| As the main low pathogenic avian influenza virus, H9N2subtype avian influenza virus is widely prevalent in our country, it had caused the enormous economic losses to the poultry industry. In addition, H9N2subtype avian influenza virus could not only infect people, but also have a potential ability to provide internal genes for high pathogenic H5N1subtype avian influenza virus and the novel H7N9subtype avian influenza virus, which may infect the humans and then result in a serious threat to public health. Therefore, researchers are paying more concerned about the H9N2subtype avian influenza virus.To investigate the pathogenicity of H9N2subtype avian influenza virus in mammals,19H9N2strains isolated by our laboratory were used for this study. And these purified virus was applied to infect BALB/c mice by inoculated intranasally with the dose of106EID5o each mouse,6infections in each virus strain. The results showed that, there was a little pathogenicity changing of H9N2virus in mammals as time went on. Although the whole infection rate of strains isolated in2012is lower than those isolated during2010-2011in pathogenicity, virus with higher pathogenicity that isolated in2012were still existed.To study the H9N2subtype avian influenza virus genetic evolutionary trend at the gene level, and its relationships with the gene sequences of high pathogenic avian influenza virus and the novel H7N9subtype avian influenza virus.19strains of H9N2virus, as well as26strains that had been sequenced before were analyzed for the genetic evolution. HA gene evolution analysis showed that all H9N2strains above belong to the Ck/BJ/1/94-like lineage in China, and there were as many as34virus classified into a third small branch, which has not reported in the literature. So it not only reflected HA gene of H9N2virus has a rapidly changed in recent years, but also explained the phenomenon of the existing vaccines were unable to protect infection from the current epidemic strains. The analysis of six internal genetic evolution showed that the internal gene appeared recombinant diversity and had high homology with those of novel H7N9virus arose in2013. By comparison of the amino acid sites, the analysis found that amino acid sites of different ages evolved in certain rules. Meanwhile, as the years went on, the evolution of these amino acid sites tended to coincide with those of the novel H7N9subtype avian influenza virus. Based on the great threat of the H9N2avian influenza to human health and the poultry industry, this study was aimed to carry on the preliminary research of the cellular inactivated vaccines for H9N2subtype avian influenza virus. Through optimization of the culture conditions in spinning bottle, it was confirmed that the best inoculation dose to culture the MDCK HA-2cells was3×104TCID50in the spinning bottle with the maintenance medium, which was the DMEM medium contains2%New calf serum, and the best harvest condition was48h after inoculation with the final volume of50ml in the cellular incubator. By comparison with different adjuvants, it turned out that the inactivator of β-propiolactone were the better to inactivate the virus during the preparation of inactivated vaccines. Finally, used different doses of the H9N2cellular inactivated vaccine to immune3weeks old SPF chickens.The result showed that the vaccine immunizing dose of20μl per chicken had the100%protective efficacy to chickens. These results demonstrated that the cellular vaccine had a great commercial value. |
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