Indicator cells for antiviral screening for filoviruses

描述(申请人提供)：该提案的目标是开发一种基于细胞的分析的商业潜力，以筛选具有抗埃博拉和马尔堡病毒等丝状病毒活性的化合物。这种以呼吸道合胞病毒为原型的生物检测的基础是在转化细胞的细胞质中表达存在于人工病毒基因组中的报告基因，该基因与感染无关。丝状病毒包括一些最臭名昭著的人类病原体，并因严重出血热综合征而导致显著的发病率和死亡率。不幸的是，很少有治疗剂可用于治疗这些病毒的感染。寻找新的抗病毒药物的努力将需要适用于高通量检测的方法。由于所有病毒都是专性的细胞内微生物，任何确定各种因素对病毒复制影响的生物测定都必须以细胞培养为基础。这涉及到与传染性病毒的合作，这对于EBOV和MBGV这样的病原体显然是不可行的。许多致病的负链RNA病毒，包括这些丝状病毒，在识别复制过程中涉及的顺式作用元件和反式作用因子方面取得了重大进展。我们计划开发不受感染的埃博拉和马尔堡病毒微型基因组表达系统，在该系统中，一种易于测量的酶的表达依赖于病毒复制所必需的RNA复制和转录因子。我们计划将这些系统用于基于细胞的分析，以筛选抗病毒化合物。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop the commercial potential of a cell-based assay to screen for compounds with antiviral activity against filoviruses such as Ebola and Marburg viruses. The basis of this bioassay, which has been prototyped with respiratory syncytial virus, is infection-independent expression of a reporter gene from an artificial viral genome present within the cytoplasm of transformed cells. Filoviruses include some of the most notorious human pathogens and cause significant morbidity and mortality due to severe hemorrhagic fever syndromes. Unfortunately, very few therapeutic agents are available to treat infections with these viruses. Efforts to find new antiviral agents will require methods that are amenable to highthroughput assays. Since all viruses are obligate intracellular microbes, any bioassay that determines the effect of various agents on viral replication, must be based on cell culture. This involves working with infectious virus, which is obviously not feasible for agents such as EBOV and MBGV. Significant advances have been made in identifying the cis-acting elements and trans-acting factors involved in replication of many pathogenic negative-strand RNA viruses, including these filoviruses. We plan to develop infection-independent Ebola and Marburg virus minigenome-expression systems in which the expression of an easily measurable enzyme is dependent on the RNA replication and transcription factors that are essential for viral replication. We plan to use these systems in cell-based assays for screening antiviral compounds.