S蛋白介导的鸡传染性支气管炎病毒适应CK细胞的分子基础研究

The continuous change of tissue tropism caused by the high variability of IBV is one of the main problems in the prevention and control of the virus. Due to the strict cell tropism of IBV, the mechanism study of the virus at the cellular level has been greatly restricted. Although some foreign researchers have proved that S protein determines the cell tropism of IBV, the key sites that determine its tropism is not clear. In this project, the S or S1, S2 genes of different cell tropism strains were replaced by reverse genetic operating system. We found that the CK cell tropism of IBV may be determined by the S2 gene. To further prove this hypothesis, and determine its key sites, our team continues to construct the recombinant virus of different strain skeleton to verify the general area of cell tropism. In combination with the non synonymous mutations of CK cell adaptive strain, which were screened by continuous passage, the key sites will be analyzed and verified. Through the repeated verify by site directed mutation, the key sites will be finally identified. This research will provide convenience for studying the pathogenesis of IBV at the cellular level in the future, and lay a theoretical foundation for the further study of the molecular mechanism of IBV cell tropism in mammalian passage cell lines and the molecular mechanism of inter species transmission of coronavirus.

鸡传染性支气管炎病毒（IBV）的高度变异性所引起组织嗜性的不断变化是目前该病防控中的主要难题之一。由于该病毒严格的细胞嗜性，IBV在细胞水平的机制研究受到了很大局限。虽然国外已有学者证明S蛋白决定了IBV的组织细胞嗜性，但其确切的分子机理尚未阐明。本项目组前期通过反向遗传操作系统嵌合置换不同细胞嗜性毒株的S或S1、S2基因，发现IBV CK细胞嗜性可能由S2基因所决定。为进一步证明该推测，并确定其关键位点，项目组拟构建不同来源毒株骨架的嵌合重组病毒，验证决定细胞嗜性的大致区域；同时结合前期连续传代筛选的CK细胞适应株的非同义突变，分析推测与细胞嗜性相关的位点；并通过定点突变反复加以验证，从而精确定位出关键位点，最终阐明IBV适应原代CK细胞的关键分子基础，为今后IBV在细胞水平上的致病机理研究奠定基础，并为IBV在哺乳动物传代细胞系中细胞嗜性及冠状病毒跨种间传播的分子机制研究提供理论依据。

鸡传染性支气管炎病毒(Infectious Bronchitis Virus, IBV)具有严格的组织细胞嗜性，除Beaudette株外，其它毒株均无法在哺乳动物细胞中有效复制。即便是鸡源肾细胞，绝大多数初次分离的IBV流行毒株也无法进行有效感染，这给IBV细胞疫苗的研制以及细胞水平致病机理的研究造成了很大的困扰。本项目拟借助实验室反向遗传操作平台，以不同CK细胞嗜性毒株为骨架，通过不断改造并拯救嵌合表达S蛋白或其部分区域的重组病毒，比较不同重组病毒之间感染原代CK细胞能力的差异，从而确定了S2亚基是影响IBV原代CK细胞嗜性变化的主要区域。接着，项目组对上述非CK细胞适应株IBYZ进行鸡胚内连续传代，从而驯化获得一株原代CK细胞适应株。通过对其S基因突变位点的分析，构建相关位点的突变株，探明并验证了第617位突变位点对S蛋白介导IBV原代CK细胞嗜性的关键作用，而L581F与V617I同时突变可以有效提高YZ120感染原代CK细胞的效率。关键位点的发现，为IBV适应原代CK细胞改造工作提供直接的操作靶点，为今后IBV在细胞水平上的致病机理研究奠定基础，并为IBV在哺乳动物传代细胞系中细胞嗜性的分子机制以及冠状病毒跨种间传播的分子机制的进一步深入研究奠定理论依据。