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Environmental Levels of the Antiviral Oseltamivir Induce Development of Resistance Mutation H274Y in Influenza A/H1N1 Virus in Mallards
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
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2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 9, e24742- p.Article in journal (Refereed) Published
Abstract [en]

Oseltamivir (Tamiflu (R)) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008-2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 mu g/L and 80 mu g/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 mu g/L of OC and rapidly dominated the viral population at 80 mu g/L. IC(50) for OC was increased from 2-4 nM in wild-type viruses to 400-700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58-293 ng/L during seasonal outbreaks and are expected to reach mu g/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Place, publisher, year, edition, pages
2011. Vol. 6, no 9, e24742- p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-159471DOI: 10.1371/journal.pone.0024742ISI: 000294803200047OAI: diva2:445504
Available from: 2011-10-04 Created: 2011-10-03 Last updated: 2013-10-24Bibliographically approved
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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