2.6. Antiviral Assay

Plaque reduction assay was used to determine the antiviral activity of EO as previously described [32]. Briefly, Vero cells (CCL-81) were cultivated for 24 h at 5% CO₂ and 37 °C. The cells were inoculated for 1 h with individual viruses; herpes simplex type-1 virus HSV-1 (ATCC VR-1493), hepatitis A virus HAV (ATCC VR-1357) or vesicular stomatitis virus VSV (ATCC VR-1238) (1 × 10¹‒10⁷ plaque-forming unit PFU/mL). The infected cells (2 × 10³ PFU) were washed and incubated with several concentrations of EO as well as acyclovir (positive control) for 1 h. Then, cells were overlaid with nutrient agarose and incubated for 72 h. Formaldehyde (10%) in phosphate-buffer solution (pH 7.3) was used to fix cells for 1 h. Crystal violet, 0.5% in 20% ethanol, was used to stain cells and then plaques were counted. The percentage of viral inhibition was calculated as [1 − (Vd/Vc)] × 100, where Vd and Vc are the number of plaques in the presence and absence of test samples.