Preparation And Immunogenicity Preliminary Study Of Inactivated Vaccine Of Bovine Viral Diarrhea Virus JL3 Strain

Bovine viral diarrhea virus(BVDV)is an important pathogen related to diseases of the gastrointestinal tract,respiratory tract and reproductive system of cattle,causing serious economic losses to the cattle industry in China.At present,vaccination is still the most effective way to prevent and control the disease.Therefore,in this study,an inactivated vaccine was prepared by using the clinically isolated epidemic strain BVDV JL3 strain,and its immunogenicity was studied to lay a theoretical foundation for the further development of the vaccine.In order to understand the prevalence of bovine viral diarrhea virus,a Taq Man dual real-time quantitative PCR method for rapid detection of bovine viral diarrhea virus types 1 and2 was established,and the clinical samples collected from some cattle farms in Jilin Province were tested by this method.The results showed that the optimal annealing temperatures of BVDV type 1 and type 2 were 57.0℃,the optimal primer concentration was 0.5μmol/L,and the optimal probe concentration was 0.3μmol/L.The method has good specificity,sensitivity and reproducibility,with a minimum detection limit of 10~1 copies/μL.The clinical sample test results showed that a total of 36 BVDV positive samples were detected in the 156 samples,and the total positive infection rate was 23.1%,of which the BVDV type 1 positive rate was 17.9%(28/156),and the BVDV type 2 positive rate was 5.1%(8/156),indicating that the infection rate of BVDV type 1 was higher.To prepare the inactivated vaccine of BVDV1 JL3 strain.Firstly,the appropriate propagation conditions of the virus were determined by optimizing the inoculation method,the multiplicity of infection and the inoculation time;the appropriate inactivation conditions of the virus were determined by optimizing the concentration and inactivation time of the inactivating agent;selected different adjuvants to prepare the BVDV JL3 strain inactivated vaccine,and conducted dosage form,sterility and safety testing.The results showed that the suitable propagation conditions of the virus were as follows:monolayer inoculation,MOI=0.05 and virus liquid collected after 92 hours.The suitable inactivation conditions of the virus were:BEI final concentration of 3 m M,inactivation for 18～24 h.Inactivated BVDV vaccines were prepared with ISA201 adjuvant,ISA206 adjuvant and IMS1313 adjuvant respectively.The results showed that the sterility,stability,viscosity and dosage form of the vaccine prepared by the three adjuvants all met the requirements for vaccine preparation.In order to evaluate the immunogenicity of the inactivated vaccines of BVDV JL3 strain prepared with different adjuvants.Mice were immunized with inactivated vaccine prepared with three adjuvants and commercial vaccine at the same time.The neutralizing antibody titer,lymphocyte proliferation,T lymphocyte subsets and the expression levels of cytokines IL-4 and IFN-γwere detected by ELISA and flow cytometry,and the immune protective effect of the vaccine was tested by a challenge test.The results showed that neutralizing antibodies could be detected at 21 days after the first immunization in each vaccine group,and the neutralizing antibodies reached the peak at 35 days after the first immunization;The spleen lymphocytes were stimulated with Con A at 35 days after the first vaccination in mice,and the SI index of each vaccine group was significantly higher than that of the negative control group(P<0.01);The contents of CD4~+T lymphocyte subsets and CD8~+T lymphocyte subsets in the three adjuvant vaccine groups and commercial vaccine groups were significantly higher than those in the negative control group(P<0.01);The levels of IL-4 and IFN-γin each vaccine group were significantly higher than those in the negative control group(P<0.01 or P<0.05).The results of the challenge test showed that the inactivated vaccines prepared by the three adjuvants could produce better protective effects against virulent challenge,and effectively reduce the viral load of various organs of the mice and the pathological damage of virus to the duodenum.Among them,the immune protective effects of ISA206 adjuvant and IMS1313 adjuvant vaccines were better than that of ISA201 adjuvant vaccine and commercial vaccine.In conclusion,this study provides effective technical means for the clinical diagnosis,epidemiological investigation and purification of the disease.At the same time,it lays the foundation for further research on BVDV vaccines.