Environmental Levels of the Antiviral Oseltamivir Induce Development of Resistance Mutation H274Y in Influenza A/H1N1 Virus in Mallards

Abstract

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards

“…By calculations of the mutation frequency per replicative viral cycle (10 Ϫ5 per site) and the exponential replication in a new host (29), a subpopulation of viruses with substantially superior fitness would already be noticeable when the replication plateau was reached, after 2 days. This time perspective is supported by results from a previous study in this mallard model where the resistant A/51833/H274Y virus emerged rapidly and dominated over the A/51833/wt virus at a high drug pressure (23). The possibility that minimally decreased fitness of the mutant would go unnoticed under our experimental conditions cannot be excluded, but we argue that substantial evolutionary space for reversion was provided.…”

“…In this in vivo study, when we gradually decreased the level of OC exposure for mallards infected with an OC-resistant avian influenza A (H1N1)/NA-H274Y virus, and finally removed OC altogether, we found no genotypic or phenotypic decrease in resistance and no reversion to wild-type virus. Targeted deep sequencing of the resistance point mutation did not indicate any tendency for the wild-type virus to reemerge when the drug pressure was below the level of induction of the NA-H274Y mutation (0.95 g/liter [23]) or when OC had been removed. Based on the number of reads and the fact that the wildtype genotype was never more prevalent than the sequencing noise, we can rule out a wild-type subpopulation with a prevalence higher than 1%.…”

“…Between the euthanasia of one generation and the introduction of a new one, there was a 1-day gap to ensure continuous virus transmission between generations (e.g., generation 1 was removed 1 day before generation 3 was introduced, to make sure that generation 3 was infected by virus from generation 2 and not from generation 1). OC exposure levels ranging from approximately 80 g/liter (a concentration at which the mutant A/51833/H274Y virus was known to dominate completely [23]) to zero were chosen, and half of the concentrations were lower than the resistance induction level of 0.95 g/liter (23).…”

“…In vivo experiments have demonstrated that OC resistance evolves in avian IAVs of both the N1 and N2 neuraminidase groups when infected mallards (Anas platyrhynchos) are exposed to 0.95 to 12 g/liter OC in their water (23)(24)(25). However, a fundamental question for the persistence of NAI resistance in the environment is whether a resistance mutation is retained without continuous selective drug pressure.…”

Assessment of Domestic Wastewater Disposal in Some Selected Wards of Maiduguri Metropolis, Borno State, Nigeria

“…The large load and high concentration of bioactive pharmaceuticals entering the wastewater and receiving rivers from widespread human consumption and excretion during a pandemic can potentially disrupt (micro)organisms through non-target effects [6][7][8][9][10][11][12] and cause the failure of wastewater treatment plants (WWTPs) to treat effluent to the required standard [13,14], hasten the generation of antiviral resistance in wildfowl and other influenza-susceptible organisms [15][16][17][18], and accelerate the generation and spread of (novel) antibiotic resistance in the environment [2,19,20].…”