| Japanese encephalitis is a vector borne zoonotic disease,causing great damages to human healthy and pig breeding industry, and now vaccination is the primary means to prevent and treat the disease. For now, domestic Japanese encephalitis virus vaccines are made from virus of genotype III. With the advantage of genotype I has become better than genotype III gradually, developing vaccines of genotype I is particularly urgent.In this study, the vaccine candidate strains of genotype I with good immunogenicity was screened, and the vaccines were made successfully. Immunization test of mice,guinea pigs and piglets and challenge protection test in mice were performed to screen two strains with good immunogenicity.1. Identification and preliminary screen of strains1) Identification and biological characteristics of isolates : There were four stable strains of Japanese encephalitis virus (YA1?YA2?MY1?CZ1 strain) by recovery of the virus,and identified as Japanese encephalitis by RT-PCR. Four kinds of virus solution were prepared by passaging in BHK-21 cells and mouse brains. Compared with the four different viruses by TCID50 and LD50, TCID50 value of the passaged virus made from mouse brains increased significantly and the virulence of the virus enhanced further.Therefore, the virus made from mouse brains was chosen for preparing inactivated vaccine.2) Preparation of inactivated vaccine: virus solution of mouse brains was inactivated with formaldehyde, and the optimal conditions for inactivation was that 0.05%ormaldehyde,37?, inactivated for 48h. The four kinds of mouse brain vaccines were performed with good quality by formulation testing, stability testing, sterility testing and safety testing.3 ) Immunization test of mice: the immunization test of mice was carried out based on qualified vaccines. The strain with better immunogenicity was screened by intraperitoneal inoculation and monitoring serum antibody levels in mice. The results showed all of the four vaccines had good immunogenicity. Two weeks after secondary immune, antibody levels in groups of YA1 and CZ1 strain vaccines were significantly higher than that of commercialization inactivated vaccine (P<0.01) . Four weeks after secondary immune, antibody levels in all groups were significantly higher than that of commercialization inactivated vaccine (P<0.01) , however, absolute value of antibody levels was higher in group YA1 and CZ12. Preparation and immunogenicity of isolates inactivated vaccine1) Immunization test of guinea pigs: the YA1 and CZ1 strains were screened for vaccine making, and the chanllenge protection test was perforemed to evaluate the possibility.The result showed that both CZ1 and YA1 can induce guinea pigs to produce qualified hemagglutinin inhibitor antibody. The antibody titre and positive conversion rate were higer than commerchialization of inactivated vaccines. hemagglutinin inhibitor antibody lever of CZ1 strain group was significantly higher than that of YA1 strain group (P<0.05).2) Challenge protection test in mice: immune 3-weeks old mice and secondary immune was taken after 1 week. Challenge in brains using 10 times LD50 was performed 1 week after secondary immune. The result showed the protection rate of YA1 strain vaccine was 70%, the protection rate of CZ1 strain vaccine was 80%, while the protection rate of commercial inactivated mouse brain vaccine and live attenuated vaccine were 60% and 70% respectively. Both of the two strains have shown excellent protective capability, and CZ1 strain was better.3) Immunization test of piglets: The results showed the two strains can both made antibody positive conversion rate of piglets up to 100%, antibody level and neutralizing antibody titer being higher than commercial inactivated vaccines. Meanwhile, the result of antibody level was the consistent with that of guinea pig immunization test and mice challenge protection test above. Moreover, CZ1 strain was of better immunogenicity. |
PDF Full Text Request