2.5. Generation of Pseudotype Virus

VSV*∆G(FLuc), a G-deficient VSV encoding GFP and firefly luciferase, has been described previously [14] and was propagated on transgenic BHK-G43 cells expressing the VSV G protein after induction by mifepristone [15]. The trans-complemented particles (about 10⁸ focus-forming units per milliliter (ffu/mL) were stored at −70 °C in the presence of 5% FBS. Pseudotype viruses were titrated on BHK-21 cells in 96-well cell culture plates as described previously [14]. Virus titers ranged from 2500 to 10,000 ffu/mL.

For production of pseudotype virus, BHK-21 or alternatively HEK 293T cells were grown in 10-cm cell culture dishes and transfected with 10 µg of pCG1-SARS-CoV-2 (Wuhan-Hu-1) spike protein expression plasmid [5] or 10 µg of SARS-CoV-1 (Frankfurt-1) spike protein expression plasmid pCAGGS-SARS-S [5], both kindly provided by Stefan Pöhlmann (German Primate Center, Göttingen, Germany). Lipofectamine 2000 (Life Technologies, Zug, Switzerland) was employed as transfection reagent. At 20 h post-transfection, the cells were washed once with GMEM and subsequently inoculated for 60 min at 37 °C with VSV*∆G(FLuc) using an moi of 5 ffu/cell. The cells were washed twice with GMEM and subsequently incubated at 37 °C with GMEM containing 5% FBS and 10% conditioned cell culture medium of hybridoma cells secreting the VSV neutralizing monoclonal antibody Mab I1, which prevents carryover of VSV particles carrying the G protein rather than the CoV-S on their cell surface. At 20 h pi, the cell culture supernatant was harvested, cell debris removed by low-speed centrifugation (1000× g, 10 min, 4 °C), and the pseudotype viruses stored in aliquots at −70 °C. Taking advantage of the GFP reporter for detection of infected cells, the pseudotype viruses were titrated on Vero E6 cells, which are highly permissive to SARS-CoV-1 and SARS-CoV-2 [18].