Sars cov 2

SARS-CoV-2 (NR-52284 obtained from BEI-Resources) infections were conducted in AGS, AGS-ACE2 and Vero cells at the indicated MOIs and incubation durations as mentioned in the legends of Figs 8 and S11. Post infection, different assays were used to evaluate the extent of infection: Cell viability assays, Spike immunostaining and qPCR. Each assay is detailed in S1 Text. The effect of endosomal acidification inhibitors on SARS-CoV-2 infection were tested in AGS-ACE2 and Vero cells. Cells were pre-treated with inhibitors/vehicle controls at different concentrations for 1 hour, followed by infection in the presence of inhibitors/vehicle control. Virus and inhibitors were removed after indicated time of infection, cells were washed 3–4 times and incubated in virus free media with or without inhibitors. Post infection, Niclosamide treated cells were maintained in media with lower concentration of Niclosamide, while Bafilomycin treated cells were maintained in virus and drug free growth media until termination of the assay. To determine cytotoxicity of inhibitors, cells were treated with indicated concentrations of inhibitors in the absence of any virus presentation.

Bat organoids were dissociated by incubation with 350 µL TrypLE to expose the apical and basolateral epithelial surface to the virus. Dissociated organoids were transferred to a BSL3 laboratory and then inoculated with SARS-CoV-2 (strain USA-WA1/2020, BEI Resources), at a multiplicity of infection (MOI) of 0.1, 1 and 10 for 2 h at 37 °C with frequent gentle agitation. Notably, the SARS-CoV-2 strain used was shown to have a defective furin cleavage site^{91 (link)}, but readily infected inducible pluripotent stem cell-derived human intestinal organoids in control experiments. Following incubation with the virus, organoids were collected into 500 µL DMEM and centrifuged at 200 g for 5 min to wash. Then cells were resuspended in 30 µL Matrigel and plated. After 10 min to allow gelation of the Matrigel, medium was added to the organoids. This medium was removed and fresh medium added to eliminate free viral particles. Then the plates were incubated at 37 °C for the indicated intervals. Infectious particles in culture supernatants were detected for each time point by plaque assay on Vero E6 cells, as previously described⁹⁰ .

Vero-human ACE2⁺ TMPRSS2⁺ (VRC, NIAID) were cultured at 37°CC in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10mM HEPES (pH 7.3), and 10 mg/mL of puromycin antibiotic.
SARS-CoV-2 (strain USA-AZ1/2020) was obtained from BEI Resources (NR-52383) and grown on Vero human ACE2⁺ TMPRSS2⁺. The virus stocks were subjected to next-generation sequencing, and the S protein sequences were the same as the published sequence for this isolate. The virus was passaged once in Vero-human ACE2⁺ TMPRRS2 over-expressing cells and titrated by focus-forming assay (FFA) on Vero-human ACE2⁺ TMPRRS2 cells, as described previously (47 (link), 48 (link)). All virus experiments were completed in an Animal Biosafety Level 3 (ABSL-3) facility.

SARS-CoV-2 (isolate USA-WA1/2020, no. NR-52281), SARS-CoV-2_eGFP (no. NR-54002), and the Alpha variant (B.1.1.7, no. NR-54000) were obtained from BEI Resources. The Beta (B.1.351) and Delta (B.1.617.2) variants were obtained from A. Pekosz (Johns Hopkins University, USA). Viruses were propagated by the National Institute of Allergy and Infectious Diseases (NIAID) SARS-CoV-2 Virology Core Laboratory under biosafety level 3 (BSL-3) conditions using Vero (CCL-81) or Vero-overexpressing human TMPRSS2 cells, cultured in DMEM supplemented with GlutaMAX, 2% FBS, penicillin, streptomycin, and fungizone. Virus stocks were deep-sequenced and subjected to minor variants analysis by the NIAID SARS-CoV-2 Virology Core Laboratory. The median tissure culture infectious dose and plaque-forming units (PFU) of virus in clarified culture medium were determined on Vero cells after staining with crystal violet. SARS-CoV-2 infections were performed in the NIAID SARS-CoV-2 Virology Core BSL-3 laboratory strictly adhering to its standard operative procedures.

We prepared 100× TCEP/EDTA buffer (250 mM TCEP, 100 mM EDTA, and 1.15 N NaOH) (Rabe and Cepko, 2020 (link)). TCEP/EDTA buffer was added to human saliva at 1:100 volume, then samples were capped, vortexed to mix and heated in a thermocycler (95°C 5 min, 4°C hold) until ready to use for RT-LAMP. When indicated, heat-inactivated SARS-CoV-2 (BEI Resources Cat. NR-52286) or H1N1 genomic RNA (Twist Biosciences Cat. 103001) was added to inactivated saliva prior to RT-LAMP.

The Vero CCL-81 cell line (African Green Monkey Kidney) from ATCC (Manassas, Virginia, VA, USA) was used in this study. Vero cells were cultured in MEM containing 10% heat-inactivated fetal bovine serum (FBS) and were incubated at 37 °C in the presence 5% CO₂. At the time of virus inoculation and antiviral assays, the concentration of FBS was reduced to 2%. SARS-CoV-2 (NR-52281: USA-WA/2020) was provided by BEI Resources (Manassas, Virginia, VA, USA) and propagated in Vero cells followed by titration using the median tissue culture infectious dose (TCID₅₀) method [12 (link)]. The viral stocks were stored in aliquots at −80 °C until further use.

SARS-CoV-2 (isolate USA_WA1/2020) was obtained from BEI Resources. The virus was propagated in Vero E6 cells (ATCC CRL-1586) cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% fetal calf serum (FCS), penicillin (50 U/mL), and streptomycin (50 mg/mL). SARS-CoV-2 titer was determined in Vero E6 cells by plaque assay. All work with SARS-CoV-2 was performed in the biosafety level 3 (BSL3) at the Ragon Institute (Cambridge, MA) following approved SOPs. To generate heat-inactivated SARS-CoV-2 a 1mL aliquot of SARS-CoV-2 was heated to 85°C for 15 minutes.