Arbovirus midgut escape mechanisms

描述（由申请人提供）：虫媒病毒在世界范围内造成重大的健康负担，每年有超过1亿人感染登革热、西尼罗河、黄热病和基孔肯雅等病毒。虫媒病毒通过节肢动物媒介传播，如蚊子，在摄入脊椎动物宿主的病毒血症血后被感染。传播到新宿主需要虫媒病毒在载体的中肠细胞中复制，然后扩散到次级组织，最终到达唾液腺。一旦后者被感染，节肢动物就能将病毒传播给新的宿主。媒介生物学领域的一个长期存在的问题是虫媒病毒如何从中肠逃脱，绕过诸如基底层和宿主免疫机制等屏障。在某些情况下，虫媒病毒能够感染和复制中肠上皮，但不能传播到其他器官。这种所谓的中肠逃逸屏障的存在意味着病毒中肠逃逸是一个活跃的过程。然而，虫媒病毒中肠逃逸的机制几乎是完全未知的。这一建议解决了蚊媒病毒登革热、基孔肯雅和辛德比斯病毒用于逃离中肠的信号机制。研究人员先前的工作已经确定了杆状病毒用于逃离其昆虫宿主中肠的新机制。杆状病毒使用一种优雅的机制，发出蛋白酶激活的阶梯信号，其中基质金属蛋白酶被激活，并反过来激活效应半胱天蛋白酶，其直接切割基底层的成分。这导致与中肠相关的气管细胞排列的基底层的重塑，并最终导致有效的全身性感染的建立。这一提议背后的假设是，蚊子传播的虫媒病毒利用同样的途径逃离中肠，初步结果支持这一假设。具体来说，(1)将确定杆状病毒改造中肠屏障的机制是否也被虫媒病毒利用，包括基质金属蛋白酶和半胱天酶的激活；(2)通过RNA干扰、虫媒病毒转导系统和转基因埃及伊蚊，通过沉默或过表达靶基因来评估参与中肠逃逸的候选基因的贡献；(3)检测细胞凋亡及关键凋亡调控基因在虫媒病毒中肠逃逸中的作用。这些研究结果将为了解虫媒病毒与媒介的相互作用提供重要的新信息，并可能导致在该领域控制虫媒病毒传播的新策略。

DESCRIPTION (provided by applicant): Arboviruses cause a significant world-wide health burden, with well over 100 million people becoming infected each year with viruses such as dengue, West Nile, yellow fever, and chikungunya, among others. Arboviruses are transmitted by arthropod vectors such as mosquitoes that become infected after ingestion of a viremic blood meal from a vertebrate host. Transmission to a new host requires that the arbovirus replicate in the midgut cells of the vector and then spread to secondary tissues eventually reaching the salivary glands. Once the latter are infected, the arthropod is able to transmit the virus to a new host. A long standing question in the field of vector biology is how arboviruses escape from the midgut, bypassing barriers such as basal laminae as well as host immune mechanisms. In some cases, arboviruses are able to infect and replicate in midgut epithelium but are not able to disseminate to other organs. The existence of this so-called midgut escape barrier implies that virus midgut escape is an active process. However, the mechanisms involved in midgut escape by arboviruses are almost completely unknown. This proposal addresses the signaling mechanisms used by the mosquito-borne viruses dengue, chikungunya, and Sindbis viruses to escape the midgut. Previous work by the investigators has defined a novel mechanism used by baculovirus to escape the midgut of their insect host. Baculoviruses use an elegant mechanism that signals a stepwise cascade of protease activation, wherein matrix metalloproteases become activated and in turn activate effector caspases, which directly cleave components of the basal lamina. This leads to remodeling of the basal lamina which lines tracheal cells associated with the midgut and culminates in the establishment of efficient systemic infections. The hypothesis underlying this proposal is that mosquito-borne arboviruses utilize this same pathway for midgut escape, and preliminary results support this hypothesis. Specifically, (1) it will be determined whether the mechanisms used by baculoviruses to remodel the midgut barrier are also utilized by arboviruses, including activation of matrix metalloproteases and caspases; (2) the contribution of candidate genes involved in midgut escape will be evaluated by RNA interference, arbovirus transducing systems, and transgenic Aedes aegypti mosquitoes by silencing or overexpressing target genes; and (3) the contribution of apoptosis and key apoptotic regulatory genes to arbovirus midgut escape will be tested. The results of these studies will contribute important new information to the understanding of arbovirus-vector interactions and potentially lead to new strategies for control of arbovirus transmission in the field.