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

Abstract

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Coinfection with influenza A virus enhances SARS-CoV-2 infectivity

“…Pseudotyped VSV luciferase-reporter particles bearing SARS-CoV-2 spike protein (pseudo-SARS-CoV-2) were used to visualize the viral entry. 12 The cells were first infected with IAV (A/WSN/1933(H1N1)) or mock-infected for 6 h, 12 h, or 24 h and then infected with the pseudo-SARS-CoV-2 virus for another 24 h (experimental scheme shown in Fig. 1a ).…”

Neutralizing and binding antibody kinetics of COVID-19 patients during hospital and convalescent phases

“…The neutralizing activity of serum samples was measured by a previously reported pseudovirus-based neutralization assay, by which the determined neutralizing titers of serum collected from COVID-19 patients correlated well with those according to a live SARS-CoV-2 virus neutralization assay [14]. Brie y, after heat inactivation at 56 ℃ for 30 min, serum samples were serially diluted twofold, starting from 10fold dilutions, after which the dilutions were mixed with an equal volume of VSV-SARS-CoV-2-Sdel18 virus (MOI=0.05).…”

Repositioning of histamine H1 receptor antagonist: Doxepin inhibits viropexis of SARS-CoV-2 Spike pseudovirus by blocking ACE2

“…Pseudovirus is a chimeric virus particle that expresses a recombinant glycoprotein of another virus on the surface of a replication-defective virus. Pseudovirus neutralization assay can mimic the infectious process of the live virus, which has a good correlation with that measured by the wild type SARS-CoV-2 assay ( Xiong et al, 2020 ). ACE2-HEK293T cells (5 × 10 4 per well) were seeded into white 96-well plates and cultured at 37 °C until cells were adherent.…”