Figure 6.

Time-of-addition assay. (A) RDS monolayers were inoculated with EV71/CA16 and luteolin at the indicated time periods and temperatures. Procedure (a) was the virus control. In procedure (b), the monolayers were pre-treated with luteolin before EV71/CA16 infection. Procedure (c) involved pre-incubation of luteolin with the virus prior to infection. In addition, the cells were treated with the compound and virus simultaneously in procedure (d) as a control of procedure (c). In procedure (e), the cells were first exposed to the virus before adding luteolin. Procedures (f), (g), and (h) were modified from procedures (b), (c), and (d) by also adding the test compound after the 1.5 h time point, which is represented by “+” in the graph. At the end of each time period (the arrow in the graph), the monolayers were washed twice to remove the unattached virus and/or excess luteolin. (B) Antiviral effects of luteolin under the procedures (a–h) were detected via real-time RT-PCR. The results are presented as the mean ± SD of three independent experiments.