title: Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method.
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.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ruixue
authors.initials: R
authors.lastname: Wang
authors.forename: Lindsey
authors.initials: L
authors.lastname: Soll
authors.forename: Vivien
authors.initials: V
authors.lastname: Dugan
authors.forename: Jonathan
authors.initials: J
authors.lastname: Runstadler
authors.forename: George
authors.initials: G
authors.lastname: Happ
authors.forename: Richard D
authors.initials: RD
authors.lastname: Slemons
authors.forename: Jeffery K
authors.initials: JK
authors.lastname: Taubenberger
mesh_terms: D000413:Alaska; D000818:Animals; D000835:Animals, Wild; D002988:Cloaca / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D004372:Ducks; D000431:Ethanol / Q000494:pharmacology*; D005404:Fixatives / Q000494:pharmacology*; D005838:Genotype; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods
chemical_list: D017931:DNA Primers; D005404:Fixatives; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; C109963:hemagglutinin, avian influenza A virus; D000431:Ethanol