2.5. Cytopathic Effect (CPE) Inhibition Assay

Cytopathic effects (CPE) in FMDV-infected BHK-21 cells were evaluated by observing the rounding-up of infected cells and cell lysis characteristics, typically occurring within 25–26 h following FMDV infection. This experiment was aimed to determine whether terpenoid-treated, FMDV-infected BHK-21 cells exhibited reduced CPE in a dose-dependent manner. Following the incubation, the attached cells were fixed with cold absolute methanol at room temperature for 15 min, and the plates were allowed to dry. Subsequently, the cells were stained with a 0.5% crystal violet solution, and the stained cells were observed using a phase-contrast inverted microscope (Olympus IX73, Tokyo, Japan). Images of cell viability areas were recorded by photography, which were further analyzed using CellProfiler Version 4.2.5. For each tested compound, the 50% effective concentration (EC50) was calculated using GraphPad Prism, version 10.1.1.