Biological and molecular characterization of Avian influenza virus (H9N2) isolates from Iran

Three Influenza A virus (H9N2) isolates obtained from three separate broiler flocks with variable mortality rates were cloned twice in embryonated SPF chicken eggs by limiting dilution. Biological properties of these isolates were examined in 4-week-old SPF chickens and chick embryo fibroblast (CEF) cultures. The isolates neither caused mortality in the inoculated chickens nor produced CPE in cell cultures, indicating low pathogenicity. PCR products of 486 bp containing the sequences for hemagglutinin (HA) cleavage site, which were generated from the isolates, were subjected to nucleotide sequencing. Sequence analysis of the HA region containing the cleavage site of the isolates showed a similar sequence motif (PARSSRG) but different flanking regions. Phylogenetic analysis of deduced amino acid sequences revealed that the isolates were closely related to those isolated earlier, indicating a common source. Moreover. the amino acid sequences of the recent isolates were very similar to those from Saudi Arabia, Germany and Pakistan. It is postulated that, except for some Chinese isolates, the pathogenicity of Iranian isolates seems to be similar to that of other Eurasian isolates. It is possible that an elevation in mortality rate under field condition could be caused by co-infection of recent isolates with the bacteria such as mycoplasma, Escherichia coli, and Ornithobacterium rhinotracheale rather than by an emerging a pathogenic H9N2 subtype of the virus.