【作者】 赵丽丽；

【导师】 夏咸柱；

【作者基本信息】 吉林大学 ， 预防兽医学， 2013， 硕士

【摘要】 狂犬病病毒（RABV）为不分节段、单链、反义的RNA病毒，属于弹状病毒科，狂犬病毒属成员，具有典型的嗜神经性。狂犬病病毒感染能引发急性致死性脑脊髓炎。据世界卫生组织（WHO）统计，全球每年死于狂犬病的人数超过55,000人，其中亚洲占56%，非洲占44%。目前，中国的狂犬病发病率仅次于印度，位居世界第二位。严重威胁人类的生命安全，因此，狂犬病的防控形势仍很严峻。尽管狂犬病是一种古老的人兽共患病，RABV也经过数百年来的研究，但是RABV确切的致病机理仍不清楚。从细胞和分子水平研究宿主和病毒相互作用是揭示病毒致病机理的重要途径。近来研究表明， RNA干扰（RNAi）已经成为研究抗病毒固有免疫反应的重要组成部分。miRNA分子的高效表达也在调节宿主-病毒相互作用中发挥重要的作用。但是细胞内miRNA具体是如何发挥机体抗病毒作用的机制仍需进一步研究。大量的研究报告显示，细胞内miRNA能够影响病毒的趋向性、毒力和致病性。细胞内miRNA还能从实质上影响病毒的进化，调节病毒对组织的嗜性等功能。这些miRNA在宿主与病毒相互作用网络中起到至关重要的作用。在RABV与宿主或细胞的相互作用方面，尤其是对RABV感染导致宿主miRNA表达谱变化以及生物学功能改变的了解还存在一定的局限性。miRNA芯片技术可高通量检测数以万计miRNA的表达水平，借助生物信息学方法预测miRNA的靶基因，并对其进行基因表达模式分析、靶基因功能聚类、靶基因作用路径和网络分析以及qRT-PCR和DNA芯片结果的相互验证等，从而高效准确的获得病毒感染过程中的宿主反应的miRNA及其相关信息。表达差异miRNA的靶基因参与多种免疫反应信号通路。miRNA表达谱和mRNA表达谱相互协调，初步阐明了RANB感染后的调节机制，并对研究RABV的致病机理具有重要的意义。本实验中，我们检测了感染RABV街毒株FJDRV和弱毒株ERA的鼠脑脑组织中的miRNA表达谱，并对表达水平显著变化的miRNA进行靶点预测和功能富集。最终，我们通过基因芯片和qPCR验证差异miRNA相关的预测靶点在这些通路中的表达水平。功能性富集显示，这些miRNA的靶点在RABV感染过程中起到重要的作用。本研究主要分为三部分：第一部分：狂犬病病毒弱毒株（ERA）感染小鼠中枢神经系统microRNA表达谱变化。本研究利用microRNA芯片检测了RABV弱毒株感染小鼠后脑组织中的microRNA表达谱变化。结果显示有10个miRNA：miR-1894-5p, miR-290-3p,miR-1901, miR-207, miR-1896, miR-715, miR-3470b, miR-146b, miR-203,miR-770-5p表达水平显著上调，6个miRNA: miR-200a, miR-200b, miR-200c,miR-182, miR-183, miR-429表达水平显著下调。这16个表达差异的miRNA经miRanda、 TargetScan和MicroCosm三种预测软件预测6200个靶基因。利用DAVID进行Gene ontology（GO）分析和KEGG Pathways分析。结果表明差异基因参与JAK-STAT信号转导通路、抗原加工与提呈、泛素介导的蛋白降解和补体活化途径等。本实验提示，在RABV感染过程中，细胞内的miRNA能直接或者间接的调节下游的基因表达。第二部分：狂犬病病毒街毒株（FJDRV）感染小鼠中枢神经系统microRNA表达谱变化。结果显示，有9个miRNA表达水平发生显著的上调，分别是：miR-691、miR-377、miR-1935、miR-190、miR-1902、miR-135α*、miR-203、miR-2138和miR-290-5p。然而只有miR-145表达水平发生显著的下调。表达差异miRNA调节的靶基因经过TargetScan、MicroCosm和miRanda三种数据库进行预测，共预测靶基因数为3038。利用DAVID进行Gene ontology（GO）分析和KEGG Pathways分析。结果表明靶基因参与了多条免疫相关信号通路。如Jak-ATAT信号通路、MAPK信号通路、Fc-γ介导的吞噬作用以及趋化因子和细胞因子受体的相互作用等。本实验表明miRNAs在病毒感染过程中起到重要的作用，细胞内miRNAs表达水平的改变可能与病毒的毒力和致病性有关。第三部分：强弱毒株感染鼠脑microRNA表达谱的比较分析。FJDRV和ERA具有不同的嗜神经性，而且致病力具有显著差异。RABV感染导致一系列miRNA和mRNA表达谱发生变化，并且发现街毒和固定毒诱导截然不同的miRNA和mRNA表达谱。结果表明，小鼠抗病毒免疫功能可能通过ERA感染所诱导的特异性miRNA介导的免疫应答调控网络得到优化，然而，FJDRV却不能。通过分析不同毒株RABV感染后中枢神经系统microRNA表达谱变化,可以加深对RABV致病机理的理解。本研究首次研究RABV颅内感染能引发小鼠脑组织microRNA表达谱变化。miRNA作为机体生理或病理过程的重要调控分子，可能参与了RABV-宿主的相互作用。本研究结果可为探索狂犬病的预防和治疗策略奠定基础。更多还原

【Abstract】 The rabies virus (RABV), a non-segmented, single-stranded antisense RNA virusof the family Rhabdoviridae, genus Lyssavirus, is a highly neurotropic virus that cancause a fatal infection in central nervous systems (CNS) of warm-blooded animals.The World Health Organization (WHO) has estimated that the number of humandeaths from rabies was55,000annually worldwide, with56%of the death in Asia and44%in Africa. At present, China has the second-highest number of human rabies caseall over the world, after India. Although significant advances have been made in rabiesprevention and control, rabies remains a major threat to public health.Rabies is an ancient Zoonoses which experienced a long history of severalthousand years, and the rabies virus has been studied over hundred years, but theexact pathogenic mechanism underlying rabies virus infection remains unknown. Inorder to understand the viral pathogenic mechanism, study the host-virus interactionat the cellular and molecular level has been seen as an important way. Recent reportsindicate that RNA interference (RNAi) represents a vital component of the innateanti-viral immune response in plants and invertebrate animals. it serves as a hostgene-regulation mechanism that is triggered by the expression of highly structuredmiRNA molecules.However, role of cellular miRNAs in the defense against viralinfection in mammalian organisms has thus far remained elusive. A large number ofresearch reports have shown that the intracellular miRNA can affect tropism,virulence and pathogenicity of the virus. It has been proposed that cellular miRNAsmay have a substantial efect on viral evolution and have the potential to regulate thetissue tropism of viruses. Hence, miRNAs play a crucial role in the host-virusinteraction networks. Understanding on the interaction between host and RABV,especially on changes in miRNA expression profile and biological functions of hostinfected with RABV is very limited.miRNA microarray technology，in combination with bioinformatics to predictmiRNA target genes, has proved to be a very efficient high-throughput detection toestablish miRNA expression pattern, to complete functional clustering, pathway and network enrichment of target genes of differentially expressed miRNA, mutualauthentication between qRT-PCR and DNA microarray results and so on, and it offersan efficient and accurate access to uncover the host response to viral infection processthe miRNA and its related information. Understanding the miRNA expression profileof host upon RABV infection at pan-genomic level, which is very important toexplain the pathogenic mechanisms of rabies virus infection.In the current study, we created an expression profile of cellular miRNAs in theCNS of mice infected with different RABV strains and performed target predictionand functional enrichment of miRNAs found to be differentially expressed. Finally,we performed gene microarray analysis and qRT-PCR to verify the expression levelsof the predicted targets of the modulated miRNAs in these pathways. SeveralmiRNAs were modulated in RABV-infected mouse brains. Functional enrichmentrevealed that many of the predicted targets of these miRNAs are likely to play keyroles in immune response. This study is mainly divided into three parts:Part1. Changes in microRNA expression profile in mouse brains infected with ERArabies virus strain. To determine changes in miRNA expression in mouse brains inresponse to RABV infection, we evaluated miRNA expression profiles that tenmiRNAs, miR-1894-5p, miR-290-3p, miR-1901, miR-207, miR-1896, miR-715,miR-3470b, miR-146b*, miR-203, and miR-770-5p, were found to be significantlyup-regulated, and six miRNAs, miR-200a,miR-200b, miR-200c, miR-182, miR-183,and miR-429, were significantly down-regulated upon RABV infection. For thesesixteen differentially expressed miRNAs, all three systems-----miRand, TargetScanand MicroCosm predicted6200target genes. Gene ontology and KEGG Pathwaysanalyses were performed by DAVID database. Network analysis of the differentiallyexpressed miRNAs in response to RABV were performed by Ingenuity PathwayAnalysis and the results indicated that the target genes of differentially expressedmiRNAs are mainly involved in JAK-STAT signaling pathway, antigen processingand presentation, ubiquitin mediated proteolysis and complement cascades. Thisexperiment suggested that the cellular miRNA can directly or indirectly regulatedownstream gene expression during RABV infection.Part2. Changes in microRNA expression profile in mouse brains infected with streetrabies virus strain. To determine changes in miRNA expression in mouse brains inresponse to RABV infection, we evaluated miRNA expression profiles that nine miRNAs, miR-691, miR-377, miR-1935, miR-190, miR-1902, miR-135a*, miR-203,miR-2138, and miR-290-5p, were found to be significantly up-regulated. However,only one miRNAs, miR-145, was found to be down-regulated upon RABV infection.This indicates that host miRNAs were modulated in the CNS upon infection withstreet rabies virus. For these nine differentially expressed miRNAs, all three systems-----miRand, TargetScan and MicroCosm predicted3038target genes. Gene ontologyand KEGG Pathways analyses were performed by DAVID database.The predictedtarget genes miRNA were found to be involved in immune-related pathways, such asthe Jak-STAT signaling pathway, MAPK signaling pathway, Fc gamma R-mediatedphagocytosis and cytokine-cytokine receptor interactions. Present experiment showedthat miRNAs play an important role in the process of rabies virus infection, miRNAsexpression levels of cellular changes may be associated with virulence andpathogenicity.Part3. Comparative Analysis in microRNA expression profile in mouse brainsinfected with of different strains. In this study, based on comprehensive examinationof miRNA expression profiles from brains infected with FJDRV, a acute street rabiesvirus, and ERA, a non-lethal laboratory-adopted rabies virus, relative to thenon-infected mice, we identified a group of miRNAs that were differentiallyexpressed. To our knowledge, this is the first report on cellular miRNA in response toRABV infections. We found that RABV infection led to alteration of cellular miRNAsin mice and intriguingly, distinct expression pattern of host miRNAs were observed inmice infected with different strains of RABV.Analysis of microRNA expression changes in different strains of RABVinfection of the central nervous system, deepen the understanding of RABVpathogenesis. This is the first study the RABV intracranial infection can lead to braintissue of mice microRNA expression changes. miRNAs regarded as importantregulatory molecules of the host physiological or pathological processes may beinvolved in RABV-host interactions. The results of this study can lay the foundationfor exploring rabies prevention and treatment strategies.更多还原