Pathology and Immunity After SARS-CoV-2 Infection in Male Ferrets Is Affected by Age and Inoculation Route

  1. Koen van de Ven
  2. Harry van Dijken
  3. Lisa Wijsman
  4. Angéla Gomersbach
  5. Tanja Schouten
  6. Jolanda Kool
  7. Stefanie Lenz
  8. Paul Roholl
  9. Adam Meijer
  10. Puck B. van Kasteren
  11. Jørgen de Jonge

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nasal turbinates was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.

Article activity feed

  1. Version published to 10.3389/fimmu.2021.750229
  2. ScreenIT

    SciScore for 10.1101/2021.06.30.450298: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: The experiment was approved by the local Authority for Animal Welfare of the Antonie van Leeuwenhoek terrain (Bilthoven, The Netherlands) under permit number AVD3260020184765 of the Dutch Central Committee for Animal experiments.
    IACUC: The experiment was approved by the local Authority for Animal Welfare of the Antonie van Leeuwenhoek terrain (Bilthoven, The Netherlands) under permit number AVD3260020184765 of the Dutch Central Committee for Animal experiments.
    Sex as a biological variableAnimal handling: Young (9-10 months) and adult (36-48 months) outbred male ferrets (Schimmel b.v.,
    RandomizationDue to strict hierarchy present in male ferret groups, we were unable to randomly allocate individual animals to treatment groups.
    BlindingMicroscopic slides were randomized and scored blindly by an experienced pathologist.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Almost all animals displayed prior exposure against CDV and multiple animals tested positive for antibodies against NL63, FCoV and CCoV before start of the experiment (Supplementary table 1).
    NL63
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 virus was originally isolated from a Dutch patient (hCoV-19/Netherlands/ZuidHolland_10004/2020) and grown on VERO E6 cells in infection medium consisting of DMEM, 1x penicillin-streptomycin-glutamine and either 0% or 2% FBS.
    VERO E6
    suggested: RRID:CVCL_XD71)
    Upon >90% CPE, the supernatant of the infected Vero cells was centrifuged at 500x g, filtered (5um pore size) and aliquots were stored at −80°C.
    Vero
    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: We detected the following sentences addressing limitations in the study:
    Due to the global circumstances at the time of this study, there are several caveats present in the experimental set-up. The availability of (male) ferrets was limited and hence we could not investigate all groups at every timepoint. In addition, several ferrets were previously exposed to Aleutian disease and coronaviruses other than SARS-CoV-2 (details in Supplemental data). Although we did not find evidence that this infection history influenced our results, we cannot fully exclude that possibility. Finally, due to the limited availability of animals it will be difficult to model SARS-CoV-2 infections in older ferrets. However, as we did not find strong differences between young and adult ferrets upon intranasal inoculation, the use of young ferrets should suffice for future experiments. As has been discussed here, ferrets are readily infected with SARS-CoV-2 but do not present clinical symptoms. Infected ferrets might thus represent the asymptomatic COVID-19 that manifests in a significant part of the population 55. In addition, the derailed immune responses in the respiratory tract in ferrets might model the long-term respiratory effects observed in long-COVID patients, although more in-depth research is required to verify this. Lastly, with the recently developed reagents for humoral and cellular immunology in the ferret model 56–60, vaccine-induced immune responses can be quantified and their effect on viral replication and pathology can be measured. This matured ferret...

    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 26. 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.
    • No funding statement was detected.
    • 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 10.1101/2021.06.30.450298v1 on bioRxiv