Detection of avian influenza a H5N1 virus by pyrosequencing

Avian influenza A H5N1 virus causes a rapidly fatal systematic disease in domestic poultry and spreads directly from poultry to mammalian species including humans. The aim of this study was to develop a method for rapidly detecting avian influenza A H5N1 virus. Pyrosequencing is a newly developed method based on sequencing-by-synthesis. The genotype of the virus is determined by a short species-specific sequence. Two stretches in the M gene specific to avian influenza A virus and the HA gene specific to H5N1 subtype were amplified respectively and pyrosequenced for subtyping. In this report, we detected four specimens of avian influenza A, and one H5N1 subtype was confirmed by pyrosequencing results. With the method we developed, it is easy to discriminate the sequence related to the high pathogenicity avian influenza, H5N1, from avian influenza A. We can use this pyrosequencing-based technique for large-scale screening at times of avian influenza A H5N1 virus outbreaks.