Sars cov 2 isolate usa wa1 2020

SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources) was propagated using Vero E6 (ATCC) cells and infectious units quantified by plaque assay. For antibody neutralization assays, 10⁴ Vero E6 cells were seeded in wells of a 96-well plate and incubated overnight in DMEM 10% FBS with penicillin/streptomycin at 37°C 5% CO₂. The following day, titrations of mAbs were incubated with 10⁴ PFU for 1 h in DMEM 10% FBS at room temperature, then added to Vero E6 cells for up to 48 h. Infection was measured by tracking virus-induced cell death of Vero E6 cells and by intracellular staining for SARS-CoV-2 nucleocapsid, described below.

Quantitative standards were prepared using a stock of passage 6 SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources, Manassas, VA), propagated in Cercopithecus aethiops kidney cells (Vero E6; ATCC, Manassas, VA). The titer of the stock was determined by plaque assay and endpoint dilution assay using Vero E6 cells according to standard methods, and titers were determined to be 1.95 × 10⁶ (1.95e6) PFU/mL and 3.58 × 10⁶ (3.58e6) TCID₅₀/mL, respectively (29 (link), 30 (link)). Stocks were then aliquoted, heat inactivated at 65°C for 30 min, removed from high containment, and frozen at −80°C until use. All work was performed in a biosafety level 3 laboratory following institutional biosafety committee-approved and validated protocols from the Indiana University School of Medicine.

An in vitro imaging assay using SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources) and Calu-3 cells was performed as previously described [23 (link)]. Briefly, Calu-3 cells were plated in 384-well plates. The following day antiviral activity was assessed in duplicates of eight serial dilutions of galidesivir, ranging from 0.023 to 50.0 µM. DMSO was included as a negative control, and remdesivir was included as a positive control. Two hours after incubation with galidesivir, cells were infected with SARS-CoV-2 under biosafety level 3 (BSL3) containment conditions at an MOI of 0.5. Forty-eight hours after viral infection, cells were fixed and incubated overnight at 4 °C with a primary antibody specific for dsRNA (anti-dsRNA J2). Following 1 h incubation with a secondary antibody at room temperature, the cells were processed for automated microscopy. Toxicity was measured by quantifying the number of cells per well. The percentage of infected cells was calculated using the formula: (dsRNA⁺ cells/cell number)/well.

Vero-E6 (female) and Huh7.5 (male) cells were seeded at 1x10⁶ cells per T150 flask and were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 1% Penicillin/Streptomycin. Three T150 flasks were assigned per condition: 0, 1, and 2 days post-infection (dpi). The next day, the media was removed, and cells were inoculated with SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources #NR-48814) at MOI of 0.01. Flasks were incubated at 37°C for 1 h with gentle rocking every 15 min. At 0, 1, and 2 dpi, supernatant from the flasks were discarded, and cells were washed with 1X PBS twice. 4 mL of 4% of paraformaldehyde was added on each of the flasks and incubated for 30 min at room temperature. Afterward, cells were quenched with 250 μL of 2 M glycine (final concentration of 125 mM) for each flask. Cells were scraped, harvested in pre-weighed microcentrifuge tubes, and span at 1000 x g for 5 min at 4°C. All supernatants aspirated, and the final pellet were weighed. Cells were frozen at −80°C until used.

Huh7 cells were purchased from Glow Biologics (GBTC-099H) and tested negative for mycoplasma. These cells expressed human ACE2 (huh7-hACE2) after transduction by lentiviral particles derived with pWPI-IRES-Puro-Ak-ACE2 (a gift from Sonja Best; Addgene plasmid # 154985). SARS-CoV-2, isolate USA-WA1/2020 (NR-52281), was obtained through BEI Resources and propagated once on VERO E6 cells before it was used for this study.