First detection and genomic characteristics of bovine torovirus in dairy calves in China.

Bovine torovirus (BToV) is a diarrhea-causing pathogen. In this study, 92 diarrheic fecal samples from five farms in four provinces in China were collected and tested for BToV using a RT-PCR assay, and 21.73% samples were found to be BToV positive. Moreover, two complete BToV genome sequences (MN073058 and MN073059) were obtained from the clinical samples, which were 28,297 and 28,301 nucleotides in length, respectively. Sequence analysis showed that the two isolates shared 10 identical amino acid mutations in the S protein compared to the complete S sequences of BToV available in the GenBank database. In addition, seven consecutive amino acid mutations were found from aa 1,486 to 1,492 in the S protein of isolate MN073058. Moreover, the two isolates shared one identical amino acid mutation in the receptor binding sites of the HE protein. To the best of our knowledge, this is the first report on the epidemic and genomic characterization of BToV in China, which is helpful for further understanding the genetic evolution of BToV.

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm.

Recombination occurs frequently between enteroviruses (EVs) which are classified within the same species of the Picornaviridae family. Here, using viral metagenomics, the genomes of two recombinant EV-Gs (strains EVG 01/NC_CHI/2014 and EVG 02/NC_CHI/2014) found in the feces of pigs from a swine farm in China are described. The two strains are characterized by distinct insertion of a papain-like protease gene from toroviruses classified within the Coronaviridae family. According to recent reports the site of the torovirus protease insertion was located at the 2C/3A junction region in EVG 02/NC_CHI/2014. For the other variant EVG 01/NC_CHI/2014, the inserted protease sequence replaced the entire viral capsid protein region up to the VP1/2A junction. These two EV-G strains were highly prevalent in the same pig farm with all animals shedding the full-length genome (EVG 02/NC_CHI/2014) while 65% also shed the capsid deletion mutant (EVG 01/NC_CHI/2014). A helper-defective virus relationship between the two co-circulating EV-G recombinants is hypothesized.

Molecular epidemiology of Porcine torovirus (PToV) in Sichuan Province, China: 2011-2013.

BACKGROUND: Porcine torovirus (PToV) is a member of the genus Torovirus which is responsible for gastrointestinal disease in both human beings and animals with particular prevalence in youth. Torovirus infections are generally asymptomatic, however, their presence may worsen disease consequences in concurrent infections with other enteric pathogens. METHODS: A total of 872 diarrheic fecal samples from pigs of different ages were collected from 12 districts of Sichuan Province in the southwest of China. RT-PCR was done with PToV S gene specific primers to detect the presence of PToV positive samples. M gene specific primers were used with the PToV positive samples and the genes were sequenced. A phylogenetic tree was constructed based on the M gene nucleotide sequences from the 19 selected novel Sichuan strains and 21 PToV and BToV M gene sequences from GenBank. RESULTS: A total of 331 (37.96%, 331/872) samples were found to be positive for PToV and the highest prevalence was observed in piglets aged from 1 to 3 weeks old. Through phylogenetic inference the 40 PToV M gene containing sequences were placed into two genotypes (I & II). The 19 novel Sichuan strains of genotype I showed strong correlations to two Korean gene sequences (GU-07-56-11 and GU-07-56-22). Amino-acid sequence analysis of the 40 PToV M gene strains revealed that the M gene protein was highly conserved. CONCLUSIONS: This study uncovered the presence of PToV in Sichuan Province, and demonstrated the need for continuous surveillance PToV of epidemiology.

Molecular characterization and phylogenetic analysis of the genome of porcine torovirus.

In this study, we amplified and sequenced the first genome of porcine torovirus (PToV SH1 strain). The genome was found to be 28,301 bp in length, sharing 79 % identity with Breda virus. It mainly consists of replicase (20,906 bp) and structural genes: spike (4,722 bp), membrane (702 bp), hemagglutinin-esterase (1,284 bp), and nucleocapsid (492 bp). Sequence alignments and structure prediction suggest genetic differences among toroviruses, mainly in NSP1 (papain-like cysteine proteinase domain). Rooted phylogenetic trees were constructed based on the 3C-like proteinase and RNA-dependent RNA polymerase genes. PToV, Berne virus and Breda virus were clustered together, forming a separate branch from white bream virus that was distant from that of the coronaviruses.

Detection and Genetic Characterization of Bovine Torovirus in Uruguay.

Bovine torovirus (BToV) is an enteric pathogen that may cause diarrhea in calves and adult cattle, which could result in economic losses due to weight loss and decreased milk production. This study aimed to report the presence, the genetic characterization and the evolution of BToV in calves in Uruguay. BToV was detected in 7.9% (22/278) of fecal samples, being identified in dairy (9.2%, 22/239) but not beef (0.0%, 0/39) calves. BToV was detected in both diarrheic (14%, 6/43) and non-diarrheic (13.2%, 5/38) dairy calves. In addition, BToV was detected in the intestinal contents of 14.9% (7/47) of naturally deceased dairy calves. A complete genome (28,446 nucleotides) was obtained, which was the second outside Asia and the first in Latin America. In addition, partial S gene sequences were obtained to perform evolutionary analyses. Nucleotide and amino acid substitutions within and between outbreaks/farms were observed, alerting the continuous evolution of the virus. Through Bayesian analysis using BEAST, a recent origin (mid-60s) of BToV, possibly in Asia, was estimated, with two introductions into Uruguay from Asia and Europe in 2004 and 2013, respectively. The estimated evolutionary rate was 1.80 × 10-3 substitutions/site/year. Our findings emphasize the importance of continued surveillance and genetic characterization for the effective management and understanding of BToV's global epidemiology and evolution.

Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein.

Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms.

Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus.

The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection.

Molecular detection of porcine torovirus in piglets with diarrhea in southwest China.

Porcine torovirus (PToV) was detected from intestinal samples of piglets with diarrhea from 20 farms in southwest China. The total prevalence of PToV was 45% (9 out of 20 farms); it was the first detection of PToV in China, and also the study analyzed the phylogenetic relationships between the Chinese PToV and PToV reference strains as well as other representative toroviruses. Genetic and phylogenetic analysis showed the existence of genetic diversity among geographically separated PToV. Statistical analysis of the PToV positive rate as well as a survey for other enteric pathogens in diarrheic pigs suggests that PToV may play a role as a causative agent of severe diarrhea in piglets.

Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle.

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.

Genetic and antigenic characterization of newly isolated bovine toroviruses from Japanese cattle.

Torovirus, a member of the Coronaviridae family, is a gastrointestinal infectious agent that has been identified in humans, cattle, pigs, and equines. Toroviruses, except equine torovirus, are difficult to propagate in cell culture; indeed, to date, only the Aichi/2004 strain of bovine torovirus (BToV) has been isolated among the human, bovine, and porcine toroviruses. In the present study, four cytopathogenic BToVs were isolated from diarrheal feces of the cattle using the HRT-18 cell line, and their genetic and antigenic properties were compared. The cytopathogenic features of BToV isolates in HRT-18 cells were similar to those of the Aichi/2004 strain. However, none of the isolates showed cytopathogenic effects in the HRT-18 cells of different origin, suggesting that one significant factor contributing to the cytopathogenicity of BToV depends on properties of the HRT-18 cells themselves. All BToVs isolated were able to agglutinate mouse, but not chicken, erythrocytes, while they lacked receptor-destroying enzyme activity. Analysis of the N terminus of the spike gene showed that three isolates, but not the Gifu-2007TI/E strain, were phylogenetically located in cluster 1 and its analogs and revealed high cross-reactivity with each other, as demonstrated by neutralization (NT) and hemagglutination inhibition (HI) assays. The Gifu-2007TI/E strain was classified close to cluster 2 and exhibited relatively low cross-reactivity with these viruses; however, the difference was not sufficient to classify BToVs into serotypes, suggesting that at least two subtypes distinguishable by the structure of the N terminus of the spike gene and that both NT and HI tests may be exist.

Detection and molecular characterization of porcine toroviruses in Korea.

This study examined the prevalence and genetic diversity of the porcine torovirus (PToV) in Korea. Of 295 samples, 19 (6.4%) samples tested positive for PToVs by RT-PCR. A low nucleotide sequence identity of the partial S gene was detected among the Korean PToVs (73.5%) and between the Korean and European PToVs (74.0%). Phylogenetic analysis of the spike and nucleocapsid genes showed that the Korean PToVs form distinct branches with clusters corresponding to the farm of origin, which were separate from the other known foreign PToVs. These findings suggest that genetically diverse Korean PToV strains cause sporadic infections in Korea.

Epidemiological analysis of bovine torovirus in Japan.

Bovine torovirus (BToV), a member of the Coronaviridae family, is an established gastrointestinal infectious agent in cattle. No epidemiological research on BToV has been reported from Japan. In this study, we performed a survey to detect BToV in Japan in 2004 and 2005 using 231 fecal samples (167 from diarrheic cattle and 64 from asymptomatic cattle) that were analyzed by nested reverse transcription (RT) PCR using primers located in the consensus sequences of the reported BToV nucleocapsid (N), membrane (M), and spike (S) genes. BToV N, M, and S genes were detected in 6.5% (15/231), 6.1% (14/231), and 5.6% (13/231) of samples by nested-RT-PCR, respectively. In conclusion, detectability was improved compared to the results of the first round of RT-PCR. BToV was detected at a significantly higher rate in diarrheic samples than in asymptomatic samples (14/167 diarrheic samples [8.4%] and 1/64 asymptomatic samples [1.6%]), suggesting that BToV may act as a risk factor for diarrhea in Japanese cattle. The nucleotide sequence of M fragments from the BToV isolates including the newly identified Japanese isolates showed more than 97% identity. A similar degree of homology was observed in the N gene fragment among BToV isolates with the exception of BRV-1 and BRV-2. Domestic samples were classified into three clusters by phylogenetic analysis of the S gene fragment, which were considerably correlated with the geographic origin of the samples. BToV positive areas did not adjoin each other but were spread across a wide range, suggesting that BToV exists conventionally in Japan and is geographically differentiated. We also developed an RFLP method to distinguish these clusters using two restriction enzymes, HaeIII and AccI. This method should be useful for comparing newly acquired BToV-positive samples with the reported BToVs.

Detection of bovine torovirus in fecal specimens of calves with diarrhea in Japan.

The aim of this study was to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples and to determine whether a relationship exists between BoTV and diarrhea in Japan. Ninety-nine diarrheic and 114 normal fecal samples from calves in Hokkaido Prefecture and 38 diarrheic fecal samples from calves in 10 other prefectures were examined by reverse transcription (RT)-PCR with primers designed in the spike (S) gene for the presence of BoTV. The specimens were also examined for the presence of other enteric pathogens, bovine rotavirus, coronavirus and Cryptosporidium spp. BoTV RNA was detected in 15 (15.2%) of the 99 diarrheic samples from Hokkaido and in 9 (23.7%) of the 38 diarrheic samples from the other prefectures. The incidence of BoTV in control specimens was 7.0%. In 11 of the 15 BoTV-positive specimens from Hokkaido, BoTV was the only pathogen detected among those examined, and 11 BoTV-positive specimens were obtained from calves less than 2 weeks of age. Rotavirus was confirmed to be associated with calf diarrhea, but coronavirus and Cryptosporidium spp. were not. Nucleotide sequences of 17 different BoTV RT-PCR products were determined. Phylogenetic analysis based on the sequences revealed that Japanese BoTVs could be classified into at least two groups. This study showed that BoTV is a common virus in fecal specimens of calves with diarrhea in Japan and may be an important pathogen of cattle, principally in young calves less than 2 weeks of age.

Whole genome analysis of Japanese bovine toroviruses reveals natural recombination between porcine and bovine toroviruses.

Bovine toroviruses (BToVs), belong to the subfamily Toroviridae within the family Coronaviridae, and are pathogens, causing enteric disease in cattle. In Japan, BToVs are distributed throughout the country and cause gastrointestinal infection of calves and cows. In the present study, complete genome sequences of two Japanese BToVs and partial genome sequences of two Japanese BToVs and one porcine torovirus (PToV) from distant regions in Japan were determined and genetic analyses were performed. Pairwise nucleotide comparison and phylogenetic analyses revealed that Japanese BToVs shared high identity with each other and showed high similarities with BToV Breda1 strain in S, M, and HE coding regions. Japanese BToVs showed high similarities with porcine toroviruses in ORF1a, ORF1b, and N coding regions and the 5' and 3' untranslated regions, suggestive of a natural recombination event. Recombination analyses mapped the putative recombinant breakpoints to the 3' ends of the ORF1b and HE regions. These findings suggest that the interspecies recombinant nature of Japanese BToVs resulted in a closer relationship between BToV Breda1 and PToVs.

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.

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.

First detection and molecular diversity of Brazilian bovine torovirus (BToV) strains from young and adult cattle.

Bovine torovirus (BToV) is an established enteric pathogen of cattle, but its occurrence in Brazilian cattle had not been reported until now. This article describes a survey on BToV in Brazil carried out on 80 fecal samples from diarrheic young and adult cattle, using a nested-RT-PCR targeting the nucleocapsid (N) gene. BToV was detected in 6.25% (5/80) of stool samples from three different geographic regions. Sequences analysis showed that Brazilian BToVs have a high degree of identity with European and Japanese BToVs and a lower degree of identity with North American Breda 1 strain. These results show that, albeit its low frequency and the scarce number of research on the field, BToV is still present amongst cattle populations.

Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses.

The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.

Longitudinal serological and virological study on porcine torovirus (PToV) in piglets from Spanish farms.

A study was performed to evaluate porcine torovirus (PToV) seroprevalence and infection in three multi-site farms from the North-eastern region of Spain. Serum samples from 120 piglets and faecal samples from 36 piglets were longitudinally collected at 1, 3, 7, 11 and 15 weeks of age. Serum samples from their dams (n=30) were also taken 1-week post-farrowing. PToV antibodies in serum were monitored by ELISA, while viral infection was assessed by real-time RT-PCR in faeces. A high seroprevalence (about 100%) was observed in animals older than 11 weeks and in adult sows. Moreover, all 1-week-old animals were seropositive, indicating maternal antibody transference through colostrum. The antibody titers declined to close to or below the ELISA cut-off value by the age of weaning (3 weeks of age). Development of a significant antibody response to PToV occurred before 7 weeks of age in about 50% of piglets, and the remaining animals developed the response by weeks 11 or 15. These results indicate that PToV infection occurred soon after weaning. Although the prevalence of infection in suckling piglets varied among the studied farms, PToV prevalences in 7 and 11-week-old pigs were between 50-67% and 58-75%, respectively, in all farms. Sequencing results indicated that more than one PToV strains were circulating in the studied farms. Present data suggest that PToV was endemic on the studied farms, and provide new insights on the epidemiology of PToV.