Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening

  1. Killian Rodriguez
  2. Rigaill Josselin
  3. Estelle Audoux
  4. Florian Saunier
  5. Elisabeth Botelho-Nevers
  6. Amélie Prier
  7. Yann Dickerscheit
  8. Sylvie Pillet
  9. Bruno Pozzetto
  10. Thomas Bourlet
  11. Paul O. Verhoeven

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Abstract

Context

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SRAS-CoV-2.

Methods

Vero E6 cells were treated with copper gluconate 18 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were maintained in fresh medium containing copper gluconate for an additional 48-hour period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT assay.

Results

The viability of Vero E6 cells treated with copper gluconate up to 200 μM was found to be similar to that of untreated cells, but it dropped below 40% with 400 μM of this agent. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3%,5.2% in cells treated with 0, 2, 10, 25, 50 and 100 μM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 μM of copper gluconate respectively (p < 0.05).

Conclusion

Copper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells. Furthers studies are needed to determine whether copper homeostasis could play a role in SARS-CoV-2 infection.

GRAPHICAL ABSTRACT

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  1. ScreenIT

    SciScore for 10.1101/2020.12.13.422548: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationThree independent experiments were performed and z-stack images were acquired in 12 random fields in duplicate wells (i.e. 6 fields per well) for each experimental condition.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells lines: Vero E6 and BHK-21 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, ref. D6429, Sigma-Aldrich, Saint Quentin Fallavier, France) supplemented with 2% foetal bovine serum (FBS) (ref. 10270106, Gibco, ThermoFisher, Courtaboeuf, France) without antibiotic.
    BHK-21
    suggested: None
    Vero E6 cells were seeded in a 96-well plate (ref. CLS3904, Corning, Sigma Aldrich) at a density of 20,000 cells per well.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistics and software: SnapGene software v5.2 (GSL Biotech, San Diego, CA) was used to determine in silico the restriction profiles of BAC/YAC containing viral cDNA of synSARS-CoV-2-GFP clone 6.2.
    SnapGene
    suggested: (SnapGene, RRID:SCR_015052)
    Statistical analysis and graphics were computed with GraphPad software v9 (Prism, San Diego, CA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The improvement of the images for publishing was performed with Fiji software (v1.53c).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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 10.1101/2020.12.13.422548 on bioRxiv