Growth, detection, quantification, and inactivation of SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.

Keywords: Coronavirus; Flow cytometry; Focus-forming assay; Plaque assay; SARS-CoV-2; Titration; Virus inactivation.

Fig. 1

SARS-CoV-2 causes cytopathic effect on Vero E6 cell monolayers. Vero E6 cells were inoculated with SARS-CoV-2 at an multiplicity of infection (MOI) of 0.01 plaque forming unit (PFU)/cell and monitored for cytopathic effect at the indicated timepoints. Images were collected using an EVOS XL Core Imaging System. Magnification is 10X for all images.

Fig. 2

Crystal violet stained plaque assay plates. Vero-furin or Vero E6 cells were inoculated with 10-fold serial dilutions of a SARS-CoV-2 stock. Plates were fixed three days post-infection and stained with crystal violet. Wells with individual plaques were used to determine the virus titer (Vero-furin 10⁻⁴, Vero E6 10⁻³). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

SARS-CoV-2 focus-forming assay. CCL81, Vero-furin, Vero E6, and MA104 cells were inoculated with 10-fold serial dilutions of a SARS-CoV-2 stock. Plates were fixed 30 h post-infection and stained with CR3022 anti-SARS-CoV-2 antibody (1 μg/mL) overnight followed by anti-human IgG-HRP (1:500) for 2 h. Foci were visualized using TrueBlue substrate and wells with discrete foci were used to determine virus titer (10⁻³ - 10⁻⁴).

Fig. 4

SARS-CoV-2 infected cell flow cytometry plots. Indicated cell types were inoculated with SARS-CoV-2 at an MOI of 0.01 PFU/cell. At each indicated timepoint post-infection, cells were collected and prepared for flow cytometry using CR3022 anti-S as the primary antibody followed by goat-anti-human IgG Alexa 647 as the secondary antibody.

Fig. 5

Virus outgrowth assay. (A) Titration of paraformaldehyde toxicity on Vero E6 cells plated in 96-well format as described in the protocol. Yellow shading indicates wells in which cytopathic effect was observed. (B) Cytopathic effect observed in Vero E6 cells following inoculation with SARS-CoV-2 infected lung homogenate, treated with or without 1% PFA for 60 min and diluted 1:15,000. Photographs show cells under phase-contrast at 20X (and 40X, inset) magnification. (C) Flow cytometric analysis of Vero E6 cells inoculated with SARS-CoV-2 following treatment with an inactivation agent or PBS (mock). Cells were dissociated to single-cell suspension once the mock-treated culture displayed CPE consistent with SARS-CoV-2 infection. Viability staining with Zombie violet was performed prior to fixation. Antibody staining was performed on 4% paraformaldehyde-fixed and permeabilized cells using the CR3022 anti-SARS-CoV-2 spike antibody followed by anti-human IgG-BV421 labelled secondary antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)