A Diagnostic Microarray for Subtyping and Pathotyping Avian Influenza Virus

Abstract

The ability to readily identify new and potentially pandemic strains of influenza virus will allow a more rapid response by health care officials to reduce the spread and human impact of the disease. We report a low-density microarray for the rapid detection and identification of potential pandemic influenza A virus subtypes H5, H7, and H9 in the present study. Subtype- and pathotype-specific probes were designed to target the hemagglutinin (HA) gene segment. The system consisted of RT-PCR using universal primers for the HA gene. RT-PCR products were hybridized against a microarray consisting of short probes 3141 nucleotides in length. Cy3-labeled DNA targets were generated with a Cy3-labeled primer. Reference strains (n=46) of different avian influenza subtypes (H1-H15, except H16), and 34 unidentified field strains of avian influenza virus (AM from wild bird habitats and live bird markets was used for validation. All referenced subtypes belonged to H5, H7, and H9 and unidentified field strains were correctly typed by their HA-subtypes and pathotypes. These included 3 highly pathogenic H5N1 avian influenza viruses which had caused South Korean outbreaks in 2003, 2005, and 2008. In addition, there were no cross-hybridization reactions with other poultry respiratory viruses, suggesting that the microarray was specific for influenza A viruses. The developed microarray was capable of subtyping and pathotyping the potential pandemic influenza A virus subtypes H5, H7, and H9.