Fig 1.
Histological analysis of pseudo-ALI cultures derived from the nasopharynx for markers of cellular differentiation and the EBV epithelial cell receptor.
Shown are pseudo-ALI cultures susceptible (bold) and non-susceptible (italics) to de novo EBV infection. Examples of ciliated cells are marked by a black box in the Hematoxylin & Eosin (H&E) stain. Mucin-producing cells are marked by a black arrow in the Alcian blue/periodic acid Schiff (AB/PAS) stain. Basal cells that are proliferating are marked by a black arrowhead in the Ki67 stain. Expression of the EBV epithelial cell receptor, Ephrin type-A receptor 2 (EphA2), is indicated by brown 3, 3’–diaminobenzidine (DAB) staining counterstained with nuclear blue.
Fig 2.
EBV de novo infection of primary nasopharynx-derived epithelial cells in pseudo-ALI culture.
A-C, Immunofluorescence staining and EBER-ISH images (red) for EBV molecular diagnostics in pseudo-ALI cultures infected with EBV-positive rAkata B-cells or EBV-negative Akata B-cells (mock), counterstained with DAPI (blue). Shown are maximum intensity projections of confocal images on the xy (square) and xz (rectangle) planes. The pixel signal intensity of the EBV markers in the unzoomed image, in mock (blue line) and EBV-infected (red line) samples, are compared in the histograms. (A) Shown are the results for four donors. Cells were harvested at days 4–5 p.i. for Zebra and days 5–7 p.i. for EBER-ISH. More extensive analysis at days 2 and 5 p.i. was performed for donors (B) no. 4 and (C) no. 7. LMP1 foci in the weakly stained sample (donor no. 7) was also scored as mean particle intensity, from the unzoomed image. Bold, susceptible donor sample; italics, non-susceptible donor sample. LUTs, look-up-tables.
Table 1.
Summary of molecular diagnostic results from EBV infection of pseudo-ALIs.
Fig 3.
Immunostaining for markers of the pseudostratified epithelium and cellular differentiation factors.
Immunofluorescence images of FFPE sections from pseudo-ALI cultures stained for the pseudostratified epithelial marker (CK7) and markers of cellular differentiation (BLIMP1, KLF4 and Inv). Sinus and tonsil sections served as control tissue for the pseudostratified and squamous epithelium, respectively. CK7, cytokeratin 7; Inv, involucrin; Low exp, low exposure.
Fig 4.
Immunostaining for cell-type specific markers unique associated with the pseudostratified epithelium.
Immunofluorescence images of FFPE sections from pseudo-ALI cultures stained for cilia (α-tubulin) and for a mucosecretory Goblet cell marker (MUC5AC). Sinus and tonsil sections served as positive and negative tissue controls, respectively. K5, keratin 5.
Fig 5.
Identification of EBV-infected cell types from scRNA-seq performed on a pseudo-ALI culture from donor no. 4.
(A) Uniform Manifold Approximation and Projection (UMAP) plots displaying the major cell clusters and assigned identity from host marker genes. Schematic displays the cell types in the pseudostratified nasal epithelium that are represented in the cell clusters. (B) Comparison of alignment methods against different EBV genome annotations by UMI counts and numbers of cells. (C) The percentage of EBV-infected cells is displayed for each cluster. Shown are the results from alignment to the fused annotation EBV genome, (D) Density plots of log10 transformed pseudocount (UMI count+1) for EBV reads against normalized cell numbers displayed for each cell cluster. Cell numbers are normalized for the total number of cells in each cluster. Cells are grouped by EBVnegative, EBVlow and EBVhigh populations designated by reads aligning to the EBV genome. Density plots with a similar profile are similarly colored. Where applicable (B-C) cells with reads aligning to the EGFP gene is also shown.
Table 2.
Summary of EBV quantitative PCR and infectious titer results for pseudo-ALI cultures from donor no.4.
Fig 6.
Assignment of cluster identity by cell type-specific marker genes for donor no. 4.
(A) Hierarchical clustering of heatmap representing cluster-specific top marker genes. Expression shows log10 transformed pseudocount (UMI count+1). (B) Dot plot representing marker gene expression by cluster. Dot size indicates the percentage of cells in each cluster expressing the marker gene. Color gradient indicates the mean expression of each marker gene, averaged from positively scored cells, for each cluster. Cluster identity enriched for cell-type specific marker genes are color-coded. Cluster 6 expresses a mixture of marker genes indicative of multiple cell types (basal, suprabasal, goblet/mucosecretory). The EBV receptor gene, CR1, is not shown as it was not expressed according to the count matrix. (C) A few of the highly cell type-specific marker genes were chosen for display by violin plots.
Fig 7.
Averaged expression of EBV genes by cluster.
Dot plot shows expression of EBV genes for epithelial cell clusters in the EBV-infected pseudo-ALI culture of donor no. 4. Genes are grouped in latent or lytic (immediate-early/early/late). Unassigned genes are in purple. Mean expression by cluster shows log10 transformed pseudocount (UMI count+1) for EBV gene averaged by cluster. Dot size indicates the percentage of cells in each cluster expressing the EBV gene. The scRNA-seq reads were aligned to the EBV genome fused annotation.
Fig 8.
Distinguishing EBV gene expression profiles by cell type.
Heatmaps show EBV gene expression by UMI count per cell, displayed for each cluster, for the EBV-infected pseudo-ALI culture of donor no. 4. To distinguish EBVhigh from EBVlow cells, total EBV counts (total EBV UMI counts per cell) are shown at the bottom of each heatmap. Only the EBV-infected cells (with reads aligning to the EBV genome) are shown with the number of EBV-infected cells displayed for each cluster indicated in parenthesis. EBV genes are color-coded into lytic: immediate-early (orange)/early (red)/late (green); latent (blue); and unassigned (purple). The scRNA-seq reads were aligned to the EBV genome fused annotation.
Fig 9.
Comparison of EBV gene count to total number of mRNAs detected per cell.
Shown are the results for the EBV-infected pseudo-ALI culture from donor no. 4. Dot plots show EBV gene pseudocount (red) and the total number of mRNAs (green) per cell. Each dot represents one cell. Cells are ordered by the EBV gene pseudocount on the x-axes. Data are organized by cluster. Dashed black box, denotes cells with high numbers of EBV transcripts (or mRNA molecules) but low total (host and viral) transcript counts representative of host shut-off. Only cells with >1000 mRNAs per cell are shown as defined by the filtering criteria (see S1 Supplementary Methods).
Fig 10.
Venn diagram of differentially expressed genes.
(A, B) NPC tumors (NPC36, NPC46 and NPC50, [39]), (A) EBVlatentlow cells from cluster 2 (C2-EBVlatentlow), and (B) EBVlatentlow cells from all epithelial clusters (Epi-EBVlatentlow) in the EBV-infected pseudo-ALI culture from donor no. 4 were compared against an uninfected pseudo-ALI culture from [41] as reference control (ALIctrl).