Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques ( Macaca fascicularis )

  1. Courtney L. Finch
  2. Ian Crozier
  3. Ji Hyun Lee
  4. Russ Byrum
  5. Timothy K. Cooper
  6. Janie Liang
  7. Kaleb Sharer
  8. Jeffrey Solomon
  9. Philip J. Sayre
  10. Gregory Kocher
  11. Christopher Bartos
  12. Nina M. Aiosa
  13. Marcelo Castro
  14. Peter A. Larson
  15. Ricky Adams
  16. Brett Beitzel
  17. Nicholas Di Paola
  18. Jeffrey R. Kugelman
  19. Jonathan R. Kurtz
  20. Tracey Burdette
  21. Martha C. Nason
  22. Irwin M. Feuerstein
  23. Gustavo Palacios
  24. Marisa C. St. Claire
  25. Matthew G. Lackemeyer
  26. Reed F. Johnson
  27. Katarina M. Braun
  28. Mitchell D. Ramuta
  29. Jiro Wada
  30. Connie S. Schmaljohn
  31. Thomas C. Friedrich
  32. David H. O’Connor
  33. Jens H. Kuhn

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques ( Macaca fascicularis ) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6–12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

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    SciScore for 10.1101/2020.05.14.096727: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: , Animal Care and Use Committee (ACUC), and were in compliance with the Animal Welfare Act regulations, Public Health Service policy, and the Guide for the Care and Use of Laboratory Animals 8th Ed. recommendations.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAll female macaques were on depot medroxyprogesterone acetate (administered intramuscularly, 150 mg/ml) while at NIHAC for several months.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Plates were then washed three times with PBS-T, and 100 μl of goat anti-human IgG Fc specific (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody conjugated to horseradish peroxidase (HRP; diluted 1:20,000 in ELISA diluent) were added to each well.
    anti-human IgG
    suggested: None
    The secondary antibody was goat α-rabbit IgG (H+L), Alexa Fluor 594 Conjugate (Thermo Fisher Scientific) prepared at 1:2,500 in 1X PBS.
    α-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A master virus stock (designated IRF_0394) was grown under high (biosafety level 3) containment conditions at the IRF-Frederick by inoculating grivet (Chlorocebus aethiops) Vero cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA; #CCL-81) maintained in Dulbecco’s Modified Eagle Medium with L-glutamine (DMEM, Lonza, Walkersville, MD, USA) supplemented with 2% heat-inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA) at 37°C in a humidified 5% CO2 atmosphere, harvested after 72 h, and quantified by plaque assay in Vero E6 cells (ATCC #CRL-1586) using a 2.5% Avicel overlay with a 0.2% crystal violet stain at 48 h following a previously published protocol (47).
    Vero
    suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
    Vero E6
    suggested: None
    Before removal from the maximum containment laboratory, virus in samples was inactivated using a cobalt irradiation source with a target dose of 50 kGy (JLS 484R-2 Cobalt-60 [60Co] Irradiator, JLShephard & Associates) following standard inactivation protocols.
    Cobalt-60
    suggested: None
    Software and Algorithms
    SentencesResources
    A master virus stock (designated IRF_0394) was grown under high (biosafety level 3) containment conditions at the IRF-Frederick by inoculating grivet (Chlorocebus aethiops) Vero cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA; #CCL-81) maintained in Dulbecco’s Modified Eagle Medium with L-glutamine (DMEM, Lonza, Walkersville, MD, USA) supplemented with 2% heat-inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA) at 37°C in a humidified 5% CO2 atmosphere, harvested after 72 h, and quantified by plaque assay in Vero E6 cells (ATCC #CRL-1586) using a 2.5% Avicel overlay with a 0.2% crystal violet stain at 48 h following a previously published protocol (47).
    SAFC
    suggested: (SAFC, RRID:SCR_008554)
    Reciprocal endpoint titers were determined in GraphPad software version 8.4.2 (Prism, La Jolla, CA, USA), using a sigmoidal 4 parameter-logistic fit curve.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The data was exported to Bio-Results Generator version 3.0 and Bio-Plex Manager software version 6.2 (BioRad).
    Bio-Plex Manager
    suggested: None

    Results from OddPub: Thank you for sharing your code.

    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 46. 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

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  2. Version published to 10.1101/2020.05.14.096727v1 on bioRxiv