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Abstract

Background: Although mainly causing a respiratory syndrome, numerous neurological symptoms have been identified following of SARS-CoV-2 infection. However, how the virus affects the brain and how the mutations carried by the different variants modulate those neurological symptoms remain unclear.

Methods: We used primary human pericytes, foetal astrocytes, endothelial cells and a microglial cell line to investigate the effect of several SARS-CoV-2 variants of concern or interest on their functional activities. Cells and a 3D blood-brain barrier model were infected with the wild-type form of SARS-CoV-2, Alpha, Beta, Delta, Eta, or Omicron (BA.1) variants at various MOI. Cells and supernatant were used to evaluate cell susceptibility to the virus using a microscopic assay as well as effects of infection on (i) cell metabolic activity using a colorimetric MTS assay; (ii) viral cytopathogenicity using the xCELLigence system; (iii) extracellular glutamate concentration by fluorometric assay; and (iv) modulation of blood-brain barrier permeability.

Results: We demonstrate that productive infection of brain cells is SARS-CoV-2 variant dependent and that all the variants induce stress to CNS cells. The wild-type virus was cytopathic to all cell types except astrocytes, whilst Alpha and Beta variants were only cytopathic for pericytes, and the Omicron variant cytopathic for endothelial cells and pericytes. Lastly wild-type virus increases blood-brain barrier permeability and all variants, except Beta, modulate extracellular glutamate concentration, which can lead to excitotoxicity or altered neurotransmission.

Conclusions: These results suggest that SARS-CoV-2 is neurotropic, with deleterious consequences for the blood-brain barrier integrity and central nervous system cells, which could underlie neurological disorders following SARS-CoV-2 infection.

Keywords: Astrocytes; Blood–brain barrier; Brain; Brain microvascular endothelial cells; Central nervous system; Excitotoxicity; Microglia; Pericytes; SARS-CoV-2.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

👁 Fig. 1

Fig. 1

Variation in susceptibility of CNS cells to SARS-CoV-2. A Astrocytes, B hCMEC/d3, C HBVP and D HMC3 were infected for 24 h at various MOI (0.1 – 1 – 2.5 – 5) with WT (blue), Alpha (pink), Beta (green), Delta (brown), Eta (orange) or Omicron (yellow-green) viruses. The uninfected condition of each CNS cell (grey) is shown and form the background. The mean and SEM for 3 different donors/passages are shown. The normalised plaque area (fluorescent viral areas (µm²) normalised over fluorescent nuclei areas (µm²)) is presented. Asterisks denote statistically significant data as determined by two-way analysis of variance (ANOVA) with corrections for multiple comparisons (Dunnett) (*P < 0.05, **P < 0.01)

👁 Fig. 2

Fig. 2

Productive infection of CNS cells is SARS-CoV-2 variant dependent. A Astrocytes, B hCMEC/d3, C HBVP and D HMC3 were incubated with WT (blue), Alpha (pink), Beta (green), Delta (brown), Eta (orange) or Omicron (yellow-green) viruses at MOI 0.1 (dashed lines) or MOI 1 (full lines) for 24 h, washed to removed unbound viruses and stained after 2, 4 or 6 days of infection. The uninfected condition is represented with a black full line. The mean and SEM for 3 different donors/passages are shown. Fluorescent viral areas (µm²) have been normalised over fluorescent nuclei areas (µm²) and the baseline (uninfected control) removed. Asterisks denote statistically significant data as determined by two-way analysis of variance (ANOVA) with corrections for multiple comparisons (Dunnett) (*P < 0.05, **P < 0.01)

👁 Fig. 3

Fig. 3

SARS-CoV-2 infection modulates the metabolic activity of CNS cells. The metabolic activity of A astrocytes, B hCMEC/d3, C HBVP and D HMC3 was assessed using an MTS assay following 2, 4 or 6 days of infection at MOI 1 (full lines) or MOI 0.1 (dashed lines) with WT (blue), Alpha (pink), Beta (green), Delta (brown), Eta (orange) or Omicron (yellow-green) viruses. The uninfected condition is represented with a black line. Results are presented in raw data (i.e. absorbance read at 490 nm). The mean and SEM for 4 to 5 different donors/passages are shown. Asterisks denote statistically significant data as determined by one-way analysis of variance (ANOVA) with corrections for multiple comparisons (Tukey) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

👁 Fig. 4

Fig. 4

SARS-CoV-2 cytopathic effect on CNS cells. A–D Astrocytes, E–H hCMEC/d3, I–L HBVP and M–P HMC3 were infected with WT (A, E, I, M), Alpha (B, F, J, N), Beta (C, G, K, O) and Omicron (D, H, L, P) viruses at MOI 0.1 (WT: light blue; Alpha: purple; Beta: light green; Omicron: yellow) or MOI 1 (WT: dark blue; Alpha: burgundy; Beta: dark green; Omicron: brown) for 6 days and the impedance was recorded in real time continuously. The mean and SEM for 3 to 5 different donors/passages are presented in percentage of the uninfected control (red)

👁 Fig. 5

Fig. 5

WT virus increases BBB permeability while WT, Alpha, Delta, Eta and Omicron infections modulate extracellular glutamate. BBB were infected at MOI 1 (full lines) or MOI 0.1 (dashed lines) with WT (blue), Alpha (pink), Beta (green), Delta (brown), Eta (orange) or Omicron (yellow-green) viruses and we measured A BBB permeability (dextran-rhodamine assay) and B extracellular glutamate at 2, 4 or 6 days post-infection. An empty control insert (grey) was used to estimate the passive diffusion of dextran-rhodamine from the upper chamber to the collector and the concentration of glutamate in the BBB medium over time. Data are presented in raw data (i.e. concentration of Dextran-rhodamine B 70 kDa in µg/mL or concentration of glutamate in µM). The mean and SEM for 3 to 4 different experiments are shown. Asterisks denote statistically significant data as defined by one-way analysis of variance (ANOVA) with corrections for multiple comparisons (Tukey) (*P < 0.05, **P < 0.01)

👁 Fig. 6

Fig. 6

SARS-CoV-2 impact on endothelial cell tight junction and adherens junction gene expression. Endothelial cell gene expression of TJP1 (also known as ZO-1), F11R (encoding JAM-A), OCLN (Occludin), CLDN5 (Claudin-5) and CDH5 (encoding VE-Cadherin) was assessed by SYBR-Green Quantitative RT-PCR following 2, 4 or 6 days of infection at MOI 1 (labelled “1”) or MOI 0.1 (labelled “0.1”) with A WT, B Alpha, C Beta, D Delta, E Eta or F Omicron viruses. Results for 3 to 5 different donors/passages are presented in ratio of the uninfected condition (dashed red line) in box and whiskers plots (median and min to max). Asterisks denote statistically significant data as defined by two-way analysis of variance (ANOVA) with corrections for multiple comparisons (Dunnett) (*P < 0.05, **P < 0.01)

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URL: https://pubmed.ncbi.nlm.nih.gov/37537664/

⇱ Differential effects of SARS-CoV-2 variants on central nervous system cells and blood-brain barrier functions - PubMed

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