spf sera as negative controls. if the detection value was lower than (mean neg + × sd), the serum was considered negative. on the other hand, a serum detection value larger than [(mean neg + sd) × ] was considered positive. of these tested samples, were positive for pdcov ( . %), were positive for pedv ( . %), and only two were positive for tgev ( . %). regarding coinfection rates, only one of the specimens ( . %) was positive for all three coronaviruses, one was positive for pedv and tgev ( . %), and of them ( . %) were positive for pdcov and pedv. based on the real-time rt-pcr (rrt-pcr) detection results, the percentage of pig farms that were positive for at least of one of the coronaviruses was % for pdcov ( / ), . % for pedv ( / ), and . % for tgev ( / ). we compared the sensitivity of the idexx rrt-pcr kit and the traditional rt-pcr used for cloning the n protein of pdcov in this study. all pdcov-positive samples were re-examined by conventional rt-pcr, which showed that only eight ( / , . %) were positive, accounting for . % ( / ) of total tested samples. the relatively low positive rate determined by conventional rt-pcr was comparable to that determined by a similar method in a recent study, in which pdcov was detected exclusively in nursing piglets, with an overall prevalence of approximate . % ( / ) in southern china [ ] . it is plausible that for the specimens containing pdcov genomic rna of low quantity or quality, most of the commercial rrt-pcr kits, which are designed to amplify short target sequences, can achieve higher sensitivity in detection compared with conventional rt-pcr. in addition, four of the eight positive samples were cloned and sequenced, and the sequence data matched the pdcov-n reference sequences. all of these four sequences covered the complete coding sequences of pdcov-n, but one of them, taiwan , had a nonsense mutation within the pdcov-n gene (data not shown). such a mutation might arise from an occasional change in rna sequence occurring during serial propagation of pdcov [ ] . despite the mutation, the result revealed the presence of the pdcov genome in the specimen in which taiwan was cloned, while it remained to be clarified if mutations occurred in other pdcov-encoded genes as well. phylogeny analysis of pdcov-n genes showed that pdcovs found in taiwan were highly similar in their nucleotide sequences to isolates from the united states, mainland china, and other countries (fig. ) . however, the aminoacid-based phylogeny results of the pdcov-n proteins revealed that taiwan isolates can be clustered into different groups (fig. ) . this might be due to missense mutations in the th (f → y), th (e → k), th (g → r/t), th (f → s), and th (g → e/d) amino acid residues of the pdcov-n protein (fig. ) . the charges of amino acids were changed in three residues ( th , th , and th ), and the remaining residue changes had altered polarity. the phylogeny results imply that pdcovs isolated in taiwan might have existed for a long time. to determine if pdcov had already existed in taiwan before , the pdcov-n protein was cloned, expressed, purified, and coated onto a -well microtiter plates for retrospective testing. swine sera collected between and were tested for their reactivity to a pdcov-n protein. the elisa results showed that of , ( . %) sera were able to react with the pdcov-n protein when [detected value > (mean neg +sd) × ] was used as positive threshold, but only of , ( . %) sera were positive when [detected value > (mean neg +sd) × ] was set as the threshold. the data indicated that pdcov has existed in taiwan since . similar elisa tests were also used to investigate the presence of pdcov in other studies [ , , , ] . based on our findings, we confirm that infection of pdcov and its coinfection with pedv in pigs have existed in taiwan between and . it is known that antibodies against pedv, tgev, or pdcov provide no crossprotection against either of the other two coronaviruses [ , [ ] [ ] [ ] [ ] . coinfection with pedv and pdcov might explain why some efforts have been ineffective in the pedv vaccination program. therefore, a divalent vaccine to control pdcov and pedv is desperately needed. 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 rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus detection and genetic characterization of deltacoronavirus in pigs porcine coronavirus hku detected in us states complete genome characterization of korean porcine deltacoronavirus strain kor/knu - porcine deltacoronavirus in mainland china pathogenecity of porcine deltacoronavirus strains in gnotobiotic pigs us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan mega : molecular evolutionary genetics analysis version . for bigger datasets a recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus occurrence and sequence analysis of porcine deltacoronaviruses in southern china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies retrospective testing and case series study of porcine delta coronavirus in us swine herds antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains two-way antigenic crossreactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications funding this study was supported by grants as- . . -bq-b and as- . . -bq-b from the bureau of animal and plant health inspection and quarantine (baphiq), council of agriculture in taiwan. tien-huan hsu has received grants from the bureau of animal and plant health inspection and quarantine. all authors declare no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. rectal swabs were collected from clinically diarrheic piglets in pig farms in taiwan, while swine sera were obtained from the animal disease control centers of various counties and the animal technology institute taiwan. key: cord- -kyavg vu authors: masters, p. s. title: localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: kyavg vu the interaction between the nucleocapsid (n) protein of mouse hepatitis virus (mhv) and rna was studied in an effort to define portions of the n molecule that participate in binding to rna. n mrnas transcribed from sp and t vectors were translated in a rabbit reticulocyte lysate. analysis of synthesized n protein in a nondenaturing gel system showed that it bound in vitro to an endogenous rna in the reticulocyte lysate but not to its own mrna. a set of deletion mutants was constructed in order to localize the rna-binding activity of the n protein. it was found that removal of as much as amino-terminal or carboxy-terminal amino acids from the molecule had little or no effect on rna binding. moreover, deletion mutants lacking both termini still retained rna-binding ability. by contrast, internal deletions or truncations extending beyond these two limits effectively abolished rna binding by n protein. thus, the rna-binding region of n has been mapped to the second (central) of the three structural domains of the molecule. coronaviruses constitute a family of viruses whose single-stranded, positivesense rna genomes have the largest coding capacities among the rna viruses [ , ] . paradoxically, coronaviruses have relatively few structural proteins. their large, roughly spherical virions are enclosed by a membrane envelope containing two (or, in some cases, three) types of membrane-bound glycoproteins. internal to this, multiple monomers of a nucleocapsid protein (n) encapsidate the viral genome, which, in the case of the prototype mouse hepatitis virus (mhv), is some , nucleotides in length [ , ] . the nucleocapsids of coronaviruses are helically symmetric [ ] , a feature seen in only one other group of positive-stranded rna viruses, those of the proposed family toro-viridae [ ] . consequently, these structures pose interesting problems with respect to genome translation as well as in understanding the processes of encapsidation and assembly. elucidation of coronavirus nucleocapsid structure and function will require description of the multiple interactions in which the n protein must participate: (i) the binding of n protein along the entire length of the rna genome; (ii) contacts between adjacent n monomers on the r n a and between n monomers that neighbor each other per helical turn; and (iii) association between n protein and the membrane glycoprotein (m), which may stabilize virion assembly [ ] . for at least some of these, it may be possible to map regions of the n molecule that are involved in a particular interaction. the work presented here focusses on the first type of interaction: the ability of n to bind to rna. coronavirus n proteins are markedly basic, and they form complexes with rna that tend to dissociate in high concentrations of salt and provide only limited protection against the action of ribonucleases [ , ] . in these respects, they resemble the np proteins of the orthomyxoviruses [ , ] . by contrast, the n -r n a complexes of the rhabdoviruses and the paramyxoviruses, which also form helically symmetric nucteocapsids, are stable in high salt and are largely resistant to ribonuclease [ , ] . few details are known about the association between n protein and rna in the coronaviral nucleocapsid. we have previously compared the amino acid sequences of the n proteins of five strains of mhv and, on the basis of this analysis, proposed a three domain structural model for the m h v n protein [ ] . in this paper it is shown that the central of these three domains is responsible for the rna-binding ability of the mhv n molecule. transcription vectors linking the n gene of mhv to the rna polymerase promoter of either bacteriophage t or bacteriophage sp were constructed by standard recombinant dna techniques [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a cdna clone, pal , containing a nearly full-length copy of the n mrna of mhv-a [ ] (embl/genbank accession no. m ) was restricted with snabi and saci, made blunt-ended, and inserted in either orientation into the vector pgem zf ( -) (promega), which had been opened and blunt-ended at the kpni and sphi sites of the polylinker. the resulting constructs, pa and pa , contained the t or sp promoter, respectively, linked to nt of the nt ' untranslated region (utr) of rna of mhv-a i- , ] , followed by the entire n protein coding region and nt of the nt ' utr ( fig. ) . hindiii-linearized pa encoded run-off transcripts that were bounded by polylinker sequences of nt and nt at their ' and ' ends; saci-linearized pa encoded run-off transcripts that were bounded by polylinker sequences of nt and nt at their ' and ' ends ( fig. ). full-length n mrnas transcribed from these two plasmids had identical translation efficiencies in vitro. truncated n mrnas were transcribed from pa that had been linearized with bsmi, scai or spei and from pa that had been linearized with acci or ecori (fig. ). an internal deletion mutant of the n gene was constructed by restriction of pa with [ ] . the n mrna initiation codon is underlined nhei and spei ( fig. ) , followed by religation of these compatible sites in the vector fragment. the resulting plasmid, pa , lacked nt - of the n gene and encoded the mutant protein n/nhe-spe. a second internal deletion mutant was generated by cleavage of pa with apai and nhei (fig. kpni site had been inserted immediately preceding the start codon. details of the construction of pa will be described elsewhere. the kpni(blunted)-hindlii fragment of pa was exchanged for the stui-hindlii fragment of the mutant vsv n sp transcription vector pn [ ] . this yielded a plasmid, pa , containing nt - and - of the coding region of the vsv n gene followed by the entire coding region of the mhv n gene; the encoded protein was designated n/fusl. a deletion mutant of pa was created by exchange of the apai(blunted)-hindlii fragment of pa for the stui-hindlii fragment of pn . the resulting plasmid, pa , contained nt - and - of the vsv n gene followed by nt - of the mhv n gene; the encoded protein was designated n/fus . during the construction of pa , there serendipitously arose a similar plasmid, pa , containing the same extent of mhv n sequence preceded only by nt - of the vsv n gene; the protein encoded by this construct was designated n/fus . all plasmid constructs were checked by restriction analysis, and all newly formed junctions were directly verified by dideoxy sequencing [ ] . dna plasmids to be used as transcription templates were purified by two cycles of equilibrium centrifugation in csci gradients in the presence of propidium iodide [ ] . capped, run-off sp and t mrnas [ ] were typically synthesized in gl reactions containing mm tris hydrochloride (pht. ), mm mgc , mm spermidine, mm nac , mm dithiothreitoi, i u rnasin (promega), gm each atp and ctp, gm each gtp and utp, iim mvgpppg (new england biolabs), gci [ , - h]utp ( ci/ mmol; amersham), gg linearized dna template, and either u sp rna polymerase or u t rna polymerase (new england biolabs). reactions were carried out for rain at either °c (sp ) or °c (t ). products were extracted twice with phenol-chloroform, twice with chloroform and were twice precipitated with ethanol. the incorporation of labeled utp was determined by binding of product rna to deae filter paper (whatman de ); the size and homogeneity of synthesized rna were verified by electrophoresis in % polyacrylamide gels containing m urea, followed by fluorography. for the synthesis of high specific activity, p-labeled n mrna, all reaction conditions were identical, except that the labeling nucleotide was ~tci [a- p]utp ( ci/mmol; amersham). translation of synthetic mrnas was carried out in a micrococcal nuclease-treated rabbit reticulocyte lysate (amersham). in the standard reaction, . to . ~tg of mrna in to ~tl of h was used to program gl of reticulocyte lysate containing u of rnasin and to gci of [ s]methionine (> ci/mmol; new england nuclear). incubations were carried out for rain at °c. in many experiments, where noted, gl of either h or rnase a was then added, and samples were incubated an additional rain at °c. bovine pancreatic rnase a (ca. u/mg; sigma type xii-a) was rendered dnase-free by heat treatment [ ] . for translation of authentic mhv mrna, total rna was purified [ ] from mhv-a -infected (as well as mock-infected) mouse clone cells at h postinfection, and gg was translated as above. in vitro-synthesized proteins were analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) [ ] and by nondenaturing page employing the standard laemmli discontinuous system in which sds was omitted from all gels and buffers, and samples were not heated prior to electrophoresis [ ] . reticulocyte lysate samples ( ) were run on sds-page gels ( % acrylamide- . % methylene bisacrylamide) and nondenaturing page gels ( . % acrylamide- . % methylene bisacrylamide) that were cm long by . mm thick. nondenaturing page was performed at v at ambient temperature (ca. h); under these conditions there was no detectable heating of the gel for the duration of the run. gels were fixed in % methanol- % acetic acid and were impregnated with m sodium salicylate prior to drying and fluorography at - °c. [ ss]methionine-labeled bands were quantitated either by (/) excision and solubilization in ml of % h for h at oc prior to counting in ml of aquasol (new england nuclear) or (ii) direct counting with a betascope blot analyzer (betagen). in order to develop in vitro assays for mhv n protein function, the n gene was inserted into bacteriophage t and sp transcription vectors. synthetic mrna containing the entire coding region of the authentic n mrna, as well as most of the ' and ' utrs, was used to program a protein synthesizing system from rabbit reticulocyte lysate. the translation product from this message, labeled with [ s]methionine, was analyzed by sds-page and had a mobility indistinguishable from that of n protein translated in vitro from total rna purified from mhv-a -infected cells (fig. a, lanes b and c) . in addition, n protein translated from synthetic mrna was immunoprecipitable by either anti-n or anti-mhv polyclonat antibodies (peng and masters, unpubl. data). in vitro-translated n protein had a slightly higher apparent molecular weight than n protein from [ s]methionine-labeled virus of mhv ( fig. a, lane a). this was possibly due to differential extents of n protein phosphorylation [ ] in vitro and in vivo, or it may indicate that the mature n protein found in mhv virions had undergone amino-terminal modification, as has been observed for the n protein of the coronavirus avian infectious bronchitis virus [ ] . this apparent difference does not bear on the present study. to examine the properties of native n protein, the in vitro translation product was analyzed by nondenaturing page. in this gel electrophoresis system, the major fraction (ca. %) of translated n protein migrated into the running gel, forming a discrete band with a mobility of . relative to bromphenol blue (fig. b, lane f). the mhv-a n protein contains a considerable excess of basic amino acid residues ( lysines and arginines as opposed to glutamates and aspartates) and has a calculated pi of . [ ] . initially it was surprising that such a molecule, expected to have a substantial net positive charge, was migrating toward the positive pole during electrophoresis. this result suggested that the n molecule was tightly bound to some polyanionic molecule and migrated as part of a complex having an overall negative charge. that this, in fact, had occurred was shown by incubation of translated n protein with rnase a prior to nondenaturing gel electrophoresis. rnase treatment abolished the ability of translated n protein to migrate into the nondenaturing gel ( fig. b, lane g) . thus, synthesized n protein had bound to some rna species present in the translation reaction, and as a consequence of this complex formation it was carried into the nondenaturing gel during electrophoresis. n protein translated from total rna purified from mhv-a -infected cells behaved in the same rnase-sensitive manner (fig. more immediately digested. the n -r n a complex was more sensitive to rnase a than to rnase t , possibly due to the different nucleotide specificities of these enzymes (rnase a cuts ' to pyrimidines; rnase t cuts ' to g residues). alternatively, the differential sensitivity may have reflected the relative accessibility of each enzyme to phosphodiester bonds within the n -r n a complex. the n proteins of coronaviruses provide their encapsidated genomes only a minimal degree of protection against the action of ribonucleases [ , ] . hence, in this respect, the n -r n a interaction in the nondenaturing gel assay resembled that found in the m h v nucleocapsid. the rnase sensitivity of the ability of n protein to migrate into nondenaturing gels was not due to possible contaminating protease activity in the rnase preparations, since the same rnase-treated samples of translated n p.s. masters protein showed no degradation when analyzed by sds-page (fig. , lanes an, lower) . moreover, inclusion of the placental rnase a inhibitor, rnasin, allowed at least partial inhibition of the effect of an intermediate concentration of rnase a (fig. , lanes p and q) . incubation with rnase-free dnase had no effect on the mobility of n protein in nondenaturing page (fig. , lane r) . it has been reported previously that the mhv n protein exhibits sequencespecific binding to nucleotides to of the mhv leader rna [ ] . since the n mrnas synthesized from the transcription vectors shown in fig. contained this portion of the leader sequence, it seemed possible that the n-rna complex observed in nondenaturing page was that of translated n protein binding to its own mrna. to test this notion, parallel translation reactions were carried out either with unlabeled n mrna or with n mrna labeled to high specific activity ( . x cpm/gg) with [a- p]utp. n protein translated from unlabeled mrna was labeled with [ s]methionine, while n protein translated from labeled mrna was left unlabeled (fig. ) . it was expected that if translated n protein were binding to its own mrna, then a p-labeled rna band migrating to the same position as that of [ s]methionine-labeled n protein should have been observed in nondenaturing page. as shown in fig. (lanes b', c', f' and g') this clearly did not occur. this was not due to failure to translate the p-labeled mrna, since unlabeled and labeled n mrnas had comparable translation efficiencies (fig. , lanes b, e, b' , and e'). during the course of the min translation reactions, the p-labeled mrna underwent limited nucleolyfic degradation, presumably due to low levels of rnase activity in the reticulocyte lysate. most of the p-labeled material produced by this limited breakdown was retained by sds-page, but virtually all of the same species migrated near the dye front in nondenaturing page. some minor p-labeled bands were seen in the nondenaturing gel at positions other than that expected for n protein-bound material. however, these could not have been due to complex formation with n protein, since the same bands were observed when n protein synthesis was abolished by inclusion of the protein synthesis inhibitor cycloheximide in translation reactions (fig. , lane i') . thus, the n-rna complex observed in the nondenaturing gel assay must have been formed by the binding of n to some endogenous rna species in the reticulocyte lysate. that the observed complex was not due to translated n protein binding to the leader portion of its own mrna was also supported by the observation that full-length n protein translated from a construct in which the mhv leader sequence was entirely deleted was also able to migrate into nondenaturing gels (data not shown). the same was observed for vsv-n : mhv-n fusion constructs which had the ' end of a heterologous mrna substituted for the mhv leader (see below). the complex reported to occur between n protein and mhv leader rna [ ] may not have formed under (a'-j') the conditions of the translation assay or may not have been detectable in the electrophoresis system used here. the identity of the r n a b o u n d by n protein in the reticulocyte lysate has not yet been determined. n protein formed a discrete band rather than a heterogeneous smear in nondenaturing p a g e , suggesting that it was binding p.s. masters to a particular rna species or at least to a set of species of fairly uniform size. moreover, a band of identical mobility was formed by n protein that had been translated in a wheat germ extract (data not shown), indicating that the unknown rna species was not unique to reticulocytes. this non-sequence-specific mode of rna binding was not unexpected. the n protein of mhv has been shown previously to bind in vitro to rna of nonviral origin by a labeled rna overlay protein blot assay [ , ] , and, indeed such non-sequence-specific binding must be the principal type of protein-rna contact along the extensive length of the coronavirus nucleocapsid. the ability of translated n protein to migrate into a nondenaturing gel was next used as an assay to map the rna-binding function of the n protein molecule. a set of n protein deletion mutants with successively larger carboxyterminal truncations (designated n/ace, n/bsm, n/sea, n/eco and n/spe) was generated by translation of run-off transcripts synthesized from transcription vectors that had been linearized with the restriction enzymes acci, bsmi, scai, ecori, or spei (fig. ) . these variants contained, respectively, deletions of , , , , and amino acids. in addition, the internal deletion contained in the n gene of a temperature-sensitive and thermolabile mhv-a mutant, albany- , was transferred to a transcription vector. the mutant n protein translated from this construct (n/alb ) contained an in-phase deletion of amino acids through but was everywhere else identical to wild type n ( [ ] ; koetzner et al., manuscript in prep.). these six mutants, as well as full-length n protein, were analyzed by sds-page and by nondenaturing page (fig. ) . on sds-page, all migrated as discrete bands of the expected relative mobilities and were largely unaffected by treatment with rnase a. the n/ace mutant showed some degree of heterogeneity in both the presence and absence of rnase a (fig. , lanes e and f). since this protein product terminated in the basic amino acid-rich sequence kpqrkgr, it possibly was rendered sensitive to degradation by trypsin-like proteolytic activities within the reticulocyte lysate. when analyzed for the ability to bind to rna, only two of the n mutants, n/acc and n/alb , were able to enter a nondenaturing get to a significant quantitative extent by comparison with full-length n protein (fig. , lanes a' to p') . this migration was completely inhibited by prior incubation with rnase a. the remainder of the carboxyterminal truncated n proteins were only minimally detectable or were undetectable by nondenaturing page. an estimate of the relative rna-binding ability of each n protein construct was made by determining the ratio of cpm of translated protein that had banded in a nondenaturing gel compared to the cpm of an identical sample that had banded in sds-page (table ) . by this analysis, the n/ace construct retained the major part of the rna-binding ability of full-length n protein, demon- strating that at least the carboxy-terminal amino acids of n were dispensable with respect to this function. however, carboxy-terminal truncation further into the n molecule, in the n/bsm, n/sca, n/eco and n/spe mutants, generally abolished the ability of n to bind to rna. the relative rna-binding ability of n/alb was intermediate between these latter and that of n/acc. thus, the left end of the albany- deletion may define the right-most boundary of the rna-binding domain of the mhv n protein, since the n/bsm mutant, which truncated two further residues, lost rna-binding ability. effect of amino-terminal or centrally located deletions on the ability of n protein to bind to r n a a set of deletion mutants collectively spanning the remainder of the n molecule was next constructed. due to the paucity of convenient restriction sites near p. s. masters defined as the average ratio of cpm of translated protein able to enter a nondenaturing gel compared to cpm of an identical sample binding in an sds gel. the number of independent determinations from which the average was obtained is indicated in parentheses b for the carboxy-terminal and internal deletions (upper set), this is calculated relative to full-length mhv n protein. for the vsv-n:mhv-n fusion constructs (lower set), this is calculated relative to the fulmength construct n/fusl the ' end of the mhv n gene, a fusion construct incorporating a portion of the ' end of a mutant vsv n gene was created in order to provide a start codon for a construct in which the ' end of the m h v n gene was then removed. consequently, both n fusion genes encoded proteins initiating with the vsvderived sequence: m a p t v k r i i n d s i i q p k l p a n e d p d r s a e d d k w l p i -yilg, corresponding to amino acids - and - of that protein [ ] . the full-length fusion protein, designated n/fusl, contained the entire m h v n protein following this sequence. the amino-terminal deletion mutant, n/ fus , lacking the first amino acids of the mhv n protein, contained the vsv sequence fused to the remainder of mhv n. two internal deletions mutants, encoding n/apa-nhe and n/nhe-spe, were generated by removal of the indicated restriction fragments from one of the original (nonfusion) n vectors (fig. ) . the resulting protein products transcribed and translated from these lacked regions of and amino acids, respectively. these mutants were analyzed by the two gel electrophoresis systems and on sds-page were observed to be homogeneous and of the anticipated relative mobilities (fig. , lanes a to k) . nondenaturing page revealed that the n/fus and n/fus proteins retained the ability to bind to rna, whereas the deletions in n/apa-nhe and n/nhe-spe abolished rna binding (fig. , lanes a' to k') . quantitation of the extent of rna binding showed that the n/fusl mutant was % as binding-competent as unaltered, full-length n protein (table ) . although the vsv n protein is, itself, an rna-binding protein, the mutant vsv n protein used in the construction of n/fus and n/fus is unable to bind indeed, for n/fus the vsv n-derived portion of the molecule appeared to somewhat impede the entry of mhv n into the nondenaturing gel. remarkably, the n/fus protein was a better rna-binding molecule than its parent, n/fusl, in spite of the loss of a large portion of the mhv n amino terminus (table ) . clearly, this deleted portion of the n molecule can play little or no role in rna binding. by contrast, the deletions in the n/apa-nhe and n/nhe-spe mutants reduced rna binding to residual levels similar to those observed for the four shortest carboxy-terminal truncation mutants ( table ). since removal of a substantial portion of either the amino or carboxy terminus of the n molecule allowed it to retain the ability to bind to rna, it became of interest to examine the effect of removal of both of these regions. this was accomplished by generating the doubly-truncated mutant n/fus /acc. included for comparison in these experiments was n/fus , an n mutant containing the same amino-terminal deletion as n/fus but having only amino acids derived from the vsv n protein (maptvkri). in addition, the corresponding accigenerated truncation of n/fus was examined. analysis of these n protein mutants and their full-length counterparts demonstrated that all were able to enter nondenaturing gels to a significant degree (fig. ) . quantitation of the gel assay showed that n/fus was at least as competent as full-length n protein and nearly twice as competent as n/fus in rna binding (table ) , thereby extending the prior result obtained with the n/fus mutant. the n mutants containing both amino-and carboxy-terminal deletions, n/fus /acc and n/fus /acc, were at least % as able as n/fusl (or at least % as able as full-length n) to bind to rna (table ) . therefore, the rna-binding domain of the mhv n protein could be localized to a portion of the molecule encompassing amino acids to . the purpose of this study was to learn what regions of the mhv n molecule participate in the most salient function of n, its binding to rna. the extent of the deletions and the rna-binding competence of the various mhv n protein mutants constructed for this study are summarized schematically in fig. . the results indicate that the rna-binding ability of the n molecule can be localized to an internal region comprising some amino acids. deletion of either amino acids from the amino terminus or amino acids from the carboxy terminus of n produced only a limited effect on the rna-binding function of this protein. moreover, mutants lacking both termini still retained fig. . effect of combined amino-terminal and carboxy-terminal deletions on the ability of n protein to bind to rna. in vitro-synthesized mrnas encoding full-length n protein, a vsv-n : mhv-n fusion protein (n/fusl), amino-terminal deletion mutants (n/fus and n/fus ), carboxy-terminal deletion mutants (n/acc and n/fusl/acc) or combined aminoterminal and carboxy-terminal deletion mutants (n/fus /acc and n/fus /acc) were translated and subsequently incubated for min at °c with either h or with gg/ml rnase a. control reactions contained no exogenous mrna (a, b, a' and b'). [ s]methionine-labeled samples were analyzed by sds-page (a-r) and by nondenaturing (nd)-page (a'-r') rna-binding ability. by contrast, deletions occurring within the internal region or entering further into it via the carboxy terminus of the molecule effectively abolished r n a binding. the regions of n defined by this analysis coincide strikingly with those of a three domain model for the molecule that we have proposed previously on the basis of a comparison of the sequences of the n proteins of five different strains of mhv [ ] . the present work suggests that these three domains of n are not only structurally distinct but are functionally separable as well. the rna-binding characteristic of the n molecule resides within domain ii, whereas domains i and iii are expendable with respect to this activity. since the deletions entering domain ii are mutations to loss of function, it [ ] or recognizable similarity to recently described arginine-rich [ ] or methionine-rich [ ] rna-binding domains. thus, the rules governing this particular mode of protein-rna interaction remain to be elucidated. in fig. , the rna-binding ability of n/alb has been scored as equivocal, based on our recent finding that the ability of this mutant protein to enter a nondenaturing gel is temperature-sensitive with respect to that of wild type n (koetzner and masters, unpubl, results) . in addition, revertants of the albany- mutant have been selected, and all of these have n proteins which no longer exhibit the rna-binding temperature-sensitivity of n/alb . for the one revertant that we have analyzed in detail, the single point mutation that causes this restoration of rna-binding ability resides in the distal third of domain ii. this finding further underscores the role of domain ii in rna binding, and supports the notion that the nondenaturing gel assay measures a biologically relevant property of the n molecule. although the n protein may recognize a specific nucleation sequence or secondary structure on the mhv genome, it must also possess a more general non-sequence-specific rna-binding capability, since it encapsidates the entire length of the , nucleotide genome, which does not appear to contain any iterated pattern of potential rna recognition sequences. this general mode of rna binding may be a common characteristic of the n (np) proteins of rna viruses having helical nucleocapsids. bacterially expressed np protein of influenza virus was shown to bind well to both viral and nonviral rna substrates when assayed in vitro [ ] . the n protein of vsv bound tightly to cellular rnas when synthesized in the absence of other viral proteins in an in vivo expression system [ ] , and it bound to endogenous reticulocyte rnas or added rna species when expressed by in vitro translation [ ] . the measles virus np protein, expressed in vivo by a vaccinia virus recombinant, has recently been shown to assemble into structures very similar to authentic measles nucleocapsids, as determined by both electron microscopy and equilibrium sedimentation on csc gradients [ ] . for mhv, it has been demonstrated previously that the n protein of this virus can bind to a variety of viral and nonviral rna species following separation of n by sds-page and blotting onto nitrocellulose [ , ] . thus, it is not unexpected that the mhv n protein should bind in vitro to nonviral rna species in the nondenaturing gel assay reported here. it should be noted that an alternative interpretation of the nondenaturing gel assay is that the mhv n protein was not directly bound to rna but, rather, to a protein constituent of an rnase-sensitive ribonucleoprotein in the reticulocyte lysate. this seems less likely in light of the previously demonstrated rna-binding activity of n [ , ] , but it cannot be ruled out until an mhv n rna-binding assay using purified components is established. if such an n protein-ribonucteoprotein complex is shown to occur, then this may indicate a previously unappreciated interaction between n protein and host cell components. the work presented here does not address the problem of sequence-specific rna recognition by n protein. only full-length genomic rna of mhv is packaged into virions. recently, analysis of defective interfering particles of mhv has identified the rna packaging signal as falling within a ( nucleotide locus near the ' end of the huge viral polymerase gene [ , ] , which is unique to genomic r n a and is not found in subgenomic r n a species. others have reported sequence-specific binding of n protein to nucleotides to of the m h v leader [ ] , which is c o m m o n to all m h v genomic and subgenomic rnas. this latter sequence was present on many of the synthesized m r n a s used in our study, but selective binding to this r n a region was not observed (fig. ) . it is possible that the concentrations of n protein and n o n -m h v r n a in the reticulocyte lysate in our assay were too high to allow demonstration of a selective recognition of m h v r n a . we are currently seeking to establish the assay conditions and the correct r n a substrate to examine sequence-specific r n a binding by the m h v n protein. nucleocapsids of large rna viruses as functionally active units in transcription coronavirus ibv: partial amino terminal sequencing of spike polypeptide $ identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m heterogeneous nuclear ribonucleoprotein particles and the pathway of mrna formation assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein mechanism of interferon action. interferon-mediated inhibition of simian virus- early rna accumulation zinc fingers": a novel protein motif for nucleic acid recognition cleavage of structural proteins during the assembly of the head of bacteriophage t characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus sequence-specific recognition of rna hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif the complete sequence ( kilobases) of murine coronavirus gene i encoding the putative proteases and rna polymerase plus and minus strand leader rnas in negative strand virus-infected cells ribonucleoprotein-like structures from coronavirus particles rna-binding domain of mhv n protein analysis of efficiently packaged defective interfering rnas of murine coronavirus: localization of a possible packaging signal molecular cloning: a laboratory manual resolution of multiple complexes of phosphoprotein ns with nucleocapsid protein n of vesicular stomatitis virus complex formation with vesicular stomatitis virus phosphoprotein ns prevents binding of nucleocapsid protein n to nonspecific rna structure and function studies of the nucleocapsid protein of mouse hepatitis virus efficient in vitro synthesis of biological active rna and rna hybridization probes from plasmids containing a bacteriophage sp promoter molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain a sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein a common rna recognition motif identified within a defined u rna binding domain of the k u snrnp protein rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay the -kd protein of signal recognition particle contains a methionine-rich rna binding domain dna sequencing with chain terminating inhibitors identification and primary structure of the gene encoding the berne virus nucleocapsid protein coronaviruses: structure and genome expression assembly of nucleocapsidlike struct~es in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprorein expression of a recombinant dna gene coding for the vesicular stomatitis virus nucleocapsid protein deans ill ( ) specific interaction between coronavirus leader rna and nucleocapsid protein synthesis and subcellular localization of the murine coronavirus nucleocapsid protein the molecular biology of coronaviruses i thank lawrence sturman for numerous valuable discussions and cynthia ricard, who first suggested the rnase experiment. i am grateful to monica parker for expert technical assistance and to arlene ramsingh for critically reading the manuscript.this work was supported in part by public health service grant gm from the national institutes of health. key: cord- -e jrhycj authors: dong, wei; chen, qianqian; hu, yihong; he, dongping; liu, jia; yan, huajie; lan, ke; zhang, chiyu title: epidemiological and clinical characteristics of respiratory viral infections in children in shanghai, china date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: e jrhycj acute respiratory tract infections (artis) due to various viruses are not only the most common causes of upper and lower respiratory infection but are also major causes of morbidity and mortality in children. in this study, we investigated the prevalence and clinical characteristics of children with virus-related artis and determined the spectrum of respiratory viruses and their correlation with meteorological variables in jiading district, shanghai, china. nasopharyngeal swabs from children with artis were collected from august to december , and used for detection of respiratory viruses by multiplex rt-pcr. seventeen respiratory viruses were detected among ( . %) of patients. the highest prevalence of respiratory viruses was detected in the age group of less than year ( . %), and the prevalence decreased with age. this suggests that children less than one year old are the most susceptible to infection. influenza virus (ifv) was the most frequently detected virus ( . %), followed by parainfluenza virus (piv) ( . %), enterovirus (ev) ( . %), and respiratory syncytial virus (rsv) ( . %). statistical analysis showed that epidemics of ifv, piv and ev had distinct seasonal variations. mean monthly temperature appeared to be the only meteorological factor associated with ifv and piv infection. these findings will provide valuable information for decision-making, prevention and treatment of artis in children. electronic supplementary material: the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. acute respiratory tract infections (artis) are major risk factors associated with morbidity and mortality of infants and children worldwide and are caused by respiratory viruses and/or bacteria [ , ] . the majority of artis are ascribed to respiratory viruses; however, antibiotics were often used in the clinical treatment of artis in some developing countries (e.g., china). investigations of the prevalence of artis and its correlation with viral pathogens are critical for improving the prevention and treatment of artis. there are more than respiratory viruses that can cause artis. respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (piv), human enterovirus (ev), influenza virus (ifv), human coronavirus (cov), adenovirus (adv), and human bocavirus (bov) are the most common viral agents associated with artis, accounting for around % of artis [ , ] . as children with artis often have similar clinical presentations, it is difficult for doctors to make decisions and prescriptions simply based on the physical symptoms. therefore, investigating the clinical characteristics of children with virus-related artis and the spectrum of respiratory viruses will facilitate the development of precise treatments for artis. w. dong and q. chen contributed equally to this work. electronic supplementary material the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. the prevalence of respiratory viruses among children with artis differs in different regions, and varies over time [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it has been relatively difficult to provide realtime surveillance of artis in china because of its large population, vast territory and multivariate climatic factors. in several previous studies, the prevalence of respiratory viruses with artis in shanghai [ , ] , wuhan [ ] , harbin [ ] , lanzhou [ , ] , nanjing [ ] , zhuhai [ ] and shantou [ ] were investigated. the positive rates of respiratory viruses among patients with artis in these cities were from . % to . %. rsv, hrv, and ifv-a were the most common respiratory viruses. in this study, we report the clinical characteristics of respiratory viral infections in children in shanghai, china, from august to december , and investigated the etiologic agents. we found that the respiratory virus positive rate was the highest in children less than year old, and the positive rate decreased with age without gender difference. furthermore, ifv, piv, ev and rsv were most frequently observed in children with artis and displayed seasonal epidemic patterns. from august to december , children with artis who had been admitted to shanghai nanxiang hospital, a district-level general hospital in shanghai, china, were recruited in this study. the study was approved by the medical ethics committee of shanghai nanxiang hospital. demographic information and clinical characteristics were recorded for each patient. nasopharyngeal swabs were collected from these patients after obtaining informed consent from their parents or guardians, and the samples were transported to the laboratory at institut pasteur of shanghai, chinese academy of sciences, in virus transport medium (including hank's buffer, bsa, hepes and antibiotics) within hours for the detection of respiratory viruses. viral rna was extracted from each clinical sample using a qiaamp viral rna mini kit (qiagen, germany). respiratory viruses were detected by a multiplex rt-pcr assay as described previously [ , ] . in brief, viruses were detected in a five-tube mrt-pcr assay. tube targeted ifv-a, ifv-b, rsv, hmpv; tube , piv - ; tube , ev, hrv and ifv-c; tube , cov- e, cov-oc , cov-nl and cov-hku ; and tube , adv and bov. for each tube, we selected one of the targeted viruses and used the culture supernatant of the standard strain of the selected virus as a positive control for rna extraction. as a result, rsv, piv- , hrv, co- e and adv were selected for tubes - , respectively. the amplification reaction was performed using a qiagen onestep rt-pcr kit (qiagen, germany). for quality control in the multiple rt-pcr, a mixture of plasmid templates was used as a positive control, and distilled water was used as a negative control. a -ll pcr mix including qia-gen onestep rt-pcr buffer, dntp mix (final concentration of each dntp, lm), primers f and r (final concentration, . lm), qiagen onestep rt-pcr enzyme mix ( ll), rna template ( . ll), and rnasefree water was prepared. the reaction steps included reverse transcription at °c for min, initial pcr activation at °c for min, cycles at °c for s, °c (tube and tube ) or °c (tube , tube and tube ) for s, and °c for min, and a final extension at °c for min. to increases the amplification in tube and tube , an additional eight cycles of °c for s, °c for s, and °c for min were added after the initial pcr activation. the respiratory viruses were distinguished according to the product sizes in % agarose gel electrophoresis. the data on mean monthly temperature (°c), rainfall (mm) and relative humidity (%) of jiading district of shanghai were obtained from the jiading weather bureau of shanghai. the meteorological station is located at ° ' n, ° ' e. statistical analysis was performed using spss software (version . ; spss, inc., chicago, il, usa). statistical comparisons of viral incidence between different groups (i.e., genders, ages and seasons) were performed using the chi-square (v ) test. bivariate correlation and multiple stepwise regression analysis were used to analyze the associations between viral infection and meteorological parameters. a probability (p) value less than . was considered statistically significant. from august to december , nasopharyngeal swabs were collected from children with artis at shanghai nanxiang hospital. their ages ranged from months to years. the majority of them ( , . %) were between and years old. the ratio of boys to girls was . (table ) . among clinical samples from the recruited children with artis, ( . %) samples were found to be respiratory virus positive. there was no significant difference in the incidence of respiratory viral infection between boys ( / ; . %) and girls ( / ; . %) (v = . , p = . ). the respiratory virus positive rate was significantly different among the different age groups (v = . , p = . ). the positive rate appeared to decrease with age. children less than one year old had the highest positive rate of . % ( / ). the positive rate decreased to . % ( / ) and . % ( / ) for the age groups of - and - years old, respectively. the lowest positive rate was observed in the age group of more than years old ( / ; . %) ( table ) . a statistically significant difference was observed between the age group of less than year old and the age group of more than years old (v = . , p = . ). no statistically significant difference was observed among the other age groups. the clinical symptoms of virus-positive artis children included cough, fever, sore throat, expectoration, runny rose, rales, diarrhea, rash, headache, muscular soreness, stomachache, dyspnea, and chest pain ( table ). of these, cough ( . %), fever ( . %), and sore throat ( . %) were the most commonly observed symptoms. these viruspositive children were often diagnosed with tonsillitis ( . %), bronchitis ( . %), upper respiratory tract infections (urtis, . %) or pneumonia ( . %) ( table ). seventeen respiratory viruses, including ifv (ifv-a/b/c), piv - , ev, rsv, cov ( e, hku , oc , nl ), adv, hmpv, bov, and hrv were detected among of children with artis in nanxiang district of shanghai (table ) . multiple viral infections were detected in ( . %) patients ( with two pathogens and with three pathogens). influenza viruses, including ifv-a, ifv-b and ifv-c, were the most frequently detected viruses ( / , . %), followed by piv ( / , . %), ev ( / , . %), and rsv ( / , . %). the detection rate of cov, adv, hmpv, bov and hrv was . %, . %, . %, . %, and . %, respectively. among these respiratory viruses, most had a similar distribution among the different age groups (fig. ) . interestingly, the prevalence of ifv appeared to increase with age, while the prevalence of piv appeared to decrease with age. we further investigated the seasonal distribution of the respiratory viruses (table ). of the analyzed viruses, ifv, piv and ev appeared to have seasonal patterns (p \ . ) ( table and supplemental figure ). for ifv, the highest prevalence was observed in winter ( / , . %), while the lowest prevalence occurred in summer ( / , . %). distinct from ifv, the highest prevalence of piv occurred in summer ( / , . %), and it was only . % ( / ) in winter. the prevalence of ev was similar in spring ( / , . %), summer ( / , . %), and autumn ( / , . %), substantially higher than that in winter ( / , . %) ( table ). rsv and cov were relatively active throughout the whole year and have similar prevalence in different seasons (table and supplemental figure ). adv, hmpv, bov, and hrv were excluded from the analysis because their sample sizes were too small. the seasonal patterns of ifv, piv and ev prevalence (table and supplemental figure ) suggest a correlation between the respiratory virus epidemic and meteorological variables. we performed regression analysis to determine whether meteorological factors (including mean monthly temperature, humidity, and rainfall) were associated with prevalence of various respiratory viruses. the nanxiang district of shanghai has a mean monthly temperature of . ± . °c, humidity of . ± . %, and rainfall of . ± . mm. bivariate correlation showed that the incidence of influenza virus infection was significantly negatively correlated with mean monthly temperature (spearman's rho: - . , p = . ), but it was not fig. the distribution of respiratory viruses in different age groups the chi-square (v ) test was performed using spss. a p-value \ . is indicated by * and is considered statistically significant (in bold) correlated with mean monthly humidity (spearman's rho: - . , p = . ) or mean monthly relative rainfall (spearman's rho: - . , p = . ) ( table ). when multiple stepwise regression analysis was performed, mean monthly temperature appeared to be the only factor associated with influenza virus infection. in contrast, the incidence of piv infection was significantly positively correlated with mean monthly temperature (spearman's rho: . , p \ . ) and mean monthly rainfall (spearman's rho: . , p = . ). multiple stepwise regression analysis showed that mean monthly temperature was the only factor associated with piv infection. no significant correlation was observed between the prevalence of rsv, ev, and cov and meteorological variables (table ). infections with respiratory viruses are the most common causes of artis in children. they can lead to serious diseases such as bronchiolitis and pneumonia and sometimes even cause death in infants and children worldwide [ ] . in china, the positive rates of respiratory viruses in children with artis or influenza-like illness (ili) range from . to . % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the positive rates appear to be slightly different in different regions, which may be attributed to different years and regional variation, as well as a lack of unified methodology. however, a large-scale surveillance based on , children with artis in central china showed that the prevalence was only . %, substantially lower than that found in other studies [ ] . similarly, in this study, we found that the prevalence of respiratory viruses was . % among children with artis from august to december in jiading district of shanghai, significantly lower than those ( . % and . %) obtained from two previous studies based on children with artis during to [ ] (v = . , p. ), and children with artis from may to july [ ] (v = . , p. ) in shanghai. this may imply that it is important to have a large sample size in the investigation. we found that the prevalence of respiratory viruses was similar between boys and girls, suggesting that there was no bias for respiratory viral infection with regard to gender. however, the prevalence appeared to decrease with age, with the highest prevalence of . % in the age group of less than year old ( table ). one possible reason is that children younger than year old are more susceptible to infection with respiratory viruses than older children are. another reason might be that children younger than year old are more likely to be brought to the hospital when exhibiting symptoms of artis than older children are. cough and fever were the two most common symptoms in our enrolled patients with artis. the majority of febrile infants in shanghai are hospitalized and prescribed antibiotics without accurate identification of bacterial infections, which may delay treatment and cause unnecessary side-effects. for precise treatment, it is urgently required that doctors know which kind of pathogens the children are infected with in order to give them proper medical treatment while avoiding the overuse of antibiotics. the proliferation of respiratory viruses among children with artis differed among different regions and often varied over time. ifv, rsv and hrv were the most commonly detected respiratory viruses among children with artis or ili in most regions of china. in shanghai, a previous study based on cases showed that piv, hrv, ifv and rsv were the top four common respiratory viruses during - [ ] . in this study, we investigated children with artis and found that ifv, piv and rsv were still the most common viruses, together with ev. compared to the previous study [ ] , we detected a decreasing prevalence ( . %) of hrv. one possible reason was that relatively severe cases (e.g., sudden onset of fever [ , cough or sore throat and difficulty breathing) were enrolled in the previous study [ , ] , which resulted in a higher detection rate. on the other hand, the genotypes of certain respiratory viruses also vary over time. a genotype shift of rsv was observed in chongqing and shanghai from to [ , , ] . for piv, the proportions of four genotypes (piv- , - , - , - ) were consistent with those reported previously [ , , , ] . some previous studies have shown that the prevalence of certain respiratory viruses (e.g. hmpv, rsv) was prevalence of respiratory viruses in children in shanghai correlated with meteorological variables [ , ] . we observed seasonal variations for ifv, piv and ev. the prevalence of ifv appeared to be significantly negatively correlated with mean monthly temperature, while the prevalence of piv was significantly positively correlated with mean monthly temperature. since , a nationwide surveillance network for influenza has been implemented in the people's republic of china. sentinel hospitals of the network are required to upload their daily influenza-like illness case numbers to the database of chinese centers for disease control and prevention. the national data will help to draw a definite conclusion on the correlation of the prevalence of respiratory viruses with the meteorological variables. winter and spring are the high-incidence season of rsv. here, we detected a total of rsv-positive samples. the prevalence of rsv in autumn ( . %) and winter ( . %) was slightly higher than that in spring ( . %) and summer ( . %) ( table ). the high prevalence of rsv in summer differed from what has been observed in previous studies [ ] , and the majority of the cases detected in summer were from july , suggesting a partial outbreak of rsv during a short period. because rsv sequences were not obtained from the samples from july [ ] , we were unable to determine the reason for this unusual rsv outbreak. in summary, we performed an epidemiological investigation on respiratory viruses in children with artis in jiading district of shanghai from to . we found that the prevalence of respiratory viruses decreased with age and that the highest prevalence ( . %) occurred in children less than year old, suggesting that children younger than year old are the most susceptible to infection with respiratory viruses. multiple respiratory viruses were detected in children with artis in jiading district of shanghai. ifv, piv, ev, and rsv were the dominant respiratory viruses, with prevalence of . %, . %, . %, and . %, respectively. the prevalence of ifv, piv and ev exhibited distinct seasonal variation. the prevalence of ifv and piv was significantly negatively and positively correlated with mean monthly temperature, which was the only meteorological factor associated with ifv and piv infection. conflict of interest none. estimates of world-wide distribution of child deaths from acute respiratory infections emerging respiratory agents: new viruses for old diseases? role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study multiplex real-time pcr for detection of respiratory tract infections the epidemiology and etiology of influenza-like illness in chinese children from molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in shanghai respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china viral etiology of acute respiratory infection in gansu province newly identified respiratory viruses associated with acute lower respiratory tract infections in children in lanzou respiratory virus infections among children in south china epidemiological analysis of respiratory viral etiology for influenza-like illness during surveillance of respiratory viruses in patients with influenzalike illness in nanjing genetic variation of human respiratory syncytial virus among children with fever and respiratory symptoms in shanghai molecular epidemiology of respiratory viruses in virus-induced asthma prevalence and correlation of infectious agents in hospitalized children with acute respiratory tract infections in central china genetic variability of subgroup a and b respiratory syncytial virus strains circulating in southwestern china from genetic variability of respiratory syncytial viruses (rsv) prevalent in southwestern china from to : emergence of subgroup b and a rsv as dominant strains parainfluenza viruses viral etiology and clinical profiles of children with severe acute respiratory infections in china relationship between meteorological conditions and respiratory syncytial virus in a tropical country effect of climatological factors on respiratory syncytial virus epidemics key: cord- - qyee authors: mase, m.; tsukamoto, k.; imai, k.; yamaguchi, s. title: phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: qyee to define the origin and evolution of recent avian infectious bronchitis virus (ibv) in japan, a genetic analysis was performed. by phylogenetic analysis based on the s gene including the sequence of the hypervariable regions, ibv isolates in japan were classified into five genetic groups, which included two already-known groups (mass and gray). among them, three major genetic groups were associated with the recent outbreaks of ib in japan. one group is indigenous to japan and could not be placed within the known existing groups in other countries. the remaining two groups, which have emerged recently, are related to isolates in china and taiwan. m. mase et al. post-translational cleavage of two separate polypeptide components, designated s and s [ ] . of these, the s glycoprotein is associated with virus attachment and is a major target of the neutralizing antibodies in chickens, so serotypic evolution in ibv is associated primarily with the sequences of the s glycoprotein [ , ] . hence, the molecular characterization of ibv is based mainly on analysis of the s gene [ , ] . classically, ibv strains have been placed into serologically related groups on the basis of virus neutralization tests (vnt). however, vnts are very laborious and time-consuming. in addition, there is considerable evidence that serologic relationships among ibv isolates, determined by in vitro vnts, have not been reflected by the results of in vivo cross-immunity studies [ ] . moreover, the antigenic variants emerging from wild-type or vaccine viruses by point mutation or rna recombination cannot be detected by vnts [ ] . recently, genetic grouping of ibv on the basis of nucleotide sequences have been applied for virus classification [ , ] . since anyone can apply the genetic information of viruses deposited into the genbank, genetic comparison with other virus strains for epidemiological analysis can be performed quickly. since the first isolation of ibv was recorded in japan in [ ] , the outbreaks have been ongoing. however, the epidemiological analysis of ibv isolates in japan has not been thorough except for a few strains [ , ] . the relationships between japanese ibv isolates and foreign ibv isolates have also remained unknown. we were particularly interested to know whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, to define the origin and evolution of recent ibv in japan, we determined the nucleotide sequences of ibv isolated in japan using the reverse transcriptase-polymerase chain reaction (rt-pcr) method coupled with direct sequencing, and analyzed the sequences phylogenetically with viruses isolated in other countries. this information is important for determining strategies to control ib. the ibv isolates employed in this study are listed in table . most of the ibv isolates were obtained from regional laboratories in japan. most of the specimens of ibv were isolated by two or three passages using embryonated specific pathogen-free (spf) eggs. the materials were submitted to our laboratory, and were propagated once in spf eggs. the presence of ibv in the inoculated embryos was initially determined by immunofluorescence assay of allantoic cells using anti-ibv chicken serum and observation of characteristic embryo changes such as dwarfing, stunting or curling, according to the procedures of a previous report [ ] . the following commercial attenuated live vaccine strains in japan were also used in this study: c- , miyazaki, on/ , kita- , ku and tm . they were derived from field cases of ib in japan. viral rna was extracted from infected allantoic fluids using a kit (isogen-ls, nippon gene, tokyo, japan). a reverse transcriptase (rt) reaction was carried out with superscript ii (life technologies, gaithersburg, md, u.s.a.) using random mers. the n-terminus of the s glycoprotein, which includes [ ] : country/region of origin/year of isolation except jp/kh/ strain. jp, japan important structures such as hypervariable regions (hvr) associated with antigenic properties [ , , ] , was selected for this analysis. the following primers were used: ibv-s (forward), agg-aat-ggt-aag-ttr-ctr-gtw-aga-g , and ibv-s (reverse), gcg-cag-tac-crt-tra-yaa-aat-aag-c . (the expected product was about base pairs). these were designed based on a comparison and alignment of the genbank sequences of several known ibv strains. pcr products were purified using the qiaquick pcr purification kit (qiagen, hilden, germany) according to the manufacturer's instructions. purified pcr products were used as a template for sequencing on an applied biosystems s automated dna sequencer using dye terminator cycle sequencing chemistry (perkin-elmer/applied biosystems, foster city, ca, u.s.a.). purified pcr products were sequenced from both directions. the derived nucleotide sequences were analyzed using genetyx-mac ver. . (software development corp., tokyo, japan), and through genbank searches. the phylogenetic analysis with available sequences from genbank was conducted using the clustal [ ] , and the tree was constructed by the neighbor-joining (nj) method [ ] . the expected sizes of dna fragments were successfully amplified by rt-pcr from all the employed ibv samples with our designed primers. the determination of nucleotide sequences of obtained pcr products revealed the diversity of those lengths ( - bp) ( table ) . by phylogenetic analysis, the ibv isolates in japan used in this study were divided into five genetic groups (fig. ). in particular, recent isolates in japan were assigned to three major genetic groups. among them, one group (jp-i) is indigenous to japan and is not present in other countries. the two other groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. some isolates that were mainly isolated in the s, such as jp/ishida/ or jp/nerima/ , and two local attenuated vaccine strains, kita- and ku, were classified into the mass group. another local attenuated vaccine strain, c- , was classified into the jp-i group, and miyazaki and tm strains were classified into the jp-ii group. only one strain (on/ ), which was also a local attenuated vaccine strain, was classified into the gray group. the deduced amino acids from obtained nucleotide sequences of pcr products were aligned with various ibv strains isolated in different geographic regions. the results are shown in fig. . many insertions or deletions of amino acids among ibv strains including the japanese isolates were found in the amplified region. the generated pcr products were employed in restriction endonuclease analysis for the development of a simple and rapid classification of genetic groups. after comparing the obtained sequences in this study, we selected two endonucleases, hae ii and ecor i (takara, tokyo, japan), for this analysis. the restriction profiles fig. . dendrogram of the s glycoprotein of infectious bronchitis viruses. amino acids - (in genbank accession number p ) of the beaudette strain s glycoprotein were used for phylogenetic analysis at the amino acid level. the dendrogram is rooted to murine hepatitis virus ml- strain. horizontal distances are proportional to the minimum number of amino acid differences required to join nodes and sequences of the pcr products of the five genetic groups in this study, as revealed by two restriction endonucleases are shown in table and fig. . each japanese genetic group has a specific profile pattern. the other genetic groups represented, such as connecticut, d , uk/ / , and ark , do not have sites for these two enzymes in their reported sequences. hence, the three major genetic groups in japan were easily differentiated from other genetic groups. recently, novel ibv strains have been proliferating abroad, and genetic analysis of these strains have been mainly based on the s gene [ ] . ibv strains in japan have been considered variants different from foreign ibvs such as massachusetts or connecticut types by vnt or pcr-restriction enzyme fragment length polymorphism based on the s gene [ , , ] . still, it remains unknown whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, it was revealed that five genetic groups, which could be differentiated by simple restriction endonuclease analysis, were present in japan. although sequencing is recommended to obtain precise genetic information, restriction endonuclease analysis is simple, easy and convenient for primary characterization in routine diagnosis. among these genetic groups, three major genetic groups were associated with recent outbreaks of ib in japan. a previously published study divided ibv strains into several distinct genetic groupings by analyzing the s gene [ ] . however, most ibv isolates in japan formed genetic groups distinct from these. among three major genetic groups of ibv in japan, one group (jp-i) has not been found in other countries; this group may be indigenous and has been prevalent in japan for a long time, from at least the s. the other two groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. recent ibv isolates in china and taiwan also form distinct genetic groups [ , ] . it is unknown whether these genetic groups originated in japan or in these neighboring countries. interestingly, recently isolated newcastle disease viruses in japan were also closely related, genetically, to those isolated in taiwan and china [ ] . furthermore, it was recently revealed that the coronavirus isolated from pheasants was genetically closely to ibv [ ] . one possibility for the migration of ibv strains into this region is dissemination by some kind of wild birds close to chickens, such as pheasants. the virus neutralization antibodies that form the basis for comparison of ibv isolates are induced largely by the n-terminus in the s glycoprotein [ , ] . in this study, prevalent ibv genetic groups in japan were shown to be completely distinct from other known serotypes from europe and north america by comparing amino acid sequences of the n-terminus in the s glycoprotein. on the basis of the relationships between genotypes and serotypes of ibv [ ] , it was suggested that the serotypes of recent ibv in japan are novel. further analysis would be required to examine the antigenic property of isolates for establishment of a control strategy for ib outbreaks in japan. on the other hand, the variation of ibv may also be attributed to recombination following co-infection of a few distinct strains [ , ] . previously, the kb strain isolated in japan [ ] was revealed to be genetic recombinant [ ] . in japan, in addition to major massachusetts (h strain) and connecticut (l strain) type live vaccines, local attenuated vaccines isolated from outbreaks of ib in japan have been developed and used. they proved to derive from the genetic groups, mass, gray, jp-i or jp-ii, although the genetic backgrounds of these attenuated vaccines have not been clarified. so far, it has been suggested that the recombination events occur between vaccine strains and field strains [ ] . it is unknown whether or not vaccine strains in japan are associated with the emergence of recombinant viruses. to detect recombinant viruses, it would be required to analyze two or more different genetic regions of isolates. further analysis of viruses would clarify whether recombinant variants have become prevalent in japan. all sequences used in this study were sent to ddbj, and their accession numbers areab to ab . completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus coronavirus ibv: structural characterization of the spike protein amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes infectious bronchitis a nomenclature for avian coronavirus isolates and the question of species status coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys use of allantoic cells for the detection of avian infectious bronchitis virus humoral antibody response and assessment of protection following primary vaccination of chicks with maternally derived antibody against avian infectious bronchitis virus serotypes of avian infectious bronchitis virus isolates from field cases in japan cross-immunity in chickens using seven isolates of avian infectious bronchitis virus a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide phylogeny of antigenic variants of avian coronavirus ibv sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism typing of recent infectious bronchitis virus isolates causing nephritis in chicken phylogenetic analysis of newcastle disease virus genotypes isolated in japan a virus isolated from infectious bronchitis-like diseases of chickens the neighbor-joining method: a new method for reconstructing phylogenetic trees ibv genotypes in japan cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools evidence of natural recombination within the s gene of infectious bronchitis virus evolutionary implications of genetic variations in the s gene of infectious bronchitis virus relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia author's address: dr. masaji mase, department of infectious diseases, national institute of animal health, - - kannondai redaktion: sachsenplatz - , wien, austria. -satz und umbruch we thank drs. t. imada and n. yuasa, national institute of animal health, japan, for valuable discussions and suggestions. we would like to thank the veterinary officials of miyazaki, akita, mie,yamanashi, nagano, shizuoka, shimane, chiba, toyama, aichi, fukui, okayama, kanagawa, osaka, and ibaraki prefectures for their cooperation in the collection of the viral samples. key: cord- -if js s authors: cowley, j. a.; walker, p. j. title: the complete genome sequence of gill-associated virus of penaeus monodon prawns indicates a gene organisation unique among nidoviruses(*): brief report date: journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: if js s we report here a nt sequence comprising the ′-end of the (+) ssrna genome of gill-associated virus (gav), an invertebrate nidovirus of penaeus monodon prawns. the sequence extends from a subgenomic rna start site nt upstream of the nt orf gene to a ′-poly(a) tail of the nt genome of gav. the putative amino acid (aa) orf protein (mw = da, pi = . ) contains potential n-linked glycosylation sites, potential o-linked glycosylation sites and six highly hydrophobic regions predicted to represent transmembrane (tm) domains. three of the predicted tm domains occur in the amino-terminal aa, two in the central portion, and one near the carboxy-terminus of orf . only one short ( aa) open reading frame (orf ) was identified between orf and the ′-poly(a) tail. completion of the genome sequence of gav has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative orf spike glycoprotein gene resides downstream of a gene (orf ) encoding a structural protein associated with nucleocapsids. prawns [ ] . gav is morphologically identical to yellow head virus (yhv), which has caused significant losses of p. monodon farmed throughout asia [ ] . gav and yhv nucleocapsids appear as helical tubular structures (∼ nm dia.) and their rod-shaped, enveloped virions (∼ nm × nm) possess diffuse surface projections of ∼ nm [ , , , , ] . yhv contains a > kb ssrna genome [ , ] with suggested positive polarity [ ] . the gav genome -end contains a ∼ kb replicase gene encoding overlapping orf a and orf b polyproteins [ ] . translation of the orf a- b polyprotein is facilitated by a aaauuuu- ribosomal frameshift site and a predicted non-'h-type' rna pseudoknot. sequence similarity in the c-like protease domain of orf a and the 'sdd' polymerase, helicase and other domains of orf b indicate a distant evolutionary relationship to vertebrate viruses of the nidovirales [ ] . conserved sequences in the gav intergenic regions function as transcription start sites for two coterminal subgenomic (sg)rnas that, in contrast to the sgrnas of coronaviruses and arteriviruses, are not fused to -genomic leader sequences [ ] . moreover, the orf gene immediately downstream of orf a- b encodes the amino acid protein similar in size and charge to torovirus nucleocapsid (n) proteins [ , ] and which immuno-electron microscopy indicates is associated with gav nucleocapsids [ja cowley et al. unpubl.] , suggesting it is likely to represent the nucleocapsid protein. in this paper, we report a nt sequence downstream of orf to a -poly(a) tail which, together with the upstream orf sequence included in gen-bank acc: ay , completes the nt (+) ssrna genome of gav. the region contains a . kb orf gene encoding a putative amino acid ( kda) glycoprotein and only one short ( aa) potential orf in the nt region between orf and the -poly(a) tail. the gav genome organisation is, therefore, markedly different to any currently known for viruses within the nidovirales. random cdna clones were generated from a ∼ kb dsrna purified from lymphoid organ total rna of gav-infected p. monodon by rt-pcr [ ] as described previously [ , ] . three . kb clones of p / (i.e. , and ) and a truncated . kb clone (p ) that contained large portions of the gav orf gene were generated by rt-pcr using primers gav and gav as described previously [ ] . clones of the gav genome -terminus were generated using different methods. (a) a random rt-pcr method [ ] was modified to generate clones from oligo-dt-primed cdna. briefly, cdna was reverse transcribed from the purified ∼ kb dsrna using the anchored primer uni-dt -c/a/g ( gccggagctct gcagaattc(t) -c/a/g ). second strand cdna was synthesised using klenow dna polymerase and primer uni-dn ( gccggagctctgcagaatt c(n) ). primer was removed following each reaction using a sephadex hr column (pharmacia) and cdna was amplified by pcr using uni-primer ( gccg gagctctgcagaattc ) and taq dna polymerase (promega) [ ] . pcr products of variable size were purified using a qiaquick column (qiagen) and cloned into pgem-t (promega) using standard procedures [ ] . (b) a cdna clone was generated similarly using lymphoid organ total rna from p. monodon experimentally-infected with gav and cdna synthesised using primer uni-dt -c/a/g followed by pcr amplification using uni-primer and the gav sense primer gav ( tcatattacatcgctggtgatccagg ) designed to a sequence in clone p / . a predominant . kb product was excised from an agarose gel, purified using a qiaquick column and cloned into pgem-t. (c) cdna clones of the gav genome -end were also obtained from a uni-zap ® xr (stratagene) λ bacteriophage library kindly provided by dr. sigrid lehnert, csiro livestock industries. briefly, oligo-dt -primed cdna was prepared from poly(a)+ rna purified from a whole head homogenate of a p. monodon naturally-infected with gav, ligated into the uni-zap xr vector and packaged according to the zap-cdna ® gigapack ® iii gold cloning kit (stratagene) instructions. the λ phage library in sm buffer ( mm nacl, mm mgso , mm tris-hcl ph . , . % gelatin) was diluted : in water, heated at • c for min and microcentrifuged for min. an aliquot ( µl) was amplified by pcr using taq polymerase (promega), an extended t primer ( gtaatacgactcactatgggc ) and the gav sense primer gav ( gctccacacaggcactaccg ) using cycles of • c/ sec, • c/ sec, • c/ min. pcr products derived from gav-specific poly(a)+ rna were cloned into pgem-t. plasmid inserts were sequenced using fs-big dye reagent (abi), t , sp or gav-specific primers and abi model- sequencing apparatus at the australian genome research facility, brisbane. sequences were edited using seqed . . (abi) and analysed using macvector (oxford molecular). a contig of a nt sequence from the gav genomic -poly(a) tail to an intergenic -sgrna start site nt upstream of the orf start codon [ ] was deduced from overlapping cdna clones (figs. and ). the complete gav genome sequence is deposited in genbank acc no: af and ay and the orf gene sequence will be described elsewhere. the -terminal sequence contains a nt orf gene encoding a putative amino acid (aa) protein with a deduced molecular weight of da and pi of . . no polypeptides with significant similarity to the putative orf protein were identified in blast . [ ] searches of genbank. orf contains potential n-linked glycosylation sites and the netogly . program [ ] predicted potential o-linked glycosylation sites predominantly located in the n-terminal half of orf . six highly hydrophobic regions (g -f , s -t , f -a , a -l , f -a and y -i ), probably representing transmembrane (tm) domains, were predicted using tmpred [ ] (figs. and ) . prediction of membrane topology using tmhmm [ ] identified three likely orf ectodomains (fig. ) . the putative ectodomains, orf a ( cys), orf b ( cys) and orf c ( cys) following tm domains , and contained even numbers of cys residues reflecting the likelihood that they are involved in intra-domain disulphidebond formation. no heptad-repeat patterns indicative of the coiled-coil structures common to the structural glycoproteins of coronaviruses [ ] and toroviruses [ ] were predicted in orf using the coils program [ ] . the absence of the novel structure predicted for the putative gav orf glycoprotein suggests that it may be processed and function differently to the s glycoproteins of coronaviruses and toroviruses. at aa, orf is the largest glycoprotein yet identified for any virus in the nidovirales. the s glycoprotein gene of coronaviruses encodes polypeptides that range in size from aa for infectious bronchitis virus [ ] to aa for feline infectious peritoneal virus [ ] , while the cognate genes of the toroviruses berne virus and breda virus [ , ] encode polypeptides ( - aa) approaching the size of gav orf . moreover, while the s glycoproteins of these viruses contain hydrophobic regions representing a signal peptide and a single tm anchor, six hydrophobic tm domains are predicted in gav orf (fig. ) . although a cluster of basic amino acids (khlkvharhhk ) occurs in the central orf region between tm domains ᭤ fig. . nucleotide and deduced amino acid sequence of the open reading frames (orf and orf ) in the nt region of gav from the -sgrna start site in the intergenic region upstream of orf to the -poly(a) tail. the six hydrophobic orf regions predicted to represent transmembrane domains are underlined, the potential n-linked glycosylation sites are boxed and the cluster of basic amino acids between hydrophobic regions and are shaded. potential cleavage sites predicted using signalp [ ] are indicated ( ) fig. . a hydropathy plot of the amino acid gav orf generated using the method of kyte and doolittle [ ] using a window size of . b schematic diagram of the membrane orientation predicted using tmhmm [ ] showing the relative positions of the six predicted transmembrane domains, the potential n-linked glycosylation sites (•) and the two cleavage sites (arrows) predicted using signalp [ ] with potential to generate orf a, orf b and orf c proteins and , it is unlike the arg-rich 'rr(f/s/h)rr'-type proteolytic cleavage motifs involved in generating the s and s glycoprotein components of the virion spikes of coronaviruses and toroviruses [ , ] . furthermore, the predicted internal membrane orientation would preclude enzymatic cleavage in this region of gav orf . the gav genome contains conspicuously few genes (orf , orf and possibly orf ) other than the orf a- b replicase gene and immuno-electron microscopy using antibodies to the ∼ kda orf protein indicates that it forms a component of nucleocapsids [ja cowley et al. unpubl.] , suggesting it is likely to be the structural nucleocapsid protein. purified virions of yhv, which is genetically closely related to gav [ ] , contain only three major structural proteins (m r - , - and - kda), the largest of which is glycosylated [ , ] . as the gav orf protein is comparable in size to the - kda yhv protein, it can be deduced that orf must encode the virus spike glycoprotein and that post-translational processing would be required to generate polypeptides of a size estimated for the two larger yhv proteins. the gav orf sequence gives few clues to mechanisms by which such proteins might be generated. possible cleavage at sites (tfa -ke and asa -la) predicted after the third and fifth tm domains using signalp [ ] would generate three tm-anchored proteins, orf a ( aa = . kda), orf b ( aa = . kda) and orf c ( aa = . kda). the theoretical gav orf b and orf c proteins approach the size and, assuming that glycosylation could account for some additional mass, are plausibly equivalent to the two largest (i.e. - and - kda) yhv structural proteins [ , ] . however, this would require the use of these predicted tm domains as internal signal sequences as has been proposed for processing of the multiple membrane-spanning structural glycoproteins of alphaviruses [ , , ] and phleboviruses [ , ] . identification of the terminal sequences of the processed gav spike glycoproteins and experimental investigation of the six hydrophobic domains in orf is required to determine their role in processing, transport and membrane anchoring of the gav structural glycoproteins. the presence of three, and in a few cases four, membrane-spanning motifs is a structural characteristic preserved across the integral membrane (m) proteins of coronaviruses [ ] , toroviruses [ , ] and arteriviruses [ ] , even though these viruses possess distinct virion architectures. as gav appears not to possess a discrete m protein gene, it is tempting to speculate that the putative tri-membranespanning orf a portion of the orf protein might fulfil a similar function to the vertebrate nidovirus m proteins. however, no structural protein similar in size to the hypothesised . kda orf a protein of gav has been reported in purifiedyhv particles [ , ] . we are currently preparing antisera to recombinant fusion proteins of components of the predicted orf ectodomains to examine pro-polypeptide processing in gav-infected prawn tissues and the association and location of processed components in mature virions. the nt sequence downstream of orf to the gav -poly(a)-tail contains only one short ( aa) orf initiating nt downstream of the orf stop codon (fig. ) . the region between orf and orf contains a nt highly aurich sequence (a -a ) in addition to a region (a -c ) with limited homology ( / nt identical) to the highly conserved sequences encompassing the intergenic sgrna start sites upstream of orf and orf [ ] . however, the a residue corresponding to the start site of the orf and orf sgrnas is replaced by g residue in the region upstream of orf and we were unable to find evidence of an abundant sgrna for orf [ ] . further work is require to determine whether an orf protein is translated or whether the entire nt sequence downstream of orf represents the gav genome -untranslated region. oligo-dt-primed cdna prepared using three independent approaches was used to determine the -terminal sequence of the gav (+) ssrna genome. identification of the -terminus using cdna primed from the ∼ kb dsrna purified from gav-infected prawn tissue confirmed that this dsrna represents a genomic replicative intermediate and that the -end of the (+) sense strand is polyadenylated. equivalent sequences identified in clones generated using cdna primed similarly from either total or poly(a)-selected rna also supported previous data that the (+) sense genomic and sgrnas are -polyadenylated. it should be noted, however, that clones derived from a phage library produced using poly(a)+ rna from the whole head of a gav-infected p. monodon all possessed one to two point mutations (i.e. in lower case ..tgata(t/g)ga(a/c)(a/g)a n - ) within − to + nt of the poly(a) junction shown in fig. . however, these clones were generated using a non-anchored oligo-dt primer and significance of the point mutations is not yet clear. each of the five clones also possessed upstream point mutations ( to / nt) and two possessed single point deletions (c and a ) ∼ nt from the poly(a) junction. while rna from p. monodon experimentally-infected with the prototype gav isolate [ ] was used to derive the sequence shown in fig. , the more variable sequences occurred in clones derived from a prawn naturally infected with gav. thus, it is likely that the point mutations/deletions are attributable to rna quasispecies and/or virus strain differences. the genomic (+) rna -untranslated regions of coronaviruses and arteriviruses contain promoter sequences for transcription of negative-sense genomic and sgrnas [ , , , , ] . cell lines in which gav will replicate and/or reverse genetics systems will be required to gain an understanding of the -terminal sequences that are important to the genomic replication process in this crustacean nidovirus. the data reported here demonstrates that the (+) ssrna genome of gav is polyadenylated similarly to genomes of all viruses of the nidovirales. however, due to the apparent absence of discrete membrane (m) and nucleocapsid (n) protein genes downstream of the gav orf glycoprotein gene, independent approaches were employed to be confident that we had identified the true genomic -end. completion of the genome sequence of gav has revealed a gene organisation that is unique among the nidoviruses currently described. significantly, gav contains no discrete gene encoding an integral m protein and the gene (orf ) most likely to encode the viral n protein resides upstream of a gene (orf ) predicted to encode a large complex glycoprotein(s). only two subgenomic (sg)rnas are transcribed in abundance in gav-infected cells [ ] compared to five to nine sgrnas in vertebrate nidoviruses. moreover, similarly to toroviruses [ ] , there is no evidence of the complex leader-primed discontinuous transcription mechanism employed by coronaviruses [ ] and arteriviruses [ ] . these characteristics, taken together with the fact that the nt gav genome comprises relatively few genes, suggests that this crustacean nidovirus is evolutionary more primitive than its vertebrate counterparts. in this regard, it may be relevant to note that the natural penaeid host of gav has changed little from ancestral crustaceans that date back at least to the cambrian period > million years ago [ ] . we have previously suggested that gav, and the closely related yhv, should be considered as members of a new genus, okavirus, within the order nidovirales [ ] . the number and organisation of genes, the characteristic rod-shaped virion morphology [ , ] and the distinct transcription process [ ] supports the placement of the genus okavirus in a family distinct from the coronaviridae and arteriviridae, for which the name roniviridae (sigla, rod-shaped nidovirus) is proposed. gapped blast and psi-blast: a new generation of protein database search programs cloning and sequencing of the gene encoding the spike protein of the coronavirus ibv another triple-spanning envelope protein among intracellularly budding rna viruses: the torovirus e protein non-occluded baculo-like virus, the causative agent of yellow-head disease in the black tiger shrimp (penaeus monodon) the coronavirus surface glycoprotein 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nucleocapsid protein of breda virus a random-pcr method (rpcr) to construct whole cdna library from low amounts of rna nucleotide sequence of cdna coding for semliki forest virus membrane glycoproteins complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase elevating virus (ldv) netoglyc: prediction of mucin type o-glycosylation sites based on sequence context and surface accessibility evidence for a separate signal sequence for the carboxy-terminal envelope glycoprotein e of semliki forest virus tmbase -a database of membrane spanning protein segments complete sequence of the glycoproteins and m rna of punta toro phlebovirus compared to those of rift valley fever virus a simple method for displaying the hydropathic character of a protein coronavirus: organization, replication and expression of genome the molecular biology of coronaviruses handbook for cultivation of black tiger prawns predicting coiled coils from protein sequences yellow-head virus: a rhabdovirus-like pathogen of penaeid shrimp identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites nucleotide sequence of the s mrna of sindbis virus and deduced sequence of the encoded virus structural protein complete nucleotide sequence of the m rna segment of uukuniemi virus encoding the membrane glycoproteins g and g molecular cloning: a laboratory manual a new model for coronavirus transcription a phosphatocopid crustacean with appendages from the lower cambrian identification and primary structure of the gene encoding the berne virus nucleocapsid protein primary structure and post-translational processing of the berne virus peplomer protein a -coterminal nested set of independently transcribed mrnas is generated during berne virus replication the molecular biology of arteriviruses a hidden markov model for predicting transmembrane helices in protein sequences proc sixth international conference on intelligent systems for molecular biology lymphoid organ virus of penaeus monodon from australia gill-associated virus (gav), a yellow head-like virus from penaeus monodon cultured in australia a yellow head virus probe: application to in situ hybridization and determination of its nucleotide sequence arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences yellow head complex viruses: transmission cycles an topographical distribution in the asia-pacific region yellow head virus infection in the giant tiger prawn penaeus monodon cultured in taiwan yellow-head virus of penaeus monodon is an rna virus author's address: jeff cowley, csiro livestock industries, meiers road we kindly thank dr. sigrid lehnert for providing the prawn cdna phage library and dr. ross tellam for helpful discussions on glycoprotein processing.genome sequence of gill-associated virus key: cord- - sj rm authors: bald-blume, niklas; bergervoet, jan h. w.; maiss, edgar title: development of a molecular assay for the general detection of tospoviruses and the distinction between tospoviral species date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: sj rm a luminex xtag-based assay for plant-infecting tospoviruses was developed. the test enables the detection of tospoviruses in general and the differentiation of the four important member species of this genus: tomato spotted wilt virus, impatiens necrotic spot virus, the proposed ‘capsicum chlorosis virus’ and watermelon silver mottle virus. the generic tospovirus primers used in this method are also applicable for detection of tospoviruses by basic rt-pcr. we also describe an economic alternative method for the distinction of the four tospoviruses mentioned and of additional member viruses, based on a restriction fragment length polymorphism (rflp). the sophisticated luminex xtag technology allows the simultaneous detection of various targets. this study is part of a project that aims to develop a method for the simultaneous detection of various plant pathogens (viral, bacterial and fungal) in plant material. the genus tospovirus comprises all plant-infecting viruses of the family bunyaviridae. the other four genera of this family (orthobunyavirus, phlebovirus, nairovirus and hantavirus) contain animal infecting viruses. the genus tospovirus obtained its name from the type species tomato spotted wilt virus and contains approved species and tentative species [ ] [ ] [ ] . these viruses have quasi-spherical particles, with a diameter of - nm, enveloped by a host-derived membrane. the two glycoproteins gn and gc are embedded in this membrane. particles contain a tripartite single-stranded rna genome with negative-or ambi-sense polarity. the three rnas differ in size and are thus called large (l), medium (m) and small (s). all three rnas are incorporated in one particle but independently packaged by many copies of the nucleoprotein and a few copies of the viral rna-dependent rna polymerase [ , ] . tospoviruses have a large host range: tomato spotted wilt virus (tswv) infects for example , plant species belonging to families, including many economically important crop plants and numerous weed species [ ] . on the other hand, other tospoviral species have a more restricted host range. some of the members, such as tswv, impatiens necrotic spot virus (insv) and iris yellow spot virus (iysv), are found worldwide, while others are restricted to certain regions: for example watermelon silver mottle virus (wsmov) is restricted to asia, capsicum chlorosis virus (cacv) to asia and australasia and polygonum ringspot virus (polrsv) to europe [ ] . the same applies to the thrips vector species (insect order: thysanoptera): some have a worldwide distribution like frankliniella occidentalis [ ] , while others are restricted to single countries: for example thrips setosus can be found only in japan [ ] . both species of thrips are vectors of tswv [ ] . overall, fifteen species of thrips transmit tospoviruses in a persistent and propagative manner [ ] and are critical for their epidemiology [ ] . the host range and geographical distribution of the established tospovirus species have increased in recent years [ ] . additionally, new species belonging to the tospovirus genus have been proposed in recent years, such as 'alstroemeria necrotic streak virus' [ ] , 'pepper necrotic spot virus' [ ] and 'tomato necrotic spot virus' [ ] . based on the large loss of produce and economic damage caused, tospoviruses are thought to be the most devastating plant viruses [ , ] . this makes the diagnosis of tospoviral infections an important issue, especially for plant protection services trying to confine further spread of associated diseases. elisa, rt-pcr and quantitative or real time rt-qpcr can be used to detect tospoviruses and to discriminate different species. because of its robustness and sensitivity, elisa is often the standard method of choice for plant virus diagnostics [ ] . tospoviruses can be serologically detected by polyclonal antibodies against the nucleocapsid protein and distinguished into four major serogroups with tswv, wsmov, iysv and groundnut yellow spot virus (gysv) being the type species [ , ] . some tospoviruses have no clear serological relationship to the serogroups and are seen as distinct mono-serotypes [ ] . in most cases, monoclonal antibodies can also be used to classify tospoviral species in serogroups [ ] , but some tospoviruses with high amino acid sequence similarity such as cacv, wsmov, groundnut bud necrosis virus (gbnv) and watermelon bud necrosis virus (wbnv), are difficult to distinguish even with monoclonal antibodies [ , ] . this cross-reactivity has also been described when using commercial antibodies or elisa tests supplied by the leibniz institute dsmz-german collection of microorganisms and cell cultures (dsmz; braunschweig, germany), agdia (elkhart, usa) and loewe biochemica (sauerlach, germany). for tswv antibodies, possible cross-reactivity with groundnut ringspot virus (grsv), tomato chlorotic spot virus (tcsv) and chrysanthemum stem necrosis virus (csnv) are known. this cross-reactivity among species complicates distinction of different tospoviruses by elisa. additionally, an elisa test has to be performed independently for every species, since multiplexing is not possible. multiplex rt-pcrs can be a solution for this and have been described for the detection of tswv, insv, csnv, iysv and cacv [ ] as well as for detection of tswv, melon yellow spot virus (mysv), wsmov, insv and iysv [ ] . in this study a new method for plant virus diagnosis is described using the luminex xtag technology to test for tospoviruses in general and for the four species tswv, wsmov, insv and cacv. virus samples of tospovirus isolates from eight different species were obtained from the dsmz. the nucleic acid-based assay platform of the luminex xtag technology allows the simultaneous detection of theoretically up to analytes in one sample. tospoviral rna is transcribed into cdna and amplified in a first rt-pcr. the products are then subjected to a target specific primer extension (tspe) reaction, for which tagged primers and biotinylated dctp are used. the primer tags allow the hybridization of tspe products to complementary anti-tags coupled to luminex magplex-tag microspheres. these paramagnetic polystyrene ''beads'' are filled with a mix of two to three fluorescent dyes at different ratios, enabling their later identification through excitation and measured fluorescence and thus the identification of bound tspe products. the presence of hybridized tspe products is revealed through the binding of streptavidin-r-phycoerythrin to the incorporated biotin, its excitation and detection of the resulting fluorescence. the test procedure is described in detail by van der vlugt et al. [ ] . this technique has been used in human medicine for diagnosis of respiratory viruses such as influenza and coronaviruses, among others [ ] . in recent years, this technique has been adapted for the diagnostics of plant pathogens. van brunschot et al. detected and distinguished different begomoviruses and their whitefly vectors [ ] and different pospiviroids [ ] applying this method. lim et al. [ ] used it for the simultaneous detection of three lily-infecting viruses. together, these studies demonstrate the advantage of this approach which allows combination of tests for various pathogens to detect the most important diseases of a particular crop. our work should contribute to this end, as part of a project for the development of a simultaneous detection method for various viral, bacterial and fungal plant pathogens. additionally, this study describes a more economical approach for generic tospovirus detection by rt-pcr and for the differentiation of tospoviral species by restriction fragment length polymorphism (rflp). infected, dried plant material of tospoviral isolates from eight different species was obtained from the dsmz including single isolates of alstroemeria necrotic streak virus (ansv), cacv, grsv, iysv, tcsv and wsmov as well as three isolates each of insv and tswv. information about the viral origin (host plant, country of origin and provider) was supplied by the dsmz (table ) primers for general tospovirus detection, for pre-amplification and for tspe reactions were designed on the basis of alignments of nucleotide sequences. sequences of segment m were retrieved from genbank (national center for biotechnology information), imported into clc main workbench (clc bio, aarhus, denmark) and aligned. for the general detection primers (tospo_gens/as), the preamplification primers (tospo_outs/tospo_genas) and the generic tspe primers (ttospo_gens/as), the alignments were analyzed for conserved regions and corresponding sequences were chosen for the primers. some degenerate bases were inserted into primers sequences. for the species-specific tspe primers ttswvs/as, tinsvs/as, twsmovs/as and tcacvs/as, conserved regions for each of the species were identified in the alignments and relevant sequences used for the primers. as internal control primers we used the pre-amplification primers (nad s/as) for the nadh dehydrogenase subunit gene (nad ) from menzel et al. [ ] and the tspe primer (tnad ) for nad from van brunschot et al. [ ] and adapted from botermans et al. [ ] . the tag sequences were added to all tspe primers by luminex (austin, usa) and were complementary to antitags on the microspheres' surfaces. the primers were synthesized by eurofins scientific (luxembourg). table displays their characteristics. viral rna was transcribed into cdna and then amplified using the access rt-pcr system kit (promega, fitchburg, usa), using the concentrations specified by the manufacturer in a volume of ll, in covered -well multiply pcr plates (sarstedt, nuembrecht, germany). the following incubations were carried out: min at °c, min at °c, cycles of s at °c, s at °c and s at °c, followed by a final extension of min at °c. the internal control primers (nad s/as) and the degenerate primers (tospo_outs/tospo_genas) were used, enabling the production of a bp fragment for all plant samples and a bp fragment for all tospoviruses. after rt-pcr, ll of each product were stained with ll of loading buffer ( . % glycerol, . % bromophenol blue, x gelred [biotium, hayward, usa]) and loaded on an agarose gel ( %) to visualize the expected fragments. the pre-amplification products were directly used for a multiplex tspe reaction. primers were extended and biotin- -dctp (thermo fisher scientific, waltham, usa), instead of normal dctp, was incorporated, alongside with the remaining unmodified nucleotides (datp, dgtp, dttp; thermo fisher scientific). a set of eleven primers was used: two generic primers, to detect all tospoviruses (ttospo_gens/as), eight specific primers, to identify four tospovirus species (ttswvs/as, tinsvs/as, twsmovs/as and tcacvs/as) and one plant internal control primer (tnad ) for nad . tspe mixes of ll containing ll of the pre-amplification products were prepared ( . u platinum genotype tsp dna polymerase [thermo fisher scientific], lm biotin- -dctp, lm each of after hybridization the mixtures were moved to cellstar -well cell culture plates (greiner bio-one, kremsmuenster, austria) and the microspheres were pelleted on a magnetic separator for min. the supernatants were discarded and the beads resuspended and washed twice in ll x tm hybridization buffer per well. the supernatants were removed again and ll of x tm hybridization buffer containing streptavidin-r-phycoerythrin ( lg/ml; thermo fisher scientific) were added to each well. the plates were protected from light and incubated on a shaker ( rpm) at room temperature for min. after pelleting the microspheres again, the supernatants were discarded and the microspheres resuspended in ll of x tm hybridization buffer per well. finally, ll per sample were analyzed in a luminex system with the xponent software (version . ; luminex). a red laser ( nm) excited the bead dyes and a green laser ( nm) excited the r-phycoerythrin, bound via streptavidin and biotin to the tspe products. the fluorescence of the r-phycoerythrin was recorded as the median fluorescence intensity (mfi) signal, allowing identification of samples containing amplified plant or tospoviral nucleic acids. identification of these nucleic acids could be achieved by the fluorescence of the bead dyes, which are unique for each microsphere with its specific anti-tag. three independent measurements were performed with the three rna extractions described above. for the general detection of tospoviruses, rna from infected plant material was transcribed into cdna by incubating ll of water, ll of rna, ll of primer tospo_genas ( lm) and . ll of dntp mix ( mm each) at °c for min. the reaction mix was then cooled on ice and . ll of revertaid reverse transcriptase ( u/ ll; thermo fischer scientific) and ll of x reaction buffer (revertaid; thermo fisher scientific), were added. the mixture was then incubated at °c for min. the cdna was amplified in a reaction mix consisting of ll of phusion flash high-fidelity pcr master mix (thermo fisher scientific), ll of water, ll of cdna, ll of primer tospo_gens and ll of primer tospo_genas and was incubated under the following conditions: one cycle for s at °c, cycles of s at °c, s at °c and s at °c and a final extension of min at °c. pcr products were analyzed by gel electrophoresis on an agarose gel. for distinction of tospoviruses by rflp, an rt-pcr was performed like above and the products were digested with the restriction enzymes hinfi (thermo fisher scientific) and bcli (new england biolabs, ipswich, usa). for this, ll of each pcr product were mixed with ll of hinfi ( u/ll), ll of bcli ( u/ll), ll of x fastdigest buffer (thermo fisher scientific) and ll of water and incubated at °c for one hour. rflp reactions were visualized using gel electrophoresis. we verified by conventional rt-pcr experiments that the pre-amplification and generic primers worked with tospovirus rna and that the species-specific primers were specific for the corresponding virus species (data not shown). after pre-amplification with the four primers tospo_outs/tospo_genas and nad s/as, the rt-pcr products were used in tspe reactions with all eleven tspe primers (ttospo_gens/as, ttswvs/as, tinsvs/as, twsmovs/as, tcacvs/as and tnad ). tspe products were hybridized to the corresponding eleven magplex-tag microspheres listed in table and the hybridization mix was analyzed. three independent luminex tests were conducted to verify the reliability of the method. every test included new rna extractions, pre-amplifications, tspe reactions, hybridizations and luminex measurements. the mean values of the mfis of the three luminex measurements were determined and plotted in fig. . the generic primer ttospo_gens detected most tospoviruses, except iysv and wsmov. the positive signal ranged from for one insv isolate to , for one tswv isolate. iysv-and wsmov-infected plant material produced a signal of and , respectively, not exceeding the threshold set at (light grey columns in fig. ) . the internal control tspe primer (tnad ) detected the nad gene in previously amplified plant material in almost all samples, with mfis ranging from for one insv isolate to , for cacv. only in the wsmov sample the threshold was not reached, with a value of (checkered columns in fig. ) . each species-specific sense tspe primer correctly identified the target virus species it was designed to detect. the tcacvs primer produced a high signal of , in the cacv infected samples but no signal was detected in any other sample or control (black columns in fig. ) . the tinsvs primer lead to high mfi values of , to , only in the samples infected with the three insv isolates (diagonally striped columns in fig. ). the signal induced by the twsmovs primer was slightly lower ( ) and also restricted to samples infected with wsmov (white columns in fig. ). the mfi values generated by the ttswvs primer were also slightly lower ( - ) and restricted to samples containing the three tswv isolates tested. in this case, a slight reaction (mfi of ) also occurred in plants infected with ansv (dark grey columns in fig. ). the five antisense tspe primers (ttospo_genas, ttswvas, tinsvas, twsmovas and tcacvas) were tested as alternatives for the sense primers, but they did not generate satisfactory results in luminex tests for tospovirus detection and distinction (data not shown). as a threshold for all tests, an mfi value twice the mfi value of the water control from the tnad test was chosen, because this sample showed the highest mfi value of all healthy plant and water controls. the values of the other tests were comparable, but were left out of the figure for the sake of clarity (dashed line in fig. ). the generic tospovirus primers tospo_gens/as (without tags) allowed the detection of all eight tospoviruses and of all three isolates of insv and tswv tested in rt-pcr experiments. the expected fragment of about bp was produced when using rna extracted from infected plants. the primers generated no band when using rna from healthy plants or from plants infected with other viruses like cucumber mosaic virus (cmv), plum pox virus (ppv) and pepper mild mottle virus (pmmov) (fig. ) , demonstrating the specificity of the primers. the four species-specific primer pairs (without tags) also worked in normal rt-pcrs for the identification of these species (data not shown). these primers led to the production of the expected fragments only when rna from plant material infected with the corresponding virus was used. for tswv the specific fragment was bp in size and for cacv, insv and wsmov bp. primer fig. results of a rt-pcr with rna from plants infected with tospovirus isolates, cmv, pmmov and ppv as well as rna from healthy plants and a water control using primers tospo_gens/as after gel electrophoresis. enterobacteria phage k dna digested with psti was used as molecular-weight size marker. dna fragments appear larger than the expected bp, because the dna stain gelred changes the migration speed of dna, depending on the concentrations of dna and gelred [ ] sequences can be deduced from table omitting the tag sequences in italics. most of the examined tospovirus species could be distinguished by a rflp analysis using the restriction enzymes hinfi and bcli after rt-pcr with primers tospo_gens/ as. the pcr products of the tospoviruses have different restriction enzyme recognition sites which leads to a distinct pattern for most tospoviruses after gel electrophoresis. rflp analysis was first performed in silico using the software clc main workbench and tospoviral segment m sequences from genbank, to predict fragment sizes. after rt-pcr, digestion and rflp analysis, the predicted fragments were observed for most viruses: for tswv these were and bp in size, for insv they were , and bp, for wsmov , , and bp, for cacv , and bp, for grsv and bp and for tcsv they were , and bp. for iysv, fragments of , and bp were detected, but a predicted bp fragment was not detected. for ansv no predictions could be made in silico because for this virus only a partial segment s sequence (containing the nucleocapsid protein gene) is available. nevertheless, the rflp pattern for the ansv was determined and fragments of about and bp were detected. therefore, the rflp pattern of ansv was similar to that of grsv and tswv, and these three viruses could not be clearly differentiated. cacv, insv, iysv, tcsv and wsmov could be distinguished from these viruses and from one another (fig. ). a molecular assay for the detection of tospoviruses in general and for viruses from the four species belonging to this genus (tswv, insv, cacv and wsmov), using the luminex xtag technology, was successfully developed. the generic tospovirus test with primer ttospo_gens detected six of the eight tested species. it failed to prove the presence of iysv and wsmov. however, the same primer without its tag, in combination with an antisense primer, detected these two viruses in rt-pcr experiments. these rt-pcr results suggest that with further optimization, the generic tospovirus luminex test could be improved to detect all eight species. the failure to detect iysv and wsmov might be a sensitivity problem of the generic primer in the tospovirus luminex test. the virus concentration in these samples could be too low for detection, as the tspe reaction only linearly amplifies the target, while a conventional pcr leads to an exponential amplification. alternatively, primers might not bind perfectly to target sequences or the tag sequences of the tspe primers might fig. results of a rflp after gel electrophoresis. rna from plants infected with tospovirus isolates was transcribed and amplified in a rt-pcr with primers tospo_gens/as. pcr products were then digested using hinfi and bcli. the o'generuler ultra low range dna ladder (thermo fisher scientific) was used as molecular-weight size marker. the figure was assembled from two gels interact non-specifically with plant or viral sequences. in this case, adaptations of the primer or tag sequences might solve the problem. the plant internal control primer tnad detected its target except in the case of wsmov samples. this result probably points to a low nucleic acid concentration in the wsmov samples preventing successful detection of the internal control due to a sensitivity problem in this luminex assay. by increasing the sample number, the low nucleic acid concentration and low mfi values of single samples in the hybridization mix would probably be corrected, leading to exceedance of the threshold. the species-specific tests using the primers tcacvs, tinsvs, ttswvs and twsmovs were specific for the viruses they were designed to detect. in the case of tinsvs and ttswvs, all three isolates of each of these two viruses were successfully detected. this study is one of only a few employing the luminex xtag technology for the detection of plant pathogens and the first for tospoviruses. so far it has been applied to screen for begomoviruses and pospiviroids [ , ] , for lily mottle virus (lmov) and lily symptomless virus (lsv) in lily plants [ ] and for cmv and its two subgroups [ ] . the related luminex xmap technology utilizing coupled antibodies instead of oligonucleotides has been used to identify potato virus x (pvx), potato virus y (pvy) and potato leaf roll virus (plrv) [ ] as well as plum pox virus (ppv) [ ] in plant material. however, antibodies cross reactivity between different tospoviral species has been described by researchers [ , ] and antisera suppliers (dsmz, agdia and loewe biochemica). this may be a problem in the antibody-based luminex xmap test for the differentiation of tospovirus species, and hence a nucleic acid based array is thought to be advantageous. an advantage of both these assay formats is their multiplexing capability and their ability to simultaneously detect various diseases in plant samples. the study of lim et al. [ ] is an example of this since they used a luminex xtag assay for the detection of the three viruses (cmv, lmov and lsv) infecting lily plants. the standard method for virus detection (elisa) lacks this potential and is quite labor-and time-intensive as it only allows to test for one virus at a time. charlermroj et al. [ ] have created a multiplex antibody array similar to an elisa for the simultaneous detection of the three viruses mysv, wsmov and chilli veinal mottle virus (chivmv) as well as of the fruit blotch bacterium acidovorax avenae subsp. citrulli. all four pathogens were immobilized by capture antibodies specific to the four pathogens in each well at preassigned positions, detected by fluorescently conjugated secondary antibodies and identified by their position in the microwells. such multiplexed antibody array technologies are still under development and are unlikely to be used for routine plant pathogen detection. a mixed detection method combining rt-pcr and elisa was developed and applied for the detection of the four tospoviruses cacv, mysv, tomato necrotic ringspot virus (tnrv) and wsmov. using this technique, the rna is first transcribed and amplified by rt-pcr using degenerate primers and digoxigenin (dig) labelled dutp, then the pcr products are hybridized to four species-specific biotinylated probes in streptavidincoated microtiter wells and finally the labelled and hybridized pcr products are detected by elisa using a peroxidase-conjugated anti-dig antibody [ ] . our luminex xtag test for tospoviruses could be combined with already existing and prospective tests for plant pathogens to create assays that can identify crops' most important diseases, similar to the respiratory virus panel test developed by mahony et al. [ ] that screens for different human respiratory viruses and their subtypes. for example, for tomato crops (solanum lycopersicum) we could combine the tests for tswv, cacv and insv from this study, for tomato yellow leaf curl virus (tylcv) from van brunschot et al. [ ] , for tomato apical stunt viroid (tasvd), tomato chlorotic dwarf viroid (tcdvd) and tomato planta macho viroid (tpmvd) from van brunschot et al. [ ] as well as the test for cmv from lim et al. [ ] or from bald-blume et al. [ ] . further tests could be added to cover the most important of the viruses infecting tomatoes [ , ] and also bacterial and fungal pathogens. a more economical alternative for generic tospovirus detection and species distinction was successfully developed in this study. the primer tospo_gens/as detected rna of all eight examined tospoviruses in rt-pcrs but not of viruses from other genera. this is one of the best coverages of tospoviral species obtained by one primer pair so far. hassani-mehraban et al. [ ] reported an overview of universal, degenerate and multiplex primers for tospovirus detection from eleven studies (details of covered species are reported in table of [ ] ). chen et al. [ ] designed two degenerate primer pairs that transcribed and amplified rna of species. one of the pairs binds to segment l and the other to the nsm gene of segment m. their antisense primer gm c for segment m partially coincides with our tospo_genas primer. since our primer is more degenerated, our primers might also identify rna from the additional tospoviral species gbnv, calla lily chlorotic spot virus (ccsv), mysv, wbnv and tomato yellow ring virus (tyrv) tested by chen et al. [ ] , although we have not tested this. hassani-mehraban et al. [ ] describe six primer pairs and rt-pcrs that cover assigned and tentative tospoviral species and classify them into six subgroups. also, the four species-specific primer pairs (without tags) can be used in rt-pcrs for the identification of these species. they lead to the amplification of nucleic acids only of the corresponding species and of all three isolates tested from either tswv or insv. they cannot be used for multiplex rt-pcrs, because the cacv, insv and wsmov specific primers lead to fragments of the same size. so a distinction of these species would not be possible by a plain multiplex rt-pcr. the fragments of the generic tospovirus primers and the tswv specific primers can be distinguished from these primers and from one another, and could be combined with one of the other four primer pairs for a clear multiplex rt-pcr. kuwabara et al. [ ] described such a multiplex rt-pcr for the detection of the five tospoviruses tswv, insv, csnv, iysv and cacv. alternatively, rt-pcr products of the different tospovirus species generated with the generic primers tospo_-gens/as could be differentiated by rflp using the restriction enzymes hinfi and bcli. the five species cacv, iysv, insv, tcsv and wsmov were clearly distinguished. the additional three species ansv, grsv and tswv could be discriminated from the rest but not clearly from each other. chu et al. [ ] used this method to detect and differentiate the five tospoviruses tswv, grsv, insv, wsmov and peanut chlorotic fan-spot virus (pcfv) using primers binding to segment l and the restriction enzyme xbai. the three methods described in this study for tospovirus detection and species differentiation (luminex xtag, rt-pcr and rflp) are comparable in detection efficiency. the luminex xtag assay seems to be less sensitive than the other two methods. however, an advantage of the luminex xtag technology is the high multiplexing potential leading to a reduction in labor when testing for several possible tospoviruses and other plant pathogens. virus taxonomy: ninth report of the international committee on taxonomy of viruses generic rt-pcr tests for detection and identification of tospoviruses international committee on taxonomy of viruses negative-strand rna viruses: the plant-infecting counterparts tospoviruses in the mediterranean area an update of the host range of tomato spotted wilt virus global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead the spread of the western flower thrips frankliniella occidentalis (pergande) life history study of thrips setosus thrips transmission of tospoviruses a distinct tospovirus causing necrotic streak on alstroemeria sp. colombia pepper necrotic spot virus, a new tospovirus infecting solanaceous crops in peru identification of a new tospovirus causing necrotic ringspot on tomato in china a top ten list for economically important plant viruses top plant viruses in molecular plant pathology the detection of viruses by enzyme-linked immunosorbent assay (elisa) serological relationship between melon yellow spot virus and watermelon silver mottle virus and differential detection of the two viruses in cucurbits monoclonal antibodies for differentiating infections of three serological-related tospoviruses prevalent in southwestern china serological comparison and molecular characterization for verification of calla lily chlorotic spot virus as a new tospovirus species belonging to watermelon silver mottle virus serogroup emerging problems of tospoviruses (bunyaviridae) and their management in the indian subcontinent purification and serological analyses of tospoviral nucleocapsid proteins expressed by zucchini yellow mosaic virus vector in squash improved multiplex reverse transcription-polymerase chain reaction to detect and identify five tospovirus species simultaneously a one-step reverse transcription-polymerase chain reaction system for the simultaneous detection and identification of multiple tospovirus infections multiplex detection of plant pathogens through the luminex magplex bead system development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay a beadbased suspension array for the multiplexed detection of begomoviruses and their whitefly vectors development of a multiplexed bead-based suspension array for the detection and discrimination of pospiviroid plant pathogens simultaneous detection of three lily-infecting viruses using a multiplex luminex bead array detection of four apple viruses by multiplex rt-pcr assays with coamplification of plant mrna as internal control development and validation of a real-time rt-pcr assay for generic detection of pospiviroids development of a molecular assay for the detection of cucumber mosaic virus and the discrimination of its subgroups i and ii multiplex microsphere immuno-detection of potato virus y, x and plrv use of luminex xmap-derived bio-plex bead-based suspension array for specific detection of ppv w and characterization of epitopes on the coat protein of the virus antibody array in a multiwell plate format for the sensitive and multiplexed detection of important plant pathogens development of a multiplex rt-pcr-elisa to identify four distinct species of tospovirus emerging viral diseases of tomato crops plant viruses online: descriptions and lists from the vide database genomic characterization of calla lily chlorotic spot virus and design of broad-spectrum primers for detection of tospoviruses completion of the genome sequence of watermelon silver mottle virus and utilization of degenerate primers for detecting tospoviruses in five serogroups simple and practical staining of dna with gelred in agarose gel electrophoresis acknowledgements we thank prof. dr. med. rainer blasczyk and prof. dr. med. stephan immenschuh of the institute for transfusion medicine of hannover medical school (mhh) for providing their luminex system and dr. eva zilian, susanne aufderbeck and stefanie vahlsing of the same institute for their support. funding this study was partly funded by the eu interreg iv a program gezonde kas (healthy greenhouse). the authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- -n kh u authors: percy, d. h.; williams, k. l.; paturzo, f. x. title: a comparison of the sensitivity and specificity of sialodacryoadenitis virus, parker's rat coronavirus, and mouse hepatitis virus-infected cells as a source of antigen for the detection of antibody to rat coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: n kh u sialodacryoadenitis virus (sdav) and parker's rat coronavirus (prc) are two recognized viral strains which cause spontaneous disease in the laboratory rat. currently there is no recognized practical procedure which will accurately differentiate infections with these strains. using sdav- and prc-infected l- cells as the source of antigen, and sera from rats collected post inoculation with either of these viral strains, the indirect fluorescent antibody (ifa) procedure was used to determine whether antibody titers could be used to differentiate infections from the homologous and heterologous virus. there was no detectable difference in the sensitivity or specificity of these systems in detecting antibody to the homologous or heterologous virus. thus there was no evidence that sdav- and prc-infected cells would serve to differentiate antibody to the homologous virus using the ifa technique. in addition, antibody titers were similar when mouse hepatitis virus (mhv)-infected cells were used as the source of antigen for the ifa technique. however, using mhv or sdav-infected cells as the source of antigen, there was a significant difference in antibody titers to the homologous virus detected using the immunoenzyme technique. the indirect fluorescence antibody (ifa) test is a recognized, sensitive method to detect antibody to specific rodent viruses. one method used for the detection of coronaviral antibodies in rodent sera consists of equal proportions of cells infected with the jhm and s strains of mouse hepatitis virus (mhv) as a source of antigen, together with uninfected cells. using this as a source of antigen, the indirect fluorescent antibody procedure has proven to be a test system with d.h. percy et al. good sensitivity for the detection of animals seropositive for routine coronaviruses [ ] . similarly, enzyme i m m u n o a s s a y (eia) procedures have been developed for antibody detection and titration. using the horseradish peroxidase or the urease assay, antibody titers m a y be substantially higher than those detected with the i f a technique [ ] . using the l- subline of l- cells, we have been able to replicate relatively high titers of sialodacryoadenitis virus (sdav) [ ] , and parker's rat coronavirus (prc) [ ] in vitro. the purpose of this study was to compare the sensitivity of sdav or prc-infected l- cells with mhv-infected cells for the detection and titration of antibody to s d a v or prc. the techniques used were i f a and an i m m u n o e n z y m e procedure. the l- subline of l- cells for the replication of sdav or prc was obtained from dr. v. l. morris, university of western ontario, london, ontario. mhv-infected nctc cells, prepared as previously described for l- cells [ ] were acquired from dr. a. l. smith, section of comparative medicine, yale school of medicine. the # strain of sdav (family coronaviridae) was obtained from dr. p. n. bhatt, yale school of medicine. the isolate of prc (family coronaviridae) was obtained from the american type culture collection, rockville, md. sdav and prc were replicated in l- cells as previously described [ , ] . for the preparation of viral antigen from rat coronaviruses, l- cells were propagated in x mm nunc polystyrene tissue culture dishes (gibco/brl inc, burlington, ont.) in eagle's minimal essential medium (gibco/brl inc). cells were grown in a humidified atmosphere containing % co . when approximately % confluent, monolayers were inoculated with stock virus suspension, and incubated for h at °c in % co . infected and control cells were then removed using a rubber policeman, placed in two separate centrifuge tubes, and centrifuged at rpm, then resuspended in pbs. cells were then mixed at one part of infected to . parts of noninfected cells. gl of the mixture were then dropped on each well in multiwell depression glass slides (gibco/brl inc). slides were air dried for h, fixed in acetone, and stored at - °c until tested. spf wistar rats were obtained at approximately weeks of age from a commercial supplier (charles river laboratories, st. constant, quebec). animals were housed in an isolation facility throughout the inoculation period. in separate studies and at different time periods, rats were inoculated intranasally with approximately tcids of either the atcc strain of prc or strain # of sdav (second passage in l- cells). animals were killed with an overdose of pentobarbitone sodium (mtc pharmaceuticals, mississauga, ont.) at , , , and days post-inoculation (pi), sera were collected by cardiac puncture, and tissues were collected from control and inoculated animals for histopathology. sera were tested for antibody titers using the following virus-infected cells as antigens: sdav, prc, and mhv. serum samples were diluted in pbs, then individual wells were flooded with the appropriate dilution and processed as previously described [ ] . fluorescein-labelled anti-rat globulin (antibodies inc., davis, ca) was used to identify positive samples. evans blue at . % was used as a counterstain. specimens were examined using a halogen-illuminated epifluorescence microscope at x and x magnifications. all samples were read as unknowns to avoid bias. titers were recorded as the highest dilution of the serum sample under test showing fluorescence. the differences between the mean values for the antibody titers to the homologous and heterologous viruses in each study were tested for significance using the general linear model of sas (glm). for the immunoenzyme technique, sera collected from spontaneous cases of sda were used to evaluate the sensitivity of mhv and sdav-infected cells for antibody titration. the procedure, using horse radish peroxidase-conjugate anti-rat igg, has been previously described [- ] . typical changes [ , ] cells (fig. a) . with l- cells inoculated with p r c and prepared for the i f a techniqe, cytoplasmic fluorescence was present most frequently in single or binucleated cells (fig. b) . using the i f a procedure and mhv-, sdav-, or prc-infected cells as a source of antigen and sera from rats exposed to s d a virus, antibody titers to all three antigens were similar (table ). there was no statistically-significant difference in antibody titers to the three antigens (p < . ). similarly, in a comparison of antibody titers in rats inoculated with prc, antibody titers were similar ( table ). there was no significant difference in the m e a n antibody titers to the homologous virus c o m p a r e d to the heterologous viral antigens (p < . ). using mhv-infected n c t c cells and sdav-infected l- cells as the source of antigen, serum antibody titers of sdav-infected rats were usually substantially higher with homologous antigen than with the mhv-infected cells (table ). in a comparison of the m e a n antibody titers, the antibody titers to a sialodacryoadenitis virus b parker's rat coronavirus c mouse hepatitis virus * no significant difference in the means of the antibody titers to the three viral antigens (p < . ) table . comparison of antibody titers in rats exposed to parker's rat coronavirus using sdav, prc, and mhv-infected cells * no significant difference in the means of the antibody titers to the three viral antigens (p < . ) the homologous viral antigen were significantly higher than those detected using the heterologous virus (p < . ). the ability to replicate relatively high titers of rat coronaviruses in a continuous cell line has provided the opportunity to evaluate the sensitivity and specificity of rat coronaviral-infected cells compared to mhv-infected cells as a source of antigen for the detection and titration of antibody to sdav and prc. the antigenic similarities among the rodent coronaviruses have been emphasized in previous studies [ , ], thus allowing the use of mhv-infected cells as a source of antigen for the detection of antibodies to either mouse or rat coronaviruses [ ] . based on our studies, the use of rat coronavirus-infected cells as a source of antigen offers no significant advantage over mhv-infected cells. furthermore, we were unable to demonstrate any appreciable advantage in using either sdav or prc as a source of viral antigen to detect antibody to the homologous virus, either in sensitivity or specificity. based on these data, there is no evidence that ifa staining of l- cells infected with either sdav or prc will serve to differentiate antibody to the homologous or heterologous virus. however, using m h v or sdav-infected cells as a source of antigen for the enzyme immunoassay procedure, there was a significant difference in the anti-sdav antibody titers using the homologous virus (p < . ). sda antibody titers were usually considerably higher using the enzyme immunoassay procedure with homologous antigen than with mhv-infected cells. characterization of the virus of sialodaeryoadenitis of rats: a member of the coronavirus group the laboratory rat. biology and diseases rat coronavirus (rcv): a prevalent, naturallyoccurring pneumotropic virus of rats replication of sialodacryoadenitis virus in mouse l- cells characteristics of parker's rat coronavirus (prc) replicated in l- cells an immunofluorescence test for detection of serum antibody to rodent coronaviruses two enzyme immunoassays for detection of antibody to rodent coronaviruses sialodacryoadenitis virus-associated lesions in the lower respiratory tract of rats this work was supported by natural sciences research council grant #a , the ontario ministry of agriculture and food, and usphs grant rr . the technical assistance of ted ]eaton and jean bagg are gratefully acknowledged. special thanks to dr. jeff wilson, population medicine, university of guelph, for the statistical analyses. received november , key: cord- -h fwi qn authors: li, q.-g.; lindman, k.; wadell, g. title: hydropathic characteristics of adenovirus hexons date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: h fwi qn the complete nucleotide sequence and the predicted amino acid sequence of the adenovirus type hexon gene were determined. the hydro-pathy of the hexon proteins from human adenovirus types , , , , , , , , , and , bovine adenovirus type , murine adenovirus type , and avian adenovirus types and was analysed. the presence of purines and pyrimid-ines in the second position of the codons was correlated to hydrophilicity and hydrophobicity, respectively. comparison of the hydrophilicity plots of eight hexons showed seven hypervariable regions to be distributed on the surface. a large portion of the hypervariable regions manifests hydrophilicity. the strength of the surface charge accumulated on the hydrophilic and hydrophobic regions correlated to the tissue tropism of the different adenovirus types. analysis of codon usage for adenovirus hexons showed that among synony-mous codons those with cytidine in the third position were preferably used to a great extent. analysis of the nucleotide and amino acid sequence pair distances and the phylogenetic tree of hexon proteins showed members of subgenera b, d and e to be closely related, especially ad and ad , and subgenus a to be closely related to subgenus f. the family adenoviridae comprises two genera mastadenovirus and aviadenovirus. the genus mastadenovirus consists of species that have been isolated from nine different host species [ ] . the most extensively studied group is that of the human adenoviruses. so far, human adenovirus (ad) serotypes have been identi®ed [ ] and divided into six different subgenera, a to f, which differ from each other in various characteristics such as tropism. adenovirus type (ad ) is the serotype most frequently associated with severe diseases [ ] . adenoviruses are non-enveloped icosahedral viruses. the virion contains at least eleven different structural polypeptides. the hexon is the most abundantly produced protein. each virion contains hexons which form the facets of the icosahedron. the nine central hexon capsomers in each facet are cemented together by polypeptide ix to form a group-of-nine [ ] . the hexon is a homotrimer consisting of three identical polypeptide chains. the monomer is an unusually large structural protein with a m r ranging from ± k ( table ) . as several entire or partial hexon sequences of different serotypes have been published (references cited in table ), the hexons of several different serotypes have been compared at the nucleotide and predicted amino acid sequence levels [ , , , ] . the biochemical and immunological properties and even the threedimensional structure of adenoviruses have been extensively studied. the trimeric hexon molecule has a pseudo-hexagonal base with a large central cavity and a triangular top. the base contains two pedestal domains, p and p . the top contains three long loops l , l and l [ ] . however, the hydropathic character of the adenovirus hexons and the codon usage has not been analysed. the hydropathy of a protein is very important for predicting putative antigenic regions of the protein. the antigenic determinants can be deduced by searching the amino acid sequence for the areas of greatest local hydrophilicity. generally, the highest peak of hydrophilicity correctly predicts an antigenic site [ ] . codon usage bias for codons with thymidine (t) or cytidine (c) at second positions in mitochondrial dna has been shown to be correlated with hydrophobicity [ ] . having analysed nine adenovirus hexon genes, we found the presence of purine and pyrimidine at the second codon position to be correlated to hydrophilicity and hydrophobicity, respectively [ ] . this ®nding was recently con®rmed by analysis of further data obtained from genbank. here, we report the hydropathy analysis of adenovirus hexon sequences predicted from a newly determined ad hexon dna sequence and thirteen published hexon sequences of ad , ad , ad , ad , ad , ad , ad , ad , ad , bav , mav , fav and fav . there is strong correlation between hydrophilicity and the codons with purine in the second position (cpuss), and between hydrophobicity and the codons with pyrimidine in the second position (cpyss) in adenovirus hexons. comparison of the surface charge on these hexons suggests the strength of the surface charge accumulated in hydrophilic regions or hydrophobic regions to be correlated with the tissue tropism of the different adenovirus types. the ad prototype strain gomen, originally from the american type culture collection (atcc), was obtained from dr. g. von zeipel, stockholm county council central microbiological laboratory, stockholm, sweden [ ] . this strain was propagated in a cells. the viral dna of ad strain gomen was prepared as described [ ] . the puri®ed dna was used as the template for pcr to amplify ®ve fragments which covered the entire hexon gene. the pcr products were cloned into pt blue vector. ligation, transformation and rapid screening were performed using the pt blue t-vector kit protocols (novagen, madison, wi, usa). the recombinant pt -blue plasmid dna was prepared for sequencing. as the pcr method occasionally gave rise to error when the complementary dna strand was synthesised using taq polymerase, the sequence so obtained was con®rmed with the following procedure: the ad hexon dna bam hi restriction fragments a and e, which contain the hexon gene, were cloned into plasmid vector pbr and multiplied in e. coli strain hb using conventional methods described previously [ ] . these recombinant plasmid dnas were used as the sequencing templates. the nucleotide sequences were determined for both strands with the dideoxynucleotide chain determination method, following procedure c of the autoread sequencing kit (pharmacia lkb biotech, sweden) and the manufacturer's instructions for the alf dna sequencer (pharmacia). all the sequence data were analysed with the lasergene software (dnastar inc., madison, wi) programs, editseq, megalign and protean. every hexon dna sequence was translated to protein sequence by using program editseq-translation. the codon usage data could be obtained when a protein ®le was opened by editseq. the hydrophilicity plot (kyte-doolittle method) could be shown by program protean. all the eight hydrophilicity plots could be moved together by the program microsoft powerpoint (fig. ). the isoelectric point and the net charge of a protein could be obtained by program protean-composition (table ) . a tabular data of hydrophilicity and hydrophobicity for each amino acid could be obtained by program protean-tabulardata. the accumulated charge of all hydrophilic regions of a protein could be obtained by deleting manually all the hydrophobic regions according to the tabular data. an easier way was that, ®rst, to delete the hydrophobic regions within dna sequence; second, to translate this dna sequence to an amino acid sequence; then, to obtain the accumulated charge of hydrophilic regions by program protean-composition. the accumulated charge of hydrophobic regions could be obtained in the same way ( table ) . the data of codon usage of accumulated hydrophilic and hydrophobic regions from different serotypes were analysed by the computer program microsoft excel (tables and ). the complete hexon gene dna sequence of ad prototype strain, gomen, was determined. it consists of nucleotides. this dna sequence, together with fig. . the ®gures around the boxes denote the amino acid numbers at the start and the end of each hypervariable region two short¯anking regions at each end, has been entered in the european molecular biology laboratory (embl, accession number: z ). the h anking region has a splice acceptor site. the h¯a nking region ends just before the start codon of proteinase. the sequence of the predicted protein, consisting of amino acids, was obtained with the lasergene software program editseq. the hydropathy data of hexon proteins from human adenovirus types , , , , , , , , , and , bovine adenovirus type , murine adenovirus type , and avian adenovirus types and were derived using the prediction method of kyte-doolittle in the lasergene computer program protean. analysis of the codon usage for the codons corresponding to hydrophilic and hydrophobic regions showed the presence of cpuss and cpyss to be strongly correlated with hydrophilicity and hydrophobicity, respectively. statistical analysis using the chi square test showed a highly signi®cant difference ( x y p` x ) for the total numbers of cpuss and cpyss in hydrophilic and hydrophobic regions ( table ) . the multiple sequence alignments of the complete nucleotide sequences and the amino acid sequences of adenovirus hexons were determined by the program megalign (data not shown). the nucleotide and amino acid sequence pair distances and the phylogenetic tree of hexon proteins showed serotypes of subgenera b, d and e to be closely related (table and fig. ) . they manifest . ± . % amino acid sequence homology. ad (subgenus e) is very similar to ad (subgenus b), the amino acid sequence homology between these two serotypes reaching . %. this is in agreement with the cross neutralisation seen between antihexon sera speci®c for ad and ad [ ] . the alignment data also showed ad (subgenus a) to be closely related to ad and ad (subgenus f), the level of sequence homology being . ± . %. in contrast, the sequence similarity of e a proteins between ad and ad was very low, only % in e rl and % in e rl [ ] . the alignment of the complete sequences of adenovirus hexons showed nucleotide sequence homology to be ± % within a subgenus of human adenoviruses, ± % between members of different subgenera with the exception between ad and ad ( . %); ± % between adenoviruses from the same host species and ± % between adenoviruses from different host species; and ± % between the two adenovirus genera mastadenovirus and aviadenovirus. the alignment of the complete amino acid sequences of adenovirus hexons revealed the existence of seven hypervariable regions (fig. ) . hydrophilicity plots derived from the hexon sequences of eight serotypes representing eight subgenera of mastadenovirus were compared (fig. ) . seven hypervariable regions were demonstrated (in boxes, corresponding to the boxes in fig. ) . the ®rst four hypervariable regions, a to a , covered most of the external loop (l ) of the hexon. they were separated by three short relatively conserved sequences which may stabilise the outer shell structure of the hexon. the l contains two hypervariable regions, b and b . all of the hypervariable regions in l and l contained longer or shorter deletions/insertions. the amino acid sequences of these regions were serotype speci®c) the hypervariable region d of mastadenovirus consisting of ± amino acids was located in l . amino acid sequence homology showed the region d to be subgenus speci®c. sequence homology was ± % for pairs of serotypes within a subgenus, and less than % between serotypes belonging to different subgenera. interestingly, the major portion of the hypervariable regions manifest hydrophilicity. according to the ribbon representation of the ad hexon subunit [ ] , the major portion of the hydrophilic regions in hypervariable areas was located at the surface of the hexon molecule. codons with cytidine in the third position are highly preferred analysis of codon usage for the serotypes showed that among the synonymous codons those with cytidine in the third position (nnc) were highly preferred (table ) . among cpuss the bias for nnc codons was strong. however, although the codon preference for nnc among cpyss was generally manifest, this was not the case for the nnc codons for the hydrophobic amino acids ile, leu and val. the isoelectric point and the surface charge for hexons of the serotypes were deduced with the program lasergene-protean-composition (table ) . at ph , the charges of the hexon proteins from different subgenera varied widely. the hexons of ad and ad belonging to subgenus c carried the highest negative charge, À x and À x , respectively. these charges were about . times greater than those of hexons of subgenus f, bav , mav and fav . more interestingly, the strength of the accumulated charge of hydrophilic or hydrophobic regions correlated with tissue tropism. the prototypes of ad , ad , ad and ad were isolated from respiratory tract specimens. the prototype of ad was isolated from nasal washing, ad from throat washing, and both ad and ad from adenoid tissue cultures [ ] . these adenoviruses were frequently isolated from patients with respiratory diseases [ ] . the hexons of these four serotypes were characterised by strong accumulated charges of À x to À x in hydrophilic regions, but manifestly weaker charges of only À x to À x in hydrophobic regions. the prototypes of ad and bav were isolated from conjunctival scrapings and conjunctiva, respectively [ ] , the hydrophobic regions of their hexons manifest charges of À x and À x . ad can be isolated both from the eye and the respiratory tract [ ] . the negative charges of the hydrophilic and the hydrophobic regions of the ad hexon were similar, À x and À x , respectively. the prototypes of ad , ad , ad and fav were isolated from faeces or rectal swabs [ , , ] . all four of these enteric adenoviruses were characterised by lower negative charges of À x to À x in both hydrophilic and hydrophobic regions of their hexons. mav isolated from spleen and fav isolated from allantois [ ] manifested individually unique patterns of surface charge. the hydropathic type and value of an amino acid is dependent on its codon usage and its position in a protein structure. scales for evaluating the hydropathic characteristics of amino acids have been developed by many different research groups. however, the hydrophilicity and hydrophobicity values obtained differ substantially [ , , ] . the most frequently used scales, introduced by kyte and doolittle were used in the present study. as ranked on hydropathy scales [ ] , the common amino acids can be divided into three different classes. the ®rst is the hydrophobic class including ile, val, leu, phe and cys. of these ®ve amino acids, only cys is encoded by cpuss, the other four being encoded by cpyss. the second is the hydrophilic class consisting of his, glu, gln, asp, asn, lys and arg, all of in general, a a g-rich region (a, adenosine; g, guanine) in a nucleotide sequence contains more cpus codons, and c t-rich region contains more cpys codons. therefore, the part of a protein, which is encoded by a g-rich region usually manifests hydrophilicity and a c t-rich region encoded peptide usually shows hydrophobic characteristics. the hydropathy value of an amino acid in a protein chain is dependent on the protein conformation. the residue side-chains can protrude at the interior or exterior portion of a protein chain. the hydropathy value of an amino acid was determined by averaging over a window which contains several consecutive residues surrounding the amino acid in question [ ] . therefore, the strength and even the type of the hydropathy of an individual amino acid may vary according to its location within a peptide chain. in particular, the hydropathy of the amino acids in the intermediate class was found to vary more frequently and in a more pronounced way ( table ) . the major portion of hydrophobic sequences in a protein will be found in the interior of the native structure, and the major portion of hydrophilic sequences will be found on the exterior [ ] . branden and tooze [ ] found that the hydropathy plots (kyte-doolittle method) agree with the crystal structure data in a polypeptide of r. sphaeroides. in this study, we found the major portion of the hypervariable areas to manifest hydrophilicity. the major portion of the hydrophilic regions in hypervariable areas is located at the surface of the crystal structure of the hexon. the nnc codons in adenovirus hexons manifested high usage. the result is compatible with that derived from analysis of different genes of ad (except the phe) [ ] . preference for the synonymous codon for phe, uuc was greater than uuu in hexon genes (table ) . however, the reverse result was true of different ad genes, uuc accounting for . % and uuu was . %. phe with its aromatic side chain is highly hydrophobic, nonpolar and chemically stable. the variability of the phe residue is one of the lowest during divergence of homologous proteins [ ] . the patterns of codon usage for phe in hexon genes were shown to be subgenus speci®c ( table ). the codon usage for phe in the hexon genes of the species of each subgenus is highly similar within subgenera b, c and f. the codon preferably used for phe in hexon genes of members of subgenera a, c and f is uuu, whereas, uuc is preferably used in hexon genes of the species of subgenera b, d and e. these ®ndings corroborate the relatedness of the va rna genes [ ] , and also the relatedness of the hexon genes of human adenoviruses [ ] . we found the nnc codons in adenovirus hexon genes to be highly preferred. analysis of the data obtained from genbank [ ] the c g content of different organisms is used as one of the criteria in taxonomy. among various eubacteria the c g content of the genome dna ranges from % to % [ ] . in adenoviridae the dna c g content varies from ± % for mastadenoviruses and ± % for aviadenoviruses [ ] . in the six different subgenera of human adenoviruses the c g content of genome dna ranges from % to % [ ] . the c g content of all serotypes reveals the existence of two groups: higher c g content ( . ± . %) group including ad , ad , bav , fav and fav , and lower c g content ( . ± . %) group including the remaining nine serotypes ( table ). the c g content of human adenovirus hexons was consistent with the level of c g content of whole adenovirus genome dna with the exception of subgenus c, ad and ad , which has higher c g content ( %) in genome dna. there are amino acids encoded by synonymous codons which contain nnc (table ). of these amino acids, nine manifested nnc high usage, and three (asp, ile and pro) showed at same level of nnc usage, whereas only three (cys, leu and val) showed low nnc usage. cys is a rare amino uuc uuu acid and only four cysteines in ad hexon. among the synonymous codons for val, seven gucs appeared in the hydrophobic region. therefore, the conclusion is that the nnc codons in ad hexon were frequently used although ad is one of the serotypes which contain lower level of c g content in hexon dnas. fav (celo) genes encoding for major core, hexon-associated and hexon proteins the gene for the adenovirus hexon polypeptide the re®ned crystal structure of hexon, the major coat protein of adenovirus type , at . a Ê resolution phylogenetic relationships among adenovirus serotypes introduction to protein structure adenovirues. in: american type culture collection (atcc) catalogue of animal & antisera, chlamydiae & rickettsie, th ed. american type culture collection the structure of the adenovirus capsid ii. the packing symmetry of hexon and its implications for viral architecture adenoviruses of chickens: serologic groups analysis of adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-speci®c residues reconstructing evolution from contemporary sequences (chapter . ) and the properties of liquid water and the characteristics of noncovalent interactions in this solvent prediction of 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adenovirus serotypes genomic mapping and sequence analysis of the fowl adenovirus serotype hexon gene nucleotide sequence of human adenovirus type dna: comparative functional analysis dna sequence of the adenovirus type hexon gene and predicted structure of the protein the adenovirus type hexon: sequence, predicted structure and relationship to other adenovirus hexons isolation and classi®cation of avian enteric cytopathogenic agents codon usage tabulated from the genbank genetic sequence data demonstration of three different subtypes of adenovirus type by dna restriction site mapping adenoviruses sequence and structural analysis of murine adenovirus type hexon genetic organization, size, and complete sequence of eight region genes of human adenovirus type authors' address: dr. g. wadell, department of virology, umea Ê university, s- umea Ê, sweden.received october , key: cord- -wuk arf authors: mohamed, fakry f.; mansour, shimaa m. g.; orabi, ahmed; el-araby, iman e.; ng, terry fei fan; mor, sunil k.; goyal, sagar m. title: detection and genetic characterization of bovine kobuvirus from calves in egypt date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: wuk arf kobuviruses are small non-enveloped rna viruses that probably cause diarrhea in cattle and swine. since its discovery in , few studies have addressed bovine kobuvirus (bkov; a species of aichivirus b) infections. bkov has been reported in europe, asia, and south america, suggesting a worldwide distribution. to investigate the presence of bkov in egypt, fecal specimens from diarrheic calves in two different egyptian provinces (cairo and sharkia) were screened by rt-pcr and ( . %) were found positive for bkov. rna from one of the positive samples (bkov/egy- /ky ) was subjected to next-generation sequencing to determine the complete bkov genome sequence. when compared to the only recorded bkov genome sequence (bkov/u- /ab ), the studied strain showed amino acid (aa) substitutions through its entire polyprotein ( aa), one nucleotide (nt) insertion and one nt deletion in the b gene and -nt deletions in the utrs ( each). additionally, five vp and seven d sequences were obtained from other samples by using rt-pcr and sanger sequencing. a discrepancy in the phylogenetic topography of vp and d was observed, where the egyptian vp sequences were classified as a distinct cluster within the proposed lineage (genotype a), which also contained strains from the uk, brazil, and japan. while, the d sequences from cairo were related to those of chinese strains unlike sharkia ones that were more closer to korean strains. to the best of our knowledge, this is the first detection and genomic characterization of bkov in egypt or indeed africa. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. as a taxonomically classifiable member of the family picornaviridae, kobuviruses are small, non-enveloped, and positive-sense single-stranded rna viruses. their genome consists of a single open reading frame (orf) that encodes a large polyprotein [ ] . after translation, the protein precursor is proteolytically processed into several structural and non-structural proteins. with a total size of . - . kb, the kobuvirus genome is organized sequentially as follows: vpg, ′utr, leader protein (l), three structural proteins (vp , vp , and vp ), seven non-structural proteins ( a- c and a- d), ′utr, and poly(a) tail [ ] . the d gene encodes for the rna-dependent rna polymerase (rdrp). according to the function of the encoded proteins, the kobuvirus genome can be arranged into three distinct regions, namely p (encoding structural capsid proteins), p , and p (encoding non-structural proteins) [ ] . initially, kobuviruses included aichivirus (aiv), bovine kobuvirus (bkov), and porcine kobuvirus (pkov) based on the hosts infected e.g. humans, cattle, and swine, respectively. later, the genus kobuvirus was divided into three species; aichivirus a, b and c (with strains abbreviated to aiv a, b, and c). aiv a includes human aiv and kobuviruses of dogs, cats, and mice. aiv b includes bovine, ovine, and ferret kobuviruses while aiv c includes only pkov [ ] . kobuviruses have also been detected in black goats, rabbits, european roller, and bats (http://www.picor navir idae.com/). based on vp sequence analysis, human aiv strains have been divided into three major lineages ( , , and ) with nucleotide (nt) sequence identities < . %. these three lineages were later renamed as genotypes a, b, and c [ ] . recently, chang et al. [ ] proposed four genotypes (a, b, c, and d) of bkov-vp strains based on . % nt identity as a recommended cutoff value for genotyping. in , aiv was linked to gastroenteritis outbreaks in the aichi prefecture of japan after being isolated from stools of patients who consumed raw oysters [ ] . later, the virus was classified as a member of the family picornaviridae [ ] . pkov was first detected in domestic pigs in hungary in [ ] . in , bkov/u- /ab was identified in japan as a cytopathic contaminant of hela cells, which have been used around the world for more than years. subsequently, the virus was detected in serum and fecal samples of apparently healthy animals [ ] and has been reported from europe, asia, and south america [ ] . kobuviruses are implicated in gastroenteritis of animals and humans. however, their role in the pathogenesis of clinical diarrhea is still unclear because they have been detected in both healthy and diarrheic animals. most cases of diarrhea are not routinely tested for kobuvirus and hence the full impact of this virus has not been accurately determined. as a newly recognized virus, genbank contains just a single bkov complete genome. in this study, we report bkov infections among cattle populations of egypt, the full-length genome of one egyptian strain (bkov/ egy- / ky ) as well as the phylogenetic analysis of bkov strains from cairo and sharkia provinces. massive outbreaks of calf diarrhea were reported from the sharkia and cairo provinces of egypt in late and early . clinically, calves (ages weeks to months) had severe diarrhea, fever, weakness, and dehydration. a total of diarrheic fecal specimens were collected aseptically and transferred to the diagnostic laboratory. the total rna was extracted from supernatants of % fecal homogenates using blood/liquid sample total rna rapid extraction kit (bioteke corporation, beijing, china) and stored at - °c. the rna samples (n = ) were subjected to rt-pcr for the detection of bkov using degenerate primers (univ-kobu-f and univ-kobu-r), which amplify a conserved region in the d (rdrp) gene of all kobuviruses [ ] ( table ). the pcr reaction was performed using one step rt-pcr kit (takara primescript onestep rt-pcr kit ver. dye plus; cat. #. rr a, takara bio inc, japan). briefly, each mixture contained μl of one-step enzyme mix, μl of one-step buffer, μl ( μm) each of upstream and downstream primers, μl of test rna and μl of rnase-free water. the μl-reaction mix was incubated in the thermocycler at °c for min for reverse transcription and at °c for min for initial denaturation. then, cycles of amplification (denaturation at °c for min, annealing at °c for sec and elongation at °c for min) were applied followed by a step of final extension at °c for min. part of the amplified pcr products ( μl) were analyzed by % agarose gel electrophoresis and ultraviolet illumination. to detect infection with bovine rotavirus (brv), bovine coronavirus (bcv), and bovine viral diarrhea virus (bvdv), rt-pcr was carried out using specific primers for these respective viruses (data not shown). a -μl aliquot of one rna sample from sharkia province (namely; bkov/egy- ) was selected for the next-generation sequencing (ngs) using the illumina platform to obtain the complete genome of bkov. the ngs workflow was performed at the university of minnesota genomic center (umgc). for quality control purposes, the total rna was quantified using ribogreen fluorometry and checked for purity. the library was created using illumina's truseq rna sample preparation kit (rs- - ). the flow cell was loaded on an illumina miseq and sequenced by cycles paired-end reads sequencing. based on the complete genome of bkov/egy- , three primer sets were designed to partially span the d, vp , and bc regions (table ) . two primer pairs were used to retest some of the study samples by rt-pcr to analyze the molecular phylogenetics of the d and vp genes. the last primer pair partially covered the bc region to confirm the specific mutations (one insertion and one deletion) observed in the b gene of bkov/egy- and to search for them in other study samples. the reaction mixtures and pcr thermal profiles were the same as above except for the annealing step that was performed at °c for min. after electrophoresis in a % agarose gel, products of expected sizes were visualized under a uv transilluminator. amplicons for partial regions of d (n = ), vp (n = ) and bc (n = ) were purified using qiaquick pcr purification kit (qiagen, valencia, ca, usa). the purified dna was sent to the umgc for sanger sequencing of the three regions in both directions (using the same forward and reverse primers used in rt-pcr). the ngs reads were trimmed to remove adaptor sequences followed by testing for sequence quality. the host sequences were removed and the remaining reads were assembled into contigs (minimum length = nt) using de novo default settings in the clc genomic workbench . software (http://www.clcbi o.com/produ cts/clc-genom ics-workb ench/). to determine viral sequences in the sample, extracted contigs were subjected to blastn analysis against ncbi databases (cutoff e value = . ). the orf finder tool of ncbi (https ://www.ncbi.nlm.nih.gov/orffi nder/) was used to find orfs. to determine the completeness of the genome and to predict structural and non-structural proteins, nt and amino acid (aa) sequences were aligned with reference kobuviruses. the alignments and phylograms were made using mega . software [ ] . the sanger sequences of partially-amplified genes were imported to sequencher software version . (http://genec odes.com/) for evaluating chromatograms, trimming of poor quality nucleotides, alignment of forward and reverse reads, and generation of final contigs. the partial d, vp , and bc sequences were compared to previously reported ones by using the mega . software [ ] . sequences were edited and aligned using the clustal-w method followed by constructing neighbor-joining phylogenetic trees using the kimura -substitution model with a bootstrap value of replicates. the nt and aa identities were calculated using the p-distance method. deduced aa analysis was performed on vp sequences to spot possible substitutions. study sequences were submitted to genbank under the following accession numbers: ky for the complete genome of bkov/egy- , and ky -ky , ky -ky , and ky -ky for the partial bc, d, and vp regions, respectively. of the fecal samples tested, ( . %) were positive for bkov-rna ( % ci: . % to . %). the rates of kobuvirus detection in the cairo and sharkia provinces were % and %, respectively. the infection rate was the highest in young calves and decreased with age; being . % ( / ) for calves ≤ one-month-old and . % ( / ) for older calves. some samples ( %) were positive for brv without showing bkov infection. all samples were negative for bcv and bvdv. based on rna quality, one bkovpositive sample (bkov/egy- / ky ) was selected for illumina sequencing. the full genome sequence of bkov/egy- / ky was obtained by illumina miseq. the resulting genome, excluding the poly a tail, was nt in length and contained a single orf encoding a polyprotein of aa, which started with a methionine (aug) initiation codon ( - ). it was organized as a ′utr of nt, a nt orf ( - ), and a ′utr of nt. bkov/egy- /ky starts with a leader (l) protein of nt ( aa). the genomic organization and potential polyprotein cleavage sites were verified by using several ncbi tools. the cleavage sites q/c ( / ) and q/g ( / ) were predicted to separate p from the p region and p from p region, respectively ( table ). the polyprotein of bkov/egy- / ky aligned well with that of bkov/u- /ab , including the highly conserved gppgtgks motif in the nt binding domain of the putative picornavirus helicase, within the c protein and the kdelr , ygdd and flkr motifs in the rdrp protein. a phylogenetic analysis, based on the complete coding region, clustered the bkov/egy- / ky with other members of aichivirus b (fig. s ) . the maximum comparative sequence identity for bkov/ egy- / ky was observed following comparisons with bkov/u- /ab ( . % at the nt level and . % at the aa level) and then with sheep kobuvirus/tb / gu ( . % at the nt level and . % at the aa level). in comparison to bkov/u- /ab , the p region (capsid proteins) was less conserved ( . % at the nt level and . % at the aa level) with the vp gene being highly variant (sharing only . % nt and . % aa identities) among the three structural proteins. vp had the least variation; sharing . % nt and . % aa identities. the p region ( a- d non-structural proteins) showed the highest identity ( . % at the nt level and . % at the aa level) followed by the p region (non-structural proteins ( a- c), which showed identities of . % at the nt level and . % at the aa level (fig. ) . collectively, in relation to bkov/u- /ab , bkov/egy- / ky showed total nt and aa substitutions of and , respectively through its entire genome (table s ). the ′utr of bkov/egy- / ky was nt in length ( nt shorter than that of bkov/u- /ab with . % nt identity). as many as nt were missing due to unresolved sequencing of the ′-terminus. the lengths of the pyrimidine tract (y), spacer sequence (x), and aug initiation codon were expressed as a formula (y -x -aug) with two deletions being present (a and c ). five aug codons were detected in the ′utr as well as the aug initiation codon ( - ). the ′utr was -nt long ( -nt deletions; t and t ) and had . % nt identity when compared to the reference sequence bkov/u- / ab . the partial d sequences from seven bkov strains were highly conserved (overall identities of . - . % at the nt level and . - % at the aa level). based on phylogenetic analysis of genbank reference strains, bkov sequences from cairo (n = ) clustered with chinese strains table predicted polyproteins in the bkov/egy- /ky complete genome the p region encodes for vp , vp and vp proteins, the p encodes for a- c proteins while the p region encodes for a- d proteins q for glutamine, g for glycine, h for histidine, a for alanine, c for cysteine, s for serine the end of the d protein ( - ) is the tga stop codon from xinjiang territory while those from sharkia (n = ) aligned with korean strains. cairo and sharkia strains were obviously separated (fig. ) . between each other, the partial vp sequences identified in this study (n = ) showed . - . % nt and . - % aa identities. closely related reference bkov-vp strains were included in the phylogenetic analysis and all study sequences belonged to lineage (genotype a), which contains strains from brazil, scotland (uk) and japan (fig. ) . the vp sequences from sharkia (egy- , sharkia- and sharkia- ) and cairo (cairo- , - , and - ) clustered phylogenetically in lineage but were further distinguishable into two subclades (fig. ). on contrast, the separation of the two sharkia-cairo subclades was more profound following analysis of the d gene, where the sharkia and cairo strains did not cluster with a significant bootstrap value in the phylogenetic tree. furthermore, a deduced aa analysis of bkov-vp sequences revealed that the study strain had six residue substitutions. all study strains had an aa substitution of a to s . in addition, cairo strains (bkov- , bkov- and bkov- ) had a unique aa change at position (v to i ). sharkia strains (bkov- and bkov- ) and bkov/egy- / ky had two aa substitutions t to a and i to v . except for bkov- , all study strains showed a residue change of a to v . bkov- strain had one aa exchange from a to t . four sequences representing the bc region shared . - % and . - % nt and aa identities, respectively. when compared to the reference bkov/u- /ab sequence, all sequences had one insertion (g ) and one deletion (c ) in the b gene, which were also seen in bkov/ egy- / ky . classifiable members of the family picornaviridae are known to cause a wide range of important diseases in humans and animals. two picornaviruses have been reported in egyptian farm animals; the endemic foot and mouth disease virus [ ] and the recently recognized bovine enterovirus [ ] . neonatal calf diarrhea (ncd) results in huge economic losses in both beef and dairy industries in egypt. recent studies have described the presence of different enteric viruses in egyptian cattle herds [ , ] and have raised questions about their actual contribution to ncd. kobuviruses have been incriminated in animal and human cases of gastroenteritis. they are transmitted through the fecal-oral route either directly from one animal to another or indirectly by consumption of contaminated food or water [ ] . the present study reports, for the first time, the existence of bkov (species aichivirus b) in egypt, in cattle showing clinical features of calf diarrhea. using generic kobuvirus primers, of ( . %) diarrheic fecal samples tested positive for bkov. these results are somewhat different from those reported in prior surveys. for example, kobuvirus was detected in . % of healthy cattle in japan [ ] , . % of diarrheic cattle in thailand [ ] , and . % of diarrheic cattle in brazil [ , ] . in other countries, the prevalence has been described as follows: . % in hungary [ ] , % in belgium [ ] , . - . % in korea [ , ] , . % in the netherlands [ ] , . % in italy [ ] , and . % in china [ ] . the highest rates of bkov have been observed in egypt ( . % in this study) and the netherlands ( . ) possibly due to the smaller sample size tested in these two studies (n = and n = , respectively). extensive epidemiologic surveillance should be planned to determine the endemicity of bkov in egypt. the genome sequence we characterized (bkov/egy- / ky ) was compared to that of bkov/u- /ab , which, to date, is the only available bkov complete genome fig. the percentage nucleotide and amino acid identities between the studied strain (bkov/egy- /ky ) and the reference strain (bkov/u- / ab ) sequence. the vp gene was highly divergent while the d gene was highly conserved (fig. ). the study strain had one nt insertion (g ) and one nt deletion (c ) in the b region and sanger sequencing confirmed the presence of this insertion/deletion in bkov/egy- /ky as well as in four other study strains. the insertion-deletion resulted in three successive aa substitutions (l a, r s, and t h). variant pkov strains frequently express deletions in their b genes. the significance of these mutations is not yet known. it has been assumed that the b protein has multiple functions in different picornaviruses [ ] . however, the b gene may not be responsible for kobuvirus pathogenesis [ ] . the phylogenetic analysis of sequences representing partial d regions showed that bkov strains from cairo grouped with chinese strains while those from sharkia clustered with korean strains. the cairo strains shared identities of . - . % at the nt level and . - % at the aa level while the two sharkia strains shared identities of . % and % at the nt and aa levels, respectively. similarly, chang et al. [ ] and candido et al. [ ] stated that bkov- d sequences from the same epizootic areas grouped together and shared higher identities. earlier studies have concentrated mostly on detection and sequencing of a smaller part of the bkov- d (rdrp) gene [ , , , ] . the d genes are usually conserved. thus, they are mainly used, phylogenetically, to discriminate bkov from other kobuviruses but not to differentiate bkov into distinct lineages [ , ] . on the other hand, vp is the most exterior, immunedeterminant capsid protein in bkov and is used for serotyping as it carries several neutralization domains [ , , ] . molecular typing of the vp gene correlated with serotype specificity as strains from the same serotype tend to be monophyletic. this information is valuable in assigning taxonomic groups [ ] . until now, the observed evolutionary variations among different bkov-vp strains have been studied based only on sequencing of bkov-vp gene sequences of chinese origin. four lineages (genotypes) were proposed based on phylogenetics and nt sequence identities (with an . % cutoff value) [ ] . bkov vp sequences from egypt belonged to lineage (genotype a), which includes bkovs from brazil, japan (only u- strain), and scotland. the members of genotype a shared nt identities higher than . %, validating the genotyping of chang et al. [ ] . other genotypes (b, c, and d) contained bkov-vp strains from china only, so it appears that genotype a is the most prevalent cluster worldwide. also, it was noticed that some strains from scotland (sc , n = ) formed a new group (tentatively named genotype e/ lineage ) within the phylogenetic tree ( fig. s ) with nt identities less than . % ( . - % with genotype a, . - % with genotype b, . - . % with group c, and . - . % with genotype d), indicating the presence of other genotypes that have not yet been acknowledged. it was noticed that bkov- d sequences from this study showed different phylogenetic topography than that of vp sequences, suggesting different evolutionary histories for the structural and non-structural regions. due to the limited number of bkov-vp sequences in genbank, the present study provides important data for future genotyping and classification of bkov. vp protein sequences from this study contained six non-synonymous substitutions at positions , , , , , and compared to reference sequences ( sequences in total); with one of them (v to i ) considered unique to cairo strains (bkov- , bkov- , and bkov- ). between bkov/egy- /ky and bkov/u- / ab , twelve aa substitutions were observed within the vp protein (table s ). adaptive mutations may play an important role in inducing diarrheal outbreaks through the generation of new variants that may not be recognized by host immune defenses. moreover, they may participate in the escape of kobuviruses from the gastrointestinal tract to the circulatory system [ ] . the discrepancy in the phylogenetic topography of vp (fig. ) and d (fig. ) between the cairo and sharkia strains is likely due to differences in evolutionary pressure and substitution rates between structural (vp ) and non-structural regions ( d). however, recombination events in non-structural genes, including the d gene, cannot be ruled out as it is common for all picornavirus to recombine in such regions [ ] . whole genome sequencing and the continued surveillance of bkov diversity are required to increase information about this virus in egypt and to document genetic shift caused by recombination events. bkov detection and analysis of its complete genome, particularly the vp gene, are highly significant for improving our understanding of kobuvirus biology. our findings further confirm that bkov is distributed globally and that further epidemiological studies are needed in egypt. the genetic diversity of kobuviruses and its role in ncd should be monitored carefully. rt-pcr and ngs approaches, applied in this study, should be useful in this regard. kobuviruses-a comprehensive review virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses picornaviridae: the viruses and their replication epidemiology of human and animal kobuviruses sequence analysis of the capsid gene of aichi viruses detected from japan prevalence and genetic diversity of bovine 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and characterization of porcine kobuvirus variant isolated from suckling piglet in gansu province recombination among picornaviruses acknowledgements we thank dr. hany abdalla (department of theriogenology, faculty of veterinary medicine, zagazig university) for help in collection of samples and for providing historical data. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- - q rt i authors: hussein, hosni a. m.; walker, lia r.; abdel-raouf, usama m.; desouky, sayed a.; montasser, abdel khalek m.; akula, shaw m. title: beyond rgd: virus interactions with integrins date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: q rt i viruses successfully infect host cells by initially binding to the surfaces of the cells, followed by an intricate entry process. as multifunctional heterodimeric cell-surface receptor molecules, integrins have been shown to usefully serve as entry receptors for a plethora of viruses. however, the exact role(s) of integrins in viral pathogen internalization has yet to be elaborately described. notably, several viruses harbor integrin-recognition motifs displayed on viral envelope/capsid-associated proteins. the most common of these motifs is the minimal peptide sequence for binding integrins, rgd (arg-gly-asp), which is known for its role in virus infection via its ability to interact with over half of the more than known integrins. not all virus-integrin interactions are rgd-dependent, however. non-rgd-binding integrins have also been shown to effectively promote virus entry and infection as well. such virus-integrin binding is shown to facilitate adhesion, cytoskeleton rearrangement, integrin activation, and increased intracellular signaling. also, we have attempted to discuss the role of carbohydrate moieties in virus interactions with receptor-like host cell surface integrins that drive the process of internalization. as much as possible, this article examines the published literature regarding the role of integrins in terms of virus infection and virus-encoded glycosylated proteins that mediate interactions with integrins, and it explores the idea of targeting these receptors as a therapeutic treatment option. viruses may be small in size, but they carry enough genetic material that they are capable of inflicting some of the deadliest diseases in the world. if not for their ability to enter host cells and efficiently impair them, we would not even talk about them. an efficient pathogen is one that has evolved a robust entry mechanism for delivery of genetic material into different target host cells, which is critical for replication and sustenance. over the years, viruses as obligate parasites have evolved successful ways to colonize host cells, using complicated but well-orchestrated mechanisms to enter cells. the whole process of virus entry-otherwise referred to as internalization-begins with the virus binding to target cells. binding to cells is a reversible process that does not ensure virus entry. virus binding or attachment only ensures viral proximity to cells. however, this process is the most essential step that kick-starts the whole cascade of events, resulting in the eventual internalization of the virus. several viruses utilize different glycosaminoglycans expressed on the target cells as binding receptors. glycosaminoglycans serve as good receptor molecules that promote binding, as they are expressed ubiquitously in eukaryotic cells. some of the most common binding receptors are heparan sulfate (hs) and chondroitin sulfate [ , , , , ] . virus binding to such receptors brings them closer to cells and provides the opportunity to interact with other receptor molecules that promote the actual internalization process. & shaw m. akula akulas@ecu.edu hypothesized that the initial step of virus binding to cells also induces conformational changes to the glycoproteins expressed on the target cells that are critical for the virus to interact with other receptor molecules, thus promoting internalization. the actual virus entry is complicated to the extent that a single virus may utilize different receptors to efficiently enter different target cells [ , ] . viruses have evolved such mechanisms to be effective pathogens. such variations in the entry mechanisms dictate the actual entry route. for example, epstein-barr virus (ebv) enters lymphoblastoid cells by fusion but enters b cells via endocytosis [ ] . integrins are a family of receptor molecules that serve as entry receptors for a variety of different viruses, including foot-and-mouth disease virus (fmdv) [ ] , kaposi's sarcoma-associated herpesvirus (kshv) [ ], herpes simplex virus- (hsv- ) [ ], adenovirus [ ] , human papillomavirus- (hpv- ) [ ] , reovirus [ ] , and others. our understanding of the role of integrins in promoting virus entry is still not complete. in this review, we have attempted to elaborate on the role of integrins in virus internalization. integrins play an important role in regulating a variety of cellular functions, including cell adhesion, cell migration, and critical signaling processes. this is possible because of their ability to interact with various ligands, including extracellular matrix glycoproteins (i.e., collagens, fibronectins, laminins, etc.) and cellular receptors (i.e., vascular cell adhesion molecule- and intercellular cell adhesion molecules) [ , , ] . discovered over twenty years ago, integrins are a large family of transmembrane glycoproteins found in a variety of organisms ranging from sponges, corals, nematodes, and echinoderms to mammals [ ] . there are about integrins that have been identified. these heterodimeric receptor molecules result from different pairings among a and b subunits [ ] . each integrin subunit has three domains: an extracellular, transmembrane, and cytoplasmic domain. the extracellular domain is the largest part, ranging from to kda, while the cytoplasmic domain is a short and largely unstructured domain of - amino acid (aa) residues, with the exception of the b subunit, which contains [ , aa residues [ ] . the transmembrane domains of integrins are single-spanning structures comprised of - aa residues that form a-helical coiled coils that exist as either homo-or heterodimers [ ] . high-resolution x-ray crystallography structural data are available for the extracellular domains of integrins [ , , ] , but no high-resolution experimental x-ray crystal structures are available for the transmembrane or cytoplasmic domain of any integrin heterodimer. much of the structural data of the transmembrane and cytoplasmic domains are based purely on nmr analysis. integrins can shift between high-and low-affinity conformations for ligand binding to transduce intracellular signals following ligand binding. in the inactive state, the extracellular domain of integrins is not bound to ligands and exists in a bent conformation. however, signals from the cell induce conformational changes that expose the external ligandbinding site, where ligands bind and transmit the signals from outside to inside the cell [ ] . although some integrins can bind their ligands in a resting state, there are other integrins whose binding to their ligand requires activation through alterations in the intracellular domains by signaling events, which subsequently lead to transmission of signals from inside to outside of the cell; this is commonly referred to as insideout signaling [ ] . ligand binding to extracellular domains of integrins leads to activation of integrins and subsequent transmission of cellular signals from outside to inside of the cell, which is known as outside-in signaling [ ] . these intracellular signals are very important for cell growth, differentiation, and apoptosis. additionally, intracellular signals lead to formation of the focal adhesion complex, which is a large and dynamic multi-protein complex that includes a vast number of intracellular proteins [ ] . along with proteoglycans, integrins form the major adhesion receptors for extracellular cellular matrix (ecm) proteins, making them important for signaling events that determine cell fate [ ] . cellular signaling processes depend on the pattern of expression and the composition of integrins, which determine the ecm type a cell can bind and initiate downstream signaling events [ ] . integrins provide a connection between the ecm proteins and the actin cytoskeleton that is crucial for regulating cytoskeletal organization and intracellular signaling pathways, all of which are a necessity for cell survival, proliferation, shape, attachment, migration, and angiogenesis [ ] . as adhesion molecules, integrins mediate cell-to-cell, cell-to-ecm, and cell-to-pathogen interactions, and such adhesion is regulated through the inside-out signaling process. integrin-induced adhesion is very important in the regulation of the immune system during leukocyte trafficking, migration, and phagocytosis [ ] . adhesion of integrins to a solid surface is the first step in cell migration and motility. the vital role of integrins in cell migration makes them essential for many important biological events, including embryonic development [ , ] , inflammatory responses [ , , ], wound healing [ , ] , and tumor metastasis [ ] . there are many pathogens, including viruses [ , , ] and bacteria [ , ] , that have the ability to use integrins with different mechanisms for invading cells. through regulation of several cell functions, integrins have a role in human disease. for example, tumors, cancer, and immunodeficiency disorders are all associated with altered integrin-mediated adhesion and migration [ ] . the hallmark of tumor development is cell attachment, migration, and proliferation; all of which are regulated by integrin-based cellular signaling [ , , ] . expression of particular integrins, including a b , a b , avb , a b and a b , on tumor cells in the context of activated cytokine receptors or growth factor receptors leads to increased disease progression and severity [ ] . integrins integrate the extracellular and intracellular environments by binding to ligands outside the cell and cytoskeletal components and signaling molecules inside the cell [ ] . the role of integrins in cancer initiation and progression makes them targets for several therapeutic agents in clinical trials of cancer therapy. integrins have been exploited by many pathogens, including bacteria and viruses to infect cells. penetration of the host-cell plasma membrane is a crucial step for a successful virus infection [ ] . to invade the host cell, several animal viruses physically interact with integrins to infect cells. there are many studies that demonstrate the critical role of membrane rafts in viral entry and infection [ , ] . interestingly, many integrins used by viruses for binding and internalization are localized to and associated with membrane rafts, which consolidate the role of integrin in virus internalization and infection [ , , , ] . a list of viruses and the manner by which they utilize integrins to infect cells is provided in table . many viruses, including adenoviruses and herpesviruses, have an rgd (arg-gly-asp) tripeptide motif displayed on their viral envelope glycoproteins. rgd is the minimal peptide sequence for binding integrins. as an integrinrecognition motif, rgd plays an important role in virus infection by binding one or different combinations of several integrins, which include avb , avb , avb , a b , avb , avb , and aiibb [ , , , ] . rgd binding to these integrins activates cellular signals such as phosphatidylinositol- -kinase (pi- k) and mitogen-activate protein kinase (mapk) pathways, which are critical for supporting virus infection of cells [ , ] . many serotypes of adenovirus use rgd-binding integrins to stimulate endocytosis and thereby promote virus entry [ , , ] . binding of the rgd motif on the adenovirus penton base capsid protein to integrins initiates virus internalization by stimulating endocytosis via clathrin-coated vesicles [ , , ] . philpott and colleagues demonstrated that blocking adenovirus binding to integrins using an rgd peptide resulted in a -to -fold reduction in viral dna intake [ ] . likewise, studies by shayakhmetov and colleagues revealed that the deletion of the rgd motif in the penton base did not affect virus attachment but significantly reduced the rate of virus internalization, specifically at the step that involves endosomal escape [ ] . several members of the family herpesviridae interact with integrins in an rgd-dependent manner. for example, herpes simplex virus type (hsv- ) envelope-associated gh interacts with avb , which is critical for virus entry of cells [ ] . kshv or human herpesvirus (hhv- ) interacts with a variety of cellular integrins, including a b , avb , and avb , and activates focal adhesion kinase (fak), src, pi- k, rho gtpases, and diaphanous (dia )-associated signaling, which is a necessity for the internalization of the virus [ , ] . the ability of kshv to interact with integrins is mediated by the rgd motif of envelope-associated gb [ , , ] . interestingly, the rgd motif of gb is also required to mediate attachment of cells to the endothelium [ ]. rgd-binding integrins are also important for other viruses. for example, the interaction between rgd of capsid protein vp of coxsackievirus a and avb is essential for virus binding and entry into cells [ , , ] . there are also other studies that demonstrate that a high-affinity interaction between rgd of coxsackievirus a and avb (compared to avb ) is important for cell entry and virus uncoating [ , , ] . notably, the rgd sequence is highly conserved in the vp protein of fmdv and mediates virus attachment to integrins, and thus internalization [ ] . synthetic rgd peptides have been shown to block fmdv attachment in a dose-dependent manner [ , , ] . hiv- tat protein interacts with rgd-binding avb , avb , and a b and initiates the integrin endocytic pathway, which is essential for entry of the virus [ , , ] . hiv- tat protein's interaction with rgd-binding integrin(s) is important for adhesion of target cells [ , ] . interactions of the more deadly ebola virus with a b is deemed critical for modulating infection of fibroblasts [ ] . thus, rgd interactions between viral proteins and integrins seem to regulate not only virus infection of cells but also the associated pathogenesis. not all integrins recognize and interact with the conserved rgd motif of viral proteins. there are multiple other non-rgd-binding integrins that drive virus entry and infection [ , ] . some of the non-rgd-binding integrins that promote entry and infection of hcmv [ ], kshv [ ] , simian virus (sv ) [ ] , and ross river virus (rrv) [ ] are a b , a b , a b , a b , a b and axb . a b and a b are collagen receptors that are utilized by viruses for cell entry and infection. rrv is an alphavirus that is endemic to australia and new guinea and is etiologically associated with epidemic polyarthritis [ , ] . rrv interacts with a b to infect target cells [ , ] . infection of rrv has been shown to be blocked by function-blocking antibodies to a b , soluble a b integrin, or peptides representing the a b integrin-binding site on collagen iv [ ] . rotavirus utilizes different non-rgd binding domains to interact with integrins. (i) the tyr-gly-leu (ygl) sequence of the rotavirus spike protein, vp , interacts with a b and a b and helps the rotavirus to bind and enter cells [ ] , and (ii) the gpr sequence in the rotavirus spike protein vp interacts with axb and helps the rotavirus to enter cells [ , ] . integrins also have a critical role to play in rotavirus pathogenesis. integrin a b and a b are important receptors for enterotoxin function and pathogenesis through their interactions with rotavirus nsp , which eventually results in diarrhea [ ] . human echovirus (ev ), which belongs to the family picornaviridae (a family of rna viruses) is implicated in many human diseases, including meningoencephalitis and carditis. ev successfully infects cells by interacting with a b integrin in an rgd-independent manner. in fact, a b clustering on the surface of cells is the determining factor that defines the success rate of the ev -mediated signaling pathway and virus infection of cells [ , , ] . also, hiv- interacts with the non-rgd-binding integrin a b via gp , which is critical for efficient cell-to-cell spread of the virus [ , , ]. more interestingly, cicala et al. hypothesize that gp -a b interactions play an important role in the very early events following sexual transmission of hiv and may have important implications in the design of vaccine strategies for the prevention of acquisition of hiv infection [ ]. stergiou et al., determined that a b plays a crucial role in modulating sv -induced cellular signaling and infection [ ] . there is also evidence that indicates a role for a b integrin in promoting human papillomavirus (hpv)-induced squamous epithelial dysplasia [ ] . apart from the traditional rgd motif, herpesvirus glycoprotein b (gb) possesses a disintegrin-like domain (dld) [ , ] . a role for dld in hcmv and kshv entry and infection has been described recently. hcmv interacts with a b and a b [ ], and kshv interacts with a b [ ] via the dld contained within gb to successfully enter cells. cell entry [ ] the minimum component of the disintegrin module required for integrin engagement is the -to -amino-acid disintegrin loop, for which a consensus sequence has been described: rx dlxxf [ ] . in the case of kshv gb, the dld sequence is rx - d/elxxf/lx c (aa - ; with a conservative d to e substitution). overall, viruses seem to use non-rgd-and rgd-binding integrins to a comparable extent as a means of binding and entering cells. integrins play a crucial role in cellular function through interactions with a variety of ligands. integrins are ligand specific and can be grouped into four major groups: laminin-binding integrins (a b , a b , a b , a b , a b , and a b ), collagen-binding integrins (a b , a b , a b , a b , and a b ), leukocyte integrins (alb , amb , axb , and adb ), and rgd-recognizing integrins (a b , avb , avb , avb , avb , avb , and aiibb ) [ ] . interestingly, rgd and non-rgd-binding integrins aid equally in the internalization of viruses ( table ). virus-integrin binding induces changes in the quaternary structure of the integrin resulting in clustering of subunits, which increases virus affinity, cytoskeletal rearrangement, and subsequent virus internalization [ ] . these conformational changes are critical for integrins to achieve outside-in and inside-out signaling necessary for various cellular functions, including cytoskeleton activation, endocytosis, gene expression, cell motility, attachment, cell cycle, cell growth, apoptosis, and differentiation [ , ] . on the other hand, interactions of viruses with cellular integrins induce conformational changes in the viral surface proteins, helping to expose the essential domains required for virus entry into a host cell [ ] . through integrin activation, viruses can induce fak phosphorylation which is followed by the activation of several focal adhesion-associated signal molecules, including src, pi- k, rho gtpases (rhoa, rac, and cdc ), dia , and other effector molecules, such as akt, ezrin, protein kinase c (pkc), mapk (mek, erk / ), nf-kb, and p mapk [ , ]. focal adhesion and associated molecules play critical roles in mediating the internalization of viral dna into target cells [ , ] . src is one of the cellular components that is activated immediately upon activation of fak by virusintegrin interactions. src-mediated tyrosine phosphorylation of clathrin regulates clathrin translocation to the plasma membrane, which is important for interactions of clathrin with a number of other essential proteins, including ap , eps , and dynamin. src-mediated tyrosine phosphorylation also plays a role in endocytosis by releasing the internalized endocytic vesicles and initiating the assembly of the plasma-membrane-associated ras activation complex [ , ] . pi- k and ras are directly responsible for activating rho and rab gtpases. these gtpases, along with the activated erk / , are critical for the microtubule and microfilament reorganization that determines the formation of various types of endocytic vesicles and their movements, as well as acting as a force to drive the virus inside and closer to the nuclear membrane [ ] . many viruses use microtubules and cytoplasmic dynein for nuclear targeting [ ] . virus-induced activity of rho gtpases such as rhoa and rac increase the efficiency of viral trafficking along microtubules to the nucleus. thus, inactivation of these rho proteins affects the stability of microtubules, thereby limiting the delivery of viral nucleic acids into the nucleus [ ] . ezrin is the best-studied member of the downstream effector molecules induced by rho gtpases, and it is a critical element for cross-linking the actin cytoskeleton with the plasma membrane and inducing the morphological changes that are commonly observed in cells being infected by viruses and other pathogens [ ] . rac, rho, cdc , and rab also act as switching molecules that are essential for internalization of any pathogen due to their ability to modulate actin dynamics, formation of endocytic vesicles and their fission, adenoviruses, echoviruses, fmdv, parechoviruses, parvoviruses, rotaviruses, kshv, hantaviruses, and others enter target cells via endocytosis by physically interacting with integrins, resulting in the activation of the fak-src-pi- k signaling pathway [ , , ] . pi- k-induction in a integrin-fak-src-dependent manner plays an important role in virus entry and infection via activation of the rho family of gtpases and ezrin, and mediates actin cytoskeleton reorganization. interestingly, these events initiate a cascade of intracellular signals that eventually activate the mitogen-activated protein kinase (mapk) pathways, which are very important in modulating a variety of cellular processes, including proliferation, differentiation, survival, and apoptosis [ , ] . in terms of cellular machinery, actin cytoskeleton reorganization is the crux that supports integrin-associated signaling-induced virus entry. apart from working as a structural platform stabilizing cellular signaling molecules, actin provides mechanical force for endosome formation and endocytic vesicle propulsion [ , ] . microtubules and microfilaments along with other cytoskeletal elements play an important role in controlling the intracellular movement of many viruses [ , , , , ] . successful virus infection involves multiple steps, which include initial binding to the cell surface, internalization, replication, and egress. in the initial step of virus infection, these versatile infectious agents can bind several different cellular surface molecules, such as proteins, lipids, and carbohydrates. these molecules may function in mediating attachment (i.e., concentrating virus on the cell surface) or serve as receptors or co-receptors facilitating viral endocytosis, conformational changes, and the initiation of signaling pathways associated with infection [ , ] . in addition to the protein receptor, which is generally dubbed the 'principal' receptor, the carbohydrate moiety of host-cell membrane proteoglycans, glycosphingolipids, and glycoproteins also serve as viral receptors. for instance, hiv- , via its glycoprotein subunits gp and gp , attached to cell-surface carbohydrates (i.e., glycosphingolipids, galactosylceramide, and heparan sulfate proteoglycans [hspgs]) as a means of promoting actual virus binding to cells [ , , ] . similarly, several human herpesviruses, including hsv [ ] , kshv [ ], and cmv [ ] , make their initial contact with cells by binding to cellsurface hspgs. in general, binding of viruses to carbohydrate moieties on the surface of cells is the key step that induces conformational changes in the viral structure that are critical for interactions with entry-promoting receptors such as integrins. blocking this step of virus interactions with carbohydrate moieties impairs viral entry via integrins [ ] . a list of viruses that utilize carbohydrate moieties to promote virus binding to cells is provided in table . integrins are exciting pharmacological targets because (i) they are exposed on the cell surface and are sensitive to pharmacological blockades and (ii) they regulate the interactions of cells and precisely sense their microenvironment. inhibitors of integrin functions have been successfully tested as drugs to treat several pathological conditions. psk , a nonpeptide antagonist of avb , inhibited osteoclast-mediated bone resorption in a cancer animal model of bone loss [ ] . volociximab, now known as m , is a humanized monoclonal antibody that binds specifically to a b integrin [ ] . in a phase i trial conducted by ricart et al., volociximab was shown to stabilize disease in patients with advanced solid tumors [ ] . vedolizumab, a humanized monoclonal antibody that specifically recognizes the a b heterodimer, underwent a phase trial to determine its effectiveness and safety in treating patients with ulcerative colitis [ ] . in that study, vedolizumab as both an initial and maintenance therapy for patients with active ulcerative colitis was shown to be effective in achieving a response and remission [ ] . recently, natalizumab, one of the five therapeutic drugs targeting integrins, has been approved for clinic use. this engineered pan-a antibody has been approved for recurrent multiple sclerosis (ms) patients and has been shown to yield promising results for relapsed ms patients by reducing the frequency of relapse, a unique therapeutic result [ ] . its efficacy against crohn's disease has also been demonstrated [ ] . though integrins have been targeted to treat cancers and other pathological disorders, we have not made a significant breakthrough in targeting integrins to treat virus infections. this does not seem encouraging, especially with many viruses having been shown to utilize integrins to enter cells. this may be due to the fact that (i) viruses utilize multiple receptor molecules to enter the same cell and that (ii) the receptors utilized by the same virus to enter cells in vitro and in vivo may differ [ ] . to overcome this pitfall, we may have to decipher the key elements in the motifs on the virus that interact with integrins and conduct detailed comparative studies outlining the manner by which the virus enters cells under in vitro and in vivo conditions. this will be crucial for gaining comprehensive knowledge of the receptors utilized by viruses to infect cells. such studies, we hope, will get us one step closer to developing treatment strategies targeting integrins to combat viral infections. integrins are just not receptors expressed on the surface of cells. they regulate a diverse set of cellular functions are involved in the pathology of autoimmune diseases [ ] and viral infections [ ] . viruses utilize different types of integrins, which are classified primarily based on the manner in which they interact with their ligands, ecm proteins. integrins physically recognize and interact with distinct amino acid sequences contained within the ligands or pathogens. these can be rgd or any other specific (non-rgd) sequences. whatever the amino acid recognition sequence may be, integrins (rgd and non-rgd) seem to generally aid in virus attachment and entry into cells. the function of integrins is not limited to providing anchoring for the virus. they are also critical for preparing the cells to support a permissive infection via outside-in signaling. expression of integrins seems to be of relevance in the initial infection as well as in the pathobiology of the virus-induced condition. recent growth in the field of biomedical sciences has already aided in the development of therapeutics based on integrin interactions to treat various cancers and other pathological conditions. to date, such novel therapeutics to treat virus infections are still only a dream, even though multiple viruses seem to utilize integrins to enter cells. future studies, we hope, will work toward understanding the roles of integrins in virus infection and associated pathogenesis, as such studies may result in novel treatment regimens aimed at preventing the internalization of viruses. after all, the ideal method of treating infection is to block the entry of a pathogen into cells. rotaviruses interact with alpha beta and alpha beta integrins by binding the same integrin domains as natural ligands rotavirus spike protein vp * binds alpha beta integrin on the cell surface and competes with virus for cell binding and infectivity adenovirus -fiber chimeric vector mediates efficient apical correction of the cystic fibrosis transmembrane conductance regulator defect in cystic fibrosis primary airway epithelia integrin alpha(v)beta( ) mediates rotavirus cell entry biochemical characterization of rotavirus receptors in ma cells integrin signalling during tumour progression conservation of the alpha beta lymphocyte homing receptor in hiv-infected patients with distinct transmission routes and disease progression profiles the effect of the novel tellurium compound as on autoimmune diseases kaposi's sarcoma-associated herpesvirus (human herpesvirus ) contains hypoxia response elements: relevance to lytic induction by hypoxia ross river virus transmission, infection, and disease: a cross-disciplinary review integrin activation by bacterial fimbriae through a pathway involving cd , toll-like receptor , and phosphatidylinositol- -kinase internalization of coxsackievirus a is mediated by {beta} -microglobulin, dynamin, and arf but not by caveolin- or clathrin the leukocyte beta integrins carbohydrate-related inhibitors of dengue virus entry integrin-mediated uptake of fibronectin-binding bacteria multiple roles of integrins in cell motility vaccinia virus envelope d l protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells integrins in cell migration integrins: a family of cell surface receptors integrins: bidirectional, allosteric signaling machines integrin beta mediates vaccinia virus entry through activation of pi k/akt signaling foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin alpha beta : influence of the leucine residue within the rgdl motif on selectivity of integrin binding integrin alphavbeta functions as a receptor for foot-and-mouth disease virus: role of the betachain cytodomain in integrin-mediated infection the varicella zoster virus glycoprotein b (gb) plays a role in virus binding to cell surface heparan sulfate proteoglycans attachment of bovine parvovirus to sialic acids on bovine cell membranes molecular mechanism of alpha -beta integrin interaction with human echovirus a human cytomegalovirus glycoprotein complex designated gc-ii is a major heparin-binding component of the envelope extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor focal adhesion kinase is critical for entry of kaposi's sarcoma-associated herpesvirus into target cells an arthritogenic alphavirus uses the alpha beta integrin collagen receptor examination of soluble integrin resistant mutants of foot-and-mouth disease virus crystal structure of the a domain from the alpha subunit of integrin cr (cd b/cd ) role of glycosaminoglycans for binding and infection of hepatitis b virus integrin alpha beta function is required for cell survival in developing retina getting to the site of inflammation: the leukocyte adhesion cascade updated adenovirus endocytosis requires actin cytoskeleton reorganization mediated by rho family gtpases adenovirus endocytosis via alpha(v) integrins requires phosphoinositide- -oh kinase integrin alpha(v)beta is an adenovirus coreceptor in-vitro and in-vivo phenotype of type asia foot-and-mouth disease viruses utilizing two non-rgd receptor recognition sites molecular basis of the inflammatory response to adenovirus vectors multistep entry of rotavirus into cells: a versaillesque dance structural basis of integrin regulation and signaling sendai virus utilizes specific sialyloligosaccharides as host cell receptor determinants looking beyond death: a morphogenetic role for the tnf signalling pathway signaling on the endocytic pathway epstein-barr virus enters b cells and epithelial cells by different routes integrin-regulated fak-src signaling in normal and cancer cells hiv- tat promotes integrin-mediated hiv transmission to dendritic cells by binding env spikes and competes neutralization by anti-hiv antibodies natural history of ross river virus-induced epidemic polyarthritis a possible role of lysophospholipids produced by calcium-independent phospholipase a( ) in membrane-raft budding and fission kaposi's sarcoma-associated herpesvirus induces the phosphatidylinositol -kinase-pkc-zeta-mek-erk signaling pathway in target cells early during infection: implications for infectivity kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via rhoa-gtp-diaphanous signaling and utilizes the dynein motors to deliver its dna to the nucleus antibody neutralization epitopes and integrin binding sites on nonenveloped viruses sulfated polymers inhibit the interaction of human cytomegalovirus with cell surface heparan sulfate glycoconjugate glycans as viral receptors herpes simplex virus type glycoprotein h binds to alphavbeta integrins adenovirus-induced maturation of dendritic cells through a pi kinase-mediated tnf-alpha induction pathway ligand binding to integrins natalizumab for relapsing remitting multiple sclerosis lipid rafts of primary endothelial cells are essential for kaposi's sarcoma-associated herpesvirus/human herpesvirus -induced phosphatidylinositol -kinase and rhoa-gtpases critical for microtubule dynamics and nuclear delivery of viral dna but dispensable for binding and entry pathogenic hantaviruses bind plexin-semaphorin-integrin domains present at the apex of inactive, bent alphavbeta integrin conformers volociximab, a chimeric monoclonal antibody that specifically binds alpha -beta integrin: a phase i, pharmacokinetic, and biological correlative study rgd-dependent entry of coxsackievirus a into host cells and its bypass after cleavage of vp protein by intestinal proteases entry of coxsackievirus a into host cells: specific interactions with alpha v beta integrin, the vitronectin receptor efficient rgd-independent entry process of coxsackievirus a arg-gly-asp: a versatile cell recognition signal galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (damps) and as receptors for pathogen-associated molecular patterns (pamps) integrins modulate the infection efficiency of west nile virus into cells alpha beta -integrin controls ebolavirus entry by regulating endosomal cathepsins role of the extracellular domain of human herpesvirus glycoprotein b in virus binding to cell surface heparan sulfate proteoglycans integrins alpha beta and alpha -beta are receptors for the rotavirus enterotoxin integrins aid virus entry structural and functional analysis of coxsackievirus a integrin alphavbeta binding and uncoating kaposi's sarcoma-associated herpesvirus/human herpesvirus envelope glycoprotein gb induces the integrin-dependent focal adhesion kinase-src-phosphatidylinositol -kinase-rho gtpase signal pathways and cytoskeletal rearrangements deletion of penton rgd motifs affects the efficiency of both the internalization and the endosome escape of viral particles containing adenovirus serotype or fiber knobs a novel role for -o-sulfated heparan sulfate in herpes simplex virus entry herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry a single-amino-acid polymorphism in chikungunya virus e glycoprotein influences glycosaminoglycan utilization receptor binding and membrane fusion in virus entry: the influenza hemagglutinin identification and analysis of a novel heparin-binding glycoprotein encoded by human herpesvirus a role for alpha -integrin in the pathology following semliki forest virus infection herpes simplex virus: receptors and ligands for cell entry cell extracellular matrix interaction in cancer integrin-mediated signaling induced by simian virus leads to transient uncoupling of cortical actin and the plasma membrane cell integrins: commonly used receptors for diverse viral pathogens alphavbeta integrin: a co-receptor for adeno-associated virus type infection broad distribution of the jc virus receptor contrasts with a marked cellular restriction of virus replication virus infection and lipid rafts involvement of glycoprotein c (gc) in adsorption of herpes simplex virus type (hsv- ) to the cell the integrins genogroup ii noroviruses efficiently bind to heparan sulfate proteoglycan associated with the cellular membrane rho, rac and cdc gtpases regulate the organization of the actin cytoskeleton glycosphingolipids as receptors for non-enveloped viruses perspectives in glycomics and lectin engineering hiv- tat regulates endothelial cell cycle progression via activation of the ras/erk mapk signaling pathway loss of the alpha beta integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro human parechovirus utilizes integrins alphavbeta and alphavbeta as receptors mechanisms of integrin-mediated virus attachment and internalization process lipid raft microdomains: key sites for coxsackievirus a infectious cycle herpes simplex virus types and differ in their interaction with heparan sulfate epstein-barr virus infection of polarized tongue and nasopharyngeal epithelial cells chondroitin -osulfotransferase- regulates e disaccharide expression of chondroitin sulfate required for herpes simplex virus infectivity single-particle em reveals plasticity of interactions between the adenovirus penton base and integrin alphavbeta kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (al-phavbeta , alphavbeta , and alpha beta ) and cd -xct during infection of human dermal microvascular endothelial cells, and cd -xct is essential for the postentry stage of infection alphavbeta integrin regulates heregulin (hrg)-induced cell proliferation and survival in breast cancer asymmetric synthesis of water-soluble analogues of galactosylceramide, an hiv- receptor: new tools to study virus-glycolipid interactions glycans as receptors for influenza pathogenesis a novel integrin specificity exemplified by binding of the alpha v beta integrin to the basic domain of the hiv tat protein and vitronectin disintegrin-like domain of glycoprotein b regulates kaposi's sarcoma-associated herpesvirus infection of cells structural basis of lectin-carbohydrate recognition host and virus determinants of picornavirus pathogenesis and tropism integrins alpha v beta and alpha v beta promote adenovirus internalization but not virus attachment integrin alpha v beta selectively promotes adenovirus mediated cell membrane permeabilization egf receptor signaling stimulates src kinase phosphorylation of clathrin, influencing clathrin redistribution and egf uptake integrin alpha v beta is an rgd-dependent receptor for coxsackievirus a adeno-associated virus serotypes: vector toolkit for human gene therapy structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics crystal structure of the extracellular segment of integrin alpha vbeta alpha( ) integrin is the main receptor of human papillomavirus type vlp functional atlas of the integrin adhesome integrin alpha beta mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins tumor alphavbeta integrin is a therapeutic target for breast cancer bone metastases ) beta integrins are required for vascular morphogenesis in mouse embryos key: cord- - evqo m authors: litwin, christine m.; bosley, james g. title: seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: evqo m respiratory tract infections (rtis) are a leading cause of mortality and morbidity. seasonality has been reported for many viruses, including influenza virus, respiratory syncytial virus (rsv), and the recently described human metapneumovirus (hmpv). we hypothesize that the availability of rapid, multiplex pcr diagnostics will provide better clinical care and new insights into the etiology and clinical spectrum of rtis. we conducted a retrospective analysis of the incidence of respiratory pathogens at a -bed adult and -bed pediatric hospital tertiary care center. a total of specimens from patients with an age range of days to years (median, years) were tested by a multiplex respiratory pathogen pcr from november , to november , . sixty-five percent of specimens were positive for at least one pathogen. as the age of the patient increased, the positivity rate for the pcr decreased proportionately. rhinoviruses/enteroviruses (rhino/entero) were the most prevalent ( . %) followed by rsv ( . %) and hmpv ( . %). twelve percent of the positive samples were positive for multiple analytes, with rhino/entero and rsv being the most common combination. the peak months were september and may for rhino/entero infections, january for rsv and february for coronavirus. hmpv peaked months after rsv, as has been observed recently in other studies. multiplex pcr provides rapid diagnostic information that can be used to make knowledgeable clinical decisions and potentially reduce the use of antibiotics. active respiratory pcr surveillance could also predict seasonal respiratory epidemics to allow for adequate planning of additional infection control measures. acute respiratory tract illnesses are the most common illness in all age groups and are an important cause of hospitalization and mortality, especially in the winter months. for children less than years of age, respiratory tract infections are the second leading cause of death [ ] . major causes of bronchiolitis and lower respiratory tract illnesses in children include respiratory syncytial virus (rsv), parainfluenza viruses, influenza virus, and human metapneumovirus (hmpv). understanding seasonal variations in these infections may allow optimal planning and utilization of resources in emergency departments, hospitals and clinics. past epidemiologic studies used routine diagnostic methods such as culture for the detection of viral and bacterial respiratory pathogens [ , ] . however, viral culture requires to days to detect most agents and detects rhinovirus or coronaviruses poorly or not at all. multiplex reverse transcriptase pcr has been shown to be more sensitive than standard respiratory virus culture, bacterial culture, and antigen detection methods [ , , , ] . moreover, pcr is much faster. for the recently characterized hmpv, rt-pcr-based techniques are generally the method of choice for detection [ ] . no antigen detection methods are commercially available for hmpv. expanded multiplex pcr panels now allow the detection of up to different viruses along with influenza a subtyping and the detection of three common bacterial respiratory pathogens, mycoplasma pneumoniae, chlamydia pneumoniae and bordetella pertussis in one hour [ , ] . the high sensitivity and the expanded capability of these tests, therefore, may affect our understanding of the epidemiology of respiratory tract infections. this study explores the seasonality and prevalence of respiratory viral pathogens at a tertiary care medical center using the multiplex pcr respiratory pathogen panel. testing took place from november , to november , on nasopharyngeal specimens (nps) originally sent to georgia health sciences university (ghsu; its name was recently changed to georgia regents university) clinical microbiology laboratory (augusta, ga) from the -bed adult and -bed pediatric hospital at ghsu for respiratory pathogen pcr assay by the filmarray respiratory panel (rp) (biofire diagnostics, inc., salt lake city, ut). nps specimens were obtained from patients with symptoms of a respiratory infection, collected from the patients using standard technique, and placed in viral transport medium (remel microtest m rt viral transport tube). specimens were tested as soon as possible after collection. the project was approved by the institutional review board of our institution; informed consent for the project was waived. demographic data, such as initial symptoms, chief complaint, age, gender, and secondary diagnosis were obtained for each specimen tested. the filmarray assay was performed according to the manufacturer's instructions. in brief, ml of purified water included in the kit was injected into the filmarray pouch to rehydrate the reagents. then, ll of the viral transport medium that had contained the nps specimen was mixed with ll of sample buffer and injected into the sample port of the pouch. the pouch was then placed into the filmarray instrument, and a respiratory pcr panel program was started. the first stage of the program consists of a multiplexed pcr, followed by an individual nested second-stage real-time pcr contained within a microarray chip. the filmarray rp includes two internal controls: an rna process control and controls for every step inside the pouch. results are analyzed using melting curve data. the organism/viruses detected by the filmarray included adenovirus, influenza a virus (flua), influenza b virus (flub), parainfluenza virus (para ), parainfluenza virus (para ), parainfluenza virus (para ), parainfluenza virus (para ), respiratory syncytial virus (rsv), coronavirus e (coronav e), coronav nl , coronav hku , coronav oc , human metapneumovirus (hmpv), bordetella pertussis, chlamydophila pneumoniae and mycoplasma pneumoniae. due to genetic similarity between the human rhinoviruses and enteroviruses, a positive result with pcr primers to these viruses was listed as rhino/ entero. rhinovirus a, b and c and enterovirus a, b and c are detected in the assay. both rsv subtypes a and b are detected in the assay, though not specifically assayed. the flua viruses could also be subtyped as flua/h , flua/h or flua- if present. a two-sample student's t-test between proportions was performed to determine whether there was a significant difference between the viruses with respect to the percentage of initial clinical symptoms in both single virus infections and mixed infections [ ] . statistical analysis was performed using the software package statpac for windows (pepin, wi). specimens from a total of patients were analyzed by pcr. the age range of the patients was days to years of age (median, years). the male:female ratio was . . rhino-and enteroviruses were the most prevalent ( . %), followed by rsv ( . %) and hmpv ( . %) ( table ) . the data were divided according to age groups. the largest age group in our study was children b years of age, followed by the age group - years of age ( table ) . the older age groups, - , and c years were small compared to the first two groups, with and patients in each group, respectively. the highest rate of pcr positivity was observed in children b years of age, with a rate of . % (table ) , followed by . % for the second-youngest age group, ages - . the positivity rates for the - and c age groups were much lower, at . % and . %, respectively. the prevalence of specific viruses and bacteria also differed between age groups (table ) . most viruses and bacteria showed the highest prevalence in children b , with the exception of flu a, for which the highest number of infections was in the - age group (table ) . rhino/ entero viruses were the most prevalent viruses in all age groups except in the - age group, where there were equal numbers of rhino/entero cases and para cases, although there were only three cases in each group. no positive results were detected for b. pertussis during the study period. all hmpv cases were detected in the b and - age groups, except for two cases. one case was in a -year-old female day-care worker admitted for asthma exacerbation, and the second was a -year-old female with an underlying hematologic malignancy. all rsv cases were also detected in the b and - age group, except for two cases. one case of rsv was detected in a -year-male who also tested positive for rhino/entero, and a second case was detected in a -yearold male. the patient that tested positive for both rhino/ entero and rsv was the only patient above the age of who tested positive for more than one virus. the major initial clinical symptoms/diagnoses of the patients included respiratory distress, asthma/wheezing, pneumonia, fever or bronchiolitis. the percentages of the major initial symptoms/diagnoses for the more prevalent pcr results are presented in fig. . when compared to rsv, patients with hmpv showed significantly less respiratory distress ( . %, p. ) as a presenting symptom, but more fever ( . %, p. ). there were no significant differences in the percentages of initial presentations with pneumonia, asthma/wheezing or bronchiolitis between rsv and hmpv. when single rsv or hmpv infections were compared to single rhino/enterovirus infections, there was a significantly higher percentage that presented with pneumonia ( . %, p. or . %, p. , respectively) or bronchiolitis ( . %, p. ; . %, p. ). mixed coinfections with rhino/entero and rsv or hmpv showed a significant lower percentage that presented with pneumonia ( . %, p. ), but not with bronchiolitis ( . %). there was a significant increase in the number of patients that presented with respiratory distress with a mixed coronavirus and rsv or hmpv co-infection ( . %, p. ). there were only cases for comparison, however. there were only two co-infections with both rsv and hmpv, one as part of a quadruple infection with two coronaviruses and one rsv and hmpv co-infection. the patient with the quadruple infection was admitted to pediatric intensive care with severe respiratory distress. the coinfection rsv and hmpv was an outpatient case seen for asthma exacerbation. all three cases of m. pneumoniae involved co-infections ( table ) . two of the cases were co-infected with rhino/ entero, and the third was a triple infection with adenovirus and rhino/entero. with all three cases, the initial symptom was asthma/wheezing. one of the two c. pneumoniae cases was a co-infection with rhino/entero with an initial symptom of respiratory distress. the pure infection with c. pneumoniae had an initial diagnosis of pneumonia. analysis of multi-analyte-positive samples sixty-five percent ( %) of all specimens were positive for at least one viral or bacterial organism. of the positive specimens, % ( / ) were positive for more than one analyte ( table ). the majority ( / ) of multianalyte-positive samples were from patients b years of age. the most common viruses detected in multi-analytepositive specimens were rhino/entero and rsv, which comprised % of all multi-analyte-positive samples ( / ). individually, rhino/entero and rsv were detected in all multi-analyte-positive samples, % and % of the time, respectively (fig. ). there were four triple-positive samples and two quadruple-positive samples ( table ) . additional viruses found in multi-analyte positive samples included hmpv, para and coronav (hku , nl , oc ). the majority of the hmpv multi-analytepositive samples were in conjunction with rhino/entero. there were only two samples in which hmpv was detected together with coronav (hku ) or coronav (hku , nl ) and rsv. also, the majority of the para multianalyte-positive samples were also positive for rhino/entero. there were only two para positives that were positive for rsv. seasonal prevalence of respiratory pathogens from november , to november , the prevalence of rhino/entero remained relatively high throughout the year, with several incidence peaks, the highest occurring in august/september, with two minor peaks in may and december (fig. ) . a distinct peak in rsv cases occurred in january, followed by a peak in total coronavirus cases in february, and a peak in hmpv cases in march. a distinct peak for para cases was observed in may. the eight cases of para and seven cases of para all occurred in the fall, in september-november (data not shown). the one case of para was detected in november. the eleven cases of adenovirus occurred both in the spring and the fall months. four cases of flua type h - were detected february-april . two cases of flua type h were detected march , and eight cases were detected november . one case of flu b was detected february the number in parentheses is the total number of cases in the group . no seasonal variation was noted for the eleven adenovirus cases. all three m. pneumoniae cases occurred in september, and the two c. pneumoniae cases occurred in january and november. in our study, specimens were analyzed over the course of a year using a respiratory pathogen multiplex pcr that yielded a positivity rate of % with multiple analytes detected in % of specimens, especially in children. as the age of the patient increased, the positivity rate for the pcr decreased proportionately. other multiplex respiratory pcr studies have reported lower positivity rates for adults [ , ] . studies have shown that older adult patients shed lower titers of viruses, which demands the use of a highly sensitive methodology such as rt-pcr over conventional culture or dfa [ ] . it is possible that the multiplex pcr assays now available may still not be sensitive enough to detect the lower titers of virus shed by older adult patients. it is also possible that adult patients may have other bacterial infections causing the respiratory infections that are not detected by the pcr, such as legionella or streptococcus pneumoniae. we noted a seasonal variation of the number of viral infections. rhino/entero was the most prevalent viral infection, with demonstrated peaks in the number of cases in may and september. the september peak appears to correspond to the start of school sessions. rsv was the second most prevalent virus, with a very large number of cases in january, followed two months later by an increased number of cases of hmpv. rhino/entero was the predominant virus for all age groups and was superseded only by rsv from december to march. brittain-long et al. similarly observed that rhinovirus was the most common finding in the respiratory tract in their study, at . %, and was the predominant virus regardless of season [ ] . although the rhinoviruses are the most perennial of all the seasonal viruses, peaks of rhinovirus illness are well documented in september, after school starts, and again in the spring [ , , ] . rsv and hmpv were the second-and third-most common causes of respiratory infections in our study. the majority of the rsv and hmpv infections in our study occurred in children. recently, in a study of over , children less than years of age, the burden of hmpv was determined to be % in hospitalized children and % of children in both outpatient clinics and emergency departments [ ] . we also detected a similar positivity rate of . %. rsv and hmpv pcr-positive cases were statistically much more likely than rhino/entero pcr-positive infections to initially present with pneumonia or bronchiolitis in our study. huguenin et al. similarly showed that rsv was associated with more-severe disease in infants hospitalized for bronchiolitis [ ] . rsv and hmpv are also known to cause severe respiratory infections in the elderly [ ] . some of these infections did occur in adults c in our study [ , ] . epidemiologic peaks for hmpv occurring to months later than that observed for rsv epidemics have been described in a number of studies [ , , , , ] . our study certainly supports this seasonality. the parainfluenza viruses tend to have different patterns of seasonal variation based on type. para , which can be predominant in spring in temperate climates, increased in the month of may [ ] . both para and para cases in our study were seen only in the fall months in our study, the typical season for these viruses [ ] . the epidemiology of para is largely unknown. the number of coronavirus cases peaked in february in our study, indicating a winter seasonality. there is some evidence, however, that the seasonality of the coronaviruses may be dependent on geographic location [ ] . there was an unusually low prevalence of influenza a virus ( . %) for the - season in our study. in a review of the - winter influenza season in the northern hemisphere by the who, the influenza season started unusually late in most of north america, the latest in nearly years in the u.s. [ ] . the season was remarkably mild in the u.s., where influenza a (h n ) was the predominant virus circulating. transmission remained low until the end of the year. no b. pertussis cases were detected in our hospital setting. the state of georgia has one of the lowest incidences of reported b. pertussis in the entire u.s., with an incidence of . per , [ ] . multiple respiratory pathogens were detected in % of the respiratory specimens in our study; % of the time, the second analyte was rhino/entero. recently, rand et al. [ ] detected . % co-infections in a study of patients by multiplex respiratory pcr. other studies have reported slightly lower rates of multiple-analyte-positive specimens of around % and . % [ , ] . the significance of these multi-analyte-positive specimens is not clear, especially in conjunction with rhino/ entero. it is likely that many of the dual-positive results with rsv and rhino/entero samples are due to viral shedding from a previous rhino/entero infection [ ] . rhinovirus is often found at a higher frequency than any other respiratory virus in asymptomatic carriers [ ] . there was a significant increase in the number of patients who presented with respiratory distress ( . %) with a mixed coronavirus and rsv or hmpv co-infection. it is possible that these may have represented true co-infections with coronavirus rather than viral shedding. however, the significance of this observation is limited by having only patients in this group. some studies that have concluded that more-severe disease occurs in infants with bronchiolitis with co-infections with rsv and hmpv, of which we detected only two cases, one as part of a quadruple infection and one mixed infection with rsv and hmpv [ , ] . the quadruple infection was a severe respiratory case requiring intensive care. a possible limitation of the study is that the subjects are biased toward the younger age groups, with only a modest representation of adults. however, the population reflects current physicians' choice of using pcr for the diagnosis of respiratory symptoms. young children may seek healthcare earlier in the course of disease, and the reasons for testing in young children may be different in adults compared to children and young adults. multiplex respiratory pcr has been used in some cases to reduce nosocomial infections during specific seasonal respiratory epidemics. for example, some investigators have advocated performing multiplex respiratory pcr on all symptomatic infants during the rsv season, recommending that the rsv-positive patients be cohorted and placed on gown and glove precautions, noting a reduction in nosocomial infection in newborn nurseries after putting such practices into place [ , ] . ideally, active respiratory pcr surveillance could help predict seasonal respiratory epidemics so that additional infection control measures can be instituted. a distinct advantage of the rapidity of diagnosis with the multiplex pcr is the ability to quickly and specifically treat the respiratory pathogen and facilitate therapy. some of the pathogens detected in the multiplex pcr have specific therapies [ ] . the multiplex respiratory pcr detects three bacterial pathogens that can all be treated with specific antibiotics, including c. pneumoniae, m. pneumoniae, and b. pertussis. adamantanes and neuraminidase inhibitors are available for the influenza viruses. intravenous polyclonal immune globulin enriched against rsv and palivzumab, a monoclonal antibody against the rsv fusion glycoprotein, have been recommended for high-risk infants. ribavirin has been also been approved for use with rsv and has had some apparent success in cases of infection with hmpv, adenovirus and parainfluenza viruses. most conventional immunoglobulin preparations seem to contain sufficient amounts of neutralizing titers against hmpv. in conclusion, molecular testing methods have significantly expanded our ability to diagnose respiratory infections because of their rapidity, sensitivity, and potential for the simultaneous detection of or more respiratory pathogens. the use of a rapid and accurate diagnostic test enhances clinical decision making and safely promotes implementation of cost-effective treatment strategies, including limiting the unnecessary use of antibiotics. review of the - winter influenza season, northern hemisphere treatment of respiratory virus infections human metapneumovirus subgroup changes and seasonality during epidemics biennial spring activity of human 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of respiratory viruses by molecular methods the tecumseh study of respiratory illness. v. patterns of infection with the parainfluenzaviruses epidemiology of viral respiratory infections occurrence of respiratory virus: time, place and person respiratory syncytial virus infection in adults human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms multicode-plx system for multiplexed detection of seventeen respiratory viruses pcr for detection of respiratory viruses: seasonal variations of virus infections mixed respiratory virus infections ) filmarray, an automated nested multiplex pcr system for multipathogen detection: development and application to respiratory tract infection comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp prevalence of human coronaviruses in adults with acute respiratory tract infections in beijing the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis seasonality and clinical features of human metapneumovirus infection in children in northern alberta human metapneumovirus: lessons learned over the first decade performance of diagnostic tests to detect respiratory viruses in older adults prevention of nosocomial transmission of respiratory syncytial virus in a newborn nursery viral respiratory infection in curitiba, southern brazil ten years' experience with year-round active surveillance of up to respiratory pathogens in children acknowledgments this study was supported by the department of pathology, medical college of georgia, georgia regents university, augusta, georgia. we declare that we have no conflicts of interest with respect to this study. key: cord- -adg vb authors: wanitchang, asawin; saenboonrueng, janya; srisutthisamphan, kanjana; jongkaewwattana, anan title: characterization of influenza a virus pseudotyped with the spike protein of porcine epidemic diarrhea virus date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: adg vb the coronavirus spike protein and the influenza virus hemagglutinin are class i viral membrane fusion proteins. while the two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, they share no sequence similarity. whether they are functionally interchangeable is currently unknown. in this study, we constructed sciav-s, a single-cycle influenza a virus pseudotyped with the spike protein of porcine epidemic diarrhea virus (pedv), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. treatment with endocytosis inhibitors did not affect syncytium formation by infected cells. moreover, the infectivity of sciav-s was associated with the degree of cell adaptation of pedv-s. intriguingly, sciav-s lacking functional neuraminidase (na) exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of pedv. together, sciav-s offers a robust platform for the investigation of the entry mechanism of pedv or, possibly, of other coronaviruses. porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus, is a pleomorphic enveloped virus that contains a large (approximately kb), positive-sense, single-stranded rna genome. it is a causative agent of ped, a highly contagious gastrointestinal disease in pigs with mortality reaching % in suckling piglets. while pedv replicates efficiently in porcine enterocytes in vivo, it grows poorly in cultured cells unless adapted through multiple passages [ ] . like those of other coronaviruses (covs), the pedv spike protein (pedv-s) is responsible for both receptor recognition and fusion between the virus and host cells [ , ] . notably, the identity of the receptor used by pedv is still a subject of controversy. while earlier studies have suggested that pedv uses porcine aminopeptidase n (papn) as a cellular receptor [ , , , ] , more-recent studies have supported a contrary view based on several lines of evidence [ , ] . cov-s proteins, including pedv-s, are members of the class i viral membrane fusion proteins, which also include hemagglutinin (ha) from influenza a virus (iav) [ , ] . both cov-s and ha fold into a metastable prefusion conformation and require proteolytic cleavage (priming) by host proteases to make them fusogenic. this cleavage produces a receptor-binding subunit (ha of influenza virus or s of cov-s) and a membrane-fusion subunit (ha or s ), which remain associated through non-covalent interactions. while details of the prefusion structure of pedv-s are lacking, recent studies of the spike proteins of the betacoronaviruses mhv and hku have suggested that the overall prefusion structures of cov-s and ha are similar [ ] and that they might share a common mechanism of membrane fusion. however, a few fundamental differences between the two proteins should also be considered. while the fusion peptide of ha is located at the n-terminus of ha , cov-s has an internal fusion peptide located downstream of the n-terminus of s . consequently, two proteolysis sites are usually required for the conformational change of s : one at the s /s junction and the other at the so-called s ' site located at the n-terminus of the fusion peptide [ , ] . in addition, unlike ha, many cov-s, proteins probably including that of pedv, are not sufficiently primed by proteolysis during viral packaging release and thus require further proteolysis by extracellular proteases, cell-surface proteases, or lysosomal proteases [ ] . considering these common and unique properties of the two proteins, it would be interesting to investigate whether they could functionally replace each other in a virus infection context. recombinant retrovirus pseudotyped with pedv-s has been generated and employed to investigate the pedv entry mechanism [ , ] . however, cells transduced by pseudotyped retrovirus usually display characteristics that are distinct from those of cells that are infected with pedv. for example, cell-cell fusion is rarely observed in transduced cells. notably, with the successful development of a system to construct single-cycle influenza a virus (sciav) [ , ] , it is now possible to assess whether sciav can be generated when pedv-s is provided in trans instead of ha. the resulting pseudotyped virus (sciav-s) will be beneficial especially for studying entry mechanism mediated by pedv-s. the present study was undertaken to test the hypothesis that pedv-s can functionally replace ha to drive replication of iav by constructing sciav-s and examining its ability to infect pedv-permissive cells. we also showed, using our pseudotyped virus system, that sialic acid is critical for mediating pedv entry. in addition, we demonstrated that iav carrying pedv-s derived from cell-adapted and field-isolated strains exhibited distinct characteristics in infected cells, suggesting different modes of entry among pedv strains. human embryonic kidney (hek) t, veroe -apn, and madin-darby canine kidney (mdck) cells were maintained at °c in opti-mem (thermo scientific) supplemented with % heat-inactivated fetal bovine serum (fbs) and iu of penicillin and mg of streptomycin per ml in humidified % co incubators. all sciav-pedv-s used in this study were generated in the hek t cells and stored at − °c until use. recombinant pedv avct was generated by reverse genetics and propagated in veroe -apn cells as described previously [ , ] . influenza a virus (a/ pr/ / ) was generated and titrated as described previously [ ] . the full-length pedv s derived from pedv avct (gen-bank lc . ), pedv yn (genbank kt . ) and pedv g (field isolate) were codon-optimized for expression in mammalian cells and synthesized (genscript and synbio tech). subsequently, each construct was subcloned into the pcaggs expression plasmid. to ensure optimal surface expression, er retention signals at the c-terminal end were removed from all constructs. the phw-ha-mcherry and phw-Δm plasmids were constructed as described previously [ ] . to construct phw-Δna, phw encoding the na gene of a/pr/ / was subjected to site-directed mutagenesis to introduce two consecutive stop codons at amino acids and . all plasmids were subjected to nucleotide sequencing to ensure that no unwanted mutations were inadvertently introduced. sciav-s expressing the mcherry protein were rescued using a strategy similar to one described in a previous study [ ] . briefly, hek t cells in a six-well plate were transfected with . μg each of phw plasmids encoding the seven segments (pb , pb , pa, np, na, m, and ns) from a/ pr/ / and phw-ha-mcherry together with μg of the pcaggs plasmid encoding each construct of pedv-s, using fugene hd (promega). to construct sciav-s lacking na, phw-Δna was used instead of phw -na. likewise, sciav-s lacking m was constructed by using phw-Δm instead of phw -m. at h after transfection, cell supernatants were harvested and adsorbed directly onto veroe -apn cells for further analysis. western blot assays were carried out according to the published procedure with some modifications [ ] . transfected cells were collected and lysed in μl of mammalian cell lysis buffer ( mm tris [ph . ], mm edta, mm nacl, % np- and protease inhibitor mixture) for min on ice. after centrifugation at , × g for min, lysates were separated on % sds-page gels and subsequently transferred to nitrocellulose membranes (bio-rad), followed by blocking with % non-fat milk in tbs-t for h. membranes were probed with one of the primary antibodies, including anti-pedv-s mouse polyclonal antibody (a kind gift from dr. qigai he), and anti-β-actin mouse monoclonal antibody clone c- (santa cruz biotechnology) followed by goat anti-mouse antibodies conjugated to hrp (biolegend). the signals were visualized with western blotting detection reagent (bio-rad). veroe -apn cells were grown on a lab-tek ii chamber slide (thermo scientific) in opti-mem supplemented with % fbs for h at °c. the adherent cells were inoculated with sciav-s for h at °c. after three washes with pbs, cells were cultured in serum-free opti-mem in the presence of trypsin ( μg/ml) for h. cells were fixed in % chilled acetone for min and blocked in % fbs/ %bsa/pbs for min. the slides were subsequently incubated with mouse anti-influenza-a-virus np antibodies (clone c ; southern biotech) for h, washed three times with pbs, and incubated with fitc-conjugated anti-mouse igg antibodies. after additional washes, the slides were mounted with antifade mounting medium with dapi (vector laboratories). cells were examined using an olympus ix fluorescence microscope. data were expressed as mean ± sd. statistical tests were performed using the student t-test in graphpad prism . (graphpad software). recently, we established a reverse genetics system to construct sciav in which the ha gene was replaced by the mcherry gene. the virus was recovered in hek t cells and subsequently propagated in mdck cells stably expressing the ha protein [ ] . using a similar strategy, we attempted to rescue sciav-s avct by transfecting hek t cells with plasmids necessary for iav reverse genetics together with codon-optimized s avct as depicted in fig. a . notably, we did not add trypsin to transfected cells during virus rescue to avoid the detachment of hek t cells. moreover, since cells expressing s avct underwent rapid cell-cell fusion, cells stably expressing s avct could not be obtained for the propagation of sciav-s avct . alternatively, the trypsin-free viruses released from hek t cells were directly inoculated onto veroe -apn cells treated with trypsin h after infection. at after infection, mcherry expression was assessed by fluorescence microscopy. unexpectedly, not only did we observe a strong mcherry signal, but we also detected extensive syncytia in sciav-s avct -infected cells (fig. b) . in the absence of trypsin treatment, we could still detect a few cells with an mcherry signal but no syncytium formation (fig. b) , suggesting that sciav-s avct might be able to enter cells but trypsin is essential for induction of cell-cell fusion. it should also be noted that control supernatants from hek t cells transfected with pcaggs expressing s avct alone did not yield detectable syncytium formation in veroe -apn cells, suggesting that the detected syncytia were not due to pedv-s expressed from residual plasmids (data not shown). to characterize sciav-s avct , we initially tried to purify rescued viruses from the supernatants and perform western blot analysis to detect co-expression of pedv-s and iav nucleoprotein (anp). unfortunately, despite several attempts, we could not obtain sufficient purified virus for the assay (data not shown). alternatively, we performed immunofluorescence staining to examine the expression of anp in the syncytium of sciav-s avct -infected veroe -apn cells and detected strong expression of anp (fig. c) . to further test whether the mcherry expression in infected cells required active influenza a virus polymerase activity, we attempted to construct sciav-s avct by omitting the plasmid encoding the pb polymerase and examined its infectivity in veroe -apn cells. as expected, in the absence of pb , neither an mcherry signal nor syncytium formation could be detected in infected cells (fig. d) . these results suggest that sciav can be pseudotyped with pedv-s and that the pseudovirus can efficiently infect pedv-permissive cells. it is also notable that the massive syncytium formation in sciav-infected cells was unexpected, and this observation has never been reported elsewhere. this points to the possibility that sciav-s might enter cells by a mechanism similar to that of pedv. given that sciav-s avct -infected cells formed large syncytia in the presence of trypsin (fig. b) and that s avct was not synthesized de novo in sciav-s avct -infected cells, we speculated that the observed syncytium formation might have been mediated by s avct remaining at the cell surface following internalization of sciav-s avct . in other words, replacing ha with s avct might have switched the mode of cell entry of sciav-s avct from endocytosis to direct fusion at the plasma membrane. moreover, since the m ion channel is essential for the uncoating of iav in host cell endosomes, entry of sciav-s avct by direct fusion with the plasma membrane would bypass viral-endosomal fusion, rendering the proton channel function of m unnecessary. to test this hypothesis, we constructed sciav-s avct lacking a functional m ion channel (sciavΔm -s avct ) and assessed its infectivity in veroe -apn cells. as shown in fig. , we detected undisturbed mcherry expression with large syncytium formation in sciavΔm -s avct -infected cells when infected cells were treated with trypsin. notably, we have shown previously that sciavΔm carrying iav ha showed no mcherry signal in mdck cells [ ] . previously, it was reported that pedv strain kpedv- is likely to enter host cells via endocytosis, as infectivity was remarkably impaired when cells were treated with endocytosis inhibitors [ ] . it is still not known however, whether pedv can also fuse directly with the plasma membrane without being internalized by endocytosis, as reported previously for other covs. given that sciav-s avct lacking the functional m ion channel could infect veroe -apn cells and form syncytia without de novo synthesis of s avct , we thus hypothesized that sciav-s avct and pedv avct could enter host cells by direct fusion at the plasma membrane. to this end, veroe -apn cells were pretreated with hypertonic sucrose ( . m) to impair the formation of both coated and uncoated pits at the plasma membrane [ ] before being infected with sciav-s avct and recombinant pedv avct . in contrast to what has been reported previously [ ] , we detected no changes in either syncytium formation or mcherry expression in infected cells regardless of hypertonic sucrose treatment (fig. a) . likewise, veroe -apn cells treated with nh cl ( mm for h) to neutralize the intracellular ph also displayed strong mcherry expression and extensive syncytium formation when infected with sciav-s avct (fig. a) . in line with the observation in sciav-s avct -infected cells, infection of veroe -apn cells with pedv avct was also hardly affected by treatment with hypertonic sucrose or nh cl solution (fig. b) . it is also notable that pretreatment of veroe -apn or mdck cells with hypertonic sucrose or nh cl could substantially inhibit replication of influenza a virus (fig. c) . these results collectively suggest that at least pedv avct can enter host cells via a mechanism that is independent of the clathrin-mediated endocytosis pathway. several studies have shown that naturally isolated pedv strains, mostly in the g genogroup, replicate poorly in vero cells [ ] . accumulated mutations in the s gene are strongly associated with cell adaptation [ , , ] . it was therefore of interest to assess the infectivity of sciav pseudotyped with s derived from pedv with various degrees of cell adaptation. s avct is closely related to the classical pedv strain sm - , which is considered a highly cell-adapted strain. in addition, a variant (yn ) derived from a non-adapted strain after the th passage in vero cells [ ] is deemed to be an intermediately adapted strain. we therefore synthesized a codonoptimized s gene (s g ) based on a pedv isolated from intestinal tissues of a piglet experiencing severe watery diarrhea during the outbreak in thailand in . blast analysis of the s g protein revealed that the amino acid sequence of our protein was . % identical to that of ch/gxnn/ (genbank afo . ). moreover, a codon-optimized s gene of strain yn was synthesized. as mentioned previously, both synthetic constructs were designed to exclude c-terminal er retention motifs (yxxΦ and kxhxx). when plasmids expressing each pedv-s were used to transfect veroe -apn cells in the presence of trypsin, only those expressing s avct and s yn displayed massive syncytium formation (fig. a) . the failure to form syncytia in pedv-s g -transfected cells was not due to defects in protein expression in transfected cells (fig. a) . we next attempted to construct sciav pseudotyped with s g (sciav-s g ) and with s yn (sciav-s yn ) and evaluated their infectivity in veroe -apn cells. as a control, sciav-s avct was also generated and examined. while abundant mcherry-expressing cells and large syncytia were detected in the cell cultures infected with sciav-s avct and sciav-s yn , those infected with sciav-s g displayed considerably fewer positive cells with punctate expression of mcherry and no apparent syncytium formation (fig. b) . the absence of syncytia in sciav-s g -infected cells may be due to an intrinsic property of pedv-s g , as cells transfected with pcaggs-s g also displayed less syncytium formation than those transfected with pcaggs-s avct and -s yn (fig. a) . taken together, these results suggest that pedv strains with different degrees of adaptation might utilize distinct pathways for internalization into host cells. several lines of evidence have indicated that pedv utilizes sialic acid, possibly neu ac, as a co-receptor for attachment and entry [ , ] . moreover, pretreatment of host cells with recombinant na has been shown to substantially veroe -apn cells. the mean ± sd from three independent experiments is presented. n.s., not statistically significant. c. veroe -apn or mdck cells were mock-treated or treated with . m sucrose or mm nh cl prior to infection with influenza a virus (a/pr/ / ; moi = . ). infected cells were maintained in opti-mem supplemented with tpck-trypsin ( μg/ml). at the indicated time points after infection, supernatants were collected and the amount of infectious virus (tcid ) was determined in mdck cells. the mean ± sd from three independent experiments is presented. n.s., not statistically significant; **, p < . reduce pedv infectivity, and the n-terminal domain (ntd) of pedv-s (residues - ) has been proposed to be critical for sialic acid binding [ ] . unlike other previously described pseudotyped viruses, sciav-s, produced in this study, contains active na on its particles. it is therefore ideal for investigating the role of na in infection by sciav-s. since sciav-s does not require na for release, we were able to construct an sciav-s variant bearing a defective na by introducing two stop codons in the na gene (Δna). of note, the na deletion did not compromise the packaging signal of the na gene, and therefore, packaging of the na segment should be undisturbed. as expected, in the absence of na, both sciav-s avct and -yn could infect veroe -apn cells with substantially higher infectivity (fig. a) . while sciav-s g with Δna did not trigger syncytium formation in infected cells, we observed significantly more cells displaying an mcherry signal when compared to cells infected with the virus with intact na (fig. b) . taken together, these results indicate that the na activity of sciav-s might hinder the attachment of the pedv-s on the sciav-s particles with sialic acid on the cell surface. constraints associated with in vitro culture of natural isolates of pedv have considerably hindered the study of their replication, and, consequently, they have not yet been well characterized. in particular, the mechanism underlying how pedv enters a host cell is not well understood. we demonstrate in this study that iav particles could be efficiently pseudotyped with pedv-s, and such pseudoviruses could fig. infectivity of sciav bearing pedv-s derived from viruses with different levels of cell adaptation. a. veroe -apn cells were transfected with pcaggs encoding s avct , s yn or s g ( μg each). transfected cells were maintained in opti-mem supplemented with trypsin ( μg/ml). transfected cells were examined h later for syncytium formation. arrows denote syncytia. likewise, hek t cells were transfected with pcaggs encoding pedv-s using the same conditions. at h after transfection, cells were harvested, lysed and subjected to western blot analysis using anti-pedv-s antibodies. beta actin was used as a loading control. b. sciav psedotyped with s avct , s yn or s g was constructed, and its infectivity in veroe -apn cells was evaluated. at hpi, infected cells were examined for syncytium formation and mcherry expression under a fluorescent microscope. scale bars, μm ( μm for sciav-s g -infected cells) be employed to investigate the receptor binding and cellular entry of pedv. using a strategy similar to the one used with our previously described sciav system [ ] , we successfully rescued sciav carrying the mcherry gene in hek t cells co-expressing pedv-s derived from various strains and demonstrated that sciav-s avct could infect veroe -apn cells and produce a strong mcherry signal. unfortunately, due to the high fusogenicity of s avct , we could not generate cells stably expressing the protein as a platform to propagate sciav-s avct in the current study. however, s avct is now being modified to be less fusogenic without compromising its receptor-binding function. one of the most intriguing findings is our observation that sciav-s avct -infected cells could form massive syncytia similar to those produced by pedv-infected cells. since s avct is not synthesized de novo in sciav-s avct infected cells, the syncytium formation observed at neutral ph is likely to be due to interaction between the receptor and s avct remaining on the cell surface after internalization of the virus. our data thus underscore the likelihood that sciav-s avct can enter cells by direct fusion at the plasma membrane. it is worth noting that some studies have shown that treatment with trypsin allows some covs to bypass the endosome-dependent pathway for entry [ , , ] . however, sciav-s avct was recovered in the absence of trypsin, and virus inoculation was carried out using trypsin-free viruses. notably, unless trypsin was added at hpi, we could not detect any syncytium formation. indeed, syncytium formation was entirely unexpected, as pedv has been reported to enter cells by clathrin-mediated endocytosis even in the presence of large amounts of trypsin during infection [ ] . we also showed that sciav-s avct lacking the m proton channel could infect veroe -apn cells and trigger formation of large syncytia similar to those observed in sciav-s avct -infected cells. in contrast to our previous report that sciav-ha requires functional m for infectivity [ ] , our findings here suggest that sciav-s avct can undergo replication without the need for m -mediated internal virion acidification in the endosome. that pedv can enter permissive cells by both direct fusion with the plasma membrane and endocytosis is not surprising as similar phenomena have also been observed with other covs [ , , ] . in this study, we also speculated that differences in the s proteins of different pedv strains might play a role in determining the mode of entry. in line with our hypothesis, park and colleagues have recently demonstrated that the cleavage of the s protein during viral assembly appears to affect the mode of mers-cov entry. if s is cleaved at the s /s junction during biogenesis by host proteases such as furin or proprotein convertases in producer cells, the released virions are primed and able to enter target cells by fusing at the plasma membrane. alternatively, virions released with uncleaved s need to be processed further by endocytic proteases in the target cell and therefore require endocytosis for entry [ ] . although pedv-s, like other alphacoronaviruses, is a. sciav pseudotyped with s avct or s yn in the presence or absence of a functional na gene in segment was constructed. syncytium formation was assessed in veroe -apn cells (in a six-well plate) infected with each sciav-s construct. the mean + sd from three independent experiments is presented. **, p < . . b. sciav pseudotyped with s g in the presence or absence of a functional na gene in segment was constructed. the expression of mcherry was assessed in infected veroe -apn cells at hpi. scale bars, μm believed to be present in an uncleaved form [ ] , data showing the cleavage of s in different pedv strains are lacking. s proteins of cell-adapted pedv such as pedv avct or pedv yn may be cleaved during morphogenesis of the virion, whereas those of pedv g may require further processing in the target cells. further analysis of cleavage of the s protein in cell-adapted and field-isolated strains will be critical for better understanding of the mechanisms underlying pedv entry. another important point worth mentioning is that we showed, using sciav-s, that sialic acid moieties located on the cell surface plays a role in pedv attachment. although it has been demonstrated that the ntd of pedv-s could bind to sialic acid, most data of the available have been based on assays using recombinant s proteins [ ] . because the construction of recombinant pedv bearing specific mutations in the s gene can be technically challenging, the available data concerning the binding of pedv s to sialic-acid-containing glycoproteins in the context of infection have so far been limited to the use of natural isolates such as the pedv-uu strain [ ] . with a well-established iav reverse genetics system, sciav-s serves as a robust platform to assess binding of the s protein to sialic acid in the context of infection. furthermore, since many covs, such as mers-cov [ ] , and transmissible gastroenteritis coronavirus (tgev) [ ] , have been reported to bind to sialic acids, it is also possible to construct sciav pseudotyped with cov-s of interest to investigate this interaction in more detail. it is noteworthy that cells infected with sciav-s display massive cell-cell fusion that is very similar to what is observed in cells infected with pedv but does not occur in cells transduced with other pseudoviruses. moreover, we showed here that pedv-s derived from a field isolate, despite its poor growth in vitro, can be cloned into the expression vector and subsequently used to construct sciav-s for further investigation. sciav thus serves as a new platform not only for investigating the mechanism by which pedv-s mediates cell entry but also to gain insights into the entry mechanisms utilized by cell-adapted and naturally isolated pedv. activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites mechanisms of coronavirus cell entry mediated by the viral spike protein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex potassium 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infection of porcine epidemic diarrhea virus from different pathways sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway comparison of lentiviruses pseudotyped with s proteins from coronaviruses and cell tropisms of porcine coronaviruses the n terminus of pa polymerase of swine-origin influenza virus h n determines its compatibility with pb and pb subunits through a strain-specific amino acid serine inhibition of influenza a virus replication by influenza b virus nucleoprotein: an insight into interference between influenza a and b viruses influenza b virus m protein can functionally replace its influenza a virus counterpart in promoting virus replication the role of orf accessory protein in replication of cell-adapted porcine epidemic diarrhea virus (pedv) acknowledgements we thank dr. qigai he for the kind gift of the anti-pedv-s antibodies.funding this study was funded by national center for genetic engineering and biotechnology (biotec)'s fellow grant, national science and technology development agency (p- - ) and giga impact initiative grant (p - ). human and animal rights statement neither animal nor human testing was involved in this study. key: cord- - h yyqj authors: zheng, xue-yan; xu, yan-jun; guan, wei-jie; lin, li-feng title: regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: h yyqj despite increased understanding of how viral infection is involved in asthma exacerbations, it is less clear which viruses are involved and to what extent they contribute to asthma exacerbations. here, we sought to determine the prevalence of different respiratory viruses during asthma exacerbations. systematic computerized searches of the literature up to june without language limitation were performed. the primary focus was on the prevalence of respiratory viruses, including adv (adenovirus), bov (bocavirus), cov (coronavirus), cmv (cytomegalovirus), env (enterovirus), hsv (herpes simplex virus), ifv (influenza virus), mpv (metapneumovirus), piv (parainfluenzavirus), rv (rhinovirus) and rsv (respiratory syncytial virus) during asthma exacerbations. we also examined the prevalence of viral infection stratified by age, geographic region, type of respiratory secretion, and detection method. sixty articles were included in the final analysis. during asthma exacerbations, the mean prevalence of adv, bov, cov, cmv, env, hsv, ifv, mpv, piv, rv and rsv was . %, . %, . %, . %, . %, . %, . %, . %, . %, . % and . %, respectively. env, mpv, rv and rsv were more prevalent in children, whereas adv, bov, cov, ifv and piv were more frequently present in adults. rv was the major virus detected globally, except in africa. rv could be detected in both the upper and lower airway. polymerase chain reaction was the most sensitive method for detecting viral infection. our findings indicate the need to develop prophylactic polyvalent or polyvirus (including rv, env, ifv and rsv) vaccines that produce herd immunity and reduce the healthcare burden associated with virus-induced asthma exacerbations. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. asthma is a chronic inflammatory airway disease that is susceptible to triggering factors, such as aeroallergens, air pollutants, and viral infection [ ] . despite major advances in prevention and management, asthma remains a considerable global healthcare burden [ ] . from to , the crude prevalence of asthma has increased by . % [ ] . currently, there are . million cases of asthma globally. despite dramatic reductions in age-standardized mortality, . million people died from asthma in [ ] . the management of asthma exacerbations, which have been consistently linked to upper and/or lower airway infections, is an important health issue [ ] . among school-age children, viral infection reportedly accounts for %- % of asthma exacerbations, and viruses are more frequently isolated from symptomatic patients than from asymptomatic patients [ ] . although rhinovirus (rv) has frequently been detected in asthmatic patients [ ] , asthma exacerbations have also been associated with infection with other respiratory communicated by tim skern. xue-yan zheng and yan-jun xu shared co-first authorship. the online version of this article (http s://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. * wei-jie guan battery @ .com * li-feng lin @qq.com viruses, including adenovirus (adv), bocavirus (bov), coronavirus (cov), cytomegalovirus (cmv), enterovirus (env), herpes simplex virus (hsv), influenza virus (ifv), metapneumovirus (mpv), parainfluenzavirus (piv), and respiratory syncytial virus (rsv) [ ] . unfortunately, the rate of association of viruses with asthma exacerbations remains controversial. a hypothetical explanation might be substantial differences in study design, populations, geographic regions, detection methods, and the type of respiratory tract secretions examined. despite our increased understanding of the roles that viruses play in asthma exacerbations, it is less clear which viruses are involved and to what extent they contribute to asthma exacerbations in different subgroups of subjects. we conducted a systematic review of the prevalence of viral infection in patients with asthma exacerbations, with subgroup analyses to identify the determinants of variations. our findings may offer a better overview for developing preventive interventions to minimize virus-induced asthma exacerbations. studies were searched comprehensively in embase, pub-med, cochrane central register of controlled trials and ebm reviews-cochrane database of systematic reviews, web of science, ovid and highwire up to june (no date-of-start was specified). references were checked for additional data. we restricted our search list to publications in the english language. when the same population was used for analysis in several publications, only the largest and most complete study was included. we adopted combined keywords related to virus detection (adenovirus, bocavirus, metapneumovirus, coronavirus, epstein-barr virus, influenza virus, parainfluenza virus, rhinovirus, enterovirus and respiratory syncytial virus) and the outcomes of asthma exacerbations ((((((("pulmonary disease, chronic obstructive"[mesh])) and ("disease progression" [mesh]))) or (((copd)) and (exacerbation)))) and (((("viruses"[mesh])) or (respiratory viral infections)) or (respiratory virus))) and ((("polymerase chain reaction"[mesh])) or (virus pcr)). cross-sectional, prospective studies, cohort studies and case-control studies detailing the prevalence of respiratory virus(es) in asthma exacerbations were included. all eligible asthmatic patients were diagnosed as having viral infection based on polymerase chain reaction (pcr)/enzyme-linked immunosorbent assay (elisa)/cell culture/direct fluorescent antibody assay (dfa)/ indirect fluorescent antibody assay (ifa), which were evaluated during exacerbations. these studies investigated asthma exacerbations in children and adults. we excluded animal studies, ex vivo and toxicological studies, summaries, commentaries and editorials, case reports and case series, duplicate publications, samplerelated or laboratory-based studies, studies in intensive care settings, patients with concomitant chronic obstructive pulmonary disease, bronchiectasis or immunosuppression, non-peer reviewed articles (a potential source of bias), nonoriginal data, nosocomial infections (hospitalization within four weeks, or delayed sample collection [> h after hospitalization]). authors were contacted via e-mail if data were incomplete. studies were excluded if no reply was obtained despite repeated contacts with the corresponding authors. this study complied with preferred reporting items for meta-analysis [ ] . the quality of individual studies was assessed based on the revised quality assessment of diagnostic accuracy studies- tool [ ] . the highest total score was , with a higher score indicating a lower risk of bias. two independent reviewers (x.y.z. and y.j.x.) screened abstracts and titles. full texts were reviewed to determine eligibility. disagreement was resolved by discussion. if consensus was not reached, another reviewer (w.j.g.) was consulted to vote for final decisions. a standardized form was used for data extraction, including the main characteristics (author, year of publication, sample size, age, definition of exacerbation, quality, detection method, study design and season), primary outcome (the prevalence of viral infection during asthma exacerbations), and secondary outcomes (the prevalence of viruses in different strata). data extraction was done by two independent reviewers (x.y.z. and l.f.l.). disagreement was resolved by consultation with the third reviewer (w.j.g.). we did a meta-analysis to obtain point estimates of the rate of asthma exacerbations associated with viral infection across the whole age spectrum globally [ ] . the der-simonian and laird random-effect model [ ] was applied because the heterogeneity among the studies was greater than %. the variance of raw proportions reported in each study was stabilized by using the untransformed proportion, logarithmic transformation, logit transformation, arcsine transformation or freeman-tukey-type arcsine square-root transformation, according to shapiro-wilk's test for normality of data distribution. we also did a subgroup analysis to assess the weight of viral infection on asthma exacerbations with respect to geographic region, population, type of respiratory tract secretion examined, and detection method. if only one study was available, we calculated the % confidence interval ( %ci) using the clopper-pearson exact test. because difference in the geographic regions, age, study population, type of respiratory tract secretion, and detection method significantly confound the determination of the prevalence of individual viruses, heterogeneity was not assessed in this study. analyses were conducted with r software (version . . ). of articles relevant to our objectives, were excluded. no additional studies were identified through citations within articles. figure summarizes the selection process. sixty articles ( studies) were analyzed to assess the association of virus infection with asthma exacerbations. the characteristics of the patient population, study design, country, sample size, asthma definition, quality score, detection method, study design, type of respiratory tract secretion examined, and viruses involved varied substantially (e-table ). of the eligible studies, were cross sectional studies, were case-control studies, were cohort studies, and seven prospective studies. the studies were conducted between and , with the duration varying between one month and years. asthma was diagnosed by a physician in studies in which the data reported on the burden of asthma. the mean age of asthmatic patients varied among the different studies, and therefore, children and adults were dichotomized for reporting. upper/lower airway specimens were screened by different methods, including pcr, dfa assay, ifa assay, virus culture, elisa, rapid enzyme immunoassay and virus neutralization test. the criteria for quality assessment and risk of bias of individual studies are summarized in e- table . the mean score of quality assessment was . (range, to ). all included studies were, at least partially, prone to reporting bias. overall, the mean prevalence ( %ci) bov and env were most frequently detected in oro-/ naso-pharyngeal aspirates, whereas adv was primarily detected in induced sputum. cov, ifv, mpv and piv were most frequently detected in oro-/naso-pharyngeal lavage. rv and rsv were the viruses most frequently detected in spontaneous oro-/naso-pharyngeal secretions. (table ) overall, pcr was the most sensitive technique for detecting adv, bov, env, ifv, mpv and piv, but not for other respiratory viruses, when compared with ifa, elisa, dfa and virus culture. elisa yielded the highest detection rates for cov and rsv (table , fig. d ). in this study, we gathered the latest data on the prevalence of viruses detected during asthma exacerbations. asthma exacerbations were mainly associated with rvs ( . %), highlighting the priority for interventions to reduce the substantial healthcare burden caused by these viruses. this finding has been echoed by the high prevalence of rvs (~ %) among unselected populations residing in north america [ ] . in the netherlands, rvs were most prevalent among symptomatic children during wheezing episodes ( %) and convalescence ( %), as well as in healthy children ( %) [ ] . a possible explanation is that rvs are the most common resident pathogens and are prone to cause upper airway infections (e.g., common cold). consistently, rvs were isolated in . % of pharyngeal exudate samples from mexicans with acute upper respiratory tract infections [ ] . the prevalence of viruses varied considerably among different studies. for example the range was - % for rsv and - % for env) [ ] (for details, see the online supplement). intriguingly, the mean estimated prevalence of envs, hsv, ifv and rsv was greater than %, whereas other individual respiratory virus had a mean prevalence of less than %. in patients with other respiratory diseases, the prevalence of these viruses partially mirrored our findings. rsv was reportedly found in % of elderly patients with acute respiratory illnesses [ ] and in . % in mexicans with acute upper respiratory tract infection [ ] , env was isolated from . % of young infants with sepsis-like illnesses [ ] , and bov was detected in . % of nasopharyngeal aspirates from hospitalized children in spain [ ] . however, the prevalence of mpv was higher ( . %) in mexicans with acute upper respiratory tract infection [ ] , and the prevalence of adv was also higher ( . %) among hospitalized children in spain [ ] than that in our pooled analysis. the substantial heterogeneity of the prevalence data was probably due to differences in study design, ethnicity, geographic region, the site of sample collection, and detection methods. therefore, direct comparisons remained challenging, and therefore subgroup analysis was done to identify possible determinants of variation. we next stratified the prevalence data by age. env, mpv, rv and rsv were more prevalent in children, whereas adv, bov, cov, ifv and piv were more prevalent in adults. env and mpv were associated with asthma exacerbations in children [ , ] , rv were frequently associated with asthma exacerbations associated with upper respiratory tract infection in children, and rsv was commonly linked to onset of wheezing in childhood [ , ] . the significant variation in the viruses involved probably reflected a major shift during the development of host-defense mechanisms. the association with specific viruses varied considerably across geographic regions. rv was the most common virus in asia, europe, america and oceania. rv is probably the dominant resident virus colonizing the upper and lower airways (online supplement), irrespective of meteorological factors such as temperature and humidity. rsv has been reported to be more common among populations in africa, which could be partially related to its generally higher prevalence in low-income countries [ ] . however, the reasons for the variation in the prevalence of other common respiratory viruses are not known. further research is warranted to determine whether ethnicity-related susceptibility or meteorological factors play a significant role in shaping the prevalence of viral infections. consistent with literature reports (online supplement text), rv can be detected in both the upper and lower airways. for rsv, apart from the greater likelihood of initial infection of the nasopharyngeal mucosa, followed by spreading of the virus to the lower airways [ ] , the extensive use of nasal swabs, but not spontaneous or induced sputum for sample collection from children might also have resulted in a higher apparent prevalence in upper airway secretions. furthermore, the higher prevalence of bov and env detected in upper airway secretions might be associated with fecalto-oral transmission [ ] or transmission from oral mucosa or salivary glands [ ] , which would be detected more frequently in infants and toddlers. in accordance with literature reports [ , ] , pcr was the most sensitive method for detecting viruses in asthma exacerbations. it is a simple, rapid and cost-effective technique that has become the mainstay for viral detection. whilst virus isolation in cell culture remains the gold standard, it is time-consuming and labor-intensive, rendering it unsuitable for rapid batch detection in clinical settings [ ] . due to their limited sensitivity and specificity, other techniques such as dfa and ifa are less frequently applied clinically. our findings confirm that viruses of various species are closely associated with asthma exacerbations. the mechanisms are multifaceted, including ) necrosis of airway epithelium, ciliostasis and loss of cilia [ ] [ ] [ ] , ) excessive release of pro-inflammatory cytokines and chemokines [ ] , ) airway hyper-responsiveness and remodeling due to epithelial injury [ ] , and ) mucus hypersecretion, which impedes mucociliary clearance [ ] . unfortunately, there are few effective treatment options (e.g., immunoglobins, nebulized antivirals) for direct elimination of viral infections [ , ] . preemptive approaches should be directed to the prevention of viral infection, particularly in susceptible populations (e.g. children, pregnant women, the elderly, and patients with comorbidities). this has been exemplified by the vaccine development for prevention of rsv and influenza virus infection [ , ] . priority should be given to the most common viruses (e.g., rv, rsv, hsv, env, and ifv), which have caused a significant healthcare burden. recently, a polyvalent vaccine for rv was developed in animal models [ ] , indicating the possibility of establishing herd immunity using polyvalent or multiple-virus vaccines in future community-based practice, which might help to achieve greater reductions in the prevalence of virus-induced asthma exacerbations. our results may not be readily extrapolated to all countries. for instance, documentation of the prevalence of multiple viruses in africa is still lacking, and little is known regarding the distribution of specific viral strains. due to the small number of studies relevant to some of our subgroup analyses (e.g., cell culture and dfa assay) even slight variations in individual studies might have had a significant effect on modified our global estimates. we have summarized the prevalence of respiratory viruses associated with asthma exacerbations. rv, env and rsv are the most prevalent of these, and therefore preemptive prophylaxis with effective vaccines or novel antiviral agents is needed to minimize the healthcare burden of asthma exacerbation resulting from viral infection. fig. the prevalence of the five most common viruses in the stratified analysis a. the prevalence of the five most common viruses when stratified by age (adults vs. children). b. the prevalence of the five most common viruses when stratified by geographic region (from top to bottom: africa, oceania, american, asia, and europe). c. the prevalence of the five most common viruses when stratified by the type of respiratory tract sample analyzed (lower vs. upper airway). d. the prevalence of the five most common viruses when stratified by method of detection (from top to bottom: culture, ifa, elisa, dfa, and pcr). the horizontal axis represents the prevalence (%) of individual viruses. pcr, polymerase chain reaction; ifa, indirect fluorescent antibody assay; dfa, direct fluorescent antibody assay; elisa, enzyme-linked immunosorbent assay; rv, rhinovirus; adv, adenovirus; bov, bocavirus; cov, coronavirus; cv, cytomegalovirus; env, enterovirus; hsv, herpes simplex virus; ifv, influenza virus; mpv, metapneumovirus; piv, parainfluenzavirus (piv); rsv, respiratory syncytial virus asthma exacerbations: pathogenesis, prevention, and treatment global initiative for asthma (gina) program. the global burden of asthma: executive summary of the gina dissemination committee report global, regional, and national deaths, prevalence, disabilityadjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, - : a systematic analysis for the global burden of disease study community study of role of viral infections in exacerbations of asthma in - year old children preferred reporting items for systematic reviews and meta-analyses: the prisma statement quadas- : a revised tool for the quality 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in young children rhinovirus-induced first wheezing episode predicts atopic but not nonatopic asthma at school age rsv global epidemiology network ( ) global, regional and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study detection of respiratory syncytial virus (rsv) at birth in a newborn with respiratory distress frequent detection and genetic diversity of human bocavirus in urban sewage samples modelling person-to-person transmission in an enterovirus a orally infected hamster model of hand-foot-and-mouth disease and encephalomyelitis high-throughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses prospective comparison of the detection rates of human enterovirus and parechovirus rt-qpcr and viral culture in different pediatric specimens bronchial mucus transport pathological changes in virus infections of the lower respiratory tract in children structure and function of the polymeric mucins in airways mucus the role of viruses in acute exacerbations of asthma human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling shotgun proteomic analysis of humaninduced sputum respiratory syncytial virus network (resvinet) ( ) lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics enterovirus infection-association with asthma study group ( ) the safety, immunogenicity, and acceptability of inactivated influenza vaccine delivered by microneedle patch (tiv-mnp ): a randomised, partly blinded a polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques key: cord- -i w huke authors: xue, yong; sun, xiaohua; li, yinghui; liu, xin; dong, chen title: increased risk of hepatitis e virus infection in schizophrenia date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: i w huke until now, the risk of hev infection in schizophrenia was unknown. the present results showed that the seroprevalence of anti-hev igg and anti-hev igm in schizophrenia were significantly higher than that in healthy controls. anti-hev igg positivity increased with age and with the duration of disease in schizophrenia patients. moreover, schizophrenia patients with increased cd (+)/cd (+) t-cell ratios (> . ) had higher anti-hev igg detection rates than those with normal ratios ( . - . ). compared with the schizophrenia patients who tested anti-hev igg negative, the levels of interleukin- and interleukin- (th cytokines) were significantly higher, while the interleukin- (th cytokine) level was significantly lower, in those with anti-hev igg positivity. of five schizophrenia patients who were anti-hev igm positive, four had elevated cd (+)/cd (+) t-cell ratios. hev rna was isolated from one of these four patients and classified as genotype . anti-hev igm positivity was not detected among healthy controls. therefore, schizophrenia patients exhibited a higher risk of hev infection than controls. hepatitis e virus (hev) is a non-enveloped, single positive-strand rna virus in the family hepeviridae with a worldwide distribution [ ] . hepatitis e, caused by hev, is an important public-health problem in many developing countries and is the cause of sporadic acute hepatitis in developed countries. although the overall mortality of hepatitis e is less than % in the general population, it can reach up to % in pregnant women in the third trimester [ ] . hev infection has four documented routes of transmission: waterborne, foodborne, bloodborne and vertical transmission from mother to child [ , , , ] . epidemiological studies indicate that a higher prevalence of hev antibodies is observed in the elderly and people who live in rural areas with poor sanitation [ , ] . moreover, isolation of animal strains of hev and the existence of other animal species that tested seropositive for anti-hev igg and igm indicate that hepatitis e is a zoonotic disease [ , ] . schizophrenia is a complex mental disease that is highly heritable, and it involves interplay between genetic and environmental factors. immune system abnormalities have been reported in patients with schizophrenia, as evidenced by increased expression of proinflammatory cytokines, t-cell activation and high levels of autoantibodies [ ] . the alterations of circulating inflammatory cytokines, such as interleukin- (il- ), interleukin- (il- ) and interferon-c (ifn-c), reflecting the functions of t help type (th ), and interleukin- (il- ), interleukin- (il- ) and interleukin- (il- ), reflecting the functions of t help type (th ), were also observed in previous studies [ , ] . moreover, the previous results suggested there was an imbalance between th and th with a shift toward the th system in schizophrenia [ ] . epidemiological studies have already shown that the rates of hepatitis b and hepatitis c virus infection in patients with schizophrenia were five and times higher, respectively, than the estimated general population rates [ ] . however, the risk of hepatitis e virus infection in schizophrenia is still unknown. in the current study, we found that the detection rates for hev antibodies in schizophrenia patients were much higher than those in healthy controls. anti-hev igg positivity increased with age and with the duration of schizophrenia. moreover, schizophrenic patients with increased ratios of cd ? /cd ? t cells had higher anti-hev igg detection rates than those with normal ratios. compared with patients who tested anti-hev igg negative, the levels of il- and il- were significantly higher, while the il- level was significantly lower in those with anti-hev igg positivity. this study was carried out between august and march . two hundred sixty-nine individuals with schizophrenia (sz) were enrolled in the present study. the diagnosis of schizophrenia was based on criteria defined by the diagnostic and statistical manual of mental disorders (fourth edition, text revision) (dsm-iv tr). the mini international neuropsychiatric interview (mini) version . . dsm was used for screening patients for schizophrenia during data collection. all of the patients who were enrolled were inpatients and were diagnosed by the department of psychiatry, huai'an third hospital in jiangsu, china. moreover, all of the patients were free of acute infections and allergic reactions for at least two weeks prior to the collection of serum samples. two hundred sixty healthy controls (hc) were randomly recruited for age (± years) and gender matching from the annual health examination population ( , subjects) living in the same region. all of the healthy controls were found to be free of schizophrenia by screening using the mini test. moreover, the inclusion criteria for the healthy controls were the absence of any psychiatric and autoimmune disorders and the lack of a history of these disorders among immediate family members. additionally, healthy controls had to declare themselves free of acute infections and allergic reactions for at least two weeks prior to the collection of serum samples. this study conformed to the declaration of helsinki, and it was approved by the research ethics committee of the huaian third hospital, following established guidelines. all participants provided written informed consent after the study procedures were explained. anti-hev igg and igm were assayed using commercial immunoassay elisa kits (wantai biopharmaceutical, inc., beijing, china) as described previously [ ] . the sensitivity and specificity of the present assays were . % and . % for anti-hev igg detection, and . % and . % for anti-hev igm detection, respectively. in brief, plates precoated with recombinant hev orf proteins were incubated with test sera, as well as reactive and nonreactive controls at °c for min, washed five times and incubated with ll of human anti-igg-or anti-igm-horseradish peroxidase conjugate. after incubation at °c for min, the plates were washed and incubated at room temperature for min with enzyme substrate. absorbance was read at nm, and the cutoff value was determined according to the manufacturer's instructions. heparinized whole blood ( ll) was incubated with anti-cd (fitc), anti-cd (pe) and anti-cd (percp) and analyzed by four-color flow cytometry using a fluorescenceactivated cell sorter facscalibur (becton-dickinson, mountain view, ca) as described elsewhere [ ] . a total of , events were collected in the lymphocyte gate using morphological parameters. data were processed using cellquest pro software (becton-dickinson) and analyzed using the summit software. the cd ? t-cell, cd ? t-cell and cd ? t-cell counts were expressed as the proportion of all pbmcs. the levels of serum il- , il- , il- and ifn-c were assayed using commercially available elisa kits (yuanxiang biotech inc., shanghai, china). each cytokine assay was optimized, and a standard curve was established using standard procedures. in brief, plates were precoated with anti-il- , anti-il- , anti-il- or anti-ifn-c capture antibody. plates were washed and incubated with serial dilutions of standards and with test specimens for h at °c. plates were washed five times, incubated with appropriate biotinylated detection antibody for h at °c, and then washed and incubated for min with streptavidin-conjugated horseradish peroxidase. the reaction was developed with tetramethylbenzidine-h o substrate after the final wash. in all assays, the samples were analyzed in duplicate, and the case-control pairs were analyzed on the same plate. the lower limits of detection for il- and ifn-c were pg/ml, and for il- and il- , they were pg/ml, respectively. hev rna isolation, sequencing and analysis total rna from five anti-hev igm-positive samples as well as negative and positive controls was extracted from ll of serum using trizol-ls reagent (invitrogen, shanghai, china), reverse-transcribed and subjected to nested pcr to amplify the orf region as described previously [ ] . the limit of hev-rna detection in the present assay was copies/ml ( copies/reaction). after purification using a qiaquick gel extraction kit (qiagen, valencia, ca, usa), the pcr products were sent to invitrogen and sequenced using an abi genetic analyzer. genetic analysis was performed with mega version . (tempe, az, usa). an unpaired student's t-test was used to test for differences between sz and hc groups of continuous variables. differences in qualitative variables were compared using v or trend v tests. a two-sided p-value of . was set to denote statistical significance. all statistical analysis was performed using statistical product and service solutions . (spss; spss inc, chicago, usa). demographic and clinical characteristics for patients and controls are shown in table . there were no age or gender differences between schizophrenia patients and controls. increased levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were detected in and schizophrenia patients, respectively. six patients had elevated levels of both alt and ast. in the hc group, only two and eight individuals had elevated levels of alt and ast, respectively. none of the controls had abnormal levels of both alt and ast. in the sz group, of ( . %) patients showed activation of t cells and increased ratios of cd ? /cd ? t cells ([ . ), while only eight people in hc group had increased ratios of cd ? /cd ? t cells (table ) . a significant difference was found between the sz group and the control. correlations between the detection rates, gender and duration of illness are summarized in table . ninety-four out of ( . %) in the sz group were positive for anti-hev igg, while out of people ( . %) in the hc group were positive (p \ . ). patients with a longer course of disease ([ years) tended to show higher rates of positivity than did patients with less than years of disease (p \ . ). the detection rates of hev igg antibody increased with age in the sz group but not in the healthy population. there was no significant difference between males and females in either the sz or the hc group. additionally, five cases, four males and one female, were hev igm antibody positive in the sz group, but no individuals were anti-hev igm positive in the hc group (p \ . ). the levels of serum il- , ifn-c, il- and il- were measured by elisa. significant differences were observed in serum il- , il- and il- levels between the schizophrenia patients with higher values of cd ? /cd ? t-cell ratios ([ . ) and those with normal values ( . ± . vs. . ± . for il- , . ± . vs. . ± . for il- and . ± . vs. . ± . for il- ), while no difference in serum ifn-c level was observed ( . ± . vs. . ± . for ifn-c). in comparison with schizophrenia patients who tested hev igg negative, hev-igg-positive patients had levels of serum il- and il- that were significantly higher, while the level of serum il- was significantly lower (p \ . , p \ . and p \ . , respectively) ( table ). there was no difference in the level of serum ifn-c between hev-igg-positive and negative patients. moreover, differences in cytokine levels were not observed within the hc group regardless of the hev igg status (data not shown). of five anti-hev-igm-positive samples, one sample with increased levels of alt and ast, and an elevated cd ? / cd ? t-cell ratio was positive for hev rna. the -nt orf sequence of the pcr product was sequenced and compared with those of known hev isolates. the isolate was . - . %, similar to the genotype isolates, but only . - . %, . % and . - . %, similar to the genotype , and hev strains, respectively. therefore, this hev isolate, named hev-huaiansz (genbank no. jx ) was classified as genotype , which was widely distributed in china. our study contributes to the sparse literature on hev seropositivity in schizophrenia patients. in previous studies, higher prevalence of antibodies to hbv nucleocapsid protein was seen in patients with psychiatric disorders [ ] . epidemiological studies also proved that patients with schizophrenia were significantly more likely to be infected with hcv than those without schizophrenia [ , ] . moreover, higher seroprevalence of antibodies to cytomegalovirus, herpes simplex virus, human immunodeficiency virus, influenza virus and coronaviruses were found in psychotic patients compared to the general population [ , , , , , , ] . therefore, it was reasonable for us to carry out the present study to evaluate the risk of hev infection in patients with schizophrenia. the results confirmed our hypothesis that anti-hev igg and igm detection rates in schizophrenia were significantly higher than in healthy controls. several factors could contribute to higher detection rates of hev antibody in schizophrenia patients. first, some symptoms of schizophrenia, in particular, the symptoms of cognitive impairment, may contribute directly to an inability to safeguard against the transmission of waterborne, foodborne or bloodborne infection [ ] . second, schizophrenia patients usually reside in settings such as psychiatric facilities, where they often forge social relationships with other higher-risk individuals [ , ] . third, financial instability and poverty are also associated with increased risks of hev infection [ ] . the clinical symptoms of hepatitis e are typical of selflimiting, acute viral hepatitis in general [ , ] . severe hepatitis e can occur in the elderly, pregnant women and people with chronic hepatitis b and chronic hepatitis c [ , , ] . chronic hepatitis e infection has also been documented in patients receiving immunosuppressive therapy following organ transplantation and those infected with hiv [ ] . the immune responses to hev involve antibody-mediated immunity, cell-mediated immunity, interferon and other host defenses [ , , , ] . data from cellular immune responses to hev suggest that the cd ? t-cell count is significantly higher in recovered hev-infected individuals than in acutely infected hev patients and the general population, but the levels of cd ? t-cells are similar in these groups [ , , ] . in schizophrenia, increased levels of cd ? t cells and higher cd ? /cd ? t-cell ratios were documented previously [ ] . in the present study, the detection rate of anti-hev igg in schizophrenia patients with increased cd ? /cd ? t-cell ratios ([ . ) was significantly higher than in patients with normal ratios ( . * . ). thus, further well-designed studies should be carried out to analyze the relationships between cd ? /cd ? t-cell levels and the increased risk of hev infection in schizophrenia patients. cellular immune responses to infections can be either of the th type, which is characterized by cytokines such as il- , ifn-c and il- , or the th type, which is characterized by cytokines such as il- , il- , il- and il- . in hepatitis b and c virus infections, the predominant immune response, which is th , is activated during liver trauma [ ] . according to pal et al. [ ] , ifn-c, a th cytokine, was lower in hev-infected pregnant women than in healthy people. furthermore, the production of il- , a th cytokine, was higher in hev-infected pregnant women than in controls [ ] . these results suggest that pregnant women with acute hev infection show a shift in the th / th balance towards a th response. this shift might play a role in the particularly severe clinical course and mortality of the hev infection during late pregnancy. it should be noted that schizophrenia might also be associated with an imbalance in th /th cytokines, and also with a shift toward the th system [ ] . in the present study, the th bias was observed in schizophrenia patients, as evidenced by the increased production of il- and il- and lower il- production. moreover, significant differences of the serum il- , il- and il- levels were observed among schizophrenia patients with and without hev igg antibodies. the major implication of this study is that there may be more potential risk factors in schizophrenia patients, which may be responsible for the increased transmission of hev. also, there are several limitations in our present study. first, the various drugs used in schizophrenia may affect the immune system, and virtually all our schizophrenia patients were on this type of medication. relationships between drugs and immune bias, and the effect of this relationship on the risk of hev infection in schizophrenia should be assessed in the future. second, we did not include inflammatory markers other than serum il- , il- , il- and ifn-c, and thus, future work should be carefully interpreted in the light of this. third, larger numbers of schizophrenia patients and controls as well as long-term follow-up will be required to further analyze the increased risk of hev infection in schizophrenia. taken together, schizophrenia patients exhibited higher risk of hev infection than controls in the present study. anti-hev igg seropositivity increased with age and with the duration of illness. moreover, schizophrenia patients with increased cd ? /cd ? t-cell ratios had higher anti-hev igg detection rates than those with normal ratios. the levels of il- and il- were significantly higher, while the il- level was significantly lower in schizophrenia patients with anti-hev igg positivity than in those who were antibody negative. in order to understand the questions behind the observed associations, further work is needed, with larger samples and longitudinal follow-ups. epidemiology of hepatitis e: current status the unique riverine ecology of hepatitis e virus transmission in south-east asia antibodies to measles in individuals with recent onset psychosis identification of genetic diversity of hepatitis e virus (hev) and determination of the seroprevalence of hev in eastern china the first experimental study on a candidate combined vaccine against hepatitis a and hepatitis e restricted enzooticity of hepatitis e virus genotypes to in the united states 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clinico-epidemiological forms of hepatitis e cytokine profiles, ctl response and t cell frequencies in the peripheral blood of acute patients and individuals recovered from hepatitis e infection efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebocontrolled acknowledgments the authors thank dr. michael a. purdy, dr. maja kodani, dr. chong-gee teo and dr. saleem kamili for kindly reviewing the manuscript. we would also like to thank dr. chong shen, xianfeng cheng, yadong zhang and chunlin shi for their assistance in the present work. the authors declare no conflict of interest. key: cord- -s w yr authors: hohdatsu, t.; okada, s.; koyama, h. title: characterization of monoclonal antibodies against feline infectious peritonitis virus type ii and antigenic relationship between feline, porcine, and canine coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: s w yr seven monoclonal antibodies (mabs) with neutralizing activity against feline infectious peritonitis virus (fipv) strain - (type ii) were prepared. when the polypeptide specificity recognized by these monoclonal antibodies (mabs) was investigated by western immunoblotting, all of the mabs reacted with peplomer glycoprotein (s) of the virus. by competitive binding assay these mabs were found to recognize at least different epitopes. the reactivity of these mabs with viruses classified as fipv type i (ucd- , ucd- , ucd- , ucd- , nw- , and black), feline enteric coronavirus (fecv) type ii strain - , canine coronavirus (ccv) strain - , and transmissible gastroenteritis virus (tgev) strains to- and sh was examined by neutralization tests. all mabs neutralized fecv strain - , ccv strain - , and tgev strains to- and sh, while they did not neutralize the fipv type i viruses. moreover, the mab against tgev strain to- , which has strong neutralizing activity against tgev viruses, neutralized ccv strain - , fecv strain - , and fipv strain - , but did not neutralize the fipv type i viruses. these results demonstrated that there are at least epitopes involved in the neutralization of fipv type ii strain - , and that these epitopes are not present in fipv type i viruses but are present in fecv strain - which does not induce feline infectious peritonitis, tgev strains to- and sh, and ccv strain - . these results suggest the presence of serotypes of fipv which can be clearly distinguished by the neutralization test using mabs. the mammalian members of the family coronaviridae are divided into distinct antigenic groups on the basis of serologic tests [ , ] . one antigenic group t. hohdatsu et al. contains mouse hepatitis virus, neonatal calf diarrhea coronavirus, human coronavirus hcv-oc , hemagglutinating encephalomyelitis virus of swine, and rat coronavirus. the second antigenic group consists of human respiratory coronavirus hcv- e, transmissible gastroenteritis virus (tgev) of swine, canine coronavirus (ccv), and feline infectious peritonitis virus (fipv) [ , ] . in addition to these members, a feline enteric coronavirus (fecv) has been isolated, which is very closely related to fipv, tgev, and ccv [ , ] . feline coronaviruses are divided into fipv types i and ii, and fecv types i and ii, on the basis of the disease types, that is, whether it causes feline infectious peritonitis (fip) or not, the ability of the viruses to proliferate in cell cultures, and the antigenic relationship to tgev and ccv [ ] . among these types, fipv type ii and fecv type ii are reported to be antigenicatly closer to tgev and ccv. between tgev purdue strain and fipv - strain a high degree of homology in the primary structure of the peplomer protein (s, k) has been described ( % for amino acid residues to and % for residues to ) [ ] . the authors prepared mabs which react only with the feline coronaviruses fipv type ii strain - and fecv type ii strain - . some of these mabs reacted with either tgev or ccv in indirect fluorescent antibody assays. also, some differences in antigenically between fipv type i virus strains were noted in an analysis using mabs (unpubl. obs.). in this study, we prepared mabs with neutralizing activity against fipv strain - , and investigated the differences in the epitopes recognized by these mabs, using a competitive binding assay. using these mabs and mabs with neutralizing activity against tgev strain to- , we examined the neutralizing epitopes for differences between feline coronaviruses, and tgev and ccv viruses. feline whole fetus cells (fcwf- ), crandell feline kidney cells (crfk), and swine kidney cells (cpk) were grown in eagle's minimum essential medium (mem) containing % l- medium, % fetal calf serum, units/ml penicillin and gg/ml streptomycin. the maintenance medium was mem containing % l- and antibiotics as above. the cells were maintained in a humidified % co incubator at °c. the coronavirus isolates used in this study and their sources are shown in table . among the fipv strains used in the study, strains ucd-t, nw- , ucd- , ucd- , ucd- , and black show cell-associated growth, and are therefore regarded as type i virus strains in the classification of pedersen et al. [ ] . fipv and fecv, tgev, and ccv were passaged or times in fcwf- cells, cpk cells, and crfk cells, respectively, and were used for the study. the antigen was prepared with the fipv - strain grown in fcwf- cell cultures. infectious culture fluid concentrated about tenfold by ammonium sulfate precipitation was layered onto a discontinuous sucrose density gradient ( and %) in a rps rotor (hitachi koki co., ltd, japan) and centrifuged at , r.p.m, for h. the virus bands formed at the interface of % and % sucrose layers were collected, diluted in nte buffer ( . m nac , . m tris-hc , ph . , . m edta) and centrifuged at , x g for h. the ,arus-containing pellet was suspended in a / volume of nte buffer. for preparation of neutralizing mabs against fipv - strain, balb/c mice, about weeks of age, were inoculated intraperitoneally with a mixture of ~tg of the viral antigen prepared as above and t cells of pertussis adjuvant. four or weeks later the mice received an intravenous booster dose of gg of viral antigen, and spleen cells were obtained for fusion days later. the fusion was carried out by essentially the same method described by k hler and milstein [ . polyethyleneglycol- , (merck, federal republic of ger-many) was used as a fusing agent and the ratio of mouse spleen cells and mouse myeloma cells (p- /x- -ag - , , ) was : . the selective medium contained hypoxanthine ( - m), aminopterin ( x - m) and thymidine ( . x -sm). the fused cells, at a concentration of . x spleen cells per ml, were dispended in ~tl volumes into wells of -well, flat-bottomed microplates (coming glass works, corning, ny) and incubated at °c in a humid atmosphere containing % cq. after incubation for weeks, the wells were examined and those which contained hybridoma cultures were tested for feline coronavirus specific antibody by a neutralization (nt) test (see below). the colonies in antibody positive wells were passaged in -well multiplates (corning glass works, corning, ny) and incubated in medium containing hypoxanthine ( - m) and thymidine ( . x - m). the cells were then cloned by the soft agar method. the neutralizing mabs against tgev to- strain used were previously reported by hohdatsu et al. [ ] . the supernatant fluid of antibody-secreting hybridoma cultures were concentrated tenfold by % saturation of ammonium sulfate and used for determination of antibody class and subclass by double diffusion in % agar gel containing . % nan . rabbit antisera against mouse immunoglobulins, igg , igg a, igg b, igg , igm and iga, and • and )~ chains (miles laboratories, naperville, u.s.a.) were placed in center wells and test samples were added to peripheral wells. the plates were incubated overnight at room temperature in a humidified chamber. sds-page was performed in slab gets under a nonreducing condition. the separation gel contained % polyacrylamide and the stacking gel contained % polyacrylamide. the sample buffer contained final concentration of % (w/v) sds, % (v/v) glycerol, . % (w/v) bromphenol blue and mm tris-hc (ph . ). the electrode buffer (ph . ) contained . g/ tris, . g/ glycine and . % sds. samples were mixed with sample buffer and kept at room temperature for h without boiling. electrophoresis was performed at room temperature for h at a constant current of ma. viral antigen separated in polyacrylamide gel by sds-page were transferred to nitrocellulose sheets of . gm pore size. the transfer was carried out electrophoretically by the method adapted from towbin et al. [ ] in a transfer-blot cell apparatus (bio-rad laboratories, richmond, ca) at ma and v for h using transfer buffer consisting of g/ tris (ph . ), % methanol and . g/ glycine. the nitrocellulose sheets were then cut into strips and incubated at °c for h in pbs containing % fetal calf serum. the sur)ernatant fluid of antibody-secreting hybridoma cultures were added in ml volumes to individual strips and incubated at °c for h. the strips were then washed times with pbs containing . % tween- , and incubated at °c for h with horseradish peroxidase-conjugated rabbit antibody against mouse igg, iga, and igm (miles lab., u.s.a.) diluted : with pbs containing % fetal calf serum. the strips were then washed and treated with substrate solution containing . g diaminobenzidine, lal of % h in ml of . m tris-hc , ph . . when distinct bands appeared about min later, the reaction was stopped by pouring off the substrate solution and rinsing with distilled water. this was carried out by a modified enzyme-linked immunosorbent assay (elisa) [ ] . serial tenfold dilutions in gl volumes of each competing mab from ascitic fluid were added to wells of a flat-bottomed microelisa plate coated with virus antigen (see above), and incubated at °c for rain. after washings with washing solution ( . % nac solution containing . % tween- ), gl of biotin-labeled mab diluted so as to show an optical density (od) of . was added, and incubated at °c for min. after washings with washing solution, peroxidase conjugated avidin (cappel, cooper biomedical, inc. malvern, pa) were diluted to the optimal concentration with pbs containing % fetal calf serum and . % tween- and gl of the dilution was added to each well of the plates. after incubation at °c for min, each well received gl of substrate solution and incubated at °c for min in a dark room. after completion of the incubation, the reaction was stopped with n h so solution and the od at nm was determined. the amount of competitive binding was estimated from the od in the presence or absence of unlabeled competing antibodies. the percentage of competition was determined by the formula, (a -n)/(a -b), where a is the od in the absence of competing antibody, b is the od in the presence of homologous antibody ( elisa units), and n is the od in the presence of a competitor. serial twofold dilutions of the mab were mixed with an equal volume of a virus suspension diluted so as to contain approximately tcid / . ml. the mixtures were incubated at °c for rain. each mixture was then inoculated into cell cultures in flat-bottomed microplates (coming glass works, coming, ny), and incubated in an atmosphere of % co in air at °c for days. two wells were employed for each antibody dilution. the anitbody titer was expressed as the reciprocal of the highest dilution of mab that completely inhibited cytopathic effect in the test. using the spleen cells of mice immunized with fipv strain - , cell fusion was conducted times. as a result, mabs ( - - , - - , - - , - - , - - , - - , and - - ) with neutralizing activity against fipv strain - were obtained. the immunoglobulin isotype and polypeptide specificity of these mabs are shown in table . polypeptide specificity was determined by western immunoblotting. all mabs were found to recognize the s protein of the viruses. figure shows an example of the reaction. to determine the differences in the epitope specificity of the neutralizing mabs, competitive binding assay was performed, and the results are summarized in table . binding of the biotin-labeled mabs - - , - - , - - , and - - to virus antigen was completely blocked by all of these unlabeled mabs. - - , - - , - - , - - , and - - by .t'-~ . %. on the basis of these results, epitopes recognized by these mabs were named i, ii, and iii. reactivity of the mabs which recognize the neutralizing epitopes in fipv strain - , with feline, porcine, and canine coronaviruses was investigated with the neutralization (nt) test. as shown in table , these mabs did not neutralize the fipv type i strains, but neutralized fecv strain - , as well as the porcine and canine coronaviruses. in particular, mabs which recognize epitope iii ( - - and - - ) also showed a high neutralizing activity against these viruses, as against fipv strain - . the the results are shown in table . while mab - neutralized the fipv type ii strain - , fecv type ii strain - , and ccv strain - , it did not neutralize fipv type i viruses. mabs -a and -a did not neutralize any coronaviruses other than tgev. in this study, we prepared mabs with neutralizing activity against fipv strain - , and used them to investigate the serological relationships between feline, porcine, and canine coronaviruses. it is generally accepted that epitopes associated with neutralization are present on the s protein of viruses. fiscus and teramoto have reported this phenomenon in feline coronaviruses i- ]. all mabs with neutralizing activity were observed to recognize s protein. this finding confirms the presence of a neutralizing epitope on the s protein. however, there have been no reports on the characteristics of this epitope. the results of the competitive binding assay using the mabs revealed the existence of at least different neutralizing epitopes (i, ii, and iii) in fipv strain - (table ) . we believe that the topographical relationship between epitopes i, ii, and iii is as follows. since (fig. a) resulting in blocking by steric hindrance, or that epitope iii overlaps part of epitopes i and ii (fig. b) . differences in the epitopes recognized by the mabs were determined. the nt activity of these mabs against several feline, porcine, and canine coronaviruses was examined. these mabs did not neutralize the fipv type i viruses, but neutralized fecv strain - , tgev strains to- and sh, and ccv strain - , which do not cause feline infectious peritonitis (table ) . moreover, a mab against tgev strain to- , which neutralized the tgev strains, neutralized fipv strain - , fecv strain - , and ccv strain - (table ). these results support the classification by pedersen et al. [ ] . when the virus groups (fipv type i, fipv type ii, fecv type ii, tgev, and ccv) are serologicalty compared, fipv type ii, fecv type ii, tgev, and ccv are found to form a group. fipv type i virus seems to be a virus of a distinct serotype. in other words, fipv inducing feline infectious peritonitis seem to consist of viruses of at least serotypes. pedersen etal. [ ] have indicated that many cases of feline infectious peritonitis in cats are induced by type i virus infection. we also found that, out of cats with feline infectious peritonitis-like symptoms and antibody-positive by indirect fluorescent antibody assay, only were positive for neutralizing antibody against fipv strain - (fipv type ii) (unpubl. data). it is probable that fipv type i is more prevalent in a natural environment. in the future, examination of the effects of different virus types on the anitbody-mediated enhancement of fipv infection [ , ] , as seen in dengue virus infection [ , ] , would be valuable. at present, we are investigating the effects of the neutralizing mab prepared in this study against in vivo fipv infection. differences between tgev strain purdue and fipv type ii strain - at the gene level of s protein have been reported [ ] . analysis of fipv type i at the gene level is also desired. recovery and characterization of a coronavirus from military dogs with diarrhoea recovery and in-vitro cultivation of a coronavirus from laboratoryinduced cases of feline infectious peritonitis (fip) antigenic comparison feline coronavirus isolates: evidence for markedly different peplomer glycoproteins comparison between virulent and attenuated strains of transmissible gastroenteritis virus heterogeneity of infection enhancement of dengue strains by monoclonal antibodies studies on transmissible gastroenteritis in pigs. iii. isolation of cytopathogenic virus and its use for serological investigation antigenic variation of porcine transmissible gastroenteritis virus detected by monoctonal antibodies antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses spaan wjm (t ) the nucleotide sequence of the peplomer gene of porcine transmissible gastroenteritis virus (tgev): comparison with the sequence of the peplomer protein of feline infectious peritonitis virus (fipv) topographical analysis of antigenic determinants on envelope glycoprotein v (e) of japanese encephalitis virus, using monoclonal antibodies continuous cultures of fused cells secreting antibody of predefined specificity isolation of feline coronaviruses from two cats with diverse disease manifestations dengue virus monoclonal antibodies identify epitopes that mediate immune infection enhancement of dengue viruses attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus immunologic phenomena in the effusive form of feline infectious peritonitis experimental studies with three new strains of feline infections peritonitis virus: fipv-ucd , fipv-ucd and fipv-ucd pathogenic differences between various feline coronavirus isolates infection studies in kittens utilizing feline infections peritonitis virus propagated in cell culture an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis pathogenicity studies of feline coronavirus isolates - and - morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal cell cultures antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species electrophoretic transfer of proteins from polyacrytamide gets to nitrocellulose sheets: procedure and some applications antibody-mediated enhancement of disease in feline infectious peritonitis: comparison with dengue hemorrhagic fever this work has been funded by the kitasato research foundation under grant no. . key: cord- -dwhyx y authors: noh, ji yeong; yoon, sun-woo; kim, doo-jin; lee, moo-seung; kim, ji-hyung; na, woonsung; song, daesub; jeong, dae gwin; kim, hye kwon title: simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription pcr date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: dwhyx y since severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses (covs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. therefore, the aim of this study was to develop a duplex real-time reverse transcription (rt)-pcr method for the simultaneous detection of these viruses. primers and probes that target the conserved spike s region of human sars-cov, mers-cov, and their related bat covs were designed. the results of real-time rt-pcr showed specific reactions for each virus with adequate detection limits of – copies/ml and – copies/ml using puc -sars-ps (a template for sars-cov) and pgem-mers-s (a template for mers-cov), respectively. in addition, this real-time rt-pcr system was able to detect the target viruses sars-like bat cov and mers-cov in bat fecal samples and sputum of mers patients, respectively. therefore, this newly developed real-time rt-pcr method is expected to detect not only sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses in bats. the recent emergence of human coronaviruses (covs) causing severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) with global spread represents a significant threat to public health. both sars-cov and mers-cov cause severe respiratory diseases and belong to the genus betacoronavirus [ ] . phylogenetic analysis has shown that sars-cov belongs to lineage b, which includes sars-like bat covs and some other bat-derived covs, whereas mers-cov belongs to lineage c, which also includes bat-derived covs [ ] . sars-cov and mers-cov share similar transmission characteristics. both viruses are thought to have originated from bats, which are the primary reservoir of diverse covs [ ] . cross-species transmission of these viruses to palm civets and dromedary camels has increased the chance of their zoonotic transmission to humans [ ] . nosocomial transmission is thought to be the main cause of human-tohuman transmission of sars-cov and mers-cov [ ] . although there have been no sars cases reported since , mers cases have been reported continuously worldwide [ ] . in addition, a recent analysis of the sarslike bat covs that use the ace receptor highlighted the need for preparedness for their potential zoonotic transmission [ , ] . bat covs related to sars-cov and mers-cov have been reported continuously worldwide [ ] [ ] [ ] [ ] . given the high similarity between sars-cov and mers-cov with respect to clinical signs, etiology, and transmission, establishment of a simultaneous detection method for these viruses would be useful for their accurate diagnosis and monitoring in humans as well as their reservoir, bats. therefore, in this study, a duplex real-time reverse transcription (rt)-pcr method was developed based on primers and probes that target the conserved spike s region of sars-cov, sars-like bat covs, mers-cov, and mers-related bat covs. for the universal detection of sars-cov and sars-like bat covs, consensus primers and probes (fig. a) were designed based on the conserved sequences of the spike s region by aligning the following reference sequences: human sars-covs sino (genbank no. ay ), tor (ay ), and urbani (ay ); sars-like bat covs b - (ku ), rp (dq ), rsshc (kc ), rf (dq ), hku (dq ), and (dq ). for the detection of mers-cov and possible related bat covs, consensus primers and probes (fig. b) were designed based on the conserved sequences of the spike s region by aligning the following reference sequences: human mers-covs tha-cu (kt ), jeddah (kf ), and kor-nih (kt ); mers-related bat covs a (dq ), pml-phe (kc ), gx (kj ), hku (ef ), sc (kj ), and hku (ef ). the designed primers and probes are shown in table . the partial sequences of the s region (nucleotides - ) of the spike gene of the sars-cov sino strain (genbank no. ay ), including the primer-and probe-binding regions, were synthesized and inserted in the puc vector at cosmogenetech co., ltd. (seoul, korea). the nucleotide sequence of the mers-cov s domain (amino acids - , emc strain, genbank no. jx ) was cloned in the pgem vector by bioneer (daejeon, korea). competent escherichia coli cells (dh a) were transformed with the recombinant plasmid vectors (puc -sars-ps and pgem-mers-s ), and the amplified plasmid was extracted. these plasmids were then used as templates for the positive control as well as in the detection limit test after calculating their copy numbers. a portion of the s region of puc -sars-ps was amplified using primers containing a t promoter sequence: sars-tem-f ( '-tcg gta cct aat acg act cac tat agg gaa gag cca cca tga agg tgt ttt tgt gtt taa tgg- ') and sars-tem-r ( '-ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt tta att agt cca gca atg aag cc- '). the amplified pcr product was used as template for in vitro transcription using an mmessage mmachine t kit (ambion, usa) following the manufacturer's manual. the in vitrotranscribed mrna was treated with turbo dnase ( u/ ll) and purified with lithium chloride precipitation solution ( . m lithium chloride, mm edta). the final concentration of sars-ps -based mrna was measured using a nanodrop spectrophotometer (thermo scientific, usa). the real-time rt-pcr was performed with reverse transcription at °c for min followed by °c for min and cycling times at °c for s and °c for s. thermocycling was performed using a lightcycler system (roche, usa), and positive results were estimated according to analysis of the fluorescent curves originating from each probe within cycles. the purified recombinant plasmids were prepared at a concentration of copies/ml for the templates of sars-cov and mers-cov. each template was diluted tenfold in nuclease-free distilled water (ambion, usa). the diluted plasmids were then used as templates for the real-time rt-pcr to evaluate the detection limits of the primers and probes. the in vitro-transcribed mrna of sars-cov ps and rna extracted from mers-cov isolate (kor/knih/ _ _ ) were prepared at a concentration of pg/ll. they were then diluted tenfold with nuclease-free distilled water and used as template for the real-time rt-pcr to evaluate the detection limits of the primers and probes. to evaluate the specificity of the primers and probes, several rna viruses, including dengue virus types , , and (kbpv-vr- , - , and - , respectively; korea bank for pathogenic viruses, seoul, korea), porcine reproductive and respiratory syndrome virus (prrsv) strain cp - , porcine epidemic diarrhea virus (pedv) strain dr (green cross veterinary products, yongin, korea), and influenza a virus (h n ) strain sk from our laboratory, were tested using the newly developed realtime rt-pcr protocol. in addition, rna extracted from a mers-cov isolate (kor/knih/ _ _ ), which was kindly provided by the korea center for disease control, was tested using the real-time rt-pcr system. a recombinant plasmid including partial sequences of the s region (nucleotides - of the spike gene, merslike bat cov strain hku [genbank no. ef ]) was synthesized (puc -mers-like-cov-ps ) and used as a template for the real-time rt-pcr. agarose gel electrophoresis was performed to observe the target bands as well as for fluorescent detection. rna was extracted from bat samples (feces, urine, and oral swabs) collected between july and april in korea. among them, samples b - , b - , b - , b - , and b - , and b - were determined to be positive for covs according to consensus primers-based rt-pcr, which can detect diverse covs by targeting conserved sequences of the rna-dependent rna polymerase gene [ ] . the positive samples were found to have rna sequences that are closely related to those of alphacoronaviruses or sars-like bat cov [ ] . real-time pcr was performed with the extracted rnas and compared to the previous rt-pcr results. compared with the results of upe-based real-time rt-pcr as a reference [ ] . detection limits of the real-time rt-pcr the tenfold-diluted plasmids (puc -sars-ps and pgem-mers-s ) were tested by real-time rt-pcr as a single template or as mixed templates. the intensity of the fluorescence (fam and hex) emitted from the hydrolyzed probes was measured every cycle. based on the measured amplification curve, the results were expressed as the quantification cycle value (cq value). the cq values obtained from the diluted plasmids were found to be significantly correlated with the copy numbers of each plasmid, with an r value of . and . for puc -sars-ps and pgem-mers-s , respectively (fig. ) . the negative control (distilled water) did not yield an amplification curve. as shown in table , the detection limits of the real-time rt-pcr for puc -sars-ps and pgem-mers-s were both copies/ml in the single-template condition and were and copies/ml, respectively, in the mixed-template condition. the detection limits of the real-time rt-pcr for the in vitro-transcribed mrna of puc -sars-ps and viral rna of mers cov (kor/knih/ _ _ ) were - and ng/ml, respectively, in the single-template condition and were - and ng/ml, respectively, in the mixed-template condition ( table ) . the specificity of the real-time rt-pcr method developed in this study was evaluated using rnas from several rna viruses, including mers-cov (kor/knih/ _ _ ), a recombinant plasmid for the bat cov hku strain, and rna from a bat fecal sample containing sars-like bat cov. the designed primers and probes were found to be specific for mers-cov and sars-like bat cov (b - ) ( table ) . no positive reactions were found for the bat coronavirus strain hku , dengue virus, prrsv, pedv, or influenza a virus. in the case of the hku strain, although the hex fluorescence signal was not detected, a target band of primers for mers-cov was detected by agarose gel electrophoresis. the extracted rnas from a total of bat samples that were previously tested using consensus primer-based rt-pcr [ ] were re-evaluated by the real-time rt-pcr method developed in this study. as shown in table , six fecal samples were positive in the consensus-primer-based rt-pcr, and these were further sequenced and analyzed using blastn (http://www.ncbi.nlm.nih.gov). among the six positive samples, five were found to be closely related to alphacoronaviruses, while one sample (b - ) was closely related to sars-like bat cov [ ] . in the real-time pcr developed in this study, five of the alphacoronaviruspositive samples were negative, but only one sars-like bat cov-positive sample was positive. the rna samples from the sputum of mers patients were tested by the newly developed real-time rt-pcr method, and the results were compared to those obtained with upebased real-time rt-pcr as a reference gold-standard test ( table ) . among the samples tested, were found to be positive by upe-based real-time rt-pcr. when the single set of primers and probe for mers-cov was used, the newly developed real-time rt-pcr results showed % consistency with those of the reference test. however, when mixed sets of primers and probes for sars-cov and mers-cov were used, % of the results were consistent with those of the reference test. three falsenegative samples had cq values of . , . , and . , respectively, in the reference test. the aim of this study was to develop a duplex real-time rt-pcr method for the simultaneous detection of sars-cov and mers-cov, which are recently emerged pathogens infecting humans. as these viruses are also closely related to several bat covs (sars-like bat covs and mers-related bat covs), the primers and probes were designed based on the conserved region of the spike s domains of sars-cov, mers-cov, and their related bat covs. in a previous study, two regions within the spike protein of sars-cov were identified that showed a high degree of sequence conservation with those of other covs [ ] . by applying our new approach, we expected to detect not only sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses. indeed, the newly developed real-time rt-pcr method could detect sars-cov-and mers-cov-specific sequences when the plasmids puc -sars-ps and pgem-mers-s were used as templates. in addition, positive results were obtained when rna extracted from mers-cov (kor/knih/ _ _ ), which was isolated from a patient in korea in [ ] , was used as a template. the new real-time rt-pcr method also showed positive results for rna extracted from a fecal sample containing sars-like bat cov (b - ) [ ] . in addition, no nonspecific amplification was found when using rnas obtained from several rna viruses belonging to the families flaviviridae, arteriviridae, and orthomyxoviridae, and the genus alphacoronavirus. the specific binding of the primers and probes used in the real-time rt-pcr was evaluated to determine their detection limits using serially diluted plasmids (puc -sars-ps and pgem-mers-s ). the detection limits of the real time rt-pcr were - copies/ml and - copies/ml for puc -sars-ps and pgem-mers-s , respectively. as the previous real-time rt-pcrs were able to detect as few as copies/ml and copies/ml for sars-cov and their related bat covs and mers-cov, respectively single template - mixed template - a mrna obtained by in vitro transcription b rna from mers-cov isolate kor/knih/ _ _ a samples that were positive in the rt-pcr ( ) were sequenced and analyzed using blastn (http:// www.ncbi.nlm.nih.gov) [ , ] , the duplex real-time rt-pcr method developed in this study can be applied with an adequate limit of detection. when the real-time rt-pcr was applied to the bat samples, it could specifically detect a sars-like bat cov, and no positive reactions were obtained with the other samples, including alphacoronavirus-positive samples. the real-time rt-pcr method developed in this study could not detect the bat cov hku strain, which belongs to the same group as human mers-cov. although these viruses are in the same group, the nucleotide sequences were found to be more variable than those of sars-cov and its related bat covs (fig. ) . however, as a target band was found in the agarose gel electrophoresis of a pcr product from the hku template, the negative result obtained with hku in the real-time rt-pcr method developed in this study might have been due to inefficient binding of the probe. the new real-time rt-pcr method was also tested with rnas extracted from the sputum of mers patients in korea. the single set of primers and probe for mers-cov could detect the same positive samples detected in the reference test, showing % agreement. however, when the mixed sets of primers and probes for sars-cov and mers-cov were used, the agreement was reduced to %. three samples that tested positive in the reference test were not detected with the newly developed method. this may be due to the low amount of viral rna in the sputum samples and the reduced sensitivity due to the duplex approach. in addition to using a standard test for the diagnosis of mers-cov infection in humans, consideration of additional tests would be helpful to avoid missing true positives. collectively, the newly developed real-time rt-pcr method was demonstrated to be applicable for the simultaneous detection of sars-cov and mers-cov, showing an adequate detection limit and specificity. by testing the new method with bat samples as well as human samples, it could be applicable to survey sars-cov, mers-cov, and potentially their related bat covs in bats and human samples. it could successfully detect sars-like cov in bat samples but showed limited detection ability for the bat cov hku strain, which is related to mers-cov. however, according to a recent finding of emc-like mers-cov, which was detected in bats of saudi arabia [ ] , we assume that the new method can be helpful for screening for mers-cov in bat samples. as novel covs continue to emerge around the world, real-time-pcr-based detection methods have been developed by pioneering researchers [ , [ ] [ ] [ ] . these methods are applicable with good limits of detection and have been validated using field samples. however, simultaneous detection of sars-cov, mers-cov, and their related bat covs would be particularly useful for the detection of these viruses in humans as well as in bats, which are a known reservoir of the viruses. therefore, the duplex real-time rt-pcr approach for the simultaneous detection of sars-cov and mers-cov developed in this study might allow more-convenient and rapid detection of these viruses in clinical samples. in addition, this new method can be used to closely monitor related bat covs in bat populations, which are considered to be a primary reservoir of these viruses. epidemiology, genetic recombination, and pathogenesis of coronaviruses ecology, evolution and classification of bat coronaviruses in the aftermath of sars sars and mers: recent insights into emerging coronaviruses transmission characteristics of mers and sars in the healthcare setting: a comparative study isolation and characterization of a bat sars-like coronavirus that uses the ace receptor a sars-like cluster of circulating bat coronaviruses shows potential for human emergence detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats close relative of human middle east respiratory syndrome coronavirus in bat mers-related betacoronavirus in vespertilio superans bats isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus identification of a novel coronavirus in bats detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus a real-time pcr assay for bat sars-like coronavirus detection and its application to italian greater horseshoe bat faecal sample surveys realtime reverse transcriptase polymerase chain reaction assays for middle east respiratory syndrome development of a molecular-beacon-based multi-allelic real-time rt-pcr assay for the detection of human coronavirus causing severe acute respiratory syndrome (sars-cov): a general methodology for detecting rapidly mutating viruses middle east respiratory syndrome coronavirus in bats, saudi arabia conflict of interest the authors have declared that no competing interests exist. key: cord- -leni pa authors: müller, b.; klemm, u.; mas marques, a.; schreier, e. title: genetic diversity and recombination of murine noroviruses in immunocompromised mice date: - - journal: arch virol doi: . /s - - -y sha: doc_id: cord_uid: leni pa murine noroviruses (mnv) are newly identified pathogens which infect laboratory mice. in this study, we found a high prevalence ( . %) of mnv in various breeding colonies of immunocompromised, transgenic and wild-type mouse lines. all mice survived infection with no signs of clinical disease. faeces samples were collected from animals housed in two separate laboratory mouse colonies in berlin, germany, and screened using quantitative reverse transcription (rt)-pcr. we have determined the complete nucleotide sequences of novel mnv strains. furthermore, we sequenced two subgenomic regions within open reading frames (orfs) and that are suitable for genotyping. sequence analysis of the full-length and partial genomes obtained from naturally infected mice yielded valuable data on genetic diversity of murine noroviruses. the discordance of genotype affiliation of some mnvs shown in orf and orf suggests intertypic recombination events in vivo. the first strain of murine noroviruses (mnv- ) was isolated in from the brain of an immunodeficient mouse, lacking recombination-activating gene and signal transducer and activator of transcription (rag =stat À=À ), which succumbed to a systemic disease [ , ] . mnv is the first known norovirus to infect small animals. sequence analysis designated the virus as a member of the new genogroup v within the genus norovirus. in contrast to the lack of a culture system for human noroviruses, the murine norovirus replicates in cultured haematopoietic cells. this provides an excellent model system to study norovirus biology and pathogenesis [ , ] . noroviruses are positive-sense rna viruses that belong to the family caliciviridae. these viruses are the major cause of outbreaks of acute nonbacterial gastroenteritis in humans worldwide. the viral genome of human noroviruses is approximately . kb in length and contains three overlapping open reading frames. orf encodes the nonstructural polyprotein, which is processed by the viral c-like protease into several functional proteins, including ntpase and rna-dependent rna polymerase [ , ] . orf and orf encode the major capsid protein (vp ) and a small basic structural protein (vp ), respectively. the vp protein possesses two domains: the p (protruding, p and p ) domain and the s domain, which forms the icosahedral shell of the capsid [ ] . most of the cellular interactions and immune recognition features are thought to be located in the p sub-domain, which is exposed at the viral surface and shows the highest degree of sequence diversity within the genome [ , ] . vp is associated with upregulation of vp expression in cis and stabilization of vp in the virus structure [ ] . although varying in size, the murine norovirus exhibits an identical genomic organisation [ ] . based on rt-pcr and genomic sequencing, the genus norovirus demonstrates high levels of genetic diversity and can be differentiated into five genogroups (gg). hence, strains of ggi, ggii and ggiv are found in humans, ggiii and ggv strains are found in cattle and mice, respectively. currently, genetic clusters are classified within these genogroups: in ggi, in ggii, in ggiii, and each in ggiv and ggv [ ] . recently, three more strains have been described within ggv [ ] . the purpose of this study was to get an insight into the prevalence and genetic diversity of murine norovirus circulating among laboratory mouse colonies in germany. a total of faeces samples were collected from research mice housed in two separate colonies in berlin, germany. these specimens were obtained from different mouse lines, including immunocompetent mice, lines with muta- ( ), b-tcr À=À ( ), rag =gchain À=À ( ), b casp À=À ( ), cd =sra À=À ( ), ccl À=À ( ), lfa À=À ( ), lyz À=À ( ), psen tg ( ), bl wt ( ) a ge genome equivalents; for more than one positive sample the mean was calculated b includes partial sequences of the orfs (underlined isolates; orf , rna polymerase gene; orf , capsid gene) à contains both regions tions in a number of immune effector molecules, and transgenic mouse lines (table ) . mice were group-housed in static filter-top cages with up to animals per cage in barrier units with documented spf status. viral rna was extracted from ml of a % faecal suspension using qiaamp viral rna mini kit (qiagen, hilden, germany) according to manufacturer's recommended protocol. for detection of murine norovirus, a single-tube real-time rt-pcr assay was established by using the taqman technology (applied biosystems, foster city, usa). primers mnv-tm and mnv-tm and a fluorescence-labeled taqman probe mnv-tmp within a conserved mnv- orf gene region were selected (table ). in the final format, quantitect probe pcr kit reaction mixtures (qiagen) contained ml of template rna, each primer at a concentration of nm, and nm probe. the mixture was then subjected to a onestep assay by using the following conditions: rt for min at c, rt inactivation=dna polymerase activation for min at c, and cycles of s at c, and s at c for amplification. to quantify the number of genomes, an external standard curve was established with a -fold series of dilution from  to  copies per reaction using a plasmid that contained the capsid gene of mnv- . viral rna was extracted from either faecal suspension (isolates m and s ) or plaque-purified virus (isolates s and s ). however, all four isolates have been propagated on raw . cells as described elsewhere [ ] . single-stranded cdna was synthesized from total rna using powerscript tm reverse transcriptase (bd clontech, heidelberg, germany) with a tagged primer complementary to the poly(a) tail of the genome ( t-atag -gcc aac gac cgg gag gcc agc ttt ttt ttt ttt ttt v- , tag sequence is underlined). pcr fragments were generated using phusion dna polymerase (finnzymes, espoo, finland). the primer pair mnv- . s and mnv- . as amplifies a bp product resembling the complete orf and the end of orf . primer pair mnv- . s and a reverse primer containing the tag sequence (atag -gcc aac gac cgg gag gcc agc- ), amplifies orf , orf and the utr. thus, the complete genomic sequences were obtained by two overlapping pcr fragments, which were cloned using topo-xl cloning kit according to manufacturer's instructions (invitrogen, karlsruhe, germany). universal t forward and m reverse primers were used for sequencing of the plasmids with the big-dye terminator cycle sequencing kit (perkin elmer, wellesley, ma), and sequences were determined using an abi prism genetic analyser (applied biosystems). primer walking was applied for subsequent sequencing. amplification of partial orf and orf sequences orf (rna polymerase region) and orf (capsid) were amplified by nested rt-pcrs. partial amplification of the polymerase gene was performed using primer pair mnv- . s and mnv- . as, resulting in a -bp product, which strains that did not cluster with the same group of viruses in orf and orf were considered to be putative recombinant strains. a nested rt-pcr which produces a long genomic fragment ( -bp) encompassing the end of the polymerase region and the end of the capsid was performed using primer pairs mnv- . s=mnv- . as (first round) and mnv- . s=mnv- . as (second round). the product was cloned into a plasmid vector as described above. similarity plots were generated (simplot version . [ ] ) in order to detect recombination. sequence alignments and phylogenetic analysis were performed using bioedit (version . . [ ] ), which includes the phylogeny inference package (phylip [ ] ). evolutionary distances were calculated by kimura's -parameter method [ ] . the computed distances were utilized to construct phylogenetic trees by the neighbor-joining method (treeview version . . [ ] ). to gain an internal estimate of how well the data supported the phylogenetic trees, bootstrap resampling ( datasets) of the multisequence alignments was carried out. in this study, faeces specimens collected from two separate laboratory mouse colonies in berlin, germany, were screened for murine norovirus using a newly established one-tube real-time rt-pcr. these specimens were obtained from mouse lines with diverse genetic modifications mainly involving the immune system ( complete nucleotide sequences from strains m (ifng À=À ), s (rag À=À ), and s (yfph tg) were determined. sequence data are available in genbank (fig. ) . sequences for m and s could be successfully determined from original material, whereas s and s had to be propagated on raw . cells and plaque purified before sequence determination. the phylogenetic tree and distance plot analysis demonstrated that our strains were genetically distinct from strains mnv - (fig. ) . regarding full-length genomes, pairwise sequence comparison with the known reference sequences displayed nucleotide identities from to % for m , s and s ( table ). the highest identity was found between strains m and s ( %). overall, we found a very high homology within the orf -orf junction region of all investigated mnv strains (fig. b) , where these strains shared a -nucleotide stretch with almost % identity. furthermore, the shell regions of all capsid sequences proved to be highly conserved between the strains investigated in this study. the highest variability was shown for the p domain, where up to % distance between the sequences was calculated. this observation is consistent with the results of investigations of the human noroviruses [ , ] . interestingly, we found a significant difference within the utr sequence of the strains mnv - and s compared to m and s . the latter strains exhibited an insertion of and bases, respectively, at position nt . for extensive investigation of genetic diversity within the detected murine noroviruses, part of the rna polymerase and part of the capsid gene were amplified and sequenced. distance plotting analysis and full-length sequence alignments were used to find optimal regions for amplification. we identified two variable regions that appeared suitable for differentiation of viral strains. these regions were composed of conserved areas which were adequate for primer design. figure b shows an example for distance plotting and primer binding sites. we used a region of bases in length located within orf between nt and nt . within orf , the region was located between nt and nt , encompassing bases (table ) . represen- [ ] tative partial genome sequences have been published in genbank. the accession numbers are listed in table . phylogenetic trees were constructed to analyze the relationships of the partial sequences with the corresponding parts of the mnv-berlin strains (m , s and s ) and the reference strains mnv - ( fig. ) . strains that share a branch of a phylogenetic tree were classified as different clusters. the term ''genetic cluster'' was used as defined by ando et al. [ ] and represents a minimum classification unit consisting of strains that reproducibly group together on a distinct branch of a phylogenetic tree and are sufficiently close in nucleotide sequence to be distinguished from strains related to other groups. isolates within the same cluster showed nucleotide distances between and . for orf and and . for orf (fig. ) . intercluster nucleotide distances ranged between . and . , and . and . , respectively. for most isolates, the phylogenetic clusters observed for orf (fig. a) were reflected by orf (fig. b) . however, isolates s , s and s are classified in different clusters comparing both phylogenetic trees. concerning orf , these isolates appear to be related to s , whereas in orf they cluster with m . these three isolates were obtained from the same mouse line (tlr À=À ), but from different cages (table ). to investigate if the different branching of isolates s , s and s was due to recombination, sliding window analyses were performed. for this purpose we cloned large fragments ( bp) spanning the end of orf and the region of orf . compared to each other, the three fragments obtained from s , s and s revealed more than % nucleotide sequence identities. the fragments were analyzed with simplot software by comparing the sequences to potentially parental strains as described elsewhere [ ] . figure a shows the results obtained by comparison of the sequence from isolate s to m and s as putative parental strains. s is distant from m within the orf polymerase region except for the end, which corresponds to the conserved orf -orf junction. s showed a closer association to s within orf , with a similarity of - %, which confirms the clustering shown in fig. . in contrast, the s sequence was only distantly related to s within the capsid region, while on the other hand, the distance to m was less than %, supporting the relationship within orf . furthermore, the reference strain mnv- , which was phylogenetically divergent in orf , clustered with isolates s , s and s in orf . to confirm the genetic recombination of mnv- , simplot analysis was performed with s and m , considering the end of orf , orf , orf and the utr, respectively (fig. b) . while mnv- was distant to s within orf , the sequences were closely related in the complete part of the genomes. the relationship between the two strains, furthermore, could be confirmed regarding the utr of s , which is closely homologous to mnv- and also lacks the insertion at position nt . the reference strain m and mnv- displayed low similarity over the entire fragment except for the junction region. following the first description of murine norovirus, serological testing in north america showed that mnv is one of the most prevalent pathogens infecting research mice [ ] . to investigate the prevalence of mnv in german research mice, we established a highly sensitive real-time pcr assay. the frequency determined in this study ( . %) was much higher than expected based on the data from serological testing ( . %) [ ] . this fact could be attributed to local endemic differences. in addition, it is possible that not all infected mice develop immunity. time-related reduction of antibody titers or insufficient cross-reactivity may also play a role. studies concerning the immunity against human noroviruses have indicated that immunity seems to be short-lived and protection, furthermore, is strainspecific [ , ] . the potential to induce persistent infections in mice [ ] , the high degree of environmental stability [ ] , and the faecal-oral route of transmission [ ] all could have contributed to the high prevalence. none of the mice investigated here showed any symptoms of disease, confirming the observations regarding infection of mice with mnv - [ ] . this certainly contributed to the unnoticed spread of the virus. in contrast, mnv- infection has been shown to be associated with lethal disease in severely immunodeficient mice [ , ] . it remains unclear if the pathogenesis is dependent on the mouse genotype or specific mnv strains. in this respect, further studies are required to evaluate if mnv infection of laboratory mice affects independent experiments unrelated to research on mnv. here, viral infection was independent of the genotype of the mouse. there was no significant difference in the prevalence of mnv in immunodeficient mice compared to wild-type or transgenic mice. human noroviruses exhibit great genetic diversity. molecular methods have become major means of characterizing and understanding the relatedness of different viral strains [ , , ] . like human noroviruses, the present sequence data indicate that murine norovirus strains are also genetically diverse and can, therefore, be subdivided in genetic clusters. the mnv strains analysed in the present study were classified into genetic clusters. the cluster pattern is comparable to the diversity of the sapoviruses (sv), another genus within the family caliciviridae [ , ] . these data suggest that mnvs, like svs, could be evolutionary more stable than human noroviruses. published mnv - complete nucleotide sequences are prototypes of clusters to , whereas m , s , s and s would be the reference sequences for clusters to (fig. a) . however, the divergences of partial orf and orf sequences of mnv isolates investigated here reveal a high degree of variability among murine noroviruses. it is noticeable that the genetic relatedness of mnv isolates is not always equivalent when analyzing different genome regions. the phylogenetic analysis presented here demonstrated that recombination events might have occurred between murine norovirus strains (fig. ) . analysis of the sequences obtained from isolate s by a nucleotide identity window search indicated a phylogenetic shift (fig. a) . furthermore, reference strain mnv- clustered differentially (fig. b) . in addition to the high error rate of the viral rna-dependent rna polymerase due to the lack of proofreading activity, recombination also contributes to the diversity and the evolution of rna viruses [ ] . intertypic recombination within human svs as well as bovine and human nvs has been demonstrated by comparison of complete genome sequences and incongruent clustering of sub-genomic sequence fragments from different regions [ , , , , , ] . breakpoint analysis of recombinant viruses revealed the orf -orf overlap as a recombination site. our data confirm that a putative crossover point of mnv recombinants (fig. ) is located within nucleotides (fig. b) of this region. this junction region is highly conserved, suggesting that the recombination might be driven by homologous rna interaction, comparable to picornaviruses and coronaviruses, where the ''copy choice'' model of rna recombination is preferred [ , ] . occurrence of subgenomic rna during replication of mnv supports this model [ ] . additionally, rna promoter regions often contain stem-loop structures [ ] . we found evidence of such structures within the highly conserved junction of mnv strains. in conclusion, our study has demonstrated a high prevalence of genetically divergent murine noroviruses in the faeces of different mouse lines. these results confirm recent observations indicating that mnv commonly circulates among laboratory mice. the potential occurrence of recombinant strains has to be taken into account for phylogenetic analysis of mnv strains. therefore, sequencing of different genome regions is recommended. the pcr methods used in this study provide means for routine diagnosis and molecular characterization of mnv. characterization of new recombinant noroviruses genetic classification of ''norwalk-like viruses in vitro proteolytic processing of the md norovirus orf nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells structural requirements for the assembly of norwalk virus-like particles norovirus recombination in orf =orf overlap surrogates 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noroviruses de novo initiation of viral rna-dependent rna synthesis stat -dependent innate immunity to a norwalk-like virus phylogenetic analysis of the complete genome of norwalk-like viruses a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences the mechanism of rna recombination in poliovirus full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination high-frequency rna recombination of murine coronaviruses immunity to calicivirus infection treeview: an application to display phylogenetic trees on personal computers clinical immunity in acute gastroenteritis caused by norwalk agent polypeptide p of a norwalk-like virus is a nucleic acid-independent nucleoside triphosphatase x-ray crystallographic structure of the norwalk virus capsid molecular epidemiology of outbreaks of gastroenteritis associated with small round structured viruses in germany in = genetic classification of ''sapporo-like viruses cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells pathology of immunodeficient mice with naturally occurring murine norovirus infection replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages murine norovirus: a model system to study norovirus biology and pathogenesis evolutionary aspects of recombination in rna viruses norovirus classification and proposed strain nomenclature genetic diversity and recombination of murine noroviruses we thank christiane e. wobus and herbert w. virgin (washington university school of medicine, st louis, mo) for kindly supplying the mnv- capsid containing plasmid and the raw . cell line. we are grateful to ute buwitt and julia tesch for excellent sequencing support. key: cord- -c xpzd d authors: zhai, jun-qiong; zhai, shao-lun; lin, tao; liu, jian-kui; wang, he-xing; li, bing; zhang, he; zou, shu-zhan; zhou, xia; wu, meng-fan; chen, wu; luo, man-lin title: first complete genome sequence of parainfluenza virus isolated from lesser panda date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: c xpzd d parainfluenza virus (piv ) is widespread in mammals and humans. up to now, there is little information about piv infection in lesser pandas. in this study, a piv variant (named zjq- ) was isolated from a lesser panda with respiratory disease in guangzhou zoo in guangdong province, southern china. the full-length genome of zjq- was found to be , nucleotides and consisted of seven non-overlapping genes encoding eight proteins (i.e., np, v, p, m, f, sh, hn and l). sequence alignment and genetic analysis revealed that zjq- shared a close relationship with a piv strain of canine-origin ( - ) from south korea. the findings of this study confirm the presence of piv in lesser panda and indicate this mammal as a possible natural reservoir. furthermore they highlight the urgent need to strengthen viral surveillance and control of piv in zoo animals. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. members of the paramyxoviridae family are the causative agents of mumps and measles in humans, newcastle disease in poultry, peste des petits ruminants in sheep and goats, as well as distemper in carnivorous animals [ ] [ ] [ ] [ ] . parainfluenza virus (piv ) belongs to the genus jun-qiong zhai, shao-lun zhai and tao lin contributed equally to this study. rubulavirus in the family paramyxoviridae (http://www. ictvonline.org/virustaxonomy.asp?taxnode_id= ), and was originally known as simian virus (sv ) or canine parainfluenza virus (cpiv) [ ] [ ] [ ] . piv has a negative sense, single-stranded rna genome of , nucleotides with a virion diameter of - nm that appears circular or polymorphous under the electron microscope [ ] [ ] [ ] . piv has eight viral proteins (i.e., np, v, p, m, f, sh, hn and l). the v and p proteins are encoded by the v/p gene sharing the same genomic region [ ] . piv has a close genetic relationship with other members of genus rubulavirus, including simian virus , human parainfluenza virus , human parainfluenza virus , mumps virus, mapuera virus and porcine rubulavirus [ ] [ ] [ ] [ ] [ ] [ ] . recently, paramyxoviruses have been found to have a global presence and to be prevalent in several countries [ ] [ ] [ ] . novel paramyxoviruses that can spread among different species have resulted in outbreaks in animals [ , ] . hosts susceptible to piv include humans, pigs, dogs, cattle, cats, hamsters and guinea pigs [ ] [ ] [ ] [ ] . piv can cause central nervous and respiratory diseases in infected hosts [ , ] . up to now, there is little information about piv in lesser pandas. in this study, a novel variant of piv (designated as zjq- ) was isolated from a lesser panda with respiratory disease in guangzhou zoo in guangdong province, southern china. furthermore, the complete genome sequence of zjq- was amplified and characterized. during the years of - , fourteen representative clinical samples from ornamental animals (including four lesser pandas, one northeast tiger, one south china tiger, four panthera leo and four ring-tailed lemurs) were collected from guangzhou zoo in guangdong province, southern china. clinical manifestations of the animals included coughing with thin nasal fluid and a slightly elevated temperature. one lesser panda died, and its necropsy identified the presence of lobular pneumonia in the lungs. nasal swabs and lung samples were stored at - °c until use. fourteen samples were tested for the possible presence of three respiratory-related pathogens (including piv , canine distemper virus, and coronavirus) by rt-pcr according to previous studies [ , ] . the results revealed the positive presence of piv nucleotides in the lesser panda samples. the lung samples from the lesser panda were then homogenized in % (w/v) sterile phosphatebuffered saline (pbs, ph . ), and centrifuged at , g for min at °c. the supernatants were filtered aseptically ( . lm pore size) and the filtrates ( ll) were inoculated onto vero cell monolayers in a -cm cell culture flask. after viral attachment for h at °c, unattached viruses were removed by gentle washing with pbs and the cells were maintained in dulbecco's minimal essential medium (dmem, gibco), containing % fetal bovine serum (fbs, gibco) and % antibiotic-antimitotic, at °c in a % co atmosphere. the viral cultures were harvested when the cytopathic effects (cpe) reached % coverage and then stored at - °c [ ] . in order to observe the virus particles, the cell suspensions were centrifuged at , rpm for min, and then fixed using . * % glutaraldehyde fixation fluid for about h at °c in a refrigerator. finally, they were submitted to the electron microscope center, south china agricultural university for processing. rna extraction of positive homogenates was performed using the minibest universal rna extraction kit (takara), and viral rna was reverse transcribed into first-strand cdna with a random primer ( -nnnnnn- ) using the primescript first strand cdna synthesis kit (takara). pcr was then used to amplify the full-length genome sequences using pairs of primers listed in table s . positive pcr products were purified (minibest agarose gel dna extraction kit, takara) and cloned into the pmd -t vector (takara). positive recombinant plasmids were further sequenced using the sanger sequencing method (bgi inc., guangzhou branch). sequence alignment and phylogenetic analysis based on different rubulavirus species sequences (table ) was performed using dnastar lasergene . (madison, wi, usa) and mega (http://www.megasoftware.net) [ ] . in comparison with uninfected cells (fig. s a) , vero cells inoculated with piv -positive samples showed demonstrable cpe by passage (fig. s b) . one viral strain (named zjq- ) was thus obtained. further observation by electron microscopy found that the isolated zjq- strain was spherical and had a diameter of - nm that is similar to, and characteristic of, paramyxoviruses (fig. s c) . moreover, through sequencing, the full-length genome sequence ( , in addition, the sh protein was not present in some piv strains due to nucleotide substitution, such as kun- (kc ), ser (jq ), cc (kp ) pv -bc (km ) and ags (kx ), implying that the protein is not essential for piv infection in pigs, dogs, calves and cells [ , , , , ] . however, the zjq- strain had a sh protein and was thus different from the above-mentioned piv strains. furthermore, phylogenetic analysis ( fig. ) was performed based on the complete genome sequences of different species of rubulaviruses. in the phylogenetic tree, zjq- and - shared the closest genetic relationship and were clustered in the same branch (fig. ) . to the best of our knowledge, very little data about piv infections in zoo animals is available. in this study, we tested whether piv infections are present in lesser pandas, northeast tigers, south china tigers, panthera leo and ring-tailed lemurs. while the rt-pcr results showed that only lesser panda samples were positive for piv , due to the limited number of animal samples collected in the zoo, we believe that piv might not be restricted to lesser pandas. in fact, a previous study showed antibodies against piv exist in zoo tigers [ ] . moreover, piv nucleotide sequences were also detected in nasal swab samples from northeast tiger and south china tiger collected in in the same zoo, and their f genes were close to the lesser panda-origin piv strain described here (data not shown). this suggests that piv is a common pathogen in zoo animals, and might play an important causal role in the respiratory diseases of zoo animals. in summary, we have identified a novel piv isolate in lesser panda and performed whole genome sequencing, indicating that this mammal may act as a possible natural reservoir for this virus. this study contributes to the epidemiology and genomics of piv , and suggests an urgent need to strengthen viral surveillance and control of piv in zoo animals. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. measles, mumps, rubella, and human parvovirus b infections and neurologic disease temporal, geographic, and host distribution of avian paramyxovirus (newcastle disease virus) genome characterization and phylogenetic analysis of a lineage iv peste des petits ruminants virus in southern china diversity of susceptible hosts in canine 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epidemiological perspectives on hendra virus infection in horses and flying foxes relationships and host range of human, canine, simian and porcine isolates of simian virus (parainfluenza virus ) parainfluenza- virus. infection of man and animal isolation of novel bovine parainfluenza virus type (bpiv ) and its incidence in korean cattle parainfluenza virus as possible cause of severe respiratory disease in calves acute encephalitis and hydrocephalus in dogs caused by canine parainfluenza virus rt-pcr and sequence analysis of the full-length fusion protein of canine distemper virus from domestic dogs molecular surveillance of traditional and emerging pathogens associated with canine infectious respiratory disease stability of the parainfluenza virus genome revealed by deep sequencing of strains isolated from different hosts and following passage in cell culture the complete genome sequencing and analysis of canine parainfluenza virus strain cc- molecular characteristics of canine parainfluenza viruses type (cpiv- ) isolated in korea complete genome sequence of a novel porcine parainfluenza virus isolate in korea genome sequence of the parainfluenza virus strain that persistently infects ags cells mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods seroepidemiological investigation of caine parinfluenza virus in tiger acknowledgements this work was mostly supported by guangzhou zoo. moreover, this work was also partially supported by ministry of science and technology of the people's republic of china (grant no. ga ), guangdong provincial department of science and key: cord- -drhiqch authors: hohdatsu, t.; tokunaga, j.; koyama, h. title: the role of igg subclass of mouse monoclonal antibodies in antibody-dependent enhancement of feline infectious peritonitis virus infection of feline macrophages date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: drhiqch antibody-dependent enhancement (ade) of feline infectious peritonitis virus (fipv) infection was studied in feline alveolar macrophages and human monocyte cell line u using mouse neutralizing monoclonal antibodies (mabs) directed to the spike protein of fipv. even among the mabs that have been shown to recognize the same antigenic site, igg a mabs enhanced fipv infection strongly, whereas igg mabs did not. these igg a mabs enhanced the infection even when macrophages pretreated with the mab were washed and then inoculated with the virus. immunofluorescence flow cytometric analysis of the macrophages treated with each of the mabs showed that the igg a mabs but not the igg mabs bound to feline alveolar macrophages. treatment of the igg a mab with protein a decreased the binding to the macrophages and, in parallel, diminished the ade activity. although no infection was observed by inoculation of fipv to human monocyte cell line u cells, fipv complexed with either the igg a mab or the igg mab caused infection in u cells which are shown to express fc gamma receptor (fc γ r) i and ii that can bind mouse igg a and igg , respectively. these results suggest that the enhancing activity of mab is closely correlated with igg subclass and that the correlation is involved in binding of mab to fc γ r on feline macrophage. feline infectious peritonitis virus (fipv) is a member of the coronavirus family and causes a chronic progressive disease in its natural host. the natural route of fipv infection is unknown although cats can be experimentally infected by oral, nasal, and parenteral administration of fipv. following infection by these routes, fipv first multiplies in the epithelial cells of the upper respiratory tract and intestine [ ] . clinically apparent fip occurs after the viruses in infected t. hohdatsu et al. macrophages and monocytes cross the mucosal barrier and spread throughout the body of the cat. generally, macrophages play an important role in non-specific defense against viral infection. however, it is also known that viruses bound to antibodies invade macrophages via fc region of the bound antibody and the macrophage's fc gamma receptor (fc r) and eventually lead to enhancement of infection [ , - , , , , ] . its most typical example is a disease called dengue hemorrhagic fever/dengue shock syndrome. the antibody for fipv is also known to enhance the fipv infection and accelerate the disease onset in cats [ , , ] . following experimental fipv infection, cats with naturally occurring fipv-neutralizing antibody frequently develop fip more rapidly and with more severe clinical signs than do seronegative cats. when cats passively immunized with anti-fipv antibodies were infected with virulent fipv, severe symptoms were observed and some of them died soon after the infection [ , ] . the enhancing effect of the antibody of fipv infection impedes prophylaxis of fip by vaccination [ , , - , , ] . we previously reported that in vitro fipv infection of feline alveolar macrophages is enhanced by murine monoclonal antibodies (mabs) to the peplomer spike (s) protein or the transmembrane (m) protein of fipv [ , ] . this antibody-dependent enhancement (ade) was completely eliminated or reduced by pretreatment of the mabs with protein a or when f(ab') fragments of the mabs were used. olsen et al. have supported our findings by showing enhancement of fipv infection in primary feline peritoneal macrophages by mouse mabs to the viral s protein [ ] . in many instances virus-neutralizing mabs were shown to have strong ade activity, and it was suggested that there was some relationship between neutralizing activity and ade activity. on the other hand, corapi et al. recognized a difference in immunoglobulin subclasses between ade-inducing fipv-neutralizing mab and ade-non inducing fipv-neutralizing mab, and they showed that the majority of the ade-inducing mabs belong to the igg a subclass [ ] . these results were obtained by the experiments in which mouse mabs and feline macrophages, i.e., xenogeneic combination of mabs and macrophages, were used. in contrast, it has been reported that mouse mabs irrespective of whether the subclass is igg , igg a or igg b enhanced infection of mouse (homologous) macrophages of dengue virus [ , ] , influenza a virus [ "] and west nile virus [ ] . since ade of the disease caused by fipv has been clearly demonstrated after vaccination of cats with a variety of candidate vaccines, a more specific understanding of ade of fipv infection is needed [ , , - , , ] . it is especially important for development of vaccines to understand the relationship between fipv neutralizing activity and ade activity of anti fipv mabs. in this study, we determined the relationship between immunoglobulin subclass and ade activity of fipv-neutralizing mabs, and attempted to explore mechanisms of ade of fipv infection in in vitro system using mouse mabs and feline alveolar macrophages or human monocytes. fipv strain - was used in this study. this strain was kindly provided by dr. m. c. horzinek of the state university utrecht, the netherlands, and was passaged two or three times in feline fetal cell (fcwf- ) cultures. feline alveolar macrophages were collected from anti-coronavirus antibody-negative adult cats and cultured in eagle's minimum essential medium containing ~ leibovitz's l- medium, ~ fetal calf serum, units of penicillin per ml and ~tg of streptomycin per ml as previously described [ ] . human monocyte cell line u was kindly provided by dr. j. arikawa of the university of hokkaido, japan, and was cultured in rpmi medium containing ~o fetal calf serum, units of penicillin per ml and ~tg of streptomycin per ml. mabs - - , - - , -l-l, - - , - - , and - - used in the present study recognize s protein of the virus as demonstrated by immunoblotting. these mabs have the ability to neutralize fipv strain - , and recognize two different antigenic sites of the viral s protein [ ] . the neutralization titer, indirect immunoftuorescent antibody titer, immunoglobulin isotype and epitope specificity of these mabs are shown in table . the culture fluid of each mab-producing hybridoma was used for the experiment. f(ab') of mab . , fab of mab iv. and f(ab') of mab g were purchased from medarex, lebanon, nh. mab . is a mouse igg antibody that reacts with human fc r i [ ] . mab iv. is a mouse igg b antibody that reacts with human fc r ii [ ] . mab g is a mouse igg antibody that reacts with human fc? r iii [ ] . a mixture of mabs that recognize peplomer protein (s), transmembrane protein (m) and nucleocapsid protein (n) of fipv strain - , respectively, was used as the primary antibody. the mabs were added to acetone-fixed fipv-infected cells and allowed to stand at °c for rain. after the specimens were washed with phosphate-buffered saline (pbs) three times, they were stained with rabbit anti-mouse igg, iga and igm serum conjugated with fluorescein isothiocyanate (miles laboratories, naperville, u.s.a). after being held at °c for min, they were washed with pbs, mounted in ~ glycerol buffer and observed under a fluorescence microscope. confluent fcwf- cell monolayers in -min plastic petri dishes were inoculated with virus dilutions of specimen to be assayed for amount of infectious virus in . ml amounts. after virus adsorption at °c for rain, the inoculated cultures were covered with ml of agar overlay medium which consisted of ~o bacto agar in minimal essential medium. the cultures were incubated in a co incubator at °c for days, and stained by incubating at °c for h under a second overlay medium containing . ~o neutral red. the infectious titer was expressed in plaque-forming units (pfu). ade assay of fipv infection of feline alveolar macrophages was performed by two methods. in the first method, equal amounts of mab and viral suspension were allowed to react at t. hohdatsu et al. °c for h before inoculation onto the macrophages. in the second method, mabs alone were allowed to react with the macrophages at °c for h before viral inoculation. in both methods, the virus-inoculated cells were examined for viral antigen by ifa using mixture of anti-fipv mabs after incubation for h, and the percent of ade was determined by the following formula: where a is the rate of ifa positive cells in the culture infected in the presence of mab and b is that in the culture infected in the absence of mab. in ade assay of fipv infection of human monocyte cell line u , virus suspension was allowed to react with mab at °c for h and then inoculated to a cell pellet of u cells. the inoculated cells were incubated at °c for h to allow virus adsorption, and then washed with hanks' balanced salt solution (hbss). after washing, the cells were cultured and h later, were examined for infecting fipv antigen by ifa. at the same time, the level of infectious virus in the culture supernatant was determined by plaque assay. undiluted mab ( - - , - - , - - , - - , - - or - - ) or a mixture of either one of the mab and fipv was added to x feline alveolar macrophages and held at °c for h. after three washings with hbss containing . % n,n , the cells were allowed to react with -fold diluted fitc-conjugated fab of goat anti-mouse igg antibody at °c for h. after being washed three times, the stained cells were analyzed by counting about , cells on a facs (becton dickinson co., u.s.a.). the relationship between ig subclass and a d e activity was investigated with the m a b s shown in table . as shown in fig. , despite the fact that they a d e activity of these m a b s was also determined by inoculating f i p v to macrophages pretreated with each m a b (fig. ) . m a b s - - , - - and - - , whose subclass is i g g a, enhanced the infection in the same way as in the case when mixture of f i p v and m a b was inoculated to macrophages. in contrast, none of the subclass igg mabs ( - - , - - and - - ) showed ade activity (fig. ) . these results, together with those shown in fig. , clearly indicate the relationship between ig subclass of mabs and the ade activity. binding of mouse anti-fipv mabs to the fc r on feline alveolar macrophages was determined by indirect immunofluorescence flow cytometry of the mabtreated macrophages. figure a shows the percentages of positively stained cells in the macrophages treated with different mab. percentage of the stained macrophages which had been treated with either one of the igg t mabs was very low and almost the same as that of control macrophages which had been treated with hbss instead of mab. in contrast, percentage of the stained macrophages which had been treated with either one of the igg a mabs was distinctly increased• figure b next, to determine ade by mouse anti-fipv mabs of f i p v infection in h u m a n u cells, f i p v alone or a mixture of f i p v and either one of the mabs was inoculated to u cells. the inoculated cells were cultured, and h later, viral proliferation was assessed by ifa of the cells with mixture of anti-fipv mabs and virus plaque assay of the culture supernatant. in table , when f i p v alone was inoculated to u cells, viral proliferation did not occur at all, whereas it occurred when the virus complexed with either one of the mabs was inoculated. notably, f i p v infection was established in u cells even with the igg mabs that did not show a d e of the infection in feline macrophages, even though the degree of the a d e was far less than that by the igg a mabs. in vitro studies using a n t i -f i p v mabs and in vivo studies of cats immunized with recombinant vaccinia virus expressing f i p v s protein have indicated that antibody against f i p v s protein induces a d e of f i p v infection [ , , , , , ] . some studies showed that mabs having virus neutralizing activity caused intense a d e of f i p v infection, and suggested that epitope of f i p v to be recognized for a d e is closely related to that for virus neutralization [ , , , , -i. in the present study, it was d e m o n s t r a t e d that even a m o n g mouse f i p v neutralizing mabs that have been shown to recognize the same antigenic site by competitive binding assay, igg a m a b s but not igg m a b s enhanced f i p v infection of feline alveolar m a c r o p h a g e s (fig. ) . these igg a mabs enhanced the infection even when macrophages pretreated with the mab were washed and then inoculated with the virus (fig. ) . therefore we assumed that mouse igg a antibodies can bind fc ? r on feline alveolar macrophages, whereas igg antibodies can not. this was confirmed by the flow cytometric analysis of binding of the mabs to feline macrophages (fig. ). in addition, it was found that the igg a mabs complexed with fipv bound to macrophages more intensely than the mab alone did (figs. a and b ). in accordance with these findings, ade of the fipv infection was intenser in the macrophages infected by the former pathway than in the virus-inoculated latter macrophages ( figs. and ) . flow cytometric analysis of the binding of protein a-treated igg a mab to macrophages and determination of ade of fipv infection by the treated mab revealed that the binding of the mouse igg a mabs to feline macrophages and their ade activity on fipv infection of the macrophages are mediated by fc region of the mabs (fig. ) . since fc y rs on feline cells have not been defined, we explored the fc-mediated ade of fipv infection by using human cells, of which fc y rs are well defined, as targets of the virus. it is known that there are three types of human fc y r, i.e., fc y r i, ii and iii [ ] . it has also been shown that fc r i and ii are present on human macrophages and that mouse igg a and igg bind well to the fc ? r i and ii, respectively. the human monocyte cell line u cells used in the present study were shown to express fc r i and ii to a similar degree on their surface from the reactivity to mab specific to the respective human fc y r (fig. ) . fipv strain - did not infect u cells at all. however, when the fipv complexed with either one of the mouse anti-fipv mabs was inoculated onto u cells, distinct viral proliferation was observed. notably, igg mabs, which did not bind to feline macrophages and did not cause ade of fipv infection of feline macrophages, caused viral proliferation in u cells. these results suggest that u cells of human origin have no virus receptor for fipv, but fc ? rs, i.e., fc y r i and ii, act as receptors for the mouse igg a and igg mabs complexed with the virus and thus allow the virus to enter and proliferate in the cells. some mouse anti-fipv igg mabs devoid of neutralizing activity were reported to exert ade of fipv infection of feline macrophages [ , ] , though the ade activity of these igg mabs was far less than that of mouse igg a mabs used in the present study. reason for the discrepancy between these studies and our study, in which mouse anti-fipv igg mabs were not found to show ade of fipv infection of feline macrophages, is not clear. there are the following possibilities: fc y r for mouse igg is expressed on feline macrophages in very small number, or fc y r for mouse igg on feline macrophages has very low affinity. in relation to the latter possibility, u cells of human origin express fc y r i and ii at a similar level, but ade activity of mouse anti-fipv igg mab on fipv infection of these cells was found to be far weaker than that of anti-fipv igg a mab. it is also known that human igg and igg have weak affinity for fc y r on human macrophages [ ] . one study of ade of dengue virus infection of mouse macrophages has shown that treatment of macrophages with neuraminidase increased affinity of their fc r ii for mouse igg mab and resulted in increased ade of the infection by mouse anti-dengue virus igg mab [ ] . this might be verified by a similar experiment with fipv system. taken all results of the present study together, it is indicated that ade of fipv infection is mediated by binding of fc region of anti-fipv antibody to fc receptor expressed on the target cells of fipv. this is supported by finding that any fipv-neutralizing mabs could not enhance fipv infection of crandell feline kidney cells which are devoid of fc r. the present study, however, explored ade of fipv infection by in vitro experiments in which xenogeneic combination of virus targets and anti-fipv antibody, i.e., feline alveolar macrophages or human monocyte u cells and mouse anti-fipv s protein mabs, was used. therefore, the findings in the present study could not be applied directly to ade of natural infection in cats. schultz et al. [ ] reported two subclasses of feline igg. however, biological properties of feline igg subclasses have not yet been clarified. further study of the feline igg subclasses and exploration of fc r on feline cells are mandatory, and what igg subclass of anti-fipv antibody in the serum of naturally infected cats enhances the fipv infection should be investigated. these studies are worthwhile for determination of basic pathogenesis of fip and development of vaccines for prevention of fipv infection. monoclonal antibodies to fc receptors for igg on human mononudear phagocytes: antibody characterization and induction of superoxide production in a monocyte cell line experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus interaction of west nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement monoclonal antibodies to sindbis virus glycoprotein e can neutralize, enhance infectivity, and independently inhibit haemagglutination or haemolysis monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus human neutrophilic fcq, receptor distribution and structure pathogenesis of dengue: challenges to molecular biology antibody-enhanced dengue virus infection in primate teukocytes dengue viruses and mononuclear phagocytes. i. infection enhancement by nonneutralizing antibody heterogeneity of infection enhancement of dengue strains by monoclonal antibodies characterization of monoelonal antibodies against feline infectious peritonitis virus type ii and antigenic relationship between feline, porcine and canine coronaviruses a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoctonal antibodies enhancement and neutralization of feline infectious peritonitis virus infection in feline macrophages by neutralizing monoclonal antibodies recognizing different epitopes studies on the mechanism of antibody-mediated enhancement of getah virus infectivity human monocytes and u cells bear two distinct fc receptors for igg neuraminidase augments fc y receptor ii-mediated antibody-dependent enhancement of dengue virus infection dengue virus monoctonal antibodies identify epitopes that mediate immune infection enhancement of dengue viruses antibody-mediated growth of influenza a nws virus in macrophagelike cell line p d identification of antigenic sites mediating antibody-dependent enhancement of feline infectious peritonitis virus infectivity monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibodydependent enhancement of infection of feline macrophages attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus immunologic phenomena in the effusive form of feline infectious peritonitis pathogenicity studies of feline coronavirus isolates - and - an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis virologic and immunologic aspects of feline infectious peritonitis virus infection antibody-mediated enhancement of flavivirus replication in macrophage-like cell lines growth of d yellow fever virus in a macrophage-like cell line, u : role of fc and viral receptors in antibody-mediated infection feline immunoglobulins attempted immunisation of cats against feline infectious peritonitis using canine coronavirus the sites of early viral replication in feline infectious peritonitis antibody-enhanced infection by hiv- via fc receptor-mediated entry function and heterogeneity of human fc receptors for immunoglobulin g early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses this work was supported by private contributions of sankyo co., ltd., ajinomoto general foods, inc. and dainippon pharmaceutical co., ltd., japan. authors' address: dr. t. hohdatsu, department of veterinary infectious diseases, school of veterinary medicine and animal sciences, kitasato university, towada, aomori , japan.received may , key: cord- -ckm ol authors: zhong, qiong; yang, zhanqiu; liu, yuanyuan; deng, haiying; xiao, hong; shi, liqiao; he, jing title: antiviral activity of arbidol against coxsackie virus b in vitro and in vivo date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ckm ol we investigated the antiviral activity of arbidol, an antiviral chemical compound, against coxsackie virus b (cvb( )) in vitro and in vivo. arbidol not only prevented the cytopathic effect (cpe) of cvb( ), as demonstrated in an mtt colorimetric assay, when added during or after viral infection, with a % inhibitory concentration (ic( )) from . to . μg/ml, but it also decreased the cvb( )-rna level in infected host cells, as shown in semi-quantitative rt-pcr. balb/c mice were used as an animal model to test the arbidol activity in vivo. orally administered arbidol at mg/kg body weight/day (once a day) significantly reduced mean virus yields in the lungs and heart as well as mortality after infection for days. our results demonstrate that in vitro and in vivo infection with cvb( ) can be effectively treated by arbidol. coxsackieviruses are members of the genus enterovirus, family picornaviridae, and are divided into two groups on the basis of pathogenicity in mice: serotypes of coxsackie a viruses (cav) and serotypes of coxsackie b viruses (cvb). cvb had been implicated in childhood myocarditis by the end of the s. since then, six cvb serotypes (cvb - ) have been shown to cause not only myocarditis but also a wide variety of human diseases, including common colds, cardiomyopathy, diabetes, neurological disorders, and inflammation [ ] [ ] [ ] [ ] . numerous outbreaks of cvb infections have been noted at the community level, but only coxsackie virus b (cvb ) is known to have caused widespread nationwide epidemics in the united states, particularly in , , and [ ] . moreover, cvb is associated with several human diseases, including encephalitis and myocarditis in immunocompromised children and cns disease in older adults. since direct viral cytotoxicity is one possible cause of damage, targeting the causative agent should limit the extent of damage, especially with early treatment. until now, there has been no enterovirus-specific vaccine or therapeutic reagent available for clinical use [ ] , although some reports have presented effective candidates for cvb in a murine model [ , , ] . a great number of picornavirus replication inhibitors have been described in vitro, but few of them have shown effectiveness in vivo [ ] , and none has been approved for clinical use. gancyclovir and cidofovir have been studied in myocarditis caused by cytomegalovirus in mice [ ] , but there is no evidence that it also works for enterovirus-induced myocarditis. acyclovir has been successfully used to treat myocarditis associated with epstein-barr virus [ , ] and varicella [ ] , but there are no reports demonstrating that acyclovir is effective in cvb myocarditis. arbidol is an antiviral molecule first developed in russia. its chemical name is ethyl- -bromo- -[(dimethylamino)-methyl]- -hydroxy- -methyl- -[(phenylthio) methyl] -indole- -carboxylate hydrochloride monohydrate. arbidol was first found to have wide-spectrum antiviral activity against human influenza virus. it was shown to inhibit the reproduction of rimantadine-resistant human influenza virus a, avian viruses h n and h n , and influenza viruses b and c [ ] . in many studies, arbidol also exhibited broad and potent antiviral activity against a number of viruses including respiratory syncytial virus [ ] , adenovirus [ ] , parainfluenza virus type , rhinovirus type , avian coronavirus, infectious bronchitis virus and marek disease virus [ ] , hepatitis b virus [ ] , and hepatitis c virus [ ] . in our previous study, arbidol showed broad-spectrum antiviral activity against a number of respiratory viruses, including flu-a, rsv, hrv , and cvb in vitro [ ] . the extensive list of arbidolsensitive viruses includes rna and dna viruses, enveloped and non-enveloped viruses, and ph-dependent and -independent viruses, emphasizing a broad spectrum of arbidol antiviral activity. at the same time, the wide spectrum of arbidol's activity suggests that arbidol targets common critical step(s) in virus-cell interaction. in this study, we systematically investigated the antiviral activity of arbidol against cvb in vitro and in vivo. arbidol was synthesized at qianjiang pharmaceutical co. ltd, hubei, china. the compound was initially dissolved in dimethyl sulfoxide (dmso) and further diluted with complete test medium. the final maximum dmso concentration was . %, which showed no effect on cell cultures. therefore, . % dmso was also added to all no-compound control samples. the efficacy of these preparations did not appear to change upon freezing and short-term storage ( month at °c). for the in vivo experiment, the compound was dissolved in . % methylcellulose solution. hep- (human laryngeal carcinoma) cells were maintained in our laboratory and were routinely grown in eagle's minimal essential medium plus nonessential amino acids (eagle's mem; sigma) supplemented with % fetal bovine serum, . % l-glutamine, u/ml penicillin, and . mg/ml streptomycin. the test medium used for the cytotoxic assay as well as for antiviral assays contained % of the appropriate serum. cvb was maintained in our laboratory and propagated in hep- cells. the virus was stored in small aliquots at - °c until use. virus titration was performed by the limited dilution method, using a -well plate with six wells per dilution. the virus titer was estimated from cytopathogenicity of cells induced by viral infection and expressed as % tissue culture infectious doses/ml (tcid /ml) [ ] . the cytotoxicity and antiviral activity of the compound were determined by quantitative colorimetric mtt [( -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide)] assay [ , ] . briefly, hep- cells were seeded at cells per well in -well plates and grown to subconfluence. after removal of the growth medium, serial twofold dilutions of the compound in ll test medium were added. at each dilution, four wells were infected with tcid / . ml of virus, while four wells were left uninfected for toxicity determination. the same dose of arbidol was added to the plate daily, since its halflife in cultured cells is about h [ ] . the plates were incubated at °c, and the development of cytopathic effect (cpe) was monitored daily by light microscopy until the virus-infected, untreated cells showed cpe up to %. at this time point, the culture medium was removed and ll of the mtt solution ( mg/ml in phosphatebuffered saline, pbs) was added to each well. the plate was further incubated for h to allow mtt formazan formation. after removal of the supernatant, ll of dmso was added for solubilization of formazan crystals, which were homogenized on a microplate shaker for min. the optical densities (ods) were then read using a microplate spectrophotometer at double wavelengths of and nm. results were expressed as a percentage of od value of treated cell cultures with respect to untreated ones. all data were analyzed with spss . , and the % cytotoxic (cc ) and % inhibitory (ic ) concentrations of the agent were determined. thus, the therapeutic index (ti) for the compound was also determined from cc /ic . each dilution was tested in triplicate, and in each set of experiments, three control wells without drug were included. serial twofold dilutions of the compound were dissolved in mem and incubated with hep- cells in -well microtiter plates for h at °c in a % co atmosphere. after removal of the compound, the cells were washed twice with pbs and challenged with tcid / . ml of cvb . after a -h incubation, the cells were washed twice with pbs and further incubated with test medium until typical cpe was visible. the inhibition of the virusinduced cpe was scored by light microscopy and measured by the mtt assay. four untreated virus controls and four uninfected, untreated cell controls were included in all assays. all data presented are results of experiments performed in triplicate. viral suspensions were cocultured with serial twofold dilutions of the compound at °c for h and then added to confluent cells. after a -h incubation, the solutions containing both compound and viruses were removed; the cells were rinsed carefully with pbs and further incubated with test medium. the assay was performed following the protocol described above. the experiment was carried out as above with the following difference: confluent cell monolayers were infected with tcid / . ml viruses for h. the cell sheets were washed with pbs and overlaid with different doses of the compound in ll test medium. semi-quantitative detection of viral rna by rt-pcr rna was extracted from both supernatants and cells after compound treatment and virus infection using trizol reagent (invitrogen). we also purified the rna of the uninfected cells, the infected cells without treatment and the test medium as negative, positive and blank controls. rna was dissolved in rnase-free water, and the concentration was measured using a spectrophotometer (bio-rad). all samples were adjusted to lg/ll according to the method of yamamoto et al. [ ] . reverse transcription was carried out at °c for min, then at °c for min. random primers were used for the preparation of complementary dna templates for pcr. the forward and reverse primers used for rt-pcr were -ccccggactgagtatca ata- ( - ) and -gcagttaggattagccgc at- ( - ), respectively. b-actin was used as internal control, and the sequences were -ggcgggaccaccatgtaccct- and -agg ggccggactcgtcatact- . twenty-eight cycles of pcr were carried out in -ll reaction mixtures containing ll synthesized cdna, ll x pcr buffer, ll m dntps, ll of primer , ll of primer , ll b-actin p , ll b-actin p , ll of taq dna polymerase, and ll of mineral oil, which was placed on top of the pcr mixture to prevent evaporation. a programmed thermal cycler was set as follows: °c for s, °c for s, and °c for s, °c for s, and °c for min. then, ll of each pcr product was loaded onto an agarose gel containing ethidium bromide and separated by electrophoresis. the gel was scanned using the sx- photoanalysis system and analyzed using four stars bioimage software, which calculates the ratio of peak value adsorption of cvb to that of b-actin. specific-pathogen-free male balb/c mice, - weeks old ( - g), obtained from animal center of wuhan university, were used in all experiments. sixty-four balb/c mice ( mice/group, four groups) were used in this experiment. forty-eight mice were infected intraperitoneally with cvb ( tcid / . ml), and the remaining were used as normal control and injected intraperitoneally with the same volume of pbs. after infection for hours, mice were treated by oral gavage (p.o.) with . ml arbidol, corresponding to approximately and mg/kg body weight/day, and treatment was continued daily for days unless otherwise indicated. the virus control group and the normal (pbs) control group received . % methylcellulose solution instead of the compound. eight mice from each group were sacrificed by cervical dislocation on day after viral exposure. the body weights of the mice were recorded daily until the animals were killed. the lungs and hearts were harvested and weighed. the organs from four mice of each group were subsequently homogenized to % (weight/volume) suspensions in test medium. the homogenates were frozen and thawed twice to release the virus and centrifuged at , rpm for min. virus titration was determined by plaque assay, and the organs from another four mice were used for pathological examination (he staining).the lung index was expressed as the ratio of mean lung weights to mean body weights [ ] . the remaining eight animals of each group were observed daily for changes in body weight and for any deaths. the titers of infectious virus particles were determined by the standard plaque formation assay. replicate aliquots ( ll/well) of serial -fold dilutions were inoculated onto hep- cell monolayers in -well plates and incubated at °c in % co . two days later, the cpe was read; results were expressed as pfus. arbidol showed significant inhibitory activity against cvb when added after infection, with an ic of . lg/ml, ti of . , and mild inhibitory activity against cvb when added before infection, with an ic of . lg/ml, which is close to the cc of the compound ( . lg/ml), and ti of . (see table ). to investigate the direct inactivating effect of arbidol, virus was treated for h with concentrations of arbidol ranging from . to lg/ml. as shown in table , arbidol was virucidal for cvb , with an ic of . , and it inhibited the cpe of cvb on hep- cells, with a ti of . . rna was extracted from the supernatants and cells in each well of the plates that were treated with the compound after virus infection, using trizol (invitrogen) according to the manufacturer's protocol. specific amplified products of cvb ( bp) and b-actin ( bp) by the two pairs of primers were identified as described in ''materials and methods''. figure a is the gel picture of the pcr products. the gel was scanned using the sx- photoanalysis system and analyzed using four stars bioimage software. the ratio of peak value adsorption of cvb to that of b-actin was calculated by the software (shown in fig. b) . there was a quantitative relationship between the ratio and dose of arbidol. the ratio decreased with increasing dose of arbidol, suggesting that the synthesis of cvb rna decreased with the increasing dose of compound when treatment was done after infection. in the viral control group, animals showed a tendency to huddle; diminished vitality, ruffled fur, and weight loss (fig. a) were observed on day after infection. on day , this group of animals began to die, and by day, all of them had died (fig. b) . in the compound-treated group, the animals receiving mg/kg body weight/day began to show behavior changes and lose weight by day (fig. a) and began to die on day after challenge. by day , half of them had survived (fig. b) . the animals receiving mg/kg body weight/day and the normal control animals did not show abnormalities during the -day period, except that the former group had a slight weight loss (fig. a) . the i.p. infection with cvb led to an increase in mean lung weight and a decrease in mean body weight on day after virus exposure. lung indexes of the -and mg/kg body weight/day groups were significantly lower than that of the viral control group, with the -mg group almost the same as the normal group. there was no significant difference between the -mg and normal group in lung index (fig. c) , and there was no significant difference among the groups of animals in mean heart weight (data not shown). four animals of each group were sacrificed on day after viral exposure, and tissue samples were obtained from the lungs, kidneys, liver, heart, and spleen for pathological examination. as shown microscopically, the lungs of the viral control animals had thickened alveoli walls due to edema of alveolar epithelia, proliferation of interstitial cells, interstitial lymphocytes and macrophage inflammatory infiltration, and hemorrhage (fig. a) . the hearts of the viral control animals had denaturation of cardiac muscle cells (fig. c) . tissue samples from the remaining animals did not have changes, except that the lungs of animals receiving mg/kg body weight/day showed local hemorrhage (data not shown). four animals of each group were sacrificed on day after viral exposure, and tissue samples were obtained from the lungs and hearts for virological examination. the virus titers of lung and heart were significantly lower in mice receiving oral administration of arbidol daily for days than in the viral control group. in the groups treated with arbidol at and mg/kg body weight/day, the mean virus yields of lungs were around and viral plaques/ lung, respectively, whereas the yields in virus control mice were viral plaques/lung (fig. a) . the mean virus yields of hearts were reduced to and viral plaques/heart, respectively, whereas the yields in viruscontrol mice were viral plaques/heart (fig. b) . arbidol is a russian-made broad-spectrum antiviral compound that has been shown to inhibit the fusion of influenza a and b viruses within endosomes [ ] . influenza-virusinduced fusion needs an acidic environment (ph . ). a slight increase in ph can abolish the fusion process [ ] . since arbidol is a weak base, it may elevate endosomal ph and abrogate virus-endosome fusion. arbidol has also been shown to exert antiviral activity against other ph-dependent viruses, such as hepatitis b virus [ ] and rhinovirus [ ] . moreover, arbidol has demonstrated low toxicity upon single-dose oral administration in many animal models. the animal ld ( % lethal dose) was mg/kg for mice, , mg/kg for rats, and , mg/kg for guinea pigs [ ] . a long-term arbidol intake study also did not show pathologic changes in animals, as confirmed by clinical, hematological, biochemical, and pathological data. at therapeutic doses, arbidol possessed no mutagenic or teratogenic activity [ ] . in this study, we tested the antiviral activity of arbidol against cvb , both in vitro and in vivo. like other enteroviruses, cvb is a small rna virus that replicates at °c, lacks a lipid envelope, and is stable at acid ph. when primarily cultured hep- cells infected with cvb were treated with arbidol by different methods, arbidol was found to present potent antiviral activity against cvb when added before and after infection. in a virucidal assay, arbidol exhibited a strong effect against cvb , and inhibited the cpe of cvb on hep- cells with a ti of . , which is higher than other treatments. those data suggested that arbidol had a strong antiviral effect against cvb during the adsorption step. we also cocultured hep- cells with different doses of arbidol after infection to study whether it still had an antiviral effect after virus entry into the host cell. the mtt assay showed that arbidol had inhibitory activity against cvb , with an ic of . lg/ml and a ti of . (see table ). semiquantitative rt-pcr data suggested that arbidol prevented the synthesis of cvb rna in a dose-dependent manner. in short, our data demonstrate that arbidol can induce durable antiviral activity in host cells, not only blocking the adsorption of viral particles onto cells but also inhibiting the late stage of the viral replication cycle. to study further whether arbidol has antiviral ability against cvb in vivo, we established a mouse model of infection. mice infected with cvb had some symptoms of circulatory failure such as cyanosis and lack of blood perfusion in the tail and auricle, and there was evidence of virus replication in the heart and lungs. pathological findings were consistent with the results from virus isolation, and all viral control mice died by day after infection. thus, cvb infection in balb/c mice is a suitable in vivo model to study in vivo antiviral activity. orally administered arbidol at mg/kg body weight/ day (once a day) after infection with coxsackievirus b for days significantly enhanced survival rate, reduced virus yields and released pathological abnormalities in lungs and hearts. in summary, our results suggest that arbidol can elicit protective antiviral activity against cvb in vitro and in vivo. the exact antiviral mechanism of arbidol is still arbidol prevents the increase of both lung index and viral titer in cbv -infected mice. four mice from each group were sacrificed on day post-infection, and body weight and lung weight were used to calculate lung index (a). viral titer of lungs (b) and hearts (c) were determined by plaque assay. asterisk means p \ . , ns no statistical difference unknown. arbidol may play a significant role in medical countermeasures against cvb infections. high yield production of an inactivated coxsackie b adjuvant vaccine with protective effect against experimental myocarditis evidence for enteroviral persistence in humans serological and molecular evidence of enterovirus infection in patients with end-stage dilated cardiomyopathy coxsackieviruses and diabetes reemergence of an epidemic coxsackievirus b genotype treatment of picornavirus infections protection of mice against lethal coxsackievirus b infection by using dna immunization dna vaccine-mediated immune responses in coxsackie virus b -infected mice picornavirus inhibitors ganciclovir and cidofovir treatment of cytomegalovirus-induced myocarditis in mice successful treatment of epstein-barr virus infection associated with myocarditis complete heart block in a child with varicella anti-rhinovirus activity of -methylthio- -aryl- -isothiazolecarbonitrile derivatives sensitivity of various influenza virus strains to arbidol. influence of arbidol combination with different antiviral drugs on reproduction of influenza virus a study of the effect of antiviral drugs on the reproduction of the respiratory syncytial virus by enzyme immunoassay arbidol: a broad-spectrum antiviral compound that blocks viral fusion antiviral chemotherapeutic agents against respiratory viruses: where are we now and what's in the pipeline? synthesis and in vitro anti-hepatitis b virus activities of some ethyl -bromo- -hydroxy- h-indole- -carboxylates arbidol: a broadspectrum antiviral that inhibits acute and chronic hcv infection antiviral activity of arbidol against influenza a virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo a left superior vena cava draining the blood from a closed coronary sinus antiviral activity and mode of action of caffeoylquinic acids from schefflera heptaphylla (l.) frodin arbidol kinetics and its effect on influenza a virus replication in mdck cell culture production of cornoside in abeliophyllum distichum cell suspension cultures effect of l-amantanamine hydrochloride (amantadine hcl) and methyl-l-adamantanethylamine hydrochloride (rimantadine hcl) on transmission of influenza virus infection in mice membrane fusion activity of influenza virus arbidol: a new antiviral, immunomodulator and interferon-inducer acknowledgments we thank drs. wei wang and linming zhao for primers and reagents, dr. po ye for pathological analysis, and dr. gang zheng for critical reading of the manuscript. key: cord- -g dqiki authors: costantini, v.; lewis, p.; alsop, j.; templeton, c.; saif, l. j. title: respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: g dqiki porcine respiratory coronavirus (prcv), a spike (s) gene deletion mutant of transmissible gastroenteritis virus (tgev), causes mild or subclinical respiratory infections in pigs. the shedding of prcv/tgev was studied at different days post-arrival in fecal and nasal swabs from prcv/tgev seronegative sentinel pigs introduced into a prcv seropositive herd with questionable tgev serology and diarrhea. nasal shedding of prcv was detected in % and % of samples by nested-rt-pcr and cell culture immunofluorescence (ccif), respectively. however fecal shedding of prcv was detected in % of the samples by nested-rt-pcr and % by ccif. four respiratory and fecal prcv strains were isolated in swine testicle cells including nasal/fecal prcv pairs (isolated at the same time) from pigs. comparison of nasal/fecal prcv pairs from individual pigs revealed different deletions in the spike (s) gene ( or nt) in pairs and a consistent change in nt / (aa t to v) for all pairs. in preliminary studies, inoculation of gnotobiotic pigs with each plaque-purified pair of the nasal and fecal prcv isolates, revealed no clinical disease but different tropisms. the nasal isolate was shed both nasally and in feces, but the fecal isolate was shed only marginally in feces, and not nasally. our results show that nested-rt-pcr was as sensitive as ccif for prcv detection in nasal swabs, but was more sensitive than ccif for prcv detection in fecal samples; alternatively prcv shed in feces was more labile with loss of infectivity. the s-gene sequence differences found between the fecal and respiratory prcv isolates may influence their tissue tropism. these new prcv isolates should be useful to understand the molecular basis of coronavirus tropism and evolution in infected swine. porcine respiratory coronavirus (prcv) is a deletion mutant of transmissible gastroenteritis coronavirus (tgev) with altered respiratory tissue tropism [ , ] . transmissible gastroenteritis virus causes fatal diarrhea in neonatal piglets. it selectively infects and replicates in the villous enterocytes of the small intestine, causing subsequent malabsortion and dehydration characteristic of transmissible gastroenteritis (tge) [ ] . transmissible gastroenteritis virus has also been shown to replicate in the upper respiratory tract tissue of infected swine [ , ] . porcine respiratory coronavirus is genetically and antigenically related to tgev, but it has a selective tropism for respiratory tissue causing mild or subclinical respiratory infections with limited to no replication in the intestinal tissue of infected swine [ , , , ] . during routine serological surveillance of pig herds in great britain, belgium, holland and france in the s, an increase in the number of herds with antibodies to tgev was noted but without concomitant increases in clinical enteric disease. a coronavirus, prcv, was isolated in from respiratory tissues of affected pigs in belgium [ ] , great britain [ ] and later in other parts of europe. several years later another strain, prcv-ind was isolated from pigs in the u.s. [ ] . both tgev and prcv contain a single-stranded positive-sense rna genome of about kb and produce - subgenomic mrnas during viral replication. the major structural proteins, the spike (s), the integral membrane (m) glycoprotein and the nucleocapsid (n) protein are translated from mrnas , and , respectively. the mrnas , - or a, b encode two putative nonstructural proteins [ , ] . comparison of tgev and prcv strains revealed that prcv has a large deletion in the region of the s gene, and minor deletions in genes / a and - / b [ , ] . most european prcvs have an identical deletion of nt in the same position at the end region, suggesting that they were derived from the same precursor [ ] . in contrast u.s. prcv strains, have deletions of various sizes ( - nt) located in different positions, suggesting that they originated independently [ ] . because this deletion is present in all independently derived prcvs, it has been proposed that the size and position of the deletion is related to the differences observed in tissue tropism between prcv and tgev [ , ] . investigators have suggested that amino acid changes at the n-terminal region of the tgev s protein also affect the enteric tropism of the pur strain of tgev [ ] . the s protein has a glycosylated membrane anchoring domain and is thought to be the viral attachment protein that interacts with the cell receptor, porcine aminopeptidase n (apn) [ , ] . however a second region in the s protein (around amino acid ) also influences the enteric tropism of tgev [ ] . the s protein of tgev has four major antigenic sites, with site a being the major inducer of neutralizing antibodies and conserved in both tgev and prcv strains [ , ] . the s protein of prcv is smaller due to the deletion with loss of one or two antigenic sites (c and b or d depending on the nomenclature) in the deletion region [ , ] . because most virus neutralization (vn) antibodies are directed to site a, conventional antibody assays fail to differentiate between pigs infected with prcv or tgev. blocking elisa tests using monoclonal antibodies to antigenic sites in the prcv deletion region of the s protein (one to conserved site a and a second to deleted site d) are used to serologically differentiate between prcv and tgev-infected pigs [ , , ] . because site a is conserved on tgev and prcv, only sera from pigs infected with either virus will contain antibodies to this site and compete with site a mabs for binding to the viral protein in blocking elisa. in contrast, because site d is only present on tgev, but absent on prcv, sera from pigs infected with tgev compete with site d mabs [ , ] . however, sometimes these tests result in false tgev positives or borderline reactions that are difficult to interpret making it problematic to accurately define the tgev status or diagnose tgev in prcv infected swine herds [ ] . we investigated the shedding of prcv/tgev in prcv/tgev seronegative sentinel pigs introduced into a prcv seropositive herd with questionable tgev serology and diarrhea of uncertain etiology in weaning pigs. our objectives were to isolate and characterizate tgev or prcv strains from this field outbreak and to determine their genetic relationships to one another and to reference strains. because previous studies have suggested that the s gene deletion area may influence viral tissue tropism, we focused on analysis of this region [ , ] . these new prcv isolates derived from both nasal swabs and feces should serve as tools to gain a better understanding of the molecular basis and evolution of the pathogenesis of coronaviruses. we attempted to isolate and characterize tgev and prcv strains from a prcv seropositive herd in canada with questionable tgev serology (inconclusive results in blocking elisa, svanovir, uppsala, sweden) and diarrhea of uncertain etiology. thirty-one prcv/tgev seronegative weaned sentinel pigs were introduced into the herd. the herd was a sow farrow-to-finish unit. the average inventory was nursing piglets, nursery (weaned) pigs and grow-finish pigs. the sentinel pigs were all placed in one room, and then dispersed among pens with or sentinel pigs in each pen, in addition to - recently weaned resident pigs. the average weaning age in the herd was days and the sentinel pigs were - -weeks-old when introduced. we investigated the shedding of prcv/tgev in sentinel pigs introduced into the herd. although resident pigs consistently tested positive for serum antibodies to prcv in a commercial blocking elisa test, occasionally some pigs also tested tgev seropositive in this test, suggesting the presence of false positives or tgev cases in the weaning pigs with diarrhea. in an attempt to isolate and characterize tgev and prcv strains from this herd, fecal and nasal swabs were collected from of the sentinel pigs at , , , and days post-arrival (dpa). a total of nasal swabs and fecal samples were collected, with nasal swabs and fecal sample pairs collected concurrently and tested by nested-rt-pcr and cell culture immunofluorescence (ccif) to detect prcv or tgev. each swab was identified v. costantini et al. as follows: pig number-dpa-origin (fecal "f" or nasal "n" sample). for example - f: pig , dpa, fecal sample (fig. ) . four respiratory (nasal) and enteric (fecal) prcv strains, but no tgev strains, were isolated. the designation of prcv was based on the presence of the typical s-gene deletion in all the isolates (described in a subsequent section). the swine testicle cells were used for virus isolation, propagation and cell culture immunofluorescence tests (ccif) as previously described [ ] . each strain was isolated and plaque-purified once or twice in swine testicle (st) cells. fecal and nasal swabs from the field cases (sentinel pigs), cell culture isolates and fecal and nasal swabs from gnotobiotic pigs were tested by nested-rt-pcr to detect and to differentiate tgev and prcv viral rna as previously described by kim et al. [ ] . the rt-pcr primers f ( -gggtaagttgctcattagaaataatgg- ) and r ( -cttcttcaa agctagggactg- ), and the nested-pcr primers f ( -ttgtggtyttggtygtaa tkcc- ) and r ( -ggctgtttggtaactaatttrcca- ) associated with the open reading frame b and the s-gene deletion area for u.s. and european strains of prcv were used [ ] . the rna from the fecal or nasal swabs or cell culture isolates were extracted using a commercial rna extraction kit (trizol ls reagent, life technology, ny, u.s.a.) according to the manufacturer's recommendation. briefly, µl of the nasal or fecal swab fluids (diluted in mem-e) were mixed with µl of trizol and were incubated for min at room temperature. following incubation, µl of chloroform were added. the samples were incubated for min at room temperature and centrifuged at . g for min at • c. the rna was precipitated with isopropanol. purified rna was resuspended in µl of depcwater. the reference strains, isu- (prcv) and m milller (tgev) were used as positive controls and negative controls included mem-e or prcv/tgev negative fecal or nasal swabs from unexposed gnotobiotic pigs. the conditions for the nested-pcr were as follows: cycles of denaturation at • c for min, annealing at • c for . min and extension at • c for . min, followed by a final cycle of extension at • c for min. pcr products were analyzed on . % agarose gels stained with ethidium bromide [ , , ] . the predicted size of the amplified product was bp for tgev and - bp for prcv [ ] . fecal and nasal swab supernatant fluids from the sentinel field cases or the cell culture passaged prcv isolates and nasal and fecal swabs from gnotobiotic pigs were diluted in minimum essential medium (mem-e) and tested by ccif to detect infectious virus using previously described procedures [ ] . briefly, or -fold serial dilutions of the nasal and fecal swab supernatants or prcv cell culture isolate, respectively were inoculated onto st cell monolayers in -well plates and incubated for hours. the cells were fixed with % acetone, stained with hyperimmune porcine anti-tgev serum conjugated to fluorescein isothiocyanate, and analyzed by fluorescent microscopy [ ] . four respiratory ( - n, - n, - n, - n) and fecal ( - f, - f, - f, - f, - f) prcv strains (including the pairs of nasal/fecal samples from three pigs one day after diarrhea, designated - n/ - f, - n/ - f, - n/ - f) were isolated from the nasal and fecal swab fluids, respectively of the sentinel pigs in contact with the resident pigs. each isolate was first passaged once or twice in st cells and then plaque purified in st cells. sequence analysis of the partial s-gene of - of times passaged in cell culture (number of times plaque-purified)] and reference strain isu- was performed with primers f and r . the rt-pcr products were purified using a geneclean spin kit (bio , ca) and sequenced by dideoxynucleotide chain termination procedures using an automated sequencer [abi , perkin elmer, ca]. sequence data were aligned using the dnastar software program (dnastar, madison, wi) and compared with the published sequence using the clustal methods. rectal and nasal swab fluids collected from gnotobiotic pigs from to dpi were tested by das-elisa to detect virus antigen as described previously [ ] . briefly, elisa plates were coated with the monoclonal antibody (mab) c and c to the tgev s protein, and mab h to the n protein (positive coating) or with negative ascites sp / (negative coating). all samples were tested in duplicate wells, one with positive and one with negative coating. viral antigen captured on the plate was detected with the purified, biotinylated igg fraction of a tgev hyperimmune antiserum, followed by streptavidin-peroxidase and - azino-bis ( -ethylbenzthiazoline- -sulfonic acid) as substrate. two hysterectomy-derived, colostrum-deprived -day-old gnotobiotic pigs were oronasally inoculated with the cell culture adapted, plaque purified - n * ( ) prcv isolate [ × plaque forming units (pfu)/pig]. as a control a third gnotobiotic pig was oronasally inoculated with mem-e. clinical parameters including diarrhea and fecal scores ( = normal, = pasty, = semiliquid, = liquid) were recorded. fecal and nasal shedding of viral rna or virus were assayed by nested-rt-pcr, ccif and das-elisa [ , ] from days post inoculation (dpi) to . one infected and one control pig were euthanized at dpi and sections of duodenum, jejunum, ileum and lung were collected for immunofluorescence assay (ifa) and histopathology. for ifa impression smears were made on glass microscope slides, air dried, fixed in acetone and stained with fitc-conjugated anti-tgev serum. for histopathology studies, tissues were processed in prefer fixative solution, embedded in paraffin and stained with hematoxylin and eosin as previously described [ , ] . in a second experimental group, two hysterectomy-derived, colostrum-deprived -dayold gnotobiotic pigs were oronasally inoculated with either the cell culture adapted, plaquepurified - f * ( ) prcv isolate [ × plaque forming units (pfu)/pig] or with ml of a : dilution of the pooled rectal swab fluids recovered (dpi - ) from one of the initial gnotobiotic pigs inoculated with - n * ( ) prcv. in addition, a third gnotobiotic pig was oronasally inoculated with mem-e as a control. pigs were euthanized at dpi and sections of duodenum, jejunum, ileum and lung were collected for ifa and histopathology. the sentinel pigs remained prcv/tgev seronegative at and dpa, but developed diarrhea at dpa and / ( %) seroconverted to prcv but not tgev detected by blocking elisa at dpa. at dpa, one sentinel pig died but neither prcv nor tgev were detected in the tissues. nasal shedding of prcv was detected in % ( fig. a and b . the peak of prcv nasal (fig. a) shedding was detected at dpa ( day after diarrhea outbreak) by nested-rt-pcr ( %) and ccif ( %). both techniques showed similar sensitivity to detect prcv in nasal swabs, with the percentage of positive samples increasing until dpa, and decreasing at dpa ( days after diarrhea outbreak). the peak of prcv fecal shedding (fig. b) was also detected at dpa ( day after diarrhea outbreak) by nested-rt-pcr ( %) but at dpa by ccif ( %). nested-rt-pcr was more sensitive than ccif for detecting prcv in the fecal swabs. four respiratory and fecal prcv field strains were adapted to growth in st cells and plaque purified. the partial s-gene of these cell culture adapted, plaque-purified strains was sequenced (fig. ) . the strains were assigned to groups according to the size and position of the s-gene deletion (fig. ) . the group isolates [ - f * ( ) and - f * ( )] had a nt deletion in the sgene starting from nt to nt . the group isolates [ - f * ( ), - n * ( ), - f * ( ) and - f * ( )] had a nt deletion starting from nt to nt and the group isolates [ - n * ( ), - n * ( ) and - n * ( )] had a nt deletion starting from nt to nt . sequence analysis revealed that the selected region of the s-gene of the prcv field isolates had higher homology to u.s. prcv strains than to european prcv strains and the size and position of the deletion was similar to u.s. strains. analysis of the sequence of cell culture adapted, plaque-purified - f * ( ), - n * ( ), - f * ( ), - n * ( ), and - f * ( ), - n * ( ) (each fecal/nasal pair collected concurrently as nasal and fecal swabs of different pigs) showed nt differences (nt , , and region - ) between the fecal and respiratory strains isolated from the same pig on the same day. however only changes in nt , and region - resulted in amino acid changes (fig. , table ). when the nucleotide and amino acid sequences of these paired samples were compared with tgev and prcv reference strains, a fifth nt substitution (nt ) between the field and the reference strains was identified. this change resulted in another v. costantini et al. table . amino acid change, in this case between the reference and the field strains (fig. , table ). two gnotobiotic pigs were oronasally inoculated with the cell culture adapted, plaque purified - n prcv nasal isolate ( × pfu/pig) and an additional pig was mock-inoculated as a control (table ) . no clinical signs including diarrhea were evident in any of the pigs after inoculation. nasal shedding of prcv was detected until dpi by rt-pcr, ccif and elisa in both exposed pigs and until dpi in exposed pig - by nested-rt-pcr. using nested-rt-pcr, fecal shedding was detected from dpi until dpi, and by elisa from dpi until dpi in both exposed pigs, but ccif failed to detect virus shedding from rectal swab samples in either exposed pig. the control pig was negative at all times by all tests ( table ). the results of the ifa on the duodenum, jejunum, ileum, lung and nasal turbinate impression smears were negative for exposed pig - which was euthanized at dpi. the histopathology examination showed normal length villi in the duodenum, jejunum and ileum. mild multifocal subacute lymphohistiocytic bronchointerstitial pneumonia with lobular atelectasis was detected in lung. in a second experiment, one gnotobiotic pig (gp - ) was oronasally inoculated with the cell culture adapted, plaque-purified - f prcv isolate ( × pfu/pig) and a second pig (gp - ) was inoculated with ml of a : dilution of the pooled rectal swab fluids recovered from gp - described above at to dpi (table ) . neither of the inoculated pigs showed clinical signs up to dpi when they were euthanized. fecal shedding was detected on and dpi in gp - and on dpi in gp - by nested-rt-pcr and on dpi in gp - by elisa. no nasal shedding was detected by nested-rt-pcr or ccif in either pig ( table ). the control pig remained negative at all times in all tests. the ifa on impression smears from duodenum, jejunum, ileum and lung of gp - was negative. likewise for gp - , the ifa on the duodenum, jejunum, ileum and lung was negative. the histopathology results showed normal length villi in the duodenum and jejunum for both gp - and gp - , but in gp - , villi in the ileum were slightly shortened. the lung tissues did not differ from the control (data not shown). in this study we investigated prcv/tgev nasal and fecal shedding in sentinel pigs introduced into a prcv seropositive herd with questionable tgev serology and diarrhea of uncertain etiology in weaning pigs. kim et al. [ ] recently reported a similar field case in a u.s. swine herd where the presence of prcv antibodies in the herd may have complicated the diagnosis of tgev infection. in this latter case tgev infection was confirmed by isolation of tgev in st cells from the gut contents of diarrheic sentinel pigs. a prcv strain was also isolated in st cells from nasal swabs from clinically normal tgev-seronegative sentinel pigs in contact with the diarrheic pigs. in the present outbreak, prcv/tgev seronegative - -week-old sentinel pigs, were introduced into the prcv seropositive herd with diarrhea occurring in weaned pigs. the sentinel pigs remained prcv/tgev seronegative at and dpa. at dpa, both the sentinel and resident pigs developed diarrhea. one day later, one sentinel pig died and laboratory testing failed to confirm a diagnosis, but the tissues were prcv/tgev negative by the immunoperoxidase test. fiftysix percent ( / ) of sentinel pigs seroconverted to prcv at dpa without respiratory clinical signs. because investigators have reported that only tgev has an enteric tropism and causes diarrhea, but the serology suggested the presence of prcv and was questionable for tgev, our overall goal was to clarify if pigs were shedding tgev or prcv and to determine if the isolates were similar or identical to each other and to previously described tgev or prcv strains. fecal and nasal shedding of prcv/tgev was detected by ccif and a nested-rt-pcr assay was used to detect and differentiate prcv and tgev [ ] . no shedding of tgev was detected by nested-rt-pcr. nasal shedding of prcv/tgev was detected in % of the samples by ccif and % of the samples were prcv positive by nested-rt-pcr. the nested-rt-pcr was as sensitive as ccif to detect prcv and there was a good agreement between both techniques, with the peak of prcv nasal shedding at dpa ( day after diarrhea). however the detection of prcv in fecal swabs showed different results. the nested-rt-pcr was more sensitive than ccif for prcv detection in fecal samples ( % and %, respectively, fig. ). the peak of prcv fecal shedding was at dpa by nested-rt-pcr ( %) but only . % of the fecal samples were positive by ccif. previous reports showed that nested-rt-pcr is more sensitive than ccif for detection of rectal shedding of tgev. in a study by kim et al. [ ] , gnotobiotic pigs were inoculated with the cell culture adapted tgev strain bw b or the original field pig intestinal content sample. viral shedding was detected in rectal swabs from dpi until by nested-rt-pcr, but only at dpi using ccif [ ] . it is also possible that intestinal antibodies to prcv, present in the intestinal contents of the prcv seropositive pigs could interfere with the detection of fecal shedding of prcv by ccif, but not by the nested-rt-pcr assays. in addition, it is possible that prcv is more labile in feces or particles may lose their spike protein [ ] resulting in loss of infectivity detected by ccif assays, but not the rna in these viral particles, detected in nested-rt-pcr assays. the prcv has a different tissue tropism from that of tgev. the tgev replicates in both respiratory and intestinal epithelial cells and causes gastroenteritis, whereas prcv replicates to high titers in the upper respiratory tract and lung tissue of swine [ , ] . however, prcv strain ar was the first prcv strain isolated from the small intestine of a field pig from an arkansas, u.s. swine herd [ ] . cox et al., , reported the isolation of prcv strain tlm from various tissues including the intestine of hysterectomy-derived colostrumdeprived pigs which were inoculated by aerosol with tcid prcv at six days of age. virus was consistently isolated in highest titers from respiratory tract tissues, but also from stomach, small intestine and colon. however none of the pigs showed respiratory signs or diarrhea [ ] . their results showed that prcv infected only a few unidentified cells at villous or crypt sites in the small intestinal mucosa and spread from the ileum to the duodenum. although unclear, the authors suggested that the aerosol inoculation of pigs with tlm caused a respiratory infection followed by viremia and ingestion of prcv to infect the intestinal cells. in our studies the detection of prcv from nasal swabs of gnotobiotic pigs gp - and - inoculated with the respiratory - n prcv isolate confirms the respiratory tropism, but the presence of fecal shedding in both pigs in the absence of villous atrophy (gp - ) also suggests an intestinal infection like that reported for prcv strain tlm [ ] . however, when a gnotobiotic pig (gp was oronasally inoculated with the fecal - f prcv isolate (isolated from the fecal swab fluids of the same sentinel pig as - n) and a second gnotobiotic pig (gp - ) was inoculated with the fecal prcv strain recovered from gp - (originally inoculated with the respiratory strain - n), virus shedding was detected only in rectal swab fluids. because the virus was inoculated by both the oral and nasal routes and detected only from rectal swab fluids and not from nasal swab fluids, the positive signal was unlikely to be derived from the virus inoculum. the histopathology results showed normal length villi in the small intestine of gp - , but slightly shortened villi in the ileum of gp - . these collective results suggest that the detection of prcv in the rectal swab fluids from the gnotobiotic pigs inoculated with - n, is not just the consequence of a respiratory infection followed by ingestion of prcv with shedding of ingested virus in feces, but another factor may exists to account for the change in tropism of these prcv strains. failure to detect villous atrophy or prcv antigen in the small intestine of gp - given - n prcv may have been due to the later time that this pig was euthanized ( dpi) versus pigs gp - and gp - that were euthanized at dpi. however because no clinical signs were evident it was difficult to establish optimal times to euthanize these pigs for antigen or lesion detection. failure to detect prcv antigen by ifa in the intestinal tissue of any of these pigs may have been due to the timing, the presence of too few infected cells for detection or to use of impression smears which may not adequately reflect cells in the crypt regions or in the intestinal submucosal region. alternatively failure to detect fecal shedding of prcv in these pigs by ccif may reflect a lower sensitivity of this assay or the lability of prcv strains in feces. differences in the tropism between tgev and prcv strains have been attributed to deletions in the region of the s-gene [ , , ] . four respiratory [ - n * ( ) - n * ( ) - n * ( ), - n * ( )] and fecal [ - f * ( ), - f * ( ), - f * ( ), - f * ( ), - f * ( )] strains were isolated and plaque-purified in st cells and the s-gene was partially sequenced. all u.s. prcv strains reported in the literature have - nt deletions within the region of the s-gene resulting in loss of or antigenic sites [ , ] . in contrast tgev strains have an intact s-gene. sequence analysis allowed grouping of the isolates into groups according to the size and position of the s-gene deleted. the deletion size ranged between to nt, starting at nt , or to nt , and . the sequences were aligned by the clustal method and the analysis showed a high homology between the prcv isolates and the other u.s. strains of prcv. previous reports indicated that the s glycoprotein domain recognized by the cellular receptor papn is close to the antigenic sites a and d (nt - ) but this domain is present in both tgev and prcv strains [ ] indicating that its presence is not sufficient for infection of enterocytes. analysis of the tropism of tgev recombinant isolates have demonstrated that nucleotide changes of the s gene that result in amino acid changes at the n-terminal region of the tgev s protein also affect the enteric tropism of tgev. because of this observation, we focused on analysis of this region [ ] . to analyze if sequence differences between the prcv fecal and respiratory strains were involved in the enteric tropism, the sequence of the fecal and nasal pairs - , - and - were compared. ballesteros et al., described aa changes at residues (g → a) and (g → t) of the s-gene that were responsible for the loss of the enteric tropism of the ptv-ts-mad tgev strain [ ] . as shown in table , only differences were found between the fecal and nasal prcv isolates. three of them where at nt , , and the fourth difference was in the region of nt - . a fifth difference (nt ) was found when the prcv field strains were compared with the reference strains. when the amino acid (aa) sequences were analyzed, only changes in nt , and the region - resulted in amino acid changes. the nucleotides - encoded the same amino acid (aa or depending on the strain). the segment from nt - (aa - ) is inside the deletion area of the nasal prcv strains. however the possibility that the change in the tropism was a consequence of the differences found in nt - was unlikely because the same sequence was found in the respiratory prcv isu- strain which causes little or no respiratory disease or fecal shedding in infected gnotobiotic pigs [ ] . the fifth difference in sequence was detected in all prcv field strains compared to the reference prcv strains. as shown (table ) , an identical nt change (nt a → g) was found in each fecal and nasal prcv pair when they were compared with the prcv reference strains. however this change would not affect the tropism because it is within the leader peptide of the s protein and not present in the mature virus. surprisingly however, based on a limited pathogenesis study in a gnotobiotic pig, the fecal - f prcv isolate appears to have lost the respiratory tropism. the two nt changes between the respiratory and fecal isolates at nt - caused an aa change from threonine to valine, respectively ( table ) . the same aa (t) was found in this position in the tgev pur mad strain, which has both enteric and respiratory tropism and in the prcv isu- strain, which has only respiratory tropism. our results would suggest that the loss of the respiratory tropism of the - f prcv strain could be a consequence of the change in this aa. it is possible that the changes in the tropism of the present prcv isolates are a consequence of the changes in nt - . however, further genetic analysis of these strains and pathogenesis studies are needed. a better understanding of the molecular basis of virus tropism may help to clarify the mechanism of disease for tgev and prcv strains and understand their evolution in infected swine in the field. two amino acid changes at the n-terminal of the transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs new porcine coronavirus? sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of the spike glycoprotein s molecular basis of transmissible gastroenteritis virus epidemiology experimental reproduction of pneumonia in gnotobiotic pigs with porcine respiratory coronavirus isolated ar field isolates of transmissible gastroenteritis virus differ at the molecular level from the miller and purdue virulent and attenuated strains and from porcine respiratory coronaviruses development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus interferon induction in rotavirus and coronavirus infections: a review of recent results porcine respiratory coronavirus: molecular features and virus-host interactions immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (tgev) and recombinant tgev spike v. costantini et al.: respiratory and fecal shedding of prcv protein vaccines and protection of their suckling pigs against virulent tgev challenge exposure detection of transmissible gastroenteritis virus by rt-pcr and differentiation of porcine respiratory coronavirus isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis transmissible gastroenteritis and porcine respiratory coronavirus targeted recombination demostrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence genetic evolution and tropism of transmissible gastroenteritis coronavirus antigenic homology among coronavirus related to transmissible gastroenteritis virus transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity evaluation of the baculovirus-expresssed s glycoprotein of transmissible gastroenteritis virus (tgev) as antigen in a competition elisa to differentiate porcine respiratory coronavirus from tgev antibodies in pigs competition elisa, using monoclonal antibodies to the transmissible gastroenteritis virus (tgev) s protein, for serologic differentiation of pigs infected with tgev or porcine respiratory coronavirus antigenic variation among transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus strain detected with monoclonal antibodies to the s protein of tgev sequence comparison of porcine respiratory coronavirus isolate reveals heterogenecity in the s, and - genes three new isolates of porcine respiratory coronavirus with various pathogenicities and spike (s) gene deletions antigenic and biological diversity among transmissible gastroenteritis virus isolated of swine evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states we thank drs jeff smiley, mustafa hasoksuk, armando hoet, sonia cheetham and mr arden agnes for technical advice and also the staff of the oardc/osu molecular and cellular imaging center for sequencing. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development (oardc), the ohio state university. this study was supported in part by the national pork producer's council on behalf of the u.s. national pork board. key: cord- -yb mxk authors: inaba, y.; sato, k.; kurogi, h.; takahashi, e.; ito, y.; omori, t.; goto, y.; matumoto, m. title: replication of bovine coronavirus in cell line bek- culture date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: yb mxk bovine coronavirus readily multiplied and induced marked cytopathic effect in bek- cultures, thus providing a sensitive, practical assay system for viral infectivity and neutralizing antibody and a satisfactory source of the virus. v[~ibus and his associates ( -- ) have demonstrated an agent with morphologic features of coronavirus by electron microscopy in fractions prepared by density gradient ultracentrifugation from diarrheal feces of calves with natural and experimentally produced neonatal diarrhea. the agent multiplied but failed to induce readily recognizable cytopathic effect in bovine embryonic kidney cell cultures ( ) . the virus has been assayed therefore in these cultures by a rather cumbersome method in which the presence of the virus was determined by microscopic examination for syncytia of cultures stained with gentian violet, or by immunofluorescent technique ( ). this paper describes briefly our recent observation that the virus readily replicates and induces a marked cytopathic effect in cultures of a continuous cell line, bek- , derived from bovine embryonic kidney ( ), thus providing a sensitive, practical assay method and a satisfactory source of the virus. bek- cells were grown at ° c in eagle's minimum essential medium (mem) containing per cent tryptose phosphate broth (difco) (tpb), per cent bovine serum, o units/m] penicillin, ~zg/ml streptomycin, and ~xg/ml fungizone. the bovine corona~drus ( ), at the th passage level in bovine embryonic kidney cells, was kindly supplied by dr. c. a. mebus, university of nebraska, and was passaged twice in primary calf kidney cell cultures in our laboratory before use in the present study. bek- cell cultures prepared in × mm tubes were inoculated with . ml amounts of virus dilution, and incubated in a roller drum at ° c. the maintenance medium after virus inoculation was mem containing per cent tpb, per cent sodium glutamate, per cent glucose, . per cent yeast extract, . per cent bovine serum albumin (armour) and antibiotics. the virus readily multiplied and induced marked cytopathic effect. the effect was first recognized days after inoculation as rounded cells scattered in the cell sheet. as incubation progressed, round cells increased in number, fused to form syncytia, disintegrated and sloughed off the glass surface. these changes destroyed cell sheets within or days after inoculation (figs. a and b) . further passages were readily made with supernatant fluid from infected cultures developing cytopathic effect, and the viral yield attained ranged from to tcidj / . ml. the cytopathic effect was specifically inhibited by antiserum to the bovine coronavirus. the antiserum used was prepared in rabbits, which received an intravenous dose of . ml of virus suspension followed at and weeks intervals, by two intramuscular doses of . ml each of equal volume mixture of the virus suspension and freund's complete adjuvant. the virus suspension was prepared by resuspending, in / volume of phosphate buffered saline, pellets obtained by centrifugation of infected bek- culture fluid at , x g for one hour. serum was obtained from the animals weeks after the last dose. the neutralization test was carried out by the method described elsewhere ( ) . the serum neutralized the virus, the antibody titer being : , while the preimmunization serum was negative. infection of bek- cells with the bovine coronavirus was also proved by immunofluoreseent staining. coverslip cultures of bek- cells inoculated with the virus were examined after or days of incubation at ° c. the cultures were fixed with acetone at ..... ° c for minutes, and stained with the fluorescein isothiocyanate-conjugated antibody for the bovine coronavirus which was kindly donated by dr. c. a. mebus. many immunofluorescent mono-and multi-nucleated cells were observed; the cytoplasm contained granular fluorescence, in some cells most of the cytoplasm fluoresced ( fig. c) . as mebus et al. ( ) we prepared fractions from infectious culture fiuid by density-gradient ultracentrifugation, and examined them by phosphotungstic negative staining in an electron microscope. r'umerous particles, about nm across, with morphologic features of coronavirus were observed (fig. ld) . these results prove bek- cells to be a good medium for propagation and assay of bovine coronavirus, rendering consistent work with the virus feasible. isolation and properties of bovine parvovirus type i from japanese calves behavior of bovine ephemeral fever virus in laboratory animals and cell cultures neonatal calf diarrhea : propagation, attenuation and characteristics of a coronavirus-like agent pathology of neonatal calf diarrhea induced by a coronavirus-like agent neonatal calf diarrhea: results of a field trial using a reo-like virus vaccine neonatal calf diarrhea : purification and electron microscopy of a coronavirus-like agent authors' address: dr. y. i~aba, national institute of animal health, kodaira, tokyo, japan.received november , key: cord- - nwjjgul authors: atherton, j. g.; kratzing, c. c.; fisher, anne title: the effect of ascorbic acid on infection of chick-embryo ciliated tracheal organ cultures by coronavirus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: nwjjgul chick embryo tracheal organ cultures showed increased resistance to infection by a coronavirus after exposure to ascorbate, while chick respiratory epithelium and allantois-on-shell preparations showed no increase in resistance to infection by an influenza virus or a paramyxovirus. microscopically for up to days when these cultures were maintained at ° c, with medium changes twice weekly. the influenza virus, human influenza virus type a, strain v - , was obtained from commonwemth serum laboratories, melbourne. the paramyxovirus, newcastle disease virus, strain v - and the coronavirus, avian infectious bronchitis virus, strain b- - , were both isolated and identified by mr. c. simmons of the animal l%eseareh institute, brisbane. influenza virus and newcastle disease virus titrations were performed either in micro-cultures of chick respiratory epithelium or in allantois-on-shell preparations, by inoculating replicate cups with dilutions of virus made in half-log steps, then incubating the preparations for days at ° c in a humidified chamber. growth of virus was detected by testing for haemagglutinin by the addition of . ml of a per cent suspension of washed adult, fowl red blood cells, after which the plates were incubated at ° c for hour and then read for haemagglutinin using substage indirect lighting. virus end-points were then calculated as ids /ml by the reen-mue?zc~ method ( ) . titrations of avian infectious bronchitis virus were performed by inoculating replicate chick-embryo tracheal organ culture tubes previously selected for ciliat activity, with dilutions of virus made in half-log steps, then continuing to incubate the preparations on a roller drum at rev/hour at ° c. these tubes were observed daily for cihal activity. cilial activity was arbitrarialy graded from + to . the final observation was made after days incubation and cilial activity reduced to + or less taken as evidence of virus infection. virus end-points were then calculated as ceto id~ /ml by the t%eed-muench method. aseorbie acid was estimated by the method of boli~r and book ( ) . all aseerbie acid solutions were used in neutralized form, without the addition of glutathione. the uptake of aseorbic acid by cells was measured by exposing ceto cultures, chick-embryo respiratory epithelial cultures and allantois-on-shell preparations respectively to ascorbie acid in concentrations of , ~g/ml for hours at ° c. samples of the cells were then taken for ascorbie acid determination and the remainder used in viral infectivity tests. tissue ascorbate concentrations obtained in ce respiratory epithelium and aliantois-on-shell, were comparable wi~h those of ceto (table t) . cell culture medium to be tested for interferon was frozen and thawed once from -- ° c, adjusted t,o p i t . with n itc , stored at ° c for hours, brought to pyi . , and then centrifuged at , ×g for i hour. the supernatant was stored at ° c and tested for interferon activity within hours. all interferon titrations were performed against a standard preparation made by similar treatment of tissue culture supernatant from ndv-infeeted chick embryo respiratory epithelial cell cultures. interferon titres were determined using a per cent plaque reduction method similar to that described by h~[oemc~ng et al. ( ) using }veslb nile virus in primary chickembryo fibroblast monolayers. virus nomenclature used is that of f e~e~ ( ). three types of experiments were done: . ascorbic acid in concentrations from to , exg/ml was incubated with standard virus suspensions diluted in b m e for from to hours at ° c. a second series was held at ° c. l%esults of infectivity assays were plotted graphically and compared with .lbhose from virus suspensions made in b m e alone. no significant effect, of ascorbic acid on the rate of thermal inactivation of influenza virus, n d v or i b virus was observed. . i n the second type of experiment virus and ascorbic acid were added to cells at the same time and the ascorbate maintained t h r o u g h o u t incubation. five dilution series of each virus were made at ° c, one in b m e alone and the others in ascorbic acid dilutions of , , and , ~g/ml. e a c h dilution t series was then used to perform virus infectivity titrations in the cell system appropriate for the virus under test,. no significant effect on viral infectivity was observed. . in the third type of experiment, cells were exposed to aseorbic acid in concentrations of -- , vglml for hours at ° c. samples of cells were taken for determination of ascorbie acid content and the remainder used in viral infectivity tests. simultaneous parallel virus titrations were performed on cells which had not been exposed to ascorbic acid. the resistance of cells to infection was measured by exposing them to halflog dilutions of a standard virus suspension, incubating, and then calculating the virus infectivity end-point. a lower titre for virus infectivity end-point in treated cells compared with untreated cells, indicating a decrease in plating efficiency, was taken as evidence of increased resistance of the treated cells to virus infection. increasing cell content of ascorbie acid did not increase the resistance of cells to infection by influenza or newcastle disease viruses. however resistance of ceto cultures to ib virus infection rose with increasing ascorbic acid content (table t and fig. ). tissue containing . ~g/ml { ~g/ml) of ascorbate showed a virus infectivity end-point of . ceto ida /ml compared with . ceto id /ml for tissues without ascorbate, i.e. a . times larger dose was needed to infect tissues containing aseorbate, with ib virus. reports of induction of increased serum concentrations of interferon i n vivo by aseorbate ( , , , ) prompted tests for interferon in the experiments cited. ceto cultures were treated with , y.g/ml of ascorbate for hours, then infected with ib virus dilutions froln -- . zvyle ~stor~ e co, c (py/mz)------ ° c. the fluids were then assayed for interferon by a plaque-reduction test using west nile virus in chick-embryo fibroblast monolayers. no significant differences in interferon content could be detected between fluid from cells alone, cells plus ascorbate, and virus-infected cells with and without aseorbate. standard interferon prepared as described above had a per cent plaque reduction (pt~ ) titre of : . the effect of interferon-plus ascorbic acid was tested by adding an interferon preparation diluted : (twice the prs concentration) to ceto cultures exposed to varying ascorbic acid concentrations and incubating at °c for hours. these cet cultures were then infected with . cet ids /ml ib virus and the infectivity end-points determined. the results are shown irt table and figure . differences between mean titres obtained with and without interferon in the presence of ascorbate, although little greater than the s.e., suggest that interferon exerted a slight effect, i.e. about doubling cell resistance to virus. this effect is very small compared with that of ascorbate. ( ) showed that exposure of wi- cells to ascorbic acid plus glutathione mixtures for days prior to infection with rhinovirus suppressed multicyclic but not single cycle growth. the same authors found some evidence for barely detectable levels of interferon at high virus moi. murphy( et al. ( ) found that increased amounts of ascorbate were unable to prevent parainfluenza virus experimental infection or primary immune response in cotton-topped marmosets. however the onset of disease was delayed, clinical responses reduced and mortality decreased in animals fed doses of ascorbic acid equivalent to g/day for man. on the other hand, sci-zwa~tz et al. ( ) followed a number of parameters of virus infection and disease in a group of human volunteers infected with rhinovirus , but were unable to show any differences between controls and those treated with ascorbie acid. our results show t h a t aseorbic acid exerted no direct effect on the infectivity of a n y of the three viruses tested, nor did it affect the resistance of cells to infection by the r t h o m y x o v i r u s (influenza) or the p a r a m y x o v i r u s (ndv). however c e t cultures previously exposed to ascorbic acid exhibited considerably increased resistance to infection by coronavirus (ibv). these results suggest t h a t different mechanisms operate for infection of cells by viruses of these different groups. the different effects of ascorbate on experimental infection by viruses from different groups suggest that, when clinical trials of the effect of ascorbate on respiratory virus infection are conducted ( , , , ) it is i m p o r t a n t to ascertain to which group the infecting virus belongs. winter illness and vitamin c: the effect of relatively low dose oxidation of ascorbic acid to dehychloascorbic acid effects of ascorbic acid on the common cold: an evaluation of the evidence large-quantity production of chicken embryo tracheal organ cultures and use in viral and mycoplasma studies ascorbic acid and the common cold: evaluation of its efficacy and toxicity an improved assay for the infectivity of influenza viruses classification and nomenclature of viruses. second report of the international committee on taxonomy of viruses effect of ascorbic acid, sodium salicylate and caffeine on the serum interferon level in response to viral infection ascorbic acid for the common cold: a prophylactic and therapeutic trial survey of interferon production and sensiti~dty in human cell lines ascorbic acid (vitamin c) and it.s effects on parainfluenza type virus infection in cottontopped marmosets a simple method of estimating fifty per cent end-points evaluation of the efficacy of ascorbic acid in prophylaxis of induced rhinovirus infection in man effect of ascorbic acid on rhinovirus replication in %v - cells enhanced interferon response to murine leukemia virus by ascorbic acid ascorbic acid in rat lung key: cord- - rgcc h authors: pedersen, n. c.; ward, j.; mengeling, w. l. title: antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rgcc h utilizing the direct and indirect fluorescent antibody procedure, the antigenic relationship of the feline infectious peritonitis virus (fipv) to other human and animal coronaviruses was studied. fipv was found to be closely related to transmissible gastroenteritis virus (tgev) of swine. transmissible gastroenteritis virus and fipv were in turn antigenically related to human coronavirus e (hcv- e) and canine coronavirus (ccv). an interesting finding in the study was that the coronaviruses selected for this study fell into one of two antigenically distinct groups. viruses in each group were antigenically related to each other to varying degrees, but were antigenically unrelated to coronaviruses of the second group. the first antigenically related group was comprised of mouse hepatitis virus, type (mhv- ), hemeagglutinating encephalomyelitis virus n (hev- n) of swine, calf diarrhea coronavirus (cdcv), and human coronavirus oc (hcv-oc ). the second antigenically related group was comprised of fipv, tgev, hcv- e and ccv. utilizing the direct and indirect fluorescent antibody procedure, the antigenic relationship of the feline infectious peritonitis virus (fipv) to other human and animal coronaviruses was studied. fipv was found to be closely related to transmissible gastroenteritis virus (tgev) of swine. transmissible gastroenteritis virus and fipv were in turn antigenically related to human eoronavirus e (i-icv- e) and canine coronavirus (ccv). an interesting finding in the study was that the eoronaviruses selected for this study fell into one of two antigenically distinct groups. viruses in each group were antigenieally related to each other to varying degrees, but were antigenically unrelated to coronaviruses of the second group. the first antigenically related group was comprised of mouse hepatitis virus, type (mi-iv- ), hemeagglutinating encephalomyelitis virus n (hev- n) of swine, calf diarrhea coronavirus (cdcv), and human coronavirus oc (hcv-oc ). the second antigenically related group was comprised of fipv, tgev, hcv- e and ccv. the family coronaviridae is a recently characterized group of animal and human viruses ( ) . coronaviruses are to nm in diameter, have a buoyant density in sucrose of . to . g/cm , are sensitive to lipid solvents, contain a large single strand of ribonucleic acid, have regularly spaced surface projections n.c. pedersen, j. wai~d, and w. l. mesrg~ll~g: t h a t are to n m in length, and bud from profiles of endoplasmic reticulum into cytoplasmic vesicles in the infected cells ( ) . coronaviruses cause bronchitis in chickens ( ), humans ( , ) and rats ( ) , acute enteric infections in b a b y pigs ( ) , calves ( ) a n d puppies ( ) , hepatitis in mice ( ), and encephalomyelitis a n d chronic vomition and wasting in swine ( , ) . feline infectious peritonitis (fip) is a viral disease of cats t h a t is characterized b y peritonitis, pleuritis or disseminated granulomata ( ) . feline infectious peritonitis represents an uncommon secondary form of a common i n a p p a r e n t or mild p r i m a r y illness of cats ( ) . the f i p agent has strong morphologie and physical similarities to known coronaviruses ( , , , ) . a possible antigenic relationship between feline infectious peritonitis virus (fipv) and the transmissible gastroenteritis virus (tgev) of swine has been recently reported ( , , ) , which further supports the assumption t h a t f i p v is a coronavirus. the antigenic relationship of f i p v to h u m a n and animal coronaviruses other t h a n t g e v has not been studied, and confirmation of the antigenic relationship of f i p v and t g e v using monospeeific antiserum is needed. the purpose of this s t u d y is twofold: to confirm the antigenic relationship of f i p v to tgev, and to demonstrate the antigenic relationship of different animal and h u m a n coronaviruses to f i p v , and to each other. the viruses selected for this s t u d y were h u m a n coronavirus c (hcv- c ), h u m a n eoronavirus e (hcv- e), t g e v and hemagglutinating encephalomyelitis virus n (hev- n) of swine, mouse hepatitis virus t y p e (miiv- ), calf diarrheal coronavirus (cdcv), and canine coronavirus (ccv). antigenic comparisons were m a d e utilizing the direct and indirect fluorescent a n t i b o d y technique. this procedure has been utilized to s t u d y serologic differences between several h u m a n coronaviruses (/ ). monospecific antiserum to f i p v was prepared in specific pathogen free kittens (liberty laboratories, liberty corners, nit). the cats were inoculated intraperitoneally with . g equivalents of liver suspension containing approximately id of the ucd- strain of fipv. the origin of this strain and the preparation of the inoeula have been previously described ( ) . serum was harvested prior to the animms' death, from to days after inoculation. by the indirect fluorescent antibody technique ( ) this antiserum had a titer of : against fipv. mouse anti-mttv- serum was produced in specific pathogen free adult. swiss white mice. mice were inoculated intraperitoneally with a sublethal dose of a per cent mouse liver suspension containing the craig strain of mitv (mhv- ). this material was obtained from the american type culture collection, rockville, md. three weeks later the mice were challenged intraperitonemly with a second sublethal dose of mhv- , followed by challenge weeks later with a lethal dose of virus. serum was harvested weeks after the final challenge dose. this serum had a titer by the indirect fluorescent antibody technique of : against mi-iv infected nctc- cells. bovine anti-cdcv antiserum was obtained from calves that. had been experimentally infected with the virus. the globulin fraction of this serum was conjugated with fluoreseein isothiocyanate (fitc). conjugated antiserum was provided by dr. c. a. mebus, lincoln, nebraska.. the conjugated antiserum produced maximum fluorescence in cdcv infected bovine fetal lung cells at dilutions of : or less. swine anti-tgev serum was obtained from a specific pathogen free sow that was experimentally infected with the miller strain of tgev. this serum was kindly provided by dr. roger woods, ames, iowa. both swine anti-tgev and ttev serum produced maximum fluorescence by the antibody technique at di utions of t : or less. guinea pig anti,hcv- e serum was provided by dr. harold kaye, communicable diseases center, atlanta, georgia. before use, the serum was absorbed with swine testicle, human embryo flbroblasts, nctc-t , african green monkey (cv- ), bovine fetal lung cells and with cat liver homogenate. this serum produced maximum fluorescence by the indirect fluorescent antibody technique at dilutions of : or less. mouse anti-i-icv-oc ascitie fluid was kindly provided by dr. harold kaye, communicable diseases center, atlanta, georgia. ascitic fluid was collected from virus free mice that had been experimentally infected with hcv-oc . this serum produced maximum fluorescence by the indirect fluorescent antibody technique at dilutions of : or tess. canine anti-ccv globulin conjugated with fluorescein isothioeyanate was provided by dr. l. q. binn, walter reed army institute of research, washing*on, d.c. it was prepared from convalescent serum of puppies experimentally infected with ccv. the conjugated antiserum produced maximum fluorescence in ccv infected canine fetal thymus cells at dilutions of : or less. cryostat microtome sections of liver from fib¥ infected cal~s were used as the antigen source of fibv. the preparation of these slides has been previously described ( ) . prior to use, the fixed liver sections were immersed for minutes in . ~ glycine-i.ic buffer, pit . to remove immunoglobulin bound in rive. when this bound immunoglobulin was not removed, a false positive reaction was seen in the indirect fluorescent antibody test, especially when the second antibody was rabbit anti-cat igg. after treating in buffer, the slides were washed immediately in phosphate buffered saline (bbs), followed by a minute and minute wash in bbs. mhv- infected cell monolayers were prepared as follows. nctc- cells (microbiological associates, bethesda, md.), adapted to grow in eagle's minimum essential media (mem) and per cent fetal calf serum (fcs), were grown in well culture chamber slides (lab-tek, microbiological associates, bethesda, md.). when confluent, the cell monolayer was exposed ~o mhv- by placing .t nil of a : dilution of per cent infected mouse liver suspension in each well. the slides were fixed in absolute acetone when significant cytopathic effect was noticed. calf diarrhea coronavirus was obtained from dr. c. a. mebus, lincoln, nebraska.. one-tenth ml of infectious tissue culture media was placed in each well of a culture chamber slide containing a three-fourth confluent monolayer of low passage fetal bovine lung cells. the slides were fixed in absolute acetone after -- days. tgev and i.iev were cultivated in swine embryonic testicle cells {national animal diseases center, ames, iowa). tgev (miller strain) infected tissue culture fluid was provided by dr. roger woods, ames, iowa,. i.iev ( n strain) infected tissue culture fluid was obtained from the national animal disease center, ames, iowa. swine testicle cells were grown in eagle's mem in per cent fcs in well culture chamber slides. when the cultures were almost confluent they were exposed to the n strain of ttev or miller strain of tgev by placing .i ml of infected tissue culture fluid in each well. the slides were fixed in absolute acetone after to days. ttcv- e was obtained as infected tissue culture fluid from dr. harold kaye, communicable diseases center, atlanta, georgia. low passage human embryonic fibroblasts were grown in eagle's mem in per cent fcs in well culture chamber slides. when the cultures were ahnost confluent, . nil of infected tissue culture fluid was placed in each well. slide cultures were fixed after days. n.c. ped~ase~, j. ward, and w. l. mengeling: hcv-oc was obtained as a mouse brain suspension from dr. harold kaye, communicable diseases center, atlanta, georgia. high passaged african green monkey kidney cells (cv- ) were grown in eagle's mem with per cent fcs in well culture chamber slides. when nearly confluent, . ml of a : dilution of brain suspension was placed in each well. slides were fixed in absolute acetone after to days. canine eoronavirus (i- ) in tissue culture fluid was obtained from the american type culture collection, izockville, ,md. low passage dog thymus cells were cultivated in well culture chamber slides. when nearly confluent, . ml of infected tissue culture fluid was placed in each well. cultures were fixed in absolute acetone after eytopathie effect became noticeable. rabbit anti-mouse igg globulin-fitc, rabbit a~nti-cat igg globulin-fitc, rabbit anti-pig igg-fitc, and goat anti-guinea pig igg-fitc were obtained from antibodies incorporated, davis, ca. conjugated anti-igg globulins were free of anticoronavirus activity as determined by reacting antigen substrates with the diluted conjugates alone. goat anti-guinea pig igg-fitc was absorbed with eat liver homogenate and human embryo cells. indirect fluorescent antibody staining was carried out as follows. antigen substrate slides were overlaid with a : dilution in pbs of the appropriate antiserum and incubated at ° c for hour in a humidified chamber. the slides were immediately rinsed with pbs and then washed for minutes in pbs. the slides were blotted dry and then overlaid with the appropriate anti-igg conjugate diluted : in pbs. the slides were incubated for i hour at ° c in humidified chamber and then washed in pbs. this treatment was followed by a minute wash in pbs containing a : dilution of a per cent stock solution of aqueous evans blue, and a minute wash in pbs. slides were then blotted dry, and eoverslips mounted with per cent glycerol in pbs. direct fluorescent antibody staining was carried out essentially as above, except tha~ the first antibody reaction was omitted. slides were photographed with a zeiss reflected light fluorescent microscope, powered by a watt ac mereury~vapor bulb, using exciter and barrier filters specific for fluorescein isothioeyanate. photomicrographs were made using ektachrome mm daylight slide film, asa (kodak co., l%oehester, ny), with second exposures. all photomicrographs were prepared from black and white negatives made from the colored slides. the cross-reactivity by immunofluorescence of antisera to different coronaviruses is listed in table . antigenic cross-reactivity varied from nondetectable, barely detectable, weak, moderate, to very strong (equal to that produced by the homologous serum). cross-reactivity, varying from barely detectable to very strong, was seen between mhv- , cdcv, hev- n and hcv-oc . there was no detectable antigenic cross-reactivity between these viruses and fipv, tgev, hcv- e, or ccv. antiserum to fipv reacted strongly with tgev, and vice versa. antiserum to both of these viruses had barely detectable to weak antigenic cross-reactivity with hcv- e, and antiserum to iicv- e had weak to moderate cross-reactivity with fipv and tgev. antiserum to ccv did not react with any of the other coronaviruses, although antiserum to fipv and tgev reacted very strongly with ccv. photomicrographs of some of the strongly positive eross-reae~ions are shown in figures and . it was concluded from these studies that, miiv- , hcv- c , cdcv, and hev- n are all antigeniemly interrelated to varying degrees, but a~ not related antigenieally to any of the other [ eoronaviruses. similany, fipv, tgev, hcv- e and ccv share antigens to varying degrees with each other, but appear antigenieally unrelated to mhv- , hcv-oc , cdcv, and hev- n. in the ease of ccv, however, the reactivity was largely in. one direction only, in t, hat antiserum to ccv showed no reactivity with any of the other eoronaviruses, whereas antiserum to fipv and tgev reacted strongly with ccv. these studies demonstrate conclusively that the fip virus has antigenic similarities to known coronaviruses, namely tge virus of swine, e virus of humans, and canine eoronavirus. this finding, coupled with the morphologic and it was interesting that the coronaviruses selected for this study segregated into distinct groups on the basis of antigenic cross-reactivity by immunefluorescence. although viruses within each group were antigenieally related to each other, there appeared to be no antigenic relationship of viruses from one group with viruses of the other group. the first antigenically related group was comprised of mhv- , hev- n, cdcv and i-icv-oc , and the second group was comprised of tgev, fipv, hcv- e and ccv. these findings confirm a number of published reports on antigenic relationships among recognized coronaviruses. on the basis of serum neutralization or complement fixation tests, antigenie relationship has been previously reported between rat eoronavirus and mi-iv ( ) , hcv-oc and mhv ( ), hev- n and ci)cv ( ) , hcv-oc and tiev- n ( ), and ccv and tgev ( ) . it has also been previously reported that hcv- e appeared to be antigenically unrelated to mhv- and hcv-oc ( ). the lack of relationship by immunofluorescence of hcv- c and hcv- e has also been reported ( ) . although the results of our studies were in agreement with most of the published literature on antigenic relationships among various corona viruses, there were several reports that we could not confirm. we could find no antigenic relationship by immunofluoreseenee between mhv- and hcv- e, and i-icv-oc and hcv- e. a relationship between these viruses has been previously described ( ). we also found no antigenic relationship between hev- n and tgev, although a relationship using the immnnopreeipitation technique has been reported ( ) . it has been reported that antiserum to tgev does not react against fipv in the fluorescent antibody test ( ) . in contrast, we found that ~ntiserum to tgev reacted strongly with fipv. finally, we are at a loss to explain the failure of anti-ccv globulin to react with tgev and fipv with immunefluorescence, especially considering the strong reaction against ccv demonstrated by both anti-fipv and tgev serum. dog anti-ccv serum will apparently neutralize tgev ( ), and it is strange that this reaction was not detected with immnnofluoreseence. recovery and characterization of a eoronavirus from military dogs with diarrhea the detection of transmissible gastroenteritis viral antibodies by immunodiffusion antigenic relationships amongst eoronaviruses a virus related to that causing hepatitis in mice (mi-iv) studies on the infectious bronchitis virus of chickens isolated in finland a hemagglutinating virus producing encephalomyeiitis in baby pigs a new virus isolated from the human respiratory tract feline infectious peritonitis virus. zbl antigenic relationship between human corona virus strain c and hemagglutinating encephalomyelitis virus strain n of swine: antibody responses in human and animal sera i~ecovery in tracheal organ cultures of novel viruses from patients with respiratory disease antigenic relationship among the coronaviruses of man and between human and animal coronaviruses detection of eoronavirus infection of man by immunofluorescence seroepidemiology of feline infectious peritonitis virus infections using transmissible gastroenteritis virus as antigen rat corona virus (rcv) : a prevalent, naturally occurring pneumotropic virus of rats serologic studies of naturally occurring feline infectious peritonitis morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal ceil cultures feline infectious peritonitis: something old, something new characteristics of a coronavirus causing vomition and wasting in pigs detection of transmissible gastroenteritis virus neutralizing antibody in cats characterization of a calf diarrheal coronavirus characterization of a calf diarrheal coronavirus morphology-of transmissible gastroenteritis virus of pigs morphogenesis of a virus in cats with experimental feline infectious peritonitis untersuchungen fiber die antigenverwandtschaft der viren der felinen infekti ser peritonitis und der transmissiblen gastroenteritis des sehweines ultrastruetural evidence for the viral etiology of feline infectious peritonitis received october , key: cord- -tycuoslv authors: storz, j.; zhang, x. m.; rott, r. title: comparison of hemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: tycuoslv hemagglutinating and acetylesterase functions as well as the kda glycoprotein were present in the highly cell-culture adapted, avirulent bovine coronavirus strain bcv-l , in the norden vaccine strain derived from it, and in wild-type, virulent strains that multiplied in hrt- cells but were restricted in several types of cultured bovine cells. the bcv-l and the wild-type strain bcv-ly- agglutinated chicken and mouse erythrocytes. the acetylesterase facilitated break-down of the bcv-erythrocyte complex with chicken but only to a minimal extent with mouse erythrocytes in the receptor-destroying enzyme test. purified preparations of the vaccine and the wild-type strains agglutinated chicken erythrocytes at low titers and mouse erythrocytes at to times higher titers whereas receptor destroying enzyme activity was detectable only with chicken erythrocytes. when wild-type strains were propagated in hrt cells at low passage levels, they produced × ( ) to . × ( ) plaque forming units per µl which agglutinated erythrocytes from mice but not from chickens. diisopropylfluoro-phosphate moderately increased the hemagglutination titers, but completely inhibited the receptor destroying enzyme of purified virus of all strains. it had virtually no influence on the plaque-forming infectivity of the different bcv strains. the acetylesterase of strain bcv-l reacting in the receptor-destroying enzyme test was stable for h at and °c. it was inactivated within min at °c while the hemagglutinin function of this strain was stable for h at , , and °c, but it was inactivated at °c within h. bovine coronavirus (bcv) has a hemagglutinin (ha) for some erythrocytes with an approximate molecular mass of kda and kda in the reduced and the nonreduced forms. this structural protein composes the short spikes of the viral envelope [ , , i] . acetylesterase activity (ae) was found associated with this glycoprotein of a bcv strain isolated from calves in the netherlands and of the strain bcv-l [ , , ] . this glycoprotein is now referred to as the hemagglutinin-esterase (he) of bcv [ ] . the ae inactivates the receptors for bcv of susceptible cells by hydrolyzing an ester bond to liberate acetate from c- of sialic acid [ , , , an enzyme function first detected in the influenza c virus by herrler and coworkers [ , . the gene of the he glycoprotein of bcv is located upstream of the s gene and predicts a protein with amino acids [ , , , . as an enteropathogen bcv causes severe diarrhea in neonatal calves, and it is also considered to be etiologically involved with winter dysentery of adult cattle [ , , ] . bcv represents one of the better characterized coronaviruses with ha properties. four major structural proteins are associated with infectious bcv. the he as well as the spike or s glycoprotein ( kda) and the integral membrane glycoprotein m ( ) ( ) ( ) ( ) are associated with the viral envelope while the phosphorylated n protein ( - kda) functions as a nucleocapsid [ , , , ] . proteolytic cleavage of the s glycoprotein precursor into s and s of and kda is required for cell fusion activity [ , , . the s /$ glycoproteins facilitate virus attachment to susceptible cells, and also binding to erythrocytes, cell fusion, and induction of neutralizing antibodies [t , , , , , . the exact functions of he and s /$ and their interplay in the infectious process in vitro and in vivo are not fully defined [ , ] . the presence and function of the he was analyzed in two cell-adapted bcv strains [ , , , ] . some of the bcv isolates from winter dysentery agglutinated rodent erythrocytes and others did not [ , . the objective of our investigations was to identify the he of wild-type bcv strains at low passage levels in h r t cell cultures and to compare it with the he of prototype bcv-l and vaccine strains by relating its functions to the interaction with different erythrocytes, the effects of enzyme inhibitors, and to plaque-forming infectivity. the cell culture-adapted prototype bcv-l was originally isolated in bovine fetal kidney (bfk) cells from diarrhea fluid of a calf [ , ] . our virus strain had been passaged times in bfk cells, times in bovine fetal brain cells, times in bovine fetal spleen (bfs) cells, and to times in human rectal tumor (hrt- ) cells [ ] [ ] [ ] . five other wildtype bcv isolates, initially maintained by calf inoculation, were adapted to hrt- cells from diarrhea fluid or intestinal mucosal scrapings of calves with clinical diarrhea and electron microscopic evidence of coronavirus infection [ , ] . these strains are: bcv-ly- , bcv-c- , bcv-miller, bcv-meeker, and bcv-fisher. the vaccine strain of bcv was cultured in hrt- cells from the vaccine of norden laboratories, omaha, nebraska [ , ] . importantly, cultivation of the bcv wild-type strains remained impossible for us until it was demonstrated that the cytopathic expression of bcv-l in cultured bovine cells was enhanced by trypsin [ ] and that hrt- cells were susceptible to bcv [ ] . these wild-type strains were completely restricted in a variety of cultured bovine cells even in the presence of trypsin [ ] . the hrt- cells were cultured with dulbecco's modified earle's medium (dmem) with % bovine fetal calf serum which was omitted from cultures used for virus propagation. a plaque assay in hrt- cells overlaid with ml of . % (w/v) agarose in dmem containing gg/ml final trypsin concentration was used to measure infectivity [ , ] . samples of the bcv strains were propagated in hrt cells and purified as described [ , , ] . the bcv-l -infected cell sediment of the first step in the virus purification was suspended in . ml phosphate buffered saline (ph of . ), treated with sound times for s at a power setting of (branson sonifyer), and centrifuged at x g for min. the supernatant fluid represented the bcv-l -infected cell lysate. uninfected hrt- cells were treated the same way and were used in control tests. acetylesterase activity of purified bcv preparations was determined according to vlasak et al. [ ] . purified preparations of the different bcv strains were added in gl quantities to ml of phosphate buffered safine containing i mm p-nitrophenylacetate (pnpa). the pnpa was initially dissolved in ethyl alcohol. hydrolysis of the substrate was monitored at nm in a beckman spectrometer with multiple cuvette sets and a chart recorder. the reaction was measured at min intervals for min. the rain value was used for comparisons [ , ] . the ha test was employing . % suspensions of erythrocytes from adult chickens or mice [ ] . the tests with -fold bcv dilutions in gl quantities were initially incubated at °c for h to assess the ha titers. thereafter, the microtiter plates were shifted to °c for h to monitor inactivation of receptors reflected by the breakdown of the bcv-erythrocyte complexes mediated by the ae in the rde assay. the extent of disappearance of the bcverythrocyte aggregates was recorded in a final evaluation made following an additional to h at room temperature when unequivocal interpretation of the results was possible [ ] . diisopropylfluorophosphate (dfp) was used as an inhibitor of serine esterase activity to assess the effect of ae from different bcv strains on the ha and rde functions as well as on bcv infectivity. a sample of gl of each purified bcv strain was treated with mm of dfp in gl for rain at room temperature. the virus samples were then diluted in fold steps and tested for ha and rde activities. untreated samples were tested in parallel. similarly, gl aliquots of the purified bcv strains were treated with mm of dfp in gl for min at room temperature. samples were then mixed with ~ of pbs, and further decimal dilutions were made. the infectivity of dfp-treated and untreated bcv preparations was assayed in the plaque test [ ] . bovine submaxillary muein (bsm) was tested with -fold dilutions of the purified virus preparations by adding gl of the mg/ml bsm. alternatively, the indicated inhibitor concentrations was diluted -fold and ha units of the strains bcv-l or bcv-ly- were added. chicken erythrocytes as well as mouse erythrocytes were employed in these tests. aliquots of the bcv-l -infected cell lysate having ha and rde titers of with chicken erythrocytes were incubated at °c, °c, °c and °c. samples were withdrawn at intervals of min for h and every min for the next h to be tested for ha and rde activities. sodium dodesyl sulfate-polyacrylamide gel electrophoresis (sds-page) was performed in % slab gels under denaturing and under mildly denaturing conditions. detailed procedures for the analysis of bcv proteins by sds-page were described [ , ] . the different viral functions were assessed on purified bcv samples containing infectivities of . x t o . x pfus per p. (table ) . ae activity was detected in the pnpa test and the gp he structural protein was seen in page of all bcv strains tested. the highest ae activity was detected in the bcv-ly- strain and this strain had also the highest infectivity even though it was only in the th passage in hrt- cells. purified virus preparations of the strains bcv-l and bcv-ly- agglutinated chicken erythrocytes at titers of and , respectively, but the ha titers were and fold higher with mouse erythrocytes. in contrast, the vaccine and three wild-type bcv strains had ha titers of to with chicken erythrocytes, and to fold higher titers were recorded with mouse erythrocytes. rde activity was detected at titers of to with chicken erythrocytes whereby h a : rde ratios of : were evident for the strains bcv-meeker, bcv-fisher and bcv-calf- . the rde activity was minimal or not detectable in tests involving mouse erythrocytes. the pfus per ha unit ranged from . × to . x for chicken erythrocytes and x to . x for mouse erythrocytes (table ) . the ha pattern of the highly cell-culture adapted and the wild-type bcv strains differed. the bcv-l strain was in the th cell culture passage while the wildtype strains had been passed only to times in hrt- cells. strains bcv-l and bcv-ly- agglutinated chicken erythrocytes while the other bcv strains did not ( table ). all strains agglutinated mouse erythrocytes. the infectivity of the preparations ranged from x to . x pfus. rde activity for receptors on chicken erythrocytes was detected in samples from bcv-l and bcv-ly- . the bcv infectivity was virtually unaffected by d f p (table ). in contrast, the p d e activity for chicken erythrocytes was completely inhibited. the h a titers for chicken erythrocytes were to times higher after d f p treatment. the r d e activity for mouse erythrocytes was eliminated by d f p and an enhancing effect on h a was not noticed. bsm inhibited h a by bcv-l and the vaccine strain, while it reduced the h a titers of the other strains for chicken erythrocytes (table ). mouse erythrocytes became aggregated in the control tests by bsm at the concentration used. the bcv-l -infected cell lysate was used to assess thermal inactivation of the h a and r d e functions for chicken erythrocytes. as documented in figs. and these activities remained unaffected at temperatures of °c and °c for h. the r d e titer was reduced from to < within min at °c while h a remained constant. this activity was lost within min at °c. all bcv strains tested had ae activities when purified preparations were assayed in the p n p a test [ ] . this enzyme was inhibited by the serine esterase inhibitor d f p when assayed in the ae-mediated breakdown of bcv-chicken erythrocyte complexes yet the h a titers of purified preparations of wild-type bcv strains were increased to fold at °c. the infectivity of identically dfp-treated table . (table ). these results indicate that the ae of the bcv is probably not directly involved in viral uptake during the infectious process. receptor binding and viral attachment to susceptible cells in infections of non-ha and ha coronaviruses are mediated by the s glycoprotein [ ] . the he protein is not expressed in some murine coronavirus strains [ ] . our finding contrasts a previous report of a -fold reduction of infectivity by dfp of another bcv strain plaque-assayed in madin-darby bovine kidney cells [ ] . infectivity assayed by the plaque test does not address the potential significance of ae in viral spread within an infected cell culture or animal. the ae activity of the he protein may play a role in facilitating virus release from infected cells and viral spread. bcv particles were seen adsorbed abundantly to the plasmalemma or microvilli. adsorbed bcv covered the entire exposed surfaces of enterocytes in infections of calves [ ] and hrt cells [i ] . further insight into the role of he will be gained through investigations of the interactions of he with cellular receptors in vitro as well as with the neuraminidate-containing glycokalyx protecting mucous membranes of the respiratory and intestinal tracts of hosts. the strongly inhibitory function of bsm on ha points in this direction (table ) . one difference between the bcv-l and bcv-ly- strains and the vaccine, meeker, fisher and calf- strains involves their interaction with different erythrocytes. chicken erythrocytes were agglutinated by bcv-l and bcv-ly- and the rde functioned. these strains also agglutinated mouse erythrocytes, but elution through rde activity was minimal. the approximately -fold concentrated purified preparations of the other bcv strains agglutinated chicken erythrocytes at low titers which were increased by dfp. the ha titers with mouse erythrocytes were to fold higher, but virtually no rde function was detectable (table ) . this difference was further substantiated x v x "" x x in ha tests with fluids from hrt- cell cultures infected with the different bcv strains at tow passages. the vaccine, meeker, fisher, calf and miller bcv strains agglutinated mouse but not chicken erythrocytes at infectivity yields of x to . x pfu s/ gl (table ). the difference in the ha pattern of the avirulent and virulent strains may be determined by straindependent receptor binding properties, or possible differences between neu , acz-containing receptors on chicken and mouse erythrocytes, or the greater abundance of receptors on mouse erythrocytes [ ] . further differences between the avirulent strain bcv l and the virulent strains are host cell restriction [ , ] , epitopes for s-specific monoclonal antibodies [ ] , and amino acid substitutions among the s and he proteins [ , ] . adaptation of wild-type bcv strains to cell cultures and the associated viral selection clearly involve significant genetic and functional changes. the difference in thermal sensitivity separated ae or r d e activity from the h a function. these studies do not define the structural proteins of bcv involved in ha. monoclonal antibodies against h e as well as against s inhibited h a by bcv-lg. the activity of anti he monoclonal antibodies was predominately against r d e [ ] . purified h e of bcv-l had a e activity but agglutinated only mouse erythrocytes while purified s was a more powerful h a for chicken and mouse erythrocytes [ ] . the functions of s and he, and their interplay in receptor binding, ha, and the infectious process of bcv need to be defined. cell culture propagation of a coronavirus isolated from cows with winter dysentery recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas, and genes of coronaviruses structural proteins of bovine coronavirus and their intracellular processing morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves the receptordestroying enzyme of influenza c is virus neuraminidate-o-acetyl-esterase comparison of bovine coronavirus (bvc) antigens: monoclonal antibodies to glycoprotein gp distinguish between vaccine and wild-type strains structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein bovine coronavirus structural proteins bovine coronavirus hemagglutinin protein neonatal calf diarrhea: propagation, attenuation, and characteristics of coronavirus-like agents hemagglutinating and acetylesterase activities of bovine coronavirus strains cloning and in vitro expression of the gene for the e hemagglutinin glycoprotein of bovine coronavirus scanning electron microscopic characterization of bovine coronavirus plaques in hrt cells winter dysentery in adult dairy cattle: detection of coronaviruses in the feces hemagglutinating encephalomyelitis virus attaches to n-acetyt- - -acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza c virus isolated he protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity the s protein of bovine coronavirus is a hemagglutinin recognizing - -acetylate sialic acid as a receptor determinant characterization of a calf diarrheal coronavirus coronaviruses: structure and genome expression structural proteins of bovine coronavirus strain l : effects of the host cell and trypsin treatment bovine coronavirus-induced cytopathic expression and plaque formation: host cell and virus strain determine trypsin dependence enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment on enteropathogenic bovine coronavirus monoclonal antibodies differentiate between the hemagglutinating and the receptordestroying activities of bovine coronavirus the molecular biology of coronaviruses proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum the e protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity human and bovine coronaviruses recognizes sialic acid-containing receptors similar to those of influenza c viruses structural polypeptides of the murine coronavirus jhm hemaggtutinating and acetylesterase activities in bcv strains ture, and biological activities of envelope protein gp ofmurine coronavirus comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent and avirulent strains of bovine coronaviruses the hemagglutinin/esterase glycoproteins of bovine coronaviruses: sequence and functional comparisons between virulent and avirulent strains we thank dr. georg herrler for enhancing discussions and help with the acetylesterase tests. the dedication and excellent technical assistance of eva kroell is greatly appreciated. these investigations were made possible through a sabbatical leave in the institut ffir virologie, justus liebig universitfit, giessen, granted to j. s. by louisiana state university, a reinvitation grant by the alexander von humboldt foundation, through research grants -csrs- - and - - from the united states department of agriculture, and a grant from the louisiana education quality support fund. received august , key: cord- - b rgqa authors: xie, qian; cao, yujuan; su, juan; wu, jie; wu, xianbo; wan, chengsong; he, mingliang; ke, changwen; zhang, bao; zhao, wei title: two deletion variants of middle east respiratory syndrome coronavirus found in a patient with characteristic symptoms date: - - journal: arch virol doi: . /s - - -x sha: doc_id: cord_uid: b rgqa significant sequence variation of middle east respiratory syndrome coronavirus (mers cov) has never been detected since it was first reported in . a mers patient came from korea to china in late may . the patient was years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with mers, and had had close contact with individuals with confirmed cases of mers.after one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. a nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of mers cov. deletions of and nt occurred between orf and the e protein, resulting in a partial protein fusion or truncation of orf and the e protein. functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package mers cov. however, the mechanism of how these deletions occurred and what effects they have need to be further investigated. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. a nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of mers cov. deletions of and nt occurred between orf and the e protein, resulting in a partial protein fusion or truncation of orf and the e protein. functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package mers cov. however, the mechanism of how these deletions occurred and what effects they have need to be further investigated. qian middle east respiratory syndrome coronavirus (mers cov) has been reported in more than countries [ ] since the first case was identified in [ ] . infection with this virus leads to a mortality rate of about %, but its origin is still not known [ ] [ ] [ ] [ ] [ ] . mers cov belongs to lineage c of the betacoronaviruses and has a single-stranded, positivesense, . -kb rna genome. the viral genomic rna encodes four structural proteins, i.e., spike glycoprotein (s), envelope (e), matrix (m) and nucleocapsid (n), as well as several nonstructural proteins, including orf - and orf b [ ] . recently, individuals were confirmed to be infected with mers cov in korea. during the epidemic, one person who was in close contact with a mers cov patient started to show mers symptoms shortly after he traveled to guangdong province of china and was confirmatively diagnosed with mers cov by lab tests. the patient was cured after days of treatment with antiviral, anti-infection, and immune-enhancing agents. in order to better understand the transmission and evolution of this virus [ ] , viral rna was isolated from a nasopharyngeal swab sample of the korean patient and sequenced. in addition to the wild-type (wt) virus, two deletion variants of mers cov were detected in this patient. the cdna was amplified using pairs of primers (supplemental table ). each fragment amplified by rt-pcr was about bp in length. after electrophoresis, pcr products were recovered using a pcr purification kit and sequenced on an ab sequencer (life technologies, guangzhou, china). the sequences obtained from pcr products were assembled into a fulllength genome sequence using dnastar (version . , dnastar inc., madison, wi, usa). [ ] . rna was extracted from nasopharyngeal swab specimens collected on days , , , and after onset of fever. reverse transcription of rna into cdna was performed as described previously. the cdna was used as the template for pcr amplification with la-taq mix (takara) and primer pair no. . pcr products were analyzed by % agarose gel electrophoresis. protein sequences were aligned using mega (version . ) [ ] . transmembrane software was used to predict the transmembrane domain of the orf protein (http://www.cbs.dtu.dk/services/ tmhmm/) [ ] . rna secondary structure was predicted using rnafold software, available at http://rna.tbi.uni vie.ac.at/cgi-bin/rnafold.cgi [ ] . all products yielded usable sequences except those produced using primer pair no. . two specific products obtained by nested pcr (fig. a) were purified, cloned and sequenced. the lower-molecular-weight band was composed of two variants that differed by bp. variant was longer than variant , with the sequence tatgg adjacent to the sequence ctcatgg). the upper band (wt) was bp longer than variant after the sequence ctcatggtatgg. all fragments of the sequences were assembled into three contigs of wt, variant and variant . the genomic sequences have been uploaded to genbank as kt [ ] , kt and kt , and the main differences in their nucleotide sequences are shown in fig. b . the predicted changes in the primary structures of the orf and e proteins are shown in (fig. c) . variant encodes a fusion protein of the orf and e proteins (orf -e) with an -amino-acid (aa) deletion at the c-terminus of orf and a -aa deletion at the n-terminus of the e protein. variant encodes two truncated proteins: a -aa fragment of the n-terminus of orf with an additional aa (fpygy), and a -aa fragment of the c-terminus of the e protein. until now, no such variant has been found in the ncbi database. although the function of the s protein has been examined previously [ ] [ ] [ ] [ ] [ ] , our knowledge of orf and e protein functions in mers cov is limited [ ] . moreover, the effects of orf and e protein mutations on viral packaging, infection and disease development have not been evaluated. based on studies of other coronaviruses, it is believed that the e protein is important for virus packaging and replication [ ] [ ] [ ] . the conserved hydrophobic transmembrane n-terminal domain of the e protein is necessary for cov to be implanted in the membrane. even single point mutations in the transmembrane protein of the infectious bronchitis virus (ibv) e protein [ ] , or amino acid changes in the n-terminus of the sars-cov e protein can result in attenuation of virulence [ ] . to predict the function of the e protein of mers cov, we aligned the e and orf -e protein sequences of mers cov with those of two other coronaviruses, sars-cov and china rattus coronavirus hku , using mega software (version . ) [ ] . the results showed that the e protein of mers cov shares high similarity with the other two coronavirus ( % for sars cov; % for hku cov) in the n-terminal, c-terminal and transmembrane domains (fig. a) . the truncated e protein with a deletion of aa - lacks the n-terminus and a major part of the hydrophobic transmembrane domain in mers cov variant , which might directly impair virus packaging and replication [ ] . the putative fusion orff -e protein (fig. b ) encoded by variant is predicted to have three transmembrane regions (transmembrane hidden markov models [ ] ), and it remains unclear whether it is able to function like the wildtype e protein. almazán et al. reported that mers cov with a deletion in the e gene produced replication-competent but propagation-defective virus particles and proposed that this defective virus should be a potential vaccine candidate for preventing mers cov infection [ ] . the two variants identified in this study carried mutations in the n-terminal domain, which is dispensable for the function of the e protein. however, variations in this region lead to changes in the location of this protein, and therefore, the virulence of these two variants might be impaired to some extent. this needs to be investigated using a recombinant virus. the orf gene of both variants of mers cov in this study was truncated and fused with the e protein. the effect of these variations on the virus could not be predicted because the function of the orf gene is not well understood. however, scobey et al. found that the effect of orf deletions on the viral replication is minimal, but deletion of the whole orf gene significantly enhances s protein expression [ ] . more investigations are required to determine the effects of the orf mutant in these two variants. intragenomic sequence deletions have been found in some coronavirus [ , ] . it has been proposed that this occurs by a copy-choice or template-strand-switching mechanism [ ] . one important condition is for there to be a specific leader sequence flanked by the deletion region and a stem-loop structure [ ] . leader sequences corresponding to the ucuaaac sequence of murine hepatitis virus (mhv) or the cuuaaca sequence of infectious bronchitis virus (ibv) were not found in mers cov in this study. maori et al. have found that inverted repeats facilitate looping out of the middle genomic sequences during rna replication, resulted in a defective rna genome [ ] . an rna secondary structure predicted using the rnafold webserver [ ] suggested that the inverted repeat sequence contains long complementary sequences at each end and forms a strong stem-loop structure in the deletion region (fig. c) . the deleted sequence was closely adjoined, characterized by a -bp nearly complete inverted repeat sequence consisting of -gtcata-cacaccaa- and -ttggtgtgtatggc- , which would result in rna replicase jumping from one segment to another distant segment. whether this feature is linked to rna intramolecular recombination remains to be investigated. wild-type mers cov and two variants were isolated for the first time from a patient who had traveled from korea to china. genomic sequencing revealed -bp and -bp deletions between orf and the e protein that would result in partial fusion or truncation of these proteins. whether this finding is a special case or not needs to be investigated by sequencing more samples. based on previous studies of e protein localization [ - , , ] , we conclude that the two variants might affect virus packaging, which could result in the attenuation of virulence and therefore be relevant for studies related to vaccine development, pathogenesis and viral evolution. zx - . conflict of interest the author of this study has no conflict of interest. ethical approval the mers patient was informed of an urgent clinical test. this study was approved by the medical ethics committee of southern medical university on may and is in accordance with the helsinki declaration and its later amendments or comparable ethical standards. open access this article is distributed under the terms of the creative commons attribution . international license (http://crea tivecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. an update on middle east respiratory syndrome: years later severe respiratory illness caused by a novel coronavirus mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in bats, saudi arabia close relative of human middle east respiratory syndrome coronavirus in bat evidence for camel-tohuman transmission of mers coronavirus lack of middle east respiratory syndrome coronavirus transmission from infected camels the input sequence was based on kt , nt - 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