Identifying Mutations in HAV Genome

It was previously reported that the antiviral effect of favipiravir and ribavirin was correlated with the incorporation of a large number of mutations into viral genomes in other viruses [36 (link),74 (link),75 (link)]. To explain the mechanisms of HAV inhibition by favipiravir and ribavirin, we examined nucleotide mutations in the HAV genome by next-generation sequencing. We first extracted cellular RNA from HAV-infected Huh7 cells treated with or without favipiravir, ribavirin, or foscarnet sodium at 100 μM each for 72 h. We next amplified the target site by linker-added specific primers (Table 2) using a PrimeScript II High Fidelity One Step RT-PCR Kit (Takara). Each reaction was performed at 45 °C for 10 min and 94 °C for 2 min, followed by 45 cycles at 98 °C, 10 s for denaturation, 1 min at 60 °C for annealing, and 10 s at 68 °C for extension. The products were purified using a QIAquick PCR purification kit (Qiagen). Targeted deep sequencing was performed using an Illumina MiSeq System (Illumina K.K., Tokyo, Japan) at the instruction of Hokkaido System Science Co. Ltd. (Sapporo, Hokkaido, Japan).