鸡传染性支气管炎病毒胚化致弱株毒力减弱的分子机制研究

Infectious bronchitis virus (IBV) is the causal agent of infectious bronchitis (IB), which still remains one of the most important poultry diseases worldwide because of numerous serotypes and variants. A virulent wild-type strain of IBV can be attenuated by serial passages in embryonated eggs, but the mechanism of their attenuation is not well understood. In the previous study, a nephropathogenic strain of infectious bronchitis virus (IBYZ) was propagated in embryonated eggs. Following 120 serial passages in embryonated eggs, an attenuated strain of IBV (YZ120) was established that does not induce histopathological lesions in the tracheae and kidney of infected chicks. To identify sequence changes responsible for the attenuation of IBV, complete genome sequences of virulent IBYZ and attenuated viruses YZ120 were obtained. For further precise clarify the molecular mechanisms of virulence attenuation of embryo-adapted YZ120, this project is intended to strain IBYZ and YZ120 for materials, with the mature reverse genetic system, through in vitro splicing and virus rescue, the pathogenicity of the recombinant viruses as the main research content. This study will not only contribute to the understanding of virulence variationg mechanisms of IBV, but also open up a new way for the development of safer attenuated vaccines by precisely modifying the IBV genome using reverse genetics.

鸡传染性支气管炎（IB)是由传染性支气管炎病毒(IBV)引起的一种严重危害世界养禽业的病毒性传染病。变异株的不断出现和流行，给该病防控带来了巨大挑战。IBV在鸡胚上连续传代可以使毒力减弱，但致弱的分子机制尚未阐明。在前期研究中，项目组将肾型强毒株IBYZ经鸡胚传代120次后培育为致弱株YZ120，在明确IBYZ与YZ120之间全基因组序列差异的基础上，项目组拟借助自主建立的IBV反向遗传操作系统，首先构建强弱毒株之间主要功能区域交叉置换的嵌合病毒，通过致病性实验确定决定病毒毒力的主要区域；然后对毒力决定区域内的核苷酸差异位点逐个进行突变，拯救获得一系列定点突变病毒，通过致病性实验精确定位YZ120胚化致弱的关键位点；进一步通过生物信息学分析及功能验证探明决定IBV毒力的关键结构域。本研究有望从分子水平揭示IBV毒力变异的分子机制，为分子流行病学调查及新型IB减毒活疫苗的设计奠定理论依据。

鸡传染性支气管炎病毒属于冠状病毒科冠状病毒属的成员，目前国内外对其分子致病机制仍然缺少深入的认识。IBV不同分离株感染易感宿主如鸡后，其致病性差异较大，目前仍缺少统一的标准用于评定该病毒的毒力，给该病的防控造成了很大的困难。本项目开展了鸡胚传代致弱株YZ120基因组全长cDNA的构建及病毒拯救，拯救获得重组病毒rYZ120株、rIBYZ-Rep-YZ120-Struct和rYZ120-Rep-IBYZ-Struct，致病性结果表明YZ120的致弱与病毒的复制酶区域与结构蛋白区域均存在相关性；借助IBYZ株的反向遗传操作系统，构建辅助蛋白基因3a、3b或3a3b基因表达缺失的重组病毒，并对其致病性进行检测，结果表明辅助蛋白基因3a、3b和3a3b的表达缺失对病毒在鸡胚中的增殖没有产生显著影响，但3b或3a3b的表达缺失会显著减弱该毒株的致病性；借助H120株的反向遗传操作系统，采用IBYZ株的S1基因片段替换H120株的相应区域，拯救获得重组病毒rH120-S1/YZ并对其生物学特性与致病性进行了检测，结果表明rH120-S1/YZ株具有良好的鸡胚增殖能力，对1日龄SPF雏鸡无明显的致病性；将YZ120在Vero细胞上连续传代，培育获得QX型鸡传染性支气管炎病毒Vero细胞适应株MJ，通过交叉置换主要的功能区域，部分功能区域及S基因，拯救获得不同嵌合病毒株并对其致病性进行检测，结果表明除了MJ株的S基因、其它区域对病毒的致病性都存在影响。以上结果表明，IBV的S基因、辅助蛋白基因乃至其它非编码区的改变，均能造成该病毒毒力的改变，影响其对天然宿主的致病性，不同方式致弱的毒株其机制可能存在差异。