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 97.5th 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.