Cytotoxicity Assay

Viral cytotoxicity was determined by measuring cell viability with the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt)/phenazine methosulfate assay. The oncolytic activity of EEV was evaluated based on the viability of cells treated with the culture medium of infected cells. Ovarian carcinoma cell lines were infected with LC16mO VGF⁻/O1⁻ (B5R) or LC16mO ΔSCR VGF⁻/O1⁻ (ΔSCR) at an MOI of 0, 0.001, 0.01, 0.1, or 1 at 37°C for 1 h, and virus diluents were removed and replaced with the appropriate culture medium. After 48 h, 50 μL medium (including EEV) was recovered from each culture and pipetted onto the newly seeded ovarian cancer cells, which were the same as the EEV-producing cells; 120 h later, cells were photographed under a fluorescence microscope, and viability was assessed with the CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega, Madison, WI, USA). Whole viral cytotoxicity was evaluated in cells infected with B5R or ΔSCR at the same MOI, which were also photographed and assessed for viability 120 h after infection.