Antiviral immunity within neural stem cells distinguishes Enterovirus-D68 strain differences in forebrain organoids

Figures

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  Fig. 1. Prototypic Fermon strain replicates in SH-SY5Y cells. A Alignment and phylogenetic tree of several EV-D68 non-neuropathogenic and neuropathogenic strains, with the prototypic Fermon strain serving as reference strain. This schematic was adapted from [11]. Phylogenetic tree from viral genome sequences was done using the NCBI phylogenetic tree software, and the protein sequences were aligned using Clustal Omega. B Representative immunofluorescence images of undifferentiated SH-SY5Y cells mock-infected or infected with Fermon or MO viruses at the indicated multiplicity of infection (MOI) at 24 h post infection (hpi). Cells were stained for DAPI (nuclei, blue), VP1 (green), and neurofilament-H (NFH, magenta). Scale bar—100 µm. C RT-qPCR analysis of RNA harvested from undifferentiated SH-SY5Y cells mock-infected or infected with Fermon or MO at the indicated MOIs for 24 h. The data are normalized to mock-infected cells and are presented as relative expression of EV-D68 to HPRT1 with Mock set to 1. Data are presented as means ± SEM (n = three biological replicates). D Plaque-forming assay of supernatants from undifferentiated SH-SY5Y cells infected with either Fermon or MO at the…

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  Fig. 2. Differential Enterovirus-D68 replication in early and late forebrain organoids. A Schematic of organoid structures. B Representative bright field images taken at 40X from organoids at day in vitro (DIV) 35 (early) and DIV85 (late) (C–H). Early and late organoids were infected mock-infected or infected with Fermon, MO, and MD viruses for 7 days. At 7 dpi, lysates and organoids were harvested for downstream analysis. C RT-qPCR analysis of DIV35 organoids. The data are normalized to mock-infected cells and are presented as relative expression of EV-D68 to GAPDH with Mock set to 1. Data are presented as means ± SEM (n = four biological replicates). D Plaque-forming assay of supernatants from DIV35 organoids. Data are presented as means ± SEM (n = three biological replicates). ND = not detected; LOD = limit of detection E RT-qPCR analysis of RNA harvested of DIV85 organoids. The data are normalized to mock-infected cells and are presented as relative expression of EV-D68 to GAPDH with Mock set to 1. Data are presented as means ± SEM (n = three biological replicates). F Plaque-forming assay of supernatants harvested from DIV85 organoids. Data are presented as means ± SEM (n = three…

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  Fig. 3. Enterovirus-D68 replication in forebrain organoids is partially dependent upon sialic acid expression. A Representative immunofluorescence images of DIV35 or DIV85 forebrain organoids for sialic acid protein expression using the Click-iT reaction. Scale bar—200 µm (B). Quantification of the relative mean fluorescence intensity (MFI, alkyne-555/DAPI) of the immunofluorescence images from (A). Three sections from three independent biological replicates for either early or late organoids were quantified, and data are presented as the means ± SEM (n = three biological replicates, 9 total replicates). Statistical analysis performed with unpaired student’s t-test. C RT-qPCR analysis of lysates harvested from DIV35 forebrain organoids treated with neuraminidase for 1 h and then infected with Fermon, MO, or MD viruses for 24 h. D RT-qPCR analysis of lysates harvested from DIV85 forebrain organoids treated with neuraminidase for 1 h and then infected with Fermon, MO, or MD viruses for 24 h. The data are normalized to untreated organoids and are presented as relative expression of EV-D68 to GAPDH with untreated set to 1. Data are presented as means ± SEM (n = three biological replicates).…

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  Fig. 4. Early and late forebrain organoids display different morphology, protein expression, and protein localization. A Representative immunofluorescence images of early and late forebrain organoids. DAPI (blue) stains nuclei, MAP2 (magenta) stains maturing neurons, and SOX2 (grey) indicates neural stem cells. Scale bar of the whole organoid—200 µm and scale bar of zoom—50 µm B Schematic of cell type expression as organoids mature from DIV35 to DIV85 (C). RT-qPCR analysis of RNA harvested from uninfected forebrain organoids at DIV35 (early) and DIV85 (late). The data are normalized to the early samples and are presented as relative expression of the respective genes to GAPDH. Data are presented as means ± SEM (n = five biological replicates). Statistical analysis was performed with unpaired student’s t-test. D, E Representative immunofluorescence images of early (D) and late (E) uninfected forebrain organoids. DAPI (blue) stains nuclei, PAX6 (cyan) marks ventricular radia glia cells, and FAM107A (red) indicates outer radial glia cells. Scale bar—50 µm

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  Fig. 5. Distinct antiviral signaling pathways are basally expressed in early forebrain organoids. A Uniform manifold approximation and projection (UMAP) of previously published single cell transcriptomics data representative of 393 cells from ventricular and sub-ventricular zone human fetal brain samples [25]. B Heatmap displaying distinct neural cell populations. Arrows highlight radial glia specific genes. C, D Dot plot displaying NF-kB genes (C) and violin plots displaying interferon stimulated genes (ISGs) (D) identified using differential gene expression analysis of cluster 1 (radial glia) compared to other clusters (cutoff = log2 fold-change = 1.9) (E). RT-qPCR analysis of lysates harvested at 7 dpi from DIV35 forebrain organoids untreated or treated with Ruxolitinib and simultaneously infected with Fermon, MO, or MD viruses. The data are normalized to untreated organoids and are presented as relative expression of EV-D68 to GAPDH with untreated set to 1. Data are presented as means ± SEM (n = four biological replicates). Statistical analysis performed with unpaired student’s t-test. F Plaque-forming assay of supernatants harvested at 7 dpi from forebrain organoids mock-infected or…

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  Fig. 6. Differential interferon-stimulated gene expression during Enterovirus-D68 infection of early and late forebrain organoids. A Heat map of fold change values from RT-qPCR of lysates from early and late forebrain organoids for several interferon-stimulated genes. B Representative immunofluorescence micrographs of early and late forebrain organoids. Organoids were stained for DAPI (nuclei, blue), PAX6 (grey, ventricular radial glia cells), and IFITM3 (red). Dashed boxes indicate the location of the zoom images. Scale bar (tiled image)—200 µm; Scale bar (zoom image)—25 µm. C RT-qPCR analysis of lysates harvested at 7 dpi from DIV35 forebrain organoids mock-infected or infected with Fermon, MO, or MD viruses. The data are normalized to mock-infected organoids and are presented as relative expression of the respective interferon-stimulated gene to GAPDH with mock-infected set to 1. Data are presented as means ± SEM (n = four biological replicates). D Representative immunofluorescence images of early organoids infected with Fermon or MO viruses at 7 dpi. Organoids were stained for DAPI (blue, nuclei), J2 (green, virus), and IFITM1 (red). Scale bar—50 µm. E RT-qPCR analysis of lysates…