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.

Influenza A/H4N2 mallard infection experiments further indicate zanamivir as less prone to induce environmental resistance development than oseltamivir

“…Prior to inclusion in the experiment, all birds tested negative for IAV by enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription polymerase chain reaction (RRT-PCR). The shedding kinetics of IAV () was similar across all experiments and similar to previous studies using this model system [14, 15, 19, 20].…”

“…The shedding patterns were similar in all five experiments, and they were also similar to those in previous observations in the mallard model [14, 15, 19, 20]. Hence, neither exposure to 1–100 µg l −1 of ZA nor the presence of the H274N or E119G substitutions appear to impact on IAV shedding.…”

“…For H274N, the mutated virus dominated the viral population, but by the last day in the second and third generations, the substitution was detected in the mixed genotype with wild-type. This indicates reduced fitness for the substitution in N2 and is in contrast to H274Y in N1, where an H1N1 LPAIV carrying the H274Y substitution has been replicating for multiple generations in the mallard model without any reversion to a mixed genotype [17, 19]. The reduced fitness is in line with previous work on the exposure of a N1 LPAIV to ZA [17] and another N2 LPAIV exposed to OC [20].…”

“…In previous mallard in vivo OC exposure experiments, the H274Y substitution emerged in H1N1 and persisted in the absence of OC, while the R292K substitution emerged in H6N2 but did not persist [13, 14, 19, 20].…”

RNA Viruses and RNAi: Quasispecies Implications for Viral Escape

“…Although many of the observed mutations lower replicative fitness in these resistant viruses, secondary compensatory mutations can eliminate fitness losses while also retaining drug resistance [ 31 ]. Oseltamivir-resistant strains of influenza have recently been identified in reservoir bird populations as well, even in the absence of continuous selective pressure, which could prove to be troubling if it leads to newly-emerging resistant strains [ 32 ]. Additionally, mutations which confer drug resistance have been studied and mapped extensively in HIV against a large array of antiviral drugs [ 33 ], including resistance to the relatively new small interfering peptides [ 34 ].…”