利用反向遗传学技术研究乙脑疫苗株SA14-14-2的减毒机制

Japanese encephalitis (JE) is one of the most important mosquito-borne infectious diseases in Asia-Pacific region. Currently, JE has emerged and re-emerged in many areas and multiple genotypes of Japanese encephalitis virus (JEV) co-circulate globally, and this situation poses great challenge for the prevention and control of JE. The live JE vaccine strain SA14-14-2 was obtained by continuous passages from the wild type strain SA14, and exhibits excellent safety profile and efficacy. Previously, we have successfully developed the full-length infectious cDNA clone of SA14-14-2 and compared all the differences of genome sequences between SA14 and SA14-14-2. In this study, we will firstly construct a chimeric JEV containing the structural proteins of SA14 using the infectious clone of SA14-14-2 as backbone. Comparing the virulence between the chimeric JEV and SA14-14-2 in mice will identify the major regions that determine the virulence. Further, site-specific mutagenesis will be manipulated in the above mentioned regions by using standard reverse genetics technology. All the rescued mutant viruses will be carefully characterized and compared with the parental virus by viological assays and animal experiments to confirm the potential virulence determinants. Finally, each mutant virus carrying the virulence determinants will be assayed by receptor binding, fusion inhibition,virus-like particle package system, and other biochemical assays to clarify the molecular mechanism of attenuation. Our project will identify all the virulence determinants in the live vaccine strain, which is of highly importance for the design and quality control of JEV vaccine.

流行性乙型脑炎是一种重要的蚊媒传染病，在亚太地区广泛流行。近年来，乙脑流行区域不断扩大，不同基因型病毒交替流行，给疾病防控带来了巨大挑战。乙脑减毒活疫苗SA14-14-2株由野毒株SA14连续传代而来，安全性和有效性俱佳。最近，我们成功构建了SA14-14-2的感染性全长cDNA克隆，在此基础上，本研究拟结合疫苗株与野毒株病毒的基因组序列差异，首先通过同源片段置换的方法构建携带野毒株结构蛋白的乙脑嵌合病毒，通过比较恢复病毒对小鼠的致病性，确定决定乙脑毒力的主要区域；进一步对毒力决定区域内的每个核苷酸差异位点进行定点突变，拯救获得一系列点突变病毒，通过小鼠感染实验确认相应关键毒力位点，并通过受体结合、膜融合抑制、复制子以及病毒样颗粒包装系统等技术手段研究突变病毒与亲本病毒的差异，阐明上述位点减毒的分子机制。本研究将能够阐明乙脑病毒的减毒机制，对于新型减毒活疫苗的设计和质量控制具有重要意义。

我国广泛使用的乙脑减毒活疫苗SA14-14-2株由野毒株SA14连续传代而来，安全性和有效性俱佳，但是其减毒的机制目前未完全明确。本研究利用SA-14-14-2的全长感染性克隆，通过反向遗传学操作以及其他多种实验方法来研究疫苗株的减毒机制。首先通过同源片段置换的方法构建携带野毒株结构蛋白的乙脑嵌合病毒，通过比较恢复病毒对小鼠的致病性，确定决定乙脑毒力的主要区域为包膜糖蛋白E。进一步通过基因比较分析，对毒力决定区内的7个氨基酸差异位点进行突变，拯救并获得一系列的单点以及多点突变病毒。通过小鼠的感染实验测定点突变病毒的神经毒力以及神经侵袭力，结果发现L107，E138和A315位点为影响病毒的毒力关键位点，其中E138影响最大。进一步通过病毒一步生长曲线，病毒吸附，膜融合抑制等技术手段阐明上述位点减毒的分子机制。结果发现，L107和A315位点可以影响病毒介导的细胞膜融合过程，而E138主要与病毒对细胞的吸附相关，将其突变后会改变对细胞的吸附能力。同时，我们发现L107及E138位点对疫苗株SA14-14-2对干扰素的敏感性起着重要作用。另外，Q264位点为影响病毒稳定性的关键氨基酸位点。本研究阐明了乙脑病毒的减毒机制，可以为新型减毒活疫苗的设计和质量控制提供了参考。