Proteogenomics Uncovers Critical Elements of Host Response in Bovine Soft Palate Epithelial Cells Following In Vitro Infection with Foot-And-Mouth Disease Virus

Figure 1. Experimental setup of RNA sequencing and exploratory data analysis. (A) Primary soft palate (SP) cell cultures were obtained from two animals. Baseline samples for RNA sequencing were collected immediately before inoculation (circle). Subsequently, the cell cultures were inoculated with foot-and-mouth disease virus (FMDV) (orange symbols) or mock-infected for use as controls (blue symbols). Cells were then harvested for sequencing at 24 h post infection (hpi) (triangles) or 28 days post infection (dpi) (squares), representing acute and persistent infection, respectively. (B) Principal components analysis based on normalized gene counts of the 1000 most variable genes. (C) The variance of normalized gene counts was calculated for each gene and the 45 genes with the highest variance were selected and visualized in a heat map. The color of the cells indicates the difference from the mean normalized gene count of the corresponding gene. Samples and genes were clustered according to these differences (trees). The transcript cluster highlighted in green is similarly activated by acute and persistent FMDV infection, while the blue cluster highlights transcripts that are only active…

Figure 2. Differential gene expression of SP cells during FMDV infection. (A) Volcano plot showing the log2 fold change (x-axis) and the adjusted p-value (y-axis) for all differentially expressed genes during the acute (24 hpi, green) and persistent (28 dpi, orange) phase of FMDV infection. The log2 fold change and adjusted p-value are calculated relative to a non-infected control from the same time point. The grey dotted lines indicate the cutoff values: adjusted p-value < 0.001 and |log2 fold change| > 1. (B) The histogram summarizes the number of genes that have a certain log2 fold change. (C) The total number of differentially expressed genes is visualized in a Venn diagram. The overlap indicates the 63 genes that are differentially expressed during both phases of infection. (D) The log2 fold change of these 63 genes is compared for acute and persistent infection.

Figure 3. Pathway enrichment analyses of significantly differentially expressed genes during acute and persistent FMDV infection of bovine soft palate cells. (A) The number of genes that contribute to a significantly enriched Reactome pathway is shown for 24 hpi (blue dots) and 28 dpi (orange dots). Black lines connect pathways that are enriched in both datasets and the size of the circles represents the ratio of genes found for a certain pathway to the overall gene number. (B) The log2 fold change of genes that contribute to selected enriched Reactome pathways are highlighted (24 hpi—blue dots) and 28 dpi—orange dots). The grey dotted lines indicate a |log2 fold change| > 1. The numbers on the right correspond to the number of genes associated with each pathway.

Figure 4. Expression analysis for selected genes by RT-qPCR at different time points. (A) Gene expression in FMDV infected soft palate cells compared to uninfected controls, at 24 hpi (blue panel) and 28 dpi (orange panel). The change in gene expression was analyzed for six selected target genes and two reference genes (not shown) using RT-qPCR. Log2 fold changes were calculated in comparison to a non-infected control from the same time points. Grey and white bars indicate the log2 fold change of gene expression using results from RT-qPCR and RNA sequencing (RNASeq), respectively. The grey dotted lines indicate a |log2 fold change| > 1. (B) The gene expression of the six selected genes was traced in a time course 2, 24, and 48 hpi; and 7 and 28 dpi. Statistically significant changes in comparison to control samples are highlighted with asterisks (p-value < 0.05: *; p-value < 0.01: **). Time points 24 hpi and 28 dpi are illustrated in blue and orange, respectively.