Transcriptome analysis reveals common differential and global gene expression profiles in bluetongue virus serotype 16 (BTV-16) infected peripheral blood mononuclear cells (PBMCs) in sheep and goats

Abstract

Bluetongue is an economically important infectious, arthropod borne viral disease of domestic and wild ruminants, caused by Bluetongue virus (BTV). Sheep are considered the most susceptible hosts, while cattle, buffalo and goats serve as reservoirs. The viral pathogenesis of BTV resulting in presence or absence of clinical disease among different hosts is not clearly understood. In the present study, transcriptome of sheep and goats peripheral blood mononuclear cells infected with BTV-16 was explored. The differentially expressed genes (DEGs) identified were found to be significantly enriched for immune system processes - NFκB signaling, MAPK signaling, Ras signaling, NOD signaling, RIG signaling, TNF signaling, TLR signaling, JAK-STAT signaling and VEGF signaling pathways. Greater numbers of DEGs were found to be involved in immune system processes in goats than in sheep. Interestingly, the DEHC (differentially expressed highly connected) gene network was found to be dense in goats than in sheep. Majority of the DEHC genes in the network were upregulated in goats but down-regulated in sheep. The network of differentially expressed immune genes with the other genes further confirmed these findings. Interferon stimulated genes - IFIT1 (ISG56), IFIT2 (ISG54) and IFIT3 (ISG60) responsible for antiviral state in the host were found to be upregulated in both the species. STAT2 was the TF commonly identified to co-regulate the DEGs, with its network showing genes that are downregulated in sheep but upregulated in goats. The genes dysregulated and the networks perturbed in the present study indicate host variability with a positive shift in immune response to BTV in goats than in sheep.

Keywords: Bluetongue; Bluetongue virus serotype-16; DEGs; DEHC genes; PBMCs; RNA-seq; transcriptome.

Fig. 1

Confirmation of viral infection and viral transcript quantification: A) Amplification of NS1 gene by PCR at 24 h, 48 h and 72 h p.i in BTV-16 infected sheep PBMCs (M-1 kb DNA ladder; 1-Negative control; 2-Infected PBMC at 24 h p.i.; 3-Infected PBMC at 48 h p.i.; 4-Infected PBMC at 72 h p.i) B) Amplification of NS1 gene by PCR at 24 h, 48 h and 72 h p.i in BTV-16 infected goats PBMCs (M-1 kb DNA ladder; 1-Negative control; 2-Infected PBMC at 24 h p.i.; 3-Infected PBMC at 48 h p.i.; 4-Infected PBMC at 72 h p.i) C) Viral copy number across time points in sheep and goats by Real time PCR. Students t-test and Tukey HSD, were done in JMP 9.0., to compare the NS1 gene expression between sheep and goats at each time point and to compare the NS1 gene expression between time points within species, respectively. Levels not connected by same letter are significantly (P < 0.05) different within a time point between species. Across time points the viral copy number was significantly different between all time points in sheep. In goats, the viral copy number was found to be significantly different at 72 h p.i. w.r.t 24 and 48 h p.i.

Fig. 2

Functional enrichment of differentially expressed genes in BTV-16 infected sheep and goats PBMCs.

Fig. 3

Pathways enriched in mapping the differentially expressed genes to canonical KEGG pathways in ClueGO. Venn diagrams show the genes involved in different pathways in sheep and goats. The bar chart indicates that more number of genes are involved in different pathways in goats than in sheep.

Fig. 4

Differentially expressed highly connected networks and immune - differentially expressed gene interactions in sheep and goats. The upregulated genes are shown in green and downregulated genes are shown in red with the gradient showing the extent of expression. The size of the node indicates connectivity (i.e. degree).

Fig. 5

Predicted transcription factors that co-regulate DEGs involved in immune processes: A) Heat Map showing the TFs binding to DEGs involved in immune processes in sheep. B) Heat Map showing the TFs binding to DEGs involved in immune processes in goats. The columns represent the DEGs involved in immune processes (Y-axis) and the rows represent the predicted TFs binding to the DEHC genes. C) Motif alignment of STAT2 that was commonly predicted to regulate DEGs involved in immune processes in sheep and goats D) network depicting the interaction of STAT2 with DE genes involved in immune processes in sheep E) Network depicting the interaction of STAT2 with DE genes involved in immune processes in goats.

Fig. 6

Quantitative real-time PCR to validate the RNA-seq experiment. Fold change (2^− ΔΔCT) with control as the calibrator is represented along with the standard error of difference in both the species.

Fig. 7

Flow chart of steps depicting the analysis of transcriptome data.