Oseltamivir-resistant influenza A (H1N1) virus strain with an H274Y mutation in neuraminidase persists without drug pressure in infected mallards

Abstract

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.

FIG 1

Viral shedding in feces from mallards was quantified by RRT-PCR of the IAV matrix gene. C_T values of >45 were considered to indicate a negative sample. Whiskers indicate standard errors of the means for two samples. The number of days after inoculation (for generation 1 [g1]) or after introduction to the experimental room (for g2 to g10) is shown along the x axis. Each generation consisted of two mallard ducks.

FIG 2

Inhibition of early and late samples of the mutated A/51833/H274Y virus and of the original A/51833/wt virus by OC and ZA. Results for early (generations 1 and 2) and late (generation 10) samples are shown as mean IC₅₀ for 8 duplicate samples. The IC₅₀ for the original virus are means from 6 repeated assays. Boxes indicate the 95% CIs of the means, and whiskers indicate minimum and maximum values.