Rc581 nfdr080i

Frozen lungs and nasal turbinates fragments from the golden Syrian hamsters were weighted and homogenized with 1 mL of ice-cold DMEM (Gibco) supplemented with 1% penicillin/streptomycin (Thermo Fisher) in Lysing Matrix M 2 mL tubes (MP Biomedicals) using the FastPrep-24™ system (MP Biomedicals).
For viral titration, the tissue homogenate supernatants were titrated on Vero-E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581-NFDR080I, DuPont) and expressed and PFU/100 mg of tissue⁶² .
For RNA isolation, the tissue homogenate supernatants were mixed with Trizol LS (Invitrogen) and the total RNA from nasal turbinates was extracted using the Direct-zol RNA MicroPrep Kit (Zymo Research) and from lungs with the (Zymo Research). The presence of SARS-CoV-2 RNA in these samples was evaluated by Superscript III Platinum One-Step RT-qPCR (Invitrogen) containing the nCoV_IP2 and the nCoV_IP2 probe (S.Table 2) according to the manufacturer’s protocol. Viral load quantification (RNA copy number/mg of tissue) was assessed by linear regression using a standard curve of RNA transcripts containing the RdRp sequence (ranging from 10⁷ to 10² copies).

The isolate BetaCoV/France/IDF00372/2020 (EVAg collection, Ref‐SKU: 014V‐03890) was kindly provided by Sylvie Van der Werf. Viral stocks were produced on Vero‐E6 cells infected at a multiplicity of infection of 1 × 10⁻⁴ plaque‐forming units (PFU). The virus was harvested 3 days post‐infection, clarified, and then aliquoted before storage at −80°C. Viral stocks were titrated on Vero‐E6 cells by classical plaque assays using semisolid overlays (Avicel, RC581‐NFDR080I, DuPont) (Baer & Kehn‐Hall, 2014 (link)).

The strain 2019-nCoV/IDF0372/2020 (EVAg collection, Ref-SKU: 014V-03890) was provided by Sylvie Van der Werf, Institut Pasteur, Paris. Viral stocks were produced on Vero-E6 cells infected at a multiplicity of infection of 1.10⁻⁴ PFU (plaque-forming units). The virus was harvested 3 days post-infection, clarified and then aliquoted before storage at -80°C. Viral stocks were titrated on Vero-E6 cells by classical plaque assay using semisolid overlays (Avicel, RC581-NFDR080I, DuPont) (50 (link)).