A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition

  1. M. Eugenia Dieterle
  2. Denise Haslwanter
  3. Robert H. Bortz
  4. Ariel S. Wirchnianski
  5. Gorka Lasso
  6. Olivia Vergnolle
  7. Shawn A. Abbasi
  8. J. Maximilian Fels
  9. Ethan Laudermilch
  10. Catalina Florez
  11. Amanda Mengotto
  12. Duncan Kimmel
  13. Ryan J. Malonis
  14. George Georgiev
  15. Jose Quiroz
  16. Jason Barnhill
  17. Liise-anne Pirofski
  18. Johanna P. Daily
  19. John M. Dye
  20. Jonathan R. Lai
  21. Andrew S. Herbert
  22. Kartik Chandran
  23. Rohit K. Jangra

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Abstract

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  1. Version published to 10.1016/j.chom.2020.06.020
  2. ScreenIT

    SciScore for 10.1101/2020.05.20.105247: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: After obtaining informed consent, serum was obtained by venipuncture (BD Vacutainer, serum), centrifuged, aliquoted and stored at -80°C prior to use.
    IRB: Protocol approval was obtained by the Institutional Review Board (IRB) of the Albert Einstein College of Medicine.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Detection of hACE2 in BHK21 transfected cells: To stain for surface-expressed hACE2, BHK21-hACE2 or the control BHK21 cells seeded onto fibronectin-coated glass coverslips were incubated with 0.4 μg/mL of hACE2-specific goat antibody (#AF933, R&D systems) at 4°C in media containing 25 mM HEPES.
    antibody
    suggested: (R and D Systems Cat# AF933, RRID:AB_355722)
    Anti-hACE2 antibody blocking assay: Huh7.5.1 cells were seeded into a 384-well plate.
    Anti-hACE2
    suggested: None
    Nex day, goat anti-human ACE2 antibody (#AF933, R&D Systems) was serially diluted and applied to Huh 7.5.1 cells.
    anti-human ACE2
    suggested: None
    Cells were blocked for ∼2 h, and then immunostained with SARS-1 nucleocapsid protein-specific antibody (SinoBiologic; 40143-V08B) and Alexa Fluor-488 labeled secondary antibody.
    SARS-1 nucleocapsid protein-specific
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero-E6 cells were grown in MEM supplemented in 10% FBS and Gentimicine.
    Vero-E6
    suggested: None
    Retroviruses were produced by transfecting 293FT cells with the hACE2 and TMPRSS2 expressing pBabe-puro plasmids along with those expressing the Moloney murine leukemia virus (MMLV) gag-pol and VSV G proteins.
    293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Detection of hACE2 in BHK21 transfected cells: To stain for surface-expressed hACE2, BHK21-hACE2 or the control BHK21 cells seeded onto fibronectin-coated glass coverslips were incubated with 0.4 μg/mL of hACE2-specific goat antibody (#AF933, R&D systems) at 4°C in media containing 25 mM HEPES.
    BHK21
    suggested: None
    Serum:virus mixtures were then added in duplicate to 384-well plates (Corning) containing Huh7.5.1 cells or 96-well plates (Corning) containing Vero cells.
    Huh7.5.1
    suggested: RRID:CVCL_E049)
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    SARS-CoV-2 stock preparation: Vero-76 cells were inoculated with SARS-CoV-2 (GenBank MT020880.1) at an MOI of 0.01 and incubated at 37°C with 5% CO2 and 80% humidity.
    Vero-76
    suggested: None
    Software and Algorithms
    SentencesResources
    Viral infectivity was measured by automated enumeration of GFP-positive cells from captured images using a Cytation5 automated fluorescence microscope (BioTek) and analyzed using the Gen5 data analysis software (BioTek).
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    The half-maximal inhibitory concentration (IC50) of the mAbs was calculated using a nonlinear regression analysis with GraphPad Prism software.
    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

  3. Version published to 10.1101/2020.05.20.105247v1 on bioRxiv