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Environmental levels of oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards – implications for the risk of an oseltamivir resistant influenza pandemic
University of Kalmar, School of Pure and Applied Natural Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Resistance in influenza is a growing problem. Oseltamivir carboxylate (OC), the active substance of the most widely used antiviral drug oseltamivir (Tamiflu ®), is poorly degraded in sewage treatment plants and surface water. OC has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to it. To test if resistance can occur in this situation, we infected mallards with influenza A/H1N1 virus and exposed the birds to 0.08 μg /L, 1.00 μg/L and 80.00 μg/L of OC. The resistance mutation H274Y occurred at 1 μg/L and rapidly dominated the viral population at 80 μg/L. The environmental levels of OC are expected to reach this magnitude. IC50 for OC was increased from 1-4 nM to 400-700 nM in H274Y-positive isolates, confirming a resistant phenotype. As influenza viruses can cross the species barrier, resistance to oseltamivir can spread to human-adapted strains with pandemic potential disabling one of the cornerstones in pandemic preparedness planning.

National Category
Microbiology in the medical area
Research subject
Natural Science, Zoonotic Ecology
Identifiers
URN: urn:nbn:se:lnu:diva-10936OAI: oai:DiVA.org:lnu-10936DiVA: diva2:400053
Projects
influenza, mallards, oseltamivir, Tamiflu, resistance
Available from: 2011-02-24 Created: 2011-02-24 Last updated: 2012-01-03Bibliographically approved
In thesis
1. Resistance to neuraminidase inhibitors in influenza A virus isolated from mallards
Open this publication in new window or tab >>Resistance to neuraminidase inhibitors in influenza A virus isolated from mallards
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Influenza A virus belongs to the Orothomyxoviridae family of viruses and is one of the most common pathogens that cause infections of the respiratory tract. The aim of this thesis was to investigate if neuraminidase inhibitor (NAI) Tamiflu® (oseltamivir, OC) and Relenza® (zanamivir, ZA) - related resistance mutations exist in the neuraminidase (NA) gene of viruses collected from wild birds.

A new set of degenerate primers was designed for the sequencing procedure, which resulted in a protocol that reduced time and costs of NA sequencing. This protocol was employed for subtyping of 120 NA genes (i.e. influenza viruses). Altogether, 230 NA sequences from avian influenza viruses originating from wild mallards (Ottenby, Sweden) were scanned for NAI-related mutations together with 5,490 avian, 379 swine and 122 environmental NA sequences from the NCBI dataset. The screening showed a distinction between the numbers of mutants found in avian virus sequences derived from NCBI (2.4%) as compared to virus sequences form mallards (6.5%). This is the first report of NAI resistance mutations in viruses isolated from wild birds.

The mutants carrying NAI resistance-related and resistance-unrelated mutations were screened using NA inhibition assay (NAIA) with ZA and OC inhibitors. The majority of mutations assayed showed IC50 values indicating an inhibitor sensitive phenotype. One H12N3 mutant showed a cross-resistant phenotype, i.e. insensitive to both ZA and OC treatment. Protein structure homology-modeling indicated that this cross-resistance might be associated to a D151K mutation, possibly supported by changes in NA residue 149, 150, 152 and 153. In addition, an OC resistance-related emergence of H274Y mutants was revealed in an experimental set up where mallard ducks, subjected to different concentrations of OC ( 0.28, 3.5 and 280 nM)  in their water pool, were infected with avian H1N1 virus.

In conclusion, this thesis provides new insights into the field of NAI resistance in avian influenza virus as well as indicating the evolutionary forces modern drug design has to confront. This thesis also emphasizes the importance of a continuous search for new means of protecting the human population from this potentially devastating pathogen. 

Place, publisher, year, edition, pages
Kalmar/Växjö: Linnaeus University Press, 2011. 144 p.
Series
Linnaeus University Dissertations, 38/2011
Keyword
influenza, mallards, neuraminidase, PCR, sequencing, mutation, oseltamivir, zanamivir, resistance, NAI, antivirals, pandemic
National Category
Microbiology in the medical area Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Research subject
Natural Science, Zoonotic Ecology; Biomedical Sciences, Virology; Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-10973 (URN)978-91-86491-66-6 (ISBN)
Public defence
2011-04-08, Fullriggaren, Landgången 4, 391 82 Kalmar, 08:30 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilFormas
Available from: 2011-03-14 Created: 2011-02-28 Last updated: 2014-05-12Bibliographically approved

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