2.9. Cytopathic Effect Inhibition Assay

The cytopathic effect (CPE) inhibition assay with slight modifications was used as previously described [42]. Time of addition tests were applied, where the CrFK cells were treated with different exposures (pre-, co-, and posttreatment) of the indicated concentrations of curcumin and Cur-CS nanoparticles. In pretreatment, the compounds were included in the cell culture medium for 8 h and were removed prior to virus infection. In cotreatment, the compounds were mixed with virus in the medium, added simultaneously to the cells, and left on the cells throughout. For posttreatment, the compounds were added to the cells 2 h postinfection (hpi) and remained throughout the time of infection. Briefly, CrFK cells were incubated in 96-well plates and incubated with predetermined concentrations of curcumin and Cur-CS nanoparticles under different types of exposure with 100 tissue culture infective dose 50 (TCID₅₀) FIPV. After incubation at 37°C for 48 and 72 h, the cell viability was determined by MTT.

The percentage of protection was calculated based on the OD using the following formula:

where T is the treated sample, V is the virus-infected control, and C is the negative control.

The median effective concentration (EC₅₀) is the concentration of compound required to achieve 50% protection against virus-induced CPE. The selective index (SI) was calculated for curcumin and Cur-CS nanoparticles against FIPV by dividing the appropriate CC₅₀ value by the corresponding EC₅₀.