Sequence and phylogenetic analysis of the haemagglutinin genes of H9N2 avian influenza viruses isolated from commercial chickens in Markazi province, Iran

Avian influenza viruses of the H9N2 subtype have seriously affected the industry of the Middle East and Asian countries since the 1990s and are considered to be one of the potential candidates for the next human pandemic. In the present study, to determine the genetic relationship of Iranian viruses, the haemagglutinin (HA) genes from two isolates of H9N2 viruses from commercial chickens in Markazi province (central Iran) during 2013-2014 were amplified and sequenced. Samples were collected and viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesise cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Purified fragments were sequenced from both directions. Finally, sequence analysis and phylogenetic studies were conducted by comparing each isolate with those of the available H9N2 strains at Gen Bank. All of the isolates possessed the same amino acid motif P-A-R-S-S-R/G-L at the HA cleavage site. Amino acid sequence comparisons of HA genes of two isolates showed 93.6% identity. Phylogenetic analysis revealed that all isolates belonged to the G1-like sublineage and one isolate showed some degree of homology with Pakistani isolates. Two isolates had leucine (L) at position 226 instead of glutamine (Q) which indicated the potential of binding to human-type receptors. The results of this study suggest that Iranian H9N2 viruses could infect mammalian species, including humans and have the potential to emerge as highly pathogenic influenza viruses in Iran.