【作者】 宁蓬勃；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2013， 博士

【摘要】 猪瘟（CSF）是由猪瘟病毒（CSFV）引起的猪的一种高度接触性传染病。在过去的几十年中，我国通过采取兔化弱毒疫苗免疫接种与扑杀相结合等措施，有效地控制了猪瘟的流行，但近年来我国猪瘟疫情又有所抬头，尤其是慢性猪瘟和非典型猪瘟成为该病的主要发生形式，给猪瘟防控工作造成了新的困难。虽然近年来对CSFV致病机理的研究取得一定进展，但之前的研究工作中，主要是对全病毒或病毒的某个或某些基因对宿主细胞的一些致病作用及其机理进行研究，而缺乏对CSFV感染后宿主细胞转录水平调控应答的系统分析，不清楚哪些应答才是CSFV强毒株引起宿主细胞发生的致病性及病理性改变的关键所在。为此，本论文开展猪瘟病毒石门株与猪瘟病毒C株感染猪静脉血管内皮细胞（SUVEC）及巨噬细胞后宿主细胞基因表达的差异性研究，并通过对猪瘟病毒感染后宿主细胞mRNA转录组水平的基因调控应答特征分析，为阐明猪瘟病毒的致病机理提供有价值的科学依据。研究工作取得了以下结果：（1）建立了区分猪瘟病毒石门株与猪瘟病毒C株的高分辨率熔解曲线（HRM）检测方法。与以往PCR技术比较，该方法具有单管操作、无须设计特异性探针等优势，既可以实现对猪瘟病毒石门株与猪瘟病毒C株的单独定量检测，也可以实现对猪瘟病毒石门株与C株的鉴别检测，为致病机理研究中获得单纯病毒感染或无病毒感染的细胞样本提供了检测手段，也为猪瘟流行病学调查的大量临床样本筛查提供了可选择的检测方法。（2）应用高通量测序技术，对猪瘟病毒石门株感染24h与未感染病毒的SUVEC基因在转录水平的表达差异开展了比较研究，数据分析结果说明，猪瘟病毒石门株可能利用改变宿主细胞复制周期、抗凋亡、抗炎症等多种策略实现自身的复制增殖；并致使宿主细胞未能对之形成有效清除，最终造成猪体产生多组织器官的病变。进一步的分析显示，PDIA3、AXL基因在猪瘟病毒石门株感染早期发生上调，而NAMPT、PDGF基因在猪瘟病毒石门株感染早期发生下调，提示这些基因对于猪瘟病毒石门株的感染具有重要意义。（3）对猪瘟病毒石门株、C株感染SUVEC和未感染病毒对照组进行DGE检测数据库两两比较，采用GO和KEGG富集显著性分析体系，分析了猪瘟病毒石门株与C株感染SUVEC后72h转录组的应答差别，在mRNA水平提出了猪瘟病毒石门株感染猪血管内皮细胞的可能机制，即病毒通过对RNA剪接机制、蛋白合成与降解机制的调控，影响细胞正常的功能，特别是对细胞骨架结构、细胞黏附、细胞周期产生影响，从而实现病毒感染复制。GO基因功能分析和聚类分析结果显示，猪瘟病毒石门株感染SUVEC后72h，SRPX2、SOD2、HMGB1、TYMS基因转录受到抑制，而VEGFC和CAV1基因的表达发生上调。初步揭示了猪瘟病毒石门株引发急性猪瘟的分子机制。（4）DGE分析显示，VEGFC基因是猪瘟病毒石门株感染后SUVEC特异上调表达的基因，进一步验证猪瘟病毒石门株的感染确能引起VEGF-C在mRNA转录及蛋白合成水平的增高。初步揭示了猪瘟病毒石门株引起血管通透性增加而导致广泛性出血的发病机制。（5）采用DGE技术对猪瘟病毒石门株、猪瘟病毒C株感染巨噬细胞和未感染病毒对照组进行GO和KEGG分析，结果显示p53信号通路在猪瘟病毒石门株感染巨噬细胞的过程中具有重要作用。试验表明，猪瘟病毒石门株感染巨噬细胞后，促进了phospho-p53(Ser15)蛋白的磷酸化进程，p21蛋白表达升高，14-3-3蛋白表达降低。研究结果提示，猪瘟病毒石门株的感染激活巨噬细胞p53蛋白，上调p21蛋白并抑制14-3-3蛋白进行双向调控，最终导致细胞周期阻滞，为实现病毒自身复制、造成持续性感染创造了可能。更多还原

【Abstract】 Classical swine fever (CSF) is a highly contagious infection of swine, caused by classicalswine fever virus (CSFV). Over the past few decades, the epidemic of CSF has beeneffectively controlled by takingC-strain vaccine and culling. But CSF begins to be increasingagain in our countryin recent years, especially being a chronic and atypical CSF, which bringsgreat difficult to prevention and control work. Although CSFV pathogenesis research hasmade some progress, previous research work mainly aimed on the whole virus or viral geneswhich could produce pathogenic to some genes of host cell, and lacked systems analysis ontranscription regulatory response of host cell. Meanwhile, it is unclear what response iscaused by virulent strain of CSFV. For this purpose, this paper carried out study ondifferentially expressed genes in swine umbilical vein endothelial cell (SUVEC) andmacrophages infected by CSFV Shimen strain and C strain, which could find out the generegulation characteristic on transcriptome of host cell in response to CSFV infection, furtherto provide scientific data and new starting point for the study on molecular mechanisms ofacute CSF and persistent infection of CSFV.This work has obtained the following results.(1) To the best of our knowledge, this work offered a first report regarding the successfulapplication of high resolution melting (HRM) analysis for simultaneous, one-step detectionand differentiation of CSFV C and Shimen strains. As a single-tube, probe-free, closeddetection system being superior to previous PCR technology, this method not only detects butalso identify CSFV C and Shimen strains. It could provide a detection method to obtain singlevirus infection or no virus infection cell sample for pathogenesis studies and also to screenlarge number of clinical samples in CSF epidemiological study of CSFV.(2) By the application of digital gene expression tag profiling (DGE), a kind ofhigh-throughput sequencing technology, a comparative study first was carried out thatdifferences of the gene expression of SUVEC were showed on CSFV Shimen strain infection24hours and the uninfected group. Data analysis results indicated that CSFV Shimen strainbrought out anti-apoptotic, anti-inflammatory and altering cell cycle to achieve their ownproliferation. So, host cell could be failed to effectively kill virus, and ultimately resulted in damages of tissues and organs. Research further identified PDIA3, AXL were up-regulatedand PDGF, NAMPT was down-regulated in early stages of CSFV Shimen infection,suggesting that these genes were involved in the acute infection mechanism caused byvirulent strains of CSFV.(3)Three groups of DGE detection database, CSFV Shimen strain infection, CSFV ShimenC strain infection, uninfected control group, were comparatively analyzed each other to findresponse differences of SUVEC presented at the mRNA level by using GO and KEGGenrichment analysis. The analysis results showed a possible mechanism of CSFV Shimenstrain72h infection to SUVEC in which virus altered the mechanism of RNA splicing, proteinsynthesis and degradation, and regulated normal cell function, especially in cytoskeleton, celladhesion and cell cycle, in order to achieve virus replication. GO gene function analysis andcluster analysis showed that SRPX2, SOD2, HMGB1and TYMS transcription were inhibitedand VEGFC and CAV1were unregulated after CSFV Shimen strain72h infection in SUVEC.The study provides a new research idea for CSFV to illustrate the pathogenesis of acute CSFin the future.(4) In this work, DGE detection and analysis techniques identified VEGFC expression waseffected after CSFV Shimen strain infection in SUVEC. For validating this result, molecularbiology tests were carried out on mRNA transcription and protein synthesis of VEGFC. Thefinal result showed VEGFC were significantly up-regulated as a result of CSFV Shimen straininfection. The result could give a better understanding for the pathogenesis of the virulentstrain of CSFV.(5) Another three groups of DGE detection database from the macrophage, CSFV Shimenstrain infection, CSFV Shimen C strain infection, uninfected control group, werecomparatively analyzed each other to find different response to CSFV infection by using GOand KEGG enrichment analysis. The result showed that p53signaling pathway played animportant role on the infection of CSFV Shimen strain in macrophages. Further tests showedthat the CSFV Shimen strain infection in macrophages promoted phospho-p53(Ser15) proteinphosphorylation process, increased p21protein expression, and decreased14-3-3proteinexpression. CSFV Shimen strain could activate p53signaling pathway to arrest cell cycle ofmacrophages for achieving their replication, eventually resulting in persistent infection ofCSFV in pigs.更多还原