Bigdye terminator v3.1 cycles sequencing kit

PCR products were sequenced using a BigDye Terminator v3.1 Cycles Sequencing Kit on an automated ABI Prism 3130 Genetic Analyzer (Thermo Fisher Scientific, Carlsbad, CA, USA). Each genomic sequence from the 99 bovine RVA strains determined herein was submitted to the DNA Data Bank of Japan (DDBJ); the sequences are retrievable from GenBank (LC590915–LC591102, Table 1). The sequence data were aligned using the ClustalW method in the MEGA X program [30 (link)]. Phylogenetic analyses were conducted using the maximum-likelihood method with the general time reversible nucleotide substitution model and 1000 bootstrap replicates. Genotype classifications of the VP7 and VP4 genes from the 99 bovine RVA strains were conducted using 80% cutoff values calculated in a previous study [31 (link)].

The partial genomes of 5′-UTR (untranslated region) were amplified using the SuperScript III One-step RT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher Scientific) and primers, 324 (Forward: 5′-ATGCCCWTAGTAGGACTAGCA-3′) and 326 (Reverse: 5′-TCAACTCCATGTGCCATGTAC-3′) that were previously reported [34 (link)]. PCR products were determined their nucleotide sequences using the BigDye Terminator v3.1 Cycles Sequencing Kit on an automated ABI Prism 3130 XL Genetic Analyzer (Thermo Fisher Scientific, Carlsbad, CA, USA). Each genomic sequence from the eight BVDVs determined herein was submitted to the DNA Data Bank of Japan; the sequences are retrievable from GenBank (accession numbers: LC600219-LC600226). Phylogenetic analysis using the data added to genomic sequences of other BVDV strains available in GenBank into genomic sequences from the eight BVDV strains was determined using the maximum-likelihood method and 1000 bootstrap replicates on MEGA7 software [35 (link),36 (link)]. Nucleotide sequences of other BVDV strains used in this study were as follows: BVDV1 strains, Nose (AB019670) and NADL (AF039181), BVDV2 strains, 1373 (AF145967), 890 (U18059), and KZ91-CP (AB003619). Moreover, this analysis also included four BVDV2 strains from PI animals from Iwate Prefecture between 2012 and 2018 (GenBank accession numbers: LC600227-LC600230).