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Viruses 2013, 5(8), 1964-1977; doi:10.3390/v5081964
Article

Avian Influenza: Mixed Infections and Missing Viruses

1
, 2
, 1
, 3
, 3
, 3,†
, 3
 and 1,*
1 Department of Pathology, Microbiology and Immunology, University of California, One Shields Avenue, Davis, CA 95616, USA 2 One Health Institute, University of California, 1089 Veterinary Medicine Drive, Davis, CA 95616, USA 3 Craig Venter Institute, Rockville, MD 20850, USA Current affiliation: Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD 20892, USA.
* Author to whom correspondence should be addressed.
Received: 7 June 2013 / Revised: 20 July 2013 / Accepted: 23 July 2013 / Published: 5 August 2013
(This article belongs to the Section Viruses of Plants, Fungi and Protoza)
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Abstract

A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes) were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.
Keywords: avian influenza; surveillance; hemagglutinin; virus isolation; embryonated chicken egg; sequencing; genome avian influenza; surveillance; hemagglutinin; virus isolation; embryonated chicken egg; sequencing; genome
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Lindsay, L.L.; Kelly, T.R.; Plancarte, M.; Schobel, S.; Lin, X.; Dugan, V.G.; Wentworth, D.E.; Boyce, W.M. Avian Influenza: Mixed Infections and Missing Viruses. Viruses 2013, 5, 1964-1977.

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