MVA-T7 was titrated on DF-1 cells grown in 96-well microtiter plates. The cells were inoculated with tenfold serial virus dilutions for 90 min and overlayed with medium containing 0.9% methylcellulose. Following incubation for 48 h at 39uC, the cells were fixed with 3% paraformaldehyde, permeabilized with 0.25% Triton X-100, and subsequently incubated with the TW2.3 monoclonal antibody directed to the vaccinia E3L protein (kindly provided by Jonathan Yewdell, NIH, Bethesda, USA) and anti-mouse IgG conjugated to horseradish peroxidase (DAKO). Infected cell foci were visualized with the AEC peroxidase substrate and expressed as plaque-forming units per milliliter (pfu/ml). Using this assay, the final VSV replicon particle preparations proved to be free of MVA-T7.