JCM Accepts, published online ahead of print on 18 June 2008

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J. Clin. Microbiol. doi:10.1128/JCM.00466-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Universal primer set for amplification and sequencing of HA₀ cleavage sites of all influenza A viruses

Astrid Gall, Bernd Hoffmann, Timm Harder, Christian Grund, and Martin Beer^*

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald - Insel Riems, Germany

^* To whom correspondence should be addressed. Email: martin.beer{at}fli.bund.de.

Sequence analysis of the endoproteolytic cleavage site within the haemagglutinin precursor protein HA₀ is fundamental for studies on the molecular biology of influenza A viruses, in particular molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5, H7 or even other subtypes which escape detection by commonly used RT-PCR protocols. Here, the first pan haemagglutinin reverse transcription polymerase chain reaction (PanHA RT-PCR) targeting the HA₀ cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA₀ cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluids and 11 swab diagnostic samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR - followed by cycle sequencing - can complement existing methods and strengthen the reliability of influenza A virus diagnostics allowing both molecular pathotyping (H5, H7) and subtyping (non H5/H7) within a single approach.

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