The R292K mutation that confers resistance to neuraminidase inhibitors leads to competitive fitness loss of A/Shanghai/1/2013 (H7N9) influenza virus in ferrets

Abstract

Background: Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment.

Methods: We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo.

Results: Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs.

Conclusions: The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus.

Keywords: H7N9 influenza virus; R292K NA mutation; ferrets; neuraminidase inhibitors; resistance; transmission.

Figure 1.

Transmission and nasal wash titers (log₁₀TCID₅₀/mL) detected in ferrets inoculated or infected by the plaque-purified A/Shanghai/1/2013 (H7N9) wild-type virus (A), or the plaque-purified A/Shanghai/1/2013 (H7N9) NA-R292K mutant virus (B). Nasal washes were collected daily from inoculated donor ferrets (days 2–8 post-inoculation), direct contact ferrets, and respiratory droplet contact ferrets (days 1–11 post-contact); the lower limit of detection was 1.789 log₁₀TCID₅₀/mL. Abbreviation: TCID_50, 50% tissue culture infectious dose.

Figure 2.

Total amount of virus shedding was approximated by calculating the area under the curve for each ferret using the virus titers determined at different days post-inoculation or post-contact. The mean ± SD area under the curve were plotted for donors, direct contacts, or respiratory droplet contact ferrets infected with the A/Shanghai/1/2013 wild-type or NA-R292K mutant strains. P values showed the results from Wilcoxon rank-sum tests. Abbreviation: SD, standard deviation.

Figure 3.

Temperature changes (°C, mean ± SD) of donors (A), direct contact ferrets (B), or respiratory droplet contact ferrets (C) over the course of transmission experiments with A/Shanghai/1/2013 wild-type or NA-R292K mutant viruses. The baseline temperature was determined at −1 day post-inoculation (−2 day post-contact). Abbreviation: SD, standard deviation.

Figure 4.

Genotyping assay to determine the percentage of R292/ K292 in the nasal washes of donors (A), direct contact ferrets (B), and respiratory droplet contact ferrets (C) infected with the plaque-purified A/Shanghai/1/2013 NA-R292K mutant virus. The percentages of the mutant K292 genotype are shown using diagonally striped bars, and the percentages of the wild-type R292 genotype are shown using solid black bars.