Distinct upper airway epithelium interferon-stimulated and profibrotic gene expression between adult and infant rhesus macaques infected with SARS-CoV-2

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   ScreenIT

   Feb 16, 2022

   SciScore for 10.1101/2022.02.12.480218: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: The Institutional Animal Use and Care Committee approved these experiments (study protocol# 21702). Field Sample Permit: Trachea collection and cell processing: Upon necropsy, tracheobronchial tissues were collected.
   Sex as a biological variable	All animals were challenged through combined intratracheal (IT, 2.0 mL for dams, 1 ml for infants) and intranasal (IN, 0.2 5 mL per nostril) inoculation with an infectious dose of 2.5 × 10^6 PFU for the dams and 1.5 × 10^6 PFU for the infants of SARS-CoV-2 (2019-nCoV/USA-WA1/2020).
   Randomization	not detected.
   Blinding	Radiographs were scored for the presence of pulmonary infiltrates by a board-certified veterinary radiologist, who was blinded to the …

   SciScore for 10.1101/2022.02.12.480218: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: The Institutional Animal Use and Care Committee approved these experiments (study protocol# 21702). Field Sample Permit: Trachea collection and cell processing: Upon necropsy, tracheobronchial tissues were collected.
   Sex as a biological variable	All animals were challenged through combined intratracheal (IT, 2.0 mL for dams, 1 ml for infants) and intranasal (IN, 0.2 5 mL per nostril) inoculation with an infectious dose of 2.5 × 10^6 PFU for the dams and 1.5 × 10^6 PFU for the infants of SARS-CoV-2 (2019-nCoV/USA-WA1/2020).
   Randomization	not detected.
   Blinding	Radiographs were scored for the presence of pulmonary infiltrates by a board-certified veterinary radiologist, who was blinded to the experimental group and time point, according to a standard scoring system (0: normal; 1: mild interstitial pulmonary infiltrates; 2: moderate pulmonary infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; 3: severe interstitial infiltrates, large areas of pulmonary consolidation, alveolar patterns and air bronchograms).
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Paraffin sections were treated in an antigen unmasking solution (Vector) at 100°C for 20 minutes before incubation with a primary antibody.	antigen unmasking solution (Vector) suggested: (Vector Laboratories Cat# H-3301, RRID:AB_2336227)
   Experimental Models: Cell Lines
   Sentences	Resources
   293 T/ACE2 cells were provided by M. Farzan and H. Mu at Scripps Florida.	T/ACE2 suggested: None
   HEK 293T cells expressing ACE2 receptors were suspended using TrypLE Select Enzyme solution (Thermo Fisher Scientific) and immediately added to all wells (10,000 cells in 100 μl of growth medium per well).	HEK 293T suggested: None
   Recombinant DNA
   Sentences	Resources
   Pseudovirions were produced in HEK293 T/17 cells (American Type Culture Collection, catalog no. CRL-11268) by transfection using Fugene 6 (Promega, catalog no. E2692) and a combination of S plasmid, lentiviral backbone plasmid (pCMV ΔR8.2), and firefly Luc reporter gene plasmid (pHR’ CMV Luc) (78) in a 1:17:17 ratio.	pCMV ΔR8.2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Analysis of the bulk RNA-seq data: RNA-Seq data was quality checked with FastQC (59) and preprocessing was carried out using TrimGalore (60) toolkit to trim low-quality bases and Illumina adapter sequences using default settings.	FastQC suggested: (FastQC, RRID:SCR_014583)
   Reads were aligned to the ENSEMBL Homo_sapiens.GRCh38.dna.primary_assembly genome using the ENSEMBL Homo_sapiens.GRCh38.100 transcript (61) annotation file with STAR (62) splice-aware RNA-seq alignment tool in paired mode allowing maximum multimapping of 3.	ENSEMBL suggested: (Ensembl, RRID:SCR_002344) STAR suggested: (STAR, RRID:SCR_004463)
   Gene level counts were quantified using FeatureCounts (63) tool, counting unique features in non-stranded mode and retaining both gene ID, gene name, and gene biotype mapping from the ENSEMBL annotation file.	FeatureCounts suggested: (featureCounts, RRID:SCR_012919)
   Normalization and differential expression were carried out with DESeq2 (64)	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Bioconductor (65) package, utilizing the ‘apeglm’ Bioconductor package (66) for log fold change shrinkage, in R statistical programming environment.	Bioconductor suggested: (Bioconductor, RRID:SCR_006442)
   The RNA-seq data was processed using the TrimGalore toolkit1 which employs Cutadapt2 to trim low quality bases and Illumina sequencing adapters from the 3’ end of the reads.	TrimGalore suggested: None
   Gene counts were compiled using the HTSeq tool5.	HTSeq suggested: (HTSeq, RRID:SCR_005514)
   Normalization and differential expression was carried out using the DESeq26 Bioconductor7 package with the R statistical programming environment8.	DESeq26 suggested: None Bioconductor7 suggested: None
   Gene set enrichment analysis9 was performed to identify gene ontology terms and pathways associated with altered gene expression for each of the comparisons performed.	Gene set enrichment suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)

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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: We detected the following sentences addressing limitations in the study:

   Our study has limitations. Firstly, we only infected two dam-infant macaque pairs and more animal numbers are needed to determine statistical differences. Additionally, a mock-inoculated control group is necessary to decipher whether the differences observed are due to SARS-CoV-2 infection alone, the age-dependent maturation of tissues and the immune response, and/or experimental procedures. Finally, the time of euthanasia was not focused on evaluating acute inflammatory responses in tissues, as at 2 weeks, virus replication is mainly gone and tissue responses reflect repair in this animal model, and we evaluated trachea instead of lung responses. However, our study is valuable in that it agrees with currently published data in SARS-CoV-2-infected pediatric and adult cohorts and furthers our understanding of why younger populations are less susceptible to severe COVID-19 compared to adults. Additionally, this model will allow further definition of molecular mechanisms of age-dependent SARS-CoV-2 pathogenesis and assess efficacy of medical countermeasures.

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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 10.1101/2022.02.12.480218v1 on bioRxiv

   Feb 14, 2022