SARS-CoV-2 Virus Isolation and Characterization

We used Vero CCL-81 cells for isolation and initial passage. We cultured Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.com). We used both NP and OP swab specimens for virus isolation. For isolation, limiting dilution, and passage 1 of the virus, we pipetted 50 μL of serum-free DMEM into columns 2–12 of a 96-well tissue culture plate, then pipetted 100 μL of clinical specimens into column 1 and serially diluted 2-fold across the plate. We then trypsinized and resuspended Vero cells in DMEM containing 10% fetal bovine serum, 2× penicillin/streptomycin, 2× antibiotics/antimycotics, and 2× amphotericin B at a concentration of 2.5 × 10⁵ cells/mL. We added 100 μL of cell suspension directly to the clinical specimen dilutions and mixed gently by pipetting. We then grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO₂ and observed for cytopathic effects (CPEs) daily. We used standard plaque assays for SARS-CoV-2, which were based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) protocols (9 (link),10 (link)).
When CPEs were observed, we scraped cell monolayers with the back of a pipette tip. We used 50 μL of viral lysate for total nucleic acid extraction for confirmatory testing and sequencing. We also used 50 μL of virus lysate to inoculate a well of a 90% confluent 24-well plate.