Identification and validation of Zika virus receptor(s) in the midgut lumen of Aedes aegypti

摘要 该项目的中心目标是识别寨卡病毒受体(S)，产生寨卡病毒的传播 缺乏受体编码基因的无能转基因蚊子及其新传播途径的鉴定 阻断疫苗候选(S)。节肢动物传播的病毒(虫媒病毒)导致广泛和 全球范围内的致衰性疾病。在缺乏有效的许可疫苗或治疗方法的情况下 包括ZIKV在内的虫媒病毒，在媒介水平上的控制是最有效的减轻负担的方法 这些病毒。在这些病毒传播给人类之前，它们必须首先感染下面的蚊子 一顿血餐。因此，了解ZIKV与蚊子相互作用的调查是至关重要的。在这 提议，我们将识别埃及伊蚊中肠中的特定分子，作为受体和 ZIKV结合和感染建立的促进剂。已确定的候选受体将在体内得到验证 通过使用CRISPR/Cas9基因编辑进行遗传消融和随后口服感染ZIKV 血餐。这项研究产生的数据将有助于1)制定有效的疾病控制措施， 例如，通过产生缺乏受体编码基因的转基因蚊子，这些蚊子将不能 以及2)识别蚊子中肠中的ZIKV受体分子将提供新的 对支持媒介能力和蚊子能力的基本生物学和分子机制的见解 虫媒病毒病原体相互作用。拟议的实验也具有重要意义，因为它们提供了 为未来的实验奠定基础，以确定相同的受体(S)是否被其他虫媒病毒使用 和其他蚊子物种。

SUMMARY The central goal of this project is to identify Zika virus (ZIKV) receptor(s), generation ZIKV transmission incompetent transgenic mosquitoes that lack the receptor-encoding gene and identification of novel transmission blocking vaccine candidate(s). Transmission of arthropod-borne viruses (arboviruses) causes widespread and debilitating diseases across the globe. In the absence of effective licensed vaccines or therapeutic treatments of arboviruses, including ZIKV, control at the vector level is the most effective method to ameliorate the burden of these viruses. Before these viruses can be transmitted to humans, they must first infect the mosquito following a blood meal. Thus, investigations to understand ZIKV-mosquito interactions are of critical importance. In this proposal, we will identify specific molecules in the Aedes aegypti mosquito midgut that act as receptors and facilitators for ZIKV binding and establishment of infection. Identified candidate receptors will be validated in vivo by genetic ablation using CRISPR/Cas9 gene editing and subsequent oral challenge with an infectious ZIKV blood meal. Data generated in this study will assist in 1) the development of effective disease control measures, e.g. via the generation of transgenic mosquitoes that lack the receptor-encoding gene and would be incompetent for ZIKV transmission; and 2) identification of ZIKV receptor molecules in the mosquito midgut will provide new insights into the basic biology and molecular mechanisms underpinning the vector competency and mosquito- arboviral pathogen interactions. The proposed experiments are also significant because they provide the groundwork for future experiments that will determine whether the same receptor(s) is used by other arboviruses and other mosquito species.