SARS-CoV-2 Omicron efficiently infects human airway, but not alveolar epithelium

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   ScreenIT

   Jan 22, 2022

   SciScore for 10.1101/2022.01.19.476898: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512)
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Next, cells were incubated in FACS buffer (2mM EDTA, 2.5% bovine serum albumin (BSA) in PBS) on ice for 5 min, stained with the AT2 marker antibody HTII-280 (1:40; Terrace Biotech) on ice for 15 min, and with goat anti-mouse IgM Alexa Fluor 488 (1:400; Invitrogen) for 5 min.	AT2 suggested: None anti-mouse IgM suggested: (Bethyl Cat# A90-140P, …

   SciScore for 10.1101/2022.01.19.476898: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512)
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cell lines were tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Next, cells were incubated in FACS buffer (2mM EDTA, 2.5% bovine serum albumin (BSA) in PBS) on ice for 5 min, stained with the AT2 marker antibody HTII-280 (1:40; Terrace Biotech) on ice for 15 min, and with goat anti-mouse IgM Alexa Fluor 488 (1:400; Invitrogen) for 5 min.	AT2 suggested: None anti-mouse IgM suggested: (Bethyl Cat# A90-140P, RRID:AB_10630983)
   Cells were incubated with primary antibodies overnight at 4°C in blocking buffer, washed twice with PBS, incubated with corresponding secondary antibodies (Alexa488- and 594-conjugated anti-rabbit IgG, anti-mouse IgG, or anti-mouse IgG1 (1:500; Invitrogen)) in blocking buffer for 2 hours at room temperature, washed two times with PBS, incubated with indicated additional stains (hoechst or phalloidin), washed twice with PBS, and mounted in Prolong Antifade mounting medium.	anti-rabbit IgG suggested: None anti-mouse IgG suggested: None anti-mouse IgG1 suggested: None Antifade mounting medium . suggested: None
   Spike was stained using mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody).	S2 suggested: None NP suggested: None mouse-anti-VSV-N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Material and Methods: Cell lines: Vero and VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS), HEPES (20 mM, Lonza), sodium pyruvate (1 mM, Gibco), penicillin (100 IU/mL), and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.	Vero suggested: None VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   TMPRSS2, GFP11 and GFP1-10 stable cells were maintained in a medium containing hygromycin (Invitrogen), puromycin (Invivogen) and geneticin (Invitrogen), respectively.	GFP1-10 suggested: None
   To determine the 8-hour infectious titer, virus stocks were thawed and diluted in 10-fold serial dilution in 200μl Opti-MEM I (1X) + GlutaMAX. 100μl of each dilution was added to monolayers of 1 × 105 VeroE6, VeroE6-TMPRSS2 or Calu-3 cells in a 96-well plate.	Calu-3 suggested: None
   30 μl of each dilution was transferred to 2 × 104 VeroE6 and VeroE6-TMPRSS2 cell monolayers or 8 × 104 Calu-3 cell monolayers in the same medium in a 96-well plate.	VeroE6-TMPRSS2 suggested: None
   Transfected HEK-293T cells were transferred in three technical replicates to GFP1-10 expressing VeroE6, VeroE6-TMPRSS2 and Calu-3 cell monolayers in a ratio of 1:80 (HEK-293T cells : GFP1-10 expressing cells).	HEK-293T suggested: None
   Recombinant DNA
   Sentences	Resources
   The pGAGGS-Omicron expression plasmid was kindly provided by Dr. Berend Jan Bosch.	pGAGGS-Omicron suggested: None
   The pGAGGS-β-Actin-P2A-7xGFP11-BFP plasmid was cloned into pQCXIP and used for retrovirus production and subsequent generation of the GFP11 stable cells as previously described by Mykytyn et al. (42).	pGAGGS-β-Actin-P2A-7xGFP11-BFP suggested: None pQCXIP suggested: RRID:Addgene_15714)
   Briefly, 1.5 μg pGAGGS-spike DNA and pGAGGS-β-Actin-P2A-7xGFP11-BFP DNA or empty vector DNA were transfected into HEK-293T cells with PEI (Polysciences) in a ratio of 1:3 (DNA: PEI)	pGAGGS-spike suggested: None
   Software and Algorithms
   Sentences	Resources
   To assess the effect of camostat mesylate (Sigma) and E64D (MedChemExpress) on viral replication, 2D cultures were pretreated in the basal and apical compartment for 1 hour with 10μM of either compound or the combination of both, or DMSO in the negative control Submerged 2D monolayers of VeroE6 and Calu-3 cells were washed in 300 μl PBS and subsequent infection was performed in AdDF +++ medium. 3D AT2 cultures were incubated with TrypLE Express for 5 minutes at room temperature and washed three times with 5 ml AdDF+++ medium.	AdDF suggested: None
   All confocal imaging was performed on a LSM700 confocal microscope using ZEN software.	ZEN suggested: None
   Read 2 was aligned to the CRCh38 human RefSeq transcriptome, with the addition of SARS-CoV-2 (Ref-SKU: 026V-03883; MW947280) genomes, using BWA using standard settings (67).	BWA suggested: (BWA, RRID:SCR_010910)
   Differential expression analysis were performed using the DESeq2 package(68).	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Data was stitched, uploaded, shared and annotated using Omero (annotated using Omero) and PathViewer.	Omero suggested: (OMERO, RRID:SCR_002629)
   Data was analyzed using the ImageQuant TL 8.2 image analysis software (GE Healthcare) by calculating the sum of all GFP+ pixels per well.	ImageQuant suggested: (ImageQuant, RRID:SCR_014246)
   Statistical analysis: Statistical analysis was performed with the GraphPad Prism 9 software.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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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 10.1101/2022.01.19.476898 on bioRxiv

   Jan 20, 2022