4.3. Antiviral Activity Assay

Briefly, 96-well plates were prepared with 5.10⁵ cells/mL of Vero E6 (200 µL per well), as previously described. Doxycycline and chloroquine concentrations were added 4 h before infection. Vero E Cells were infected with IHUMI-3 strain at an MOI of 0.25. After 48 h post-infection, the replication was estimated by RT-PCR using the Superscrit III platinum one step with Rox kit (Invitrogene) after extraction with the BIoExtract SuperBall kit (Biosellal, Dardilly, France). The primers used were previously described [69]. The percentage of inhibition of SARS-CoV-2 replication was estimated for each drug concentration as following: (mean CT_{drug concentration} − mean CT_{control 0%})/(mean CT_{control 100%} − mean CT_{control 0%}) × 100. EC₅₀ (median effective concentration) and EC₉₀ (90% effective concentration) were calculated with the inhibitory sigmoid E_max model, which estimated the EC₅₀ and EC₉₀ through nonlinear regression by using a standard function of the R software (ICEstimator version 1.2). EC₅₀ and EC₉₀ values resulted in the mean of 10 different experimentations.