Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus

Abstract

Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

Keywords: antiviral immune response; apoptosis; cell cycle; orf virus; transcriptomic profiles.

Figure 1. A number of differentially expressed genes were induced by ORFV

(A) Volcano plot for the samples 8 hours post infection with mRNA expression differences, compared to hour 0. Log2(fold change) is plotted as the abscissa and log10(q) is plotted as the ordinate. Significantly up-regulated genes are indicated in red and down-regulated genes are indicated in blue. (B) Scatter plot of the the 0 h.p.i and 8 h.p.i groups is shown. (C) Graph showing the number and fold change of up- and down-regulated genes. (D) A Venn diagram presents the number of differentially expressed genes that are unique or shared in every paired group. (E) The expression patterns of 11 genes that were shared in the three paired groups in (D). q= FDR corrected P-values, FPKM= fragments per kilobase of exon model per million mapped reads.

Figure 2. Functional analyses of the differentially expressed genes by Gene Ontology (GO) classifications for the three paired groups

The abscissa represents the enriched gene number. Blue = group 3 h.p.i. vs. group 0 h.p.i, red = group 8 h.p.i. vs. group 3 h.p.i, green = group 8 h.p.i. vs. group 3 h.p.i.

Figure 3. Function enrichment of the DEGs from the transcriptome data by GO analysis

The figure presents the GO enrichment of the samples at 3 h.p.i. (A) or 8 h.p.i. (B), compared to uninfected cells. (C) GO enrichment is shown for group 8 h.p.i. against group 3 h.p.i. Enrichment factor is plotted on the x axis. The size of each bubble represents the number of genes.

Figure 4. KEGG classifications of differentially expressed genes for the three paired groups

Blue = 3 h.p.i. group vs. 0 h.p.i. group, red = 8 h.p.i. group vs. 0 h.p.i. group, green = 8 h.p.i. group vs. 3 h.p.i. group.

Figure 5. Transcription heatmap of 1194 DEGs for the three groups, with subcluster classification

Color scale is shown in the right side. Up-regulated or down-regulated genes are presented by red or green bars.

Figure 6. Trend analysis of the 1194 differentially expressed genes

(A) Different expression patterns of 1194 DEGs were partitioned into 8 profiles. The number of the DEGs and p value assigned to each profile is shown. (B) Characteristic expression patterns represented in profiles 2, 4 and 7 (p<0.001). Experimental points are indicated on the x axis: 0 for the uninfected group, 3 for 3 h.p.i, and 8 for 8 h.p.i. The y axis shows the normalized expression values. (C) The percentages of the differentially expressed genes related to the GO classifications for the three paired groups are shown. Blue = profile 2, green = profile 4, red = profile 7.

Figure 7. ORFV induced a range of differentially expressed genes associated with “growth and death” and “immune response”

(A) Significantly (fold change>2 or <-2 and corrected p-value<0.05) up-regulated (red) or down-regulated (green) genes for the different pathways associated to growth and death are shown. (B) Significantly up-regulated or down-regulated genes associated with immune response are shown.

Figure 8. Validation of differentially expressed genes by real-time PCR (RT-PCR)

(A) RT-PCR was performed with the same samples used in RNA-SEQ. The average value and standard deviation of the independent experiments for the paired group 0 h.p.i vs. 3 h.p.i are plotted in comparison with the fold increase data obtained from Illumina sequencing. (B) The results of the paired group 0 h.p.i. vs. 8 h.p.i. are shown.