Understanding innate immune responses of insect vectors to intracellular pathogens.

该提案将解决埃及伊蚊（登革病毒（DENV）的昆虫载体）与人类之间的分子相互作用。具体来说，我们将研究蚊子用于消除DENV的免疫过程，以及病毒如何规避这些反应。最近的研究已经解决了调节或决定DENV在蚊子中细胞内发育的分子和途径的作用，但这些研究中的大多数都使用了对DENV易感的实验室蚊子品系。与哥伦比亚的合作者，我们已经确定了现场收集的Ae。埃及人对 ** DENV具有天然抵抗力。这些已在实验室中维持，并通过暴露于DENV进一步选择，从而产生对DENV具有不同易感性的选定品系。我们的初步数据表明，这两种耐药菌株使用细胞凋亡（一种细胞死亡形式）来消除DENV。利用这些数据，我们提出：1：使用高通量测序（RNA-seq）比较暴露于DENV后在抗性（Cali-MIB）和易感（Cali-S）昆虫中表达的基因; 2：使用真实的时间定量PCR确认和定量暴露于登革病毒后所选基因的差异表达。3)使用基因敲低策略（RNAi）来确定/确认特定分子在确定表型中的作用，以及4：评估田间收集的昆虫对DENV的基因表达和凋亡反应，以确定我们基于实验室的假设是否代表田间真正发生的情况，并评估我们选择的实验室菌株对所有4种DENV血清型的免疫反应。这些研究的结果将确定那些决定田间种群表型的基因和蛋白质，并为理解载体和病原体基因组之间的相互作用和分子相互作用铺平道路。

This proposal will address the molecular interactions between Aedes aegypti, the insect vector of Dengue viruses (DENv) to humans. Specifically we will look at the immune immune processes used by mosquitoes to eliminate DENv, and how the viruses circumvent these responses. Recent studies have addressed the role of molecules and pathways that regulate or determine the intracellular development of DENv in mosquitoes, but the majority of these studies have used lab strains of mosquitoes that are susceptible to DENv.****With collaborators in Colombia we have identified field-collected Ae. aegypti that are naturally resistant to**DENv. These have been maintained in the lab and further selected by exposure to DENv, resulting in selected lines with differential susceptibility to DENv. Our initial data indicate that the 2 resistant strains use apoptosis, a form of cell death, to eliminate DENv. With these data in hand we propose to: 1: Compare the genes expressed in the resistant (Cali-MIB) and susceptible (Cali-S) insects after exposure to DENv using high throughput sequencing (RNA-seq); 2: Confirm and quantify, using Real Time quantitative PCR, the differential expression of selected genes after exposure to Dengue virus. 3) Use gene knockdown strategies (RNAi) to determine/confirm the role of specific molecules in determining the phenotype, and 4: Evaluate the gene expression and apoptotic responses of field collected insects to DENv to determine if our lab-based hypotheses represent what truly happens in the field, and evaluate the immune responses of our selected lab strains to all 4 DENv serotypes.****The results of these studies will identify those genes and proteins that determine the phenotype in field**populations and will pave the way to understanding the interplay and molecular interactions between the genomes of vector and pathogen.**