Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host-factors having implication in the disease pathogenesis and severity

  1. Voddu Suresh
  2. Varshasnata Mohanty
  3. Kiran Avula
  4. Arup Ghosh
  5. Bharti Singh
  6. R. Rajendra Kumar Reddy
  7. Amol Ratnakar Suryawanshi
  8. Sunil K. Raghav
  9. Soma Chattopadhyay
  10. Punit Prasad
  11. Rajeeb Kumar Swain
  12. Rupesh Dash
  13. Ajay Parida
  14. Gulam Hussain Syed
  15. Shantibhusan Senapati

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Abstract

Syrian golden hamsters ( Mesocricetus auratus ) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology that resembles human COVID-19 patients. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo . Analysis of clinical parameters and tissue samples shows a similar type of pathophysiological manifestation of SARS-CoV-2 infection as reported earlier in COVID-19 patients and hamsters infected with other isolates. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14dpi. Here, we carried out quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters at day 4 and day 14 post infection. This resulted in the identification of 1,585 differentially expressed proteins of which 68 proteins were significantly altered among both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in the identification of candidate biomarkers and understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.09.434371: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All the experiments were done with prior approval of Institutional Biosafety Committee (IBSC) and Institutional Animal Ethical Committee (IAEC).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Horse serum (Vector Laboratories) was used for blocking the sections for 30 min at room temperature and incubated with Ki-67 antibody (#VP-RM04; Vector Laboratories, 1:100) overnight at 4°C.
    Ki-67
    suggested: None
    #VP-RM04
    suggested: (Vector Laboratories Cat# VP-RM04, RRID:AB_2336545)
    Sections were washed twice with 1x PBS and treated with biotinylated anti-rabbit/mouse IgG secondary antibody (Vector Laboratories) for 45 minutes, followed by ABC reagent for 30 min.
    anti-rabbit/mouse IgG
    suggested: None
    The whole-cell lysates (WCL) were subjected to SDS-PAGE and transferred to nitrocellulose membrane (Thermo Scientific), followed by blocking and immunoblotting with antibodies specific for SARS-CoV-2 Nucleocapsid protein (#11-2003, Abgenex) or β-actin (#4970, CST) [15]. qRT-PCR: RNA isolation from culture supernatant was performed using QIAamp Viral RNA Mini Kit (#52906, Qiagen), and for hamster tissue samples, TRizol reagent (#10296010, Invitrogen) was used.
    SARS-CoV-2 Nucleocapsid protein (#11-2003, Abgenex)
    suggested: None
    β-actin
    suggested: None
    Software and Algorithms
    SentencesResources
    Bioinformatics and statistical analysis: The Proteome Discoverer 2.3 (Thermo Scientific, Bremen, Germany) was used to carry out protein identification and quantitation.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Gene Ontology (GO) and pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed using the EnrichR online tool http://amp.pharm.mssm.edu/Enrichr/) [19].
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    EnrichR
    suggested: (Enrichr, RRID:SCR_001575)
    Data availability: The raw data files and the MSF files were submitted to the PRIDE partner repository [20] with dataset identifier PXD024547.
    PRIDE
    suggested: (Pride-asap, RRID:SCR_012052)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. 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.
    • 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 10.1101/2021.03.09.434371 on bioRxiv