蓝舌病病毒感染绵羊胚胎睾丸细胞转录组测序数据的基因集富集分析及验证

doi:10.16431/j.cnki.1671-7236.2025.09.029

摘要/Abstract

摘要： 【目的】从促炎细胞因子和细胞凋亡涉及的生物过程和信号通路角度,探讨在蓝舌病病毒(Bluetongue virus，BTV)感染进程中将促炎细胞因子-炎症反应-凋亡三者串联起来的关键宿主细胞因子或蛋白，以期获得BTV致病机制相关宿主因子或蛋白线索。【方法】对BTV-1血清型毒株Y863感染的绵羊胚胎睾丸细胞(OA3.Ts)总RNA进行转录组测序(RNA-Seq)；利用R package edgeR(v 3.14.0)包统计差异表达基因，对差异表达基因进行基因集富集分析(GSEA)，获得其在基因集C2：curated gene sets下的KEGG基因子集，基因集C5:GO下的生物过程、细胞组分和分子功能基因子集，以及基因集Hallmark下的基因富集图谱；利用实时荧光定量RT-PCR、ELISA和Western blotting对关键生物过程或通路的10个领头亚集(LS)基因的表达进行验证；并通过Annexin V-FITC/碘化丙啶(PI)染色法检测被感染细胞的凋亡水平。【结果】 GSEA结果表明，差异表达基因分别显著富集在基因集C2：curated gene sets的KEGG基因子集下的凋亡通路，基因集C5：GO的生物过程基因子集下的对肿瘤坏死因子(TNF)的反应、对白细胞介素1(IL-1)的反应、细胞对IL-1的反应、对γ-干扰素(IFN-γ)的反应、细胞对IFN-γ的反应和IL-12 产生等条目，以及基因集Hallmark下的炎症反应通路。实时荧光定量RT-PCR结果显示，GSEA结果中关键促炎细胞因子和蛋白中的IL-1α、IL-6、C-X-C趋化因子8(CXCL8)、半胱天冬酶7(CASP7)、CASP8、TNF受体超家族成员5(CD40)和核苷酸结合寡聚化结构域样受体热蛋白结构域亚家族成员3(NLRP3)的转录水平显著上调，log₂差异倍数(FoldChange,FC)值分别为0.34、1.03、7.42、3.98、3.61、1.00和1.74。ELISA和Western blotting结果显示，与对照组相比，感染组CXCL8、CD40、CASP7和CASP8表达水平显著或极显著上升(P＜0.05；P＜0.01)。Annexin V-FITC/PI染色结果表明，凋亡的OA3.Ts细胞由对照组的1.89%上升至感染组的12.78%。【结论】本研究分析预测高表达的CD40和CXCL8是串联促炎细胞因子-炎症反应-凋亡三者的关键因素，对BTV感染导致的多组织出血、水肿、弥散性血管内凝血和组织坏死等临床症状及雄性绵羊睾丸退化和无精子症具有重要潜在贡献。

关键词: 蓝舌病病毒(BTV); 转录组测序; 基因集富集分析; CD40; CXCL8

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

中图分类号:

S852.65

鲁丹枫, 李占鸿, 张振兴, 朱沛, 李卓然. 蓝舌病病毒感染绵羊胚胎睾丸细胞转录组测序数据的基因集富集分析及验证[J]. 中国畜牧兽医, 2025, 52(9): 4319-4333.

LU Danfeng, LI Zhanhong, ZHANG Zhenxing, ZHU Pei, LI Zhuoran. Gene Set Enrichment Analysis and Verification of RNA Sequencing Data Obtained from Sheep Embryonic Testicular Cells Infected with Bluetongue Virus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4319-4333.

参考文献

[1] GONDARD M,POSTIC L,GARIN E,et al.Exceptional Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) circulation in France in 2023[J].Virus Research,2024,350:199489.
[2] DE KLERK J,TILDESLEY M,ROBBINS A,et al.Parameterisation of a Bluetongue virus mathematical model using a systematic literature review[J].Preventive Veterinary Medicine,2024,232:106328.
[3] MCLAUGHLIN B E,DEMAULA C D,WILSON W C,et al.Replication of Bluetongue virus and Epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle,sheep,and deer[J].American Journal of Veterinary Research,2003,64(7):860-865.
[4] HOWERTH E W.Cytokine release and endothelial dysfunction:A perfect storm in Orbivirus pathogenesis[J].Veterinaria Italiana,2015,51(4):275-281.
[5] DEMAULA C D,JUTILA M A,WILSON D W,et al.Infection kinetics,prostacyclin release and cytokine-mediated modulation of the mechanism of cell death during Bluetongue virus infection of cultured ovine and bovine pulmonary artery and lung microvascular endothelial cells[J].Journal of General Virology,2001,82(4):787-794.
[6] DEMAULA C D,LEUTENEGGER C M,BONNEAU K R,et al.The role of endothelial cell-derived inflammatory and vasoactive mediators in the pathogenesis of bluetongue[J].Virology,2002,296(2):330-337.
[7] POURCELOT M,DA SILVA MORAES R A,LACOUR S,et al.Activation of inflammasome during Bluetongue virus infection[J].Pathogens,2023,12(6):801.
[8] TAKEUCHI O,AKIRA S.Pattern recognition receptors and inflammation[J].Cell,2010,140(6):805-820.
[9] STEWART M E,ROY P.Role of cellular caspases,nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate[J].Virology Journal,2010,7:362.
[10] RATINIER M,SHAW A E,BARRY G,et al.Bluetongue virus NS4 protein is an interferon antagonist and a determinant of virus virulence[J].Journal of Virology,2016,90(11):5427-5439.
[11] RODRÍGUEZ-CALVO T,ROJAS J M,MARTÍN V,et al.Type Ⅰinterferon limits the capacity of Bluetongue virus to infect hematopoietic precursors and dendritic cells in vitro and in vivo[J].Journal of Virology,2014,88(2):859-867.
[12] DEMAULA C D,LEUTENEGGER C M,JUTILA M A,et al.Bluetongue virus-induced activation of primary bovine lung microvascular endothelial cells[J].Veterinary Immunology and Immunopathology,2002,86(3-4):147-157.
[13] PENG X,CHAN E Y,LI Y,et al.Virus-host interactions:From systems biology to translational research[J].Current Opinion in Microbiology,2009,12(4):432-438.
[14] SUBRAMANIAN A,TAMAYO P,MOOTHA V K,et al.Gene set enrichment analysis:A knowledge-based approach for interpreting genome-wide expression profiles[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(43):15545-15550.
[15] ZHU J,YANG H,LI H,et al.Full-genome sequence of Bluetongue virus serotype 1 (BTV-1) strain Y863,the first BTV-1 isolated of eastern origin found in China[J]. Genome Announcements,2013,1(4):e00403-13.
[16] RAMAKRISHNAN M A.Determination of 50% endpoint titer using a simple formula[J].World Journal of Virology,2016,5(2):85.
[17] CHEN S,ZHOU Y,CHEN Y,et al.Fastp:An ultra-fast all-in-one FASTQ preprocessor[J].Bioinformatics,2018,34:i884-i890.
[18] KIM D,LANGMEAD B,SALZBERG S L.HISAT:A fast spliced aligner with low memory requirements[J].Nature Methods,2015,12:357-360.
[19] PERTEA M,PERTEA G M,ANTONESCU C M,et al.StringTie enables improved reconstruction of a transcriptome from RNA-Seq reads[J].Nature Biotechnology,2015,33:290-295.
[20] PERTEA G,PERTEA M.GFF utilities:GffRead and GffCompare[J]. F1000Research,2020,9:ISCB Comm J-304.
[21] ROBINSON M D,MCCARTHY D J,SMYTH G K.edgeR:A bioconductor package for differential expression analysis of digital gene expression data[J].Bioinformatics,2010,26:139-140.
[22] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2^-ΔΔCt method[J].Methods,2001,25(4):402-408.
[23] ZARATE M A,WESOLOWSKI S R,NGUYEN L M,et al.In utero inflammatory challenge induces an early activation of the hepatic innate immune response in late gestation fetal sheep[J].Innate Immunity,2020,26(7):549-564.
[24] LI H,CUNHA C W,DAVIES C J,et al.Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep[J].Journal of General Virology,2008,89(7):1699-1708.
[25] VAN BA H,HWANG I C.Role of caspase-9 in the effector caspases and genome expressions,and growth of bovine skeletal myoblasts[J].Development Growth & Differentiation,2014,56(2):131-142.
[26] EFE U,DEDE S,YVKSEK Ü,et al.Apoptotic and oxidative mechanisms in liver and kidney tissues of sheep with fluorosis[J].Biological Trace Element Research,2021,199(1):136-141.
[27] MACHUGH D E,TARAKTSOGLOU M,KILLICK K E,et al.Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis[J].Veterinary Research,2012,43:25.
[28] ZHAO Z,LI Y,CAO J,et al.Early pregnancy modulates expression of the Nod-like receptor family in lymph nodes of ewes[J].Animals,2022,12(23):3285.
[29] SPRAGUE A H,KHALIL R A.Inflammatory cytokines in vascular dysfunction and vascular disease[J].Biochemical Pharmacology,2009,78(6):539-552.
[30] BENVENISTE E N,NGUYEN V T,WESEMANN D R.Molecular regulation of CD40 gene expression in macrophages and microglia[J].Brian,Behavior,and Immunity,2004,18(1):7-12.
[31] CAGNONI F,ODDERA S,GIRON-MICHEL J,et al.CD40 on adult human airway epithelial cells:Expression and proinflammatory effects[J].The Journal of Immunology,2004,172(5):3205-3214.
[32] GIROUARD J,REYES-MORENO C,DARVEAU A,et al.Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression[J].Molecular Immunology,2005,42(7):773-780.
[33] RUSSO R C,GARCIA C C,TEIXEIRA M M,et al.The CXCL8/IL-8 chemokine family and its receptors in inflammatory diseases[J].Expert Review Clinical Immunology,2014,10(5):593-619.
[34] CAMBIER S,GOUWY M,PROOST P.The chemokines CXCL8 and CXCL12:Molecular and functional properties,role in disease and efforts towards pharmacological intervention[J].Cellular & Molecular Immunology,2023,20(3):217-251.
[35] LU D,LI Z,ZHU P,et al.Whole-transcriptome analyses of sheep embryonic testicular cells infected with the Bluetongue virus[J].Frontiers in Immunology,2022,13:1053059.
[36] ZHOU S W,WANG J,CHEN S Y,et al.The substrate stiffness at physiological range significantly modulates vascular cell behavior[J].Colloids and Surfaces B:Biointerfaces,2022,214:112483.
[37] ELIOPOULOS A G,DAVIES C,KNOX P G,et al.CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily[J].Molecular and Cellular Biology,2000,20(15):5503-5515.
[38] YANG P,TAN J,YUAN Z,et al.Expression profile of cytokines and chemokines in osteoarthritis patients:Proinflammatory roles for CXCL8 and CXCL11 to chondrocytes[J].International Immunopharmacology,2016,40:16-23.
[39] LYONS H E,ARMAN B M,ROBERTSON S A,et al.Immune regulatory cytokines in seminal plasma of healthy men:A scoping revies and analysis of variance[J].Andrology,2023,11(7):1245-1266.
[40] PUGGIONI G,PINTUS D,MELZI E,et al.Testicular degeneration and infertility following Arbovirus infection[J].Journal of Virology,2018,92(19):10.1128.
[41] LOBODIN B,DIKSTEIN R.So close,no matter how far:Multiple paths connecting transcription to mRNA translation in eukaryotes[J].EMBO Reports,2020,21(9):e50799.