Serial twofold or fourfold dilutions of type I IFN were prepared with cell culture medium containing 5% FBS. The IFN dilutions (100 ml) were added in quadruplicates to confluent cells grown in 96-wells (5610 4 cells/well) and incubated for either 1, 2, 4, or 20 hours at 39uC (DF-1 cells) or 37uC (all other cell lines). The cells were infected with VSV*DG(Luc) (m.o.i. of 5) and incubated for 5 hours at 37uC. The medium was aspirated and 30 ml of luciferase lysis buffer (Biotium Inc., Hayward, CA) was added to the cells. The cell lysates were stored at 220uC. Firefly luciferase activity was determined with a Centro LB 960 luminometer (Berthold Technologies). Luminescence was recorded for 1 s following injection of 30 ml of D-luciferin substrate (Biotium) to white 96-well plates containing 6-ml aliquots of cell lysate. The relative antiviral activity was calculated according to the following formula: Antiviral Activity (%) = 100 -[(RLU +IFN -Blank)6100/ (RLU 2IFN -Blank)]. Mock-infected cell lysates served as blanks and relative light units (RLU) detected with these samples were subtracted from the readings taken from VSV*DG(Luc)-infected cell lysates. RLU +IFN represents the RLU values from IFN-treated cells and RLU 2IFN the readings taken from reference cells, which had not received any type I IFN.