The Genome Sequence Analysis Of Japanece Encephlitis Virus Isolated From Swine And Research On Construction Of Recombinant Adenovirus Co-expressing The NS1and E Gene And Humoral Immunity

Japanese encephalitis virus belongs to Flaviviridae Flavivirus,it is a zoonosis which isspread by the Mosquito as a media. Japanese Encephalitis can cause swine reproductivedisorders,such as abortion，barren，dead fetus,mummified fetus and weak fetus appearing.Whenhuman infected,people can cause central nervous system disease. infected weakly can causepermanent nervous system sequelae, infect severely can cause death.In this study, analyzed the complete genome sequences of WLS-1and WLS-2strains.Theresults showed that the WLS-2is a virulent strain,and belongs to genotype III. We obtained theNS1and E genes of the WLS-2strain by RT-PCR method and used the gene encoding the2Aprotein of the foot and mouth disease virus as a linker,tandemed the NS1and E gene toconstruct the recombinant adenovirus,and reseached its humoral immune level.1Analysis of the complete genome sequences of WLS-1and WLS-2strainAfter sequence alignment for JEV on the Gene bank, eight pairs of primers were designedin the conserved regions to cover the full length of the genome. Using RT-PCR amplificationtechnology, we obtained the corresponding gene sequences, then connected sequences topMD18-T Simple vector and sequenced. We analyzed the amino acid sequences of the isolatedstrains WLS-1and WLS-2, virulent strain SA14, BeiJing-1, JaGAr01and attenuated strainSA1414-2, SA14-12-1-7, as well as the homology analysis of JEV Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴgenotypeand constructed the evolutionary tree. The result showed: at the key acid sites of the virulenceof JEV,such as E107,E138,E176,E177, E264,E279,E315.The WLS-1was same to theattenuated strain SA14-14-2,and WLS-1was same to the virulent strain，such as SA14,BeiJing-1-2.WLS-1and WLS-2have the highest homology with JEV Ⅲgenotype by the homologyanalysis. WLS-1,WLS-2and JEV Ⅲ genotype on the same branch in molecular evolutionarytree anslysis,which can be concluded that the WLS-1and WLS-2strains belong to genotypeⅢ,and WLS-1was the attenuated strain and WLS-2virulent strong.2Construct the recombinant adenovirus and evaluate its humoral immune levelIn order to prepare a recombinant adenovirus co-expressing NS1and E protein of Japaneseencepahlitis virus (JEV)，the sequence encoding2A peptide of foot-and-mouth disease viruswas introduced as a linker between NS1and E gene to construct the shuttle plasmid ofpShuttle-JEV-NS1-2A-E, which was transformed into E.coli BJ5183to produce thepAd-JEV-NS1-2A-E by homologous recombination. Then the recombinant adenovirus ofrAd-JEV-NS1-2A-E was generated by transfection of pAd-JEV-NS1-2A-E into AD293cells.The indirect immunofluorescence and western blot assay showed that E-2A-NS1protein wasexpressed and self-cleavaged into E and NS1proteins in rAd-JEV-NS1-2A-E infected AD293 cells. In addition, the antibodies against both adenovirus and WNV were efficiently producedin mice immunized with the rAd-JEV-NS1-2A-E detected by indirect ELISA. This studyprovided the basis for the development of recombinant adenovirus vaccine against JEVinfection.