Human iPS cell-derived sensory neurons can be infected by SARS-CoV-2

Abstract

COVID-19 has impacted billions of people since 2019 and unfolded a major healthcare crisis. With an increasing number of deaths and the emergence of more transmissible variants, it is crucial to better understand the biology of the disease-causing virus, the SARS-CoV-2. Peripheral neuropathies appeared as a specific COVID-19 symptom occurring at later stages of the disease. In order to understand the impact of SARS-CoV-2 on the peripheral nervous system, we generated human sensory neurons from induced pluripotent stem cells that we infected with the SARS-CoV-2 strain WA1/2020 and the variants delta and omicron. Using single-cell RNA sequencing, we found that human sensory neurons can be infected by SARS-CoV-2 but are unable to produce infectious viruses. Our data indicate that sensory neurons can be infected by the original WA1/2020 strain of SARS-CoV-2 as well as the delta and omicron variants, yet infectability differs between the original strain and the variants.

Keywords: Cellular neuroscience; Neuroscience; Virology.

Graphical abstract

Figure 1

SARS-CoV-2 infects a subset of human sensory neurons (A) UMAP of single-cell RNA sequencing (scRNA-seq) from hiPSC-sensory neurons co-cultured with HEK293T cells exposed to heat-inactivated virus or SARS-CoV-2 strain WA1/2020 for 48h. Mock is used as a control. (B) Feature plot of average gene expression for sensory markers NTRK2 (Trk.B), NTRK3 (Trk.C), pan-neuronal marker MAP2 and 293T cell marker AMOT in all samples combined (mock, heat-inactivated and SARS-CoV-2). (C) Feature plot of average gene expression for SARS-CoV-2’s Nucleocapsid in hiPSC-sensory neurons co-cultured with HEK293T cells exposed to WA1/2020 SARS-CoV-2 for 48h. (D) Differential gene expression analysis between SARS-CoV-2 positive and negative sensory neurons. The dot size indicates the proportion of sensory neurons expressing the corresponding gene. The color gradient of each dot indicates the p-value for the change in gene expression (more red = more significant). Genes with a negative Log2FC have reduced expression in SARS-CoV-2 positive neurons compared to SARS-CoV-2 negative neurons.

Figure 2

Infected sensory neurons synthesize SARS-CoV-2 RNA but do not replicate progeny virion (A) Fraction of negative strand SARS-CoV-2 RNA compared to the total number of SARS-CoV-2 RNA. Negative strand SARS-CoV-2 RNA was detected in sensory neurons and 293T cells positive for SARS-CoV-2 RNA in scRNA-seq. (B) Plaque forming assay on hiPSC-derived cortical and sensory neurons and Calu-3 cells exposed to WA1/2020 SARS-CoV-2 for 24, 48 and 72 h. N = 3; t-test ∗p value<0.05. Mean ± S.E.M. (C) Detection of viral dsRNA and neuronal marker beta3-Tubulin (TUBB3) by immunofluorescence in hiPSC-sensory neurons co-cultured with HEK293T cells exposed to WA1/2020 SARS-CoV-2 for 48h. Scale: 20μm. C′ zoom of the inset presented in C. (D) Detection of SARS-CoV-2’s Nucleocapsid and neuronal marker beta3-Tubulin (TUBB3) by immunofluorescence in hiPSC-sensory neurons co-cultured with HEK293T cells exposed to WA1/2020 SARS-CoV-2 for 48h. Scale: 20μm. D′ zoom of the inset presented in D. (E) Boxplot of average Nucleocapsid staining intensity in the axons of hiPSC-sensory neurons co-cultured with HEK293T cells exposed to WA1/2020 SARS-CoV-2 for 48h. Average with min and max, +/− SD.

Figure 3

Infection of sensory neurons by SARS-CoV-2 variants delta and omicron (A) SARS-CoV-2 genes expression in hiPSC-sensory neurons exposed to mock, heat-inactivated or active WA1/2020 SARS-CoV-2 or the delta and omicron variants. (B) SARS-CoV-2 genes expression in HEK293T cells exposed to mock, heat-inactivated or active SARS-CoV-2 strain WA1/2020 and variants delta and omicron. (C) Heatmap of average gene expression for the top 20 most differentially expressed genes between SARS-CoV-2-positive (SARS pos.) and -negative (SARS neg.) hiPSC-sensory neurons and 293T cells upon exposure to WA1/2020 SARS-CoV-2 or the delta or omicron variants.