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Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water
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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2015 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 59, no 9, 5196-5202 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2015. Vol. 59, no 9, 5196-5202 p.
National Category
Infectious Medicine
Identifiers
URN: urn:nbn:se:uu:diva-265062DOI: 10.1128/AAC.00886-15ISI: 000364343900014PubMedID: 26077257OAI: oai:DiVA.org:uu-265062DiVA: diva2:862357
Funder
Swedish Research Council Formas, 211-2013-1320Swedish Research CouncilNIH (National Institute of Health), HHSN266200700010C
Available from: 2015-10-21 Created: 2015-10-21 Last updated: 2017-12-01Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1184
Keyword
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
Identifiers
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)
Opponent
Supervisors
Available from: 2016-03-17 Created: 2016-02-21 Last updated: 2018-01-10

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Gillman, AnnaNykvist, MarieOlsen, BjörnJärhult, Josef D

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