A HTS Assay for Identifying Anti-Viral Drugs Against Arbovirus Infections

描述(由申请人提供)：在这项建议中，我们建议转移我们的HTS就绪，基于荧光的caspase-3/7检测方法，以根据BTV感染模型系统中不同的宿主凋亡反应来鉴定新的抗病毒药物。节肢动物传播病毒(虫媒病毒)是重要的人类和/或动物病原体，可引起严重疾病和/或死亡的急性病毒感染。最近的几次人类和/或动物疫情是由虫媒病毒引起的，包括亚洲的登革病毒(DNV)、北美的西尼罗河病毒(WNV)和欧洲的蓝舌病毒(BTV)。虫媒病毒是独一无二的，因为它们是通过节肢动物载体传播给脊椎动物宿主的，因此，它们必须能够在两个非常不同的宿主类群--脊椎动物和昆虫--中复制。虽然虫媒病毒在脊椎动物和昆虫细胞中都能有效复制，但各自的宿主细胞反应却截然不同。例如，BTV感染在脊椎动物细胞中诱导了快速的凋亡反应，而在昆虫细胞中这种凋亡反应并不明显，尽管病毒在两种宿主细胞中都有有效的复制。虽然在受感染的动物/人类中，细胞凋亡与虫媒病毒疾病呈正相关，但昆虫没有表现出任何疾病的迹象。很少有人注意到这种不同的宿主细胞反应的意义，以及将这种药物发现策略应用于保护脊椎动物宿主的可能性。我们假设，阻止BTV诱导脊椎动物细胞凋亡的化合物可能通过抑制细胞凋亡或通过干扰病毒生命周期发挥作用。二次化验将使我们能够将抗病毒药物从细胞凋亡抑制剂中分离出来。这些抗病毒化合物可能还可以保护宿主细胞免受其他虫媒病毒诱导的宿主细胞凋亡反应，包括DNV和WNV。我们的长期目标是开发新的预防和控制人类虫媒病毒疾病的措施。这项应用的目标将通过实现以下特定目的来实现：在脊椎动物细胞中使用所开发的高效的HTS方法来筛选指定的化合物文库。基于我们的初步数据显示BTV通过内在的凋亡途径诱导脊椎动物细胞的凋亡，我们设计、开发、优化和验证了一种高效的HTS检测方法。我们建议将这一检测转移到指定的筛选中心，以复制、小型化和自动化检测。还将使用包括斯塔罗孢菌素在内的凋亡诱导剂进行二次检测，以确认HITS并排除包括凋亡抑制物在内的假阳性。还将研究这些命中的可能机制，以确定这些命中的优先顺序，以便进一步研究DNV和WNV感染。节肢动物传播病毒(虫媒病毒)是一种重要的人类和/或动物病原体，可引起严重疾病和/或死亡的急性病毒感染，几种虫媒病毒可引起最近的人或动物疫情，包括亚洲的登革病毒、北美的西尼罗河病毒和欧洲的蓝舌病毒。在这项建议中，我们建议转移我们的HTS就绪的，基于荧光的caspase-3/-7检测来识别新的抗虫媒病毒感染的靶点，基于虫媒病毒感染中不同的宿主凋亡反应，以保护宿主免受虫媒病毒的感染，包括登革病毒和蓝舌病毒。我们的长期目标是开发新的预防和控制人类和动物虫媒病毒疾病的措施。

DESCRIPTION (provided by applicant): In this proposal, we propose to transfer our HTS-ready, florescence based caspases-3/7 assay to identify novel anti-viral drugs against arboviruses infection based on the different host apoptotic responses in BTV infection model system. Arthropod borne viruses (arboviruses) are important human and/or animal pathogens that cause acute virus infections with severe diseases and/or death. Several recent human and/or animal epidemics are caused by arboviruses, including Dengue virus (DNV) in Asia, West Nile virus (WNV) in North America and Bluetongue virus (BTV) in Europe. Arboviruses are unique because they are transmitted to their vertebrate hosts by arthropod vectors, therefore, they must be capable of replicating in two very divergent host taxa--vertebrates and insects. Although arboviruses replicate efficiently in both vertebrate and insect cells, the respective host cellular responses are quite different. For example, BTV infection induces a rapid apoptotic response in vertebrate cells, whereas such apoptotic response is unapparent in insect cells, despite the productive virus replication in both host cells. While apoptosis has been positively linked to arbovirus diseases in infected animals/human, insects show no detectable signs of any diseases. Little attention has been paid to the significance of such different host cellular responses and the possibility of adapting such strategy for drug discovery to the protection of vertebrate hosts. We hypothesize that compounds preventing BTV-induced apoptosis in vertebrate cells could act either via inhibiting apoptosis or via interfering viral life-cycle. A secondary assay will allow us to separate anti-viral hits from the apoptosis inhibitors. These anti-viral compounds could presumably also protect host cells from host apoptotic response induced by other arboviruses, including DNV and WNV. Our long term goal is to develop new prevention and control measures for arbovirus diseases in human. The objectives of this application will be achieved by carrying out the following Specific Aim: To screening designated compound library using the developed efficient HTS assay in the BTV infection model in vertebrate cells. Based on our preliminary data showing that BTV-induced apoptosis in vertebrate cells via intrinsic apoptotic pathway, an efficient HTS assay have been designed, developed, optimized and validated using this model system. We propose to transfer this assay to the designated screening center to reproduce, miniaturize and automate the assay. A secondary assay using apoptosis inducers including Staurosporine will also be implemented to confirm hits and exclude false positives including apoptosis inhibitors. The possible mechanism of these hits will also be examined to prioritize these hits for further investigations against DNV and WNV infection. Arthropod borne viruses (arboviruses) are important human and/or animal pathogens that cause acute virus infections with severe diseases and/or death, several arboviruses cause recent human or animal epidemics, including Dengue virus in Asia, West Nile virus in North America and Bluetongue virus in Europe. In this proposal, we proposed to transfer our HTS-ready, florescence based caspases- 3/-7 assay to identify novel targets against arboviruses infection based on the different host apoptotic responses in arbovirus infection to protect hosts from arboviruses infections, including Dengue virus and Bluetongue virus. Our long term goal is to develop new prevention and control measures for arbovirus diseases in human and animals.