Geneart ag technology

rVSV-luc pseudotypes were generated following a published protocol (Whitt, J Virol Methods 2010). First, BHK-21 were transfected to express the S protein of SARS-CoV-2 (codon optimized, kindly provided by J. García-Arriaza, CNB-CSIC, Spain), Ebola virus Makona Glycoprotein (EBOV-GP) (KM233102.1) was synthesized and cloned into pcDNA3.1 by GeneArt AG technology (Life Technologies, Regensburg, Germany) or VSV-G following the manufacturer’s instructions of Lipofectamine 3000 (Fisher Scientific). After 24 h, transfected cells were inoculated with a replication-deficient rVSV-luc pseudotype (MOI: 3–5) that contains firefly luciferase instead of the VSV-G open reading frame, rVSVΔG-luciferase (G*ΔG-luciferase) (Kerafast, Boston, MA). After 1 h incubation at 37°C, cells were washed exhaustively with PBS and then DMEM supplemented with 5% heat-inactivated FBS, 25 μg/mL gentamycin and 2 mM L-glutamine were added. Pseudotyped particles were collected 20–24 h post-inoculation, clarified from cellular debris by centrifugation and stored at -80°C [20 (link),21 (link),95 (link)]. Infectious titers were estimated as tissue culture infectious dose per mL by limiting dilution of rVSV-luc-pseudotypes on Vero E6 cells. Luciferase activity was determined by luciferase assay (Steady-Glo Luciferase Assay System, Promega).

rVSV-luc pseudotypes (PSV) were generated following a published protocol (Whitt, 2010 (link)). First, BHK-21cells were transfected using Lipofectamine 3000 (Fisher Scientific) to express the different viral envelope glycoproteins: Ebola virus Makona Glycoprotein (EBOV-GP, GenBank KM233102.1) and S protein of MERS-CoV (NC_019843) were synthesized and cloned into pcDNA3.1 by GeneArt AG technology (Life Technologies, Regensburg, Germany); S protein of SARS-CoV-2 (codon-optimized; kindly provided by J. García-Arriaza, CNB-CSIC, Spain) or S protein of SARS-CoV (SinoBiological, Cat: VG40150-G-N) were cloned into a pCMV/hygro vector. After 24 h, transfected cells were inoculated with a replication-deficient rVSV-luc pseudotype (MOI: 3–5) (Kerafast, Boston, MA). After 1 h incubation at 37°C, cells were washed exhaustively with PBS and then DMEM supplemented with 5% heat-inactivated FBS, and 25 μg/mL gentamycin and 2 mM L-glutamine were added. PSV were collected 24 h post-inoculation, clarified from cellular debris by centrifugation, and stored at -80°C. Infectious titers were estimated as tissue culture infectious doses by limiting dilution of rVSV-luc-pseudotypes on Vero E6 cells. Luciferase activity was determined by luciferase assay (Steady-Glo Luciferase Assay System, Promega).