Antiviral effects of miRNAs in extracellular vesicles against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mutations in SARS-CoV-2 RNA virus

  1. Jae Hyun Park
  2. Yuri Choi
  3. Chul-Woo Lim
  4. Ji-Min Park
  5. Shin-Hye Yu
  6. Yujin Kim
  7. Hae Jung Han
  8. Chun-Hyung Kim
  9. Young-Sook Song
  10. Chul Kim
  11. Jisook Moon

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 2019 (COVID-19). No treatment is available. Micro-RNAs (miRNAs) in mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are potential novel therapeutic agents because of their ability to regulate gene expression by inhibiting mRNA. Thus, they may degrade the RNA genome of SARS-CoV-2. EVs can transfer miRNAs to recipient cells and regulate conditions within them. MSC-EVs harbor major therapeutic miRNAs that play important roles in the biological functions of virus-infected host cells. Here, we examined their potential impact on viral and immune responses. MSC-EVs contained 18 miRNAs predicted to interact directly with the 3’ UTR of SARS-CoV-2. These EVs suppressed SARS-CoV-2 replication in Vero E6 cells. In addition, five major miRNAs suppressed virus activity in a luciferase reporter assay by binding the 3’ UTR. MSC-EVs showed strong regenerative effects and potent anti-inflammatory activity which may prevent lethal cytokine storms. We confirmed that EVs regulated inflammatory responses by several cell types, including human brain cells that express the viral receptor ACE2, suggesting that the brain may be targeted by SARS-CoV-2. miRNAs in MSC-EVs have several advantages as therapeutic agents against SARS-CoV-2: 1) they bind specifically to the viral 3’ UTR, and are thus unlikely to have side effects; 2) because the 3’ UTR is highly conserved and rarely mutates, MSC-EV miRNAs could be used against novel variants arising during viral replication; and 3) unique cargoes carried by MSC-EVs can have diverse effects, such as regenerating damaged tissue and regulating immunity.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.27.190561: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Isolation of human brain neuronal stem cells and placental MSCs: Neuronal stem cells were generated from the central nervous system tissue of spontaneously fetus from ectopic pregnancy at during the gestational week (GW) 8 with mother’s consent (IRB
    IRB: Sample collection and use for research purposes were approved by the Institutional Review Board (IRB, 2015-08-130-016) of Bundang CHA General Hospital (Seongnam, Korea)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Immunofluorescence staining was performed using anti-human CD 63 (1:100; SC-5275, Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.
    anti-human CD 63
    suggested: (Fitzgerald Industries International Cat# 10R-CD63aHU, RRID:AB_1283602)
    Antibodies specific for the following proteins were obtained from the indicated suppliers: CD81 (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), CD9 (1:1,000, Santa Cruz Biotechnology), CD63 (1:1,000, Santa Cruz Biotechnology) TSG 101 (1:500
    CD81
    suggested: None
    CD9
    suggested: None
    CD63
    suggested: None
    Next, 10 μg of protein was loaded onto 10% SDS-PAGE gels, transferred to a PVDF membrane (Merck Millipore), and analyzed by western blotting with antibodies specific for p65 (1:1,000, Cell Signaling Technology, Danvers, MA USA), phosphor-p65 (1:1,000
    p65
    suggested: None
    phosphor-p65
    suggested: None
    Primary antibodies were diluted in TBS-T and incubated with blots overnight at 4°C, followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson immunoresearch, West Grove, PA, USA) for 1 h.
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    Following washing with PBS three times to remove excess primary antibody, the cells were further incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2,000; Invitrogen) for 1 h at room temperature, washed with PBS, and stained with Gold Antifade reagent containing DAPI (Invitrogen; Thermo Fisher Scientific.) for 5 min prior to the position and quantification of ProLong nuclei.
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, Vero cells were grown in a 96-well plate (1 × 104 cells/well) at 37°C/5% CO2 until 80% confluent.
    Vero
    suggested: RRID:CVCL_ZW93)
    Cell culture: The Human Lung fibroblast cell line LL24, Human Bronchial epithelial cell line Beas-2B, Mouse microglial cell line BV2 were obtained from ATCC (Manassas, VA, USA)
    BV2
    suggested: BCRJ Cat# 0356, RRID:CVCL_0182)
    Immunocytochemistry (ICC): Beas-2B cells were cultured and fixed in 4 % PFA for 30 min.
    Beas-2B
    suggested: None
    Human neuroblastoma SK-N-BE(2)C cells were maintained in DMEM supplemented with 10% FBS.
    SK-N-BE(2)C
    suggested: NCBI_Iran Cat# C577, RRID:CVCL_0529)
    Software and Algorithms
    SentencesResources
    Reads were aligned to a human reference genome (GRCh38) using subread aligner [48] and the featureCounts tool was used to obtain miRNA read counts [49]
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    . miRNA read counts were normalized, and miRNA expressed at low levels was filtered using the edgeR R package.
    edgeR
    suggested: (edgeR, RRID:SCR_012802)
    The PITA tool was used to predict binding sites for miRNAs.
    PITA
    suggested: (PITA, RRID:SCR_010853)
    To investigate the biological function of miRNAs, GO term and KEGG pathway analysis were performed using the DAVID Bioinformatics Resource 6.8 [55].
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    The results of functional analyses were visualized using the Cytoscape 3.8 and Pathview R packages [56].
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Pathview
    suggested: (Pathview, RRID:SCR_002732)
    The default value was used for MUSCLE analysis.
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    P values for GO term analysis and KEGG pathway analysis were corrected for multiple comparisons using the Benjamini–Hochberg method.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT01960348CompletedAPOLLO: The Study of an Investigational Drug, Patisiran (ALN…

    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.

  2. ScreenIT

    SciScore for 10.1101/2020.07.27.190561: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll donors provided written, informed consent.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Immunofluorescence staining was performed using anti-human CD 63 (1:100; SC-5275, Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.
    anti-human CD 63
    suggested: (Fitzgerald Industries International Cat# 10R-CD63aHU, RRID:AB_1283602)
    Antibodies specific for the following proteins were obtained from the indicated suppliers: CD81 (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), CD9 (1:1,000, Santa Cruz Biotechnology), CD63 (1:1,000, Santa Cruz Biotechnology) TSG 101 (1:500
    CD81
    suggested: None
    CD9
    suggested: None
    CD63
    suggested: None
    Next, 10 μg of protein was loaded onto 10% SDS-PAGE gels, transferred to a PVDF membrane (Merck Millipore), and analyzed by western blotting with antibodies specific for p65 (1:1,000, Cell Signaling Technology, Danvers, MA USA), phosphor-p65 (1:1,000
    p65
    suggested: None
    phosphor-p65
    suggested: None
    Primary antibodies were diluted in TBS-T and incubated with blots overnight at 4℃, followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson immunoresearch, West Grove, PA, USA) for 1 h.
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    Following washing with PBS three times to remove excess primary antibody, the cells were further incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2,000; Invitrogen) for 1 h at room temperature, washed with PBS, and stained with Gold Antifade reagent containing DAPI (Invitrogen; Thermo Fisher Scientific.) for 5 min prior to the position and quantification of ProLong nuclei.
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The recombinant plasmid was transfected into human neuroblastoma SK-N-BE(2)C cells, and luciferase activity was measured 48 h later.
    SK-N-BE(2)C
    suggested: ATCC Cat# CRL-2268, RRID:CVCL_0529
    Then, the SARS-CoV-2 were serially diluted 10-fold and 0.1 ml of each dilution was added to triplicate wells of 96-well plates containing confluent Vero cells (final volume 0.2 mL/well).
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059
    Cell culture The Human Lung fibroblast cell line LL24, Human Bronchial epithelial cell line Beas-2B, Mouse microglial cell line BV2 were obtained from ATCC (Manassas, VA, USA)
    BV2
    suggested: BCRJ Cat# 0356, RRID:CVCL_0182
    Immunocytochemistry (ICC) Beas-2B cells were cultured and fixed in 4 % PFA for 30 min.
    Beas-2B
    suggested: None
    Software and Algorithms
    SentencesResources
    Reads were aligned to a human reference genome (GRCh38) using subread aligner [48] and the featureCounts tool was used to obtain miRNA read counts [49]
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    . miRNA read counts were normalized, and miRNA expressed at low levels was filtered using the edgeR R package.
    edgeR
    suggested: (edgeR, RRID:SCR_012802)
    Read quality control was performed using qrqc in R package and the distribution of small RNAs was calculated using sRNAtoolbox [50]. miRNA target prediction and functional analysis The PITA tool [51] was used to investigate miRNA binding sites in the 3' UTR.
    PITA
    suggested: (PITA, RRID:SCR_010853)
    To investigate the biological function of miRNAs, GO term and KEGG pathway analysis were performed using the DAVID Bioinformatics Resource 6.8 [55].
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    The results of functional analyses were visualized using the Cytoscape 3.8 and Pathview R packages [56].
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Pathview
    suggested: (Pathview, RRID:SCR_002732)
    The 3' UTR sequence of the coronaviruses were aligned using the MUSCLE tool [27].
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)

    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 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.

    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. ScreenIT

    SciScore for 10.1101/2020.07.27.190561: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementMaterials and Methods Isolation of human brain neuronal stem cells and placental MSCs Neuronal stem cells were generated from the central nervous system tissue of spontaneously fetus from ectopic pregnancy at during the gestational week (GW) 8 with mother's consent (IRB 2009-06-074)Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableBecause ACE2 receptors are expressed primarily in the liver, kidneys, male reproductive tissue, muscle, and the gastrointestinal tract (GI), these organs can be damaged by such viruses (Suppl. Fig. 2A, B).Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Immunofluorescence staining was performed using anti-human CD 63 (1:100; SC-5275, Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.
    anti-human CD 63
    suggested: (Fitzgerald Industries International Cat# 10R-CD63aHU, AB_1283602)
    Antibodies specific for the following proteins were obtained from the indicated suppliers: CD81 (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), CD9 (1:1,000, Santa Cruz Biotechnology), CD63 (1:1,000, Santa Cruz Biotechnology) TSG 101 (1:500
    CD81
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>CD9</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CD63</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, 10 μg of protein was loaded onto 10% SDS-PAGE gels, transferred to a PVDF membrane (Merck Millipore), and analyzed by western blotting with antibodies specific for p65 (1:1,000, Cell Signaling Technology, Danvers, MA USA), phosphor-p65 (1:1,000</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>p65</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>phosphor-p65</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies were diluted in TBS-T and incubated with blots overnight at 4℃, followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson immunoresearch, West Grove, PA, USA) for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-rabbit</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-mouse</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following washing with PBS three times to remove excess primary antibody, the cells were further incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2,000; Invitrogen) for 1 h at room temperature, washed with PBS, and stained with Gold Antifade reagent containing DAPI (Invitrogen; Thermo Fisher Scientific.) for 5 min prior to the position and quantification of ProLong nuclei.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-mouse IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</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">These EVs suppressed SARS-CoV-2 replication in Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The recombinant plasmid was transfected into human neuroblastoma SK-N-BE(2)C cells, and luciferase activity was measured 48 h later.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SK-N-BE(2)C</b></div>
        <div>suggested: ATCC Cat# CRL-2268, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0529">CVCL_0529</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the SARS-CoV-2 were serially diluted 10-fold and 0.1 ml of each dilution was added to triplicate wells of 96-well plates containing confluent Vero cells (final volume 0.2 mL/well).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero</b></div>
        <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture The Human Lung fibroblast cell line LL24, Human Bronchial epithelial cell line Beas-2B, Mouse microglial cell line BV2 were obtained from ATCC (Manassas, VA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BV2</b></div>
        <div>suggested: BCRJ Cat# 0356, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0182">CVCL_0182</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunocytochemistry (ICC) Beas-2B cells were cultured and fixed in 4 % PFA for 30 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Beas-2B</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">Of the 2698 targets, we selected 83 genes associated with the GO term ‘inflammatory response’, and then performed KEGG pathway JH Park et al., 27 July 2020 – preprint copy - BioRxiv analysis to further identify the pathways in which they are involved: these were interleukin production and regulation, cell chemotaxis, and response to external stress (Suppl. Fig. 4A and B).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>KEGG</b></div>
        <div>suggested: (KEGG, <a href="https://scicrunch.org/resources/Any/search?q=SCR_012773">SCR_012773</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>BioRxiv</b></div>
        <div>suggested: (bioRxiv, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003933">SCR_003933</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reads were aligned to a human reference genome (GRCh38) using subread aligner [48] and the featureCounts tool was used to obtain miRNA read counts [49]</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>featureCounts</b></div>
        <div>suggested: (featureCounts, <a href="https://scicrunch.org/resources/Any/search?q=SCR_012919">SCR_012919</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">. miRNA read counts were normalized, and miRNA expressed at low levels was filtered using the edgeR R package.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>edgeR</b></div>
        <div>suggested: (edgeR, <a href="https://scicrunch.org/resources/Any/search?q=SCR_012802">SCR_012802</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Read quality control was performed using qrqc in R package and the distribution of small RNAs was calculated using sRNAtoolbox [50]. miRNA target prediction and functional analysis The PITA tool [51] was used to investigate miRNA binding sites in the 3' UTR.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PITA</b></div>
        <div>suggested: (PITA, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010853">SCR_010853</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To investigate the biological function of miRNAs, GO term and KEGG pathway analysis were performed using the DAVID Bioinformatics Resource 6.8 [55].</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>DAVID</b></div>
        <div>suggested: (DAVID, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001881">SCR_001881</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The results of functional analyses were visualized using the Cytoscape 3.8 and Pathview R packages [56].</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Cytoscape</b></div>
        <div>suggested: (Cytoscape, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003032">SCR_003032</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Pathview</b></div>
        <div>suggested: (Pathview, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002732">SCR_002732</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 3' UTR sequence of the coronaviruses were aligned using the MUSCLE tool [27].</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MUSCLE</b></div>
        <div>suggested: (MUSCLE, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011812">SCR_011812</a>)</div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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.

  4. Version published to 10.1101/2020.07.27.190561v1 on bioRxiv