Propagation, inactivation, and safety testing of SARS-CoV-2

  1. Alexander S. Jureka
  2. Jesus A. Silvas
  3. Christopher F. Basler

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. Because work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable safe study of RNA, DNA and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.05.13.094482: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    2.1 Cells and Virus: Vero E6 (ATCC# CRL-1586), Calu-3 (ATCC# HTB-55), Caco-2 (ATCC# HTB-37), Huh7, A549 (ATCC# CCL-185), and 293T cells were maintained in DMEM (Corning) supplemented with 10% heat inactivated fetal bovine serum (FBS; GIBCO).
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Caco-2
    suggested: None
    Huh7
    suggested: None
    A549
    suggested: None
    293T
    suggested: None
    VeroE6 cells were inoculated in duplicate with a dilution of 1:100 with an adsorption period of 1 hour at 37°C and shaking every 15 minutes.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    After blocking in 5% non-fat dry milk in TBST (10 mM Tris, 150 mM NaCl, 0.5% Tween-20, pH8) for one hour, membranes were incubated overnight at 4C with antibodies targeting SARS-CoV N (Novus Biologicals; NB100-56576) or SARS-CoV S (Sino Biological; 40150-T62).
    Novus Biologicals
    suggested: (Novus Biologicals, RRID:SCR_004286)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.05.13.094482v1 on bioRxiv