Studies On Biological Characterization And Gene Characterization Of HA,NA,NS For H3N2 Subtype Swine Influenza Viruses

Virul samples were isolated from nasal tract and lungs of virus infected dead pigs of some eares of GuangDong in 2004.Theses virus isolates were propagated in specific-pathogen-free chicken embryos.The positive cultures were further examined by serological and biological methods. Biological characterizations of these isolates were studied,can agglutinate red blood cells coming from different animals,but their hemagglutinin were not stable.These isolates are highly infectious to chicken embryo.These mice experimently infected with swine influenza displayed classic respiratory syndromes and visible pathology.The primers of HA,NA,NS,NS1 gene segments were designed according to the sequences from GeneBank by Primer Premier 5.0. The nucleic acids of the all swine influenza viruses conducted in the research were extracted from the viruses propagated in specific-pathogen-free chicken embryos. HA,NA,NS segments were amplified by RT-PCR (Reverse transcription-polymerase chain reaction). The cDNAs were cloned into pMD18-T vector and identified. The sequencing products were purified and run gel on sequencing machine.Finally, DNAStar software was used for data analysis, sequence translation, phylogenetic tree drawing and molecular evolution analysis. Sequencing analysis indiated that three fragment highly shared homology and the amino acid,the amino acid sequence at the HA cleavage site is PEKQTR*G,not belong to the HPIV.The analysis of phylogenetic tree showed that these isolates had the close relationship with HongKong SIV after 1999 and the IV in North American after 2000.According to sequence analysis,the NS1 gene of one SIV from the pMD18-T-NS vector cloned into prokaryotic expression vector.The result of recombinant vector identified showed that the NS1 gene cloned into the expression vector. Establish the expressiom condition of NS1 gene in the E.coli.In conclusion, this study elucidated the Biological characterization and Molecular Evolution and Prokaryotic Expression of H3N2 SIVs isolated from the GuangDong, clarified the category of the recent epidemical strains of H3N2 SIVs circulating in the GuangDong, and made a foundation for the further investigation. Of particular significance, these would provide the theory support for the possibility and development trend of influenza occurring in China even the whole globe. It's important to study the Biological Characterization,Molecular Evolution and Prokaryotic Expression of H3N2 Subtype Swine Influenza Viruses in virology or veterinary medicine.