Since they were first identified 30-40 years ago, the filoviruses (Ebola viruses and Marburg viruses) have caused a number of outbreaks of severe hemorrhagic disease in humans that are characterized by a very high mortality rate. Little is understood regarding the transmission of these zoonotic viruses, mostly due to the sporadic nature of the outbreaks and their geographic isolation to regions of equatorial Africa. There are currently no vaccines or drugs approved for the prevention and treatment of filovirus infections and as a result of this lack of therapy, combined with the high mortality and human-to-human transmission, all material containing infectious filovirus is handled under the highest level of biosafety (BSL4). This places a severe restriction on antiviral drug discovery efforts, as technology for high-throughput screening cannot easily be transferred to a BSL4 setting; certainly not on the scale normally used for drug screening programs in industry. The impetus for identifying filovirus antivirals has been further heightened by concerns that these viruses may be used as bioterror agents. Consequently both Ebola and Marburg viruses are classified as NIAID priority A pathogens for biodefense, and development of diagnostics, vaccines and therapeutics for these agents is emphasized as a research priority. Towards this goal we describe a plan to develop a high-throughput screening assay for inhibitors of Ebola virus replication that can be performed at BSL2, and therefore be compatible for use in specialist small molecule screening facilities. The assay is based on the Zaire Ebola virus minigenome assay, which because it is plasmid-based and does not involve infectious virus, can be performed under BSL2 conditions. The assay measures viral genome replication and transcription and to date, no inhibitors of the Ebola virus replication complex (e.g. polymerase inhibitors) have been described. In addition to being potential drug candidates, such molecules will serve as useful probes to further our understanding of how this viral complex functions. In Aim 1 the assay will be optimized and miniaturized to meet the criteria for use in a small molecule high-throughput screen. In Aim 2 we will conduct a pilot screen of biologically active molecules to judge assay performance and establish the criteria for hit selection. Secondary assays performed with the Reston Ebola and Marburg virus minigenome systems will assist in identifying those compounds that have potential pan-filovirus antiviral activity. Successful completion of this project will allow for expanded small molecule screening campaigns with increased possibility of discovering compounds with potent anti-filovirus activity.