Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water

Abstract

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

FIG 1

Experimental mallard model with IAV shedding and NA 222 amino acid residues. The values 2.5 ± 0.28, 7.2 ± 0.79, and 24 ±1.4 μg/liter are the means ± standard deviations of 11, 9, and 7 water samples, respectively. The dashed horizontal lines indicate changes in OC concentrations. G1 represents generation one consisting of two mallards, G2 represents the second pair of mallards introduced in the experimental room, etc. Closed rectangles represent the presence of mallards in the experimental room and dotted lines indicate days. Note that birds were introduced into and removed from the experiment room in the daytime such that each bird was part of the experiment during 5 ′ 24 h, with a 24-h gap between every second generation. Shading indicates detection of IAV by RRT-PCR of the matrix gene from daily fecal samples (C_T values of ≥45 were determined to be negative). I, isoleucine at NA residue 222; T*, threonine in shared proportions with isoleucine at NA residue 222; T, threonine at NA residue 222, as determined by Sanger sequencing of fecal samples; W, water from the 170-liter pool in the experimental room. Closed rectangles represent change of water and shading indicates detection of IAV by RRT-PCR of the matrix gene.

FIG 2

Shedding of A(H7N9) virus determined by RRT-PCR of the matrix gene from daily fecal samples. The x axis displays the number of days that each pair of birds had been present in the experimental room at the time of sampling. The y axis displays cycle threshold (C_T) values of the RRT-PCR as a semiquantitative measure of excreted IAV. C_T values of ≥45 were determined to be negative. G1 represents mallard generation one of the experiment, consisting of two mallards, G2 represents the second pair of mallards introduced in the experimental room, etc. Values in the graph indicate mean C_T values from two samples, error bars indicate standard errors of the mean (SEM). For clarity, if one of the two samples of a generation from 1 day was IAV negative (C_T of ≥45), only the positive sample was included in the graph, i.e., G1, day 1; G2, day 5; G3, day 2; G4, day 3; G5, day 1 (see also Fig. 1).