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Comparative Study
. 2008 Aug;46(8):2561-7.
doi: 10.1128/JCM.00466-08. Epub 2008 Jun 18.

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

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Comparative Study

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

Astrid Gall et al. J Clin Microbiol. 2008 Aug.

Abstract

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA(0) 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 HA(0) 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 HA(0) 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.

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Figures

FIG. 1.
FIG. 1.
Assay sensitivity for influenza A virus of subtypes H5 and H7. (A) RNA from four influenza A virus isolates of the H5 subtype was tested in 10-fold dilution series with the PanHA primers as well as H5-specific primer pair H5-kha-1 and H5-kha-3 and primer pair J3 and B2a. (B) RNA from four influenza A virus isolates of the H7 subtype was tested in 10-fold dilution series with the PanHA primers as well as H7-specific primer pair GK7.3 and GK7.4. CT values for the M gene obtained by real-time RT-PCR are shown beneath the agarose gel image. Marker, GeneRuler 100-bp DNA Ladder Plus (Fermentas). ntc, no template control.

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References

    1. Aamir, U. B., U. Wernery, N. Ilyushina, and R. G. Webster. 2007. Characterization of avian H9N2 influenza viruses from United Arab Emirates 2000 to 2003. Virology 36145-55. - PMC - PubMed
    1. Anonymous. 2005. Avian influenza, chapter 2.7.12. In Manual of diagnostic tests and vaccines for terrestrial animal OIE. World Organisation for Animal Health, Paris, France. http://www.oie.int/eng/normes/mmanual/A_00037.htm.
    1. Anonymous. 2007. Avian influenza, chapter 2.7.12. In Terrestrial animal health code OIE. World Organisation for Animal Health, Paris, France. http://www.oie.int/eng/normes/mcode/en_chapitre_2.7.12.htm.
    1. Anonymous. 2006. Commission decision of 4 August 2006 approving a diagnostic manual for avian influenza as provided for in council directive 2005/94/EC [notified under document number C(2006) 3477 (2006/437/EC)]. Official Journal of the European Union, Luxembourg. http://eur-lex.europa.eu/LexUriServ/site/en/oj/2006/l_237/l_23720060831e....
    1. Butt, K. M., G. J. D. Smith, H. Chen, L. J. Zhang, Y. H. C. Leung, K. M. Xu, W. Lim, R. G. Webster, K. Y. Yuen, J. S. Malik Peiris, and Y. Guan. 2005. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J. Clin. Microbiol. 435760-5767. - PMC - PubMed

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