title: 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.
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 anna.gillman@medsci.uu.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Gillman
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: Marie
authors.initials: M
authors.lastname: Nykvist
authors.affiliation: Zoonosis Science Centre, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden 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: Department of Chemistry, Umeå University, Umeå, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Söderström
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: Department of Pathology and Wildlife Disease, Swedish Veterinary Institute, Uppsala, Sweden.
authors.forename: Caroline
authors.initials: C
authors.lastname: Bröjer
authors.affiliation: Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Waldenström
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: Björn
authors.initials: B
authors.lastname: Olsen
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; D004372:Ducks; D064766:Influenza A Virus, H7N9 Subtype / Q000187:drug effects* / Q000201:enzymology*; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D053139:Oseltamivir / Q000031:analogs & derivatives / Q000494:pharmacology*; D014867:Water / Q000737:chemistry*
chemical_list: D014867:Water; D053139:Oseltamivir; D009439:Neuraminidase; C535162:oseltamivir carboxylate