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Detection of Resistance Mutations to Antivirals Oseltamivir and Zanamivir in Avian Influenza A Viruses Isolated from Wild Birds
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. Uppsala University. (Zoonotisk Ekologi och Epidemiologi)
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (Bioorganic and Biophysical Chemistry Laboratory)
Uppsala University.
Uppsala University.
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 1, e16028Article in journal (Refereed) Published
Abstract [en]

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are the first-line of defense against potentially fatal variants of influenza A pandemic strains. However, if resistant virus strains start to arise easily or at a high frequency, a new anti-influenza strategy will be necessary. This study aimed to investigate if and to what extent NA inhibitor–resistant mutants exist in the wild population of influenza A viruses that inhabit wild birds. NA sequences of all NA subtypes available from 5490 avian, 379 swine and 122 environmental isolates were extracted from NCBI databases. In addition, a dataset containing 230 virus isolates from mallard collected at Ottenby Bird Observatory (Öland, Sweden) was analyzed. Isolated NA RNA fragments from Ottenby were transformed to cDNA by RT-PCR, which was followed by sequencing. The analysis of genotypic profiles for NAs from both data sets in regard to antiviral resistance mutations was performed using bioinformatics tools. All 6221 sequences were scanned for oseltamivir- (I117V, E119V, D198N, I222V, H274Y, R292K, N294S and I314V) and zanamivir-related mutations (V116A, R118K, E119G/A/D, Q136K, D151E, R152K, R224K, E276D, R292K and R371K). Of the sequences from the avian NCBI dataset, 132 (2.4%) carried at least one, or in two cases even two and three, NA inhibitor resistance mutations. Swine and environmental isolates from the same data set had 18 (4.75%) and one (0.82%) mutant, respectively, with at least one mutation. The Ottenby sequences carried at least one mutation in 15 cases (6.52%). Therefore, resistant strains were more frequently found in Ottenby samples than in NCBI data sets. However, it is still uncertain if these mutations are the result of natural variations in the viruses or if they are induced by the selective pressure of xenobiotics (e.g., oseltamivir, zanamivir).

Place, publisher, year, edition, pages
2011. Vol. 6, no 1, e16028
Keyword [en]
Influenza A, antiviral resistance, mallard, oseltamivir, zanamivir
National Category
Microbiology in the medical area
Research subject
Ecology, Zoonotic Ecology
Identifiers
URN: urn:nbn:se:lnu:diva-10274DOI: 10.1371/journal.pone.0016028OAI: oai:DiVA.org:lnu-10274DiVA: diva2:390316
Funder
Swedish Research CouncilSwedish Research Council Formas
Available from: 2011-01-21 Created: 2011-01-21 Last updated: 2016-07-20Bibliographically 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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