脑组织特异microRNA靶向EV71减毒株的构建及免疫效果评价

Human Enterovirus type 71 (EV71) is the major pathogen of severe Hand,Foot,and Mouth disease (HFMD) in children. EV71 infection has been associated with the severe neurological complications, including encephalitis, aseptic meningitis, as well as death. miRNAs have emerged as key post-transcriptional regulator and have unique expression profile in different tissues, which provides an opportunity for the development of live attenuated vaccines of neurotropic viruses. Recently, we constructed the infectious cDNA clone of EV71 Chinese isolates and established the murine model of EV71 infection. In this proposal, we aim to screen for the brain tissue specific miRNAs in EV71 infected cells by using microarray and reporter plasmids. Then, the target sequences of brain-specific miRNAs will be introduced into the infectious clone of EV71 with different styles. All the rescued EV71 mutant viruses will be characterized by standard virological and biochemical assays, and the growth properties of the rescued viruses in neural cells and mice brains will be further compared with the parental viruses. Finally, the characteristics of attenuation and immnue protection efficacy of the mutant viruses will be evaluated in murine model. This study will produce a novel live attenuated EV71 strain by using the reverse genetic technology, providing critical information for the vaccine development of neurotropic viruses.

肠道病毒71型（EV71）是婴幼儿重症手足口病的主要病原体，主要侵犯患者的中枢神经系统，可导致脑炎、脑膜脑炎等严重疾病。miRNA是一类重要的转录后调控因子，在不同组织中具有特异的表达谱，这为嗜神经病毒减毒活疫苗的研究提供了契机。最近，我们成功构建了EV71国内分离株的感染性全长cDNA克隆，建立了EV71病毒感染的小鼠模型，在此基础上，本研究将首先通过miRNA芯片、报告质粒等方法筛选脑组织特异miRNA靶序列；进一步通过反向遗传学技术将上述miRNA靶序列以多种方式插入EV71感染性克隆的特异位点，拯救获得携带特异miRNA靶序列的EV71突变病毒，通过一系列病毒学和生化方法鉴定其生物学特征，观察其在神经细胞以及鼠脑中的增殖能力，并在小鼠模型中评价其减毒特征和免疫保护效果。本研究将能够获得基于反向遗传学技术的新型EV71减毒株，对嗜神经病毒疫苗的研究具有重要意义。

手足口病是我国近年来流行于婴幼儿的丙类传染病，每年发病率和病死率均较高，是重要的公共卫生问题。虽然对于EV71灭活疫苗的研究目前已完成三期临床试验，但EV71病毒的不断进化变异以及多种其他肠道病毒的新发与重组影响了常规灭活疫苗的实施效果，因此及时研发新型抗病毒疫苗对于防治手足口病具有现实意义和价值。本研究首先分析了EV71病毒AH08/06株感染SH-SY5Y神经细胞的特征并对其细胞miRNA表达谱进行了差异分析；在此基础上，选择神经特异性miRNA-124和miRNA-125进行组合，将其插入EV71病毒基因组，构建了含有miRNA靶序列的EV71病毒全长克隆，随后纯化制备了恢复病毒和筛选鉴定出正确改造的突变病毒。对miRNA靶向EV71突变病毒在接种RD、Vero、SK-N-SH等细胞系的增殖特征进行了研究，还对其在小鼠体内的乳鼠神经毒力、遗传稳定性、组织嗜性和免疫原性开展了研究，测定了抗体效价水平和细胞免疫水平。制备鉴定了miRNA靶向EV71突变病毒高效价免疫血清，并对EV71减毒株在小鼠模型中的免疫保护效果进行了评价研究。以上实验结果表明miRNA靶向突变病毒免疫小鼠可产生高效价免疫血清并且具有对野生病毒感染的保护效果。本课题研究工作为深入了解miRNA的减毒机制并研发新型肠道病毒疫苗提供了重要资料。