SARS-CoV-2 D614G Pseudovirus Cytotoxicity

Due to D614 SARS-CoV-2 viruses were the majority of the earliest variants detected in Spain within clade 19B [36 (link)], a mutant clone with D614G change was created by site-directed mutagenesis in pNL4-3Δenv_SARS-CoV-2-SΔ19(G614)_Ren pseudovirus [37 (link)]. For analysis of direct cellular cytotoxicity (DCC), Vero E6 cells were infected with equal amounts of both one cycle pseudotyped viruses D614 and G614 (100 ng p24 Gag/well) and then plated onto 48-well plates. After 48 h of incubation, Vero cells were co-cultured for 1 h with PBMCs from patients and healthy donors (ratio 1:10). After detaching Vero monolayer with trypsin-EDTA solution (Sigma Aldrich-Merck, Darmstadt, Germany), caspase-3 activity was measured by luminescence using Caspase-Glo 3/7 Assay system (Promega). Cytotoxic cell populations such as Natural Killer (NK), NKT and TCRγδ+ cells were analyzed in the supernatants using specific conjugated antibodies: CD3-PE, CD56-BV605, CD16-PercP, CD8-APC H7, and TCRγδ-FITC (BD Biosciences). Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer and FACS Diva software (BD Biosciences). FlowJo software (Tree Star Inc.) was used for data analysis.