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Journal of Clinical Microbiology, February 2009, p. 327-334, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01330-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Design and Validation of a Microarray for Detection, Hemagglutinin Subtyping, and Pathotyping of Avian Influenza Viruses{triangledown} ,{dagger}

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

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

Received 13 July 2008/ Returned for modification 11 August 2008/ Accepted 25 November 2008

Continuing threats of devastating outbreaks in poultry and of human infections caused by highly pathogenic avian influenza virus (HPAIV) H5N1 emphasize the need for the further development of rapid and reliable methods of virus detection and characterization. Here we report on the design and comprehensive validation of a low-density microarray as a diagnostic tool for the detection and typing of avian influenza virus (AIV). The array consists of one probe for the conserved matrix gene and 97 probes targeting the HA0 cleavage-site region. Following fragment amplification by a generic PCR approach, the array enables AIV detection, hemagglutinin (HA) subtyping, and pathotyping within a single assay. For validation, a panel of 92 influenza A viruses which included 43 reference strains representing all 16 HA subtypes was used. All reference strains were correctly typed with respect to their HA subtypes and pathotypes, including HPAIV H5N1/Asia, which caused outbreaks in Germany in 2006 and 2007. In addition, differentiation of strains of the Eurasian and North American lineages of the H5 and H7 subtypes was possible. The sensitivity of the microarray for the matrix gene is comparable to that of real-time reverse transcription-PCR (RT-PCR). It is, however, 10- to 100-fold lower than that of real-time RT-PCR with respect to HA subtyping and pathotyping. The specificity of the array was excellent, as no pathogens relevant for differential diagnosis yielded a positive reaction. Validation with field samples included 19 cloacal swab specimens from wild and domestic birds. Influenza A virus was verified in all samples, whereas the HA subtypes could be determined for 14 samples. The results demonstrate that the microarray assay described complements current methods and can accelerate the diagnosis and characterization of AIV.

* Corresponding author. Mailing address: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, Greifswald-Insel Riems 17493, 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 3 December 2008.

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

Journal of Clinical Microbiology, February 2009, p. 327-334, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01330-08
Copyright © 2009, 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]  
  • He, J., Bose, M. E., Beck, E. T., Fan, J., Tiwari, S., Metallo, J., Jurgens, L. A., Kehl, S. C., Ledeboer, N., Kumar, S., Weisburg, W., Henrickson, K. J. (2009). Rapid Multiplex Reverse Transcription-PCR Typing of Influenza A and B Virus, and Subtyping of Influenza A Virus into H1, 2, 3, 5, 7, 9, N1 (Human), N1 (Animal), N2, and N7, Including Typing of Novel Swine Origin Influenza A (H1N1) Virus, during the 2009 Outbreak in Milwaukee, Wisconsin. J. Clin. Microbiol. 47: 2772-2778 [Abstract] [Full Text]  


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