Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression

  1. Mariano Carossino
  2. Paige Montanaro
  3. Aoife O’Connell
  4. Devin Kenney
  5. Hans Gertje
  6. Kyle A. Grosz
  7. Maria Ericsson
  8. Bertrand R Huber
  9. Susanna A. Kurnick
  10. Saravanan Subramaniam
  11. Thomas A. Kirkland
  12. Joel R. Walker
  13. Kevin P. Francis
  14. Alexander D. Klose
  15. Neal Paragas
  16. Markus Bosmann
  17. Mohsan Saeed
  18. Udeni B. R. Balasuriya
  19. Florian Douam
  20. Nicholas A. Crossland

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Abstract

Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCE

COVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis.

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  1. Version published to 10.1101/2021.01.13.425144v3 on bioRxiv
  2. Version published to 10.1101/2021.01.13.425144v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.01.13.425144: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Biosafety: All aspects of this study were approved by the Institutional Biosafety Committee and the office of Environmental Health and Safety at Boston University prior to study initiation.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableIntranasal inoculation with SARS-CoV-2: At 4 months of age, K18-hACE2 mice of both sexes were intranasally inoculated with 1 × 106□PFU of SARS-CoV-2 in 50 µl of sterile 1X PBS (n=47 [n=25 male and n=12 female), or sham inoculated with 50 µl of sterile 1X PBS (n=3; female).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    To generate the passage 1 (P1) virus stock, Vero E6 cells, pre-seeded the day before at a density of 10 million cells, were infected in T175 flasks with the master stock, diluted in 10 ml final volume of Opti-MEM (ThermoFisher Scientific).
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Heterozygous K18-hACE2 C57BL/6J mice of both sexes (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from the Jackson Laboratory (Jax, Bar Harbor, ME).
    C57BL/6J
    suggested: RRID:MGI:3589388)
    Intranasal inoculation with SARS-CoV-2: At 4 months of age, K18-hACE2 mice of both sexes were intranasally inoculated with 1 × 106□PFU of SARS-CoV-2 in 50 µl of sterile 1X PBS (n=47 [n=25 male and n=12 female), or sham inoculated with 50 µl of sterile 1X PBS (n=3; female).
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Furthermore, an unstained section was used to create an autofluorescence signature that was subsequently removed from whole-slide images using InForm software version 2.4.8 (Akoya Biosciences).
    InForm
    suggested: (inForm, RRID:SCR_019155)
    Statistical analysis: Descriptive statistics and graphics as well as Kaplan-Meier (survival) curves and statistical tests were performed using GraphPad Prism statistical analysis software (GraphPad, San Diego, CA).
    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 53. 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.

  4. Version published to 10.1101/2021.01.13.425144v1 on bioRxiv