Figure 7

Time-of-addition analysis of the antiviral activity of theaflavin. A Schematic illustration of the virucidal assay (top). Theaflavin (10, 20 and 30 μM) and LSDV-ΔTK/EGFP (MOI = 0.1) were mixed and incubated at 37 °C for 1 h and then diluted 50-fold before adding to MDBK cells. The possible inactivating effect of theaflavin on the expression of EGFP was determined by fluorescent microscope assay at 72 hpi (middle) and the number of EGFP expressing cells is calculated (bottom). B Schematic illustration of the primary time-of-addition experiment (upper left). MDBK cells were treated with theaflavin (30 μM) and LSDV-ΔTK/EGFP (MOI = 0.1) at different times as indicated, and the possible inactivating effect of theaflavin on the expression of EGFP was determined by fluorescent microscope assay at 72 hpi (right) and the number of EGFP expressing cells is calculated (lower left). C Schematic illustration of the second time-of-addition experiment (upper left). MDBK cells were infected with LSDV-ΔTK/EGFP at an MOI of 0.1 and treated with theaflavin (30 μM) at different time points (1, 4, 8, 16, and 24 hpi). The possible inactivating effect of theaflavin on the expression of EGFP was determined by fluorescent microscope assay at 72 hpi (right) and the number of EGFP expressing cells is calculated (lower left). Data represented three independent experiments with three technical replicates (shown as mean ± SEM), and T-tests was performed. ns, not significant (P > 0.05), ***P < 0.001.