High-Titer SARS-CoV-2 Virus Production

SARS-CoV-2 (BetaCoV/Australia/VIC01/2020 (Passage 2)) was obtained from the Victorian Infectious Diseases Reference Laboratory at the Peter Doherty Institute for Infection and Immunity in Melbourne, Australia [20 (link)]. A Master stock (Passage 3) and Working stocks (Passage 4) were propagated in Vero/hSLAM cell line. To generate high titre stocks for aerosol challenge, the virus was first concentrated using Amicon^® Ultra-15 100 k centrifugal filter units for 25 min at 4000× g, at 4 °C (UFC910024, Merck Millipore, Watford, UK) prior to a final purification step through a 30% (w/v) sucrose cushion at 179,200× g for 2 h, at 4 °C. Genome stability was verified following the passage using an amplicon-based Illumina whole genome sequencing approach with the Liverpool protocol/primers [21 (link)]. Sub-consensus variant analysis was conducted using LoFreq [22 (link)]. Virus was enumerated using plaque assay or Reed & Muench TCID₅₀ method [23 (link)] in Vero C1008 cells, as indicated. The presence of replicating virus was assessed in the lungs, liver, spleen, kidney, brain, and blood by plaque assay and a further blind passage in Vero C1008 cells for 7 days.