Administration of aerosolized SARS-CoV-2 to K18-hACE2 mice uncouples respiratory infection from fatal neuroinvasion

  1. Valeria Fumagalli
  2. Micol Ravà
  3. Davide Marotta
  4. Pietro Di Lucia
  5. Chiara Laura
  6. Eleonora Sala
  7. Marta Grillo
  8. Elisa Bono
  9. Leonardo Giustini
  10. Chiara Perucchini
  11. Marta Mainetti
  12. Alessandro Sessa
  13. José M. Garcia-Manteiga
  14. Lorena Donnici
  15. Lara Manganaro
  16. Serena Delbue
  17. Vania Broccoli
  18. Raffaele De Francesco
  19. Patrizia D’Adamo
  20. Mirela Kuka
  21. Luca G. Guidotti
  22. Matteo Iannacone

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Abstract

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2–infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

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  1. Version published to 10.1126/sciimmunol.abl9929
  2. ScreenIT

    SciScore for 10.1101/2021.08.06.455382: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces.
    Euthanasia Agents: Mice were euthanized by cervical dislocation.
    Sex as a biological variableSocial scent-discrimination test: The social scent-discrimination test was used to assess hyposmia/anosmia in IN- infected or AR-infected male mice compared to PBS-treated control male mice, as previously described (Zheng et al., 2021).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    In vivo treatment: In selected experiments, K18-hACE2 mice were injected intraperitoneally with 2 mg per mouse of α-IFNAR1 blocking antibody (BioXcell, #BE0241, clone MAR1-5A3) 1 day before infection.
    α-IFNAR1
    suggested: None
    #BE0241
    suggested: (Bio X Cell Cat# BE0241, RRID:AB_2687723)
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 cells were seeded into 96 wells plates and infected at 95% of confluency with base 10 dilutions of virus stock.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (referred to in the text as K18-hACE2) were purchased from The Jackson Laboratory.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    As control, K18-hACE2 mice received the same volume of aerosolized PBS (125 μL per mouse).
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Flow cytometry analysis was performed on BD FACS Symphony A5 SORP and analyzed with FlowJo software (Treestar)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    RNA-seq data processing and analysis: Raw reads were aligned to mouse genome build GRCm38 using STAR aligner (Dobin et al., 2013)
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Gene counts were generated using featureCounts (part of the Subread package (Liao et al., 2019)), based on GENCODE gene annotation version M22.
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    Differentially Expressed Genes (DEGs) between AR- and IN-infected mice were identified by generating a linear model using LIMMA R package (Ritchie et al., 2015) (
    LIMMA
    suggested: (LIMMA, RRID:SCR_010943)
    Bright-field images were acquired with an Aperio Scanscope System CS2 microscope and the ImageScope program (Leica Biosystem) following the manufacturer’s instructions.
    ImageScope
    suggested: (ImageScope, RRID:SCR_014311)
    , anti-Iba1 (Synaptic system, #234006), anti-CD68 (Abcam #ab53444), and anti-iNOS (Abcam #ab3523).
    Synaptic system
    suggested: (Synaptic Systems, RRID:SCR_013612)
    Flow and imaging data were collected using FlowJo Version 10.5.3 (Treestar) and Imaris (Bitplane), respectively.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    Statistical analyses were performed with GraphPad Prism software version 8 (GraphPad).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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:
    • 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.

    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 10.1101/2021.08.06.455382v1 on bioRxiv