伪狂犬病毒编码的miRNA簇在其感染过程中的表达谱及功能研究

Pseudorabies virus (PRV), an important member of alpha herpesvirus family, is characterized by neurotropic property and latent infection in peripheral ganglia, and causes huge economic loss worldwide. Previously we reported that a microRNA (miRNA) cluster, which encompasses 11 mature miRNAs, was generated from the intron of large latency transcript (LLT) gene of PRV wild type (wt) in infected epithelial cell line. However, the expression profile and function of the miRNA cluster in vivo are not known. Eventhough protein-coding genes play important role in the pathogenesis of alpha herpesvirus, many issues are not addressed. For example, VP16 protein participates the transcription of immediate early genes in acute infection; However,without VP16 protein, how does alpha herpesviruses reactivate from latency, and how does it maintain latency in peripheral ganglia, etc? Our hypothesis is that PRV miRNA cluster plays important role during infection. In this project, a mutant virus, in which LLT intron is deleted, will be used to infect piglets to study whether or not it could establish latent infection and whether or not it could be reactivated. At acute infection, latency, and reactivation stage, trigeminal ganglia (TG) from PRV wt- and mutant virus-infected pigs will be collected and total RNA will be investigated by high-throughput sequencing. Ago CLIP-seq and 2DE-MS will be utilized to explore miRNAs and target genes. Then the mutant virus and wt will be used to infect PK-15 cells and neural cell line N2a. In addition, LLT intron and individual miRNA will be cloned and expressed in PK-15 cells and / or N2a, and checked by northern blot、western blot、real-time PCR、flow cytometry and dual-luciferase reporter gene assay. Combinding knowledge and methods of virology, neurology, immunology, and bioinformatics, this project is aimed to determine the expression profile and function of PRV-encoded miRNA in vivo, and help to understand the mechanism how alpha herpesviruses establish and maintain latent infection, and reactivate from latent infection.

伪狂犬病毒（PRV）是α疱疹病毒的重要成员，其主要特征为嗜神经性及潜伏感染。前期体外试验显示，PRV的潜伏相关基因（LLT）的内含子编码11个成熟miRNA并聚集成簇；我们推测该miRNA簇在PRV建立、维持潜伏感染和再激活中发挥调节作用，进而通过构建缺失LLT内含子的突变株，研究突变株能否建立潜伏感染及能否再激活；感染仔猪，在急性期、潜伏期及再激活期分别取三叉神经节，用高通量测序法确定miRNA表达谱，用Ago CLIP-seq和2DE-MS技术发掘miRNA及其靶基因；克隆LLT的内含子、miRNA及靶基因，用northern blot、western blot、FCM、定量PCR及双荧光素酶报告试验等，研究miRNA与病毒靶基因及宿主靶基因的互作；综合病毒学、神经生物学、免疫学及生物信息学等知识，从miRNA簇的角度揭示PRV神经致病性机制，验证上述假设，并为其它病毒的研究提供借鉴。

伪狂犬病毒（ Pseudorabies virus, PRV）是α疱疹病毒亚科的成员之一，具有嗜神经性和潜伏感染性。本课题从整体角度分析intron的生物学功能。主要研究结果如下: .1.伪狂犬病毒LLT基因内含子缺失突变株（PRV LLTΔintron）的构建。.利用同源重组的原理获得intron缺失突变株。空斑大小比较发现重组病毒形成的空斑与野毒显著变小。PRV-∆Intron-EGFP增殖能力显著低于野毒。PRV-∆Intron-EGFP诱导MDBK细胞凋亡的能力显著增强。Intron可以负调控IE180与EP0的表达。.2.LLTΔintron对PRV毒力的影响.选用5-7周龄的Balb/c小鼠进行腹股沟皮下攻毒，结果表明Intron缺失后病毒毒力显著下降。并且与野毒相比，相同剂量的重组病毒引起小鼠死亡的时间显著延迟，说明Intron与PRV毒力密切相关。.Intron缺失后，PRV-ΔIntron-EGFP在仔猪中仍能建立潜伏感染，但其引起的感染仔猪各时期的临床症状水平、排毒水平、基因组拷贝数水平、宿主免疫应答水平都比PRV Ea低。因此，我们认为，PRV LLT Intron具有增强PRV在宿主中的致病力的能力。.3. LLT intron的表达及功能研究.通过Northern Blot、茎环PCR及poly(A)加尾法等多种方法检测miRNAs的体外表达情况。Intron能提高免疫小鼠的抗病毒能力。因此，我们认为，Intron可以作为一个新的毒力因子影响宿主的抗病毒反应。.4. PRV miRNA簇的重要靶基因研究.PRV编码的prv-mir-LLT7可以调控猪的MHC Ⅰ基因和病毒的VP16 基因的表达。该病毒编码的prv-mir-LLT1、prv-mir-LLT9 和 prv-mir-LLT11可以下调IE180基因的表达。我们推测α疱疹病毒进化了一个精妙和经济的维持合适感染状态策略。.5.PRV变异株新型疫苗研究.以当前流行的PRV变异株（CH/HNSMX/2012株）为亲本，通过基因工程技术构建gI、gE、TK三基因缺失毒株rSMXΔgI/gEΔTK，通过一系列动物试验，研究了该毒株的安全性和免疫效力；为PRV变异株弱毒疫苗的研发与应用奠定了基础。.6. 发表SCI文章4篇，申请专利2项。培养研究生8名。