Generation of SARS-CoV-2 pseudotyped variants
rVSV-ΔG-G* (G*: VSV glycoprotein) and VSV-ΔG-Spike (carrying Spike
protein of coronavirus instead of VSV glycoprotein) virus were produced
according to the protocol provided by Whitt in 2010 (Whitt, 2010).
BHK-21/WI-2 cells (Baby Hamster Kidney-21/ Clone WI-2, Kerafast, EH1011)
were infected with vaccinia virus (ATCC, strain vTF7-3,
VR-2153TM) for 1 h, followed by co-transfection of
five plasmids: VSV-ΔG, together with VSV accessory plasmids encoding for
VSV-N, P, L, and G proteins (Kerafast), all of which were under T7
promoter control. The primary transfection was performed using
Lipofectamine 2000. BHK-21/WI-2 cells were transfected with Spike
plasmid to assist in creating passage 1 (P1). Forty-eight hours
following primary transfection, supernatant containing the recovered
VSV-Spike was collected, centrifuged at 1300 × g × 5 min to remove cell
debris. Total supernatant was collected, centrifuged, and used for
sequential passaging in Vero E6 cells to eliminate parental virus
(Yahalom-Ronen et al., 2020), as shown in Figure 1 .
VSV-ΔG-Spike was propagated in DMEM containing 5% FBS (reduced serum
media), MEM NEAA, 2mM L-glutamine, and 1% P/S.