Abstract
Pseudotyped viruses are useful virological tools because of their safety and versatility. On the basis of a vesicular stomatitis virus (VSV) pseudotyped virus production system, we developed a pseudotyped virus-based neutralization assay against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in biosafety level 2 facilities. Compared with the binding antibody test, the neutralization assay could discriminate the protective agents from the antibody family. This protocol includes production and titration of the SARS-CoV-2 S pseudotyped virus and the neutralization assay based on it. Various types of samples targeting virus attachment and entry could be evaluated for their potency, including serum samples derived from animals and humans, monoclonal antibodies and fusion inhibitors (peptides or small molecules). If the pseudotyped virus stock has been prepared in advance, it will take 2 days to get the potency data for the candidate samples. Experience in handling cells is needed before implementing this protocol.
Highlights
More than 18 million cases of the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had been diagnosed globally by 6 August 2020
Cell transfection and pseudotyped virus production (Steps 15–26) SARS-CoV-2 pseudotyped viruses are prepared by SARS-CoV-2 S plasmid transfection providing membrane proteins on the surface of cells, and G*ΔG-vesicular stomatitis virus (VSV) infection providing genomes of VSV (Fig. 1)
The two systems differ in their backbones; the former is derived from VSV, whereas for the latter, the backbone comes from lentiviruses such as HIV36 or murine leukemia virus[38]
Summary
More than 18 million cases of the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had been diagnosed globally by 6 August 2020. During the preparation of pseudotyped virus, 293T cells are transfected with the SARS-CoV-2 S protein expression plasmid (Fig. 1). Cell transfection and pseudotyped virus production (Steps 15–26) SARS-CoV-2 pseudotyped viruses are prepared by SARS-CoV-2 S plasmid transfection providing membrane proteins on the surface of cells, and G*ΔG-VSV infection providing genomes of VSV (Fig. 1). The detected raw data are imported into the pseudoviral titration macro TCID50_SARS-CoV-2 (Supplementary Method 1 and Box 1) to calculate the pseudoviral titer and the recommended dilution for the neutralization test (Fig. 3a–c). When the raw data for control and samples are exported from the luminometer and pasted into the macro Neutralization_SARS-CoV-2 (Supplementary Method 2 and Box 1), the half maximal effective concentration (EC50) is calculated for the tested samples (Fig. 3d–f). In addition to being used to evaluate products such as neutralizing antibodies and entry inhibitors in the process of virus infection, the assay can be used to study virus cell tropism[36] and receptor recognition patterns
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