Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes

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1. Version published to 10.1128/jvi.02209-20

   Mar 10, 2021
2. 

   ScreenIT

   Nov 19, 2020

   SciScore for 10.1101/2020.11.16.386003: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Protocols containing SARS-CoV-2 were approved by the Texas Biomedical Research Institute’s Institutional Biosafety Committee (IBC).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, confluent monolayers of Vero E6 cells (1.2 × 106 cells/well, 6-well plate format, triplicates) were transfected, using lipofectamine 2000 (LPF2000, Thermo Fisher) with 4 μg/well of pBeloBAC11-SARS-CoV-2/WT, pBeloBAC11-SARS-CoV-2-del7a/Venus, - del7a/mCherry, or - del7a/Nluc plasmids.	Vero E6 suggested: …

   SciScore for 10.1101/2020.11.16.386003: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Protocols containing SARS-CoV-2 were approved by the Texas Biomedical Research Institute’s Institutional Biosafety Committee (IBC).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, confluent monolayers of Vero E6 cells (1.2 × 106 cells/well, 6-well plate format, triplicates) were transfected, using lipofectamine 2000 (LPF2000, Thermo Fisher) with 4 μg/well of pBeloBAC11-SARS-CoV-2/WT, pBeloBAC11-SARS-CoV-2-del7a/Venus, - del7a/mCherry, or - del7a/Nluc plasmids.	Vero E6 suggested: RRID:CVCL_XD71)
   Software and Algorithms
   Sentences	Resources
   Raw reads were filtered using Trimmomatic v0.39 (23).	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848)
   Reads were mapped to the modified SARS-CoV-2 templates with Bowtie v2.4.1 (24), and the total genomic coverage was quantified using MosDepth v0.2.6 (25).	Bowtie suggested: (Bowtie, RRID:SCR_005476) MosDepth suggested: (mosdepth, RRID:SCR_018929)
   Allele frequencies were estimated with LoFreq* v2.1.3.1 (26) and low frequency variants with less than a 100x read depth or a 1% minor allele frequency were eliminated.	LoFreq* suggested: None
   All sequence data has been deposited in the NCBI Short Read Archive (BioProject: PRJNA678001).	NCBI Short Read Archive suggested: None BioProject suggested: (NCBI BioProject, RRID:SCR_004801)
   Mean values and standard deviation (SD) were determined using GraphPad Prism software (version 8.2).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Individual wells from three independent experiments conducted in quadruplicates were used to calculate the average and standard deviation (SD) of viral inhibition using Microsoft Excel software.	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Another limitation of green fluorescent proteins during in vivo imaging is the absorption of the fluorophores’ excitation and emission by hemoglobin and autofluorescence of tissues (52–55). Recombinant viruses expressing red fluorescent proteins represent a better option to combine with genetically modified GFP-expressing cell lines and/or animals and, based on their reduced autofluorescence background, to more accurately capture the dynamics of viral infection and replication. Reporter-expressing replicating competent viruses can be used to monitor viral infections, assess viral fitness, evaluate and/or identify antivirals and/or NAbs, where reporter gene expression can be used as a valid surrogate for viral detection in infected cells. Expression of Venus, mCherry, or Nluc from our rSARS-CoV-2 were confirmed by directly visualizing fluorescence expression under a fluorescent microscope (Venus and mCherry) or luciferase activity (Nluc) using a microplate reader (Figures 2 and 3). Western blot analyses using specific antibodies against each of the reporter genes further confirm expression from their respective rSARS-CoV-2 (Figures 2 and 3). Notably, despite deletion of the 7a ORF and insertion of a reporter gene, the three reporter-expressing rSARS-CoV-2 displayed similar growth kinetics and plaque phenotype than their WT counterpart (Figure 3). As expected, viral infection was visualized in real time, without the need of secondary approaches (e.g. MAbs) to detect the presenc...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   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).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 43. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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   Read the original source
3. 

   Version published to 10.1101/2020.11.16.386003v1 on bioRxiv

   Nov 18, 2020