Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant protect Vero E6 cells against infection by viral pseudotyped particles expressing SARS-CoV-2, SARS-CoV or MERS-CoV Spike protein

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   ScreenIT

   Mar 26, 2022

   SciScore for 10.1101/2022.03.22.485373: (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	47 Reverse transcription reactions were performed in a 20-µl reaction mixture that contained the specified amount of RNA transcripts produced in vitro or 1 µg of RNA extracted from either HEK-293T cells or S- pseudovirus preparations, 2 µl of random primer mix (60 µM; 25 µM oligo(dT) and 35 µM random hexamers), 2 µl 10X RT buffer, 1 µl dNTP mix (10mM) (Thermo Fisher - Invitrogen), 0.2 µl RNase inhibitor (40 U/µl), and 0.2 µl MMuLV RT (200 U/µl).
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After electrophoresis, samples were analyzed by …

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   SciScore for 10.1101/2022.03.22.485373: (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	47 Reverse transcription reactions were performed in a 20-µl reaction mixture that contained the specified amount of RNA transcripts produced in vitro or 1 µg of RNA extracted from either HEK-293T cells or S- pseudovirus preparations, 2 µl of random primer mix (60 µM; 25 µM oligo(dT) and 35 µM random hexamers), 2 µl 10X RT buffer, 1 µl dNTP mix (10mM) (Thermo Fisher - Invitrogen), 0.2 µl RNase inhibitor (40 U/µl), and 0.2 µl MMuLV RT (200 U/µl).
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After electrophoresis, samples were analyzed by immunoblot assays using the following primary antibodies: monoclonal anti-p30 antibody Ab130757 (Abcam); monoclonal anti-C9 antibody Ab5417 (Abcam) (used for C9-tagged S proteins of SARS-CoV and MERS-CoV); polyclonal anti- VSV-G antibody EPR12997 (Abcam) and polyclonal anti-SARS-CoV-2 S antibody 40590-T62 (SinoBiological).	anti-p30 suggested: None anti-C9 suggested: None anti- VSV-G suggested: None anti-SARS-CoV-2 S suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Transfection of HEK-293T cells was carried out using Lipofectamine 2000 (Thermo Fisher Scientific - Invitrogen)	HEK-293T suggested: None
   The filtrate containing pseudoviral particles (200 µl) was inoculated onto Vero E6 cells and the cells were then incubated on a rocker for 2 h prior to addition of complete DMEM (300 µl).	Vero E6 suggested: None
   Recombinant DNA
   Sentences	Resources
   DNA constructs: Plasmid pcDNA3.1-SARS-CoV-S contained a short coding sequence corresponding to the CD5 signal peptide (codons 1-24) that replaced the first eleven N-terminal codons in the original SARS-CoV-S sequence.	pcDNA3.1-SARS-CoV-S suggested: None
   The resulting PCR products were digested with HindIII and EcoRI or NotI and ApaI, and were then cloned into the HindIII-EcoRI-digested pcDNA3.1-MERS-CoV-S or NotI-ApaI-digested pcDNA3.1-SARS- CoV-2-S-Δ19 vectors.	pcDNA3.1-MERS-CoV-S suggested: None pcDNA3.1-SARS- suggested: None
   The resulting plasmids were named pcDNA3.1-CD5-MERS-CoV-S and pcDNA3.1-CD5-SARS-CoV-2-S-Δ19, respectively.	pcDNA3.1-CD5-MERS-CoV-S suggested: None
   Similarly, the first eleven codons of SARS-CoV-2-S- Δ19 were replaced by the CD5 signal in the pcDNA3.1-CD5-SARS-CoV-2-S-Δ19 plasmid.	pcDNA3.1-CD5-SARS-CoV-2-S-Δ19 suggested: None
   Synthesis of Luciferase (LUC) RNA molecules for production of standard curves: Using plasmid pTG-Luc as a template, the DNA sequence encoding LUC was isolated by PCR with primers containing KpnI and BamHI sites.	pTG-Luc suggested: None
   36 The PCR product was cloned into the corresponding sites of pBluescript SK in which a T7 promoter sequence is found immediately next to the cloning site on the 5’ end.	pBluescript suggested: None
   Using BamHI-linearized pSK-Luc (5 µg per reaction), in vitro run-off transcription with T7 RNA polymerase was performed using buffer T (80 mM HEPES, pH 7.5, 20 mM MgCl2, 2 mM spermidine, 40 mM DTT and 10 mM NaCl) that was supplemented with pyrophosphatase (0.1 U/µl) as described previously.	pSK-Luc suggested: None
   Software and Algorithms
   Sentences	Resources
   Relative luciferase units (RLU) were determined using the Glomax 20/20 luminometer (Promega) and infectivity values were analyzed as described previously 36 and plotted using GraphPad Prism 9.3.1 software.	GraphPad Prism 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: 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:

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

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   Version published to 10.1101/2022.03.22.485373v1 on bioRxiv

   Mar 23, 2022