title: 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.
  
  
  OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section for Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; Zoonosis Science Centre, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Gillman
authors.affiliation: Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Shaman
authors.initials: S
authors.lastname: Muradrasoli
authors.affiliation: Section for Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; Zoonosis Science Centre, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mårdnäs
authors.affiliation: Department of Chemistry, Umeå University, Umeå, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Söderström
authors.affiliation: Department of Chemistry, Umeå University, Umeå, Sweden; Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodnany, University of South Bohemia in Ceske Budejovice, Ceske Budejovice, Czech Republic.
authors.forename: Ganna
authors.initials: G
authors.lastname: Fedorova
authors.affiliation: Section for Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; Zoonosis Science Centre, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
authors.forename: Max
authors.initials: M
authors.lastname: Löwenthal
authors.affiliation: Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden.
authors.forename: Michelle
authors.initials: M
authors.lastname: Wille
authors.affiliation: Department of Virology, Immunobiology and Parasitology, Swedish Veterinary Institute, Uppsala, Sweden.
authors.forename: Annika
authors.initials: A
authors.lastname: Daggfeldt
authors.affiliation: Section for Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; Zoonosis Science Centre, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
authors.forename: Josef D
authors.initials: JD
authors.lastname: Järhult
mesh_terms: D000818:Animals; D024882:Drug Resistance, Viral; D004372:Ducks / Q000821:virology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009980:Influenza A virus / Q000235:genetics*; D005585:Influenza in Birds / Q000821:virology*; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D053139:Oseltamivir; D009439:Neuraminidase