【作者】 李么明；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2009， 博士

【摘要】 乙型脑炎(Japanese encephalitis, JE)(简称乙脑)是由乙型脑炎病毒(Japanese encephalitis virus, JEV)感染引起的一种危害严重的人兽共患虫媒传染病。该病是危害我国养猪业的重大繁殖障碍性疫病之一。同时,它还对人类健康造成巨大威胁,是人类中枢神经系统最常见的虫媒病之一。但长期以来,该病的致病和免疫机理尚不太清楚,临床中针对该病原的快速检测方法也很缺乏。因此,开展JEV与免疫细胞的互作机制和快速检测方法研究,对于揭示该病毒的致病与免疫机理、新型疫苗开发和临床快速检测具有重要科学和实践意义。本论文主要围绕JEV树突状细胞(Dendritic cells, DCs)载体疫苗、JEV强、弱毒株与小鼠DCs互作、JEV胶体金快速检测方法等三个方面展开研究,具体内容如下：1.JEV树突状细胞载体疫苗研究利用灭活的JEV P3株体外刺激小鼠骨髓来源的树突状细胞(bmDCs),制备DC疫苗并免疫BALB/c小鼠,并于二次免疫后3周使用强毒攻击。结果显示：虽然DC疫苗免疫后诱导的JEV中和抗体低于灭活疫苗,但其诱导的IFN-γ和TNF-α水平却显著高于灭活疫苗,而且显著提升抗原特异性CD8+T细胞水平。攻毒保护性试验表明：尽管DC疫苗的保护效力略低于灭活疫苗(100%),但仍然能提供90%的保护。本研究首次表明JEV负载的DC疫苗可诱导机体产生较高的细胞免疫水平,并能提供有效的免疫保护力,是一种具有潜在应用前景的新型细胞疫苗。2.JEV强毒株(P3株)与小鼠树突状细胞的相互作用研究本研究通过探讨DC感染JEV P3株后的表型功能的变化,来揭示JEV强毒感染与机体免疫逃逸机制之间的关系。RT-PCR、Realtime-PCR、Western blot、空斑实验、混合淋巴细胞反应(MLR)、ELISpot和流式细胞检测结果表明：JEV强毒株可感染DC,并可在DC中复制,JEV P3株感染不仅抑制DC的MHCⅡ、CD40、CD80和CD86等免疫共刺激分子和抗原提呈相关分子的表达,显著降低DC刺激淋巴细胞增殖和抗原提呈能力,而且还可促进T调节细胞的分化。该研究结果显示：JEV强毒株感染抑制DC的成熟,并降低其抗原提呈能力,导致机体的免疫抑制。本研究从病毒与DC互作的角度揭示了JEV强毒株一种可能的免疫逃避机制,并为解析JEV强毒株的致病机理提供重要线索。3.JEV疫苗株(SA14-14-2)与小鼠树突状细胞的相互作用研究本研究通过研究DC感染JEV疫苗株后表型功能的变化,从而探讨疫苗株接种诱导机体强大的抗病毒免疫的内在机制。结果表明：JEV SA14-14-2株也可以感染并可在DC中复制,但是JEV SA14-14-2株感染不仅促进DC表面的免疫共刺激分子和抗原提呈相关分子的表达,而且还可显著增强DC刺激淋巴细胞增殖和抗原提呈能力,抑制T调节细胞的分化。说明：疫苗株可以促进DC成熟,增强DC抗原提呈能力并诱导免疫激活。本研究首次从疫苗毒与DC互作的角度揭示了JEV疫苗株感染引发强大的免疫激活效应的一种可能机制,并为揭示JEV疫苗株的免疫保护机理提供科学依据。4.检测JEV的胶体金方法的建立本研究制备了JEV E蛋白的单克隆抗体,并利用其中两株单克隆抗体建立了针对JEV的胶体金快速检测方法。特异性试验结果显示,该方法与伪狂犬病毒(PRV)、繁殖与呼吸综合征病毒(PRRSV)、口蹄疫病毒(FMDV)和禽流感病毒(AIV)均无交叉反应。同时,用该方法对临床188份送检病料进行检测,结果表明：该方法与RT-PCR检测结果的特异性和敏感性分别为99.3%和85.7%。说明：本研究建立检测方法可以作为一种新型检测方法用于JEV的临床快速检测。更多还原

【Abstract】 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 MHCⅡ. 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.更多还原