Determining the Mutation Bias of Favipiravir in Influenza Using Next-generation Sequencing

Favipiravir is a broad spectrum antiviral drug that may be used to treat influenza. Previous research has identified that favipiravir likely acts as a mutagen but the precise mutation bias that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and quantitatively detect favipiravir-induced mutations and to sample orders of magnitude more mutations than would be possible through Sanger sequencing. We demonstrate that favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue and secondarily as an adenine analogue resulting in the accumulation of transition mutations. We also use a standard NGS pipeline to show that the mutagenic effect of favipiravir can be measured by whole genome sequencing of virus.