Figures and data
Rubella virus infects primary human microglia in cultured brain slices.
A. Schematic for brain slice infection. Mid-gestation (GW18-23) human brain slices were infected with RV for 72 hours. B,C. Immunostaining for RV capsid and Iba1 on cultured cortical slices at 72 hpi, at 10x with scale bar 100 μm (B) and at 40x objective with scale bar 50 μm (C). D. Quantification of RV capsid-positive cells co-labeled with microglial marker Iba1: 764/819 (93.3%) of RV+ cells were microglia based on Iba1 staining across four biological replicates. Error bars denote standard deviation. E. Diagram of viral genome of GFP-expressing RV (RV-GFP). Cortical brain slices were infected with RV-GFP for 72 hours. F. Examples of GFP fluorescence and Iba1 immunostaining at 72 hpi of cultured cortical slices with GFP-RV, at 62x with scale bar 20 μm. GFP expression of modified rubella virus is localized to Iba1-positive microglia cells (arrows).
Rubella infection of microglia is dependent on the presence of other cells.
A. Schematic of rubella infection. Primary prenatal brain tissue was dissociated and different cell types were purified using MACS. Microglia cells were cultured alone or in combination with neurons, glial cells, or all cell types. 2D cultures were infected with RV for 72 hours and processed for immunostaining. B-E. Representative images of microglia cultured with different cell types. Cell cultures were stained for microglia marker Iba1 (red), RV capsid (green) and DAPI (grey; on the overlay Merge channel). B. Purified microglia only. C. Microglia and neurons co-cultured at 1:5 ratio. D. Microglia and non-neuronal cell types cultured together at 1:5 ratio. E. Microglia cultured with non-microglial cells (flow-through from a MACS purification) at 1:5 ratio. F. Quantification of RV capsid immunopositivity among microglia (Iba1+) for conditions in B-E. FT: flow through after microglia MACS purification. Error bars denote SEM. Each data point represents a field of view from the same experimental batch. G. Quantification of microglia (Iba1+) among RV capsid-positive cells.
Direct cell-cell contact is not required for microglia infection by rubella.
A. Schematic for experimental set up. Primary human brain tissue was dissociated, and microglia were cultured with or without microglia-depleted flow through portion. Cells were co-cultured in direct contact or in solution-permeable chambered transwells (TW). B. Representative images of microglia-enriched cultures (top row), microglia cultured with other cell types in the same well (middle row), and microglia cultured in the bottom compartment with other cell types cultured in a permeable transwell chamber (bottom row) infected with RV for 72 hours. C. Quantification of RV capsid immunopositivity among microglia (Iba1+). Three fields of view across the same experiment were quantified for each condition. Error bars represent SEM. p-value between microglia and co-culture condition is 0.0479. p-value between microglia and trans well condition is 0.0159. D. Quantification of microglia (Iba1+) among RV capsid-positive cells.
Rubella infects microglia in brain organoids and leads to interferon response.
A. Primary human microglia were transplanted into brain organoids, resulting neuro-immune organoids were infected with RV and 72 hours post-infection were processed for downstream analysis. B. Immunofluorescence imaging of brain organoids including markers of radial glial cells (Sox2), transplanted microglia (Iba1) and RV capsid (RV). C. Single cell RNA sequencing analysis identified 13 clusters, including neurons and glial cells (Div.: dividing cells, RG: radial glia, Astros: astrocytes). D. Dot plot depicting cluster marker genes for each cluster. E. UMAP plots of organoids colored by condition. Left: organoids with or without microglia. Right: organoids that were infected with RV or controls. F. Feature plot (left) and violin plot (right) for Interferon Alpha Inducible Protein 6 (IFI6) across different conditions. G. Differentially expressed genes in different cell types in response to RV infection without (top panel) and with microglia (bottom panel). In the presence of microglia, fewer differentially expressed genes in response to RV infection were identified across all major cell types. Kolmogorov-Smirnov test was used on DEGs with p-value<0.05. *** <0.001, NS – not significant, * <0.05. H. Violin plots for select genes differentially expressed in response to RV and presence of microglia.
