Screening of Botanical Drugs against SARS-CoV-2 Entry

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1. 

   ScreenIT

   Jun 7, 2021

   SciScore for 10.1101/2021.06.03.447021: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   SARS-CoV-2 S was detected using a rabbit anti-S2 subunit mouse monoclonal antibody (GeneTex, GTX632604;1:2000 dilution).	anti-S2 subunit suggested: None
   Horseraddish peroxidase-linked goat anti-mouse IgG antibody (Proteintech, CHI, USA, 1:5000).	anti-mouse IgG suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)
   Cells were permeabilized using PBS with 0.2% Triton X-100 for 15 min and blocked with 5% FBS (Gibco), followed by treatment with the primary antibody anti-SARS-CoV-2 NP …

   SciScore for 10.1101/2021.06.03.447021: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   SARS-CoV-2 S was detected using a rabbit anti-S2 subunit mouse monoclonal antibody (GeneTex, GTX632604;1:2000 dilution).	anti-S2 subunit suggested: None
   Horseraddish peroxidase-linked goat anti-mouse IgG antibody (Proteintech, CHI, USA, 1:5000).	anti-mouse IgG suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)
   Cells were permeabilized using PBS with 0.2% Triton X-100 for 15 min and blocked with 5% FBS (Gibco), followed by treatment with the primary antibody anti-SARS-CoV-2 NP (GeneTex GTX635678, USA) at a 1:500 dilution overnight at 4°C.	anti-SARS-CoV-2 NP suggested: None
   After six rinses with PBS, the cells were stained with DyLight 488-labeled antibody against rabbit IgG (KPL, Gaithersburg, MD, USA).	antibody against rabbit IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells and viruses: HEK 293T, Vero E6, Caco-2, and BHK-21 cells were cultured in Dulbecco’s modified Eagle medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA).	HEK 293T suggested: None Vero E6 suggested: None
   Briefly, 293T cells transfected with pcDNA3.1-SARS-CoV-2 S (ct19), pcDNA3.1-SARS-CoV-2 S (wt), or pcDNA3.1-MERS-CoV S (ct16) for 48 h were infected with pseudotype VSV (described below), in which the G gene was replaced with the luciferase gene, at an MOI of 0.1 for 2 h.	293T suggested: None
   After 48 h, the supernatants were filtered to remove the vaccinia virus and inoculated into BHK-21 cells that had been transfected with pCAGGS-VSV G 24 h previously.	BHK-21 suggested: None
   Drug-drug interactions of remdesivir with hits: Caco-2 cells were seeded at a density of 2.4×104 cells per well in 96-well plates.	Caco-2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Briefly, 293T cells transfected with pcDNA3.1-SARS-CoV-2 S (ct19), pcDNA3.1-SARS-CoV-2 S (wt), or pcDNA3.1-MERS-CoV S (ct16) for 48 h were infected with pseudotype VSV (described below), in which the G gene was replaced with the luciferase gene, at an MOI of 0.1 for 2 h.	pcDNA3.1-MERS-CoV suggested: None
   After 45 min, the cells were transfected with 11 μg of mixed plasmids with a 5:3:5:8:1 ratio of pVSVΔG-eGFP-GPC (pVSVΔG-Rluc to generate pseudotype VSV), pBS-N, pBS-P, pBS-G, and pBS-L.	pVSVΔG-eGFP-GPC suggested: None pVSVΔG-Rluc suggested: None pBS-N suggested: None pBS-P suggested: None pBS-G suggested: None pBS-L suggested: None
   After 48 h, the supernatants were filtered to remove the vaccinia virus and inoculated into BHK-21 cells that had been transfected with pCAGGS-VSV G 24 h previously.	pCAGGS-VSV suggested: None
   The titer of the pseudotype virus was measured by infecting BHK-21 cells previously transfected with pCAGGS-VSV G and determined by plaque assay 24 hours post-infection (h.p.i).	pCAGGS-VSV G suggested: None
   All point mutants (D614G, K417N/E484K/N501Y/D614G) were made from pcDNA3.1-SARS-CoV-2 S (ct19) by using a fast mutagenesis system (Transgene Biotech), and mutations were confirmed by sequencing (Sangon, Shanghai).	pcDNA3.1-SARS-CoV-2 suggested: None
   Membrane fusion assay: Vero E6 cells were co-transfected with pcDNA3.1-SARS-CoV-2S (ct19) (0.25 μg) and pEGFP-N1 (0.25 μg) by using lipo2000 in 24-well plates.	pcDNA3.1-SARS-CoV-2S suggested: None pEGFP-N1 suggested: RRID:Addgene_111933)

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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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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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:

   • 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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   Results from scite Reference Check: We found no unreliable references.

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   Read the original source
2. 

   Version published to 10.1101/2021.06.03.447021v1 on bioRxiv

   Jun 4, 2021