传染性法氏囊病病毒感染DF-1细胞的转录组学分析
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摘要
传染性法氏囊病病毒(Infectious bursal disease virus, IBDV)感染鸡(Gallus gallus)引起的IBD是一种严重的、高度传染的免疫抑制性疾病。为探索IBDV感染所引起的宿主细胞转录组变化,本研究以IBDV感染鸡成纤维细胞系DF-1,提取细胞总RNA,使用TruSeq~?RNA LT Sample Prep Kit v2构建RNA文库,采用Illumina HiSeq 3000平台进行高通量测序,分析IBDV感染12 h引起的DF-1细胞转录组变化,筛选显著变化基因。实验结果显示,差异表达基因总共有3 417条,其中在IBDV感染组中相对高表达的基因有1 887条,相对低表达的基因有1 530条(差异倍数(Fold change, FC)>2且P≤0.05)。对差异基因进行基因本体(Gene Ontology, GO)注释及KEGG信号通路富集分析,发现IBDV感染能够引起DF-1细胞中免疫、凋亡、代谢等20多条信号通路发生显著变化;其中细胞免疫和细胞凋亡通路可能在IBDV感染和免疫调控机制中具有重要作用。随机选择9条差异表达基因进行qRT-PCR验证,变化趋势与转录组数据一致。本研究为揭示IBDV致病及免疫抑制的分子机制提供了参考信息。
Infectious bursal disease virus(IBDV) infection cause IBD in chicken(Gallus gallus), which is an acute, highly contagious and immunosuppressive poultry disease. In order to explore the transcriptome changes in host cells infected by IBDV infection, cellular RNAs extracted from IBDV or mock infected DF-1 cells were used to build a library by using Tru Seq~?RNA LT Sample Prep Kit v2, and then performed highthroughput sequencing on the Illumina HiSeq 3000 instrument. The transcriptome changes of DF-1 cells were analyzed at 12 h after IBDV infecting, and significantly altered genes were screened. The results showed that there were a total of 3 417 differentially expressed genes with 1 887 upregulated and 1 530 downregulated((Fold Change, FC) >2 and P≤0.05). Systematic bioinformatics analysis with Gene Ontology(GO) and KEGG, revealed that IBDV infection could cause significant changes in more than 20 signaling pathways,such as immunity, apoptosis and metabolism in DF-1 cells. Among them, cellular immunity and apoptosis pathway might play important roles in IBDV infection and immune regulation mechanisms. Nine differentially expressed genes were randomly selected for q RT-PCR verification, and the changing trend was consistent with that of transcriptome data. This study provides reference information for revealing the molecular mechanism of IBDV pathogenesis and immunosuppression.
Infectious bursal disease virus(IBDV) infection cause IBD in chicken(Gallus gallus), which is an acute, highly contagious and immunosuppressive poultry disease. In order to explore the transcriptome changes in host cells infected by IBDV infection, cellular RNAs extracted from IBDV or mock infected DF-1 cells were used to build a library by using Tru Seq~?RNA LT Sample Prep Kit v2, and then performed highthroughput sequencing on the Illumina HiSeq 3000 instrument. The transcriptome changes of DF-1 cells were analyzed at 12 h after IBDV infecting, and significantly altered genes were screened. The results showed that there were a total of 3 417 differentially expressed genes with 1 887 upregulated and 1 530 downregulated((Fold Change, FC) >2 and P≤0.05). Systematic bioinformatics analysis with Gene Ontology(GO) and KEGG, revealed that IBDV infection could cause significant changes in more than 20 signaling pathways,such as immunity, apoptosis and metabolism in DF-1 cells. Among them, cellular immunity and apoptosis pathway might play important roles in IBDV infection and immune regulation mechanisms. Nine differentially expressed genes were randomly selected for q RT-PCR verification, and the changing trend was consistent with that of transcriptome data. This study provides reference information for revealing the molecular mechanism of IBDV pathogenesis and immunosuppression.
引文
胡伯里.2014.传染性法氏囊病病毒结构蛋白VP2和VP3调控细胞自噬的分子机制研究[D].博士学位论文,浙江大学,导师:周继勇,pp.1-3.(Hu B L.2014.Molercular mechanisms of autophagy regulation by infectious bursal disease virus structural proteins VP2 and VP3[D].Thesis for Ph.D.,Zhejiang University,Suppervisor:Zhou J Y,pp.1-3.)
佟美姣,高雪丽,李博韬,等.2013.传染性法氏囊病毒对SPF雏鸡免疫器官细胞凋亡及bcl-2 mRNA表达的影响[J].畜牧兽医学报,44(04):617-621.(Tong M J,Gao X L,Li B T,et al.2013.Effect of infectious bursal disease virus on apoptosis and expression of bcl-2 mRNAin immune organs of SPF chicks[J].Acta Veterinaria et Zootechnica Sinica,44(04):617-621.)
谢珲,张欢欢,马月,等.2016.甲型流感病毒感染小鼠肺组织中环状RNA的表达变化及其靶基因功能分析[J].国际流行病学传染病学杂志,43(6):382-387.(Xie H,Zhang H H,Ma Y,et al.2016.CircRNA profiles in the lung tissue of influenza a virus-infected mice and biological function of their target genes[J].International Journal of Epidemiology and Infectious Disease,43(6):382-387.)
叶承金.2014.传染性法氏囊病病毒抑制宿主细胞干扰素-β表达的机制研究[D].博士学位论文,浙江大学,导师:周继勇,pp.19-20.(Ye C J.2014.Mechanism of inhibiting interferon-βinduction by infectious bursal disease virus infection[D].Thesis for Ph.D.,Zhejiang University,Suppervisor:Zhou J Y,pp.19-20.)
张倩,李平华,马翔,等.2019.高通量测序技术在鉴别影响猪产仔数QTL及候选基因中的应用[J].畜牧与兽医,51(01):118-124.(Zhang Q,Li P H,Ma X,et al.2019.Application of high-throughput sequencing techniques in identifying QTL and candidate gene of litter size of sow[J].Animal Husbandry&Veterinary Medicine,51(01):118-124.)
Broz P,Monack D M,2013.Newly described pattern recognition receptors team up against intracellular pathogens[J].Nature Reviews Immunology,13(8):551-565.
Cubas-gaona L L,Diaz-beneitez E,Ciscar M,et al.2018.Exacerbated apoptosis of cells infected with infectious bursal disease virus upon exposure to interferon alpha[J].Journal of Virology,92(11):e00364.
Dobos P,Hill B J,Hallett R,et al.1979.Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes[J].Journal of Virology,32(2):593-605.
Fuller D M,Zhu M,Ou-yang C W,et al.2011.A tale of two TRAPs:LAT and LAB in the regulation of lymphocyte development,activation,and autoimmunity[J].Immunologic Research,49(1-3):97-108.
Guo X,Wang L,Cui D,et al.2012.Differential expression of the Toll-like receptor pathway and related genes of chicken bursa after experimental infection with infectious bursa disease virus[J].Archives of Virology,157(11):2189-2199.
Kawai T,Akira S.2011.Toll-like receptors and their crosstalk with other innate receptors in infection and immunity[J].Immunity,34(5):637-650.
Lee C C,Wu C C,Lin T L.2014.Chicken melanoma differentiation-associated gene 5(MDA5)recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity[J].Archives of Virology,159(7):1671-1686.
Müller H,Islam M R,Raue R,2003.Research on infectious bursal disease-the past,the present and the future[J].Veterinary Microbiology,97(1-2):153-165.
Maddaly R,Pai G,Balaji S,et al.2010.Receptors and signaling mechanisms for B-lymphocyte activation,proliferation and differentiation-insights from both in vivo and in vitro approaches[J].FEBS Letters,584(24):4883-4894.
Moresco E M,Lavine D,Beutler B,2011.Toll-like receptors[J].Current Biology,21(13):R488-R493.
Ou C,Wang Q,Yu Y,et al.2017.Chemokine receptor CCR5and CXCR4 might influence virus replication during IBDV infection[J].Microbial Pathogenesis,107:122-128.
Rauf A,Khatri M,Murgia M V,et al.2012.Fas/FasL and perforin-granzyme pathways mediated T cell cytotoxic responses in infectious bursal disease virus infected chickens[J].Results in Immunology,2:112-119.
Wang B,Duan X,Fu M,et al.2018.The association of ribosomal protein L18(RPL18)with infectious bursal disease virus viral protein VP3 enhances viral replication[J].Virus Research,245:69-79.
Ye C J,Jia L,Sun Y,et al.2014.Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5[J].Journal of Virology,88(19):11154-11165.
佟美姣,高雪丽,李博韬,等.2013.传染性法氏囊病毒对SPF雏鸡免疫器官细胞凋亡及bcl-2 mRNA表达的影响[J].畜牧兽医学报,44(04):617-621.(Tong M J,Gao X L,Li B T,et al.2013.Effect of infectious bursal disease virus on apoptosis and expression of bcl-2 mRNAin immune organs of SPF chicks[J].Acta Veterinaria et Zootechnica Sinica,44(04):617-621.)
谢珲,张欢欢,马月,等.2016.甲型流感病毒感染小鼠肺组织中环状RNA的表达变化及其靶基因功能分析[J].国际流行病学传染病学杂志,43(6):382-387.(Xie H,Zhang H H,Ma Y,et al.2016.CircRNA profiles in the lung tissue of influenza a virus-infected mice and biological function of their target genes[J].International Journal of Epidemiology and Infectious Disease,43(6):382-387.)
叶承金.2014.传染性法氏囊病病毒抑制宿主细胞干扰素-β表达的机制研究[D].博士学位论文,浙江大学,导师:周继勇,pp.19-20.(Ye C J.2014.Mechanism of inhibiting interferon-βinduction by infectious bursal disease virus infection[D].Thesis for Ph.D.,Zhejiang University,Suppervisor:Zhou J Y,pp.19-20.)
张倩,李平华,马翔,等.2019.高通量测序技术在鉴别影响猪产仔数QTL及候选基因中的应用[J].畜牧与兽医,51(01):118-124.(Zhang Q,Li P H,Ma X,et al.2019.Application of high-throughput sequencing techniques in identifying QTL and candidate gene of litter size of sow[J].Animal Husbandry&Veterinary Medicine,51(01):118-124.)
Broz P,Monack D M,2013.Newly described pattern recognition receptors team up against intracellular pathogens[J].Nature Reviews Immunology,13(8):551-565.
Cubas-gaona L L,Diaz-beneitez E,Ciscar M,et al.2018.Exacerbated apoptosis of cells infected with infectious bursal disease virus upon exposure to interferon alpha[J].Journal of Virology,92(11):e00364.
Dobos P,Hill B J,Hallett R,et al.1979.Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes[J].Journal of Virology,32(2):593-605.
Fuller D M,Zhu M,Ou-yang C W,et al.2011.A tale of two TRAPs:LAT and LAB in the regulation of lymphocyte development,activation,and autoimmunity[J].Immunologic Research,49(1-3):97-108.
Guo X,Wang L,Cui D,et al.2012.Differential expression of the Toll-like receptor pathway and related genes of chicken bursa after experimental infection with infectious bursa disease virus[J].Archives of Virology,157(11):2189-2199.
Kawai T,Akira S.2011.Toll-like receptors and their crosstalk with other innate receptors in infection and immunity[J].Immunity,34(5):637-650.
Lee C C,Wu C C,Lin T L.2014.Chicken melanoma differentiation-associated gene 5(MDA5)recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity[J].Archives of Virology,159(7):1671-1686.
Müller H,Islam M R,Raue R,2003.Research on infectious bursal disease-the past,the present and the future[J].Veterinary Microbiology,97(1-2):153-165.
Maddaly R,Pai G,Balaji S,et al.2010.Receptors and signaling mechanisms for B-lymphocyte activation,proliferation and differentiation-insights from both in vivo and in vitro approaches[J].FEBS Letters,584(24):4883-4894.
Moresco E M,Lavine D,Beutler B,2011.Toll-like receptors[J].Current Biology,21(13):R488-R493.
Ou C,Wang Q,Yu Y,et al.2017.Chemokine receptor CCR5and CXCR4 might influence virus replication during IBDV infection[J].Microbial Pathogenesis,107:122-128.
Rauf A,Khatri M,Murgia M V,et al.2012.Fas/FasL and perforin-granzyme pathways mediated T cell cytotoxic responses in infectious bursal disease virus infected chickens[J].Results in Immunology,2:112-119.
Wang B,Duan X,Fu M,et al.2018.The association of ribosomal protein L18(RPL18)with infectious bursal disease virus viral protein VP3 enhances viral replication[J].Virus Research,245:69-79.
Ye C J,Jia L,Sun Y,et al.2014.Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5[J].Journal of Virology,88(19):11154-11165.