Impact of PEDV infection on the biological characteristics of porcine intestinal exosomes

Abstract

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection primarily affecting pigs. It is caused by the porcine epidemic diarrhea virus (PEDV). PEDV targets the villus tissue cells in the small intestine and mesenteric lymph nodes, resulting in shortened intestinal villi and, in extreme cases, causing necrosis of the intestinal lining. Moreover, PEDV infection can disrupt the balance of the intestinal microflora, leading to an overgrowth of harmful bacteria like Escherichia coli. Exosomes, tiny membrane vesicles ranging from 30 to 150 nm in size, contain a complex mixture of RNA and proteins. MicroRNA (miRNA) regulates various cell signaling, development, and disease progression processes. This study extracted exosomes from both groups and performed high-throughput miRNA sequencing and bioinformatics techniques to investigate differences in miRNA expression within exosomes isolated from PEDV-infected porcine small intestine tissue compared to healthy controls. Notably, two miRNA types displayed upregulation in infected exosomes, while 12 exhibited downregulation. These findings unveil abnormal miRNA regulation patterns in PEDV-infected intestinal exosomes, shedding light on the intricate interplay between PEDV and its host. This will enable further exploration of the relationship between these miRNA changes and signaling pathways, enlightening PEDV pathogenesis and potential therapeutic targets.

Keywords: exosomes; microRNA; porcine epidemic diarrhea virus; porcine intestinal tissue damage; ultracentrifugation.

Figure 1

Piglet autopsy lesions. (A) Anatomy of the PEDV-infected group. (B) Anatomy of healthy piglets.

Figure 2

Porcine intestine exosome identification. (A) Photomicrographs of exosomes acquired using transmission electron microscopy. (B) NanoFCM study of exosome particle size. (C) Western blotting was utilized to identify the expression of biomarker proteins, such as TSG101, HSP70, and CD9. Lane 1, Healthy piglets' intestinal tissue exosome. Lane 2, PEDV-infected piglets' intestinal tissue exosomes.

Figure 3

The cytotoxicity of exosomes in Vero cells. Three independent experiments were carried out.

Figure 4

(A) Volcano plot of differentially expressed miRNAs. Two miRNAs (ssc-miR-132 and ssc-miR-181d-5p) were up-regulated in small intestinal tissue of healthy piglets, while the remaining 12 were down-regulated. (B) Venn diagram of miRNAs shared and unique between groups. It depicts a Venn diagram of the number of common and unique miRNAs found in the PEDV-infected and control groups. Each number denotes the number of miRNAs observed in each group. The number in the overlap area represents the miRNAs shared by the two comparison groups. Each non-overlapping region's number represents the unique miRNA in each group (P < 0.05). It can be seen that there were 336 known miRNAs in both the PEDV-infected and control groups, with the infected group having fewer miRNAs than the control group. There were 14 new miRNAs, with the infected group having more than the control group. Blue: miRNAs of PEDV-infected intestinal exosomes. Yellow: miRNAs of healthy intestinal exosomes.

Figure 5

Cluster analysis of differential miRNA expression.

Figure 6

Fluorescence quantitative PCR validation.

Figure 7

(A) Gene Ontology classification of target genes. (B) KEGG enrichment pathway of target genes.