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Evolution of SARS-CoV-2 in the murine central nervous system drives viral diversification

Nature Microbiology volume 9, pages 2383–2394 (2024)Cite this article

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Abstract

Severe coronavirus disease 2019 and post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral divergence were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titres and decreased morbidity compared with the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but dissemination from the CNS to the lung required the intact FCS. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization.

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Fig. 1: Viral RNA from the CNS has a higher degree of genetic divergence regardless of vaccination status.
Fig. 2: BALB/c mice infected with mouse-adapted SARS-CoV-2 have increased viral diversity in the CNS.
Fig. 3: Mutation of the FCS blocks spike S1/S2 cleavage and mitigates utilization of an alternative entry pathway.
Fig. 4: ΔFCS virus is attenuated after intranasal infection.
Fig. 5: ΔFCS virus replicates faster in the CNS after intracranial inoculation.
Fig. 6: There is selective pressure for the deletion of the FCS in the CNS.

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Data availability

All data supporting the findings of this study are available within the paper, extended data or in the source data files. Viral whole-genome sequencing data have been deposited to NCBI as Bioproject PRJNA1017824. SARS-CoV-2 reference genome accession MN908947.3. Source data are provided with this paper.

Code availability

No unique code was generated in the course of the data acquisition. To determine consensus RNA sequence, reads were trimmed to remove adaptors and low-quality sequences using Trimmomatic v0.36. Trimmed reads were aligned to the reference genome sequence of SARS-CoV-2 (accession MN908947.3) using bwa v0.7.15. Pile-ups were generated from the alignment using samtools v1.9 and consensus sequence determined using iVar v1.2.2. For phylogenetic analysis, consensus sequences assembled for each sample were aligned using MAFFT v7.453 software. A maximum likelihood (ML) phylogeny with all consensus sequences were inferred with IQ-Tree v2.0.5. We performed probabilistic inference of intra-host viral quasispecies of the spike gene for each sample using QuasiRecomb. All final tree representation was performed with the R package ggtree v3.2.1. Viral diversity was determined using lme4 version 1.1-34 in R version 4.0.3.

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Acknowledgements

Infectious clones provided by the Vineet Menachery lab from University of Texas Medical Branch. This research was supported, in part, through the computational resources and staff contributions provided by the Quest high-performance computing facility at Northwestern University, which is jointly supported by the Office of the Provost, the Office for Research and Northwestern University Information Technology. Tissue embedding and imaging was performed by the Research Histology and Tissue Imaging Core at UIC Research Resources Center. Funding for this work was provided by: NIH grants R01 AI150672 and R56 DE033249 (J.M.R.), DOD grant MS200290 (S.E.L.), NIH grant R21 AI163912 (J.F.H.), NIH grant U19 AI135964 (J.F.H., E.A.O. and R.L.R.), and through institutional support for the Center for Pathogen Genomics and Microbial Evolution (J.F.H., E.A.O. and R.L.R.). The funding sources had no role in the study design, data collection, analysis, interpretation or writing of the report. Figures 4a and 5a were created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Jacob Class, Lacy M. Simons.

  2. These authors jointly supervised this work: Judd F. Hultquist, Justin M. Richner.

Authors and Affiliations

  1. Department of Microbiology and Immunology, College of Medicine, University of Illinois Chicago, Chicago, IL, USA

    Jacob Class, Jazmin Galván Achi, Laura Cooper, Lijun Rong & Justin M. Richner

  2. Department of Medicine, Division of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Lacy M. Simons, Ramon Lorenzo-Redondo, Egon A. Ozer & Judd F. Hultquist

  3. Center for Pathogen Genomics and Microbial Evolution, Havey Institute for Global Health, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Lacy M. Simons, Ramon Lorenzo-Redondo, Egon A. Ozer & Judd F. Hultquist

  4. Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Tanushree Dangi & Pablo Penaloza-MacMaster

  5. Department of Anatomy and Cell Biology, College of Medicine, University of Illinois Chicago, Chicago, IL, USA

    Sarah E. Lutz

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Contributions

Conceptualization was performed by J.F.H., J.M.R. and P.P.-M.; methodology was carried out by J.C., L.M.S., L.C., J.G.A., S.E.L, T.D. and R.L.-R.; software and validation was carried out by L.M.S.; formal analysis was performed by J.C., L.M.S. and R.L.-R.; investigation was carried out by J.C., L.C., T.D. and L.M.S.; resources were provided by J.M.R., L.R., P.P.-M., J.F.H., R.L.-R. and E.A.O.; data curation was carried out by J.C., L.M.S. and R.L.-R.; writing of the original draft was performed by J.C., J.F.H. and J.M.R.; writing, review and editing, was performed by L.M.S., J.F.H., L.C., P.P.-M., L.R. and J.M.R; visualization was carried out by R.L.-R., J.F.H., S.E.L., J.C. and J.M.R.; supervision was carried out by J.F.H., R.L.-R., E.A.O., J.M.R. and L.R.; project administration was carried out by J.M.R., J.F.H., R.L.-R., E.A.O. and L.R.; and funding acquisition was carried out by J.M.R., J.F.H., R.L.-R., E.A.O. and L.R.

Corresponding authors

Correspondence to Judd F. Hultquist or Justin M. Richner.

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Competing interests

J.F.H. has received research support paid to Northwestern University from Gilead Sciences and is a paid consultant for Merck. All other authors declare no conflicts of interest.

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Nature Microbiology thanks Kei Sato, Shan-Lu Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Ad5-S and Ad5-N vaccines elicit robust humoral and cellular immunity against target antigens.

Mice were vaccinated with PBS, or Ad5 vaccines encoding for spike or nucleocapsid viral proteins (see reference below). 21 days post vaccination, mice were euthanized, and serum and spleens were collected. a, Antigen specific CD8 T cells were quantified with tetramers to the spike or nucleocapsid protein. Serum was analyzed for antibodies against spike (b) or nucleocapsid (c) recombinant protein via ELISA assay. P-values of Student’s t-test is reported24.

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Unprocessed histology and in vitro images.

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Source Data Extended Data Fig. 1 (download XLSX )

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Class, J., Simons, L.M., Lorenzo-Redondo, R. et al. Evolution of SARS-CoV-2 in the murine central nervous system drives viral diversification. Nat Microbiol 9, 2383–2394 (2024). https://doi.org/10.1038/s41564-024-01786-8

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