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PubMed1991

cDNA probes for the diagnosis of bovine torovirus (Breda virus) infection.

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.

Animals↗
PubMed2001

Enteric infections with coronaviruses and toroviruses.

Many enteric viruses are difficult or impossible to propagate in tissue culture. Coronaviruses and toroviruses are large, enveloped, plus-strand RNA viruses in the order Nidovirales that cause enteric disease in young pigs, cows, dogs, mice, cats and horses. Two different serogroups of mammalian coronaviruses cause frequent respiratory infections in humans, and coronaviruses and toroviruses have been implicated in human diarrhoeal disease by immunoelectron microscopy. However, there is as yet no consensus about the importance of these enveloped viruses in human diarrhoea, and little is known about their genetic variability. The large spike (S) glycoprotein is an important determinant of species specificity, tissue tropism and virulence of coronavirus infection. To infect enterocytes, both S glycoproteins and the viral envelope must resist degradation by proteases, low and high pH, and bile salts. One specific site on the S glycoprotein of bovine coronavirus must be cleaved by an intracellular protease or trypsin to activate viral infectivity and cell fusion. S glycoprotein binds to specific receptors on the apical membranes of enterocytes, and can undergo a temperature-dependent, receptor-mediated conformational change that leads to fusion of the viral envelope with host membranes to initiate infection. Analysing spike-receptor interactions may lead to new ways to propagate these enteric viruses as well as new strategies for development of novel antiviral drugs.

Coronavirus↗
PubMed2004

Bovine torovirus (Breda virus) revisited.

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.

Animals↗
PubMed1993

The Coronaviridae now comprises two genera, coronavirus and torovirus: report of the Coronaviridae Study Group.

At the April 1992, mid-term meeting of the International Committee on Taxonomy of Viruses (ICTV) a proposal from the Coronaviridae Study Group (CSG) to include the torovirus genus in the Coronaviridae was accepted. Following another proposal, the arterivirus genus was removed from the Togaviridae but not assigned to another family. The arteriviruses have some features in common with the Coronaviridae but also have major differences. After much debate, culminating in September 1992, it was decided that the CSG would not recommend inclusion of arterivirus in the Coronaviridae. It was agreed that (a) the nomenclature used for coronavirus genes, mRNAs and polypeptides (Cavanagh et al., 1990) should be used for toroviruses, (b) that the small (about 100 amino acids) membrane-associated protein, which is distinct from the integral membrane glycoprotein M, associated with virions of infectious bronchitis (Liu & Inglis, 1991) and transmissible gastroenteritis (Godet et al., 1992) coronaviruses would be referred to by the acronym sM (lower case 's') and (c) that 'pol' (polymerase) should be used as a working term for gene 1, which comprises open reading frames (ORFs) 1a and 1b in both genera of the Coronaviridae.

Arterivirus↗
PubMed1997

Hemagglutinin-esterase, a novel structural protein of torovirus.

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.

Amino Acid Sequence↗
PubMed1990

The polymerase gene of corona- and toroviruses: evidence for an evolutionary relationship.

In this paper we demonstrate that the organization of the polymerase gene of toroviruses and coronaviruses is similar. The polymerase gene of both virus families consists of at least two large ORFs (1a and 1b). Four domains of conserved amino acid sequences have been identified in nearly identical positions in the 3' ORF of the pol gene of toroviruses and coronaviruses. The most 3' conserved domain which is still unique for these viruses encodes a 33-kDA protein in MHV-A59, which is cleaved from a precursor protein. Expression of ORF1b of the pol gene of both virus families occurs by ribosomal frameshifting. A predicted stem-loop structure and pseudoknot are conserved in the ORF1a/ORF1b overlap of toro- and coronaviruses. On the basis of these results we postulate that toro- and coronaviruses are ancestrally more related to each other than to other families of positive stranded RNA viruses.

Amino Acid Sequence↗
PubMed2003

Comparison of ELISA and RT-PCR versus immune electron microscopy for detection of bovine torovirus (Breda virus) in calf fecal specimens.

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.

Animals↗
PubMed2003

Detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle.

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 (< or = 6 months old), 27 young adults (52 years), 125 adults (> or = 2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent 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.

Aging↗
PubMed2002

Enteric and nasal shedding of bovine torovirus (Breda virus) in feedlot cattle.

OBJECTIVE: To assess fecal and nasal shedding patterns of bovine torovirus (BoTV) in cattle at time of arrival and periodically throughout the first 21 days after arrival at a feedlot. ANIMALS: 57 steers. PROCEDURE: Fecal and nasal-swab samples collected on days 0, 4, 14, and 21 after arrival were tested for BoTV, using ELISA. A subset of samples from calves testing positive and negative for BoTV was analyzed, using reverse transcriptase-polymerase chain reaction (RT-PCR). Paired serum samples were collected on days 0 and 21 and tested for BoTV antibodies, using a hemagglutination inhibition assay. RESULTS: Overall rate of fecal shedding of BoTV was 21 of 57 (37%) by ELISA and 40 of 42 (95%) by RT-PCR with peak shedding on day 4. Diarrhea was more common in calves shedding BoTV than those not shedding the virus (odds ratio, 1.72). Overall rate of nasal shedding of BoTV was 15 of 57 (26%) by ELISA and 42 of 42 (100%) by RT-PCR, with peak shedding on day 0. Specificity of the RT-PCR product was confirmed by sequence analysis. Approximately 93% of the calves seroconverted to BoTV (> 4-fold increase in titer). Differences were not detected between calves shedding BoTV and nonshedders in relation to disease and treatments, perhaps because of the low number of cattle in the study. CONCLUSIONS AND CLINICAL RELEVANCE: This study confirmed BoTV infections in feedlot cattle, including BoTV antigen and viral RNA in nasal secretions, and the shedding pattern during the first 21 days after arrival in a feedlot.

Animals↗
PubMed2003

Association of enteric shedding of bovine torovirus (Breda virus) and other enteropathogens with diarrhea in veal calves.

OBJECTIVE: To determine the prevalence, fecal shedding pattern, and association of bovine torovirus (BoTV) with diarrhea in veal calves at time of arrival and periodically throughout the first 35 days after their arrival on a veal farm. ANIMALS: 62 veal calves. PROCEDURE: Fecal samples collected on days 0, 4, 14, and 35 after arrival were tested for BoTV by use of ELISA and reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Paired serum samples obtained from blood collected on days 0 and 35 were analyzed for BoTV antibodies with a hemagglutination inhibition assay. Fecal samples were also screened for other enteric pathogens, including rotavirus, coronavirus, and Cryptosporidium spp. RESULTS: Fecal shedding of BoTV was detected in 15 of 62 (24%) calves by use of ELISA and RT-PCR assay, with peak shedding on day 4. A significant independent association between BoTV shedding and diarrhea was observed. In addition, calves shedding > or = 2 enteric pathogens were more likely to have diarrhea than calves shedding < or = 1 pathogen. Calves that were seronegative or had low antibody titers against BoTV (< or = 1:10 hemagglutination inhibition units) at arrival seroconverted to BoTV (> 4-fold increase in titer); these calves were more likely to shed virus than calves that were seropositive against BoTV at arrival. CONCLUSIONS AND CLINICAL RELEVANCE: Shedding of BoTV was strongly associated with diarrhea in neonatal veal calves during the first week after arrival at the farm. These data provide evidence that BoTV is an important pathogen of neonatal veal calves.

Animals↗
PubMed2006

Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses.

Virus attachment to host cells is mediated by dedicated virion proteins, which specifically recognize one or, at most, a limited number of cell surface molecules. Receptor binding often involves protein-protein interactions, but carbohydrates may serve as receptor determinants as well. In fact, many different viruses use members of the sialic acid family either as their main receptor or as an initial attachment factor. Sialic acids (Sias) are 9-carbon negatively-charged monosaccharides commonly occurring as terminal residues of glycoconjugates. They come in a large variety and are differentially expressed in cells and tissues. By targeting specific Sia subtypes, viruses achieve host cell selectivity, but only to a certain extent. The Sia of choice might still be abundantly present on non-cell associated molecules, on non-target cells (including cells already infected) and even on virus particles themselves. This poses a hazard, as high-affinity virion binding to any of such "false'' receptors would result in loss of infectivity. Some enveloped RNA viruses deal with this problem by encoding virion-associated receptor-destroying enzymes (RDEs). These enzymes make the attachment to Sia reversible, thus providing the virus with an escape ticket. RDEs occur in two types: neuraminidases and sialate-O-acetylesterases. The latter, originally discovered in influenza C virus, are also found in certain nidoviruses, namely in group 2 coronaviruses and in toroviruses, as well as in infectious salmon anemia virus, an orthomyxovirus of teleosts. Here, the structure, function and evolution of viral sialate-O-acetylesterases is reviewed with main focus on the hemagglutinin-esterases of nidoviruses.

Amino Acid Sequence↗
PubMed1997

Electronmicroscopic evidence of torovirus like particles in children with diarrhoea.

Pleomorphic virus-like particles approximately 90 nm in diameter with short fringe (7-9 nm long) were observed in faecal specimens of two diarrhoeic children in the course of routine screening of samples for viruses by electronmicroscopy. No other viral, bacterial or parasitic pathogens were detected in the same samples. Immune electronmicroscopic examination showed that these virus like particles were agglutinated by immunesera raised against Breda I and Breda II viruses of calves. This observation suggests that torovirus may be a new viral pathogen of humans.

Child, Preschool↗
PubMed1991

Another triple-spanning envelope protein among intracellularly budding RNA viruses: the torovirus E protein.

The nucleotide sequence of the Berne virus envelope (E) protein gene was determined and its 26.5K translation product was identified by in vitro transcription and translation. Computer analysis of the protein sequence revealed the characteristics of a class III membrane protein lacking a cleaved signal sequence but containing three successive transmembrane alpha-helices in the N-terminal half, much the same as the coronavirus membrane (M) protein. The disposition of the E protein in the membrane was studied by in vitro translation in the presence of microsomes and by subsequent proteinase K digestion. Only small portions of either end of the polypeptide were found to be exposed on opposite sides of the vesicle membranes. Experiments with a hybrid E protein (EM) containing the C-terminal tail of a coronavirus M protein, to which an anti-peptide serum was available, showed that this C-terminus was present at the cytoplasmic side of the membrane, which is another similarity to the coronavirus M protein. Immunofluorescence experiments indicated that the EM protein, expressed by a recombinant vaccinia virus, accumulated in intracellular membranes, predominantly those of the endoplasmic reticulum. The common features of the torovirus E and the coronavirus M protein support our hypothesis that an evolutionary relationship exists between these groups of intracellularly budding viruses.

Amino Acid Sequence↗
PubMed1990

[New types of virus infections of domestic animals in the German Democratic Republic. 1. Serologic survey studies of the distribution of equine torovirus infections in the GDR].

Sera collected from 124 horses were checked by means of the serum neutralisation test against equine Bern virus. Torovirusspecific antibodies were recordable from 35 percent of all horses tested. These results are likely to suggest that toroviruses are widespread in the GDR and occur not only in horses but in other domestic animals and in man, as well.

Animals↗