Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters

  1. Bertrand Bryche
  2. Audrey St Albin
  3. Severine Murri
  4. Sandra Lacôte
  5. Coralie Pulido
  6. Meriadeg Ar Gouilh
  7. Sandrine Lesellier
  8. Alexandre Servat
  9. Marine Wasniewski
  10. Evelyne Picard-Meyer
  11. Elodie Monchatre-Leroy
  12. Romain Volmer
  13. Olivier Rampin
  14. Ronan Le Goffic
  15. Philippe Marianneau
  16. Nicolas Meunier

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Abstract

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-COV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria .

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  1. ScreenIT

    SciScore for 10.1101/2020.06.16.151704: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingThis evaluation was performed by three experienced investigators blinded to the animal treatment.
    Power Analysisnot detected.
    Sex as a biological variableTwelve female golden Syrian hamsters (eight weeks old) were purchased from Janvier’s breeding Centre (Le Genest, St Isle, France).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The sections were then incubated overnight with primary antibodies directed against SARS Nucleocapsid Protein (1:200; rabbit polyclonal, NB100-56576, NOVUS, France); olfactory marker protein - OMP (1:500; goat polyclonal; 544-10001, WAKO, USA), cytokeratin-18 - CK18 (1:50; mouse polyclonal; MAB3234 – RGE53, Sigma-Aldrich, France), ionized calcium-binding adapter molecule 1 - Iba1 (1: 1000; goat polyclonal; ab178846, Abcam, France); G alpha s/olf - Golf (1:500; rabbit polyclonal; Santa Cruz Biotechnology (sc383), Dallas, TX, USA).
    SARS Nucleocapsid Protein
    suggested: (LSBio (LifeSpan Cat# LS-C59557-500, RRID:AB_1510641)
    cytokeratin-18 - CK18
    suggested: None
    ionized calcium-binding adapter molecule 1
    suggested: (Abcam Cat# ab178846, RRID:AB_2636859)
    Iba1
    suggested: (Abcam Cat# ab178846, RRID:AB_2636859)
    Fluorescence staining was performed using goat anti-rabbit Alexa-Fluor-488 (1:1000; Molecular Probes – A32731; Invitrogen, Cergy Pontoise, France), donkey anti-goat Alexa-Fluor-555 (1:1000; Molecular Probes A32816; Invitrogen, Cergy Pontoise, France) and donkey anti-mouse Alexa-Fluor 555 (1:500; Molecular Probes – A32773; Invitrogen, Cergy Pontoise, France) secondary antibodies.
    anti-rabbit
    suggested: (Molecular Probes Cat# A-21429, RRID:AB_2535850)
    anti-goat
    suggested: (Molecular Probes Cat# A-21432, RRID:AB_141788)
    anti-mouse
    suggested: (Molecular Probes Cat# A-21127, RRID:AB_141596)
    Experimental Models: Cell Lines
    SentencesResources
    A volume of 400 μl was homogenized and microfiltered (0.5 μm) previous to inoculation on Vero cells CCL-81 (passage 32, from ATCC, USA) grown at 80% confluence in a BSL3 virology laboratory (Virology Unit, CHU de Caen, France).
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Images were quantified using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2012) to threshold specific staining.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.06.16.151704 on bioRxiv