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Journal of Clinical Microbiology, August 2008, p. 2561-2567, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00466-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Universal Primer Set for Amplification and Sequencing of HA0 Cleavage Sites of All Influenza A Viruses{triangledown} ,{dagger}

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

Received 9 March 2008/ Returned for modification 22 April 2008/ Accepted 9 June 2008

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA0 is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay 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 fluid samples and 11 diagnostic swab 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 assay—followed by cycle sequencing—can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.

* Corresponding author. Mailing address: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald—Insel Riems, Germany. Phone: 49 (0) 38351 7200. Fax: 49 (0) 38351 7151. E-mail: martin.beer{at}fli.bund.de

{triangledown} Published ahead of print on 18 June 2008.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

Journal of Clinical Microbiology, August 2008, p. 2561-2567, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00466-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gall, A., Hoffmann, B., Harder, T., Grund, C., Ehricht, R., Beer, M. (2009). Rapid and Highly Sensitive Neuraminidase Subtyping of Avian Influenza Viruses by Use of a Diagnostic DNA Microarray. J. Clin. Microbiol. 47: 2985-2988 [Abstract] [Full Text]  
  • Gall, A., Hoffmann, B., Harder, T., Grund, C., Hoper, D., Beer, M. (2009). Design and Validation of a Microarray for Detection, Hemagglutinin Subtyping, and Pathotyping of Avian Influenza Viruses. J. Clin. Microbiol. 47: 327-334 [Abstract] [Full Text]  


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