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Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA(1) genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA(1) had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.

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]