1. SciScore for 10.1101/2021.10.20.465121: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The use of clinical samples (sera) was approved by the Institutional Review Board at Charité -Universitätsmedizin Berlin (EA1/068/20, EA2/092/20 and EA2/066/20) and is in accordance with the Berlin State Hospital Law, allowing for pseudonymized scientific analysis of routine patient data.
    Consent: All other experiments were conducted with hBAECs isolated from explanted lungs which were obtained from the Hannover Lung Transplant Program after patients informed consent, ethical vote 2923-2015.
    Sex as a biological variablePer group, nine male and female Roborovski dwarf hamsters (Phodopus roborovskii) obtained via the German pet trade were used.
    RandomizationHamsters of the same sex were randomly distributed into experimental groups and individually marked with a subcutaneously implanted IPTT-300 transponder (BMDS, Seaford (DE), USA) that allows remote identification and measurement of body temperature.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Blocked membranes were incubated with the following antibodies: mouse anti-HIV-1 p24 capsid (ExBio, 1:1000), rabbit anti-S2 spike (Novusbio, NB100-56578, 1:1000), mouse anti-HA (Sigma, H3663, 1:1400), rabbit anti-tubulin (Cell Signaling Technology, 2144S, 1:1000).
    suggested: None
    suggested: (Sigma-Aldrich Cat# H3663, RRID:AB_262051)
    suggested: None
    anti-tubulin ( Cell Signaling Technology , 2144S
    suggested: None
    Blocked membranes were incubated with the following antibodies: rabbit anti-S2 spike (Novusbio, NB100-56578, 1:1000), rabbit anti-SARS-CoV-2 nucleocapsid (GeneTex, GTX135361, 1:1000).
    suggested: None
    suggested: (Novus Cat# NB100-56578, RRID:AB_838846)
    anti-SARS-CoV-2 nucleocapsid ( GeneTex , GTX135361
    suggested: None
    Experimental Models: Cell Lines
    , NCI-H1299 (ATCC CRL-5803),
    suggested: NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)
    HEK-293T (ATCC CRL-3216),
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    Virus infection and virus growth kinetics in cell cultures: Vero E6, Caco-2, NCI-H1299 and A549 cells were seeded at a densitiy of 350,000 cells per ml and Calu-3 cells at a density of 500,000 cells per ml one day prior to infection.
    suggested: None
    suggested: None
    All samples were titrated on Vero E6 cells by plaque assay to determine infectious titers.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Competition assay: Calu-3 cells were infected in 24-well plates with a mixture of two SARS-CoV-2 variants, using three different ratios (1:1 and 9:1 and 1:9) and an initial, total infectious dose of 10.000 PFU (corresponding to an MOI of 0.04).
    suggested: None
    CHO cells were transiently transfected with expression plasmids for HIV-1 Tat and individual pCG-spike-HA or empty vector control for 48 hours, using Lipofectamine LTX Reagent with PLUS™ Reagent (Invitrogen)
    suggested: None
    TZM-bl cells, stably expressing LTR-driven luciferase, were transfected with a plasmid encoding human ACE2 and human myc-TMPRSS2.
    suggested: RRID:CVCL_B478)
    Lentivirus production and transduction experiments: SARS-CoV-2 spike-HA-pseudotyped lentiviral particles were produced in triple-transfected HEK293T cells.
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Experimental Models: Organisms/Strains
    Cells and culture conditions: A549 parental (ATCC CCL-185), A549-ACE2, A549-TMPRSS2, A549-ACE2 + TMPRSS2 (Widera et al. 2021), Caco-2 (ATCC HTB-37), Calu-3 (HTB-55), CHO (HIV Reagent Program ARP-2238)
    A549-TMPRSS2, A549-ACE2 + TMPRSS2
    suggested: None
    Recombinant DNA
    Codon-optimized, C-terminally tagged spike cDNAs in pCG were generated using pCG-SARS-CoV-2 spike Wuhan as a template (Hoffmann et al., 2020) in which the N-terminus was repaired and D614G was introduced via site-directed mutagenesis.
    suggested: RRID:Addgene_51476)
    For generation of the D614G mutant we performed site-directed mutagenesis PCR (NEB) on synthetic viral subgenomic fragments cloned into pUC57 vectors (Thi Nhu Thao et al., 2020).
    suggested: RRID:Addgene_40306)
    CHO cells were transiently transfected with expression plasmids for HIV-1 Tat and individual pCG-spike-HA or empty vector control for 48 hours, using Lipofectamine LTX Reagent with PLUS™ Reagent (Invitrogen)
    suggested: None
    Cells were transfected with individual pCG-SARS-CoV-2 spike-HA plasmids, the HIV-1-based packaging plasmid deltaR8.91 (Zufferey et al., 1997) and the luciferase transfer plasmid pCSII-luciferase (Agarwal et al., 2006) via calcium phosphate precipitation.
    pCG-SARS-CoV-2 spike-HA
    suggested: None
    suggested: None
    suggested: None
    Software and Algorithms
    The processed sequence reads were mapped to the BetaCoV/Munich/ChVir984/2020 genome (here referred to as SARS-CoV-2 2019-nCoV strain) in Geneious (version 9.1.8).
    suggested: (Geneious, RRID:SCR_010519)
    Quantification was done by the use of ImageJ 1.48v software.
    suggested: (ImageJ, RRID:SCR_003070)

    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: We detected the following sentences addressing limitations in the study:
    While caveats and limitations of neoplastic cell lines apply, NCI-H1299 cells yielded higher levels of replication and infectious virus production not only for B.1.1.7, but also for B.1.617.2. This cell line of epithelial morphology is devoid of detectable ACE2 protein and remains susceptible to SARS-CoV-2 infection even in the presence of ACE2-neutralizing antibodies, suggesting the existence of an alternative, but not variant-specific mode of entry. This finding is reminiscent of a recent study (Puray-Chavez et al., 2021) that identified SARS-CoV-2 infection of the H522 lung cell line in an ACE2-independent fashion (in this study the alternative way of infection could only be utilized by viruses carrying an E484D substitution within the spike protein RBD). Surprisingly, while experiments with reciprocal chimeras established that the B.1.1.7 spread advantage in NCI-H1299 cells is mediated to a large extent by its spike, the entry process per se was similarly or even less efficient for B.1.1.7 than for B.1. Notably, a virus consisting of the B.1.1.7 backbone with B.1 spike displayed a slightly better replication efficiency than the original B.1 virus in type-I IFN-competent Calu-3 cells, confirming that other genetic determinants of B.1.1.7 beyond spike contribute to enhanced replication. These findings suggest a post-entry, yet largely spike-dependent replication advantage of B.1.1.7 that manifests itself in NCI-H1299 cells. Remarkably, we conducted preliminary studies of re...

    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.

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