A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2

  1. Aurora Kraus
  2. Elisa Casadei
  3. Mar Huertas
  4. Chunyan Ye
  5. Steven Bradfute
  6. Pierre Boudinot
  7. Jean-Pierre Levraud
  8. Irene Salinas

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Abstract

The COVID-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with SARS-CoV-2 and allow rapid and high throughput testing of drugs and vaccines. Exposure of larvae to SARS-CoV-2 Spike (S) receptor binding domain (RBD) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ACE inhibitor, reverted this effect. Intranasal administration of SARS-CoV-2 S RBD in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. Zebrafish intranasally treated with SARS-CoV-2 S RBD became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. Single cell RNA-Seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. Combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of SARS-CoV-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model.

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    SciScore for 10.1101/2020.11.06.368191: (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 variableThere were no significant differences in olfactory responses between males and females, hence responses of both sexes were averaged together.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Electro-olfactogram recordings: Adult AB zebrafish were anesthetized and received 2 µl of recombinant SARS-CoV-2 S RBD protein (50 ng/µl) in PBS or PBS alone.
    AB
    suggested: RRID:BDSC_203)
    Software and Algorithms
    SentencesResources
    Sections were observed under a Nikon Ti inverted microscope (brightfield), and images were acquired with the Nikon Elements Advanced Research software and analyzed using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Gene ontology (GO) analysis was done with web-based GUIs Metascape and ShinyGO v0.61 which draw multiple currently maintained databases (ensembl, ENTRZ, KEGG among others).
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Biological process webs were created using biological process output from ShinyGO v0.61 in Prism GraphPad.
    ShinyGO
    suggested: (ShinyGO, RRID:SCR_019213)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Biological processes bar graphs were produced by Metascape.
    Metascape
    suggested: (Metascape, RRID:SCR_016620)
    Exposure of zebrafish larvae to SARS-CoV-2 S RBB recombinant protein and captopril treatment: 5 dpf or 7 dpf zebrafish (n=5/well, total of n=7-8 replicate wells) were placed in a 12 flat-bottomed well plate (FALCON) with 2 ml of tank water per well.
    FALCON
    suggested: (Falcon, RRID:SCR_016089)

    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: We detected the following sentences addressing limitations in the study:
    Zebrafish can overcome many of the limitations of mammalian models thanks to their transparent bodies, short life-span, low maintenance costs and production of large numbers of embryos. We therefore performed the simplest infection procedure, where SARS-CoV-2 was added to the water of zebrafish larvae. In this manner, BSL-3 trained personnel with no experience in zebrafish microinjection can readily expose larvae to SARS-CoV-2 without the need of animal protocols in a similar fashion to in vitro cell culture infections. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in the water did not however result in any detectable viral replication. This finding supports that, despite the general conservation of ACE2 molecules in vertebrates, the amino acid differences identified in zebrafish Ace2, are sufficient to prevent infectivity. Whether infection via microinjection as it was previously done in Influenza A infection models (Gabor et al., 2014) would result in SARS-CoV-2 replication remains to be elucidated. Further, our findings support that, similar to mice, the generation of transgenic larvae expressing human ACE2 may be the best approach in order to support active replication. Despite the absence of active viral replication, our model provided important insights into the transcriptional innate immune responses to SARS-CoV-2. Pathological cytokine responses are detected in severe disease COVID-19 patients highlighting the need to identify interventions that control such mal...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04355429Not yet recruitingEfficacy of Captopril in Covid-19 Patients With Severe Acute…

    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.

    About SciScore

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  3. Version published to 10.1101/2020.11.06.368191v1 on bioRxiv