Rubella virus tropism and single-cell responses in human primary tissue and microglia-containing organoids
- Department of Neurological Surgery, University of California, San Francisco, United States
- Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, United States
- Department of Anatomy, University of California, San Francisco, United States
- Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, United States
- Weill Institute for Neurosciences, University of California, San Francisco, United States
- Department of Biochemistry and Biophysics, University of California, San Francisco, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 1 supplement
Rubella virus (RV) 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 hr. (B, C) Immunostaining for RV capsid and Iba1 in cultured cortical slices at 72 …
Figure 1—figure supplement 1
GFP expression in rubella virus (RV)-infected Vero cells.
(A) Co-localization of RV viral capsid and GFP expression in Vero cells. (B) Heat-inactivated wild type RV, parental wild type RV strain, and GFP-expressing RV construct in Vero cells. RV-GFP image …
Figure 2 with 2 supplements
Rubella virus (RV) 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 magnetic-activated cell sorting (MACS). Microglia cells were cultured …
Figure 2—figure supplement 1
Rubella virus (RV) inoculum dilution in mixed co-cultures of microglia and non-microglia cells.
(A) Representative images of co-cultures inoculated with mock virus or with RV viral stock diluted at 1:50. (B) Quantifications of cells immunopositive for RV capsid across different inoculum …
Figure 2—figure supplement 2
Rubella infection in non-microglia cells.
(A) Representative images of different cell types depleted of microglia. Cell cultures were stained with rubella virus (RV) capsid (green) and DAPI. (B) Quantification of total cells that are …
Figure 3
Direct cell-cell contact is not required for microglia infection by rubella virus (RV).
(A) Schematic for experimental setup. Primary human brain tissue was dissociated, and microglia were cultured with or without microglia-depleted flow-through portion. Cells were co-cultured in …
Figure 4
Microglia in neuroimmune organoids are infected with rubella virus (RV).
(A) Primary human microglia were transplanted into brain organoids and resulting neuroimmune organoids were exposed to RV. After 72 hr or 2 weeks organoids were processed for immunofluorescence …
Figure 5 with 1 supplement
Rubella virus (RV) exposure of brain organoids leads to interferon response.
(A) Single-cell RNA sequencing analysis identified 13 clusters, including neurons and glial cells (Div.: dividing cells, RG: radial glia, Astros: astrocytes, IPCs: intermediate progenitor cells). (B)…
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Figure 5—source data 1
Cluster marker genes for brain organoid single-cell RNA sequencing (scRNAseq) dataset, related to Figure 5.
- https://cdn.elifesciences.org/articles/87696/elife-87696-fig5-data1-v1.xlsx
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Figure 5—source data 2
Differentially expressed genes detected in brain organoid single-cell RNA sequencing (scRNAseq) dataset, related to Figure 5.
- https://cdn.elifesciences.org/articles/87696/elife-87696-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Single-cell RNA sequencing (scRNAseq) analysis of brain organoids.
(A) Uniform manifold approximation and projection (UMAP) of brain organoids colored by condition. (B) UMAP colored by individual cell line. Two iPSC lines, 13325 and WTC10, were used for the …
Figure 6
NOVA1 expression is reduced in response to rubella virus (RV) exposure.
(A) Differentially expressed genes in different cell types in response to RV treatment without (top panel) and with microglia (bottom panel). IPCs – intermediate progenitor cells. In the presence of …
Author response image 1
Rubella virus titering experiment performed in Vero cells (positive control) or dissociated microglia co-cultures.
In primary microglia co- cultures, viral titer falls below detection levels after several days of infection.
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Modest increase in RV RNA over time in brain slice infections.
Rubella virus RNA measured by qPCR relative to GAPDH gene, in n=3 samples (2 technical replicates each condition). Brain slices were exposed to RV, then collected at end of inoculation (4 hours post …
Author response image 4
After 2 weeks post infection, microglia remain positive for RV capsid.
Author response image 5
After 2 weeks post infection, microglia remain positive for RVOrganoids without microglia do not show positive RV immunofluorescence.
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P2RY12 co-localizes with Iba1 in primary brain tissue from gestational week 17.5, including cells with more ameboid morphology (arrows).
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Examples of microglia with round (top) and ramified (bottom) morphology that co-localize with RV capsid staining.
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Rubella infection in non-microglia cells.
(A) Representative images of different cell types depleted of microglia. Cell cultures were stained RV capsid (green) and DAPI. (B) Quantification of total cells that are positive for RV capsid …
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Organoids without microglia do not show positive RV immunofluorescence.
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Data composition depending on condition.
A. Cell number contribution from organoids with and without microglia. B. Contribution of each condition to each cluster composition.
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Quantification of RV+ cells that are not microglia across different cell populations.
No statistically significant difference was detected in RV infectivity in cells cultured with or without microglia.
Author response image 12
Rubella infection in non-microglia cells.
(A) Representative images of different cell types depleted of microglia. Cell cultures were stained RV capsid (green) and DAPI. (B) Quantification of total cells that are positive for RV capsid …
Author response image 13
Rubella virus titering experiment performed in Vero cells (positive control) or dissociated microglia co-cultures.
In primary microglia co- cultures, viral titer falls below detection levels after several days of infection.
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J2 antibody labels dsRNA in both RV-exposed and control heat- inactivated virus conditions, presumably due to native dsRNA that is not unique to the viral replication.
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Microglia in organoids co-localize with RV capsid staining.
Author response image 16
Rubella virus titering experiment performed in Vero cells (positive control) or dissociated microglia co-cultures.
In primary microglia co- cultures, viral titer falls below detection levels after several days of infection.
Author response image 17
Modest increase in RV RNA over time in brain slice infections.
Rubella virus RNA measured by qPCR relative to GAPDH gene, in n=3 samples (2 technical replicates each condition). Brain slices were exposed to RV, then collected at end of inoculation (4 hours post …
Author response image 18
J2 antibody labels dsRNA in both RV-exposed and control heat- inactivated virus conditions, presumably due to native dsRNA that is not unique to the viral replication.
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FISH probes to positive strand (genomic) and negative strand (replication template) RV RNA in Vero cells and microglia co-cultures.
(A) Representative images of Vero cells infected with RV (top row) or Zika virus as control (bottom row). At 72hpi, cells were fixed and processed for immunofluorescence with anti-RV capsid antibody …
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Rubella infection in non-microglia cells.
(A) Representative images of different cell types depleted of microglia. Cell cultures were stained RV capsid (green) and DAPI. (B) Quantification of total cells that are positive for RV capsid …
Author response image 23
Organoids labeled with splice regulator NOVA1 (magenta), neuronal marker NeuN (green) and intermediate progenitor cell marker EOMES (cyan).
Author response image 22
Microglia in organoids co-localize with RV capsid staining.
Organoid with microglia were exposed to RV for 72 hrs or two weeks.
