Research On The Bivalent Vaccines Against Porcine Japanese Encephalitis And Pseudorabies

Japanese encephalitis (JE) and pseudorabies are the main causes of infectious reproductive failure in swine, and make significant economic losses in the pig industry. Also, JE is a serious mosquito-borne viral disease of major public health importance in Asia. Pigs are considered the main vertebrate host and represent an important amplifier and reservoir for JEV. Therefore prevention of JE in swine can benefit for both swine production and humans' health. In the present, vaccination is the most effective prophylactic measure to prevent and control these diseases. However, there are problems in both inactivated and attenuated JEV vaccines that are used currently. So E and E-NS1 genes of JEV were cloned and sequenced, furthermore, E gene was expressed in E.coli and BHK-21 cell. Recombinant pseudorabies virus expressing NS1 or E protein of Japanese encephalitis virus was constructed, and the research of their biological characteristic, safety and immunogenicity was carried out.1. Cloning and sequence analysis of E and E-NS1 genes of JEV SA14-14-2E and E-NS1 genes were amplified from JEV SA14-14-2 strain by RT-PCR and were further cloned and sequenced. The results indicate that there was no nucleotide difference between the cloned E gene and E of SA14-14-2 strain, while there were 3 nucleotides and 2 amino acids differences between the cloned E-NS1 gene and E-NS1 of SA14-14-2 strain.2. Expression of E gene in E.coli and BHK-21 cellThe cloned plasmid pSK-E was digested with EcoRI and BamHI, then the fragment of E was cloned into a prokaryotic expression vector and a eucaroytic expression vector, and named pKG-E and pcDNA3.1E, respectively. The pKG-E plasmid was then transfected into E.coli BL21 (DE3). SDS-PAGE and western blotting showed that E could be highly expressed as a fusion protein whose molecular weight was about 83KD and E protein was specific to antisera against JEV. Plasmid pcDNA3.1E was transfected into BHK-21 cells with lipofectamine. After 48h, the above BHK-21 cells were harvested to analyze. Western blot indicated that E was expressed in BHK-21 cells.3. Construction of recombinant pseudorabies virus expressing NS1 or E protein of Japanese encephalitis virusE and NS1 were respectively subcloned into vector pgG-Uni to form recombinant plasmid (designed as pgG-E and pgG-NS1, respectively). A co-transfection experiment was performed in porcine kidney (PK-15) cells with pgG-E or pgG-NSl and the genome of the vector virus (PRV TK~-/gG~-/LacZ~+ mutant). By plaque purification, PCR detectionand southern hybridization, recombinant pseudorabies virus expressing E or NS1 protein oi JEV was acquired and named TK7gG"/E+ and TK7gG7NSl+, respectively. Western blot analysis and ELISA demonstrated the E or NS1 protein could be expressed in recombinant PRV strains. Studies on their biological characteristics confirmed that the propagation of recombinants in cells was not affected and their genetic stability was high. 4. Study on the immunogenicity of recombinant pseudorabies virus TK7gG7NSl+and TK/gG/E+Four to six-week-old female Balb/c mice and forty to sixty-day-old piglets that were serologically negative to both pseudorabies and Japanese encephalitis were inoculated with TK7gG7NSl+ or TKVgGTE* to evaluate their safety and immunogenicity. The results of the animal tests showed that TK7gG7NSl+ and TK7gG7E+ are safe to both mice and piglets. Furthermore, The vaccinated mice could acquire protective immunity against lethal challenge of the virulent PRV. PRV neutralizing antibody can be induced in immunized piglets, and a good humoral and cellular immune response against JEV could be induced in the mice and piglets immunized with TK7gG7E+ or TK7gG7NSl+. Although compared with JEV SA14-14-2, the specific immune responses induced by TK/gG7E+ or TK7gG7NSl+ were little lower, there was significant difference in JEV-specific CTL activity and antibody to JEV between the group immunized with TKT/gGTE* or TK/gG7NSl+ and the negative control (P>0.05). The above results revealed that the two recombinant viruses could be suitable candidate vaccine strains for developing a novel genetic vaccine to combat pseudorabies and Japanese encephalitis in the pig industry.