T-705 (Favipiravir) Induces Lethal Mutagenesis in Influenza A H1N1 Viruses <i>In Vitro</i>

TLDR

Several novel anti‑influenza compounds are in development, including T‑705 (favipiravir), whose mechanism is thought to target influenza RNA‑dependent RNA polymerase. We investigated the mechanism of T‑705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. The study applied serial drug pressure in MDCK cells, monitoring viral replication and mutation patterns across passages. T‑705 did not select resistant mutations but caused a dramatic drop in infectious titers, increased G→A/C→T transversions, and reduced specific infectivity, demonstrating lethal mutagenesis as its key antiviral mechanism.

Abstract

ABSTRACT Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly ( P &lt; 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.