Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method

[Sally Furniss]

Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method




<!-- articleText --> Ruixue Wang^a, Lindsey Soll^a, Vivien Dugan^a, Jonathan Runstadler^b, George Happ^b, Richard D. Slemons^c and Jeffery K. Taubenberger^a^,^,

<!-- authorsNoEnt --> ^aLaboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
^bInstitute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775, USA
^cDepartment of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210, USA

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Received 26 November 2007;
<!-- graphText, refText -->revised 4 January 2008;
<!-- graphText, refText -->accepted 29 January 2008.
<!-- graphText, refText -->Available online 4 March 2008.
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<!-- refMsg --> Abstract

This study presents an interconnected approach for circumventing two inherent limitations associated with studies defining the natural history of influenza A viruses in wild birds. The first limiting factor is the ability to maintain a cold chain from specimen collection to the laboratory when study sites are in more remote locations. The second limiting factor is the ability to identify all influenza A virus HA subtypes present in an original sample. We report a novel method for molecular subtyping of avian influenza A virus hemagglutinin genes using degenerate primers designed to amplify all known hemagglutinin subtypes. It was shown previously that templates larger than 200 bp were not consistently amplifiable from ethanol-fixed cloacal swabs. For this study, new primer sets were designed within these constraints. This method was used to perform subtyping RT-PCR on 191 influenza RNA-positive ethanol-fixed cloacal swabs obtained from 880 wild ducks in central Alaska in 2005. Seven different co-circulating hemagglutinin subtypes were identified in this study set, including H1, H3, H4, H5, H6, H8, and H12. In addition, 16% of original cloacal samples showed evidence of mixed infection, with samples yielding from two-to-five different hemagglutinin subtypes. This study further demonstrates the complex ecobiology of avian influenza A viruses in wild birds

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