Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19

  1. Jordan J. Clark
  2. Rebekah Penrice-Randal
  3. Parul Sharma
  4. Anja Kipar
  5. Xiaofeng Dong
  6. Shaun H. Pennington
  7. Amy E. Marriott
  8. Stefano Colombo
  9. Andrew Davidson
  10. Maia Kavanagh Williamson
  11. David A. Matthews
  12. Lance Turtle
  13. Tessa Prince
  14. Grant L. Hughes
  15. Edward I. Patterson
  16. Ghada Shawli
  17. Daniele F. Mega
  18. Krishanthi Subramaniam
  19. Jo Sharp
  20. Lynn McLaughlin
  21. En-Min Zhou
  22. Joseph D. Turner
  23. Giancarlo Biagini
  24. Andrew Owen
  25. Julian A. Hiscox
  26. James P. Stewart

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Abstract

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that rapidly caused a pandemic. Coalescence of this virus with seasonal respiratory viruses, particularly influenza virus is a global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, they exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by infection with IAV is responsible for the observed inhibition of SARS-CoV-2, however, infection with attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest that the concept of ‘twinfection’ is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.

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  1. Version published to 10.1101/2020.10.13.334532v3 on bioRxiv
  2. Version published to 10.1101/2020.10.13.334532v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.10.13.334532: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics and clinical information: The patient from which the virus sample was obtained gave informed consent and was recruited under the International Severe Acute Respiratory and emerging Infection Consortium (
    IACUC: Mice: Animal work was approved by the local University of Liverpool Animal Welfare and Ethical Review Body and performed under UK Home Office Project Licence PP4715265.
    RandomizationVirus infection: Animals were randomly assigned into multiple cohorts.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following primary antibodies were applied: rabbit anti-human ACE2 (Novus Biologicals; clone SN0754; NBP2-67692), goat anti-IAV (Meridian Life Sciences Inc., B65141G), rabbit anti-Iba-1 (antigen: AIF1; Wako Chemicals; macrophage marker)
    anti-human ACE2
    suggested: None
    anti-Iba-1
    suggested: None
    Briefly, after deparaffination, sections underwent antigen retrieval in citrate buffer (pH 6.0; Agilent) for ACE2 and SARS-CoV NP detection, and Tris/EDTA buffer (pH 9) for IAV for 20 min at 98 °C, followed by incubation with the primary antibodies (diluted in dilution buffer, Agilent; anti-IAV 1:200
    anti-IAV
    suggested: None
    This was followed by blocking of endogenous peroxidase (peroxidase block, Agilent) for 10 min at RT and incubation with the secondary antibodies, EnVision+/HRP, Rabbit (Agilent) for ACE2 and SARS-CoV, and rabbit anti-goat Ig/HRP (Agilent) for IAV), for 30 min at RT, and EnVision FLEX DAB+ Chromogen in Substrate buffer (Agilent) for 10 min at RT, all in an autostainer (Dako).
    SARS-CoV
    suggested: None
    anti-goat Ig/HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Titres were determined by an influenza plaque assay using MDCK cells.
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    UK strain of SARS-CoV-2 (hCoV-2/human/Liverpool/REMRQ0001/2020), which was cultured from a nasopharyngeal swab from a patient, was passaged a further 4 times in Vero E6 cells 24.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice carrying the human ACE2 gene under the control of the keratin 18 promoter (K18-hACE2; formally B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from Jackson Laboratories.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Lungs and brain from an age-matched wild type BALB-C mouse stained for ACE2 served to assess any differences in the ACE expression pattern in the transgenic mice, and two wild type C57BL6/J mice infected intranasally with 102 PFU IAV X31 in 50 μl sterile PBS and sacrificed at 6 days post infection served to assess any effect of hACE2 expression in the course of IAV infection.
    C57BL6/J
    suggested: None
    Software and Algorithms
    SentencesResources
    The Illumina reads were mapped to the England/2/2020 genome using HISAT and the consensus genome was called using an in-house script based on the dominant nucleotide at each location on the genome.
    HISAT
    suggested: (HISAT2, RRID:SCR_015530)
    mAB rabbit anti-mouse CD3 (clone SP7: Spring Bioscience, Ventana Medical Systems, Tucson, USA; T cell marker) and rat anti-mouse CD45R (clone B220, BD Biosciences; B cell marker), following previously published protocols 46–48.
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Alignment files were sorted and indexed with samtools before counting reads using Salmon with the corresponding annotation file (Mus_musculus.GRCm38.gtf) from Ensembl using –noErrorModel -l U parameters 26,50.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Salmon
    suggested: (Salmon, RRID:SCR_017036)
    Ensembl
    suggested: (Ensembl, RRID:SCR_002344)
    The edgeR package was used to normalise sequencing libraries and identify differentially expressed genes, defined as at least a 2-fold difference from the mock infected group (n=5) and a false discovery rate (FDR) less than 0.05 51.
    edgeR
    suggested: (edgeR, RRID:SCR_012802)
    Principle component Analysis (PCA), volcano plots, heatmaps and Venn diagrams were produced in R studio using the following packages: edgeR, ggplot2 and pheatmap.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Differential gene expression data was used for gene ontology enrichment analysis of biological process terms in each group using enrichGO in the ClusterProfiler programme in R 52.
    ClusterProfiler
    suggested: (clusterProfiler, RRID:SCR_016884)

    Results from OddPub: Thank you for sharing your data.

    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.

    About SciScore

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  4. Version published to 10.1101/2020.10.13.334532v1 on bioRxiv