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Oseltamivir- and Zanamivir-Resistance Related Mutations in Influenza A Viruses Isolated from Wild Mallards in Sweden Studied by a Colorimetric Neuraminidase Inhibition Assay
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]


Resistance to neuraminidase inhibitors (NAIs) is a growing problem in the battle against influenza A viruses. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the influenza virus. To date, no low-pathogenic avian influenza (LPAI) virus functionally resistant to NAIs has been detected. The aim of this study was to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC)- or zanamivir (ZA)-related resistance mutations. In this work, 21 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The R118K mutation was the most frequently observed; it was seen in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA previously observed in the N1 and N2 groups of NAs. Furthermore, both negative controls (NCs) in the N6 and one NC in the N9 subtype were less sensitive to ZA than were genotypically related mutants of the respective subtypes. The presence of OC- and ZA-related mutations in the NA of viruses isolated from wild birds did not result in a NAI-resistant phenotype in this study. The R118K and R152K mutations seemed to somewhat increase the sensitivity to both NAIs compared to WT viruses which supports the thought that the mutations were a result of natural NA variation and not induced by NAI residuals in the environment. Although not resulting in a NAI resistant phenotype, the finding of the mutations D151N and I222V shows that mutations with the potential to enhance NAI resistance exist in the pool of LPAI viruses which stresses the need for surveillance of NAI resistance in wild birds.

National Category
Microbiology in the medical area Microbiology
URN: urn:nbn:se:uu:diva-160966OAI: oai:DiVA.org:uu-160966DiVA: diva2:453742
Available from: 2011-11-03 Created: 2011-11-03 Last updated: 2013-10-25
In thesis
1. Tamiflu® - Use It and Lose It?
Open this publication in new window or tab >>Tamiflu® - Use It and Lose It?
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Influenza A viruses cause seasonal and pandemic outbreaks that range from mild infections to the disastrous Spanish Flu. Resistance to neuraminidase inhibitors (NAIs) is a growing problem as these drugs constitute a vital part of treatment strategies and pandemic preparedness plans worldwide. Oseltamivir (Tamiflu®) is the mostly used NAI. Its active metabolite, oseltamivir carboxylate (OC), is excreted from treated patients and degrades poorly in sewage treatment plants and surface water. Thus, OC can enter aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance and resistance could develop. If NAI resistance is established in influenza viruses circulating among wild birds, the resistance can form part of a virus re-entering the human population either by reassortment or by direct transmission.

In this thesis, evidence is presented that OC is present in the waterways during a seasonal influenza outbreak in Japan, a country in which oseltamivir is liberally used. Furthermore, when mallards were infected with an influenza A/H1N1 virus and subjected to low, environmental-like concentrations of OC, resistance developed through acquisition of the well-known resistance mutation H274Y. The influenza infection in the mallards was mainly intestinal, had a rapid onset and was progressing in a longitudinal fashion in the intestine. Finally, influenza A viruses isolated from wild mallards in Sweden and containing resistance-related mutations were examined by a neuraminidase inhibition assay. The viruses did not have a decreased sensitivity to NAIs, but had mutations with a resistance-enhancing potential.

Thus, OC is present in the environment and environmental-like concentrations of OC induce resistance in influenza viruses of dabbling ducks. The present resistance situation among wild birds is not well understood but the existence of H274Y among wild birds, though rare, and the spread of the former seasonal A/H1N1 virus containing H274Y among humans indicate that resistance mutations could establish themselves also among wild birds. An oseltamivir-resistant pandemic or a human-adapted highly-pathogenic avian influenza virus are frightening scenarios as oseltamivir is a cornerstone in the defense in those situations. There is a need for further studies, surveillance in wild birds and for a prudent use of antivirals.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 60 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 725
influenza, oseltamivir, Tamiflu, resistance development, H274Y, environment, pharmaceuticals, mallard, dabbling duck, avian influenza, influensa, resistensutveckling, miljö, läkemedel, gräsand
National Category
Infectious Medicine
Research subject
Infectious Diseases
urn:nbn:se:uu:diva-160974 (URN)978-91-554-8225-1 (ISBN)
Public defence
2011-12-17, Gustavianum, Auditorium minus, Akademigatan 3, Uppsala, 10:00 (English)
Available from: 2011-11-25 Created: 2011-11-03 Last updated: 2012-02-29Bibliographically approved

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