Differentially Expressed miRNA Profiles in Serum-Derived Exosomes from Cattle Infected with Lumpy Skin Disease Virus

Abstract

Exosomal miRNAs from individual cells are crucial in regulating the immune response to infectious diseases. In this study, we performed small RNA sequencing (small RNA-seq) analysis to identify the expressed and associated exosomal miRNAs in the serum of cattle infected with lumpy skin disease virus (LSDV). Cattle were infected with a 10^6.5 TCID50/mL LSDV Vietnam/HaTinh/CX01 (HT10) strain and exosomal miRNA expression in the serum of infected cattle was analyzed using small RNA sequencing (small RNA-seq). We identified 59 differentially expressed (DE) miRNAs in LSDV-infected cattle compared to uninfected controls, including 18 upregulated and 41 downregulated miRNAs. These 59 miRNAs were used to predict 7656 target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the target genes were enriched in several biological processes and pathways associated with viral replication, immune response, virus-host interactions, and signal transduction. Additionally, we identified 708 potentially novel cattle miRNAs corresponding to 710 genomic loci. The transcription levels of five miRNA genes (bta-miR-11985, bta-miR-1281, bta-miR-12034, bta-miR-let-7i, and bta-miR-17-5p) were validated using reverse transcription quantitative real-time PCR, showing consistency with the small RNA-seq results. Overall, these findings provide significant insights into the immune and protective responses during LSDV infection in cattle, offering valuable information on identifying new biomarkers and understanding the pathogenesis of LSDV.

Keywords: exosome; lumpy skin disease; miRNA; pathway; small RNA-seq.

Figure 1

Characterization of bovine exosomes isolated from serum in the control and treatment groups. (A) Exosome size distribution: red and blue represent serum-derived exosomes from the control and LSDV-infected groups, respectively. (B) Western blot analysis: exosomes from both the control and infected groups were analyzed via Western blotting using exosomal markers (CD9, CD63, and CD81).

Figure 2

Volcano plot and heatmap illustrating miRNA expression levels in control and LSDV-infected groups. (A) Volcano plot: Log2 fold changes and p-values were computed by comparing the two groups and visualized in the plot. The X-axis represents the log2 fold change, and the Y-axis depicts the negative logarithm (base 10) of the p-value. Yellow dots indicate a fold change ≥ 2 and a raw p-value < 0.05. Blue dots indicate a fold change ≤ −2 and a raw p-value < 0.05. (B) Heatmap: hierarchical clustering analysis was conducted using the Euclidean method and complete linkage. The heatmap represents the similarity of expression levels (normalized values) of 59 mature miRNAs across six samples. The control group is denoted by a red box, and the infected group is indicated by a blue box.

Figure 3

Gene Ontology (GO) enrichment analysis of target genes from 59 DE miRNAs. GO analysis was used for target genes across three categories: (A) biological processes, (B) cellular components, and (C) molecular functions.

Figure 4

Pathway analysis of differentially expressed (DE) miRNAs. (A) Top 20 enriched KEGG pathways and (B) Reactome pathways for target genes of the 59 known DE miRNAs. Dot size reflects the gene count, and the gene ratio indicates the proportion of target genes associated with a specific KEGG/Reactome term compared to the total number of genes in that term.

Figure 5

Validation of DE miRNAs using quantitative real-time polymerase chain reaction (RT-qPCR). Bar graphs depict the average fold changes observed across individual samples. miRNA expression levels were normalized to bovine U6 expression. Error bars represent the standard error of the mean (*** p < 0.001). Experiments were conducted in triplicate using individual samples.