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

The fluorescent intensity assay that quantifies the ZsGreen expression from the ZIKV-ZsGreen vector was used in the time-of-addition assay. Briefly, 10 4 Vero B4 cells per well were seeded in a 96-well black plate with transparent bottoms 1 day prior to the experiment. To perform the assay with pre-treatment conditions (−2 to 0 h), the media in wells were replaced with 10 µM benzavir-2 in DMEM, containing 1% FBS for 2 h at 37 • C. Then, all the wells were infected with 0.2 MOI of rZIKV-ZsGreen for 2 h at 37 • C and replaced with fresh DMEM. In the 0−2 h wells, 10 µM benzavir-2 was added together with the virus. At 2 hpi, the virus was removed from all of the wells and 10 µM benzavir-2 was added every second hour in 2 h pulses (0 to 2 h, 2 to 4 h, 4 to 6 h, or 6 to 8 h). For the 8 h sample (0 to 8 h), the cells were treated with 10 µM benzavir-2, together with the virus, for 2 h, and the medium was replaced with 10 µM benzavir-2 in DMEM for an extra 6 h. After 24 h of infection, the cells were fixed with 4% paraformaldehyde and washed with PBS. The fixed cells were counterstained with 300 nM DAPI in PBS. The Trophos plate runner HD (Trophos, Roche Group, Basel, Switzerland) identified all individual ZsGreen-expressing cells and quantified the fluorescence intensity in each well.