INTERFERENCE TO DENGUE VIRUS REPLICATION IN MOSQUITOES

干扰蚊子中的登革热病毒复制

• 批准号: 2069088
• 负责人: CAROL D BLAIR
• 金额: $ 13.3万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2069088
• 关键词: Aedes; Sindbis virus; antisense nucleic acid; communicable disease control; dengue; dengue virus; disease vectors; gene expression; gene mutation; genetic manipulation; genetic transcription; genetically modified animals; immunofluorescence technique; immunoprecipitation; molecular cloning; nucleic acid sequence; polymerase chain reaction; site directed mutagenesis; structural genes; transfection /expression vector; virus RNA; virus protein; virus replication

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.

这项研究的长期目标是定义分子方法 促进细胞内干扰登革热 (DEN) 病毒感染 Ae 内。埃及蚊子。 基于的新型表达系统 蚊媒甲病毒的传染性克隆 [Sindbis (SIN)； 披膜病毒科]将用于表达特定的 DEN-2 病毒基因或 可能对病毒产生干扰的RNA序列 在蚊子细胞和蚊子中复制。 这些方法将 使用已经成功的病毒干扰策略 产生抗病转基因植物。 这种方法有 这在蚊子中是前所未有的，并且在这项工作中得到了利用 代表了破坏病毒载体宿主的前所未有的机会 循环。 被证明有希望的策略最终可以 用于产生抗病毒转基因蚊子。 我们建议使用的 SIN 病毒表达载体采用二进制系统 产生仅对单一个体具有感染性的重组病毒 复制周期。 这是通过共转染易感基因来完成的 含有从两种不同的细胞（体外）转录的 RNA 的细胞 质粒。 一个质粒的 RNA 由非结构基因组成 以及SIN病毒26S 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并且 取代病毒结构基因。 来自第二个质粒的RNA 由 SIN 感染性克隆组成，其中 NSP1 衣壳结合位点 已被删除，但编码完整的结构基因区域 病毒。 只能包装来自第一个质粒的有缺陷的 RNA 形成具有传染性的病毒体。 当考虑到时，这有明显的好处 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形式插入该载体中 方向。 初步研究将针对感染了 重组病毒并用DEN-2病毒进行攻击。 如果 事实证明策略能够成功地引起干扰，然后 类似的实验将在蚊子身上进行。 我们还计划 使用 SIN 双启动子表达质粒进行分析 对蚊子的干扰。 这些病毒表达载体产生 完全感染性的重组病毒体，这将允许传播 将病毒传播到整个蚊子体内。 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，还将提供相关信息 目前转基因节肢动物生产的研究 载体能力降低。