Antiviral Activity of Benzavir-2 against Emerging Flaviviruses

Gwon, Yong-Dae; Strand, Mårten; Lindqvist, Richard; Nilsson, Emma; Saleeb, Michael; Elofsson, Mikael; Överby, Anna K.; Evander, Magnus

Monolayers of Vero B4 cells were infected with ZIKV, TBEV, WNV, JEV, YFV, or DENV2 with a multiplicity of infection (MOI): 0.1 in DMEM, containing either DMSO, 2.5 µM benzavir-2, or 50 µM ribavirin, for 1 h at 37 • C 5% CO2. After 1 h of infection, the inoculum was replaced with DMEM 1% penicillin-streptomycin and 2% FBS, supplemented with either DMSO, 2.5 µM benzavir-2, or 50 µM ribavirin. Supernatants were harvested at 24 hpi (TBEV and ZIKV) or 48 hpi (WNV, JEV, YFV, and DENV2), and titers were determined using focus-forming assay, as previously described [28, 29] . In brief, viruses were diluted in 10-fold and used for infection of Vero B4 cells. Viral foci were detected using primary monoclonal mouse antibodies, diluted by 1:1000, and directed against TBEV (19/1493) [30] , flavivirus E (WNV, JEV, DENV2-HB112 ATCC), or YFV (CRC1689 ATCC) [29] [30] [31] . After primary antibody incubation, secondary horseradish peroxidase-conjugated anti-mouse antibodies (1:2000, Thermo Fisher Scientific) were added, followed by TrueBlue peroxidase substrate (KPL, Gaithersburg, MD, USA).