Validation of luciferase virus entry assay for ZEBOV pseudotypes and VLPs.

<p>(A) The luciferase entry assay was performed using EVP (open bars) or VSVP (solid bars) or ZEBO-VLP (diagonally hatched bars) on HEK293-mCAT-1 cells in the presence of anti-Ebola virus neutralizing antibody (KZ52, 0.3 µg/ml) or a control antibody of irrelevant specificity (anti-HA, 12CA5; Roche, 0.3 µg/ml). Data were normalized to luciferase activity in cells incubated with untreated virus. Each data point represents the mean±SD of 3 independent experiments. (B) Luciferase entry assays were performed using VSVP, EVP, or FrVP on HEK293-mCAT-1 cells in the presence of either 20 mM ammonium chloride (open bars) or 50 nM bafilomycin A1 (solid bars). Effect of ammonium chloride was also tested on ZEBO-VLP. Data were normalized to luciferase activity for untreated cells. Each data point represents the mean±SD of 3 independent experiments. (C) Kinetics of entry was measured for EVP (open squares), VSVP (closed squares), and ZEBO-VLP (open circles). Each was incubated with HEK293-mCAT1 cells at room temperature. After 10 min, cells were washed to remove unbound virus and incubated at 37°C. At indicated time intervals, aliquots of cells were withdrawn, and luciferase signals were measured. For each virus, luciferase activity at different time points was normalized to the maximum luciferase activity for that virus. Each data point represents the mean±SD of 2 independent experiments.