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

Abstract

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.

Keywords: Bovine torovirus; Japan; Natural recombination; Porcine torovirus.

Fig. 1

Similarity plot analyses of the entire genome length of PToV NPL/2013 (pink line), PToV SH1 (red line), BToV Breda1 (green line), and BToV Ishikawa/2010 (A), BToV Kagoshima/2014 (B), and BToV Tochigi/2–13 (C) as query sequences, using a sliding window of 200 nt and a moving step size of 20 nt.

Fig. 2

Detection of recombination breakpoint in Japanese BToV genomes. (A) Genome organization of ToV. Presumed recombinant breakpoints were mapped to the 3′ end of ORF1b and of HE coding regions. Pink boxes indicate sequence regions originating from PToV. Green boxes indicate sequence regions originating from BToV Breda1 strain.(B) Bootscan analysis of BToV Ishikawa/2010 vs PToV NPL/2013 (purple line), BToV Ishikawa/2010 vs BToV Breda1 (green line) and PToV NPL/2013 vs BToV Breda1 (yellow line). Cut-off of the bootstrapping test (> 70%) is indicated by the break-point. (C) Sequence of recombination junctions of Japanese BToVs. Dots indicate sequences similar to that of BToV Ishikawa/2010. Identities between Japanese BToVs and PToV (NPL/2013 and SH1) are highlighted in pink; identities between Japanese BToV and BToV Breda1 are highlighted in green.

Fig. 3

Phylogenetic trees based on sequences of 5′UTR (A), ORF1a (B), ORF1b (C), S (D, E), M (F), HE (G), N (H), and 3′UTR (I). Phylogenetic trees were constructed using the maximum likelihood method in MEGA5.22 with bootstrap values (1000 replicates). Scale bar indicates nucleotide substitutions per site. The BToV strains are represented by green (BToV Breda1 is indicated by boldface), whereas the PToV strains are represented by red. The BToV strains analyzed in this study are shown by black open square. *: Length of nucleotide sequences using analysis. **: Nucleotide position of BToV Ishikawa/2010.