INTERFERENCE TO DENGUE VIRUS REPLICATION IN MOSQUITOES

这项研究的长期目标是确定分子方法， 促进对登革（DEN）病毒感染的细胞内干扰 在Ae。埃及蚊子。 新的表达系统基于 蚊媒甲病毒的感染性克隆[Sindbis（SIN）; 披膜病毒科]将用于表达特异性DEN-2病毒基因或 可潜在诱导干扰病毒的RNA序列 在蚊子细胞和蚊子中复制。 这些办法将 使用病毒干扰策略， 产生抗病转基因植物。 了这种方针 这在蚊子身上是不可能的， 这是一个前所未有的机会来破坏病毒载体宿主 周期 被证明是有前途的战略最终可以 用于产生抗病毒的转基因蚊子。 我们建议使用的SIN病毒表达载体采用二元系统 以产生仅感染单个细胞的重组病毒， 复制周期 这是通过收集敏感的 细胞与RNA已经转录（体外）从两个不同的 质粒。 来自一个质粒的RNA由非结构基因组成 和SIN病毒26 S mRNA的内部起始位点。 一个 外源基因插入内部起始位点的下游， 取代了病毒的结构基因。 来自第二个质粒的RNA 由SIN感染性克隆组成，其中NSP 1衣壳结合位点 已被删除，但它编码的完整结构基因区， 病毒 只有来自第一个质粒的缺陷RNA可以被包装 形成传染性病毒体 这有明显的好处时，考虑到 与重组病毒相关的安全问题。 我们建议 将DEN病毒序列以有义和反义两种方式插入该载体中 方向。 最初的研究将是用感染了 用DEN-2病毒攻击重组病毒。 如果 策略被证明是成功的干扰，然后 将在蚊子中进行等同的实验。 我们还计划 使用SIN双启动子表达质粒分析 干扰蚊子。 这些病毒表达载体产生 完全感染性的重组病毒体， 病毒在蚊子体内传播。 这些研究将提供 关于干扰机制的大量信息， 基因表达水平，此外，将提供相关信息 转基因节肢动物生产的研究， 减少载体能力。

The long term goal of this research is to define molecular approaches for promoting intracellular interference to dengue (DEN) virus infections within Ae. aegypti mosquitoes. Novel expression systems based on infectious clones of mosquito-borne alphaviruses [Sindbis (SIN); Togaviridae] will be used to express either specific DEN-2 viral genes or RNA sequences which can potentially induce interference to virus replication in both mosquito cells and mosquitoes. These approaches will use virus interference strategies which have been successful in generating disease resistant transgenic plants. Such an approach has never before been possible in mosquitoes and its utilization in this work represents an unprecedented opportunity to disrupt the virus-vector-host cycle. Strategies which prove to be promising can eventually be used to generate virus resistant transgenic mosquitoes. The SIN virus expression vectors we propose to use employ a binary system to generate recombinant virus which is infectious for only a single replicative cycle. This is accomplished by cotransfecting susceptible cells with RNAs that have been transcribed (in vitro) from two different plasmids. The RNA from one plasmid consists of the nonstructural genes and internal initiation site for the 26S mRNA of SIN virus. An exogenous gene is inserted downstream of the internal initiation site and replaces the viral structural genes. RNA from the second plasmid consists of a SIN infectious clone in which the NSP1 capsid binding site has been deleted but which encodes the complete structural gene region of the virus. Only the defective RNA from the first plasmid can be packaged to form an infectious virion. This has obvious benefits when considering safety issues related to work with recombinant viruses. We propose to insert DEN virus sequences into this vector in both sense and antisense orientations. Initial studies will be with mosquito cells infected with the recombinant viruses and challenged with DEN-2 virus. If the strategies prove to be successful in causing interference, then equivalent experiments will be performed in mosquitoes. We also plan to use the SIN double-promoter expression plasmids for analysis of interference in mosquitoes. These viral expression vectors generate fully infectious recombinant virions which will allow dissemination of the virus throughout the mosquito. These studies will provide considerable information about the mechanisms of interference at the level of gene expression and in addition, will give information pertinent to current research on the production of transgenic arthropods with reduced vector competence.