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Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 9Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2015. Vol. 10, no 9
National Category
Infectious Medicine
URN: urn:nbn:se:uu:diva-265061DOI: 10.1371/journal.pone.0139415ISI: 000362175700114PubMedID: 26422258OAI: oai:DiVA.org:uu-265061DiVA: diva2:862356
Swedish Research Council, 211-2013-1320
Available from: 2015-10-21 Created: 2015-10-21 Last updated: 2016-04-04Bibliographically approved
In thesis
1. Tamiflu in the Water: Resistance Dynamics of Influenza A Virus in Mallards Exposed to Oseltamivir
Open this publication in new window or tab >>Tamiflu in the Water: Resistance Dynamics of Influenza A Virus in Mallards Exposed to Oseltamivir
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened.

The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected.

We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic.

We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay.

Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants.

We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 114 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1184
Influenza A virus, avian influenza, oseltamivir, neuraminidase inhibitors, resistance, environmental, Mallard, waterfowl
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Microbiology in the medical area Pharmacology and Toxicology
Research subject
Medical Science
urn:nbn:se:uu:diva-277610 (URN)978-91-554-9484-1 (ISBN)
Public defence
2016-04-08, Auditorium minus, Museum Gustavianum, Akademigatan 3, Uppsala, 09:15 (English)
Available from: 2016-03-17 Created: 2016-02-21 Last updated: 2016-04-04

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Gillman, AnnaJärhult, Josef D
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