2.6. Antiviral CPE Reduction and Cytotoxicity Assay

Vero cells were seeded at a density of 2.5 × 10⁴ in 96-well tissue culture plates and were allowed to adhere overnight in the incubator. The next day, dilution series of the compounds were prepared in 2% FBS assay medium, after which the cultures were infected with MAYV (MOI 0.01). At day 3 pi, the inhibition of CPE was quantified using the MTS/PMS method. The cells were checked microscopically for minor signs of virus-induced cytopathogenic effects or compound-induced adverse effects on cell monolayer morphology. The 50% effective concentration (EC₅₀), which is defined as the concentration of compound that is required to inhibit virus-induced cell death by 50%, was determined using logarithmic interpolation. In parallel, the toxicity of the compounds was determined by exposing uninfected cells to the same serial dilutions of the compound used in the antiviral assay. At day 3 pi, the 50% cytotoxic/cytostatic concentration (CC₅₀), i.e., the concentration of compound that is required to reduce cell viability by 50%, was determined microscopically or by using the MTS/PMS method.