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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]

The aim of the present study was to evaluate the potential effect of flagellin as adjuvant in Newcastle disease virus (NDV) vaccine on the cellular and humoral immunity in chickens. Fifty-six specific pathogen-free chickens were assigned to seven groups of eight chickens and immunized twice with a two-week interval, intramuscularly. Group 1, received phosphate buffered saline as control (C), groups 2, 3, 4, 5, 6 and 7 were immunized with inactivated NDV [Ag], Ag + full FliC protein [AgF], Ag + truncated Flic protein [AgT], Ag + native Flic protein [AgN], commercial NDV vaccine [Vac] and Vac + N [VacN], respectively. After 45 days, spleen and bursa of Fabricius samples were collected and analyzed by flow cytometry and responses in control/vaccinated chickens were studied by immunophenotyping. Humoral response was also, evaluated by ELISA during the experiment. Results showed that immunized chickens with Ag + flagellin proteins had significantly higher frequency of circulating CD3+, CD4+ and CD8+ T cells in bursa of Fabricius in AgF, AgT and AgN, respectively, compared with other groups. Similar results were observed for spleen; however, the highest frequency of circulating CD3+, CD4+ and CD8+ T cells belonged to AgT and AgF, respectively. ELISA results showed that all flagellin-adjuvanted groups had higher antibody titers than other groups with the highest antibody response in VacN. It can be concluded that flagellin may induce both humoral and cellular immune responses against ND and is suggested for use as an efficient adjuvant. • Flagellin-adjuvanted groups had higher frequency of CD4 and CD8 T cells. • All flagellin-adjuvanted groups had higher antibody titers than other groups. • Native flagellin enhanced antibody response in commercial vaccine. [ABSTRACT FROM AUTHOR]

• The most IL-6 and TNF-α gene expression was observed in full flagellin adjuvanted group. • The recombinant full flagellin adjuvanted group showed the highest cellular activity. • The highest HI titer was observed in commercial vaccine and recombinant flagellin adjuvanted groups. The present study was designed to investigate the potential application of native (N) and recombinant (truncated modified [tmFliC] and full-length [flFliC]) flagellin proteins along with inactivated Newcastle disease virus (NDV). Fifty six SPF chickens were immunized twice with PBS (control), inactivated NDV (Ag), inactivated NDV/flFliC (AgF), inactivated NDV/tmFliC (AgT), inactivated NDV/N (AgN), commercial vaccine containing Montanide (Vac) and Vac/N (VacN), with a two-week interval. Blood was collected weekly and spleens were harvested after chickens were sacrificed. Interleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) gene expression in peripheral blood mononuclear cells were analyzed by Real-Time PCR. Antibody response was assessed by haemagglutination inhibition (HI). Cellular activity was quantified by MTT assay. Results showed that the most IL-6 and TNF-α gene expression was observed in AgF group (P < 0.01). The lowest gene expression among vaccinated groups was observed in Ag group for IL-6 and Ag and Vac group for TNF-α. The highest HI titer was observed in Vac, VacN, AgF and AgT groups. The AgF group showed the highest cellular activity (P < 0.01). In conclusion, flagellin-adjuvanted groups showed a pro-inflammatory effect and acted similarly to or better than the Vac group. Hence, flagellin can be proposed as a potential adjuvant for ND vaccine. [ABSTRACT FROM AUTHOR]