Cytotoxicity assays.

Cell viability of infected HCC cell lines (Huh7 and HepG2) and primary human hepatocytes was analyzed by measuring levels of released lactate dehydrogenase (LDH) in cell culture supernatant. The cells were plated, infected, and washed as described for the growth curve experiments. At 24, 48, and 72 h postinfection, aliquots of supernatant were collected, and LDH release was quantified using the CytoTox 96 nonradioactive cytotoxicity assay protocol (Promega, Madison, WI). For each time point, LDH release following virus infection was calculated as a percentage of the maximum control LDH release. Baseline LDH levels detected in the supernatant of mock-treated cells were subtracted from the values obtained from the experimental wells.

Cell viability of neurons was analyzed with an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carbooxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay using the CellTiter 96 AQueous One Solution cell proliferation assay (Promega). Neurons were seeded in collagen-coated 96-well dishes at a density of 5 × 10⁴ cells per well and mock treated or infected with rVSV, rNDV, or rVSV-NDV at an MOI of 0.01. At 24, 48, and 72 h postinfection, cell viability was measured according to the manufacturer’s protocol. Cytotoxicity was quantified as the difference in cell viability between the experimental samples and the uninfected controls.