Fig 1.
Histopathological staining and immunofluorescence analysis.
(a) Histopathological staining of tissues. In the kidney section, the black square highlights immune cell infiltration, while the black arrows indicate tubular cell casts and obstructions. The bursa of Fabricius section shows damage and depletion of lymphoid follicles, as indicated by black arrows. In the trachea section, arrows point to immune cell infiltration. (b) Immunofluorescence using IBV-N antibody in kidney, trachea, and Bursa tissues. Green fluorescence indicates viral positivity.
Fig 2.
Single-cell profiling of cell populations in the kidney collected from IBV infected and control chickens.
(a) UMAP projection representing the cell clusters identified by specific molecular markers. (b) Cell type annotation and dot plot representing characteristic genes (row) in each cluster (column). Dot size represents the proportion of cells in the cluster that express the gene; intensity indicates the expression level (Z-score) relative to those from other clusters. (c) Heatmap depicting the expression levels (Z-scores) of signature genes across each cluster. (d) Proportion of cell types in renal tissue before and after infection. (e) Components and structure of the nephron. (f) Expression of inflammatory genes and salt excretion genes in immune cells and distal nephron cells. The dot size represents the change in the percentage of gene-expressing cells (infect group cell percentage / control group cell percentage), while the color reflects changes in gene expression levels (infected group average expression / control group average expression).
Fig 3.
Dual immunofluorescence identifying cell types infected by IBV in chicken kidney and changes in cell-cell communication after IBV infection.
(a) White arrows indicate the localization of IBV N protein in AQP2-expressing collecting duct cells. Green fluorescence shows positive staining for IBV N and red fluorescence shows staining for AQP2. (b) White arrows indicate the localization of IBV N protein in CALB1-expressing distal tubule cells. Red fluorescence shows positive staining for IBV N and green fluorescence shows staining for CALB1. (c) Changes in cell communication numbers: The top network diagram shows cell clusters as nodes, with line thickness indicating changes in communication numbers. The lower heatmap details these changes, with rows representing signal-sending cells and columns indicating signal-receiving cells. The color scale reflects the inter-group differences in signal communication frequency between different cell types (number of communications in the infected group—number in control group). The bar plots at the top and right side represent the overall differences in the number of signals sent/received by specific cell clusters. (d) Changes in cell communication strength: Similar to (c), with the top network diagram displaying changes in communication strength (communication strength in the infected group—strength in the control group). In the lower heatmap, the color scale reflects the inter-group differences in signal communication strength between different cell types. The bar plots at the top and right side represent the overall differences in the strength of signals sent/received by specific cell clusters (infected group—control group). (e) Inter-group differences in the communication strength of specific signaling pathways (receptor-ligand pairs) across cell clusters. Rows represent signal pathways and columns correspond to cell clusters, with heatmap colors depicting the strength variation of signals (infect group vs. control group). Upper left triangles for signal sent and lower right triangles for signal received. The bar plot on the right side shows the overall difference in communication strength of these signaling pathways between the IBV-infected group and the control group.
Fig 4.
Single-cell transcriptomic profiles of bursa tissues from IBV-infected and control chickens.
(a) UMAP dimensional reduction and clustering, cells are categorized into subpopulations based on molecular specificity markers. (b) Bubble plot illustrating the expression of cell type-specific molecular markers and viral RNA. The rows display gene names, and the columns represent cell populations. Bubble size indicates the proportion of gene expression in each cell type, while color denotes normalized gene expression levels (Z-score). (c) Heatmap depicting the expression levels (z-score) of characteristic genes in each cluster. (d) Bar graph showing the proportion of various cell types before and after infection. (e) Schematic representation of the Bursa of Fabricius structure. IFE, interfollicular epithelium; FAE, follicle associated epithelial cell; ERC, reticular epithelial cell; BSDC, bursa secretory dendritic cell.
Fig 5.
Dual immunofluorescence identifying cell types infected by IBV in bursa and changes in cell-cell communication after IBV infection.
(a) Dual immunofluorescence identification of IBV infection in FAE cells; white arrows indicate the localization of IBV N protein in CSF1R-expressing FAE cells. Yellow arrows represent positive staining for IBV N protein in IFE cells. (b) Dule immunofluorescence detection of TIMD4 and IBV N protein. (c) Changes in cell communication numbers: The top network diagram shows cell clusters as nodes, with line thickness indicating changes in communication numbers. The lower heatmap details these changes, with rows representing signal-sending cells and columns indicating signal-receiving cells. The color scale reflects the inter-group differences in signal communication frequency between different cell types (number of communications in the infected group—number in control group). The bar plots at the top and right side represent the overall differences in the number of signals sent/received by specific cell clusters. (d) Changes in communication strength: Similar to (c), with the top network diagram displaying changes in communication strength. (e) Inter-group differences in the communication strength of specific signaling pathways (receptor-ligand pairs) across cell clusters. Rows represent signal pathways and columns correspond to cell clusters, with heatmap colors depicting the strength variation of signals (infect group vs. control group). Upper left triangles for signal sent and lower right triangles for signal received. The bar plot on the right side shows the overall difference in communication strength of these signaling pathways between the IBV-infected group and the control group.
Fig 6.
Single-cell transcriptomic profiles of trachea tissues from IBV-infected and control chickens and changes of cell-cell communication after IBV infection.
(a) UMAP dimensional reduction and clustering, cells are categorized into subpopulations based on molecular specificity markers. (b) Bar graph showing the proportion of various cell types before and after infection. (c) Bubble plot illustrating the expression of cell type-specific molecular markers and viral RNA. The rows display gene names, and the columns represent cell populations. Bubble size indicates the proportion of gene expression in each cell type, while color denotes normalized gene expression levels(Z-score). (d) Heatmap depicting the expression levels (z-score) of characteristic genes in each cluster. (e) Changes in cell communication numbers: The top network diagram shows cell clusters as nodes, with line thickness indicating changes in communication numbers. The lower heatmap details these changes, with rows representing signal-sending cells and columns indicating signal-receiving cells. The color scale reflects the inter-group differences in signal communication frequency between different cell types (number of communications in the infected group—number in control group). The bar plots at the top and right side represent the overall differences in the number of signals sent/received by specific cell clusters. (f) Changes in communication strength: Similar to (e), with the top network diagram displaying changes in communication strength. (g) Inter-group differences in the communication strength of specific signaling pathways (receptor-ligand pairs) across cell clusters. Rows represent signal pathways and columns correspond to cell clusters, with heatmap colors depicting the strength variation of signals (infect group vs. control group). Upper left triangles for signal sent and lower right triangles for signal received. The bar plot on the right side shows the overall difference in communication strength of these signaling pathways between the IBV-infected group and the control group.
Fig 7.
Identification of gene modules and expression patterns via weighted gene co-expression network analysis (WGCNA).
(a) Module tree illustrating the hierarchical clustering of nodes (genes); vertical distances represent dissimilarities between nodes. Gene modules delineated through Dynamic Tree Cut are indicated, with modules merged based on a correlation threshold >0.7. (b) The number of genes in each module post-merging. (c) Enrichment results of immune-related gene modules. (d) Expression differences of immune-related gene modules across various cell clusters. Heatmap depicting the differences in gene module eigengene values between infected and control groups. The color scale represents the eigengene value differences for each gene module (infected group eigen- control group eigen). Columns representing gene modules and rows representing cell clusters. Numbers to the right of cell cluster names represent the subdivision of cell clusters.
Fig 8.
(a) The top heatmap displays the expression patterns of genes within the steelblue gene module across different cell clusters in the virus-infected and control samples. Rows represent genes, and columns represent cell clusters. This heatmap displays the 10–12 cell clusters exhibiting the most significant upregulation of module eigen after viral infection. The bar plots above the heatmap represent the module eigen values for each cell cluster. Below, the Gene co-expression network diagram displaying nodes sized by gene connectivity within the module. Node colors represent the results of differential gene expression analysis after merging the cell clusters shown in the above heatmap (infected group vs. control group, p.adjust < 0.05).). log2FC = log2 (Infected group gene expression / Control group gene expression). Transcription factors are highlighted. Con, Control group; Kid, Kidney; IBV, IBV infected group; BF, Bursa of Fabricius; Tra, Trachea; PT, Proximal Tubule. Numbers to the right of cell cluster names represent the subdivision of cell clusters. (b) Gene expression heatmap and co-expression network of darkorange module. (c) Gene expression heatmap and co-expression network of genes in the steelblue module.
Fig 9.
Differential gene expression and pathway activation patterns in cells infected with IBV.
(a) Viral load in IBV-infected distal-collecting duct cells of kidney, IFEs of bursa, and tracheal epithelial cells. (b-d) Gene expression differences in IBV-infected distal-collecting duct cells, IFEs, and tracheal epithelial cells compared to their uninfected cells within the same cell clusters. (e) Venn diagram illustrating shared upregulated genes across infected distal-collecting duct cells, IFEs, and tracheal epithelial cells. (f-h) Enrichment results of KEGG pathways for genes upregulated in IBV-infected cells.
Table 1.
Antbodies and reagents used in present study.