Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

Hualong Xiong; Yangtao Wu; Jiali Cao; Ren Yang; Jian Ma; Xiaoyang Qiao; Xiangyang Yao; Baohui Zhang; Yali Zhang; Wangheng Hou; Yang Shi; Jingjing Xu; Liang Zhang; Shaojuan Wang; Baorong Fu; Ting Yang; Shengxiang Ge; Jun Zhang; Quan Yuan; Baoying Huang; Zhiyong Li; Tianying Zhang; Ningshao Xia

To construct VSV pseudovirus carrying the spike protein of SARS-CoV-2, the spike gene of Wuhan-Hu-1 strain (GenBank: MN908947) was codon-optimized for expression in human cells and cloned into the eukaryotic expression plasmid pCAG to generate recombinant plasmid pCAG-nCoVS. The spike gene mutant of SARS-CoV-2 that with C-terminal 18 aa truncation was also cloned into plasmid pCAG and generate pCAG-nCoVSde18. Plasmid pCAG-nCoVS and pCAG-nCoVSde18 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.026948 doi: bioRxiv preprint were transfected into Vero-E6 respectively. 48 hours post transfection, the VSVdG-EGFP-G (Addgene, 31842) [15] virus were inoculated to the cell expressing SARS-CoV-2 spike protein or SARS-CoV-2 Sde18 truncated protein and incubated for one The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.026948 doi: bioRxiv preprint were analyzed by Columbus system (PerkinElmer) and the numbers of GFPactivated cell for each well were counted to represent infection performance. The reduction (%) of mAb treatment GFP-activated cell numbers in comparing with nontreated control well was calculated to show the neutralizing potency.