Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium

  1. Aleksandra Milewska
  2. Anna Kula-Pacurar
  3. Jakub Wadas
  4. Agnieszka Suder
  5. Artur Szczepanski
  6. Agnieszka Dabrowska
  7. Katarzyna Owczarek
  8. Alessandro Marcello
  9. Marek Ochman
  10. Tomasz Stacel
  11. Zenon Rajfur
  12. Marek Sanak
  13. Pawel Labaj
  14. Wojciech Branicki
  15. Krzysztof Pyrc

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.

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  1. Version published to 10.1128/jvi.00957-20
  2. ScreenIT

    SciScore for 10.1101/2020.03.01.971499: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The study was approved by the Bioethical Committee of the Medical University of Silesia in Katowice, Poland (approval no: KNW/0022/KB1/17/10 dated 16.02.2010).
    Consent: Written consent was obtained from all patients.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    A mouse monoclonal anti-TMPRSS2 antibody (clone P5H9-A3; 1:500 dilution; Sigma-Aldrich, Poland), followed by incubation with a horseradish peroxidase-labeled anti-mouse IgG (65 ng/ml; Dako, Denmark) diluted in 5% skimmed milk / TBS-Tween (0.1%).
    anti-TMPRSS2
    suggested: (Santa Cruz Biotechnology Cat# sc-101847, RRID:AB_2205599)
    anti-mouse IgG
    suggested: None
    A horseradish peroxidase-labeled anti-His tag antibody (1:25000 dilution; Sigma-Aldrich, Poland) diluted in 5% skimmed milk / TBS-Tween (0.1%) was used to detect the His-tagged HmuY proteins.
    anti-His tag
    suggested: None
    anti-His tag antibody
    suggested: None
    A horseradish peroxidase-labeled anti-His tag antibody (1:25000 dilution; Sigma-Aldrich, Poland) diluted in 5% skimmed milk / TBS-Tween (0.1%) was used to detect the His-tagged HmuY proteins.
    anti-His tag
    suggested: None
    anti-His tag antibody
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    RD cells grown in 90% confluency were infected with HCoV-HKU1 (108 RNA copies per ml) in Dulbecco’s MEM (Thermo Fisher Scientific, Poland
    RD
    suggested: None
    Lentivirus production and transduction: 293T cells were seeded on 10 cm2 dishes, cultured for 24 h at 37°C with 5% CO2 and transfected with psPAX, pMD2G and third transfer plasmid (pWPI/KLK13, pLKO.1-TRC/shrnaKLK13 or Lego-G2) using polyethyleneimine (Sigma-Aldrich, Poland). psPAX
    293T
    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:
    As CleavEx technique is a convenient surrogate system allowing for precise mapping of the cleavage site, it has some limitations. To ensure the reliability of results, purified full-length HCoV-HKU1 S protein was subjected to the proteolytic cleavage. Also here we observed efficient cleavage of the HCoV-HKU1 S protein by KLK13. While the results presented here show that KLK13 is able to process the HCoV-HKU1 S protein, one may question whether the cleavage is sufficient for HCoV-HKU1 entry. It was previously presented for MERS-CoV that two consecutive enzymatic scissions are required for activation of the S protein. In this scenario, KLK13 would prime the HCoV-HKU1 S at S1-S2 site, enabling scission at S2-S2’ site by the TMPRSS2 or another host protease (38, 40, 93). This may be one of the factors limiting the HCoV-HKU1 replication in RD_KLK13 cells, as only minimal replication is observable. Summarizing, we show that KLK13 is a key determinant of HCoV-HKU1 tropism. This may explain why, since its first identification in 2004, all efforts to culture HCoV-HKU1 in standard cell lines have failed. We believe that this study increases our knowledge of HCoV-HKU1 and may promote the future in-depth investigation of coronaviruses. Considering the increasing number and diversity of coronaviruses, and the proven propensity of coronaviruses to cross the species barrier and cause severe diseases in humans, further research on the role of different proteases in coronaviral infections is ...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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

    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 10.1101/2020.03.20.999029v1 on bioRxiv