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PubMed2003

Phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events.

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.

Animals↗

PubMed1993

Enzyme-linked immunosorbent assay reactivity of torovirus-like particles in fecal specimens from humans with diarrhea.

Toroviruses are recognized enteric pathogens of cattle and horses; in humans, similar pleomorphic particles have been described, but doubt has been raised concerning their identity as viruses. We screened fecal samples from humans with diarrhea for the presence of torovirus-like particles (TVLPs) by electron microscopy and subsequently used an enzyme-linked immunosorbent assay (ELISA) with bovine torovirus reference reagents to test for the presence of torovirus antigens. To add another selection criterion to this heterologous ELISA, we enriched the TVLPs from the stool specimens by using sucrose density gradients before testing. The results of ELISA and EM correlated significantly, the ELISA having a sensitivity of 68% and a specificity of 86% (chi-square, P < 0.0001). In the gradient, peaks of ELISA reactivity were found at a buoyant density of 1.16 g/ml and were parallel to those found when using bovine torovirus. Furthermore, in 50% of the ELISA-positive gradients, a hemagglutinin for human group O erythrocytes comigrated with the peaks of ELISA reactivity. We were unable to isolate human TVLPs in human colonic tumor or rectal tumor cells. We cloned and sequenced amplification products obtained by low-stringency polymerase chain reaction amplification using consensus primers mapping to the 3' end of the genome of animal toroviruses, but found no significant homologies with animals torovirus sequences. Rabbits were inoculated with material from the gradient peak fractions of human stool specimens, and their sera were assayed for immunologic comparison with bovine torovirus as a reference. A two-way antigenic cross-reactivity was seen between human TVLP and bovine torovirus reagents when tested by ELISA. The rabbit antisera to human TVLP detected a higher number of electron microscopy-positive stool specimens than did the rabbit antisera to bovine torovirus. The application of these assays and reagents should help to elucidate the roles of TVLPs and toroviruses in diarrheal disease in humans.

Animals↗

PubMed1998

Human torovirus: a new nosocomial gastrointestinal pathogen.

Studies were undertaken to determine if human torovirus is associated with gastroenteritis and to examine the clinical features of torovirus illness in children. The fecal excretion of torovirus in patients with gastroenteritis was compared with that in matched asymptomatic controls in a case-control study. Toroviruses were identified in 72 (35.0%) of 206 gastroenteritis cases compared with 30 (14.5%) of 206 controls (P<.001). Clinical features of torovirus gastroenteritis in 172 patients positive for torovirus were compared with those of 115 patients infected with rotavirus or astrovirus. Persons infected with torovirus were more frequently immunocompromised (43.0% vs. 15.7%) and nosocomially infected (57.6% vs. 31.3%). They also experienced less vomiting (46.4% vs. 66.7%) but had more bloody diarrhea (11.2% vs. 1.8%). An antibody response to torovirus developed mainly in older, nonimmunocompromised children (P<.01). These studies demonstrate an association between torovirus excretion and gastroenteritis in the pediatric population among immunocompromised hospitalized patients and in previously healthy patients.

Adolescent↗

PubMed1998

Detection of bovine torovirus in fecal specimens of calves with diarrhea from Ontario farms.

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.

Animals↗

PubMed1997

Association of torovirus with acute and persistent diarrhea in children.

OBJECTIVE: To study the etiologic role of toroviruses as a cause of gastroenteritis in humans. METHODS: The design was a case-control study. We compared the rate of torovirus detection in fecal specimens from a selection of children with acute or persistent diarrhea and controls without diarrhea from a study of childhood diarrhea in an urban Brazilian slum. Stool samples were coded and tested in a blinded fashion for the presence of torovirus antigen by enzyme-linked immunosorbent assay, other enteropathogens, toxins and fecal leukocytes. RESULTS: Thirty-three children with acute diarrhea, 41 children with persistent diarrhea and 17 controls were enlisted in the study. Torovirus antigen was detected in 9 (27%) samples from children with acute diarrhea, 11 (27%) samples from children with persistent diarrhea and none of the samples from controls (P < 0.05). In addition the presence of enteroaggregative E. coli was associated with persistent diarrhea and the presence of Cryptosporidium oocysts was common although not significant (P = 0.08); torovirus and Cryptosporidium occurred in different subsets of samples, whereas torovirus and enteroaggregative Escherichia coli were commonly found in combination. CONCLUSIONS: These data indicate that toroviruses, alone or in combination with enteroaggregative E. coli, may play a pathogenic role in acute and possibly persistent diarrhea. Further studies are warranted to determine the etiologic role of toroviruses in gastroenteritis.

Acute Disease↗

PubMed2002

Torovirus detection in faecal specimens of calves and pigs in Hungary: short communication.

Bovine torovirus is an established aetiological agent of disease in cattle, while porcine torovirus has only been isolated from healthy animals. Evidence for the presence of torovirus has been described in several European countries and also in the United States. A survey was performed to detect toroviruses in Hungary by means of sampling ten swine and nine bovine herds. Rectal swabs and faecal specimens were collected from diarrhoeic calves and from weaned piglets. The samples were tested by the reverse transcription-polymerase chain reaction (RT-PCR) using torovirus-specific primers and the positive samples were further examined by electron microscopy (EM). Torovirus was detected in 4 diarrhoeic calves (out of 111) and in 10 healthy weaned pigs (out of 200 tested), representing two of the 9 calf herds and two of the 10 pig herds tested. This is the first report of exact diagnosis of torovirus in Hungary.

Animals↗

PubMed1999

The novel hemagglutinin-esterase genes of human torovirus and Breda virus.

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.

Acetylesterase↗

PubMed1998

Identification and characterization of a porcine torovirus.

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.

Amino Acid Sequence↗

PubMed1998

Bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of Breda virus.

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.

Amino Acid Sequence↗

PubMed1991

Association of diarrhea in cattle with torovirus infections on farms.

An epidemiologic survey was performed to determine the incidence of torovirus infections in 2 disease entities of cattle: diarrhea of replacement calves up to 2 months old, and winter dysentery of adult cattle. Samples were obtained from 187 diarrheal and 115 healthy calves from 15 farms, as well as 149 diarrheal and 67 healthy cows from 27 farms with or without winter dysentery. Enzyme-linked immunosorbent assays for detection of torovirus, rotavirus, and coronavirus antigen in feces, and of torovirus and coronavirus antibodies in serum were used to monitor infections in these groups. Torovirus was detected in 9 of the 15 farms in the study, and in 6% of calves with diarrhea, which was significantly higher than in healthy calves (2%). Seroconversion to torovirus was found significantly more often after winter dysentery episodes than on farms without a disease history; coronavirus seroconversion was less common.

Animals↗

PubMed1997

Characterization of torovirus from human fecal specimens.

The toroviruses, Berne virus (BEV) and Breda virus (BRV), are recognized pathogens of horses and cattle, respectively. Torovirus-like particles (TVLPs) that are immunologically related to BRV have been reported as etiological agents of gastroenteritis in humans. Of the toroviruses, only BEV has been shown to replicate in cell culture. Hence, these agents can be routinely detected only by electron microscopy (EM), although serological testing has been used as well. Our studies have provided supporting evidence that the TVLPs detected in the stool specimens of pediatric patients with gastroenteritis are human toroviruses. By EM, these particles are morphologically similar to BEV and BRV. Thin-section electron microscopy revealed that TVLPs contain toroidal-shaped nucleocapsids. Viruses purified from human fecal specimens agglutinate rabbit erythrocytes. BRV antiserum as well as convalescent sera from patients with gastroenteritis whose stools contain TVLPs were shown to contain antibodies that react with purified TVLPs as demonstrated by hemagglutination inhibition, immunoelectron microscopy, and immunoblotting. RNA extracted from partially purified TVLP preparations is amplifiable by RT-PCR using primers bracketing a 219-base region at the 3' end of the Berne virus genome. Sequence analysis of amplicons from five isolates showed a high degree of identity with the corresponding BEV sequence.

Animals↗

PubMed1998

Cell culture-grown putative bovine respiratory torovirus identified as a coronavirus.

A putative bovine respiratory torovirus (BRTV) was propagated in bovine fetal diploid lung and human colonic tumour cells, and fringed pleomorphic particles were detected in the culture supernatants by electron microscopy. Antisera directed against a bovine (Breda strain) and equine (Berne strain) torovirus failed to react with BRTV-infected cells in immunofluorescence assays and did not neutralise BRTV. No toroviral RNA was found in the supernatants of infected cells by means of a reverse transcriptase-polymerase chain reaction with torovirus-specific primers. On the other hand, bovine coronavirus-specific antisera and monoclonal antibodies did neutralise the cytopathic effects, and coronaviral antigen was detected in the cultures by immunofluorescence. Furthermore, bovine coronavirus RNA was detected in the supernatants of BRTV-infected cells after nucleic acid amplification. It is concluded that the cytopathic BRTV isolate is a coronavirus.

Animals↗

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↗

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↗