Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells

  1. Darrell R. Kapczynski
  2. Ryan Sweeney
  3. Erica Spackman
  4. Mary Pantin-Jackwood
  5. David L. Suarez

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Abstract

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  1. Version published to 10.1016/j.virol.2022.01.014
  2. ScreenIT

    SciScore for 10.1101/2021.08.18.456916: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary monoclonal antibodies included mouse anti-human ACE2 (Origen), rabbit anti-human TMPRSS2 (Abcam, Cambridge, UK) and mouse anti-beta actin (Invitrogen).
    anti-human ACE2
    suggested: None
    anti-human TMPRSS2
    suggested: (Abcam Cat# ab82146, RRID:AB_1658352)
    anti-beta actin
    suggested: None
    Secondary antibodies included rat anti-mouse IgG1 HRP (Southern Biotech, Birmingham, AL), and mouse anti-rabbit IgG1 HRP (Southern Biotech).
    anti-mouse IgG1
    suggested: None
    AL
    suggested: None
    anti-rabbit IgG1
    suggested: None
    Primary antibodies against SC2 included rabbit anti-Nucleoprotein MAb (Origene) and rabbit anti-Spike MAb (Origene), diluted as above, were added for 1 hour at room temperature.
    anti-Nucleoprotein MAb (Origene)
    suggested: None
    anti-Spike
    suggested: None
    Cells were washed 3 times with PBS and incubated in the secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor® 555) (ABCAM) diluted 1:20,000 in TBS, for one hour at room temperature.
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    RNA extraction and RT-PCR for human ACE2 and TMPRSS2: Total RNA was extracted from 2.5 x 105 cells in one well of a 6 well plate from Vero, DF1, DF1 +-, DF1 -+, DF1 ++, MDCK, MDCK +-, MDCK -+ and MDCK ++.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Software and Algorithms
    SentencesResources
    PCR products were visualized on 1% agarose gel (Bio-Rad Laboratories, Hercules, CA) containing SYBR Safe (Invitrogen) using a documentation system (Syngene International Ltd, Bengaluru, India).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Statistical analysis: Viral titers at 48 hpi were compared with the two-way ANOVA with Tukey multiple comparison (Prism 9.1.0 GraphPad Software, San Diego, CA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    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: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.08.18.456916v1 on bioRxiv