JIB-04 has broad-spectrum antiviral activity and inhibits SARS-CoV-2 replication and coronavirus pathogenesis

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   ScreenIT

   Sep 28, 2020

   SciScore for 10.1101/2020.09.24.312165: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethics statement: Animal experiments were approved by the Committee on the Ethics of Animal Care and Use of the Science and Technology Agency of Jiangsu Province.
   Randomization	The pigs were randomly separated into three groups, housed in separate rooms, and fed the same artificial milk substitutes that meet the nutrient and energy recommendations of the National Research Council [NRC, 2012] at the animal facility of the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Fifteen neonatal male pigs at birth were obtained from a …

   SciScore for 10.1101/2020.09.24.312165: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethics statement: Animal experiments were approved by the Committee on the Ethics of Animal Care and Use of the Science and Technology Agency of Jiangsu Province.
   Randomization	The pigs were randomly separated into three groups, housed in separate rooms, and fed the same artificial milk substitutes that meet the nutrient and energy recommendations of the National Research Council [NRC, 2012] at the animal facility of the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu Province.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Fifteen neonatal male pigs at birth were obtained from a TGEV-free farm in Nanjing without suckling.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Proteins were resolved in SDS-PAGE and detected as described (47) using the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).	GAPDH suggested: (BioLegend Cat# 631402, RRID:AB_2107422)
   Secondary antibodies were anti-rabbit (7074, Cell Signaling) or anti-mouse (7076, Cell	anti-rabbit (7074, Cell Signaling) suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) anti-mouse (7076 suggested: None
   Serum samples were collected from each pig at 24 and 48 hpi to detect specific anti-TGEV antibodies.	anti-TGEV suggested: None
   For immunofluorescence analysis, samples were incubated with rabbit anti-TGEV-N antibody (1:500, DA0224, Shanghai YouLong Biotech) for 30 min at 37 °C.	anti-TGEV-N suggested: None
   After three washes, samples were stained with Cy3-conjugated goat anti-rabbit secondary antibody (Beyotime) and DAPI (Invitrogen).	anti-rabbit suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell cytotoxicity assay: The viability of Vero E6 and MA104 cells after drug treatment was determined using the Cell Counting Kit 8 (ab228554, Abcam).	Vero E6 suggested: None
   Small interfering RNA transfection: HEK293 cells were transfected using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher).	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   Flow cytometry: Vero E6-TMPRSS2 cells were inoculated with VSV-GFP or VSV-SARS-CoV-2 at an MOI of 3 for 1 hr at 37°C.	Vero E6-TMPRSS2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Image analysis was performed using ImageJ and customized R scripts.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Absorbance at 460nm was measured using Gen5 software and BioTek ELx800 Microplate Reader.	Gen5 suggested: (Gen5, RRID:SCR_017317)
   All siRNA used in this study were designed and synthesized by GenePharma (Shanghai, China) and their sequences of siRNAs are listed in Table S2.	GenePharma suggested: None
   Percentage of GFP positive cells and GFP intensity were determined by BD LSRFortessa™ X-20 cell analyzer and analyzed by FlowJo v10.6.2 (BD).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   RNA sample quality was measured by both NanoDrop spectrophotometer (Thermo Fisher) and Bioanalyzer 2100 (Agilent).	NanoDrop suggested: None
   Differential gene expression analysis was performed using DESeq2.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   The RNA-seq raw and processed datasets were deposited onto NCBI Gene Expression Omnibus database (GSE156219)	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
   2E, 2F, 3C, S3D, S3F, 4C, and 4D was calculated by Student’s t test using Prism 8.4.3 (GraphPad).	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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

   Identifier	Status	Title
   NCT04341493	Recruiting	Hydroxychloroquine vs Nitazoxanide in Patients With COVID-19
   NCT04348409	Recruiting	Efficacy and Safety of Nitazoxanide for the Treatment of Hos…

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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: 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 10.1101/2020.09.24.312165 on bioRxiv

   Sep 25, 2020