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.