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Resistance to neuraminidase inhibitors in influenza A virus isolated from mallards
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (Zoonotic Ecology and Epidemiology)
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 [en]
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: urn:nbn:se:lnu:diva-10973ISBN: 978-91-86491-66-6 (print)OAI: oai:DiVA.org:lnu-10973DiVA: diva2:400967
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
List of papers
1. 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
Open this publication in new window or tab >>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
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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:nbn:se:lnu:diva-10936 (URN)
Projects
influenza, mallards, oseltamivir, Tamiflu, resistance
Available from: 2011-02-24 Created: 2011-02-24 Last updated: 2012-01-03Bibliographically approved
2. Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene
Open this publication in new window or tab >>Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene
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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.

Keyword
Influenza A, Neuraminidase, Sequencing, PCR, Degenerate primers
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Zoonotic Ecology
Identifiers
urn:nbn:se:lnu:diva-10007 (URN)10.1016/j.jviromet.2010.09.006 (DOI)
Funder
Swedish Research CouncilFormas
Available from: 2011-01-15 Created: 2011-01-15 Last updated: 2011-03-14Bibliographically approved
3. Detection of Oseltamivir-and Zanamivir-Resistant Influenza A (H12N3) Virus from Wild Mallards in Sweden
Open this publication in new window or tab >>Detection of Oseltamivir-and Zanamivir-Resistant Influenza A (H12N3) Virus from Wild Mallards in Sweden
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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
influenza, mallards, oseltamivir, zanamivir, aniviral, resistance
National Category
Microbiology in the medical area
Research subject
Natural Science, Zoonotic Ecology
Identifiers
urn:nbn:se:lnu:diva-10932 (URN)
Funder
Swedish Research CouncilFormas
Available from: 2011-02-24 Created: 2011-02-24 Last updated: 2012-01-03Bibliographically approved
4. Detection of Resistance Mutations to Antivirals Oseltamivir and Zanamivir in Avian Influenza A Viruses Isolated from Wild Birds
Open this publication in new window or tab >>Detection of Resistance Mutations to Antivirals Oseltamivir and Zanamivir in Avian Influenza A Viruses Isolated from Wild Birds
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).

Keyword
Influenza A, antiviral resistance, mallard, oseltamivir, zanamivir
National Category
Microbiology in the medical area
Research subject
Ecology, Zoonotic Ecology
Identifiers
urn:nbn:se:lnu:diva-10274 (URN)10.1371/journal.pone.0016028 (DOI)
Funder
Swedish Research CouncilSwedish Research Council Formas
Available from: 2011-01-21 Created: 2011-01-21 Last updated: 2016-07-20Bibliographically approved

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