2.11. Plaque Assay

The plaque assay was performed as described [31]. Briefly, cell culture supernatants were collected after 48 h of ZIKV infection and fatty acid treatment (48 hpi) and diluted serially 10⁻³ to 10⁻⁴ using virus infection media in duplicates. The diluted supernatant samples were kept for virus adsorption for 1 h over 90% confluent Vero cells and the cells were washed with PBS prior to virus adsorption. After virus adsorption, 1:1 ratio of 2% low melting agarose and plaque assay media containing 2× DMEM, 1× penicillin and streptomycin, 4% FBS, sodium bicarbonate 7.5%, 20 mM HEPES, sodium pyruvate, 1X nonessential amino acids, and 0.01% plasmocin were added to each well and incubated at 37 °C for 4 days. Fixation of cells was done by using 10% formalin in PBS for 1 h. After the removal of agarose overlaid in each well, fixed cells were stained with 0.1% crystal violet staining solution for 1 h, followed by washing the plates with distilled water and allowing it to air dry. Plaques were counted in each well and expressed plaque forming units/mL (pfu/mL).