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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, N.I.H., Intramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alaska
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Animals
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Animals, Wild
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Cloaca / virology*
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DNA Primers / genetics
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Ducks
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Ethanol / pharmacology*
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Fixatives / pharmacology*
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Genotype
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Hemagglutinin Glycoproteins, Influenza Virus / genetics*
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Influenza A virus / genetics*
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Influenza A virus / isolation & purification
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Molecular Sequence Data
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Phylogeny
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Polymorphism, Genetic*
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RNA, Viral / genetics*
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RNA, Viral / isolation & purification
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Reverse Transcriptase Polymerase Chain Reaction / methods*
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Sequence Analysis, DNA
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Specimen Handling / methods
Substances
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DNA Primers
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Fixatives
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Hemagglutinin Glycoproteins, Influenza Virus
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RNA, Viral
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hemagglutinin, avian influenza A virus
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Ethanol
Associated data
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GENBANK/EU264565
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GENBANK/EU264566
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GENBANK/EU264567
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GENBANK/EU264568
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GENBANK/EU264569
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GENBANK/EU264570
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GENBANK/EU264571
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GENBANK/EU264572
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GENBANK/EU264573
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GENBANK/EU264574
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GENBANK/EU264575
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GENBANK/EU264576
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GENBANK/EU264577
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GENBANK/EU264578
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GENBANK/EU264579
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GENBANK/EU264580
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GENBANK/EU264581
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GENBANK/EU264582
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GENBANK/EU264583
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GENBANK/EU264584
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GENBANK/EU264585
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GENBANK/EU264586
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GENBANK/EU264587
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GENBANK/EU264588
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GENBANK/EU264589
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GENBANK/EU264590
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GENBANK/EU264591
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GENBANK/EU264592
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GENBANK/EU264593
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GENBANK/EU264594
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GENBANK/EU264595
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GENBANK/EU264596
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GENBANK/EU264597
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GENBANK/EU264598