Transcriptional landscape of the kidney in chickens infected by virulent or avirulent infectious bronchitis virus

Fig. 1. Workflow. Forty-five 10-day-old specific pathogen-free (SPF) chickens were equally divided into three groups. Each chicken in the infected groups was administered either the GD17/04 or 4/91 vaccine strains at a dose of 105 EID50 via oculonasal administration, while PBS was administered as a negative control. Five chickens from each group were euthanized at 3, 5, and 7 dpi. Kidney tissue from the same region was homogenized, followed by total RNA extraction, transcriptome sequencing, and bioinformatics analysis.

Fig. 2. Volcano map. The horizontal axis represents the ratio of expression levels in the treated sample compared to the control sample. The vertical axis indicates the statistical test value associated with changes in gene expression levels, specifically the p-value. Each point corresponds to a specific gene: red points indicate significantly upregulated genes, while blue and grey points represent downregulated genes and genes with non-significant differences, respectively. The top 10 genes, identified based on their p-values, are labeled with their gene names; however, some genes may not be labeled due to the absence of a designated gene name.

Fig. 3. GO enrichment analysis. GO enrichment analysis of DEGs within the gene set was conducted using Goatools software, employing Fisher's exact test as the analytical method. A significant enrichment of a GO function is indicated when the adjusted p-value (Padjust) is less than 0.05. The color of the circles represents the Padjust value and the size indicates the gene number; the vertical axis denotes the GO Term, while the horizontal axis illustrates the Rich factor, defined as the ratio of the number of genes or transcripts enriched in this GO term (Sample number) to the total number of annotated genes or transcripts (Background number). A. The GO enrichment of DEGs in the GD17/07 strain of infected chickens on the 3rd, 5th, and 7th days post-infection is labeled as G3, G5, and G7, respectively. B. The GO enrichment for the 4/91 vaccine strain is represented by V3, V5, and V7, corresponding to the 3rd, 5th, and 7th days post-infection.

Fig. 4. KEGG enrichment analysis. The vertical axis represents the various pathways, while the horizontal axis denotes the Rich factor, which is the ratio of the number of genes or transcripts enriched in a specific pathway (Sample number) to the total number of annotated genes (Background number). A higher Rich factor indicates a greater degree of enrichment. The size of each point corresponds to the number of genes present in that pathway, whereas the color of the point reflects different ranges of adjusted p-values (padjust). A. The KEGG enrichment of DEGs in the GD17/07 strain of infected chickens on the 3rd, 5th, and 7th days post-infection, labeled as G3, G5, and G7, respectively. B. The KEGG enrichment for the 4/91 vaccine strain, represented by V3, V5, and V7, which correspond to the 3rd, 5th, and 7th days post-infection.

Fig. 5. WGCNA analysis for GD17/04. A. The scale-free tolerance curve and average connectivity curve are presented. The horizontal axis represents the power-exponential weighted β value, while the vertical axis depicts the average connectivity (k/degree/connectivity) of each node within the network, which is derived from the adjacency matrix corresponding to the respective β value. B. The gene dendrogram illustrates the clustering of genes into modules based on their expression trends. Each branch represents a gene, and colors denote specific modules. A gray color indicates that the gene has not been assigned to a particular module. The GD17/07 strain of infected chickens is analyzed on the 3rd, 5th, and 7th days post-infection, labeled as G3, G5, and G7, respectively. C represents the control group. In this context, green indicates low expression, while red indicates high expression. C. The correlation between modules and traits is displayed, showing the relationship between specific modules and phenotypes. The horizontal axis represents different groups, while the vertical axis represents various modules. The left column of numbers indicates the number of genes within each module, and…

Fig. 6. WGCNA analysis for 4/91 vaccine. A. The scale-free tolerance curve and average connectivity curve. B. The gene dendrogram illustrates the 4/91 vaccine strain's effect on infected chickens at 3, 5, and 7 dpi, labeled as V3, V5, and V7, respectively, while C denotes the control group. C. The correlation between modules and traits. D. GO enrichment analysis of genes in each module. E. The Venn diagram analysis illustrates the intersection of genes within the brown module as depicted in Fig. 6C, alongside the genes from each module shown in Fig. 5C. F. The Venn diagram for DEGs in the brown modules. G. The heatmap for DEGs in the brown module.

Fig. 7. The immune response in the chicken host against the virulent or avirulent IBV strain. A. KEGG analysis for DEGs in the brown module in Fig. 6C and DEGs in the turquoise module in Fig. 5C. B. The Venn analysis illustrates the intersection of DEGs in the brown module in Fig. 6C and the DEGs in the turquoise module in Fig. 5C. C. A heatmap depicting 69 DEGs, selected from immune-related genes regulated by both virulent and avirulent IBV strains, is provided. The clustering of the relevant genes is illustrated in the clustering tree within the circle. D. The clustering trend of 59 DEGs. These 59 DEGs are derived from Fig. 7C and are clustered into a large branch as enclosed by a green arc shape. E. KEGG enrichment analysis for the core 69 genes. The right vertical axis represents the number of genes in this pathway, corresponding to different points on the broken line. Different colors represent the various branches of the KEGG pathway. F. The network of protein interactions for the core 69 genes was analyzed using STRING.

Fig. 8. Validation of RNA-sequencing data by RT-qPCR. Chicken kidneys infected by the GD17/04 strain at 3, 5, and 7 dpi were homogenized, and RNA was extracted. The relative mRNA expression levels of the indicated genes were quantified using RT-qPCR, with GAPDH serving as the internal reference gene. The PBS group was utilized as the control group for calculating the log2 (fold change). Data are presented as the mean with standard error of the mean (SEM) from three independent experiments and are highlighted in red. The blue bar chart represents the sequencing data.

Fig. 9. TRIM25 promotes the proliferation of IBV in vitro. A and B. DF-1 cells were transfected with siRNA (RNA-1 to RNA-3) targeting TRIM25, alongside a control group. After a transfection period of 48 hours, the mRNA and protein levels of TRIM25 were evaluated using RT-qPCR (A) and Western blot (B), respectively. C and D. Following transfection with TRIM25 siRNA or the Flag-TRIM25 overexpression vector for 24 hours, DF-1 cells were subsequently infected with GD17/04 at a multiplicity of infection (MOI) of 0.5 for 18 hours. DF-1 cells were then lysed to assess the IBV-N mRNA levels via RT-qPCR (C) and the IBV-N protein levels through Western blot (D). All experiments were conducted with three independent biological replicates.