SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients

Article activity feed

1. Version published to 10.1093/jmcb/mjac021

   Apr 1, 2022
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethical statement and COVID-19 patients: The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440). Consent: Written informed consent was obtained for both patients and healthy controls. IACUC: The protocol for production of mouse hyperimmune serum were carried out with 8-week-old male mice following the institutional guidelines on ethics in animal experiments and was approved by the University of São Paulo Ethics Committee for Animal Experimental Research -CETEA (Protocol no. 001/2020-1).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Production …

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethical statement and COVID-19 patients: The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440). Consent: Written informed consent was obtained for both patients and healthy controls. IACUC: The protocol for production of mouse hyperimmune serum were carried out with 8-week-old male mice following the institutional guidelines on ethics in animal experiments and was approved by the University of São Paulo Ethics Committee for Animal Experimental Research -CETEA (Protocol no. 001/2020-1).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Production of mouse anti-SARS-CoV-2 hyperimmune serum: Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with detection of specific IgM or IgG antibodies to SARS-CoV-2.	IgG suggested: None
   In parallel, PBMCs were treated with 0.5ug/ml Camostat (Sigma Aldrich, cat. SML005) or with 10 uM of anti-ACE2 antibody (Rhea Biotech, cat.	anti-ACE2 suggested: None
   To determine the phenotype of SARS-CoV-2-infected cells, we used primary antibodies for CD4 (Abcam cat. ab133616), CD8 (Abcam cat. ab4055), CD14 (Abcam cat. ab133335), CD19 (Abcam cat. ab134114), CD20 (Abcam cat. ab103573).	CD4 suggested: (Abcam Cat# ab133616, RRID:AB_2750883) CD8 suggested: (Abcam Cat# ab4055, RRID:AB_304247) CD14 suggested: None CD19 suggested: (Abcam Cat# ab134114, RRID:AB_2801636) CD20 suggested: (Abcam Cat# ab103573, RRID:AB_10859291)
   Secondary antibodies used were polyclonal anti-rabbit conjugated with 488 (Thermo Fisher cat.	anti-rabbit suggested: None
   Flow cytometry: Unseparated whole blood leukocyte samples from COVID-19 patients or healthy donors infected in vitro with SARS-CoV-2 were surface stained with Fixable Viability Dye eFluor™ 780 (eBioscience) and monoclonal antibodies specific for CD3 (APC eBioscience cat.	CD3 suggested: None
   SARS-CoV-2 antibodies were detected with secondary anti-Mouse Alexa488.	SARS-CoV-2 suggested: None anti-Mouse suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat. ab133616), CD20 (Abcam cat. ab103573), CD14 (Abcam cat. ab133335) and IL-6 (BD cat. 554400).	IL-6 suggested: (BD Biosciences Cat# 554400, RRID:AB_398549)
   Experimental Models: Cell Lines
   Sentences	Resources
   In order to confirm inactivation, titration of the inactivated product was done both by TCID50 and by plaque assay in Vero-E6 cells with 5-day incubation, without any cytopathic effects.	Vero-E6 suggested: None
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	Caco-2 suggested: None
   To detect viral antigens in cells, we used serum from a recovered COVID-19 patient, which was first tested for specificity by immunofluorescence in SARS-CoV-2 infected Vero CCL81 cells (Supplementary Fig 1B).	Vero CCL81 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Production of mouse anti-SARS-CoV-2 hyperimmune serum: Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.	C57Bl/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Results of viral RNA quantifications by one-step qRT-PCR were plotted with GraphPad® Prism 8.4.2 software.	GraphPad® Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The quantity of SARS-CoV-2-positive cells of different phenotypes was quantified by using the analyze particles tool from Fiji by ImageJ.	Fiji suggested: (Fiji, RRID:SCR_002285)
   All data were acquired using a Verse or Canto flow cytometers (BD Biosciences) and subsequent analysis was done using FlowJo (TreeStar) software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat. ab133616), CD20 (Abcam cat. ab103573), CD14 (Abcam cat. ab133335) and IL-6 (BD cat. 554400).	SIMPLE suggested: (SIMPLE, RRID:SCR_009389)
   Images were pseudocolored and overlaid in the first image of the preparation counterstained with hematoxylin using ImageJ v1.50b (NIH, USA) and Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).	ImageJ suggested: (ImageJ, RRID:SCR_003070) Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
   Statistical analysis: All descriptive statistics, patient stratification, and positive cell frequencies were done using GraphPad Prism Software, version 6.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 35, 37, 30 and 31. 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.

   ---

   Read the original source
3. 

   Version published to 10.1101/2020.07.28.225912v2 on bioRxiv

   Aug 7, 2020
4. 

   ScreenIT

   Aug 6, 2020

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with …

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with detection of specific IgM or IgG antibodies to SARS-CoV-2.	IgG suggested: None SARS-CoV-2 suggested: None
   To determine the phenotype of SARSCoV-2-infected cells, we used primary antibodies for CD4 (Abcam cat. ab133616), CD8 (Abcam cat.	CD4 suggested: (Abcam Cat# ab133616, RRID:AB_2750883) CD8 suggested: None
   Secondary antibodies used were polyclonal anti-rabbit conjugated with 488 (Thermo Fisher cat.	anti-rabbit suggested: None
   Unseparated whole blood leukocyte samples from COVID-19 patients or healthy donors infected in vitro with SARS-CoV-2 were surface stained with Fixable Viability Dye eFluor™ 780 (eBioscience) and monoclonal antibodies specific for CD3 (APC eBioscience cat.	CD3 suggested: None
   SARS-CoV-2 antibodies were detected with secondary anti-Mouse Alexa488.	anti-Mouse suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)
   Experimental Models: Cell Lines
   Sentences	Resources
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	Caco-2 suggested: None
   P1 was diluted 1:1000 in Dulbecco’s modified Eagle’s medium (DMEM) and inoculated in Vero-E6 cells monolayers to produce the P2 stock.	Vero-E6 suggested: None
   To detect viral antigens in cells, we used serum from a recovered COVID-19 patient, which was first tested for specificity by immunofluorescence in SARS-CoV-2 infected Vero CCL81 cells (Supplementary Fig 1B).	Vero CCL81 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.	C57Bl/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Results of viral RNA ® quantifications by one-step qRT-PCR were plotted with GraphPad® Prism 8.4.2 software.	GraphPad® Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The quantity of SARS-CoV-2-positive cells of different phenotypes was quantified by using the analyze particles tool from Fiji by ImageJ.	Fiji suggested: (Fiji, RRID:SCR_002285)
   All data were acquired using a Verse or Canto flow cytometers (BD Biosciences) and subsequent analysis was done using FlowJo (TreeStar) software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat.	SIMPLE suggested: (Simpleaffy, RRID:SCR_001302)
   Images were pseudocolored and overlaid in the first image of the preparation counterstained with hematoxylin using ImageJ v1.50b (NIH, USA) and Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).	ImageJ suggested: (ImageJ, RRID:SCR_003070) Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
   All descriptive statistics, patient stratification, and positive cell frequencies were done using GraphPad Prism Software, version 6.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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 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: Please consider improving the rainbow (“jet”) colormap used on pages 29, 30, 32, 34 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   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.

   Read the original source
5. 

   ScreenIT

   Jul 30, 2020

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with …

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with detection of specific IgM or IgG antibodies to SARS-CoV-2.	IgG suggested: None
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	anti-mouse suggested: None
   To determine the phenotype of SARSCoV-2-infected cells, we used primary antibodies for CD4 (Abcam cat. ab133616), CD8 (Abcam cat.	CD4 suggested: (Abcam Cat# ab133616, RRID:AB_2750883) CD8 suggested: None
   Secondary antibodies used were polyclonal anti-rabbit conjugated with 488 (Thermo Fisher cat.	anti-rabbit suggested: None
   Unseparated whole blood leukocyte samples from COVID-19 patients or healthy donors infected in vitro with SARS-CoV-2 were surface stained with Fixable Viability Dye eFluor™ 780 (eBioscience) and monoclonal antibodies specific for CD3 (APC eBioscience cat.	CD3 suggested: None
   Tissue sections from paraffin-embedded lung fragments obtained from two COVID-19 fatal cases were tested by immunohistochemistry (IHC) using anti-SARS-CoV-2 polyclonal antibody for in situ detection of SARS-CoV-2.	anti-SARS-CoV-2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	Caco-2 suggested: None
   P1 was diluted 1:1000 in Dulbecco’s modified Eagle’s medium (DMEM) and inoculated in Vero-E6 cells monolayers to produce the P2 stock.	Vero-E6 suggested: None
   To detect viral antigens in cells, we used serum from a recovered COVID-19 patient, which was first tested for specificity by immunofluorescence in SARS-CoV-2 infected Vero CCL81 cells (Supplementary Fig 1B).	Vero CCL81 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Three C57Bl/6 mice were inoculated intramuscularly with an emulsion containing the equivalent of 106 TCID50 of inactivated SARS-CoV-2 in complete Freund's adjuvant (CFA, BD, cat. 263810) diluted 1:1 in PBS.	C57Bl/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Results of viral RNA ® quantifications by one-step qRT-PCR were plotted with GraphPad® Prism 8.4.2 software.	GraphPad® Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The quantity of SARS-CoV-2-positive cells of different phenotypes was quantified by using the analyze particles tool from Fiji by ImageJ.	Fiji suggested: (Fiji, RRID:SCR_002285) ImageJ suggested: (ImageJ, RRID:SCR_003070)
   All data were acquired using a Verse or Canto flow cytometers (BD Biosciences) and subsequent analysis was done using FlowJo (TreeStar) software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat.	SIMPLE suggested: (Simpleaffy, RRID:SCR_001302)
   Images were pseudocolored and overlaid in the first image of the preparation counterstained with hematoxylin using ImageJ v1.50b (NIH, USA) and Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).	Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
   All descriptive statistics, patient stratification, and positive cell frequencies were done using GraphPad Prism Software, version 6.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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 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: Please consider improving the rainbow (“jet”) colormap used on pages 29, 30, 32, 34 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   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.

   Read the original source
6. 

   ScreenIT

   Jul 30, 2020

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with …

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with detection of specific IgM or IgG antibodies to SARS-CoV-2.	IgG suggested: None
   To determine the phenotype of SARSCoV-2-infected cells, we used primary antibodies for CD4 (Abcam cat. ab133616), CD8 (Abcam cat.	CD8 suggested: None
   Secondary antibodies used were polyclonal anti-rabbit conjugated with 488 (Thermo Fisher cat.	anti-rabbit suggested: None
   Unseparated whole blood leukocyte samples from COVID-19 patients or healthy donors infected in vitro with SARS-CoV-2 were surface stained with Fixable Viability Dye eFluor™ 780 (eBioscience) and monoclonal antibodies specific for CD3 (APC eBioscience cat.	CD3 suggested: None
   SARS-CoV-2 antibodies were detected with secondary anti-Mouse Alexa488.	anti-Mouse suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)
   Tissue sections from paraffin-embedded lung fragments obtained from two COVID-19 fatal cases were tested by immunohistochemistry (IHC) using anti-SARS-CoV-2 polyclonal antibody for in situ detection of SARS-CoV-2.	anti-SARS-CoV-2 suggested: None
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat.	CD4 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	Caco-2 suggested: None
   P1 was diluted 1:1000 in Dulbecco’s modified Eagle’s medium (DMEM) and inoculated in Vero-E6 cells monolayers to produce the P2 stock.	Vero-E6 suggested: None
   To detect viral antigens in cells, we used serum from a recovered COVID-19 patient, which was first tested for specificity by immunofluorescence in SARS-CoV-2 infected Vero CCL81 cells (Supplementary Fig 1B).	Vero CCL81 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.	C57Bl/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Results of viral RNA ® quantifications by one-step qRT-PCR were plotted with GraphPad® Prism 8.4.2 software.	GraphPad® Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The quantity of SARS-CoV-2-positive cells of different phenotypes was quantified by using the analyze particles tool from Fiji by ImageJ.	Fiji suggested: (Fiji, RRID:SCR_002285) ImageJ suggested: (ImageJ, RRID:SCR_003070)
   All data were acquired using a Verse or Canto flow cytometers (BD Biosciences) and subsequent analysis was done using FlowJo (TreeStar) software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat.	SIMPLE suggested: (Simpleaffy, RRID:SCR_001302)
   Images were pseudocolored and overlaid in the first image of the preparation counterstained with hematoxylin using ImageJ v1.50b (NIH, USA) and Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).	Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
   Correlation analysis, one-way ANOVA, two-way ANOVA, linear regressions, Holm-Sidak, and Bonferroni post-tests were also performed using GraphPad Prism.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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 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: Please consider improving the rainbow (“jet”) colormap used on pages 29, 30, 32, 34 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   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.

   Read the original source
7. 

   ScreenIT

   Jul 30, 2020

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with …

   SciScore for 10.1101/2020.07.28.225912: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   The study was approved by the National Ethics Committee (CONEP, CAAE: 30248420.9.0000.5440 and 31797820.8.0000.5440).

   Randomization

   not detected.

   Blinding

   not detected.

   Power Analysis

   not detected.

   Sex as a biological variable

   Male C57Bl/6 mice were bred and maintained under specific pathogen-free conditions at the animal facility of the Ribeirão Preto Medical School (FMRP) at University of São Paulo.

   Cell Line Authentication

   not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A total of 29 hospitalized patients were enrolled, all with clinical and radiological features of COVID-19 and confirmed SARS-CoV-2 infection by RT-PCR in respiratory secretions, with detection of specific IgM or IgG antibodies to SARS-CoV-2.	IgG suggested: None
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	anti-mouse suggested: None
   To determine the phenotype of SARSCoV-2-infected cells, we used primary antibodies for CD4 (Abcam cat. ab133616), CD8 (Abcam cat.	CD8 suggested: None
   Secondary antibodies used were polyclonal anti-rabbit conjugated with 488 (Thermo Fisher cat.	anti-rabbit suggested: None
   Unseparated whole blood leukocyte samples from COVID-19 patients or healthy donors infected in vitro with SARS-CoV-2 were surface stained with Fixable Viability Dye eFluor™ 780 (eBioscience) and monoclonal antibodies specific for CD3 (APC eBioscience cat.	CD3 suggested: None
   SARS-CoV-2 antibodies were detected with secondary anti-Mouse Alexa488.	SARS-CoV-2 suggested: None
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat.	CD4 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Animal serum conversion was evaluated by indirect immunofluorescence using slide preparations of SARS-CoV-2 infected Caco-2 cells, fixed with 4% paraformaldehyde and AlexaFluor 488-labelled rabbit anti-mouse secondary antibody.	Caco-2 suggested: None
   P1 was diluted 1:1000 in Dulbecco’s modified Eagle’s medium (DMEM) and inoculated in Vero-E6 cells monolayers to produce the P2 stock.	Vero-E6 suggested: None
   To detect viral antigens in cells, we used serum from a recovered COVID-19 patient, which was first tested for specificity by immunofluorescence in SARS-CoV-2 infected Vero CCL81 cells (Supplementary Fig 1B).	Vero CCL81 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Three C57Bl/6 mice were inoculated intramuscularly with an emulsion containing the equivalent of 106 TCID50 of inactivated SARS-CoV-2 in complete Freund's adjuvant (CFA, BD, cat. 263810) diluted 1:1 in PBS.	C57Bl/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Results of viral RNA ® quantifications by one-step qRT-PCR were plotted with GraphPad® Prism 8.4.2 software.	GraphPad® Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The quantity of SARS-CoV-2-positive cells of different phenotypes was quantified by using the analyze particles tool from Fiji by ImageJ.	Fiji suggested: (Fiji, RRID:SCR_002285)
   All data were acquired using a Verse or Canto flow cytometers (BD Biosciences) and subsequent analysis was done using FlowJo (TreeStar) software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Sequential immunoperoxidase labeling and erasing (SIMPLE) [12] was then performed to determine the immunophenotypes of SARS-CoV-2 infected cells, using antibodies to CD4 (Abcam cat.	SIMPLE suggested: (Simpleaffy, RRID:SCR_001302)
   Images were pseudocolored and overlaid in the first image of the preparation counterstained with hematoxylin using ImageJ v1.50b (NIH, USA) and Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).	ImageJ suggested: (ImageJ, RRID:SCR_003070) Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
   Correlation analysis, one-way ANOVA, two-way ANOVA, linear regressions, Holm-Sidak, and Bonferroni post-tests were also performed using GraphPad Prism.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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 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: Please consider improving the rainbow (“jet”) colormap used on pages 29, 30, 32, 34 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   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.

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8. 

   Version published to 10.1101/2020.07.28.225912v1 on bioRxiv

   Jul 29, 2020