Plaque reduction assay

The antiviral activity was evaluated by plaque reduction assay [29] at different infection stages: pre-infection and post-infection, as well as direct inactivation of the viruses. Briefly, the direct effect of peptides on virus per se was analyzed by the following procedures. Monolayer of PK-15 was prepared in 12-well tissue culture plates. Prior to infection, 300 μl of different virus stocks with the titer of 10³ pfu/ml were incubated with 33 μl of peptides diluted in DMEM at concentrations of 25, 5, 1, 0.2 μg/ml at 37 °C for 1 h. After incubation, the peptide-virus mixture was further diluted in DMEM and was inoculated to the monolayer of PK-15. The absorption proceeded at 37 °C for 1 h. The virus-peptide mixture was then removed and replaced with the overlaid medium (4% FBS and 1.5% carboxymethyl cellulose in DMEM). The plaque formation was measured on the 3-4th days later by crystal violet solution staining.

To analyze the post-infection effect of the peptides on virus growth, cells were infected with the same amount of virus mentioned above at 37 °C for 1 h. Subsequently, the cells were washed 3 times with DMEM and treated by the peptides at different concentrations for 1 h at 37 °C. The peptides were removed from the cell culture and replaced with the overlaid medium. The resulting PFU titer was determined as described above.

To understand the pre-infection effect of the peptide on virus, the cells in 12-well tissue culture plates were treated with peptides at serial concentrations at 37 °C for 1 h. Then, cells were washed with DMEM for 3 times, followed by infection with viruses. The cells were covered with the overlaid medium for plaque assays.