山羊副流感病毒3型感染MDBK细胞的转录组分析
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摘要
本研究旨在探究山羊副流感病毒3型(CPIV3)感染MDBK细胞后的转录组基因变化情况,丰富CPIV3转录组信息。取1MOI CPIV3JSHA2014-1病毒液感染MDBK细胞,设非感染正常细胞为对照,于24h后收获细胞,提取总RNA,利用Illumina HiSeqTM2500对感染组与对照组进行高通量测序,并用测序评估、基因注释等生物信息学方法进行分析。结果显示,差异表达基因共261个,其中表达上调140个,表达下调121个,经RT-qPCR方法验证8个差异表达的干扰素信号通路相关基因,结果与高通量测序一致。进一步GO分类结果显示,差异表达基因主要涉及细胞生物学进程、构成细胞的组分以及实现的分子功能三个方面,KEGG分析显示这些基因参与代谢、生物系统、细胞进程、基因信息进程和环境信息进程。本研究为深入探究CPIV3的致病机制奠定了基础。
To explore the cellular transcriptional gene differentially expressed in MDBK cells infected with caprine parainfluenza virus type 3(CPIV3),we analyzed the mRNA expression profiles in MDBK cells infected with 1 MOI CPIV3 JSHA2014-1 strain at 24 hpi using high-throughput sequencing.The results indicated that a total of 261 genes were obviously differentially expressed in the infected MDBK cells including 140 up-regulated genes and 121 down-regulated genes.The eight of these genes were verified by RT-qPCR assay,and the results were consistent with those of high-throughput sequencing.GO analysis classification showed that differentially expressed genes were mainly involved in biological processes,cellular components and molecular functions.KEGG analysis shows that the signaling pathways involved in these genes included metabolism-related signaling pathways,biological systems,cellular processes,gene information processes and environmental information processes.This study laid the foundation for further exploring the pathogenesis of CPIV3.
To explore the cellular transcriptional gene differentially expressed in MDBK cells infected with caprine parainfluenza virus type 3(CPIV3),we analyzed the mRNA expression profiles in MDBK cells infected with 1 MOI CPIV3 JSHA2014-1 strain at 24 hpi using high-throughput sequencing.The results indicated that a total of 261 genes were obviously differentially expressed in the infected MDBK cells including 140 up-regulated genes and 121 down-regulated genes.The eight of these genes were verified by RT-qPCR assay,and the results were consistent with those of high-throughput sequencing.GO analysis classification showed that differentially expressed genes were mainly involved in biological processes,cellular components and molecular functions.KEGG analysis shows that the signaling pathways involved in these genes included metabolism-related signaling pathways,biological systems,cellular processes,gene information processes and environmental information processes.This study laid the foundation for further exploring the pathogenesis of CPIV3.
引文
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[26]HARADA H,FUJITA T,MIYAMOTO M,et al.Structurally similar but functionally distinct factors,IRF-1and IRF-2,bind to the same regulatory elements of IFN and IFN-inducible genes[J].Cell,1989,58(4):729-739.
[27]SUN K,METZGER D W.Inhibition of pulmonary antibacterial defense by interferon-γduring recovery from influenza infection[J].Nat Med,2008,14(5):558-564.
[28]钟纯燕,李基棕,毛立,等.MiR-222对山羊副流感病毒3型复制的影响[J].畜牧兽医学报,2018,49(12):2664-2671.ZHONG C Y,LI J Z,MAO L,et al.The molecular mechanism study of miR-222inhibit caprine parainfluenza virus type 3replication[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2664-2671.(in Chinese)
[29]MCSHARRY J J.Analysis of virus-infected cells by flow cytometry[J].Methods,2000,21(3):249-257.
[30]FREDERICKSEN F,DELGADO F,CABRERA C,et al.The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells(MDBK)infected with the Bovine Viral Diarrhea Virus 1(BVDV-1)[J].Gene,2015,569(1):95-103.
[31]ZHANG Q Z,SHI K C,YOO D.Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1[J].Virology,2016,489:252-268.
[32]CHEN J,YANG Y F,YANG Y,et al.AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling[J].Nat Microbiol,2018,3(3):302-309.
[2]YANG L L,LI W L,MAO L,et al.Analysis on the complete genome of a novel caprine parainfluenza virus 3[J].Infect Genet Evolut,2016,38:29-34.
[3]LI J Z,LI W L,MAO L,et al.Rapid detection of novel caprine parainfluenza virus type 3(CPIV3)using a TaqMan-based RT-qPCR[J].J Virol Methods,2016,236:126-131.
[4]COSTA V,ANGELINI C,DE FEIS I,et al.Uncovering the complexity of transcriptomes with RNA-Seq[J].J Biomed Biotechnol,2010,2010:853916.
[5]CONG F,LIU X L,HAN Z X,et al.Transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection[J].BMCGenomics,2013,14(1):743.
[6]LI M Y,TAN H W,WANG F,et al.De Novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley(Petroselinum crispum)[J].PLoS One,2014,9(9):e108977.
[7]ZHU S Y,TANG S W,TANG Q M,et al.Genome-wide transcriptional changes of ramie(Boehmeria nivea L.Gaud)in response to root-lesion nematode infection[J].Gene,2014,552(1):67-74.
[8]刘倩宏.深度测序技术对羊布鲁菌16M株感染小鼠巨噬细胞转录组学的研究[D].长春:吉林大学,2012.LIU Q H.Deep sequencing-based expresssion transcriptional profiling changes of mouse macrophages during Brucella melitensis 16Minfection[D].Changchun:Jilin University,2012.(in Chinese)
[9]陈达香,陈瑜,郝文波,等.羊口疮病毒作为免疫调节剂在疾病治疗中的研究进展[J].生物技术通讯,2017,28(2):182-187.CHEN D X,CHEN Y,HAO W B,et al.Progress in Orf virus as an immunomodulator in diseases treatment[J].Letters in Biotechnology,2017,28(2):182-187.(in Chinese)
[10]李基棕,李文良,毛立,等.山羊副流感病毒3型感染MDBK细胞的miRNA表达谱变化分析[J].畜牧兽医学报,2017,48(5):896-906.LI J Z,LI W L,MAO L,et al.Identification and analysis of the miRNA expression profiles in MDBKcells in response to the infection of caprine parainfluenza virus type 3(CPIV3)[J].Acta Veterinaria et Zootechnica Sinica,2017,48(5):896-906.(in Chinese)
[11]COCK P J A,FIELDS C J,GOTO N,et al.The Sanger FASTQ file format for sequences with quality scores,and the Solexa/Illumina FASTQ variants[J].Nucleic Acids Res,2010,38(6):1767-1771.
[12]KIM D,LANGMEAD B,SALZBERG S L.HISAT:a fast spliced aligner with low memory requirements[J].Nat Methods,2015,12(4):357-360.
[13]ERLICH Y,MITRA P P,DELABASTIDE M,et al.Alta-Cyclic:a self-optimizing base caller for nextgeneration sequencing[J].Nat Methods,2008,5(8):679-682.
[14]MORTAZAVI A,WILLIAMS B A,MCCUE K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nat Methods,2008,5(7):621-628.
[15]ANDERS S,HUBER W.Differential expression analysis for sequence count data[J].Genome Biol,2010,11(10):R106.
[16]WESTFALL P H,YOUNG S S.P value adjustments for multiple tests in multivariate binomial models[J].J Amer Statist Assoc,1989,84(407):780-786.
[17]XING S S,DU J Z,GAO S D,et al.Analysis of the miRNA expression profile in an Aedes albopictus cell line in response to bluetongue virus infection[J].Infect Genet Evolut,2016,39:74-84.
[18]岳桂东,高强,罗龙海,等.高通量测序技术在动植物研究领域中的应用[J].中国科学:生命科学,2012,42(2):107-124.YUE G D,GAO Q,LUO L H,et al.The application of high-throughput sequencing technology in plant and animal research[J].Scientia Sinica(Vitae),2012,42(2):107-124.(in Chinese)
[19]SUN Y,HAN M Y,KIM C,et al.Interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus[J].Viruses,2012,4(4):424-446.
[20]UEMATSU S,SATO S,YAMAMOTO M,et al.Interleukin-1receptor-associated kinase-1plays an essential role for Toll-like receptor(TLR)7-and TLR9-mediated interferon-αinduction[J].J Exp Med,2005,201(6):915-923.
[21]MEYERS J A,MANGINI A J,NAGAI T,et al.Blockade of TLR9agonist-induced type I interferons promotes inflammatory cytokine IFN-γand IL-17secretion by activated human PBMC[J].Cytokine,2006,35(5-6):235-246.
[22]KAWAI T,TAKAHASHI K,SATO S,et al.IPS-1,an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction[J].Nat Immunol,2005,6(10):981-988.
[23]AU W C,YEOW W S,PITHA P M.Analysis of functional domains of interferon regulatory factor 7and its association with IRF-3[J].Virology,2001,280(2):273-282.
[24]RUVOLO V,NAVARRO L,SAMPLE C E,et al.The epstein-barr virus SM protein induces STAT1and interferon-stimulated gene expression[J].J Virol,2003,77(6):3690-3701.
[25]LEAMAN D W,CHAWLA-SARKAR M,JACOBSB,et al.Novel growth and death related interferonstimulated genes(ISGs)in melanoma:greater potency of IFN-βcompared with IFN-α2[J].J Interf Cytok Res,2003,23(12):745-746.
[26]HARADA H,FUJITA T,MIYAMOTO M,et al.Structurally similar but functionally distinct factors,IRF-1and IRF-2,bind to the same regulatory elements of IFN and IFN-inducible genes[J].Cell,1989,58(4):729-739.
[27]SUN K,METZGER D W.Inhibition of pulmonary antibacterial defense by interferon-γduring recovery from influenza infection[J].Nat Med,2008,14(5):558-564.
[28]钟纯燕,李基棕,毛立,等.MiR-222对山羊副流感病毒3型复制的影响[J].畜牧兽医学报,2018,49(12):2664-2671.ZHONG C Y,LI J Z,MAO L,et al.The molecular mechanism study of miR-222inhibit caprine parainfluenza virus type 3replication[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2664-2671.(in Chinese)
[29]MCSHARRY J J.Analysis of virus-infected cells by flow cytometry[J].Methods,2000,21(3):249-257.
[30]FREDERICKSEN F,DELGADO F,CABRERA C,et al.The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells(MDBK)infected with the Bovine Viral Diarrhea Virus 1(BVDV-1)[J].Gene,2015,569(1):95-103.
[31]ZHANG Q Z,SHI K C,YOO D.Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1[J].Virology,2016,489:252-268.
[32]CHEN J,YANG Y F,YANG Y,et al.AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling[J].Nat Microbiol,2018,3(3):302-309.