Hepatitis B Virus Genotyping Protocol

All 9 human samples were genotyped and examined for the presence of mutations, as described previously [19 (link)]. PrimeSTAR (Takara Bio) PCR reaction was performed following the manufacturer’s instructions and using the following primers: HBV_MAFFT.Bf.41-18, CCTAGGACCCCTGCTCGT; and HBV_MAFFT.Br.601-16, ACAGACTTGGCCCCCA. Thermal cycling conditions included preincubation at 95 °C for 30 s, followed by 50 cycles at 98 °C for 10 s, 60 °C for 5 s, and 72 °C for 36 s, and extension at 72 °C for 5 min. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Tokyo, Japan) and processed for DNA sequencing using ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems, Waltham, MA, USA) with the same forward or reverse primer. Sequence data were generated using the ABI PRISM 3730 DNA Analyzer (Applied Biosystems). These sequences were compared to the consensus genotypes sequences using Clustal Omega [31 (link)] and their genotypes were assessed using HBVdb online tools [25 (link)].