Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease

  1. Joseph W. Golden
  2. Curtis R. Cline
  3. Xiankun Zeng
  4. Aura R. Garrison
  5. Brian D. Carey
  6. Eric M. Mucker
  7. Lauren E. White
  8. Joshua D. Shamblin
  9. Rebecca L. Brocato
  10. Jun Liu
  11. April M. Babka
  12. Hypaitia B. Rauch
  13. Jeffrey M. Smith
  14. Bradley S. Hollidge
  15. Collin Fitzpatrick
  16. Catherine V. Badger
  17. Jay W. Hooper

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Abstract

The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two different doses of SARS-CoV-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and the assessment of MCMs.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.09.195230: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Animal experimental protocols were approved by a standing internal institutional animal care and use committee (IACUC).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Then the sections were incubated with primary antibodies: rabbit polyclonal anti-SARS-CoV Spike at a dilution of 1:200 (40150-T62-COV2, Sino Biological, Chesterbrook, PA, USA), mouse monoclonal anti-SARS-CoV NP at a dilution of 1:200 (40143-MM05, Sino Biological), mouse monoclonal anti-pan cytokeratin at a dilution of 1:100 (sc-8018, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-e-cadherin at a dilution of 1:100 (33-4000, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-myeloperoxidase (MPO) at a dilution of 1:200 (A039829-2, Dako Agilent Pathology Solutions, Carpinteria, CA, USA), rabbit polyclonal anti-CD3 antibody at a dilution of 1:200 (A045229-2, Dako Agilent Pathology Solutions), rat monoclonal anti-CD45 antibody at a dilution of 1:100 (05-1416, Millipore Sigma, Burlington, MA, USA), rabbit polyclonal anti-CD68 at a dilution of 1:200 (ab125212, Abcam, Cambridge, MA, USA), mouse monoclonal anti-NeuN at a dilution of1:200 (MAB377, Millipore Sigma), and/or chicken polyclonal anti-GFAP at a dilution of 1:200 (ab4674, Abcam) for 2 hours at room temperature.
    anti-SARS-CoV
    suggested: (Rockland Cat# 200-401-A51, RRID:AB_828457)
    anti-SARS-CoV NP
    suggested: (RayBiotech Cat# MD-05-0425, RRID:AB_951740)
    anti-pan cytokeratin
    suggested: (Santa Cruz Biotechnology Cat# sc-8018, RRID:AB_627396)
    sc-8018
    suggested: (Santa Cruz Biotechnology Cat# sc-8018, RRID:AB_627396)
    anti-e-cadherin
    suggested: (Thermo Fisher Scientific Cat# 13-1800, RRID:AB_2533004)
    anti-myeloperoxidase (MPO
    suggested: (MBL International Cat# JM-3831-100, RRID:AB_1279154)
    anti-CD3
    suggested: (Agilent Cat# A0452, RRID:AB_2335677)
    anti-CD45
    suggested: (Millipore Cat# 05-1416, RRID:AB_10562966)
    anti-CD68
    suggested: (Abcam Cat# ab125212, RRID:AB_10975465)
    anti-NeuN
    suggested: (Millipore Cat# MAB377, RRID:AB_2298772)
    anti-GFAP
    suggested: (Abcam Cat# ab4674, RRID:AB_304558)
    After rinses with PBT, the sections were incubated with secondary goat anti-rabbit or anti-chicken Alexa Fluor 488 at dilution of 1:500 (Thermo Fisher Scientific) and goat anti-mouse or anti-rat Alexa Fluor 568 at a dilution of 1:500 (Thermo Fisher Scientific) antibodies, for 1 hour at room temperature.
    anti-rabbit
    suggested: None
    anti-chicken
    suggested: (Thermo Fisher Scientific Cat# A-11041, RRID:AB_2534098)
    anti-mouse
    suggested: None
    anti-rat
    suggested: (Thermo Fisher Scientific Cat# A-11077, RRID:AB_2534121)
    Experimental Models: Cell Lines
    SentencesResources
    A master challenge stock of virus was propagated by making two passages in Vero76 cells in Modified Eagles Medium with Earle’s Salts (EMEM) (Corning) supplemented with 1% GlutaMAX, 1% NEAA, and 10% heat inactivated fetal bovine serum (FBS) (Gibco).
    Vero76
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: C57BL/6J (BL6), Rag2 KO mice and K18-hACE2 mice [B6.Cg-Tg(K18-hACE2)2Prlmn/J] (6-8 weeks old) were purchased from the Jackson Laboratory.
    C57BL/6J
    suggested: None
    Rag2 KO
    suggested: RRID:NSRRC_0035)
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    B6.Cg-Tg(K18-hACE2)2Prlmn/J
    suggested: None
    Software and Algorithms
    SentencesResources
    Images were captured on a Zeiss LSM 880 confocal system (Zeiss, Oberkochen, Germany) and processed using ImageJ software (National Institutes of Health, Bethesda, MD).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Data from each NanoString panel were first processed independently using nSolver (v.4.0) software (NanoString) as follows: following quality control checks on the individual RCC files, raw counts across samples were normalized to the mean counts for spiked synthetic DNA-positive controls present in the hybridization reactions to mitigate platform-associated sources of variation.
    nSolver
    suggested: None
    Candidate reference genes were selected using the nCounter advanced analysis (nCAA) module (v.2.0.115), which implements the geNorm algorithm for downselection(52).
    geNorm
    suggested: (geNORM, RRID:SCR_006763)
    All analyzes were performed using Prism software.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Supplemental Materials and Methods:
    Methods
    suggested: None

    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 pages 33 and 37. 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.

  2. Version published to 10.1101/2020.07.09.195230 on bioRxiv