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

描述（由申请人提供）：在本提案中，我们建议将我们的HTS-ready，基于荧光的caspase -3/7实验转移到基于BTV感染模型系统中不同宿主凋亡反应的虫媒病毒感染鉴定新的抗病毒药物。节肢动物传播的病毒（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/或动物流行病是由虫媒病毒引起的，包括亚洲的登革热病毒（DNV）、北美的西尼罗河病毒（WNV）和欧洲的蓝舌病病毒（BTV）。虫媒病毒是独特的，因为它们通过节肢动物媒介传播给脊椎动物宿主，因此，它们必须能够在两个非常不同的宿主类群中复制——脊椎动物和昆虫。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大不相同。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，尽管病毒在两种宿主细胞中都有大量复制，但这种凋亡反应并不明显。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，但昆虫没有显示出任何可检测到的疾病迹象。很少有人注意到这种不同宿主细胞反应的重要性，以及将这种策略用于药物发现以保护脊椎动物宿主的可能性。我们推测，防止btv诱导的脊椎动物细胞凋亡的化合物可能通过抑制细胞凋亡或干扰病毒生命周期来起作用。第二次分析将使我们能够从细胞凋亡抑制剂中分离出抗病毒命中。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（包括DNV和WNV）诱导的宿主凋亡反应。我们的长期目标是为人类虫媒病毒疾病制定新的预防和控制措施。本应用程序的目标将实现以下具体目标：在脊椎动物细胞BTV感染模型中，使用开发的高效HTS方法筛选指定的化合物库。基于btv通过内在凋亡途径诱导脊椎动物细胞凋亡的初步数据，我们设计、开发、优化并验证了基于该模型系统的高效HTS实验。我们建议将此分析转移到指定的筛选中心，以复制，小型化和自动化分析。使用细胞凋亡诱导剂（包括Staurosporine）的二次检测也将实施，以确认命中并排除假阳性（包括细胞凋亡抑制剂）。还将审查这些命中的可能机制，以便在进一步调查DNV和西尼罗河病毒感染时优先考虑这些命中。节肢动物传播的病毒（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，最近有几种虫媒病毒引起人类或动物流行病，包括亚洲的登革热病毒、北美的西尼罗河病毒和欧洲的蓝舌病病毒。在本研究中，我们提出利用基于hts的荧光caspases- 3/-7检测方法，根据虫媒病毒感染中不同的宿主凋亡反应来鉴定新的虫媒病毒感染靶点，以保护宿主免受包括登革热病毒和蓝舌病病毒在内的虫媒病毒感染。我们的长期目标是为人类和动物虫媒病毒疾病制定新的预防和控制措施。

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.