Quantifying SARS-CoV-2 and Ebola Pseudotype Infectivity

SARS-CoV-2 strain JPN/TY/WK-521 (59 (link)) was propagated in Vero-TMPRSS2 cells maintained in 2%-FBS/DMEM at 37°C in 5% CO₂. Infectious titers of SARS-CoV-2 were determined in 50% tissue culture infectious dose (TCID₅₀) assays using Vero-TMPRSS2 cells. Using VSV containing the green fluorescent protein (GFP) gene instead of the VSV G gene, VSV-SARS2 and VSV-EBOV were generated as described previously (60 (link), 61 (link)). Pseudotyped VSVs were pretreated with a neutralizing MAb to VSV G (VSV-G [N] 1-9) (33 (link)) to abolish the background infectivity of parental VSV. Pseudotyped VSVs were inoculated into each cell line cultured on 96-well plates, and infectious units (IU) were determined 20 h later by counting the number of GFP-expressing cells using IN Cell Analyzer 2500HS (GE Healthcare).

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Okuya K., Hattori T., Saito T., Takadate Y., Sasaki M., Furuyama W., Marzi A., Ohiro Y., Konno S., Hattori T, & Takada A. (2022). Multiple Routes of Antibody-Dependent Enhancement of SARS-CoV-2 Infection. Microbiology Spectrum, 10(2), e01553-21.

Publication 2022

IN Cell Analyzer 2500HS

Assays Cell Cell line Green fluorescent protein Infectious Parental Protein gene Sars cov 2 infectious Sars2 Strain Tissue Tmprss2 Vero cells

Corresponding Organization :

Other organizations : Hokkaido University, National Institutes of Health, National Institute of Allergy and Infectious Diseases, National Hospital Organization Hokkaido Medical Center

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Variable analysis

independent variables

Strain of SARS-CoV-2 used (JPN/TY/WK-521)
Cell line used for virus propagation (Vero-TMPRSS2 cells)
Cell culture medium composition (2%-FBS/DMEM)
Temperature and CO2 conditions (37°C, 5% CO2)
Pseudotyped VSVs used (VSV-SARS2 and VSV-EBOV)
Pretreatment of pseudotyped VSVs with neutralizing MAb to VSV G

dependent variables

Infectious titers of SARS-CoV-2 determined by TCID50 assays
Infectious units (IU) of pseudotyped VSVs determined by counting GFP-expressing cells

control variables

Vero-TMPRSS2 cell line used for both virus propagation and TCID50 assays
Incubation time for pseudotyped VSV inoculation (20 h)

controls

Positive control: Parental VSV
Negative control: Pretreated pseudotyped VSVs to abolish background infectivity of parental VSV

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