Aglycone polyether ionophores as broad-spectrum agents inhibit multiple enveloped viruses including SARS-CoV-2 in vitro and successfully cure JEV infected mice

  1. Jia-Qi Li
  2. Minjian Huang
  3. Ya-Nan Zhang
  4. Ran Liu
  5. Zhe-Rui Zhang
  6. Qiu-Yan Zhang
  7. Yong Wang
  8. Jing Liu
  9. Zixin Deng
  10. Bo Zhang
  11. Han-Qing Ye
  12. Tiangang Liu

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Abstract

Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo . Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.27.354563: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableFemale sex C57BL/6 mice, 3–4 weeks old, were kept in biosafety level 2 laboratory and given access to standard pellet feed and water ad libitum.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody-CR9114 against the HA protein of influenza A or B virus was kindly provided by Dr. Chen,
    Antibody-CR9114 against the HA
    suggested: None
    Following blocking, membranes were subject to sequential incubation with anti-FLAG mouse antibody (1: 2000 dilution) and secondary anti-mouse IgG conjugated to HRP (Bio-Rad).
    anti-FLAG
    suggested: None
    anti-mouse IgG
    suggested: None
    After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit or mouse secondary antibody (Bio-Rad) for 1 h at room temperature, followed by washing three times with TBST.
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, virus, compounds and antibody: BHK-21 (baby hamster kidney cell line), Vero (African green monkey epithelial kidney cell line), MDCK (Mardin–Darby canine kidney) and Vero E6 cells (ATCC, no.1586) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with 10 % FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 °C with 5 % CO2.
    MDCK
    suggested: None
    Vero E6
    suggested: RRID:CVCL_XD71)
    SARS-CoV-2 (IVCAS 6.7512) was propagated on the Vero-E6 cells and titrated by single layer plaque assay with standard procedure.
    Vero-E6
    suggested: None
    Then, 100 µL of each dilution were added to individual wells of 24-well plates containing confluent BHK-21 cells.
    BHK-21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    Times of addition assay: In this experiment, a monolayer of Vero cells was grown in 12-well plate in DMEM containing 2% inactivated FBS.
    Vero
    suggested: None
    Western blot analysis: Equal amounts of WT and mutant NS4A–NS4B-FLAG expression plasmids were mock-transfected or co-transfected with NS2B-3 pro-expression plasmid into 293T cells using Lipofectamine 2000 as described above.
    293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Female sex C57BL/6 mice, 3–4 weeks old, were kept in biosafety level 2 laboratory and given access to standard pellet feed and water ad libitum.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    The CC50 was calculated by nonlinear regression using GraphPad Prism 8.0 software to determine the cytotoxic concentration at which 50 % of the cells are viable.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    In general, for metrics with multiple treatment groups with longitudinal data (e.g., mouse weight loss or pulmonary function over time), two-way ANOVA was performed with the suggested multiple comparison test as advised by Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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 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).

    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.

    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.

  2. Version published to 10.1101/2020.10.27.354563v1 on bioRxiv