SARS-CoV-2 Virus Propagation in Vero-E6 Cells

The U.S. reference strain SARS-CoV-2 USA-WA 1/2020 (BEI resources NR-52281) was propagated in African Green Monkey kidney cells (Vero-E6 ATCC CRL-1586) as described previously (60 (link)). Handling of SARS-CoV-2 was done under strict BSL3 biosafety protocols at the Center for Food Safety BSL3 laboratory. Briefly, 1- or 2-day-old 90% confluent cells were used to prepare virus stocks using a multiplicity of infection of 0.01. Infection media consisted of Dulbecco’s modified Eagle medium (DMEM) supplemented with 2% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic cocktail. Harvesting the virus was done at 72 h postinfection. Infected cells were collected from the flasks and centrifuged at 450 × g for 5 min at 4°C to pellet the cell debris, while supernatants containing the virus, were ultra-filtered through Amicon 100 kDa Ultra-15 centrifugal devices (Millipore) immediately after harvest to concentrate the viruses 10 times, while semipurifying them from cell culture lysates (51 (link)). An aliquot of the virus was immediately titrated as described below. The original viral titer generated was ~7 log₁₀ TCID₅₀/mL, while the ultrafiltered virus titer was ~8 log₁₀ TCID₅₀/mL.