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PubMed1994

Nucleotide sequence and expression of the spike (S) gene of canine coronavirus and comparison with the S proteins of feline and porcine coronaviruses.

We have cloned, sequenced and expressed the spike (S) gene of canine coronavirus (CCV; strain K378). Its deduced amino acid sequence has revealed features in common with other coronavirus S proteins: a stretch of hydrophobic amino acids at the amino terminus (the putative signal sequence), another hydrophobic region at the carboxy terminus (the membrane anchor), heptad repeats preceding the anchor, and a cysteine-rich region located just downstream from it. Like other representatives of the same antigenic cluster (CCV-Insavc-1 strain, feline infectious peritonitis and enteric coronaviruses, porcine transmissible gastroenteritis and respiratory coronaviruses, and the human coronavirus HCV 229E), the CCV S polypeptide lacks a proteolytic cleavage site present in many other coronavirus S proteins. Pairwise comparisons of the S amino acid sequences within the antigenic cluster demonstrated that the two CCV strains (K378 and Insavc-1) are 93.3% identical, about as similar to each other as they are to the two feline coronaviruses. The porcine sequences are clearly more divergent mainly due to the large differences in the amino-terminal (residues 1 to 300) domains of the proteins; when only the carboxy-terminal parts (residues 301 and on) are considered the homologies between the canine, feline and porcine S polypeptides are generally quite high, with identities ranging from 90.8% to 96.8% . The human coronavirus is less related to the other members of the antigenic group. A phylogenetic tree constructed on the basis of the S sequences showed that the two CCVs are evolutionarily more related to the feline than to the porcine viruses. Expression of the CCV S gene using the vaccinia virus T7 RNA polymerase system yielded a protein of the expected M(r) (approximately 200K) which could be immunoprecipitated with an anti-feline infectious peritonitis virus polyclonal serum and which was indistinguishable from the S protein synthesized in CCV-infected cells.

Amino Acid Sequence↗
PubMed1994

Identification of a putative cellular receptor for bovine herpesvirus 1.

Recognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.

Animals↗
PubMed1996

Characterization of functional domains in the human coronavirus HCV 229E receptor.

Human aminopeptidase N (hAPN or CD13) and porcine aminopeptidase N (pAPN) are functional receptors for human coronavirus (HCV) 229E and porcine transmissible gastroenteritis virus (TGEV), respectively. However, hAPN cannot function as a receptor for TGEV and pAPN cannot function as a receptor for HCV 229E. In this study, we constructed a series of chimeric hAPN/pAPN genes and expressed the corresponding proteins in transfected cells. Subsequently, we identified the chimeric proteins that can function as a receptor for HCV 229E. The results show that replacement of a small region of pAPN sequence (pAPN amino acids 255-348) with the corresponding hAPN sequence (hAPN amino acids 260-353) converts pAPN into a functional receptor for HCV 229E. The region of hAPN that we have defined in this way does not correspond to the region of pAPN that has been identified as essential for the TGEV-receptor interaction. We conclude that although both viruses use a homologous receptor protein, different regions of the protein are required to mediate susceptibility to infection with HCV 229E and TGEV.

Animals↗
PubMed1997

Mouse hepatitis virus strain A59 is released from opposite sides of different epithelial cell types.

Coronaviruses infect humans and animals through epithelial cells of the gastrointestinal and respiratory tracts that serve as their primary target. When studying infections in cultured polarized epithelial cells, we found previously that coronaviruses are released from specific plasma-membrane domains; thus, mouse hepatitis virus (strain A59; MHV-A59) leaves murine epithelial kidney cells from the basolateral surface, whereas release of transmissible gastroenteritis virus from porcine epithelial kidney cells is confined to the apical membrane. This observation begged the question whether a particular coronavirus is consistently shed through the same membrane, irrespective of the nature of the epithelial cell. We therefore extended our studies with MHV-A59 to Madin-Darby canine kidney (MDCK) strain I and human colon carcinoma (Caco-2) cells, both of which are naturally refractory to MHV-A59 but were made susceptible to infection by transfection with recombinant MHV receptor cDNA. The release of MHV-A59 from Caco(MHVR) cells occurred preferentially from the basolateral side, consistent with our previous observations. In contrast, release from MDCK(MHVR) cells occurred almost exclusively from the apical surface. Because of this difference, we studied MHV-A59 infection of MDCK(MHVR) cells in more detail. The virus entered the cells preferentially from the apical side, a situation similar to that in murine epithelial cells, where the highest density of MHV receptor glycoprotein was found. The results from this and previous studies show that targeting of vesicles containing MHV-A59 to a specific side of epithelial cells may vary in different epithelial cell types.

Animals↗
PubMed1997

Identification of residues critical for the human coronavirus 229E receptor function of human aminopeptidase N.

Aminopeptidase N (APN) is the major cell surface receptor for group 1 coronaviruses. In this study, we have isolated and characterized a feline APN cDNA and shown that the transfection of human embryonic kidney cells with this cDNA renders them susceptible to infection with the feline coronavirus feline infectious peritonitis virus, the human coronavirus (HCV) 229E and the porcine coronavirus porcine transmissible gastroenteritis virus. By using chimeric APN genes, assembled from porcine and feline sequences, we have shown that, analogously to the human APN protein, a region within the amino-terminal part of the feline APN protein (encompassing amino acids 132-295) is essential for its HCV 229E receptor function. Furthermore, by comparing the relevant feline, human and porcine APN sequences, we were able to identify a hypervariable stretch of eight amino acids that are more closely related in the feline and human APN proteins than in the porcine APN molecule. Using PCR-directed mutagenesis, we converted this stretch of amino acids within the porcine APN molecule to the corresponding residues of the human APN molecule. These changes were sufficient to convert porcine APN into a functional receptor for HCV 229E and thus define a small number of residues that are critically important for the HCV 229E receptor function of human APN.

Amino Acid Sequence↗
PubMed1999

A novel internal open reading frame product expressed from a polycistronic mRNA of porcine epidemic diarrhoea virus may not contribute to virus attenuation.

Cell-culture-adapted (ca) porcine epidemic diarrhoea virus (PEDV) contains three internal open reading frames (I ORF) within the nucleocapsid protein gene and lacks the downstream counterpart of porcine transmissible gastroenteritis virus ORF7 or feline infectious peritonitis virus ORF6a. To confirm whether such features also exist in wild-type (wt) PEDV, the 3' 1800 nucleotides of its genome were sequenced and were found to be identical to those of ca virus. The coding potential of I-1 ORF was ascertained by transient expression in Vero cells followed by immunofluorescence using antipeptide sera. The I-1 protein was synthesized as a 12 kDa non-phosphorylated PEDV-specific protein that was not present in detectable amounts in virions. However, a low copy number of I-1 in the virion would suggest it is a structural component. Nevertheless, identical nucleotide sequences and gene expression strategies of attenuated ca virus and its virulent parent, wt PEDV, demonstrate that the 3' 1800 nucleotides or the genes and gene products encoded therein may not contribute to virus attenuation.

Amino Acid Sequence↗
PubMed2002

Conservation of substrate specificities among coronavirus main proteases.

The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CL(pro). The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CL(pro) cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CL(pro) cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.

Amino Acid Sequence↗
PubMed1976

The isolation of reovirus-like agents (rota-viruses) from acute gastroenteritis of piglets.

Isolations of reovirus-like agents (rotaviruses) were made from nine of 23 outbreaks of piglet diarrhoea on different farms and from both weaned and unweaned piglets. The viruses were shown to be morphologically and anti-genically similar to the rotaviruses of children and calves. Gnotobiotig piglets given intranasal inoculations of five different isolates developed acute gastroenteritis, and the virus was re-isolated from the faeces or intestinal contents. The piglet virus was not adapted to replicate in cell culture. We conclude that the pig rotavirus is commonly associated with outbreaks of gastroenteritis and is probably an important aetiological factor in this disease.

Animals↗
PubMed2006

Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA.

Porcine circovirus type 2 (PCV2) infection of natural interferon producing cells (NIPCs) impairs the induction of interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha by cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides (ODNs), thereby preventing both their autocrine maturation and the paracrine maturation of myeloid dendritic cells (DCs). The present study shows that the PCV2-mediated inhibition of NIPCs was mediated by viral DNA, although it was independent of virus replication. The inhibitory effect of PCV2 DNA was more diversified than if it had simply targeted CpG-ODN-induced cytokines (IFN-alpha, TNF-alpha, interleukin-6, IL-12). A broad spectrum inhibition was noted, affecting responses induced by toll-like receptor (TLR)-7 and TLR9 agonists, as well as viruses including pseudorabies virus, transmissible gastroenteritis virus and classical swine fever virus. From these results, it would appear that PCV2 DNA can induce a dominant negative signal influencing independent pattern recognition receptor-induced activation cascades. Despite a concomitant internalization of PCV2 DNA and CpG-ODNs, no colocalization was observed, indicating that PCV2 DNA and CPG-ODNs may not target the same receptor. This study describes a novel modulation of the innate immune response, which would render the host more susceptible to secondary or concomitant microbial infections.

Animals↗
PubMed1991

Experimental induction of diarrhoea in newly-weaned piglets.

To induce diarrhoea and hypovolaemia, newly-weaned conventionally bred piglets (3- to 4-weeks-old), were either given secretagogues or were inoculated with enterotoxigenic Escherichia coli (ETEC). Choleratoxin (n = 2), E. coli heat-labile enterotoxin (n = 2) or castor oil (n = 2) were given intragastrically or piglets were intraperitoneally injected with DL 5-hydroxytryptophan (n = 3). These substances induced a transient diarrhoea without clinical symptoms of dehydration. Therefore, a combination of castor oil and DL 5-hydroxytryptophan was given two times a day during 3 consecutive days to 3 piglets. Although diarrhoea lasted for 5 days, still no hypovolaemia occurred. Probably the secretagogues have to be given continuously to mimic the continuous release of enterotoxins during secretory colibacillary diarrhoea. It was, therefore, tried to reproduce colibacillosis in the just-weaned piglets. Animals were inoculated with K88ac fimbriae producing ETEC strains (O149:K91:K88ac; LT, STa and STb positive) (n = 7), or pretreated with chloramphenicol followed by the ETEC inoculation (n = 8), or pretreated with the antibiotic, inoculated with an enteropathogenic coronavirus, transmissible gastroenteritis virus (TGEV), and subsequently inoculated with ETEC (n = 18). Only the last procedure induced a reproducible diarrhoea (93%) and dehydration resulting in a mortality of 80%. It was concluded that the latter experimental procedure could be used to study diarrhoea and hypovolaemia in newly-weaned piglets and to evaluate the effect of potentially antisecretory drugs on postweaning diarrhoea in piglets.

Animals↗
PubMed2006

A serological survey of selected pathogens in wild boar in Slovenia.

Serum samples collected from 178 shot wild boars (Sus scrofa) were tested for the presence of antibodies against classical swine fever virus, Aujeszky's disease virus (ADV), porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, swine influenza virus, porcine parvovirus (PPV), swine vesicular disease virus, Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella spp., Brucella spp. and Haemophilus parasuis (HPS) throughout Slovenia during the hunting season 2003/2004. The number of samples corresponds to 3% of the total hunting bag. By enzyme-linked immunosorbent assay (ELISA) antibodies against ADV were detected in 55 sera (31%), against PRCV in five sera (3%), PPV in 87 sera (49%), APP in 93 sera (52%), M. hyopneumoniae in 38 sera (21%), Salmonella spp. in 85 sera (47%) and HPS in 33 sera (18%).

Animals↗
PubMed2005

Feline coronavirus serotypes 1 and 2: seroprevalence and association with disease in Switzerland.

To determine the prevalence of antibodies to feline coronavirus (FCoV) serotypes 1 and 2 in Switzerland and their association with different disease manifestations, a serological study based on immunofluorescence tests was conducted with Swiss field cats using transmissible gastroenteritis virus (TGEV), FCoV type 1 and FCoV type 2 as antigens. A total of 639 serum samples collected in the context of different studies from naturally infected cats were tested. The current study revealed that, with an apparent prevalence of 83%, FCoV serotype 1 is the most prevalent serotype in Switzerland. FCoV type 1 viruses induced higher antibody titers than FCoV type 2, and were more frequently associated with clinical signs and/or feline infectious peritonitis. The antibody development in seven cats experimentally infected with FCoV type 1 revealed that, with progressing duration of infection, antibodies to FCoV type 1 significantly increased over those to FCoV type 2. There was a significant relationship between antibody titers against TGEV, FCoV 1, and FCoV 2 and TGEV antigen detected the highest proportion of seropositive cats. We conclude that a vaccine against FCoV should be based on FCoV type 1-related antigens and that for serodiagnosis of FCoV infection TGEV should be used to attain the highest diagnostic efficiency. When serology is used in addition to clinical signs, hematology, and clinical chemistry results as an aid to diagnose clinical FIP, TGEV shows a diagnostic efficiency equal to that of a FCoV antigen.

Animals↗
PubMed1974

Porcine parvovirus: natural and experimental infections of the porcine fetus and prevalence in mature swine.

Antibodies against porcine parvovirus were detected in 17 of 116 prenursing pig sera. Antibodies against transmissible gastroenteritis or ECPO-6 (an enterovirus) were not detected in prenursing sera of the pigs tested. Seventy-seven percent of 129 serum samples from 23 Ohio farms and 82% of 96 samples from slaughter plants in Ohio were serologically positive for porcine parvovirus. Mummies or other abnormalities were not observed in newly born pigs exposed to porcine parvovirus by the transuterine route 101 days after gestation. Indirect evidence suggested that the virus had not spread to other fet uses following exposure after 101 days at least not in a sufficient amount of time to stimulate detectable antibody. Direct intrafetal exposure to porcine parvovirus (i.m. injection, transutero) after 62 days of gestation resulted in dealth and mummification of the two fetuses, and apparently in the subsequent spread of the virus, as five of nine live pigs born were serologically positive for porcine parvovirus and these five pigs had not been injected with the virus. Immunoglobulin G was detected in all newborn pigs irregardless of known antigenic stimulation or the presence of specific antibody. In general, the presence of immunoglobulin M or immunoglobulin A in fetal serum was correlated with a history of antigenic stimulation or the presence of detectable antibody.

Animals↗
PubMed1976

Reovirus-like agent associated with fatal diarrhea in neonatal pigs.

Large numbers of a reovirus-like agent were visualized with electron microscopy in bacteria-free gut homogenates obtained from piglets with a fatal diarrhea resembling transmissible gastroenteritis. The syndrome, of vomiting, diarrhea, dehydration, and death, was reproduced in piglets artificially infected with these bacteria-free gut homogenates. Reovirus-like particles persisted in serial piglet passage and none was seen in uninfected, asymptomatic controls. Hyperimmune sera (made in recovered piglets) aggregated the reovirus-like particles, as judged by immunoelectron microscopy, and neutralized the infectious agent. The cytoplasm in enterocytes on infected intestinal epithelium fluoresced when this hyperimmune sera was used in an indirect fluorescent antibody test. Feeding cow colostrum or diets containing porcine gamma globulin protected infected piglets. No cytopathogenic effect was noted in infected tissue cultures, nor did this agent affect neonatal guinea pigs, hamsters, mice, and rats. The agent did not agglutinate human O or A erythrocytes.

Age Factors↗
PubMed1990

Serotypic differentiation of group A rotaviruses with porcine rotavirus gene 9 probes.

The serotypic specificities of Gottfried and OSU porcine rotavirus gene 9 probes were investigated in a dot hybridization assay. The probes were reacted with homologous and heterologous serotypes of group A rotaviruses of human and animal origin. Hybridizations were conducted under relatively low-stringency (52 degrees C, no formamide, 5 x SSC) and high-stringency (52 degrees C, 50% formamide, formamide, 5 x SSC) conditions (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Under conditions of relatively low stringency, the Gottfried and OSU gene 9 probes demonstrated broad cross-reactivity and were useful in the detection of homologous and heterologous serotypes of group A rotaviruses. Under conditions of relatively high stringency, the Gottfried and OSU gene 9 probes were serotype specific. The Gottfried gene 9 probe (serotype 4) hybridized with homologous Gottfried porcine rotavirus as well as the serotype 4 human rotaviruses ST3 and VA70. The OSU gene 9 probe (serotype 5) hybridized with homologous OSU porcine rotavirus and the serotype 5 equine rotavirus H1. Hybridization was not observed with the antigenically distinct group B and C porcine rotaviruses or with other porcine enteric viruses, including calicivirus and a coronavirus, transmissible gastroenteritis virus, regardless of stringency conditions. Analysis of 14 group A rotavirus-positive field samples resulted in the serotypic differentiation, collectively, of six serotype 4 or 5 porcine rotaviruses. No field samples reacted with both the Gottfried and OSU gene 9 probes.

Animals↗
PubMed1990

Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis.

Both genomic and subgenomic replicative intermediates (RIs) and replicative-form (RF) structures were found in 17CL1 mouse cells that had been infected with the A59 strain of mouse hepatitis virus (MHV), a prototypic coronavirus. Seven species of RNase-resistant RF RNAs, whose sizes were consistent with the fact that each was derived from an RI that was engaged in the synthesis of one of the seven MHV positive-strand RNAs, were produced by treatment with RNase A. Because the radiolabeling of the seven RF RNAs was proportional to that of the corresponding seven positive-strand RNAs, the relative rate of synthesis of each of the MHV positive-strand RNAs may be controlled by the relative number of each of the size classes of RIs that are produced. In contrast to alphavirus, which produced its subgenome-length RF RNAs from genome-length RIs, MHV RF RNAs were derived from genome- and subgenome-length RIs. Only the three largest MHV RF RNAs (RFI, RFII, and RFIII) were derived from the RIs that migrated slowest on agarose gels. The four smallest RF RNAs (RFIV, RFV, RFVI, and RFVII) were derived from RIs that migrated in a broad region of the gel that extended from the position of 28S rRNA to the position of the viral single-stranded MHV mRNA-3. Because all seven RIs were labeled during very short pulses with [3H]uridine, we concluded that the subgenome-length RIs are transcriptionally active. These findings, with the recent report of the presence of subgenome-length negative-strand RNAs in cells infected with porcine transmissible gastroenteritis virus (P. B. Sethna, S.-L. Hung, and D. A. Brian, Proc. Natl. Acad. Sci. USA 86: 5626-5630, 1989), strongly suggest that coronaviruses utilize a novel replication strategy that employs the synthesis of subgenomic negative strands to produce subgenomic mRNAs.

Animals↗
PubMed1991

Minus-strand copies of replicating coronavirus mRNAs contain antileaders.

The 5' leader sequence on mRNAs of the porcine transmissible gastroenteritis coronavirus was determined and found to be 90 nucleotides in length. An oligodeoxynucleotide with a sequence from within the leader was used as a probe in Northern analysis on RNA from infected cells, and an antileader (a minus-strand copy of the leader sequence) was shown to be present on all mRNA minus-strand species. RNase protection analysis showed the antileader to be approximately the same length as the leader. The kinetics of antileader appearance was the same as that for the appearance of minus-strand RNA species. This, along with a demonstration that viral mRNAs become packaged, gives further support to the idea that coronavirus mRNAs can undergo replication via subgenomic mRNA-length replicative intermediates, and that input mRNAs from infecting virions may serve as initial templates for their own replication. In this sense, then, coronaviruses behave in part like RNA viruses with segmented genomes.

Animals↗