Favipiravir-resistant influenza A virus shows potential for transmission

Abstract

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.

Fig 1. 4 donor ferrets were infected with 10^4 PFU of a virus mix of wildtype Eng195 and K229R+P653L.

Direct contact and indirect sentinels were exposed from day 1. Ferrets were nasal washed each day and virus infectivity in nasal wash titred by plaque assay. 2 samples were chosen for sequencing from each ferret and are denoted by the black outlined symbols.

Fig 2. Targeted sequencing of PA and PB1 using NGS showed the percentage of PB1 K229R and PA P653L mutations for donor, contact and indirect contact ferrets.

The top pie chart shows the percentage of each genotype for residue 229 in PB1 with the mutant (R229) in red and the wild type (K229) in black. The bottom pie chart shows the percentage of each genotype for residue 653 in PA with the mutant (L653) in blue and the wild type (P653) in black. The inoculum shows 5% K229 and 5% P653. For each infected ferret, two sequenced time points (as described in Fig 1) are shown. The group 4 indirect contact was not infected.

Fig 3

A) The proportion of each virus genotype are shown over 20 rounds of replication for a model with reassortment and mutation. The starting proportions are 5% Wild type and 95% K229R + P653L. Strain fitness for Wild type, K229R, P653L and K229R + P653L were set at 1, 0.01, 1.25 and 1 respectively. 10^6 viruses are modelled with 10^6 cells with a mutation rate, μ = 2 ×10⁻⁴. B) As A but the strain fitness for Wild type, K229R, P653L and K229R + P653L were set at 1, 0.01, 1 and 1 respectively. C) As A but the strain fitness for Wild type, K229R, P653L and K229R + P653L were set at 1, 1, 1.25 and 1 respectively. D) As A but there was no reassortment allowed during coinfection, only mutation. All graphs show results from 100 replicates (the line width is from the 2.5th to the 97.5^th percentile).

Fig 4. Minigenome assays were performed in 293T cells in 24 or 48-well plates.

For 24 well plates, Pol I–firefly luciferase minigenome reporter, at 0.08 μg and PCAGGS-Renilla, at 0.1 μg were transfected with PCAGGS plasmids coding for wildtype and mutated polymerase subunits (PB1, PB2 and PA) and NP at 0.08, 0.08, 0.04 and 0.12 μg respectively derived from A) Eng195 first wave and B) Eng687 third wave pH1N1 virus. Plasmid amounts were halved for 48-well plate experiments. Luciferase signal was read 24 hours post-transfection. Polymerase activity is given as a ratio Firefly to Renilla signals. One-way ANOVA with Dunnett’s multiple comparison test, *** p<0.001, **** p<0.0001, ns = not significant. N = 3. Error bars show s.d. Experiments were repeated 3 times and a representative experiment is shown.

Fig 5. Schematic explaining how virus populations change for the donor and direct contact ferrets from Group 1.

Large pie charts show the percentage of PB1 K229R + PA P653L mutant (purple) and wild-type viruses (black). Reassortment leads to the generation of the single mutant PA P653L (blue) in the donor which is transmitted to the direct contact. Smaller pie charts on each ferret show the sequencing results for PB1 and PA as in Fig 2.