2.4. Cytopathic effect (CPE) reduction, cell cycle, and virus yield

LLC-MK2 cells (3 × 10⁵ cells/well) were cultured in the 6-well plates overnight, and then infected by 3 × 10⁴ pfu (plaque formation unit) HCoV-NL63, representing as a MOI (multiplicity of infection) of 0.1 to significantly induce viral cytopathic effect (CPE) in LLC-MK2. The cells were simultaneously added with HCoV NL63, and treated with Sambucus Formosana Nakai stem ethanol extract at the concentrations of 0, 1, 10, and 50 μg/ml or the phenolic acid constituents at the concentrations of 0, 10, 50, and 100 μM, respectively. After the 36-h incubation at 37 ℃ and 5％ CO₂, HCoV NL63-induced cytopathic effect (CPE) with cell swelling, rounding, vacuoles, and eventual detachment was photographed using microscope, in which vacuoles in CPE from HCoV NL63-infected LLC-MK2 cells were more predominant at 37 °C (^{Lednicky et al., 2013}). In the cell cycle assay, the cells were harvested 36 h post infection, stained with propidium iodide, and then examined using flow cytometry, as described in our prior study (^{Wang et al., 2018}). To determine the production of progeny virions (virus yield assay), the cultured media were collected for determined the virus titers using plaque assay. LLC-MK2 cell monolayer in the well of 6-well plates was added with 100 μl of serial 10-fold dilutions of above cultured media. After a 1-h incubation, the cell monolayer was overlaid by the medium containing 0.75% agarose (3 ml per well) for 2-day incubation at 37 °C in a CO₂ incubator to allow the plaque formation. Finally, the cell monolayer was stained with the solution of naphthol blue-black dye and plaque number calculated. Fifty percent (50%) inhibitory concentration (IC₅₀) for virus yield reduction by the stem ethanol extract and the phenolic acid constituents was determined.