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The full-length non-structural (NS) gene of seven H9N2 isolates (2008-2012) was amplified and genetically analyzed. The NS gene of these isolates belongs to the Y439 sublineage and with an average of 96.4 % identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated at 0.38 for subgroup 1 and 0.42 for subgroup 2 indicates that purifying selection dominates the evolution of the NS H9 in Iran. The third position of codons values, ranging from 0.371 to 0.392, confirms that synonymous codon usage in NS of the H9N2 viruses was less biased. To investigate the evolution of the NS gene of H9N2 viruses isolated from Asia, phylogenetic analysis was conducted. The results showed that Y439 sublineage with various clusters circulated in Korea, the Middle East, and Pakistan, whereas G1 sublineage existed in China. Homology analysis showed that the NS gene of Korea and Israel isolates grouped into distinct clusters, while most of Iranian isolates were closely related to the Pakistan viruses. Prediction of RNA pseudoknot within NS1 gene using RNA folding path showed eight patterns with different free energy levels. Estimation of free energies suggests destabilization of the pseudoknot and changes in RNA secondary structure may be subject to evolution of NS gene in China. [ABSTRACT FROM AUTHOR]

Abstract: The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994–2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection. [Copyright &y& Elsevier]