利用介体昆虫细胞培养和RNAi体系研究南方水稻黑条矮缩病毒的复制机理

Southern rice black-streaked dwarf virus（SRBSDV）, transmitted by white-backed planthopper vectors in a persistent-propagative manner，causes the southern rice black-streaked dwarf disease that is one of the important rice virus diseases in southern China. The viroplasm induced by SRBSDV infection was the site for viral replication and assembly of progeny virions. It consists of a granular region and a filamentous region and is a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses. The nonstructural viral proteins P5, P6 and P9-1 are the constituents of the matrix of viropasm.In this proposal, we want to knock down the expression of each viral gene from these three nonstructural proteins by using RNA interference, to test whether viral replication and multiplication also were suppressed in virus-infected cultured insect vector cells and vector bodies. And the baculovirus expression and yeast two-hybrid assay are used to identify the mechanism of the interaction between virus core particle protein and the granular inclusion. The results will lead us to identify key factors of viral replication and to understand the molecular mechanism of the formation of matrix of viroplasm and viral replication, which would provide theory basis for designing efficient control disease strategies in future based on the blockage of viral replication and assembly of progeny virions in the body of insect vector.

由南方水稻黑条矮缩病毒（SRBSDV）引起的南方水稻黑条矮缩病是我国南方稻区重要的虫传病毒病害。SRBSDV在介体白背飞虱内形成提供病毒复制和增殖的病毒原质（viroplasm）结构，它不同于其他呼肠孤病毒viroplasm的单一颗粒状基质形态，由纤维状和颗粒状两种形态的基质组成。病毒编码的非结构蛋白P5、P6 和 P9-1是组成病毒原质的组分。本项目拟研究通过RNA干扰技术在SRBSDV侵染的白背飞虱培养细胞和虫体内抑制P5，P6或P9-1基因表达后病毒增殖和被抑制程度，明确病毒复制关键因子，利用杆状病毒表达体系和蛋白互作手段解析病毒内核组分被招募到viroplasm纤维状基质的机制，深入研究SRBSDV在其介体内组装形成viroplasm基质的分子机制，解析病毒增殖机制，为建立基于阻断SRBSDV在介体内的复制从而干扰介体传毒的新型病害调控策略提供理论依据。

南方水稻黑条矮缩病毒（SRBSDV）是呼肠孤病毒科（Reoviridae）的成员，由白背飞虱以持久性增殖型方式进行传播。它引起的南方水稻黑条矮缩病是我国南方稻区重要的病毒病害。SRBSDV编码的非结构蛋白P5、P6 和 P9-1是形成提供病毒复制和装配的病毒原质(viroplasm)的组分。病毒原质由纤维状和颗粒状两种形态的基质组成，纤维状基质是病毒复制和子代病毒粒体装配的场所，颗粒状基质则与病毒mRNA富集有关。本项目首先通过RNA干扰技术在SRBSDV侵染的白背飞虱培养细胞和虫体内分别抑制P5，P6或P9-1基因表达，根据病毒增殖和被抑制程度，明确了P6是病毒复制的最关键因子，解析了病毒增殖机制；并通过干扰Dicer-2基因抑制介体昆虫siRNA天然免疫途径，使得病毒在虫体中过量增殖，利用电镜技术观察了病毒过量增殖对于介体昆虫细胞造成的病变，探索了打破病毒与介体昆虫的“和平共处”对于病毒传播影响，为建立基于阻断SRBSDV在介体内复制从而干扰介体传毒的新型病害调控策略提供理论依据。此外，研究还对另一种水稻呼肠孤病毒水稻瘤矮病毒在其介体昆虫中突破唾液腺释放屏障的机制以及经卵垂直传播的机制进行了解析，进一步探索了昆虫传播对于病毒病害发生流行的影响，为病害防控提供了新的思路。