Study On Interaction Of Japanese Encephalitis Virus With Dendritic Cells

Japanese encephalitis (JE), which is caused by Japanese Encephalitis virus, is one of the most serious mosquitoborne infectious disease that affects the security of people and livestock. JE is also a veterinary problem, from which the pig industry suffered serious losses because of the reproduce failure. In humans, at the same time, it is one of the common mosquitoborne diseases in central nervous system (CNS). However, so far, the pathogenesis and immunological mechanism of JE are not clear and there are few clinical methods for detecting the pathogen JEV. Therefore, the researches about interaction of JEV with immunocytes and the clinical rapid detection method for JEV will play important role in revealing the pathogenesis and immunological mechanism of JE, in developing new vaccine and in clinical screening pathogen. The content of this thesis is as follows:Dendritic Cells (DCs) loaded with inactivated virus as a vaccine against JEV, interaction of DCs with JEV wild strain and attenuated strain, and the rapid detection of JEV with immunochromatographic strip.1. Dendritic Cells (DCs) loaded with inactivated virus as a vaccine against JEVMouse bone marrow-derived dendritic cells (bmDCs) were generated and stimulated with inactivated JEV in vitro. BALB/c mice were immunized with inactivated JEV-stimulated bmDCs and then challenged with JEV wild type strain. The results demonstrated that intravenous (i.v.) injection of JEV-pulsed bmDCs into each mouse produced notable levels of JEV-specific neutralizing antibodies and higher levels of CD8+ T cell, IFN-y and TNF-a compared with JEV-inactivated vaccine. Furthermore, stimulated bmDCs could elicit a highly protective efficacy (90%) against JEV challenge. It suggests that stimulated bmDCs can be utilized as an alternative strategy to develop new generation of vaccines or therapy for JEV infection.2. The interaction of Japanese encephalitic virus wild strain P3 and bmDCsIn current study, we examined the alteration of phenotype and function of DCs including bone marrow-derived DCs (bmDCs) and spleen-derived DCs (spDCs) due to JEV P3 infection in vitro and in vivo in order to unveil the relationship of JEV wild strain infection and viral immune escape. The results of RT-PCR,realtime-PCR,Western blot,standard plaque assay and FACS showed that P3 could productively infect DCs, and this infection dramatically downregulated cell surface molecule expressions, such as CD40, CD80, CD86 and MHCâ…¡. MLR and ELISpot assays revealed an impaired capacity of P3-infected DCs to activate allogeneic naive T cells. P3 inhibited the expression of TNF-a and IL-12 of DCs even upon response to LPS, but enhanced IL-10. Interestingly, viral infection of DCs expanded CD4+Foxp3+ Treg (regulatory T cells). Summarily, infection of DCs by JEV P3 impaired cell maturation and their activity for T cell activation, modulated cytokine production, and induced immune escape. It was the first time for revealing the JEV P3 strain infection induced immune escape by the interaction of virus and DC, indicating a possible mechanism of JEV P3 strain induced disease pathogenesis.3. The interaction of Japanese encephalitic virus vaccine strain SA14-14-2 with bmDCsIn this study, it would reveal the intrinsic mechanism of JEV vaccine strain SA14-14-2 vaccination and induced robust antiviral immune that the study of phenotypic and functional alteration of DCs after JEV SA14-14-2 infection. The results demonstrated that JEV SA14-14-2 could infect bmDCs, upregulated immature bmDCs maturation marker molecules and antigen presentation molecules. MLR, ELISpot and FACS revealed an enhanced allostimulatory capacity of SA14-14-2-infected bmDCs and an impaired capability of differentiating Treg. These results implied that the immune activation of DC by SA14-14-2 infection may be associated with robust immune activation against viral infection induced by attenuated vaccine immunization.4. Development of immunochromatographic strip for detecting Japanese encephalitis virusIn this study, two mAbs against JEV E protein were developed, and by which a rapid immunochromatographic strip (ICS) was established for detecting Japanese Encephalitis virus (JEV). The specificity results showed that this method had no cross-reaction with Pseudorabies viruses (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Foot and Mouth Disease virus (FMDV) and Asia Influenza Virus (AIV). A RT-PCR used as a reference test for submitted 188 clinical samples determined the specificity and sensitivity of the ICS to be 99.3% and 85.7%, respectively. It indicated that the established method could be used as a new mean for rapid screening the clinical JEV.