SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for spillback to New World rodents
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Abstract
Coronavirus disease-19 (COVID-19) emerged in late 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats ( Rhinolophus spp.) and may have infected an intermediate host prior to spillover into humans. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice ( Peromyscus maniculatus ) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 6 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood-brain barrier. Despite this, no conspicuous signs of disease were observed, and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, including IFNα, IFNβ, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8β expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission and localization into the olfactory bulb, recapitulating human neuropathology. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources determined the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 respiratory disease and neuropathogenesis, and that they have the potential to serve as secondary reservoir hosts in North America.
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SciScore for 10.1101/2020.08.07.241810: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All work with infectious virus was performed at BSL-3 with approval from the Colorado State University Institutional Biosafety Committee. Randomization not detected. Blinding Decalcified skulls and visceral organs were processed, embedded in paraffin wax and 4-5 µm sections were stained with hematoxylin and eosin for blinded evaluation by the pathologist using Nikon i80 microscope (Nikon Microscopy). Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After transfer, the blots were sectioned by lane, blocked, and individual lanes incubated with 1:100 dilutions of … SciScore for 10.1101/2020.08.07.241810: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All work with infectious virus was performed at BSL-3 with approval from the Colorado State University Institutional Biosafety Committee. Randomization not detected. Blinding Decalcified skulls and visceral organs were processed, embedded in paraffin wax and 4-5 µm sections were stained with hematoxylin and eosin for blinded evaluation by the pathologist using Nikon i80 microscope (Nikon Microscopy). Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After transfer, the blots were sectioned by lane, blocked, and individual lanes incubated with 1:100 dilutions of the indicated deer mouse sera or with mouse anti-nucleocapsid monoclonal antibody overnight. anti-nucleocapsidsuggested: NonePrimary antibodies from deer mice were detected with goat anti-P. anti-Psuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus was passaged twice on Vero E6 cells (ATCC CRL-1586) in 2% FBS-DMEM containing 10,000 IU/ml penicillin and streptomycin at 37°C under 5% CO2 to generate stock virus used in these experiments. Vero E6suggested: NoneFor western blot detection, infected Vero cell supernatants were subject to centrifugal filter concentration with molecular weight cutoff of 300 kDa (Pall Microsep 300K Omega). Verosuggested: NoneSoftware and Algorithms Sentences Resources Images were scanned with a Visioneer One Touch 9420 scanner at a gamma value of 1.0, and all contrast adjustments were uniformly applied using Adobe Photoshop. Adobe Photoshopsuggested: (Adobe Photoshop, RRID:SCR_014199)MiSeq reads were demultiplexed, quality checked by FASTQC, paired-end reads were processed to removed Illumina primers and quality trimmed with Cutadapt, duplicate reads were removed. MiSeqsuggested: (A5-miseq, RRID:SCR_012148)FASTQCsuggested: (FastQC, RRID:SCR_014583)Cutadaptsuggested: (cutadapt, RRID:SCR_011841)Alignments were further processed, and quality checked, using Geneious software, consensus sequences were determined and any gaps in sequences were filled in with the reference sequence. Geneioussuggested: (Geneious, RRID:SCR_010519)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: Please consider improving the rainbow (“jet”) colormap(s) used on page 18. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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SciScore for 10.1101/2020.08.07.241810: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement All work with infectious virus was performed at BSL-3 with approval from the Colorado State University Institutional Biosafety Committee. Randomization not detected. Blinding Decalcified skulls and visceral organs were processed, embedded in paraffin wax and 4-5 µm sections were stained with hematoxylin and eosin for blinded evaluation by the pathologist using Nikon i80 microscope (Nikon Microscopy). Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After transfer, the blots were sectioned by lane, blocked, and individual lanes incubated with 1:100 dilutions of the … SciScore for 10.1101/2020.08.07.241810: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement All work with infectious virus was performed at BSL-3 with approval from the Colorado State University Institutional Biosafety Committee. Randomization not detected. Blinding Decalcified skulls and visceral organs were processed, embedded in paraffin wax and 4-5 µm sections were stained with hematoxylin and eosin for blinded evaluation by the pathologist using Nikon i80 microscope (Nikon Microscopy). Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After transfer, the blots were sectioned by lane, blocked, and individual lanes incubated with 1:100 dilutions of the indicated deer mouse sera or with mouse anti-nucleocapsid monoclonal antibody overnight. anti-nucleocapsidsuggested: NonePrimary antibodies from deer mice were detected with goat anti-P. anti-Psuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus was passaged twice on Vero E6 cells (ATCC CRL-1586) in 2% FBS-DMEM containing 10,000 IU/ml penicillin and streptomycin at 37°C under 5% CO2 to generate stock virus used in these experiments. Vero E6suggested: NoneFor western blot detection, infected Vero cell supernatants were subject to centrifugal filter concentration with molecular weight cutoff of 300 kDa (Pall Microsep 300K Omega). Verosuggested: NoneSoftware and Algorithms Sentences Resources Images were scanned with a Visioneer One Touch 9420 scanner at a gamma value of 1.0, and all contrast adjustments were uniformly applied using Adobe Photoshop. Adobe Photoshopsuggested: (Adobe Photoshop, RRID:SCR_014199)MiSeq reads were demultiplexed, quality checked by FASTQC, paired-end reads were processed to removed Illumina primers and quality trimmed with Cutadapt, duplicate reads were removed. MiSeqsuggested: (A5-miseq, RRID:SCR_012148)FASTQCsuggested: (FastQC, RRID:SCR_014583)Cutadaptsuggested: (cutadapt, RRID:SCR_011841)Consensus sequences were aligned in Geneious. Geneioussuggested: (Geneious, RRID:SCR_010519)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 Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on page 18. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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