uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Tamiflu in the Water: Resistance Dynamics of Influenza A Virus in Mallards Exposed to Oseltamivir
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
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 [en]
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: urn:nbn:se:uu:diva-277610ISBN: 978-91-554-9484-1 (print)OAI: oai:DiVA.org:uu-277610DiVA: diva2:905183
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: 2016-04-04
List of papers
1. Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir
Open this publication in new window or tab >>Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir
Show others...
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, e71230- p.Article in journal (Refereed) Published
Abstract [en]

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-206300 (URN)10.1371/journal.pone.0071230 (DOI)000323097300103 ()23951116 (PubMedID)
Available from: 2013-08-30 Created: 2013-08-30 Last updated: 2017-12-06Bibliographically approved
2. Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water
Open this publication in new window or tab >>Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water
Show others...
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.

National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-265062 (URN)10.1128/AAC.00886-15 (DOI)000364343900014 ()26077257 (PubMedID)
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
3. Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards
Open this publication in new window or tab >>Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards
Show others...
2015 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 7, 2378-2383 p.Article in journal (Refereed) Published
Abstract [en]

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-251973 (URN)10.1128/AEM.04034-14 (DOI)000351842000013 ()25616792 (PubMedID)
Available from: 2015-05-18 Created: 2015-04-28 Last updated: 2017-10-04
4. Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure.
Open this publication in new window or tab >>Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure.
Show others...
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.

National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-265061 (URN)10.1371/journal.pone.0139415 (DOI)000362175700114 ()26422258 (PubMedID)
Funder
Swedish Research Council, 211-2013-1320
Available from: 2015-10-21 Created: 2015-10-21 Last updated: 2017-12-01

Open Access in DiVA

fulltext(2337 kB)255 downloads
File information
File name FULLTEXT01.pdfFile size 2337 kBChecksum SHA-512
0ce5e1e32549e3dc96cf1d5814c627415c707f2ce0d3e8b6f2e9b8ec87488019cb78cdec6616c2160d3a65c2025c4ac501592720a5e370eeab5d0e1e3a918539
Type fulltextMimetype application/pdf
Buy this publication >>

Authority records BETA

Gillman, Anna

Search in DiVA

By author/editor
Gillman, Anna
By organisation
Infectious Diseases
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)Microbiology in the medical areaPharmacology and Toxicology

Search outside of DiVA

GoogleGoogle Scholar
Total: 255 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 924 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf