Method for Sequencing the Whole Genome of the Highly Pathogenic Avian Influenza A(H5N6) Virus.

We wished to develop a simple method to amplify and sequence the whole genome of the highly pathogenic avian influenza A ( H5N6 ) virus. Hemagglutinin ( HA ) gene sequences of A ( H5), neuraminidase (NA) gene sequences of A (N6), as well as the internal gene sequences of subtype A (H5N1) and A (H9N2) influenza viruses of the-previous 5 years were downloaded from GenBank and GISAID, and individual genes were aligned. Thirty-two primer pairs targeted to conserved regions of these gene sequences were designed and validated. After optimization, the whole genome sequence of nine influenza A(H5N6) viruses isolated from infected humans and those circulating in the environment were obtained-with the 32 primer pairs. Viruses isolated from human respiratory specimens were amplified to produce distinct products of the polymerase chain reaction. When tested on viruses. isolated from environmental swabs, few primer pairs produced specific and non-specific products. A Sanger protocol for generating the whole genome sequence of the highly pathogenic avian influenza virus A (H5N6) was established and shown to be a rapid and easy method to provide data for phylogenetic analyses of this virus.