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

Abstract

11Favipiravir is a broad spectrum antiviral drug that may be used to treat influenza. Previous 12 research has identified that favipiravir likely acts as a mutagen but the precise mutation bias 13 that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-14 generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and 15 quantitatively detect favipiravir-induced mutations and to sample orders of magnitude 16 more mutations than would be possible through Sanger sequencing. We demonstrate that 17 favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue 18 and secondarily as an adenine analogue resulting in the accumulation of transition 19 mutations. We also use a standard NGS pipeline to show that the mutagenic effect of 20 favipiravir can be measured by whole genome sequencing of virus. 21 Importance 22New antiviral drugs are needed as a first line of defence in the event of a novel influenza 23 pandemic. Favipiravir is a broad-spectrum antiviral which is effective against influenza. The 24 exact mechanism of how favipiravir works to inhibit influenza is still unclear. We used next-25 generation sequencing (NGS) to demonstrate that favipiravir causes mutations in influenza 26 RNA. The greater depth of NGS sequence information over traditional sequencing methods 27 allowed us to precisely determine the bias of particular mutations caused by favipiravir. NGS 28 can also be used in a standard diagnostic pipeline to show that favipiravir is acting on the 29 virus by revealing the mutation bias pattern typical to the drug. Our work will aid in testing 30 whether viruses are resistant to favipiravir and may help demonstrate the effect of 31 favipiravir on viruses in a clinical setting. This will be important if favipiravir is used during a 32 future influenza pandemic. 33

Favipiravir, an anti-influenza drug against life-threatening RNA virus infections

“…Therefore, theoretical resistant mutants should be generated but are unable to replicate or replicate with reduced fidelity to replace the entire virus population. The favipiravir-resistant mutant replicates as an artificially generated clone but does not become dominant among the entire virus population that grows in the presence of favipiravir (Abdelnabi, et al, 2017;Delang, et al, 2014;Goldhill, et al, 2019). Our results are consistent with the absence of a resistant virus in the entire virus population treated with favipiravir.…”

“…Favipiravir treatment increases the frequency of transition (Delang, et al, 2014;Goldhill, et al, 2019;Vanderlinden, et al, 2016) and transversion (Baranovich, et al, 2013) in viral genomes, and these mutations are hypothesized to be caused by favipiravir, resulting in lethal mutagenesis (Baranovich, et al, 2013;Delang, et al, 2014;Goldhill, et al, 2019;Vanderlinden, et al, 2016). We observed a transition of influenza virus and poliovirus in cultures treated with favipiravir but did not assess the frequency because we were unable to J o u r n a l P r e -p r o o f Journal Pre-proof compare the transition rate with the proper antiviral agents with similar mechanisms as a control Daikoku, Yoshida, Okuda, & Shiraki, 2014).…”

“…A common feature of less susceptible viruses is high fidelity of RdRp that may distinguish favipiravir-RTP and GTP and replicate in the presence of favipiravir by avoiding the incorporation of favipiravir-RTP. Since their proliferative ability is not high, clones that are less susceptible to favipiravir grow well alone or are not viable, but an additional alteration that modulates RdRp activity restores the replication capability and the susceptibility to favipiravir (Abdelnabi, et al, 2017;Delang, et al, 2014;Goldhill, et al, J o u r n a l P r e -p r o o f Journal Pre-proof 2019). Favipiravir-resistant mutants of influenza virus have been created by reverse-engineering and replicate as a clone with a reduced growth property.…”

The mechanism of resistance to favipiravir in influenza

“…We and others have shown that favipiravir induces mutations in the viral genome ( 25 , 36 ). One possible mechanism by which the K229R mutation might confer resistance to favipiravir would be to increase the fidelity of the polymerase, as has been observed for ribavirin resistance in several viral systems ( 37 ).…”

Status of antiviral therapeutics against rabies virus and related emerging lyssaviruses

“…Favipiravir (T-705) is a broad-spectrum RNA virus inhibitor that is currently licensed in Japan for stock-piling against pandemic influenza viruses resistant to oseltamivir. T-705 has been shown to be effective against Ebola virus, also within order Mononegavirales and therefore was considered for use against RABV also [175][176][177][178][179][180][181]. When tested in mouse neuroblastoma neural 2a cells, T-705 indeed lowered RABV titers by three to four order of magnitude (EC 50 of 32.4 μM against a circulating RABV strain and 44.3 μM against the vaccine strain).…”