Full length sequencing of all nine subtypes of the neuraminidase gene of influenza A viruses using subtype specific primer sets

An RT-PCR based method was developed using subtype specific overlapping primers to obtain full length amplification of neuraminidase (NA) gene from all subtypes (N1–N9) of influenza A viruses. This method was validated using reference strains of avian influenza viruses (AIV) (N1–N9), human influenza viruses (N1 and N2), and swine influenza viruses (N1–N3). Amplification of the NA gene was obtained with all viruses tested. Additionally, 200 field isolates of AIV from wild birds were tested by this method and the NA gene was amplified in all isolates. The NA subtype of all 200 isolates was determined by further sequencing of the amplified NA genes and all sequences were submitted to GenBank. The method described in this paper can be used to determine subtype of influenza isolates as well as their evolution and mutations if any, in the NA gene.