Viral propagation and titration

The SARS-CoV-2 virus was propagated in Vero E6 cells cultured in DMEM high Glucose supplemented with 2% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin. Cells were seeded at a density of 1x10⁶ cells/mL in T175 flasks and incubated at 37°C, 5% CO₂ for 18 - 20 h. The sub-confluent cell monolayer was then washed twice with sterile Dulbecco’s PBS (DPBS). Cells were inoculated with 3,5 mL of the virus properly diluted in DMEM 2% FBS at a multiplicity of infection (MOI) of 0.001, and incubated for 1 h at 37°C in a humidified environment with 5% CO₂. At the end of the incubation, 50 mL of DMEM 2% FBS were added to the flasks. The infected cultures were incubated at 37°C, 5% CO₂ and monitored daily until approximately 80%–90% of the cells exhibited cytopathic effect (CPE). Culture supernatants were then collected, centrifuged at 4°C at 1,600 rpm for 8 min to allow removal of cell debris, aliquoted and stored at −80°C as the harvested viral stock. Viral titers were determined in confluent monolayers of Vero E6 cells seeded in 96-well plates using a 50% tissue culture infectious dose assay (TCID₅₀). Cells were infected with serial 1:10 dilutions (from 10⁻¹ to 10⁻¹¹) of the virus and incubated at 37°C, in a humidified atmosphere with 5% CO₂. Plates were monitored daily for the presence of SARS-CoV-2 induced CPE for 4 days using an inverted optical microscope. The virus titer was estimated according to Spearman-Karber formula (^{Kundi, 1999}) and defined as the reciprocal of the highest viral dilution leading to at least 50% CPE in inoculated wells.