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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Abstract

Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses (HuCoV) that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surface and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with ∼97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S- pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted Saber TM ) used at 1:16 dilution, their infectivity was dramatically reduced by ∼98%. However, the combined use of Saber TM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted Saber TM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.

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  1. 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

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomization47 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).
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    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
    SentencesResources
    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
    SentencesResources
    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
    SentencesResources
    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)

    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).


    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.

    Results from TrialIdentifier: No clinical trial numbers were referenced.


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


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


    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.

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


    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.