Analysis of hemagglutinin genes of isolates of avian influenza virus H9 subtype from Shanghai area.

In order to explore the genetic variation of the hemagglutinin(HA)gene and epidemic rule of H9 subtype avian influenza viruses in Shanghai area,seven H9 subtype avian influenza viruses(AIVs)were isolated from live poultry markets of Shanghai including one isolate from 2002,two isolates from 2006 to 2008 separately.These specimens were paired tracheal and cloacal swabs.Seven influenza A viruses were determined as H9 subtype by fluorescence RT-PCR kit and then isolated by using 10-day-old specific-pathogen-free chicken eggs.They were further identified as H9 subtype by HI test.The cDNA of HA gene were amplified by RT-PCR and sequenced.Their nucleotide and putative amino acid sequences homology was compared.The results showed that their nucleotide sequence homology was from 88.6%-99.6% and that amino acid sequence homology was 89.1%-99.1%.Genetic analysis revealed that all seven isolates had an RSSR↓GLF motif at the cleavage site of HA,representing low pathogenicity in chicken.In addition,these isolates all had 7 potential glycosylation sites.The isolate of 2002 and one from early 2006 had amino acid residue glutamine(Q)at position 226(H3 numbering),however,the other 5 isolates had leucine(L)at position 226(H3 numbering).All had glycine(G)at position 228 at the receptor-binding site.Phylogenetic analysis of the H9 HA gene showed that all of the H9 viruses are incorporated into the lineage represented by Ck/Bei/1/94.These findings may provide an important basis for establishment of scientifically preventive measurements to control H9 subtype avian influenza in Shanghai area.