The ISG15 Inhibit Replication Of JEV In Vitro And The Three Kinds Of Immunopotentiators Improving The Efficacy Of JEV Inactivated Vaccine

Japanese encephalitis(JE)virus,belongs to the familyflaviviridae,causes infection of the central nervous system in human and clinical symptoms of stillbirths,reproductive dysfunction in swine.The vaccination is effective strategy to prevent and control of the disease in swine and block the virus spread to human.Both of the attenuated or inactivated JE vaccines were effective in swine.IFN-stimulated gene 15(ISG15),an ubiquitin-like protein,is rapidly induced by IFN-a/p,and play a role on the antiviral immune response.Many studies have proved ISG15 antiviral activity in vitro and in vivo.ISG15 may be used as a potential immune adjuvant to enhance the immune response of vaccines.Other reports showed that a variety of chemical reagents could used as immunopotentiators and partial of these products have licensed in human vaccines.This study confirmed the antiviral effect of ISG15 in vitro,developed three immunopotentiators which can improve immune efficacy of porcine vaccine and effect expression of ISG15 in vivo.1.Establishment of Real Time PCR detection method for JEV,ISG15,IFN-?,IL-2,IL-4,IL-10 and ?-actinIn this study,the Real Time PCR primers of JEV,porcine ISG15,IFN-y,IL-2,IL-4,IL-10 and ?-actin were designed according to the gene sequences published in GenBank.The total RNA was extracted from the porcine PBMCs post 24 h inoculation with JEV at M.O.I.of 1.The reverse transcripted cDNA was used as the template for Real Time PCR.We optimized both of the annealing temperature and the primer concentrationin the reaction system,and established the standard curves of JEV,porcine ISG15,IFN-y,IL-2,IL-4,IL-10 and ?-actin gene,and systematically evaluated the Real Time PCR methods.The optimized annealing temperature was 60? and the primer concentration was 0.25 ?M.The parameters of the standard curve were satisfied the requirements for Real Time PCR.The coefficient of determination R2 of the standard curve were higher than 0.99.The amplifiction efficiency of the JEV,?-actin,ISG15,IFN-y,IL-2,IL-4 and IL-10 were 103.0%,103.0%,107.3%,97.3%,96.9%,100.4%,103.1%,respectively.Together with the results,we established specific and repeatable Real Time PCR reaction system which useful for next step experimental detection.2.The overexpression and interference of ISG15 influence on the replication of JEV in vitroIn this study,Real Time PCR were used to test the ISG15 mRNA level and the JEV nucleic acids copy number in swine testicular(ST)cells infected with JEV after pretreated with IFN-a.The results showed that mRNA levels of ISG15 were significantly up-regulated,the copy numbers of the JEV nucleic acids were significantly reduced.We constructed the recombinant pcDNA3.1 expression pladmid of the ISG15 gene via RT-PCR and other molecular cloning technology.ST cells were transfected with this recombinant ISG15 plasmid.After 24 h,ISG15 over expression cell infected with JEV.The mRNA levels of ISG15 were significantly up-regulated.The viral genomes and virus titers of JEV were significantly reduced,which indicated that replication of JEV was significantly inhibited by overexpression of ISG15.In contrast,we designed and constructed plasmid of shRNA to inhibit the ISG15 gene.ST cells were transfected with the shRNA plasmid,and infected with JEV post 24 h of transfection.The mRNA level of ISG15 was significantly down-regulated which indicated that the expression of ISG15 was interfered by the shRNA plasmid.The viral genomes and virus titers were significantly increased.All results showed that the replication of JEV was significantly inhibited by ISG15 in vitro.3.The immunopotentiator improving the efficacy of porcine JEV inactivated vaccine and effecting the levels of ISG15 mRNAIn this study,the JEV JS01 strain was propagated in the BHK-21 cells.The titer of the virus stock determined by cytopathic effects(CPEs)in BHK-21 cells was 107.5 TCID50/mL.Inactivated virus was prepared as inactivated vaccine.The inactivated vaccines were combined used with immunopotentiators IP-23,IP-24 and IP-26.Fouty days old healthy piglets free of JEV infection were randomly divided into five groups(n=5).Piglets in the 1st,2nd and 3rd groups were shot with inactivated vaccines containing IP-23,IP-24 or IP-26 respectively.The pig in the 4th group was immunized with JEV inactivated vaccine without immunopotentiators.The last group was injected with PBS as placebo.All piglets were boosted with the same vaccine as prime immunization at two weeks post prime.Compared to the piglets received the JEV inactivated vaccine without immunopotentiators,increased neutralizing antibody titers were observed in the 1st,2nd and 3rd groups.Specifically the 2nd group elicited highest antibody titers among all tested groups.The MTT assay was adopted to evaluate the proliferation of PBMCs which isolated at 14 and 28 days post boost vaccination.However,no significantly difference were monitored in the piglets among five groups.The potential of PBMCs expression of cytokines were examined by Real Time PCR after these cells stimulated with JEV strain of JSOlfor 24 h.The mRNA levels of ISG15 were no significantly difference.The mRNA levels of IFN-?,IL-2 were significant increased in piglets of the 2nd group and 3rd group,indicating Th1 immune response improving by IP-24 and IP-26.The mRNA levels of IL-4 were significant elevated in the group co-immunized with inactivated vaccine and IP-23,indicating IP-23 enhancement of Th2 immune response.