Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

  1. Jennifer Harcourt
  2. Azaibi Tamin
  3. Xiaoyan Lu
  4. Shifaq Kamili
  5. Senthil Kumar. Sakthivel
  6. Janna Murray
  7. Krista Queen
  8. Ying Tao
  9. Clinton R. Paden
  10. Jing Zhang
  11. Yan Li
  12. Anna Uehara
  13. Haibin Wang
  14. Cynthia Goldsmith
  15. Hannah A. Bullock
  16. Lijuan Wang
  17. Brett Whitaker
  18. Brian Lynch
  19. Rashi Gautam
  20. Craig Schindewolf
  21. Kumari G. Lokugamage
  22. Dionna Scharton
  23. Jessica A. Plante
  24. Divya Mirchandani
  25. Steven G. Widen
  26. Krishna Narayanan
  27. Shinji Makino
  28. Thomas G. Ksiazek
  29. Kenneth S. Plante
  30. Scott C. Weaver
  31. Stephen Lindstrom
  32. Suxiang Tong
  33. Vineet D. Menachery
  34. Natalie J. Thornburg

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Abstract

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures.

Article Summary

Scientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.

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  1. ScreenIT

    SciScore for 10.1101/2020.03.02.972935: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell culture, limiting dilution, and isolation: Vero CCL-81 cells were used for isolation and initial passage.
    Vero CCL-81
    suggested: None
    Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells were cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with heat inactivated fetal bovine serum(5 or 10%) and antibiotic/antimyotic (GIBCO).
    Vero E6
    suggested: None
    Vero
    suggested: None
    293T
    suggested: None
    A549
    suggested: None
    EFKB3
    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: 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:
    • 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. ScreenIT

    SciScore for 10.1101/2020.03.02.972935: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Overnight incubation with primary antibody, either rabbit polyclonal sera against the SARS-CoV spike (Sino Biological #40150-T52), β-Actin antibody (Cell Signaling Technology #4970), or a custom rabbit polyclonal sera against SARS-CoV nucleocapsid, was then performed.
    β-Actin
    suggested: None
    The renatured, full-length, SARS-CoV N protein was used to immunize rabbits to generate an affinity-purified rabbit anti-SARS-CoV N polyclonal antibody.
    anti-SARS-CoV
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin.
    Vero-CCL81
    suggested: None
    To establish a plaqu assay and determine the preferred Vero cell type for quantification, we titered our passage four stock on VeroE6 and VeroCCL81.
    Vero
    suggested: None
    In contrast, Vero CCL81 produced less clear plaques and was most easily quantitated with neutral red 3 days post inoculation.
    Vero CCL81
    suggested: None
    On the individual plaque monolayers, SARS-CoV-2 infection of Vero E6 cells produced cytopathic effect with areas of cell clearance (Figure 2C).
    Vero E6
    suggested: None
    These results are consistent with previous susceptibility findings for SARS-CoV and suggest other common culture systems including MDCK, HeLa, HEP-2, MRC-5 cells, and embryonated eggs are unlikely to support SARS-CoV-2 replication (14-16).
    MDCK
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>HeLa</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>MRC-5</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Consistent with the replication results (Figure 3A), SARS-CoV-2 showed robust N protein in both Vero cell types, less protein in HUH7.0 and 293T, and minimal signal in A549 and EFK3B cells (Figure 3B).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>EFK3B</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_GZ34">CVCL_GZ34</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Notably, the cleavage pattern to the the SARS spike positive control from Calu3 cells, a respiratory cell line, varies slightly and could signal differences between proteolytic cleavage of the spike proteins between the two viruses due to predicted insertion of a furin cleavage site in SARS-CoV-2 (10).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Calu3</b></div>
        <div>suggested: BCRJ Cat# 0264, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Replication in HUH7.0 cells also increased quickly after an initial eclipse phase, but plateaued by 24 hours post inoculation in the intracellular compartment at 2 x 103 TCID50 / ml and dropped off after 66 hours post-inoculation.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HUH7.0</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus was not detected in the supernatant of infected HUH7 cells until 36 hours post inoculation and exhibited lower titers at all timepoints (Figure 4) Significant CPE was never observed in HUH7.0 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HUH7</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture, limiting dilution, and isolation Vero CCL-81 cells were used for isolation and initial passage.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero CCL-81</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6, Vero CCL-81, HUH 7.0 293T, A549, and EFKB3 cells were cultured in Dulbecco’s minimal essential medium (DMEM) supplemented with heat inactivated fetal bovine serum(5 or 10%) and antibiotic/antimyotic (GIBCO).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>A549</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>EFKB3</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Consensus Nanopore sequences were generated using minimap 2.17 and samtools 1.9.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>samtools</b></div>
        <div>suggested: (SAMTOOLS, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002105">SCR_002105</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Consensus sequences by Sanger sequences were generated from both directions using Sequencher 5.4.6, and were further confirmed by consensus sequences generated from nanopore sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Sequencher</b></div>
        <div>suggested: (Sequencher, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001528">SCR_001528</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 6 rounds of PCR the libraries were analyzed on an Agilent Bioanalyzer and quantified by qPCR.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Agilent Bioanalyzer</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were pooled and sequenced with a paired-end 75 base protocol on an Illumina (Illumina, Inc) MiniSeq instrument using the High-Output kit.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MiniSeq</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reads were processed with Trimmomatic v0.36 (21) to remove low quality base calls and any adapter sequences.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Trimmomatic</b></div>
        <div>suggested: (Trimmomatic, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011848">SCR_011848</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Contigs greate than 400 bases long were compared against the NCBI nucleotide collection using BLAST.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BLAST</b></div>
        <div>suggested: (BLASTX, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001653">SCR_001653</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The trimmed reads were mapped to the MN985325.1 reference sequence with BWA v0.7.17 (23) and visualized with the Integrated Genomics Viewe (24) to confirm the identity to the USA-WA1/2020 strain.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BWA</b></div>
        <div>suggested: (BWA, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010910">SCR_010910</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lysates were harvested with Laemmli SDS-PAGE sample buffer (BioRAD) containing a final concentration of 2% SDS and 5% β-mercaptoethano Cell lysates were the boiled and removed from the BSL3.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BioRAD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr></table>

    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).

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.03.02.972935v2 on bioRxiv
  4. Version published to 10.1101/2020.03.02.972935v1 on bioRxiv