Cloning And Sequencing Of The Full-Length Genome Of Porcine Japanese Encephalitis Virus Strain SXBJ07 And Establishment Of ELISA Detection

Epidemic encephalitis B (Japanese encephalitis), caused by Epidemic encephalitis virus B (Japanese encephalitis virus, JEV, Epidemic encephalitis virus B), was an important mosquito-borne zoonosis. Culex mosquitoes acted as the primary media of JE, the natural focus diseases. In development of JE, swine was considered the most important natural amplifying host and amplifier among several animals and also the transmitter mostly threatening the health of human beings. The infected patients showed encephalitis or suffered from neurological sequelae even fatal. JEV was the one of main pathogens caused reproductive disturbance in sows. Infected sows showed lower fatality rate, but may suffer from abortion, stillborn delivery or mummified fetuses. JEV also caused boar acute orchitis or infertility, which has been recognized as one of the most economically diseases in the swine industry worldwide.There were the most JE cases in China, but JEV could show mutation with the changes of location and time, which can make things difficult for monitoring and control of JE. A number of geographically diverse JEV strains have been isolated at different times from humans, mosquitoes and pigs. In recent years, lots of isolates of JEV strain were obtained from swine brain, stillborn or mosquito, and several strains have been cloned and fully sequenced. To understand the epidemic status of JE in Shaanxi province, epidemiological investigation was studied in part of areas in this paper. The new JEV strain was isolated from swine brain, and the full-length genome has been cloned and analyzed. The main antigenic protein was expressed in E. coli, and established the ELISA detection. Experimental results obtained as follows.1. The JEV antibody and nucleic acid in part of Shaanxi province were detected by LAT and one-step RT-nested-PCR. The results showed that the positive rate of antibody and nucleic acid in immunized farm were 94.80% and 0.56%, respectively, which may caused by the immunized failure. The positive rate of antibody and nucleic acid in non-immunized farm and house keeper were 11.72% and 10.73%, which can diagnose as JE. Culex tritaeniorhynchus and Culex pipiens pallens were the local predominant and may acted as the primary media of JE. 2. By nude mice inoculation and isolation and culture in BHK-21, the new JEV strain of specific pathogenicity and CPE was obtained from swine brain specimens. According to the results of CPE characteristic, hemagglutination test and indirect immunofluores- cence, the mew isolate was confirmed as JEV and named SXBJ07 strain. The results of amplification and sequencing the PrM gene and E gene by RT-PCR and homology analyzing the nucleotide and amino acid can further verified the SXBJ07 strain in molecular level.3. From RT-PCR and rapid-amplification of cDNA ends (RACE), the SXB07 strain genome full sequence of 10 965 bp length was obtained. Sequences analyze and homology comparison showed that there were many deletion and insertion in the 5'NTR and 3'NTR, and there were amino acid substitutions in 13 sites of the active donmains of E protein. Compared the full genome sequences of SXBJ07 with other reference strains, there were higher homology of nucleotide and amino acid with genotypeⅠ, and JEV/sw/Mie/40 has the nearest genetic relationship in all these strains. Identification of genotype based on PrM and E gene showed that SXBJ07 strain belong to genotypeⅠ. This was the first JEV genotypeⅠstrain isolated from Shaanxi.4. Inserting the JEV envelope protein gene into prokaryotic expression vector pET-32α, recombinant expression vector was constructed. The recombinant vector was translated to E. coli DH5αand successfully expressed the envelope protein. The results of SDS-PAGE analysis indicated that fusion protein was accord with target protein. Western blot analysis showed that the expressed envelope protein from prokaryotic expression can specifically combine with positive serum antibody. The purified JEV envelope protein acted as envelope antigen, this study established a specific and sensitive indirect ELISA detection through chessboard titration, determining the threshold quantity between negative and positive, specificity test, and within-run, run to run test. The clinical detection results showed that the ELISA detection has higher specificity and sensitivity.