The Study On Cloning Purification And Immunogenicity Of Japanese Encephalitis Virus

Swine epidemic encephalitis B was a mosquito-borne viral human zoonotic diseases that caused by the Japanese encephalitis virus. It’s common that pigs have been infected by JEV. The pig infected JEV is Japanese encephalitis’s important reservoir and the host of amplification and it also is JEV’s main source of infection. Being infected pregnant sows will have abortion、stillbirth、mummy fetus, boar will suffer from orchitis or even infertility. This condition can cause grate economic loss to the pig industry development. In this study, aiming at screening a newly-prepared vaccine strain, based on JEV strains isolated from Sichuan province, we have cloned the strains to make them purer, studied their biological characteristic and immunological characteristics. The main contents are as follows:1. Purified of JEV strains by plaque clone technologyPlaques of Japanese encephalitis virus were picked up to make the virus strains purer. Finally, we got the two virus strains with stable plaque traits which were called CZ1-1,NJ1-1. The size of plaque which CZ1-1strain formed at72h was2-3mm while the size of plaque which NJ1-1strain formed at48h was1-2mm. The results of RT-PCR detected showed:the PrM/E gene nucleotide sequences were amplified. That provided purer JEV strains for studying JEV. Both of the two JEV strains had good adaptability and high pathogenicity to BHK-21cells. The TCID50of CZ1-1strain was10-652/0.1mL while NJ1-1was10-690/0.1mL. Both of the two JEV strains had good adaptability and high pathogenicity to mice. The LD50of CZ1-1strain was1O-8.55while The LD50of NJ1-1strain was10-7.56.2. Study on immunogenicity of JEV vaccineOil emulsion inactivated vaccines of JEV were prepared with CZ1-1strain and NJ1-1strain. Both of the antigen dose were7.OlgLDso. The physical character of the inactived vaccines, sterility test, safety test, inactived test and the immune efficacy test have been systematically studied. The results show that the oil emulsion inactived vaccines of JEV were sterile and safe. Mice were immunized intraperitoneal two times at one week interval.14d after the first immunity, mice were challenged against CZ1-1or NJ1-1in intracerebral or intrapertoneal inoculations. The results show that the two vaccines’ protection after challenged with CZ1-1by i.c route were70%,60%, after challenged with NJ1-1by i.c route were70%,50%, after challenged with CZ1-1by i.p route were80%,70%, after challenged with NJ1-1by i.c route were100%,90%. They have catched up with or exceeded the level of commercial inactivated vaccine.7d,14d,21d,28d after mice immuned, the blood of each group of mice tails was collected. Neutralizing antibody of JEV were detected by micro serum neutralization test.Results show that28d after the first immunity the CZ1-1vaccine’ neutralizing index was1:138, NJ1-1vaccine’ neutralizing index was1:96. In contrast, CZ1-1strain can be expected to be backup encephalitis inactivated vaccine strain.