PCR Amplification and Sequencing of hBoV VP1/VP2 Gene

Conventional PCR was performed to amplify a fragment of the hBoV VP1/VP2 gene region. Nucleic acid amplification was carried out using an Eppendorf Mastercycler instrument (Eppendorf, Stevenage, UK) and a Qiagen One-Step RT-PCR kit (Qiagen, Hilden, Germany) with primers/protocol described previously (Supplementary Table (available here)) [17 (link)]. The amplified products with a length of 576 base pairs (bp) corresponding to the nucleotide positions 3233–3808 in the genome of strain PK-5510 (accession number FJ170278) were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide. The amplicons were extracted and purified with a PureLink Quick Gel Extraction kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The purified amplicons were sequenced in both directions using the BigDye™ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) on an ABI 3730XL (Thermo Fisher Scientific) DNA Analyser.
Partial VP1/VP2 gene nucleotide sequences of hBoV strains analyzed in this study were deposited in GenBank under the accession numbers MW759050-MW759067.