Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne orthonairovirus, causes a severe hemorrhagic disease in humans (Crimean-Congo hemorrhagic fever, CCHF). Currently, no vaccines are approved to prevent CCHF; treatment is limited to supportive care and the use of ribavirin, the therapeutic benefits of which remain unclear. CCHF is part of WHO's priority list of infectious diseases warranting further research and development. To aid in the identification of new antiviral compounds, we generated a recombinant CCHFV expressing a reporter protein, allowing us to quantify virus inhibition by measuring the reduction in fluorescence in infected cells treated with candidate compounds. The screening assay was readily adaptable to high-throughput screening (HTS) of compounds using Huh7 cells, with a signal-to-noise ratio of 50:1, and Z′-factors > 0.6 in both 96- and 384-well formats. A screen of candidate nucleoside analog compounds identified 2′-deoxy-2′-fluorocytidine (EC50 = 61 ± 18 nM) as having 200 × the potency of ribavirin (EC50 = 12.5 ± 2.6 μM), as well as 17 × the potency of T-705 (favipiravir), another compound with reported anti-CCHFV activity (EC50 = 1.03 ± 0.16 μM). Furthermore, we also determined that 2′-deoxy-2′-fluorocytidine acts synergistically with T-705 to inhibit CCHFV replication without causing cytotoxicity. The incorporation of this reporter virus into the high-throughput screening assay described here will allow more rapid identification of effective therapeutic options to combat this emerging human pathogen.