Administration of aerosolized SARS-CoV-2 to K18-hACE2 mice uncouples respiratory infection from fatal neuroinvasion
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Abstract
The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2–infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.
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SciScore for 10.1101/2021.08.06.455382: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces.
Euthanasia Agents: Mice were euthanized by cervical dislocation.Sex as a biological variable Social scent-discrimination test: The social scent-discrimination test was used to assess hyposmia/anosmia in IN- infected or AR-infected male mice compared to PBS-treated control male mice, as previously described (Zheng et al., 2021). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources In vivo … SciScore for 10.1101/2021.08.06.455382: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces.
Euthanasia Agents: Mice were euthanized by cervical dislocation.Sex as a biological variable Social scent-discrimination test: The social scent-discrimination test was used to assess hyposmia/anosmia in IN- infected or AR-infected male mice compared to PBS-treated control male mice, as previously described (Zheng et al., 2021). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources In vivo treatment: In selected experiments, K18-hACE2 mice were injected intraperitoneally with 2 mg per mouse of α-IFNAR1 blocking antibody (BioXcell, #BE0241, clone MAR1-5A3) 1 day before infection. α-IFNAR1suggested: None#BE0241suggested: (Bio X Cell Cat# BE0241, RRID:AB_2687723)Experimental Models: Cell Lines Sentences Resources Vero E6 cells were seeded into 96 wells plates and infected at 95% of confluency with base 10 dilutions of virus stock. Vero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Mice: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (referred to in the text as K18-hACE2) were purchased from The Jackson Laboratory. B6.Cg-Tg(K18-ACE2)2Prlmn/Jsuggested: RRID:IMSR_JAX:034860)As control, K18-hACE2 mice received the same volume of aerosolized PBS (125 μL per mouse). K18-hACE2suggested: RRID:IMSR_GPT:T037657)Software and Algorithms Sentences Resources Flow cytometry analysis was performed on BD FACS Symphony A5 SORP and analyzed with FlowJo software (Treestar) FlowJosuggested: (FlowJo, RRID:SCR_008520)RNA-seq data processing and analysis: Raw reads were aligned to mouse genome build GRCm38 using STAR aligner (Dobin et al., 2013) STARsuggested: (STAR, RRID:SCR_004463)Gene counts were generated using featureCounts (part of the Subread package (Liao et al., 2019)), based on GENCODE gene annotation version M22. featureCountssuggested: (featureCounts, RRID:SCR_012919)GENCODEsuggested: (GENCODE, RRID:SCR_014966)Differentially Expressed Genes (DEGs) between AR- and IN-infected mice were identified by generating a linear model using LIMMA R package (Ritchie et al., 2015) ( LIMMAsuggested: (LIMMA, RRID:SCR_010943)Bright-field images were acquired with an Aperio Scanscope System CS2 microscope and the ImageScope program (Leica Biosystem) following the manufacturer’s instructions. ImageScopesuggested: (ImageScope, RRID:SCR_014311), anti-Iba1 (Synaptic system, #234006), anti-CD68 (Abcam #ab53444), and anti-iNOS (Abcam #ab3523). Synaptic systemsuggested: (Synaptic Systems, RRID:SCR_013612)Flow and imaging data were collected using FlowJo Version 10.5.3 (Treestar) and Imaris (Bitplane), respectively. Imarissuggested: (Imaris, RRID:SCR_007370)Statistical analyses were performed with GraphPad Prism software version 8 (GraphPad). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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 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.
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