Genjet reagent 2

To reconstitute active viruses from bacmids, HEK293T cells stably expressing T7 polymerase and viral N protein were seeded in T25 cell culture flasks. After 24 h confluent cells were transfected using 10 µL of purified bacmid and 5 µL of GenJet™ Reagent (II) (SL100489, SignaGen^® Laboratories, Frederick, MD, USA) according to the manufacturer’s protocol. After 3–4 days the supernatant (P0) was transferred to confluent CaCo-2 cells in T25 cell culture flasks and replaced by fresh DMEM containing 5% fetal bovine serum, Glutamax, HEPES, and non-essential amino acids after 4–6 h. Fluorescent signal was microscopically controlled after 4–5 days and supernatant was transferred 1:50 to CaCo-2 cells in T75 cell culture flasks. Active viruses were harvested and sterile filtered after detection of strong fluorescence signal or cytopathic effect. Viral titers were determined using endpoint titration (TCID₅₀).

For virus reconstitution, a coculture of HEK293T cells stably expressing either ACE2 or the viral N protein and T7-RNA polymerase was transfected with 8 µg of purified pBelo-S-CoV-2 and 2 µg of pLV-EF1a-IRES-Blast-N using 20-µL GenJet™ Reagent (II) (SL100489, SignaGen^® Laboratories, Frederick, MD, USA) according to the manufacturer’s protocols in T25 tissue culture flasks. Three days post-transfection, the supernatant (P0) was transferred on CaCo-2 or Vero E6 cells for passage 1 (P1) virus stocks. Passage 2 (P2) virus stocks were obtained after the infection of CaCo-2 cells with 1:50 volume of the P1 virus. The viral titers were calculated by endpoint titration (TCID₅₀). Positive wells were determined by in-cell immunostaining of the viral spike protein.