Analysis Of The Effect Of Japanese Encephalitis DNA Gene Vaccine On Inducing Immune Response In Dogs

Japanese encephalitis(JE)caused by Japanese encephalitis virus(JEV)is a serious zoonotic disease.Immunization is the most effective way to prevent and control the disease.In order to investigate the immune effect and safety of Japanese encephalitis DNA gene vaccine in dogs,this study used Japanese encephalitis virus(JEV)virulent GZ strain recombinant eukaryotic plasmid pcDNA3.1-pr M/E-q combined with pcDNA3.1-NS1-NS2A-q and porcine Japanese encephalitis live vaccine(SA14-14-2 strain)were inoculated to 3-month-old dogs on 0 d,14 d and 28 d,respectively,and the immune response and virus residues were detected in the immunized dogs.detection,in order to contribute to the prevention and control of Japanese encephalitis.1.Evaluation of the immune effect of Japanese encephalitis DNA gene vaccineThe levels of immunoglobulin M(IgM)and immunoglobulin G(IgG)were detected by Elisa assay before immunization(0 d)and within 242 d(8 months)after immunization;Before immunization(0 d)and within 42 d after immunization,blood was collected from the forelimb veins of all the experimental dogs in the JEV virulent strain recombinant plasmid group,the JEV attenuated vaccine group and the control group,and the blood was analyzed by flow cytometry to detect T lymphocyte subsets.Population CD4~+/CD8~+T lymphocyte levels;Tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-1β(IL-1β),and interleukin-2(IL-2),interleukin-4(IL-4),interleukin-6(IL-6),interleukin-8(IL-8)levels.The experimental results showed:(1)When inoculated with a dose of 0.2 m L,the IgM and IgG levels of the experimental dogs in the JEV virulent recombinant plasmid group and the JEV attenuated vaccine group increased significantly from 3 d to 242 d after immunization(P<0.01)Compared with the control group,the levels of IgM and IgG in the control group did not change significantly;The IgM level of the JEV virulent recombinant plasmid group was slightly lower than that of the JEV attenuated vaccine group,and the difference was not significant(P>0.05);After 7 days of immunization,the IgG level of the experimental dogs in the JEV virulent recombinant plasmid group was significantly(P<0.05)lower than that in the JEV attenuated vaccine group,and the IgG level of the experimental dogs in the JEV virulent recombinant plasmid group was also slightly lower than that in the JEV attenuated vaccine group at the rest of the time.But the difference was not significant(P>0.05);Before immunization(0 d)to 42 d after immunization,the ratio of CD4~+/CD8~+in the JEV virulent strain recombinant plasmid group and the JEV attenuated vaccine group increased,and the control group had no obvious change trend.There was no significant difference in the ratio of CD4~+/CD8~+(P>0.05),and the ratio of CD4~+/CD8~+in experimental dogs in the JEV virulent recombinant plasmid group was slightly lower than that in the JEV attenuated vaccine group,but the difference was not significant(P>0.05);The levels of TNF-α,IFN-γ,IL-2,IL-4,IL-6,IL-8 and other cytokines in the experimental dogs in the JEV virulent strain recombinant plasmid group and the JEV attenuated vaccine group increased,but there was no significant change in the control group.The level of cytokines in the JEV virulent recombinant plasmid group was slightly lower than that in the JEV attenuated vaccine group,and the levels of cytokines in the two experimental groups were significantly higher(P<0.01)than those in the control group.(2)When inoculated with a dose of 0.3 m L,the IgM and IgG levels of the experimental dogs in the JEV virulent recombinant plasmid group and the JEV attenuated vaccine group increased from 3 d to 242 d after immunization,while the IgM and IgG levels in the control group did not change significantly,and the two groups had no significant changes.The levels of IgM and IgG in the experimental group were significantly higher(P<0.01)than those in the control group;From 3 d to 182 d after immunization,the IgM level of the JEV virulent recombinant plasmid group was slightly higher than that of the JEV attenuated vaccine group,and the difference was not significant(P>0.05).The level decreased to lower than that of the JEV attenuated vaccine group,and the difference was not significant(P>0.05);The IgG level of JEV virulent recombinant plasmid group was slightly higher than that of JEV attenuated vaccine group,but the difference was not significant(P>0.05);After immunization,the ratio of CD4~+/CD8~+in experimental dogs in the JEV virulent recombinant plasmid group and the JEV attenuated vaccine group increased,but the JEV virulent recombinant plasmid group was slightly higher than the JEV attenuated vaccine group,and the difference was not significant(P>0.05).There was no significant change in the group;The levels of TNF-α,IFN-γ,IL-2,IL-4,IL-6,IL-8 and other cytokines increased in the JEV virulent strain recombinant plasmid group and the JEV attenuated vaccine group,but did not change significantly in the control group.The level of cytokines in the virulent recombinant plasmid group was slightly higher than that in the JEV attenuated vaccine group.(3)When inoculated with a dose of 0.5 m L,the IgM and IgG levels of the experimental dogs in the JEV virulent recombinant plasmid group and the JEV attenuated vaccine group increased from 3 d to 242 d after immunization.and IgG levels were significantly(P<0.01)higher than those in the control group;at 3 and 7 days after immunization,the IgM level in the JEV virulent recombinant plasmid group was significantly(P<0.05)higher than that in the JEV attenuated vaccine group;14 days after immunization On the 35th day,the IgM level in the recombinant plasmid group of virulent JEV strain was extremely significantly(P<0.01)higher than that in the attenuated JEV vaccine group;from 42 d to 152 d after immunization,the level of IgM in the recombinant plasmid group of virulent JEV strain was slightly higher than that in the attenuated JEV vaccine group.There was no significant difference in the vaccine group(P>0.05);from 182 days to 242 days after immunization,the IgM level of the JEV virulent strain recombinant plasmid immunization group decreased to a very significant(P<0.01)lower than that of the JEV attenuated vaccine immunization group;the JEV virulent strain The IgG level of the experimental dogs in the recombinant plasmid immunization group was slightly higher than that in the JEV attenuated vaccine immunization group,but the difference was not significant(P>0.05).There was no obvious change trend in the control group.The ratio of CD4~+/CD8~+in the JEV virulent recombinant plasmid group was slightly higher than that in the JEV attenuated vaccine group,but the difference was not significant(P>0.05).The levels ofα,IFN-γ,IL-2,IL-4,IL-6,IL-8 and other cytokines increased,and there was no significant change in the control group.The level of cytokines in the recombinant plasmid group of the strong JEV strain was slightly higher than that of the attenuated JEV vaccine group.2.Safety evaluation of Japanese encephalitis DNA gene vaccineHealth monitoring of all experimental dogs before and after immunization;242 days after immunization,one dog was randomly selected from each experimental group,and one dog was randomly selected from the control group,and sterile fresh cerebrospinal fluid was taken out by surgical puncture,and observed by transmission electron microscope;Take sterile fresh brain,cerebellum,heart,liver,spleen,lung,kidney and other tissues for HE staining and RT-PCR detection.The results showed that none of the experimental dogs showed symptoms such as systemic reaction,local reaction,elevated body temperature,vomiting,diarrhea,diet reduction,and disturbance of consciousness;No Japanese encephalitis virus particles were observed in the cerebrospinal fluid of the JEV virulent recombinant plasmid group,the JEV attenuated vaccine group and the control group by transmission electron microscopy;No obvious pathological changes were found after observing the tissue by HE staining of histopathological tissue sections;No virus was found in the tissue detected by RT-PCR.The experimental results showed that the recombinant plasmids pcDNA3.1-pr M/E-q,pcDNA3.1-NS1-NS2A-q and attenuated JEV vaccine(SA14-14-2 strain)had good safety in dogs.