Lassa and Ebola virus inhibitors identified using minigenome and recombinant virus reporter systems

• Creation of BSL-2 and BSL-4 platforms to screen antivirals for LASV and EBOV.
• Minigenome platforms expressing Gaussia luciferase were optimized for high-throughput screening.
• Recombinant viruses expressing fluorescent proteins were used to confirm antiviral hits.
• Includes first report of recombinant LASV expressing a reporter gene.
• Antiviral efficacy of several compounds, including 6azaU and 2′-dFC, was demonstrated.

Lassa virus (LASV) and Ebola virus (EBOV) infections are important global health issues resulting in significant morbidity and mortality. While several promising drug and vaccine trials for EBOV are ongoing, options for LASV infection are currently limited to ribavirin treatment. A major factor impeding the development of antiviral compounds to treat these infections is the need to manipulate the virus under BSL-4 containment, limiting research to a few institutes worldwide. Here we describe the development of a novel LASV minigenome assay based on the ambisense LASV S segment genome, with authentic terminal untranslated regions flanking a ZsGreen (ZsG) fluorescent reporter protein and a Gaussia princeps luciferase (gLuc) reporter gene. This assay, along with a similar previously established EBOV minigenome, was optimized for high-throughput screening (HTS) of potential antiviral compounds under BSL-2 containment. In addition, we rescued a recombinant LASV expressing ZsG, which, in conjunction with a recombinant EBOV reporter virus, was used to confirm any potential antiviral hits in vitro. Combining an initial screen to identify potential antiviral compounds at BSL-2 containment before progressing to HTS with infectious virus will reduce the amount of expensive and technically challenging BSL-4 containment research. Using these assays, we identified 6-azauridine as having anti-LASV activity, and demonstrated its anti-EBOV activity in human cells. We further identified 2'-deoxy-2'-fluorocytidine as having potent anti-LASV activity, with an EC50 value 10 times lower than that of ribavirin.