高致病性囊泡病毒HvAV-3h的DNA复制、演化及其传播机制研究

Ascovirus HvAV-3h recently discovered is a Chinese variant of Heliothis virescens ascovirus 3a. As a potential biocontrol factor, it has high pathogenicity and faster killing speed to Spodoptera litura, Spodoptera exigua, Helicoverpa armigera and so on, refer to as the important insect pests. This project is designed on basis of the conception using biodiversity to prevent the important insect pests' outbreak and disaster. Firstly, this project targets HvAV-3h by using real-time quantitative PCR, Pulsed Field Gel Electrophoresis (PFGE), Southern Blot and so on to study the DNA replication mechanism of HvAV-3h. Secondly, In order to understand the evolutionary path of HvAV-3h, phylogenetic relationships of ascovirid strains were analysized based on the genome sequences with weight assignment of the genome variable region and sequences of the original of replication according to biological evolutionism. Thirdly, the propagation mechanism of HvAV-3h among its intermediate hosts and (final) hosts is studied to systematically illuminate the role and position of HvAV-3h in the system "HvAV-3h - Spodoptera litura - Microplitis pallidipes". This will provide a theoretical foundation and scientific basis for maintaining the balance of the system in the agricultural ecosystem and building an efficient green prevention and control technology system to the important lepidopteran pests such as Spodoptera litura.

囊泡病毒HvAV-3h是最近发现的烟芽夜蛾囊泡病毒（Heliothis virescens ascovirus 3a）中国毒株，其对斜纹夜蛾、甜菜夜蛾、棉铃虫等重要害虫均具有较高的致病性和较快的致死速度，是一种应用前景较好的生防因子。本项目基于生物多样性抑制重要害虫爆发成灾的理念，以高致病性HvAV-3h为研究对象，利用实时荧光定量PCR、脉冲场凝胶电泳、Southern Blot等技术研究HvAV-3h基因组DNA复制机制；基于进化生物学理论对其基因组变异区和复制起点序列等进行权重赋值，研究囊泡病毒的系统发育关系，阐明 HvAV-3的演化历程；深入研究HvAV-3h在宿主间的传播机制，明确其在"HvAV-3h-斜纹夜蛾-淡足侧沟茧蜂"系统中的作用和地位。为维持该系统在农业生态系中的微平衡、构建针对斜纹夜蛾等重要鳞翅目害虫的高效绿色防控技术体系提供理论基础和科学依据。

本项目针对国家实施“一控两减”战略的需求，研发应用前景较好的生防因子，基于对斜纹夜蛾、甜菜夜蛾、棉铃虫等重要害虫均具有较高致病能力和较快致死速率的烟芽夜蛾囊泡病毒3h株（HvAV-3h），开展系统研究工作。以HvAV-3h感染SeEI、Sf9细胞系，通过对其感染效率和基因组拷贝数的时序动态研究，明确了病毒基因组DNA的“阶段式”复制方式；通过TEM和SEM检测了病毒粒子形态特征、454测序测定了全基因组序列，分析了HvAV-3h的基因组特征及其变异区域，并分别以MCP、DNA Polymerase以及62个核心基因的氨基酸序列开展其与近缘毒株的系统发育关系分析，揭示了烟芽夜蛾囊泡病毒3h株起源于美国的3f株，随后扩散至澳大利亚、印度尼西亚和美国，演化出3g、3e和3i株的演化历程。基于LC-MS/MS技术分析了HvAV-3h的蛋白质组学特征。进一步阐明了HvAV-3h毒株对三种重要农业害虫的致病致死规律，明确了HvAV-3h依赖于斯氏侧沟茧蜂等寄生蜂的产卵行为在斜纹夜蛾、棉铃虫等宿主幼虫个体间进行机械传播的流行致病特点。项目研究成果对昆虫囊泡病毒的地理分布、物种演化、流行致病等研究领域起到了较大的推动作用，为维持农业生态系中的微平衡、构建针对斜纹夜蛾与棉铃虫等重要鳞翅目害虫的高效绿色防控技术体系提供了理论基础和科学依据。