Effect Of Forsythiaside A On The Replication Of Bovine Viral Diarrhea Virus And Assessment Of Immune Efficacy On Mice Of The Prepared BVDV DNA Vaccine

Background:Bovine viral diarrhea(BVD)is a disease caused by bovine viral diarrhea virus(BVDV)that can cause significant loss of dairy farming.Antigen of BVD is complex and has a poor cross protection among different types of BVDV.However,there are no effective drugs and effective vaccine for prevention.Forsythiaside A(FTA)can inhibit BVDV replication,but the precise mechanism is unclear.Aims:The aims of the present study are to explore the possible mechanism of inhibition of BVDV replication by FTA,and to prepare BVDV DNA vaccine and also evaluate immunization strategy and its immune effect.Methods:Peripheral blood mononuclear cells(PBMCs)were isolated from the cattle with both negative BVDV antigen and antibody.BVDV C24V strain(MOI = 0.1)challenged PBMCs and FTA was added to PBMC at concentration of 100 ?g/mL.Virus-infected cells were incubated with FTA for 1 h under serum-free condition,followed by 10%fetal bovine serum or FTA.BVDV E2 gene from clinical strain was amplified and the DNA vaccine was constructed.One hundred fifty BALB/c mice were randomly divided into 5 groups and immunized as follows respectively:1)100 ?L PBS;2)100 ?g empty vector;3)100 ?g pcDNA3.1-E2;4)40 ?g E2 protein;5)pcDNA3.1-E2 and E2 protein).BVDV virus(6 × 106 TCID50,BVDV C24V)was injected through intraperitonea after the third immunization.The replication of BVDV was analyzed by real cell counting and absolute quantitative PCR.The expression of costimulatory factors was quantitatively analyzed by real-time PCR and Western blotting.The expression of antibody-related factors and antibody titer were quantitatively analyzed by ELISA.The effect of forsythoside A on the proliferation,activation and apoptosis of PBMCs was evaluated by flow cytometry.The virus isolation was evaluated by immunofluorescence.The protective effect of the vaccine on mice challenged by BVDV was evaluated by immunofluorescence and HE staining.Results:FTA promoted T cell activation and proliferation,and inhibited BVDV-induced apoptosis of PBMCs.FTA can regulate the,expression of CD28/CTLA-4 and promote the expression of 4-1BB during BVDV infection.In addition,FTA could increase the expression of TRAF-2 at 24 h after BVDV infection.FTA can increase the expression of IgG2a and IL-2 during BVDV infection and regulate the excessive secretion of IFN-?.FTA could inhibit the number of BVDV copies in each PBMC,with no effect on the virulence of the virus.The eukaryotic expression vector pcDNA3.1-E2 was prepared successfully.DNA vaccines and subunit vaccines emulsified with ISA 61 in the present study could stimulate high levels of antibody levels and promote the expression of CD4+ IFN-?+ and CD8+ IFN-?+ T cells.And the main antibody sub-types were IgG1,IgG2b and IgG2a.They also can increase the concentration of IL-4 in serum and protect the mice from pathological changes induced by BVDV challenge in spleen,lungs and kidney.Furthermore,DNA vaccine immunization can promote spleen lymphocyte proliferation.The prepared DNA vaccines and subunit vaccines with the prime boost strategy can stimulate neutralizing antibodies at a high level and promote effective CTL as well as Thl and Th2 cellular immune responses.They could enhance the secretion of IL-4 and TNF-a,and protected mice against BVDV infection.Conclusion:FTA promotes T cell activation and proliferation,and inhibits BVDV-induced apoptosis of PBMC,which may be related to TRAF2-dependent CD28-4-1BB signaling.The prime boost vaccination could improve the longevity and strength of immunity.