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Martin Petric - One of the best experts on this subject based on the ideXlab platform.
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the complete sequence of the Bovine Torovirus genome
Virus Research, 2006Co-Authors: Ryan Draker, Martin Petric, Rachel L. Roper, Raymond TellierAbstract:Viruses in the family Coronaviridae have elicited new interest, with the outbreaks caused by SARS-HCoV in 2003 and the recent discovery of a new human coronavirus, HCoV-NL63. The genus Torovirus, within the family Coronaviridae, is less well characterized, in part because Toroviruses cannot yet be grown in cell culture (except for the Berne virus). In this study, we determined the sequence of the complete genome of Breda-1 (BoTV-1), a Bovine Torovirus. This is the first complete Torovirus genome sequence to be reported. BoTV-1 RNA was amplified using long RT-PCR and the amplicons sequenced. The genome has a length of 28.475 kb and consisted mainly of the replicase gene ( approximately 20.2 kb) which contains two large overlapping ORFs, ORF1a and ORF1b, encoding polyproteins pp1a and pp1b, respectively. Sequence analysis identified conserved domains within the predicted sequences of pp1a and pp1b. Sequence alignments and protein secondary structure prediction data suggest the presence of a 3C-like serine protease domain with similarity to the arterivirus 3C-like serine protease and a single papain-like cysteine protease domain with similarity to the picornavirus leader protease. The ADRP (APPR-1'') domain - unique to the Coronaviridae - was also located in BoTV pp1a. In addition, several hydrophobic domains were identified that are typical of a nidovirus replicase. Within the pp1b sequence the polymerase and helicase domains were identified, as well as sequences predicted to be involved in ribosomal frameshifting, including the conserved slippery sequence UUUAAAC and two potential pseudoknot structures.
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The complete sequence of the Bovine Torovirus genome.
Virus Research, 2006Co-Authors: Ryan Draker, Martin Petric, Rachel L. Roper, Raymond TellierAbstract:Abstract Viruses in the family Coronaviridae have elicited new interest, with the outbreaks caused by SARS-HCoV in 2003 and the recent discovery of a new human coronavirus, HCoV-NL63. The genus Torovirus , within the family Coronaviridae , is less well characterized, in part because Toroviruses cannot yet be grown in cell culture (except for the Berne virus). In this study, we determined the sequence of the complete genome of Breda-1 (BoTV-1), a Bovine Torovirus. This is the first complete Torovirus genome sequence to be reported. BoTV-1 RNA was amplified using long RT-PCR and the amplicons sequenced. The genome has a length of 28.475 kb and consisted mainly of the replicase gene (∼20.2 kb) which contains two large overlapping ORFs, ORF1a and ORF1b, encoding polyproteins pp1a and pp1b, respectively. Sequence analysis identified conserved domains within the predicted sequences of pp1a and pp1b. Sequence alignments and protein secondary structure prediction data suggest the presence of a 3C-like serine protease domain with similarity to the arterivirus 3C-like serine protease and a single papain-like cysteine protease domain with similarity to the picornavirus leader protease. The ADRP (APPR-1″) domain – unique to the Coronaviridae – was also located in BoTV pp1a. In addition, several hydrophobic domains were identified that are typical of a nidovirus replicase. Within the pp1b sequence the polymerase and helicase domains were identified, as well as sequences predicted to be involved in ribosomal frameshifting, including the conserved slippery sequence UUUAAAC and two potential pseudoknot structures.
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The novel hemagglutinin-esterase genes of human Torovirus and Breda virus.
Virus Research, 1999Co-Authors: Lynn Duckmanton, Raymond Tellier, Christopher D. Richardson, Martin PetricAbstract:Abstract Human Torovirus (HTV) and Breda virus (BRV), members of the genus Torovirus in the family Coronaviridae, are established infectious agents of humans and cattle, respectively. The hemagglutinin-esterase (HE) gene of Breda virus serotype 2 (BRV-2) has been identified and the nucleotide sequence for BRV serotype 1 (BRV-1) genome which contains the open reading frames for the viral structural proteins has been reported revealing the presence of a 1.25 kb gene whose nucleotide sequence is identical to that of the BRV-2 HE gene. In this study, we amplified the 1.2kb HE gene from the HTV genome using long RT-PCR and sequenced the amplicon directly. At the nucleotide level, the HTV HE gene manifests 85% sequence identity to the HE genes of BRV-1 and BRV-2 and 89% identity with the X pseudogene sequence of BEV. The 1.25 kb amplicons which contained the HE genes of BRV-1 and HTV were cloned and expressed in a baculovirus system and the proteins purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Hyperimmune sera prepared in guinea pigs against these proteins were reactive with both Bovine Torovirus (BTV) and human Torovirus (HTV) antigens. By immunoblot, they reacted specifically with a 65 kDa protein corresponding in size to the Torovirus HE protein. Furthermore, the hyperimmune sera but not the preimmune sera reacted with a series of BTV-positive and HTV-positive fecal specimens by immunoblot and dot blot analysis. By immunoelectron microscopy (IEM) Torovirus particles from BTV-positive specimens from calves with diarrhea and HTV-positive specimens from patients were aggregated by the hyperimmune sera. Human convalescent sera and gnotobiotic calf post-infection sera reacted by immunoblot with the expressed 65 kDa protein. The expressed HE protein of HTV has important diagnostic potential.
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Detection of Bovine Torovirus in Fecal Specimens of Calves with Diarrhea from Ontario Farms
Journal of Clinical Microbiology, 1998Co-Authors: Lynn Duckmanton, Susy Carman, Éva Nagy, Martin PetricAbstract:Breda virus (BRV), a member of the genus Torovirus, is an established etiological agent of disease in cattle. BRV isolates have been detected in the stools of neonatal calves with diarrhea in both Iowa and Ohio and in several areas of Europe. However, this virus has been reported only once in Canada. Therefore, a study was performed to determine the extent to which Bovine Torovirus is present in calves with diarrhea from farms in southern Ontario. A total of 118 fecal samples from symptomatic calves and 43 control specimens from asymptomatic calves were examined by electron microscopy (EM) and reverse transcription-PCR (RT-PCR) for the presence of Torovirus. Torovirus RNA was detected in 43 of the 118 diarrheic samples (36.4%) by RT-PCR with primers designed in the conserved 3′ end of the Torovirus genome. By EM, Torovirus particles were observed in 37 of the 118 specimens (31.4%). All but one of these samples were also positive by RT-PCR. The incidence of Torovirus in the asymptomatic control specimens by RT-PCR was only 11.6%. To establish the identity of the particles observed in the diarrheic specimens, five of the amplicons from samples positive by both RT-PCR and EM were cloned and sequenced. Nucleotide sequence analysis revealed that the Bovine Torovirus found in southern Ontario manifests between 96 and 97% sequence identity to the BRV type 1 strain found in Iowa. This study shows that Bovine Torovirus is a common virus in the fecal specimens of calves with diarrhea from farms in southern Ontario and thus may be an important pathogen of cattle.
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Bovine Torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of Breda virus.
Virus Research, 1998Co-Authors: Lynn Duckmanton, Raymond Tellier, Peter P. Liu, Martin PetricAbstract:Abstract Breda virus (BRV), a member of the genus Torovirus, is an established etiological agent of diarrhea of cattle, which is found as two separate serotypes, BRV-1 and BRV-2. In this study, a 7.5 kb fragment of the BRV-1 genome that bracketed the genes for the structural proteins of BRV was amplified by long RT-PCR and the amplicon purified and sequenced directly. Sequence analysis revealed the presence of four open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, and an ORF for a novel 1.2 kb gene located between the M and N genes. This new gene was identical in nucleotide sequence to the hemagglutinin-esterase (HE) gene of BRV-2. With the exception of this new ORF, BRV-1 manifests 80% nucleotide sequence identity with the Torovirus prototype, Berne virus (BEV) in the 7.5 kb region from the 3′ end of the genome that contains the genes for the structural proteins. A 504 base segment containing the ORF for the BRV-1 N gene was amplified by RT-PCR, and cloned into an Escherichia coli expression system. The resulting protein was purified by SDS-PAGE and used to immunize guinea pigs. Hyperimmune serum was reactive with Bovine Torovirus (BTV) and human Torovirus (HTV) antigens. By immunoelectron microscopy, it was shown to aggregate broken but not intact Torovirus particles from BTV-positive fecal specimens. By immunoblot, the hyperimmune serum reacted specifically with the 20 kD N proteins of both BTV and HTV, as well as with the expressed N protein. BRV-1 and BRV-2 immune sera from gnotobiotic calves, but not human convalescent sera from HTV-infected patients, reacted with the expressed N protein by immunoblot. These findings were applied to the design of a dot blot assay that could specifically detect BTV and HTV from fecal specimens.
Fumihiro Taguchi - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth.
Journal of virology, 2021Co-Authors: Makoto Ujike, Wataru Kamitani, Yukako Kawachi, Yui Matsunaga, Yuka Etho, Hideki Asanuma, Fumihiro TaguchiAbstract:Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the Bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.
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Characterization of localization and export signals of Bovine Torovirus nucleocapsid protein responsible for extensive nuclear and nucleolar accumulation and their importance for virus growth.
Journal of Virology, 2020Co-Authors: Makoto Ujike, Wataru Kamitani, Yukako Kawachi, Yui Matsunaga, Yuka Etho, Hideki Asanuma, Fumihiro TaguchiAbstract:Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N-proteins, but little is known about ToV N-proteins. Here, we studied the subcellular localization of the Bovine ToV (BToV) N-protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with EGFP revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN-accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN predominantly accumulated in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. Understanding its unique features, BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.
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Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus
2020Co-Authors: Makoto Ujike, Y. Etoh, N. Urushiyama, Fumihiro Taguchi, Luis Enjuanes, Wataru KamitaniAbstract:Torovirus (ToV) has recently been classified in the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. Reverse genetics systems for many CoVs have been established, but none exist for ToVs. Here, we describe a reverse genetics system using a full-length infectious cDNA clone of Bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV containing genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the Hemagglutinin-esterase (HE) gene was manipulated, since cell-adapted wtBToV generally loses the full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued; these showed no significant differences in cell growth, suggesting that HE is not essential for viral growth in cells. Then, recombinant virus in which HE was replaced by the Enhanced Green Fluorescent Protein (EGFP) gene expressed EGFP in infected cells, but showed significantly reduced viral growth compared to wtBToV. Moreover, the recombinant virus readily deleted the EGFP gene after one passage. Interestingly, one variant with mutations in non-structural proteins (NSPs) showed improved EGFP expression and viral growth during serial passages, although it eventually deleted the EGFP gene, suggesting that these mutations contributed to EGFP gene acceptance. These recombinant viruses provide new insights regarding BToV and its reverse genetics will help advance understanding of this neglected pathogen.
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Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus
2020Co-Authors: Makoto Ujike, Y. Etoh, N. Urushiyama, Fumihiro Taguchi, Luis Enjuanes, Wataru KamitaniAbstract:Abstract Torovirus (ToV) has recently been classified in the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. Reverse genetics systems for many CoVs have been established, but none exist for ToVs. Here, we describe a reverse genetics system using a full-length infectious cDNA clone of Bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV containing genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the Hemagglutinin-esterase (HE) gene was manipulated, since cell-adapted wtBToV generally loses the full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued; these showed no significant differences in cell growth, suggesting that HE is not essential for viral growth in cells. Then, recombinant virus in which HE was replaced by the Enhanced Green Fluorescent Protein (EGFP) gene expressed EGFP in infected cells, but showed significantly reduced viral growth compared to wtBToV. Moreover, the recombinant virus readily deleted the EGFP gene after one passage. Interestingly, one variant with mutations in non-structural proteins (NSPs) showed improved EGFP expression and viral growth during serial passages, although it eventually deleted the EGFP gene, suggesting that these mutations contributed to EGFP gene acceptance. These recombinant viruses provide new insights regarding BToV and its reverse genetics will help advance understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens that have been detected in many species, including humans. BToV has spread worldwide, leading to economic losses. We developed the first reverse genetics system for Tobaniviridae using a BAC-based BToV. Using this system, we showed that recombinant BToVs with HEf and HEs showed no significant differences in cell growth. In contrast, clinical BToVs generally lose the HE gene after a few passages but some recombinant viruses retained the HE gene for up to 20 passages, suggesting some benefits of HE retention. The EGFP gene of the recombinant viruses was unstable and was rapidly deleted, likely via negative selection. Interestingly, one virus variant with mutations in NSPs was more stable, resulting in improved EGFP-expression and viral growth, suggesting that the mutations contributed to some acceptance of the exogenous EGFP gene without clear positive selection. The recombinant BToVs and reverse genetics developed here are powerful tools for understanding fundamental viral processes and their pathogenesis and for developing BToV vaccines.
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Hemagglutination mediated by the spike protein of cell-adapted Bovine Torovirus
Archives of Virology, 2013Co-Authors: Kozue Shimabukuro, Hiroshi Tsunemitsu, Makoto Ujike, Toshihiro Ito, Hitoshi Oshitani, Fumihiro TaguchiAbstract:Bovine Torovirus (BToV)-Aichi, recently isolated in cultured cells, showed hemagglutination (HA) activity, although the virus has a truncated hemagglutinin-esterase (HE) protein, judging from its gene structure, indicating the existence of another viral protein with HA activity. We examined whether the spike (S) protein possesses HA activity. A BToV antiserum used in this study, reactive to S but not to HE, inhibited HA activity. Furthermore, cells infected with BToV and those expressing S showed hemadsorption (HAD) activity, which was inhibited by the anti-BToV serum; however, HAD activity by expressed HE was not blocked. These data indicate that the S protein of BToV-Aichi is responsible for its HA activity.
Linda J. Saif - One of the best experts on this subject based on the ideXlab platform.
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Torovirus Pathogenesis and Immune Responses
Nidoviruses, 2014Co-Authors: Armando E. Hoet, Linda J. SaifAbstract:Toroviruses are pleomorphic viruses with a peplomer-bearing envelope containing an elongated tubular nucleocapsid with helical symmetry. They contain a linear, nonsegmented, positive- and single-stranded polyadenylated RNA genome. Currently only four species are recognized as members of the Torovirus genus in the Coronaviridae family: equine Torovirus (EToV), initially known as Berne virus; Bovine Torovirus (BToV), originally known as Breda virus; human Torovirus (HToV); and porcine Torovirus. Torovirus-like (TVL) particles have been detected in feces from other animal species, such as turkeys, dogs, and cats. In addition, antibodies against Toroviruses are frequently detected in ungulates such as horses, sheep, goats, and pigs. This chapter provides a comprehensive review of Torovirus pathogenesis and immune responses. A brief epidemiological background highlighting HToVs is also provided.
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Nidoviruses - Torovirus Pathogenesis and Immune Responses
Nidoviruses, 2014Co-Authors: Armando E. Hoet, Linda J. SaifAbstract:Toroviruses are pleomorphic viruses with a peplomer-bearing envelope containing an elongated tubular nucleocapsid with helical symmetry. They contain a linear, nonsegmented, positive- and single-stranded polyadenylated RNA genome. Currently only four species are recognized as members of the Torovirus genus in the Coronaviridae family: equine Torovirus (EToV), initially known as Berne virus; Bovine Torovirus (BToV), originally known as Breda virus; human Torovirus (HToV); and porcine Torovirus. Torovirus-like (TVL) particles have been detected in feces from other animal species, such as turkeys, dogs, and cats. In addition, antibodies against Toroviruses are frequently detected in ungulates such as horses, sheep, goats, and pigs. This chapter provides a comprehensive review of Torovirus pathogenesis and immune responses. A brief epidemiological background highlighting HToVs is also provided.
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Bovine Torovirus (Breda virus) revisited.
Animal Health Research Reviews, 2004Co-Authors: Armando E. Hoet, Linda J. SaifAbstract:Bovine Torovirus (BoTV) is a pleomorphic virus with a spike-bearing envelope and a linear, non-segmented, positive-sense single-stranded RNA genome. This kidney-shaped virus is associated with diarrhea in calves and apparently has a worldwide distribution. This review provides details of the history and taxonomy of BoTV since its discovery in 1979. Information about virion morphology and architecture, antigenic and biological properties, viral genome, protein composition, thermal and chemical stability, and pH and proteolytic enzymes resistance is also summarized. A major focus of this review is to postulate a possible epidemiological cycle for BoTV, based on epidemiological data obtained in our studies and other published data, and progressing from the newborn calf to the adult animal. The distribution, host range, pathogenesis, disease and clinical signs (under experimental and natural exposure), pathology, diagnosis, prevention, treatment and control of BoTV infections are also described. In addition, a discussion of the zoonotic implications of Torovirus-like particles detected in patients with gastroenteritis that resemble and cross-react with BoTV is presented. Hopefully, the findings described here will alert others to the existence of BoTV in cattle and its contribution to the diarrheal disease complex. This review also highlights the need for continual vigilance for potential zoonotic viruses belonging to the order Nidovirales, such as the SARS coronavirus.
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Detection of Bovine Torovirus and other enteric pathogens in feces from diarrhea cases in cattle.
Journal of Veterinary Diagnostic Investigation, 2003Co-Authors: Armando E. Hoet, Thomas E. Wittum, P. R. Nielsen, Mustafa Hasöksüz, Christopher Thomas, Linda J. SaifAbstract:The objectives of this study were to determine the prevalence of Bovine Torovirus (BoTV) in Bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (#6 months old), 27 young adults (#2 years), 125 adults ($2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosor- bent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV. Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves (P , 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV- positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent. In cattle, enteric disease is a major health problem associated with diarrhea, dehydration, and weight loss. The severity of the diarrhea may depend on the caus- ative agents, which include a number of enteropatho- gens (bacteria, viruses, fungi, protozoa, and hel- minths), and their interaction with other factors such as environment, other etiologic agents, management, and host factors (i.e., immunosuppression, malnutri- tion, and stress). 27
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Comparison of ELISA and RT-PCR versus immune electron microscopy for detection of Bovine Torovirus (Breda virus) in calf fecal specimens.
Journal of Veterinary Diagnostic Investigation, 2003Co-Authors: Armando E. Hoet, Kyeong-ok Chang, Linda J. SaifAbstract:Bovine Torovirus (BoTV) is an uncultivable enteric pathogen of cattle. Its failure to grow in vitro limits epidemiological studies, characterization of the virus, and development of diagnostic techniques. The objectives of this study were to develop and standardize an antigen-capture enzyme-linked immunosorbent assay (ELISA) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of BoTV in fecal specimens. These assays were compared with immunoelectron microscopy (IEM) to evaluate their sensitivity, specificity, and efficiency as well as their advantages and limitations. Additionally, several methods to calculate ELISA cutoff values were used and compared using a statistical approach to obtain the optimal cutoff value for the ELISA. A plate cutoff ELISA value was determined to be the best method to calculate the cutoff value. The ELISA and RT-PCR assays developed in this study identified BoTV antigen and viral nucleic acids in feces without cross-reactions with the other calf enteric viruses examined. Both assays showed good agreement with IEM, with a Kappa value of 0.86 for ELISA and 0.85 for RT-PCR. The latter exhibited the higher analytical sensitivity. On the basis of the results obtained in this study, it is recommended that no single test should be used alone in an epidemiological survey because of the observed limitations of each assay. The fast and inexpensive ELISA combined with the highly specific and sensitive RT-PCR are a practical approach for future epidemiological studies of BoTV. These results should provide other researchers with the information needed to develop similar diagnostic assays for the study of BoTV.
Hiroshi Tsunemitsu - One of the best experts on this subject based on the ideXlab platform.
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Hemagglutination mediated by the spike protein of cell-adapted Bovine Torovirus
Archives of Virology, 2013Co-Authors: Kozue Shimabukuro, Hiroshi Tsunemitsu, Makoto Ujike, Toshihiro Ito, Hitoshi Oshitani, Fumihiro TaguchiAbstract:Bovine Torovirus (BToV)-Aichi, recently isolated in cultured cells, showed hemagglutination (HA) activity, although the virus has a truncated hemagglutinin-esterase (HE) protein, judging from its gene structure, indicating the existence of another viral protein with HA activity. We examined whether the spike (S) protein possesses HA activity. A BToV antiserum used in this study, reactive to S but not to HE, inhibited HA activity. Furthermore, cells infected with BToV and those expressing S showed hemadsorption (HAD) activity, which was inhibited by the anti-BToV serum; however, HAD activity by expressed HE was not blocked. These data indicate that the S protein of BToV-Aichi is responsible for its HA activity.
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Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle
Archives of Virology, 2012Co-Authors: Masaharu Fukuda, Keito Tasei, Tsunehiko Aita, Masaji Mase, Kazufumi Kuga, Ayako Miyazaki, Makoto Sugiyama, Tohru Suzuki, Hiroshi TsunemitsuAbstract:A one-step multiplex reverse transcription ( RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: Bovine group A rotavirus (GAR), Bovine group B rotavirus (GBR), Bovine group C rotavirus (GCR), Bovine coronavirus (BCV), and Bovine Torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 10^2, 10^0, 10^1, and 10^2 TCID_50/ml, respectively, and that for GBR was 10^6 copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of Bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.
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Characterization of epidemic diarrhea outbreaks associated with Bovine Torovirus in adult cows
Archives of Virology, 2012Co-Authors: Tsunehiko Aita, Ayako Miyazaki, Masaki Kuwabara, Kazunori Murayama, Yuri Sasagawa, Shizuka Yabe, Ryohei Higuchi, Tsutomu Tamura, Hiroshi TsunemitsuAbstract: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 2007-2008, 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 3-5 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/2004. 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/2004 indicated that both strains have truncated HE genes with deletions in the 3′ 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.
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First isolation of cytopathogenic Bovine Torovirus in cell culture from a calf with diarrhea.
Clinical and Vaccine Immunology, 2007Co-Authors: Masaki Kuwabara, Ayako Miyazaki, Kazumasa Wada, Yukiko Maeda, Hiroshi TsunemitsuAbstract:A cytopathogenic virus (designated the Aichi/2004 strain) was isolated in a human rectal adenocarcinoma cell line (HRT-18) from the ileum contents of a calf with diarrhea. Oval and elongated particles, approximately 100 to 170 nm in diameter, with club-shaped projections were seen in the infected culture supernatant, and Torovirus-like (tubular and torus nucleocapsid) structures were seen in the infected cells by electron microscopy. An antiserum against Bovine Torovirus (BToV) reacted with the infected cells by immunofluorescence and neutralized the isolate. However, antisera against Bovine coronavirus (BCV) failed to react with the infected cells by immunofluorescence or did not neutralize the isolate. Further, the isolate was positive for BToV by reverse transcription-PCR (RT-PCR) targeting fragments of the nucleocapsid (N), membrane (M), and spike (S) genes. Comparison of the nucleotide sequences of the PCR products with those of the published N, M, and S genes (476 to 497, 672, and 687 to 690 nucleotides, respectively) of Toroviruses showed high sequence identities (up to 99.4%, 98.7%, and 94.9% for the N, M, and S genes, respectively) between the isolate and BToVs. In contrast, the isolate was negative for BCV by RT-PCR. In a serological survey of serum samples from 355 calves at 33 farms, 92% of calves were positive for neutralizing antibodies to the isolate. These results indicate that the isolate in this study was BToV and that BToV infection might be common in cattle in Japan. To our knowledge, this is the first isolation of BToV in tissue culture.
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Phylogenetic and Evolutionary Relationships among Torovirus Field Variants: Evidence for Multiple Intertypic Recombination Events
Journal of Virology, 2003Co-Authors: Saskia L. Smits, Marion Koopmans, Antonio Lavazza, K. Matiz, Marian C. Horzinek, R. J. De GrootAbstract:Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about Torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among Toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human Torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by Bovine Torovirus (BToV) Breda, porcine Torovirus (PToV) Markelo, equine Torovirus Berne, and the putative human Torovirus. The ungulate Toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown Toroviruses. From these observations, the existence of two additional Torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.
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Identification and Characterization of a Porcine Torovirus
Journal of Virology, 1998Co-Authors: A. Kroneman, Marian C. Horzinek, R. J. De Groot, L. A. H. M. Cornelissen, Herman EgberinkAbstract:A porcine Torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine Torovirus (ETV) and Bovine Torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a Torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.
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Hemagglutinin-esterase, a novel structural protein of Torovirus.
Journal of virology, 1997Co-Authors: L. A. H. M. Cornelissen, Marian C. Horzinek, Herman Egberink, Christel M. H. Wierda, F. J. Van Der Meer, A. A. P. M. Herrewegh, R. J. De GrootAbstract:We have characterized the 3'-most 3 kb of the genome of Bovine Torovirus (BoTV) strain Breda. A novel 1.2-kb gene, located between the genes for the membrane and nucleocapsid proteins, was identified. This gene, the 3'-most 0.5 kb of which is also present in the genome of the equine Torovirus isolate Berne virus (BEV), codes for a class I membrane protein displaying 30% sequence identity with the hemagglutinin-esterases (HEs) of coronaviruses and influenza C viruses. Heterologous expression of the BoTV HE gene yielded a 65,000-molecular weight N-glycosylated protein displaying acetylesterase activity. Serologic evidence indicates that the HE homolog is expressed during the natural infection and represents a prominent antigen. By using an antiserum raised against residues 13 to 130 of HE, the HE protein was detected in radioiodinated, sucrose gradient-purified BoTV preparations. Formal evidence that HE is a structural protein was provided by immunoelectron microscopy. In addition to the large, 17- to 20-nm spikes, BoTV virions possess shorter surface projections (6 nm on average). We postulate that these surface projections, which are absent from the BEV virion, are composed of the BoTV HE homolog. The HE gene, which has now been demonstrated in three different virus genera, is a showpiece example of modular evolution.
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Copyright © 1997, American Society for Microbiology Hemagglutinin-Esterase, a Novel Structural Protein
1996Co-Authors: Of Torovirus, Marian C. Horzinek, Herman Egberink, Christel M. H. Wierda, F. J. Van Der Meer, A. A. P. M. Herrewegh, Lisette A. H. M. Cornelissen, Raoul J. De GrootAbstract:We have characterized the 3*-most 3 kb of the genome of Bovine Torovirus (BoTV) strain Breda. A novel 1.2-kb gene, located between the genes for the membrane and nucleocapsid proteins, was identified. This gene, the 3*-most 0.5 kb of which is also present in the genome of the equine Torovirus isolate Berne virus (BEV), codes for a class I membrane protein displaying 30 % sequence identity with the hemagglutinin-esterases (HEs) of coronaviruses and influenza C viruses. Heterologous expression of the BoTV HE gene yielded a 65,000-molecular weight N-glycosylated protein displaying acetylesterase activity. Serologic evidence indicates that the HE homolog is expressed during the natural infection and represents a prominent antigen. By using an antiserum raised against residues 13 to 130 of HE, the HE protein was detected in radioiodinated, sucrose gradient-purified BoTV preparations. Formal evidence that HE is a structural protein was provided by immunoelectron microscopy. In addition to the large, 17- to 20-nm spikes, BoTV virions possess shorter surface projections (6 nm on average). We postulate that these surface projections, which are absent from the BEV virion, are composed of the BoTV HE homolog. The HE gene, which has now been demonstrated in three different virus genera, is a showpiece example of modular evolution. RNA viruses are genetically very flexible. During the repli-cation of their genomes, nucleotide substitutions occur at
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cDNA probes for the diagnosis of Bovine Torovirus (Breda virus) infection.
Journal of Clinical Microbiology, 1991Co-Authors: M Koopmans, Eric J. Snijder, Marian C. HorzinekAbstract:A genomic cDNA library of RNA from Breda virus (BRV), a Bovine Torovirus, was prepared. The nucleotide sequence of the 3' end of the genome was found to be highly conserved (93% identical) between BRV and Berne virus, the Torovirus prototype. Cross-hybridization experiments were performed to select Berne virus cDNA clones for use as probes in a dot hybridization assay; the objective was to detect heterologous Torovirus RNA in fecal material. A rapid RNA extraction method was employed to make the test applicable for routine diagnosis. Samples from calves after experimental and natural infection with BRV were assayed to establish the sensitivity and specificity of the test and to compare the test with the enzyme-linked immunosorbent assay (ELISA) for antigen detection. For this purpose, 53 samples from seven infected calves were tested with both methods. In the ELISA, BRV was detected in six fecal samples from three inoculated calves. By use of the hybridization test, 16 samples from seven calves reacted positively. With one exception, only postinoculation samples were found positive in hybridization. No signal was seen in feces from uninoculated calves or from calves infected with rotavirus or coronavirus.