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.

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... investigate RV tropism in the human brain, cultured cortical slices from mid-gestation samples were infected with M33 RV, representing a laboratory strain originally derived from a clinical isolate ( Figure 1A). At 72 hours post-infection, immunostaining for the RV capsid protein revealed numerous cells positive for the RV antigen, of which >90% were co-labeled with the microglia marker Iba1 (Figure 1B-D). ...

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... investigate RV tropism in the human brain, cultured cortical slices from mid-gestation samples were infected with M33 RV, representing a laboratory strain originally derived from a clinical isolate ( Figure 1A). At 72 hours post-infection, immunostaining for the RV capsid protein revealed numerous cells positive for the RV antigen, of which >90% were co-labeled with the microglia marker Iba1 (Figure 1B-D). To confirm functional transcription and translation of the viral genome, a new reporter construct of RV designed to express GFP within the non-structural P150 gene was generated (RV-GFP, GenBank Accession OM816675, Figure 1E) and validated by GFP expression in Vero cells ( Figure 1 -figure supplement 1). ...

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... 72 hours post-infection, immunostaining for the RV capsid protein revealed numerous cells positive for the RV antigen, of which >90% were co-labeled with the microglia marker Iba1 (Figure 1B-D). To confirm functional transcription and translation of the viral genome, a new reporter construct of RV designed to express GFP within the non-structural P150 gene was generated (RV-GFP, GenBank Accession OM816675, Figure 1E) and validated by GFP expression in Vero cells ( Figure 1 -figure supplement 1). In human primary brain slices infected with RV-GFP, GFP expression was detected predominantly in microglia, confirming the production of RV proteins inside this cell type ( Figure 1F) consistent with the wildtype M33 RV. ...

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... 72 hours post-infection, immunostaining for the RV capsid protein revealed numerous cells positive for the RV antigen, of which >90% were co-labeled with the microglia marker Iba1 (Figure 1B-D). To confirm functional transcription and translation of the viral genome, a new reporter construct of RV designed to express GFP within the non-structural P150 gene was generated (RV-GFP, GenBank Accession OM816675, Figure 1E) and validated by GFP expression in Vero cells ( Figure 1 -figure supplement 1). In human primary brain slices infected with RV-GFP, GFP expression was detected predominantly in microglia, confirming the production of RV proteins inside this cell type ( Figure 1F) consistent with the wildtype M33 RV. ...

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... confirm functional transcription and translation of the viral genome, a new reporter construct of RV designed to express GFP within the non-structural P150 gene was generated (RV-GFP, GenBank Accession OM816675, Figure 1E) and validated by GFP expression in Vero cells ( Figure 1 -figure supplement 1). In human primary brain slices infected with RV-GFP, GFP expression was detected predominantly in microglia, confirming the production of RV proteins inside this cell type ( Figure 1F) consistent with the wildtype M33 RV. Error bars denote standard deviation. ...

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... we could not detect an increase in the viral particles from microglia mixed cultures, we confirmed the presence of GFP from the RV-GFP reporter construct, and we believe it serves as a proof that the virus can infect microglia cells and lead to production of functional viral protein (Author response image 2, Figure 1E-F of the current manuscript): ...

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... Figure 1. Additional microglia markers should be used to reinforce the evidence that microglia cells are the principal RV targets. ...

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... 72hpi, cells were fixed and processed for immunofluorescence with anti-RV capsid antibody (RVcap) or Zika virus antibody (Zika4G2), and then FISH was performed using probes to positive strand (+) or negative strand (-) RV RNA. Negative strand RV RNA difficult to visualize at low-power magnification, and required quantification within cell borders defined by wheat germ agglutinin staining with results in panel B While we could not detect an increase in the viral particles from microglia mixed cultures, we confirmed the presence of GFP from the RV-GFP reporter construct, and we believe it serves as a proof that the virus can infect microglia cells and lead to production of functional viral protein (see Author response image 20, Figure 1E-F of the current manuscript) ...