Virus

The long strain of RSV was obtained from ATCC and propagated in HEp-2 cells. Briefly, the virus was added to a monolayer of HEp-2 cells and allowed to absorb for 2 h with rocking every 15 min. After 2 h of absorption, the medium was replaced with DMEM containing 2% FBS. Cells were left to grow for another 4–5 days until 80–90% cytopathic effects (CPE) were observed, and then the entire cell culture was collected. After three freeze–thaw cycles, the viruses were collected by centrifugation at 50,000 g for 1.5 h at 4 °C, and the remaining pellet was resuspended, aliquoted and stored at -80 °C before use. Plaque forming units (PFU) were determined by plaque assays with HEp-2 cells.

To determine the EC₅₀ (concentration for 50% of maximal effect) value of luteolin against RSV infection, A549 cells were pretreated with various concentrations of luteolin before infected with RSV. After infection for 12, 24, 48, 72 and 96 h, the culture medium was collected for plaque assays to determine the viral titer. The viral replication inhibition rate was expressed as the percentage of viral replication compared to that in the DMSO-treated cells, and the EC₅₀ was calculated. The selectivity indexes (SIs) for the various time points were calculated with the following equation: SI = CC₅₀/EC₅₀. The CC₅₀ value (concentration of drug required to reduce cell viability by 50%) of luteolin in A549 cells was determined by CCK-8 assay.