Transgenic Resistance of Aedes aegypti to the Four Serotypes of Dengue Virus

项目总监/首席调查员(最后、第一、中间)：Franz，Alexander，W.E. 项目总结 登革热病毒的四个血清型(DENV1-4；黄病毒；黄病毒科)是最重要的蚊子- 感染人类的虫媒病毒。在世界热带地区，登革热病毒高度流行， 约有25亿人面临疫情传播的风险。登革热病毒的主要媒介是伊蚊。 埃及伊蚊。目前，DENV的控制工作主要依赖于杀虫剂的应用，因为疫苗或抗病毒药物 药物不能广泛使用。此外，Ae对杀虫剂的抗性也在增加。埃及伊蚊 需要制定替代的遗传媒介/病毒控制战略。一本这样的小说 策略是种群置换，将易感染DENV的野生型蚊子替换为 转基因、抗病毒的蚊子。这项拨款申请的目的是为了产生转基因 天哪。埃及株，对所有四种DENV血清型都不耐药。 蚊子通过摄取病毒血粉从人类宿主那里感染登革热病毒。中肠上皮是 第一个感染DENV的蚊子组织。在中肠，DENV需要建立感染 病灶才能扩散到次级组织，包括唾液腺，这必须是 在病毒可以传播给新的人类宿主之前被感染。 核糖核酸干扰(RNAi)途径。埃及伊蚊是主要的抗病毒免疫靶向途径 虫媒病毒。我们已经证明，在中肠组织中触发针对DENV2的RNAi完全消除 在病毒可以建立感染源之前进行感染。我们最近培育出了转基因Ae。埃及伊蚊 来源于DENV2基因组的反向重复序列dsRNA在中肠组织中的表达 当感染开始时，蚊子就会被叮咬。一株DENV2抗性转基因株系(Carb109)已被保存 经过30多代的研究，已将Carb109转基因导入野生型Ae。埃及伊蚊 将这些蚊子从高度敏感的DENV2表型转变为完全耐药的 表型。 在这里，我们建议在Ae中设计RNAi介导的抗性。埃及伊蚊对所有四种DENV血清型使用 红外效应器构造的新颖设计。我们将设计190个碱基的IR结构，每个结构都针对一个高度 病毒RNA编码DENV1-4 NS5的保守区。抗病毒效应基因将是至关重要的 基于种群替换的田间DENV控制策略的组成部分。拟议的研究 这一努力将进一步加强人口替换的概念，并补充其他机构正在进行的研究 研究小组将抗病毒效应器结构转移到野生型蚊子种群中。 0925-0001/0002(08/12版)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): Franz, Alexander, W.E. Project summary The four serotypes of dengue viruses (DENV1-4; Flavivirus; Flaviviridae) are the most important mosquito- borne arboviruses infecting humans. In tropical regions of the world, DENV are hyper-endemic with approximately 2.5 billion people at risk for epidemic transmission. The principal vector of DENV is Aedes aegypti. Currently, DENV control efforts rely primarily on insecticide applications, since vaccines or antiviral drugs are not available for widespread use. Additionally, insecticide resistance is increasing among Ae. aegypti populations, requiring the development of alternative, genetic vector/virus control strategies. One such novel strategy is population replacement in which wild-type mosquitoes susceptible for DENV are replaced by genetically-modified, virus-resistant mosquitoes. The purpose of this grant application is to generate transgenic Ae. aegypti lines, which are refractory to all four serotypes of DENV. The mosquito acquires DENV from a human host by intake of a viremic bloodmeal. The midgut epithelium is the first mosquito tissue that becomes infected with DENV. In the midgut, DENV needs to establish infection foci before being able to disseminate to secondary tissues including the salivary glands, which have to be infected before the virus can be transmitted to a new human host. The RNA interference (RNAi) pathway in Ae. aegypti is the major antiviral immune pathway targeting arboviruses. We have shown that triggering RNAi against DENV2 in midgut tissue completely eliminates infection before the virus can establish infection foci. We recently generated transgenic Ae. aegypti that express an inverted-repeat (IR) dsRNA derived from the genome of DENV2 in midgut tissue of bloodfed mosquitoes as infection begins. One DENV2-refractory transgenic line (Carb109) has been maintained for more than 30 generations and the Carb109 transgene has been introgressed into wild-type Ae. aegypti converting these mosquitoes from a highly susceptible DENV2 phenotype to a completely refractory phenotype. Here, we propose to engineer RNAi-mediated resistance in Ae. aegypti to all four DENV serotypes using a novel design for the IR effector constructs. We will design 190 bp IR constructs each targeting a highly conserved region of viral RNA encoding DENV1-4 NS5. The antiviral effector genes will be critically important components for a population replacement-based control strategy of DENV in the field. The proposed research effort will further strengthen the concept of population replacement and complement ongoing studies by other research groups to move anti-viral effector constructs into wild-type mosquito populations. 0925-0001/0002 (Rev. 08/12) Page Continuation Format Page