Microglia Do Not Restrict SARS-CoV-2 Replication following Infection of the Central Nervous System of K18-Human ACE2 Transgenic Mice

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1. Version published to 10.1128/jvi.01969-21

   Feb 23, 2022
2. 

   ScreenIT

   Nov 20, 2021

   SciScore for 10.1101/2021.11.15.468761: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mice and viral infection: All experiments were performed in accordance with animal protocols approved by the University of California, Irvine Institutional Animal Care and Use Committee.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Several primary antibodies were used, including Iba1 (1:500 Wako), GFAP (1:1000 Abcam), Mac2/ Galactin-3 (1:500, CL8942AP Cedarlane), CD4 (1:200 Abcam), CD8 (1:200 Abcam), MHC I (1:200 Abcam), and MHC II (1:200 Abcam).	Iba1 suggested: (US Biological Cat# S1000-78G1, RRID:AB_2270053) GFAP suggested: None CD4 suggested: None CD8 suggested: None
   On the …

   SciScore for 10.1101/2021.11.15.468761: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mice and viral infection: All experiments were performed in accordance with animal protocols approved by the University of California, Irvine Institutional Animal Care and Use Committee.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Several primary antibodies were used, including Iba1 (1:500 Wako), GFAP (1:1000 Abcam), Mac2/ Galactin-3 (1:500, CL8942AP Cedarlane), CD4 (1:200 Abcam), CD8 (1:200 Abcam), MHC I (1:200 Abcam), and MHC II (1:200 Abcam).	Iba1 suggested: (US Biological Cat# S1000-78G1, RRID:AB_2270053) GFAP suggested: None CD4 suggested: None CD8 suggested: None
   On the second day, slides were treated with appropriate secondary antibodies (1:1000 goat anti-rat/rabbit Invitrogen, 1:1000 goat anti-chicken, Abcam) following PBS rinsing.	anti-rat/rabbit suggested: None anti-chicken , suggested: None
   Cells were incubated overnight at 4°C in blocking solution with primary antibodies anti-MAP2 (EnCor Biotech. Cat:NC0388389) and anti-SARS-CoV-2 nucleocapsid (Sino Bio.	anti-MAP2 suggested: None anti-SARS-CoV-2 nucleocapsid (Sino Bio. suggested: None
   Cells were then washed with 1X PBS and incubated in blocking solution for 2hrs at RT with secondary antibodies, Alexa Fluor 594-conjugated goat anti-chicken and Alexa Fluor 488-conjugated goat anti-rabbit.	anti-chicken suggested: (AnaSpec; EGT Group Cat# 29709-1-H488, RRID:AB_10563238) anti-rabbit suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   8-16 week-old heterozygous K18-hACE2 C57BL/6 [strain: B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice were obtained from Jackson Laboratory.	C57BL/6 suggested: None B6.Cg-Tg(K18-ACE2)2Prlmn/J] suggested: RRID:IMSR_JAX:034860)
   Software and Algorithms
   Sentences	Resources
   The relative fold change between samples used in the ddCt calculation was calculated (2-ddCt). qPCR statistical analysis: Statistical analysis was performed in Prism (GraphPad Software).	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Reads passing quality control were used for quantification using featureCounts and analyzed with the R package DESeq2 to identify DEGs	featureCounts suggested: (featureCounts, RRID:SCR_012919) DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Genes passing an FDR of 10% were used for GO enrichment analysis using GOrilla (27) (http://cbl-gorilla.cs.technion.ac.il/).	http://cbl-gorilla.cs.technion.ac.il/ suggested: (GOrilla: Gene Ontology Enrichment Analysis and Visualization Tool, RRID:SCR_006848)
   From those lists, the genes with the top 100 most variable TPM were plotted using the R package pheatmap.	pheatmap suggested: (pheatmap, RRID:SCR_016418)
   Following fixation, the 24-48hr brains were either cryoprotected in 30% sucrose, embedded in O.C.T. (Fisher HealthCare), and sliced via Cryostat in 10µm sagittal sections.	Fisher HealthCare suggested: None
   Cell quantities were determined using the spots module in Imaris.	Imaris suggested: (Imaris, RRID:SCR_007370)

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   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).

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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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04343651	Completed	Study to Evaluate the Efficacy and Safety of Leronlimab for …
   NCT04347239	Active, not recruiting	Study to Evaluate the Efficacy and Safety of Leronlimab for …
   NCT04878055	Recruiting	Study on Efficacy and Safety of Reparixin in the Treatment o…
   NCT04347226	Recruiting	Anti-Interleukin-8 (Anti-IL-8) for Patients With COVID-19
   NCT04389645	Completed	Interferon Gamma Induced Protein 10 (IP-10) in a Clinical De…

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   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).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   Results from rtransparent:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • 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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3. 

   Version published to 10.1101/2021.11.15.468761v1 on bioRxiv

   Nov 17, 2021