Resistance mutation R292K is induced in influenza A(H6N2) virus by exposure of infected mallards to low levels of oseltamivir

Abstract

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.

Figure 1. Set-up of the Mallard Model.

Each experiment consisted of 4 generations of mallards with 2 individuals in each. The first generation of mallards was infected by esophageal inoculation with the A/Mallard/Sweden/50908/2006(H6N2) virus (day 0) and were put in the experimental room where the sole water source contained OC. The following generations were introduced with 3-day intervals allowing the infection to be fecal-orally transmitted between generations during two days. Five days after introduction, each generation birds were removed and euthanized. The water in the sole water source of the birds was changed daily and contained the same level of OC during each 14 day-experiment. Three subsequent experiments were performed with OC levels of 120 ng/L, 1.2 µg/L and 12 µg/L.

Figure 2. Viral Shedding.

RRT-PCR performed on fecal samples. The Y-axis displays CT-values. The cut-off for negative results was set to a CT-value of 45. Error bars display ± standard deviation. n = 8 in 120 ng/L and 1.2 µg/L groups, n = 4 in the two groups from the 12 µg/L experiment.

Figure 3. Mutation Analysis of NA the 12 µg/L Experiment.

Sequencing of the NA gene from fecal samples, water and post egg propagation from the 12 µg/L experiment. G = generation. Pi = post infection. H2O = water. Green wt = wild type. Red R292K = mutated at 292. Red (R292K) = mixed genotype R292 and K292. * = D113N. (*) = mixed genotype D113 and N113.+ = D141N. (+) = mixed genotype D141 and N141. Purple fs = failure to sequence. Blue fe = failure to propagate in embyonated hen eggs. White ns = not sequenced. White ne = not propagated in embryonated hen eggs.