Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure

Abstract

Background: Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied.

Aim and methods: In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype.

Results and conclusion: Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.

Fig 1. Viral excretion.

IAV was detected by RRT-PCR of the matrix gene from daily fecal samples. The Y-axis displays cycle threshold (CT) values as a quantitative measure of viral excretion. Samples with CT values ≥ 45 were considered negative. The X-axis displays which days of the experiment samples were collected from the Mallards. G1 = two birds in each of three experiments (n = 6), etc. G1 and G2 were OC exposed, G3 was OC exposed day 0–2, and G4 and G5 were unexposed. Value points display mean CT values of 6 samples and error bars display standard errors of the mean (SEM). No significant difference in excretion was detected between drug-exposed (G1 and G2 with resistant genotype NA-292K) and unexposed (G4 and G5 with wild type genotype NA-292R) birds day 1, 4 or 5, while drug-exposed birds had lower virus excretion levels day 2 and 3(P = 0.034).