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Detection of Oseltamivir-and Zanamivir-Resistant Influenza A (H12N3) Virus from Wild Mallards in Sweden
University of Kalmar, School of Pure and Applied Natural Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Resistance to neuraminidase inhibitors (NAIs) is a growing problem in battle against influenza A virus. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the virus. To date, no low-pathogenic avian influenza (LPAI) virus that is resistant to NAIs has been detected. The aim of this study is to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC)- or zanamivir (ZA)-related resistance mutations. In this work, 22 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The R118K mutation was the most recurrent, as it was observed in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA observed in the N1 and N2 groups of NAs. Furthermore, both negative controls (NC) in the N6 and one NC in the N9 subtype were less sensitive to ZA than were genotypically related mutants of the respective subtypes. One H12N3 strain (80087 isolate) was cross-resistant, with an IC50 of >104 nM. This virus carried D151K and R156K mutations that were not associated with NAI resistance. Analyses of the 3D structure of NA indicated that the NAI-resistant phenotype that retained NA catalytic activity was likely to be due to the D151K mutation. However, the residues occupying positions 149, 150 (part of the 150 loop) and 153 may have contributed to this resistance. Other possible factors included the presence of the 150 cavity, the nature of the substrate (anchored or unanchored) and the interaction of the ligand with the active site. These results demonstrate that NAI-resistant viruses exist in LPAI and highlight the importance of monitoring influenza A viruses in wild birds, as these viruses can be transmitted to humans and can thus become part of a human-adapted influenza virus with pandemic potential.

Keyword [en]
influenza, mallards, oseltamivir, zanamivir, aniviral, resistance
National Category
Microbiology in the medical area
Research subject
Natural Science, Zoonotic Ecology
Identifiers
URN: urn:nbn:se:lnu:diva-10932OAI: oai:DiVA.org:lnu-10932DiVA: diva2:400045
Funder
Swedish Research CouncilFormas
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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