Time-of-addition assay.

This assay was performed as described previously (24). In short, MDCK cells were infected at a multiplicity of infection (MOI) of 0.01 and 10 μM FA-583 or FA-617 was added before infection (−1 h), at the time of infection (0 h), and at 1, 2, 4, 6, and 8 h after infection. Viral titers of corresponding time points were determined at 10 h postinfection by plaque assay. The experiments were carried out in triplicate, and two individual sets of assays were performed for confirmation.