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Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (Zoonotisk ekologi och epidemiologi)
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
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2010 (English)In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 170, no 1-2, 94-98 p.Article in journal (Refereed) Published
Abstract [en]

This study describes the design of degenerate primers and their use for synthesis of full-length avian influenza A neuramindase (NA). Each reaction was performed using either two forward primers and one reverse primer, or one forward primer and one reverse primer. Both primer combinations had comparable amplification efficiencies for all NA subtypes (1-9). A total of 11 virus strains, including both field isolates and reference strains, were amplified successfully using these degenerate primer sets. Of the sequences amplified, 108 strains (93.9%) resulted in near full-length NA cDNAs after two readings with one forward primer and one reverse primer. Of the remaining sequences, five strains (4.3%) yielded reads with enough information for subtype categorization by BLAST although they were of insufficient quality for assembly. One strain (0.9%) yielded different subtypes from both sequence reads whereas the other one (0.9%) was not possible to assemble and subtype. This successful demonstration of these degenerate primers for the amplification and sequencing of all avian NA subtypes suggests that these primers could be employed in the avian influenza surveillance program as well as studies of antiviral resistance, virus ecology or viral phylogeny.

Place, publisher, year, edition, pages
2010. Vol. 170, no 1-2, 94-98 p.
Keyword [en]
Influenza A, Neuraminidase, Sequencing, PCR, Degenerate primers
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Zoonotic Ecology
Identifiers
URN: urn:nbn:se:lnu:diva-10007DOI: 10.1016/j.jviromet.2010.09.006OAI: oai:DiVA.org:lnu-10007DiVA: diva2:387890
Funder
Swedish Research CouncilFormas
Available from: 2011-01-15 Created: 2011-01-15 Last updated: 2011-03-14Bibliographically 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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