Efficient culture of SARS-CoV-2 in human hepatoma cells enhances viability of the virus in human lung cancer cell lines permitting the screening of antiviral compounds

  1. Santseharay Ramirez
  2. Carlota Fernandez-Antunez
  3. Long V. Pham
  4. Line A. Ryberg
  5. Shan Feng
  6. Martin S. Pedersen
  7. Lotte S. Mikkelsen
  8. Sandrine Belouzard
  9. Jean Dubuisson
  10. Judith M. Gottwein
  11. Ulrik Fahnøe
  12. Jens Bukh

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Abstract

Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log 10 increase in infectivity titers (TCID 50 ) in Huh7.5 cells, with titers of ~8 Log 10 TCID 50 /mL, and >2-Log 10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log 10 TCID 50 /mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log 10 TCID 50 /mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity.

Importance

The cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.

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    SciScore for 10.1101/2020.10.04.325316: (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

    Antibodies
    SentencesResources
    Afterwards, cells were washed twice with PBST followed by 1-hour incubation with secondary antibody F(ab’)2-Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP (Invitrogen #A24476) diluted 1:2000 in PBSK.
    anti-Human IgG
    suggested: (Thermo Fisher Scientific Cat# A24476, RRID:AB_2535945)
    Experimental Models: Cell Lines
    SentencesResources
    Afterwards, all experiments in Vero E6 were performed with media supplemented only with FBS 10%, 10,000 units penicillin and 10 mg streptomycin/mL (Sigma).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Human hepatoma cells (Huh7 and Huh7.5 cells) and Calu-1 were cultured in DMEM (high glucose, GlutaMAX and pyruvate, Invitrogen ThermoFisher) supplemented with 10% fetal bovine serum (Sigma), 10,000 units penicillin and 10 mg streptomycin/mL (Sigma), and kept at 37°C in a humidified incubator with 5% CO2.
    Huh7
    suggested: None
    Huh7.5
    suggested: RRID:CVCL_YU20)
    Calu-1
    suggested: None
    A549 cells were cultured in the same conditions but with Ham’s F-12K (Kaighn’s) Medium (Gibco, ThermoFisher) supplemented with the same 10% fetal bovine serum and antibiotics.
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    Results of treated well were normalized to non-treated controls and the percentage of cell viability was plotted against the Log10 of drug concentration, followed by non-linear regression (Y=Bottom + (Top-Bottom)/(1+10^((LogCC50−X)*HillSlope)) using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.
    • No funding statement was detected.
    • Thank you for including a protocol registration statement.

    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 10.1101/2020.10.04.325316 on bioRxiv