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  3. SciScore for 10.1101/2022.02.16.480801: (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.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    The separated proteins were transferred onto the PVDF membrane and probed using mouse anti-Flavivirus envelope 4G2 primary antibody and anti-mouse-HRP conjugated secondary antibody.
    anti-Flavivirus envelope 4G2
    suggested: None
    suggested: None
    For IFA, Anti-mouse Influenza virus NP (HT103) was used as the primary antibody.
    suggested: None
    Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A11001) was used as the secondary antibody.
    anti-Mouse IgG
    suggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)
    Primary antibody incubation for western blot was done with Anti-mouse Influenza virus NP (HT103) and secondary antibody with Goat Anti-Mouse IgG - H&L Polyclonal Antibody, HRP conjugated (Abcam, ab6789).
    suggested: None
    The antibodies used for IFA include SARS-CoV-2 spike primary antibody (GTX632604, GeneTex) and goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488.
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    After a 15 min chase, cells were fixed with 4% PFA, labeled with anti-influenza virus HA antibody (PY102) followed by anti-mouse Alexa 488 secondary antibody to label the virus particles.
    anti-influenza virus HA
    suggested: None
    Expression levels of VP1 protein were detected by western blot using monoclonal mouse anti-enterovirus primary antibody Clone 5-D8/1 (Dako, M7064,) and anti-mouse antibody-HRP (Sigma Aldrich, A4416)
    suggested: (Agilent Cat# M7064, RRID:AB_2118128)
    suggested: (Agilent Cat# M7064, RRID:AB_2118128)
    anti-mouse antibody-HRP
    suggested: None
    suggested: None
    After 12hr, cells were fixed with 4% formalin and immunolabeled with primary mouse anti VP6, and anti-mouse secondary Alexa Fluor 488 antibodies to detect virus-infected cells by IFA.
    anti VP6,
    suggested: (Acris Antibodies Cat# AM10217SU-N, RRID:AB_11216263)
    Experimental Models: Cell Lines
    DMEM containing 2% FBS was used for infection in Vero E6 cells and complete DMEM was used for HEK293T-ACE2 cells.
    suggested: None
    Vero E6, Calu-3, and HEK293T-ACE2 cells were seeded in 24-well cell culture dishes to reach 80% confluency post 24hr.
    suggested: None
    IAV plaque assay: MDCK cells were seeded in 12-well plates to reach complete confluency after 24hr.
    suggested: None
    Flavivirus infection: Confluent A549 cells in a 24-well cell culture plate were pre-treated for 3hr with 2 mM PA and infected with 100 µL per well DMEM containing 0.1 MOI JEV clinical strain P20778 or ZIKV Cambodia.
    suggested: None
    Influenza polymerase Assay: HEK293293T cells were seeded in a poly-L-lysine (Sigma Aldrich, P915
    suggested: None
    Briefly, HEK293T cells were seeded in 2 X T75 flasks to reach 50-60% confluency the next day.
    suggested: None
    Infection with AAV6 particles: HEK293 293T cells were seeded in a poly-L-lysine coated 24-well dish to reach 60-70% confluency the next day.
    suggested: None
    Adenovirus 5 infection: For infection studies, HEK293 cells were pre-treated for 3hr with 2mM PA and infected with 10 MOI AAV5-eGFP in the presence of the drug.
    suggested: None
    The infectious RNA was transfected in HeLa cells and the cell culture supernatant was harvested after 48hr.
    suggested: None
    Plaque assay: Vero E6 cells were seeded in a 12-well plate at a confluency of approximately 90% and infected with serially diluted CVB3-containing cell culture supernatant and incubated at 37℃ for 1hr with gentle swirling of the medium at every 10-15 min interval.
    Vero E6
    suggested: None
    Recombinant DNA
    For each flask, the following plasmids were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, 11668019) as per manufacturer instructions: 17.7 μg pAdDeltaF6(Addgene 112867), 7.9 μg pRepCap6 (Addgene 110770), and 5.9 μg pAAV-CAG-GFP (Addgene 37825).
    suggested: RRID:Addgene_112867)
    suggested: RRID:Addgene_110770)
    suggested: RRID:Addgene_37825)
    Briefly, pCB3/T7 DNA was linearized using SalI-HF enzyme (R3138S, NEB) and CVB3 RNA was produced by in vitro transcription reaction.
    suggested: None
    Software and Algorithms
    The fluorescence intensity of Tf-647 labeled vesicles was then measured along this distance using ImageJ/Fiji.
    suggested: None
    The number of GFP positive cells was analyzed using a Cytoflex (Beckman Coulter) flow cytometer and results were analyzed using CytExpert software.
    suggested: (CytExpert Software, RRID:SCR_017217)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 54, 55, 56, 57 and 60. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

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