Research On The Bivalent Vaccines Against Porcine Japanese Encephalitis And Pseudorabies

Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are the main causes of infectious reproductive failure in swine. They are economically important diseases. Meanwhile, JE also is an enzootic disease, and swine plays an important role in viremia-amplifying host. Therefore prevention of JE in swine can benefit for both swine production and humans' health. Currently, vaccine inoculation is effective method to prevent and control these diseases. However, there is no commercial JE vaccine for swine JE control. This dissertation mainly was targeted to develop genetic vaccine of JEV for swine JE control by using PRV as a live viral vector.1. The cloning and sequencing of PrM-E genes of JEV SA14-14-2PrM-E genes were amplified from JEV SA14-14-2 strain by RT-PCR and cloned into pMD18-T vector (designated as pTPrM-E) and sequenced. The sequence analysis showed that the PrM genes of SA14 and SA 14-14-2 strains had 100% homology, while there are 4 mutantions in the E gene, and it cause 3 amino acid changes.2. The construction of three Recombinant virus strains recombinized with PRV Ea mutant strain TK^-/gG^-/1acZ⁺ and JEV dominant immunogenic genes.Three pairs of primers were designed to subclone PrM? PrM-E and NS1 genes from P^TPrMe and p^TNSl respectively. JEV PrM, PrM-E and PrM-E-NSl genes were attained. Recombinant transfer vectors were co-transfected PK-15 cells with the genome DNA of PRV Ea TK/gG7/LacZ+ stain respectively. Three recombinant psedorabies viruses TK^-/gG^-/PrM⁺, TK^-/gG^-/ PrM-E⁺ and TK^-/gG^-/PrM-E-NSl⁺ containing JEV genes were obtained and identified by plaque purification, PCR amplification and Southern blot. PrM, E and NS1 proteins expressed in recombinant PRV strains were detected respectively by western blot. It was confirmed that the propagation of recombinants in IBRS-2 cells was not affected.3. Study on the immunogenicity of three bivalent genetic engineering vaccines against JEV and PRVBalb/c mice at age of 6-8 weeks and piglets at age of 50-60 days old that were serologically negative to both pseudorabies and Japanese encephalitis were selected to determine the immunogenicity of the bivalent genetic engineering vaccines. The results of the animal tests showed that they are safe to both mice and pigs. Furthermore, the specificimmunological responses can be induced in immunized animals. TK^-/gG^-/PrM-E(+) and TK^-/gG^-/PrM-E-NSl⁺ in immunized groups induced antibody to JEV and JEV-specific CTL activity, but the specific immune response were little lower than those in JEV SA14-14-2 immunized group. TK^-/gG^-/PrM⁺ caused the weakest immune responses. There was no significant difference in JEV-specific CTL activity and antibody to JEV between TK^-/gG^-/PrM-E⁺ and TK^-/gG^-/PrM-E-NSl⁺ strains (P>0.05). There was no significant difference in PRV neutralization antibody titers between piglets immunized with recombinant viruses and vector virus either (p>0.05). Therefore the two recombinant TK^-/gG^-/PrM-E⁺ and TK^-/gG^-/PrM-E-NSl⁺ strains will potentially be used as bivalent genetic engineering vaccines against JEV and PRV to control both JEV and PRV in swine.