To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A
virus, we need reliable, simple methods that not only characterize partial sequences
but analyze the entire influenza A genome. We designed primers based on the fact that
the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved
between all influenza A viruses and unique for each segment. The primers designed
for each segment contain influenza virus specific nucleotides at their 3'-end and
non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able
to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7,
and N9 subtypes, the segment specific sequences of the neuraminidase genes are different.
Therefore, we optimized the primer design to allow the amplification of those neuraminidase
genes as well. The resultant primer set is suitable for all influenza A viruses to
generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct
expression plasmids for reverse genetics systems.