Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti
Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti
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DOI：
10.1073/pnas.0600479103
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发表时间：
2006-03-14
影响因子：
11.1
通讯作者：
Olson, KE
中科院分区：
文献类型：
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作者：
Franz, AWE;Sanchez-Vargas, I;Olson, KE
对蚊子（埃及伊蚊）进行遗传修饰以表现出对2型登革热病毒（DENV-2）的载体能力受损。我们利用蚊子中肠中的天然抗病毒RNA干扰（RNAi）途径，构建了一个效应基因，表达来自DENV-2 RNA基因组前膜蛋白编码区的反向重复序列（IR）RNA。助理埃及人羧肽酶A启动子用于在摄入血粉后在中肠上皮细胞中表达IR RNA。通过使用非自主Mariner Mosl转化系统将启动子和效应基因插入到白眼波多黎各Rexville D（Higgs'白色眼）菌株的基因组中。转基因家族Carb 77在血餐后中肠表达IR RNA。Carb 77蚊感染含DENV-2的人工血粉后，中肠和唾液腺中的病毒包膜抗原明显减少。个体蚊子的DENV-2滴定显示，大多数Carb 77蚊子对病毒复制的支持较差。与对照蚊子相比，Carb 77系的病毒体外传播显著减少。来自Carb 77中肠的RNA提取物中DENV-2衍生的siRNA的存在以及当RNAi途径中断时抗性表型的丧失证明DENV-2抗性是由RNAi应答引起的。转基因A.显示出对DENV-2的高水平抗性的埃及伊蚊为开发控制登革热病毒传播的种群替代策略提供了有力的工具。
Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner Mosl transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.