Light Sheet Microscopy-Assisted 3D Analysis of SARS-CoV-2 Infection in the Respiratory Tract of the Ferret Model

  1. Luca M. Zaeck
  2. David Scheibner
  3. Julia Sehl
  4. Martin Müller
  5. Donata Hoffmann
  6. Martin Beer
  7. Elsayed M. Abdelwhab
  8. Thomas C. Mettenleiter
  9. Angele Breithaupt
  10. Stefan Finke

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Abstract

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host–pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (>150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and suggest clustering of infection foci in close proximity. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

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  1. Version published to 10.3390/v13030529
  2. ScreenIT

    SciScore for 10.1101/2020.10.17.339051: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and chemical reagents: For the detection of SARS-CoV-2 infection, a 1:1 mixture of hybridoma cell culture supernatants of anti-SARS-CoV-1 N mouse monoclonal antibody clones 4E10A3A1 (RRID:AB_2833160) and 4F3C4 (RRID:AB_2833162) [57] at a dilution of 1:5 or a polyclonal rabbit anti-SARS-CoV-1 N (RRID:AB_838838; Novus Biologicals, USA) at a dilution of 1:250 were used.
    anti-SARS-CoV-1 N
    detected: (Novus Cat# NB100-56576, RRID:AB_838838)
    clones
    detected: (Imported from the IEDB Cat# 4E10A3A1, RRID:AB_2833160)
    4F3C4
    detected: (Imported from the IEDB Cat# 4F3C4, RRID:AB_2833162)
    anti-SARS-CoV-1 N
    suggested: None
    Alexa Fluor™ 488/568/647-conjugated antibodies against mouse IgG and rabbit IgG were used as secondary antibodies (1:500; Invitrogen, USA).
    mouse IgG
    suggested: None
    rabbit IgG
    suggested: None
    Primary antibodies against SARS-CoV N were applied for 1 h at room temperature in 1% normal donkey serum/PBS-T, followed by three washes with PBS and incubation with the secondary antibody for 1 h at room temperature in 1% normal donkey serum/PBS-T.
    normal donkey serum/PBS-T
    suggested: None
    Following a blocking step with 6% normal donkey serum/0.2% Triton X-100/10% DMSO/PBS for 2 days at 37 °C, primary antibodies were diluted in 3% normal donkey serum/5% DMSO in PTwH (0.2% Tween-20 in PBS with 10 µg/mL heparin) and applied for 4 days at 37 °C.
    normal donkey serum/0.2
    suggested: None
    normal donkey serum/5
    suggested: None
    Secondary antibodies were diluted in 3% normal donkey serum/PTwH and the samples were incubated for another 4 days at 37 °C.
    normal donkey serum/PTwH
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Infected VeroE6 cells were then fixed 24 h post-infection with 4% paraformaldehyde (PFA) for 20 min.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Tissue samples of SARS-CoV-2-infected ferrets: In a previous study on experimental transmission of SARS-CoV-2 among different animal species, ferrets were inoculated intranasally with 105 TCID50 of SARS-CoV-2 isolate 2019_nCoV Muc-IMB-1 [30].
    Muc-IMB-1
    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 24 and 25. 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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  3. Version published to 10.1101/2020.10.17.339051 on bioRxiv