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 virus concentration of p0 stocks was determined by plaque assay, as previously described [23, 24] . Briefly, 500 µL of tenfold serial dilutions of the virus samples were added to a 6-well plate containing a confluent monolayer of Vero B4 cells. After 1 h incubation with shaking every 15 min, the virus suspension was discarded and an overlay was added that contained 2 mL of Dulbecco's modified Eagle medium (DMEM) with 2% FBS, 0.5% minimum essential amino acids, 0.5% sodium pyruvate, 0.5% L-Glutamine, 1% penicillin/streptomycin, and 1% carboxymethylcellulose (CMC, Sigma Aldrich, St. Louis, MO, USA). After 4-5 days of incubation, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution (1% crystal violet powder, 20% w/v methanol in distilled H 2 O). Plaques were counted and viral titers were calculated as plaque-forming units (PFUs)/mL.