A trans -complementation system for SARS-CoV-2

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   ScreenIT

   Jan 20, 2021

   SciScore for 10.1101/2021.01.16.426970: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01). IRB: ΔORF3-E mNG virion neutralization assay: The research protocol for use of human serum specimens was approved by the University of Texas Medical Branch (UTMB) Institutional Review Board (IRB protocol number 20-0070).
   Randomization	Animals were randomized upon arrival at Washington University and housed in groups of <5 per cage in rooms maintained between 68-74°F with 30-60% humidity and day/night cycles of 12 h intervals (on 6AM-6PM).
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological …

   SciScore for 10.1101/2021.01.16.426970: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01). IRB: ΔORF3-E mNG virion neutralization assay: The research protocol for use of human serum specimens was approved by the University of Texas Medical Branch (UTMB) Institutional Review Board (IRB protocol number 20-0070).
   Randomization	Animals were randomized upon arrival at Washington University and housed in groups of <5 per cage in rooms maintained between 68-74°F with 30-60% humidity and day/night cycles of 12 h intervals (on 6AM-6PM).
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Briefly, 105 TCID50 of WT SARS-CoV-2, 6×105 TCID50 of ΔORF3-E mNG virion, or 5×103 TCID50 of S-IV-P5-Vero-P2 virion in 100 μl volume were inoculated into four- to five-week-old male Syrian golden hamsters via the intranasal route.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: Vero E6, Vero CCL-81, Calu-3, and HEK-293T cells were purchased from the American Type Culture Collection (ATCC) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/ml Penicillium-Streptomycin (P/S), and 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT).	Vero suggested: None Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   S4A) on Vero E6 cells (to remove single-round infectious virion), followed by propagation on Vero-ORF3-E cells.	Vero E6 suggested: None
   Selection of Vero-ORF3-E cell line: For packaging the lentivirus, the pLVX-ORF3-E plasmid was transfected into HEK-293T cells using the Lenti-X Packaging Single Shots kit (	Vero-ORF3-E suggested: None HEK-293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   The resulting cells were defined as Vero-ORF3-E P0 cells.	Vero-ORF3-E P0 suggested: None
   ΔORF3-E mNG virion for mAb and antiviral testing: Vero CCL-81 cells (1.2×104) or A549-hACE2 cells in 50 μl of culture medium containing 2% FBS were seeded in each well of black μCLEAR flat-bottom 96-well plate.	Vero CCL-81 suggested: None A549-hACE2 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Heterozygous K18-hACE c57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from the Jackson Laboratory.	K18-hACE c57BL/6J suggested: None
   Software and Algorithms
   Sentences	Resources
   Bioinformatics analysis: Fluorescence images were processed using ImageJ (41).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Virus sequences were download from the NCBI database and aligned using Geneious software.	NCBI suggested: (NCBI, RRID:SCR_006472) Geneious suggested: (Geneious, RRID:SCR_010519)
   Figures were created and assembled using BioRender and Adobe illustration (San Jose, CA).	BioRender suggested: (Biorender, RRID:SCR_018361)
   Statistical analysis: A linear regression model in the software Prism 9 (GraphPad) was used to calculate the NT50 and EC50 values from the ΔORF3-E virion assay.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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

   One limitation of our study is the use of Vero E6 cells for constructing the Vero-ORF3-E cell line. When propagated on Vero E6 cells, SARS-CoV-2 could accumulate deletions at the furin cleavage site in the S protein (32, 33). This cleavage deletion affects the neutralization susceptibility of SARS-CoV-2 and possibly the route of entry into cells (34). Although we did not observe furin cleavage deletions when our ΔORF3-E mNG virion was passaged on Vero-ORF3-E cells, this possibility could be minimized or eliminated by using other cell lines, such as A549-hACE2 or Vero-TMPRSS2-hACE2 cells. In summary, we have developed a trans-complementation system for SARS-CoV-2 that likely can be performed at BSL-2 laboratories for COVID-19 research and countermeasure development. Thus, the experimental system could be used by researchers in industry, academia, and government laboratories who lack access to a BSL-3 facility.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.

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   Version published to 10.1101/2021.01.16.426970v1 on bioRxiv

   Jan 19, 2021