Mild and severe SARS-CoV-2 infection induces respiratory and intestinal microbiome changes in the K18-hACE2 transgenic mouse model

  1. Brittany Seibert
  2. C. Joaquín Cáceres
  3. Stivalis Cardenas-Garcia
  4. Silvia Carnaccini
  5. Ginger Geiger
  6. Daniela S. Rajao
  7. Elizabeth Ottesen
  8. Daniel R. Perez

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and declining economies around the world. K18-hACE2 mice develop disease resembling severe SARS-CoV-2 infection in a virus dose-dependent manner. The relationship between SARS-CoV-2 and the intestinal or respiratory microbiome is not fully understood. In this context, we characterized the cecal and lung microbiome of SARS-CoV-2 challenged K18-hACE2 transgenic mice in the presence or absence of treatment with the M pro inhibitor GC376. Cecum microbiome showed decreased Shannon and Inv Simpson diversity index correlating with SARS-CoV-2 infection dosage and a difference of Bray-Curtis dissimilarity distances among control and infected mice. Bacterial phyla such as Firmicutes, particularly Lachnospiraceae and Oscillospiraceae, were significantly less abundant while Verrucomicrobiota, particularly the family Akkermansiaceae, were increasingly more prevalent during peak infection in mice challenged with a high virus dose. In contrast to the cecal microbiome, the lung microbiome showed similar microbial diversity among the control, low and high challenge virus groups, independent of antiviral treatment. Bacterial phyla in the lungs such as Bacteroidota decreased while Firmicutes and Proteobacteria were significantly enriched in mice challenged with a high dose of SARS-CoV-2. In summary, we identified changes in the cecal and lung microbiome of K18-hACE2 mice with severe clinical signs of SARS-CoV-2 infection.

IMPORTANCE

The COVID-19 pandemic has resulted in millions of deaths. The host’s respiratory and intestinal microbiome can affect directly or indirectly the immune system during viral infections. We characterized the cecal and lung microbiome in a relevant mouse model challenged with a low and high dose of SARS-CoV-2 in the presence or absence of an antiviral M pro inhibitor, GC376. Decreased microbial diversity and taxonomic abundances of the phyla Firmicutes, particularly Lachnospiraceae, correlating with infection dosage was observed in the cecum. In addition, microbes within the family Akkermansiaceae were increasingly more prevalent during peak infection, which is observed in other viral infections. The lung microbiome showed similar microbial diversity to the control, independent of antiviral treatment. Decreased Bacteroidota and increased Firmicutes and Proteobacteria were observed in the lungs in a virus dose-dependent manner. These studies add to a better understanding of the complexities associated with the intestinal microbiome during respiratory infections.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.04.20.440722: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Ethics statement: Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia (Protocol A2019-03-032-Y1-A3) and performed following the IACUC Guidebook of the Office of Laboratory Animal Welfare and PHS policy on Humane Care and of Use of Laboratory Animals.
    Field Sample Permit: Studies were performed in an animal Biosafety level 3 containment facility at the Animal Health Research Center (AHRC) at the University of Georgia. Cells and Virus: The SARS-CoV-2 (Isolate USA-WA1/2020) isolate, kindly provided by Dr. S. Mark Tompkins, Department of Infectious Diseases, University of Georgia, was used for virus challenge in the animal studies.
    Sex as a biological variableMouse experiments: Female K18-hACE2 mice (6 weeks old) were randomly distributed into six groups (n=6/group for controls and n=9/group for challenged), anesthetized and challenged intranasally with 50 µL of phosphate buffer saline (PBS), 1×103 TCID50/mouse (Low virus dose) or 1×105 TCID50/mouse (High virus dose).
    RandomizationMouse experiments: Female K18-hACE2 mice (6 weeks old) were randomly distributed into six groups (n=6/group for controls and n=9/group for challenged), anesthetized and challenged intranasally with 50 µL of phosphate buffer saline (PBS), 1×103 TCID50/mouse (Low virus dose) or 1×105 TCID50/mouse (High virus dose).
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Briefly, the virus was grown in Vero E6 Pasteur cells provided by Maria Pinto (Center for Virus research, University of Glasgow, Scotland, UK), and maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich, St Louis, MO) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO), 1% antibiotic/antimycotic (AB, Sigma-Aldrich, St Louis, MO) and 1% L-Glutamine (Sigma-Aldrich, St Louis, MO).
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse experiments: Female K18-hACE2 mice (6 weeks old) were randomly distributed into six groups (n=6/group for controls and n=9/group for challenged), anesthetized and challenged intranasally with 50 µL of phosphate buffer saline (PBS), 1×103 TCID50/mouse (Low virus dose) or 1×105 TCID50/mouse (High virus dose).
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Amplicon concentration was measured using the Qubit dsDNA HS Assay kit (ThermoFisher, Waltham, MA) on the Qubit 3.0 fluorometer (ThermoFisher, Waltham, MA).
    Amplicon
    suggested: (Amplicon, RRID:SCR_003294)
    Fragment size distribution was analyzed on a subset of samples using the Agilent Bioanalyzer 2100 DNA-HS assay (Agilent, Santa Clara, CA, USA).
    Agilent Bioanalyzer
    suggested: None
    Taxonomy was assigned in ‘dada2’ using the native implementation of the naïve Bayesian classifier using Silva v.
    Silva
    suggested: (SILVA, RRID:SCR_006423)
    Hierarchal cluster analysis of the Bray-Curtis distances was created using command hclust with agglomeration method “average” (UPGMA) producing a cophenetic correlation coefficient of 0.79.
    hclust
    suggested: (HCLUST, RRID:SCR_009154)
    The dendrogram was created using the function plot and shading/group colors were added using Adobe Illustrator (Version 25.0.1)
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Some limitations of this study must be noted. While the environment was stable and controlled, the sample size for each group at each time point was small and the potential contribution of cage effect on the microbiome was not analyzed. The high mortality observed in the high virus dose groups only allowed for collection of two time points (2 and 5 dpc, only one sample was collected at 14 dpc from the only survivor in the High/GC-376 group). As indicated above, ceca samples were only collected for mice not treated with GC-376. In contrast, lung samples from mock inoculation/vehicle treated control mice did not yield sufficient amplifiable microbial DNA for sequencing, so the comparison focused on SARS-CoV-2 dose-dependent responses in GC-376 treated mice. The microbiome of the cecum showed significant decreases in Shannon and Inv Simpson index comparing the control to the low-dose and high-dose infected groups (Fig. 2B and 2C). The low virus dose group shared a higher number of ASVs with the control group compared to the high virus dose group (Fig. 2D). These observations suggest a virus dose-dependent effect of the ceca microbial diversity in mice infected with SARS-CoV-2. While preparing the manuscript, a report was published that analyzed the small intestine microbiome of hACE2 mice among unvaccinated and vaccinated mice challenged with a high dose of SARS-CoV-2 (47). While we compare control mice to low and high doses of non-vaccinated mice challenged with SARS-CoV-2 in t...

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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

    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 10.1101/2021.04.20.440722 on bioRxiv