China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (9): 4319-4333.doi: 10.16431/j.cnki.1671-7236.2025.09.029

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Gene Set Enrichment Analysis and Verification of RNA Sequencing Data Obtained from Sheep Embryonic Testicular Cells Infected with Bluetongue Virus

LU Danfeng1, LI Zhanhong2,3, ZHANG Zhenxing2,3, ZHU Pei2,3, LI Zhuoran2,3   

  1. 1. School of Medicine, Kunming University, Kunming 650214, China;
    2. Yunnan Tropical and Subtropical Animal Virus Disease Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China;
    3. Key Laboratory of Transboundary Animal Diseases Prevention and Control (Co-construction by Ministry and Province), Yunnan Animal Science and Veterinary Institute, Kunming 650224, China
  • Received:2025-01-06 Online:2025-09-05 Published:2025-08-29

PDF (PC)

12

Abstract: 【Objective】 This study aimed to explore the key host cytokines or proteins which stringed pro-inflammatory cytokines,inflammatory responses,and apoptosis together in Bluetongue virus (BTV) infection progress from the perspectives of biological processes and signaling pathways involved in pro-inflammatory cytokines and apoptosis,and further obtain clues of host factors or proteins related to the pathogenesis of BTV. 【Method】 Total RNA of BTV-1 serotype strain (Y863) infected sheep embryonic testicular cells (OA3.Ts) was extracted for RNA sequencing (RNA-Seq).R package edgeR (v 3.14.0) was used to count differentially expressed genes.Gene set enrichment analysis of differentially expressed genes were performed to obtain their gene enrichment maps under the KEGG subset of C2:curated gene sets,biological processes (BP),cellular components (CC) and molecular function (MF) subsets of C5:GO gene sets,as well as Hallmark gene sets.Real-time quantitative RT-PCR,ELISA and Western blotting were used to validate 10 genes expression of leading-edge subsets (LS) genes in the key signaling pathways or biological process.The level of apoptosis of BTV infected OA3.Ts cells was detected by Annexin V-FITC/propidium iodide (PI) staining. 【Result】 GSEA results indicated that differentially expressed genes were significantly enriched in apoptosis pathway of KEGG subset under C2:curated gene sets,response to tumor necrosis factor (TNF),response to interleukin 1 (IL-1),cellular response to IL-1,response to interferon-γ (IFN-γ),cellular response to IFN-γ,and IL-12 production terms of BP subset under C5:GO gene sets,and inflammatory response pathway under Hallmark gene sets.The results of Real-time quantitative RT-PCR showed that the transcriptional levels of gained key pro-inflammatory cytokines and proteins based on GESA analysis were significantly up-regulated,including IL-1α,IL-6,C-X-C chemokine 8 (CXCL8),caspase 7 (CASP7),CASP8,TNF receptor superfamily member 5 (CD40),and nucleotide-binding oligomerization domain receptor family pyrin domain containing protein 3 (NLRP3),with the log2FoldChange (FC) values of 0.34,1.03,7.42,3.98,3.61,1.00 and 1.74,respectively.ELISA and Western blotting results showed that compared with control group,the expression levels of CXCL8,CD40,CASP7 and CASP8 of infected group were extremely significantly or significantly increased (P＜0.05 or P＜0.01).It was confirmed that the percentage of apoptotic OA3.Ts cells was elevated from 1.89% of control groups up to 12.78% of infected groups using Annexin V-FITC/PI staining method. 【Conclusion】 The highly expressed CD40 and CXCL8 were the key factors in stringing pro-inflammatory cytokines,inflammatory response,and apoptosis together,as well as were important potential contributors to the clinical symptoms of hemorrhage of multi-tissue,edema,disseminated intravascular coagulation,and tissue necrosis in sheep,as well as testicular degeneration and azoospermia in male sheep.

Key words: Bluetongue virus (BTV); RNA-Seq; gene set enrichment analysis; CD40; CXCL8

CLC Number: