Replicon-based screening for inhibitors of alphaviruses

描述（由申请人提供）：本研究的目标是开发一种基于细胞的检测方法，以筛选针对托加病毒家族某些成员的抗病毒活性化合物。我们特别针对的是来自甲病毒属的致病性较高的脑炎病毒，如委内瑞拉马脑炎病毒（VEEV）、东部马脑炎病毒（EEEV）和西部马脑炎病毒（WEEV），它们已被列为B类生物恐怖主义制剂。Sindbis病毒和VEEV病毒是典型的甲病毒，已被广泛用作外源基因表达载体。这些药物为我们提供了有用的系统来促进基于甲病毒复制子的抗病毒筛选程序的发展。该筛选计划将重点使用Sindbis和VEE复制子系统进行小分子的高通量筛选。使用旧大陆病毒（Sindbis）和新世界病毒（VEEV）进行筛选为我们提供了寻找广谱抗甲病毒化合物的机会，以便更好地处理种、准种和未知的甲病毒病原体。我们提出以下具体目标：1)从VEE复制子产生瞬时或组成性表达报告基因的细胞；2)验证复制子细胞进行筛选；3)利用现有的Sindbis复制子和新获得的VEE复制子筛选合成和天然化合物文库；4)确认最有效化合物的抗病毒活性。这些努力将有助于找到控制这些重要病原体的治疗方式。

DESCRIPTION (provided by applicant): The goal in this study is to develop a cell-based assay to screen compounds for antiviral activity against certain members of the Togavirus family. We are particularly targeting the more pathogenic encephalitis-causing viruses from the alphavirus genus, such as Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) which have been listed as category B bioterrorism agents. Sindbis virus and VEEV are prototypic alphaviruses that have been extensively used as vectors for expression of foreign genes. These agents provide us with useful systems to promote the development of an antiviral screening program based on alphavirus replicons. This screening program will focus on using Sindbis and VEE replicon systems for high-throughput screening of small molecules. Using an Old World virus (Sindbis) and a New World virus (VEEV) in screening provides us with an opportunity to find broad-spectrum anti-alphavirus compounds in order to better deal with species, quasispecies and unknown alphavirus pathogens. We propose the following specific aims: 1) generate cells that transiently or constitutively express a reporter gene from a VEE replicon; 2) validate the replicon cells for screening; 3) screen synthetic and natural compound libraries using both our existing Sindbis replicons and newly made VEE replicons; 4) confirm the anti-viral activity of the most potent compounds. These efforts will contribute to the goal of finding therapeutic modalities to control these important pathogens.