Influenza infection in ferrets with SARS-CoV-2 infection history

  1. Caroline Vilas Boas de Melo
  2. Florence Peters
  3. Harry van Dijken
  4. Stefanie Lenz
  5. Koen van de Ven
  6. Lisa Wijsman
  7. Angéla Gommersbach
  8. Tanja Schouten
  9. Puck B. van Kasteren
  10. van den Brand Judith
  11. Jørgen de Jonge

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Abstract

Non-pharmaceutical interventions (NPIs) to contain the SARS-CoV-2 pandemic drastically reduced human-to-human interactions, decreasing the circulation of other respiratory viruses as well. As a consequence, influenza virus circulation – normally responsible for 3-5 million hospitalizations per year globally – was significantly reduced. With downscaling the NPI countermeasures, there is a concern for increased influenza disease, particularly in individuals suffering from post-acute effects of SARS-CoV-2 infection. To investigate this possibility, we performed a sequential influenza H1N1 infection 4 weeks after an initial SARS-CoV-2 infection in the ferret model. Upon H1N1 infection, ferrets that were previously infected with SARS-CoV-2 showed an increased tendency to develop clinical symptoms compared to the control H1N1 infected animals. Histopathological analysis indicated only a slight increase for type II pneumocyte hyperplasia and bronchitis. The effects of the sequential infection thus appeared minor. However, ferrets were infected with B.1.351-SARS-CoV-2, the beta variant of concern, which replicated poorly in our model. The histopathology of the respiratory organs was mostly resolved 4 weeks after SARS-CoV-2 infection, with only reminiscent histopathological features in the upper respiratory tract. Nevertheless, SARS-CoV-2 specific cellular and humoral responses were observed, confirming an established infection. Thus, there may likely be a SARS-CoV-2 variant-dependent effect on the severity of disease upon a sequential influenza infection as we observed mild effects upon a mild infection. It, however, remains to be determined what the impact is of more virulent SARS-CoV-2 variants.

Importance

During the COVID-19 pandemic, the use of face masks, social distancing and isolation were not only effective in decreasing the circulation of SARS-CoV-2, but also in reducing other respiratory viruses such as influenza. With less restrictions, influenza is slowly returning. In the meantime, people still suffering from long-COVID, could be more vulnerable to an influenza virus infection and develop more severe influenza disease. This study provides directions to the effect of a previous SARS-CoV-2 exposure on influenza disease severity in the ferret model. This model is highly valuable to test sequential infections under controlled settings for translation to humans. We could not induce clear long-term COVID-19 effects as SARS-CoV-2 infection in ferrets was mild. However, we still observed a slight increase in influenza disease severity compared to ferrets that had not encountered SARS-CoV-2 before. It may therefore be advisable to include long-COVID patients as a risk group for influenza vaccination.

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  1. ScreenIT

    SciScore for 10.1101/2022.03.22.485425: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: The study proposal was evaluated and approved by the Animal Welfare Body of Poonawalla Science Park – Animal Research Center (Bilthoven, The Netherlands) under permit number AVD32600 2018 4765 of the Dutch Central Committee for Animal experiments.
    IACUC: The study proposal was evaluated and approved by the Animal Welfare Body of Poonawalla Science Park – Animal Research Center (Bilthoven, The Netherlands) under permit number AVD32600 2018 4765 of the Dutch Central Committee for Animal experiments.
    Euthanasia Agents: Euthanasia was performed by exsanguination via heart puncture under anesthesia with ketamine and medetomidine.
    Sex as a biological variableFerrets: Twenty outbred male ferrets (Mustela putorius furo), aged 8 months, were obtained from the colony of Euroferret (Denmark).
    Randomizationnot detected.
    BlindingHistopathology was scored blindly by a veterinary pathologist.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    All ferrets tested negative for antibody responses measured against SARS-CoV-2 spike protein (S), its receptor binding domain (RBD), and the nuclear protein of influenza virus by ELISA.
    SARS-CoV-2 spike protein (S), its receptor binding domain (RBD)
    suggested: None
    Fixation of the cells occurred with incubation of Fixation/Permeabilization buffer for 20 min/4°C, followed by two washing steps with 1x permbuffer, prior to incubation for 30 min/RT with the antibodies: FITC-conjugated anti-CD3e (MCA1477A647, clone CD3-12, BioRad) and PE- conjugated anti-IFNγ (MCA1783PE, clone CC302, Bio-rad).
    anti-CD3e
    suggested: (Creative Diagnostics Cat# CABT-48633RH, RRID:AB_2528922)
    anti-IFNγ
    suggested: None
    MCA1783PE
    suggested: (Bio-Rad Cat# MCA1783PE, RRID:AB_324003)
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 virus culture was performed in VERO E6 cells (Vero C1008, ATCC CRL-1586), first grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% of fetal bovine serum (FBS) + 1x penicillin, streptomycin and glutamine (PSG) at 37°C and 5% CO2.
    Vero
    suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
    C1008
    suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
    MDCK cells were washed twice with PBS, and H1N1 virus suspension was added to the cells in MEM + 1x PSG + 2 µg/ml L-
    MDCK
    suggested: None
    TCID50 determination: SARS-CoV-2 virus stock, swab material and tissue samples were 1:10 serial diluted in DMEM medium containing 2% FBS and 1x PSG and added on VERO E6 cells in 96-well plates.
    VERO E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Cell acquisition was performed on the BD LSRFortessa™ Cell Analyzer (BD Biosciences) and analyzed using FlowJo V10.6.2 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: All data were analyzed with GraphPad Prism 9.1.0 software (GraphPad Software, Inc).
    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: 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.

    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.

  2. Version published to 10.1101/2022.03.22.485425v1 on bioRxiv