Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease.

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Development and use of a reverse transcription insulated isothermal PCR assay for detection and characterization of bovine torovirus in yaks.

Bovine torovirus (BToV) is an important diarrhea-causing pathogen affecting bovines. To facilitate BToV detection, a reverse transcription insulated isothermal PCR (RT-iiPCR) assay was developed that targets the BToV M gene with high specificity and reproducibility. The assay has a limit of detection of 23 copies/µL. Out of 69 diarrheic fecal samples from yaks collected on six farms in Tibet and Sichuan provinces in China, 11.59% (8/69) tested positive for BToV using this assay. The full-length spike (S) and hemagglutinin-esterase (HE) genes of three positive samples were subsequently sequenced. Notably, an identical recombination event was identified in the S1 subunit of the S protein of three isolates. All of the HE genes were found to belong to genotype III and shared the same unique aa variation (P44S) in the esterase domain. This study is the first confirmation of BToV in yaks and the first report of an S gene recombination event in BToV. Our findings will enhance the current understanding of the molecular characteristics and genetic evolution of BToV.

Complete genome sequencing and genetic analysis of a Japanese porcine torovirus strain detected in swine feces.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

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.

First report and genetic characterization of bovine torovirus in diarrhoeic calves in China.

BACKGROUND: Coronaviruses are notorious pathogens that cause diarrheic and respiratory diseases in humans and animals. Although the epidemiology and pathogenicity of coronaviruses have gained substantial attention, little is known about bovine coronavirus in cattle, which possesses a close relationship with human coronavirus. Bovine torovirus (BToV) is a newly identified relevant pathogen associated with cattle diarrhoea and respiratory diseases, and its epidemiology in the Chinese cattle industry remains unknown. RESULTS: In this study, a total of 461 diarrhoeic faecal samples were collected from 38 different farms in three intensive cattle farming regions and analysed. Our results demonstrated that BToV is present in China, with a low prevalence rate of 1.74% (8/461). The full-length spike genes were further cloned from eight clinical samples (five farms in Henan Province). Phylogenetic analysis showed that two different subclades of BToV strains are circulating in China. Meanwhile, the three BToV strains identified from dairy calves, 18,307, 2YY and 5YY, all contained the amino acid variants R614Q, I801T, N841S and Q885E. CONCLUSIONS: This is the first report to confirm the presence of BToV in beef and dairy calves in China with diarrhea, which extend our understanding of the epidemiology of BToVs worldwide.

Transcriptional and Translational Landscape of Equine Torovirus.

The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5' untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames "hidden" within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase.

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3Cpro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-ß expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.

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.

Ultrastructural characterization of membranous torovirus replication factories.

Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double-membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV-infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double-stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.

Detection and molecular characterisation of bovine corona and toroviruses from Croatian cattle.

BACKGROUND: Bovine coronavirus (BCoV) together with bovine torovirus (BToV), both members of the Coronaviridae family, order Nidovirales are the most common viral enteric pathogens. Although studied separately, their joint occurrence and the molecular diversity in cattle in Croatia have not been investigated. METHODS: A survey is carried out on 101 fecal samples from diarrheic young and adult cattle during the 3-year period from i) one large dairy herd, ii) four small herds and iii) three nasal and paired fecal samples from calves with symptoms of respiratory disease. Samples were submitted to RT-PCR and sequencing for BCoV Nucleocapsid gene, BCoV Spike gene and BToV Spike gene. RESULTS: BCoV was detected in 78.8 % of fecal samples from symptomatic cattle and three nasal and paired fecal samples from calves with respiratory symptoms. BToV was detected in 43.2 % of fecal samples from symptomatic cattle and a fecal sample from calves with respiratory symptoms. Molecular characterisation of those viruses revealed some nucleotide and aminoacid differences in relation to reference strains. CONCLUSIONS: BToV should be regarded as a relevant pathogen for cattle that plays a synergistic role in mixed enteric infections.

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 of bovine torovirus in fecal specimens from calves with diarrhea in Turkey.

Bovine torovirus (BToV), a member of the family Coronaviridae, is an established gastrointestinal infectious agent in cattle. In this study, we performed a survey to detect BToV in Turkey between 2009 and 2011 using 235 fecal samples from neonatal calves with diarrhea that were analyzed by the nested reverse transcription (RT) PCR method using primers located in the consensus sequences of the BToV membrane (M) gene. The BToV M gene was detected in 4.7 % (11/235) of the samples using the nested RT-PCR method. The nucleotide sequences of partial M fragments from the BToV isolates, including the newly identified Turkish isolates, showed more than 96 % identity. The result indicates that BToV is one of the pathogens that contribute to neonatal calf diarrhea cases in Turkey.

Progress of research on coronaviruses and toroviruses in large domestic animals using reverse genetics systems.

The generation of genetically engineered recombinant viruses from modified DNA/RNA is commonly referred to as reverse genetics, which allows the introduction of desired mutations into the viral genome. Reverse genetics systems (RGSs) are powerful tools for studying fundamental viral processes, mechanisms of infection, pathogenesis and vaccine development. However, establishing RGS for coronaviruses (CoVs) and toroviruses (ToVs), which have the largest genomes among vertebrate RNA viruses, is laborious and hampered by technical constraints. Hence, little research has focused on animal CoVs and ToVs using RGSs, especially in large domestic animals such as pigs and cattle. In the last decade, however, studies of porcine CoVs and bovine ToVs using RGSs have been reported. In addition, the coronavirus disease-2019 pandemic has prompted the development of new and simple CoV RGSs, which will accelerate RGS-based research on animal CoVs and ToVs. In this review, we summarise the general characteristics of CoVs and ToVs, the RGSs available for CoVs and ToVs and the progress made in the last decade in RGS-based research on porcine CoVs and bovine ToVs.

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.

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.

A novel multiplex RT-qPCR assay for simultaneous detection of bovine norovirus, torovirus, and kobuvirus in fecal samples from diarrheic calves.

Calf diarrhea results in significant economic loss and is caused by a variety of pathogens, including enteric viruses. Many of these viruses, including bovine norovirus (BNoV), bovine torovirus (BToV), and bovine kobuvirus (BKoV), are recognized as the causative agents of diarrhea; however, they remain understudied as major pathogens. We developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) assay for rapid and simple detection of BNoV, BToV, and BKoV. Our method had high sensitivity and specificity, with detection limits of 1 × 102 copies/µL for BNoV, BToV, and BKoV, which is a lower detection limit than conventional RT-PCR for BNoV and BKoV and identical for BToV. We tested fecal samples from 167 diarrheic calves with our multiplex RT-qPCR method. Viral detection was superior to conventional RT-PCR methods in all samples. The diagnostic sensitivity of the multiplex RT-qPCR method (100%) is higher than that of the conventional RT-PCR methods (87%). Our assay can detect BNoV, BToV, and BKoV in calf feces rapidly and with high sensitivity and specificity.

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.