Development Of Japanese Encephalitis-porcine Parvovirus Combined Inactivated Vaccine

The Japanese encephalitis and porcine parvovirus infection are themajor diseases for inducing the reproductive failure of pigs. The Japanese encephalitisvirus and porcine parvovirus have been in a wide range of the world and spreadquickly endemically.They brought a so overwhelming loss to breeding anddevelopment of live pig, and retarded progress of hog industry. At present, the controlof these infectious diseases relied mainly on immunization both in China and abroad.The higher frequency of vaccination not only took time and energy but also increasedcosts and the immune stimulation of pigs because of the lack of combined vaccine.Therefore, the combined vaccine was urgently needed now. We have conducted someresearches to develop the Japanese encephalitis-Porcine parvovirus bivalentinactivated vaccine.A SYBR Green I fluorescent quantitative PCR assay was developed for rapiddetection of Japanese encephalitis virus (JEV) NS3gene. The assay developed wasfound to be more sensitive as compared to conventional RT-PCR. There were nocross-reactions with other viruses. The coefficient variation (CV) of intra/inter-assayfor the same sample was less than1.2%.The results indicated that the assay developedwas of high specificity, sensitivity and reproducibility.To investigate the proliferation dynamic of Japanese encephalitis virus inBHK-21cells, BHK-21cells were infected with Japanese encephalitis virus. Afterinfection at different time, the Japanese encephalitis virus copies and titers of the cellsupernatant and suspension were detected by the fluorescent quantitative PCR andTCID50method. The proliferation dynamic curves were drawn respectively andanalysed. The results showed that there was a parallel correlation between theproliferation dynamic of Japanese encephalitis virus in BHK-21cells detected by thefluorescent quantitative PCR assay and that by TCID50method.The virus proliferationpeak was found at28h after infection, and the similar high titer was maintained to40h after infection. This method provides a useful tool for detecting viral titers duringthe production of Japanese encephalitis vaccine.The physicochemical properties and immunogenicity of Japanese encephalitis virus LS strain were studied in the test. The results indicated that JEV LS strain wassensitive to temperature (56℃), acid (below pH5), diethyl ether and trypsin. Theviral titer of LS strain remained at the same level undergoing a repeated freeze-thawtest. The virus can coagulate the red cells of goose. The hemagglutination titer of LSstrain remained keep the high level after20serial passage in BHK-21cells. JEV LSstrain was inactivated by formaldehyde to prepare oil emulsion vaccine. The vaccinecould induce high LAT antibody titer(1:64) in mice.We have the further understandingof the characteristics of JEV LS strain, laying the foundation for developing Japaneseencephalitis vaccine.Development of Japanese encephalitis-porcine parvovirus oil emulsion inacti-vated vaccine. The JEV LS strain was cultured on BHK-21cells and the PPV HNZK-1strain was cultured on PK-15cells. The high titer JEV and PPV venom concentratedby using dialysis bags was used as a vaccine antigen. The antigen was inactivated byformaldehyde to prepare oil emulsion bivalent vaccine. After the physical propertiestest, sterile and safety test, the experimental animals were immunized with thebivalent vaccine. The result showed that the JEV LAT antibody titer reached thehighest point (1:51) at six weeks after immunization. The PPV HI antibody titer alsoreached the highest level (1:2048) at eight weeks after immunization, which washigher than the single commodity vaccine.