Bhv5-miR-B10对BoHV-5沿嗅神经通路侵入大脑的调控作用研究

Bovine herpesvirus 5 (BoHV-5) is one of the important member of alphaherpesvirus subfamily. BoHV-5 is genetically and antigenetically related to bovine herpesvirus 1 (BoHV-1). However, BoHV-5 causes fetal meningitis in calves, and is distiguished by neurovirulence with BoHV-1, which is the etiological agent of infectious bovine rhinotracheitis in cattle and abortion in pregnant cows. Previously we have reported that BoHV-5 has the capacity to encode microRNAs (miRNAs) by its genome, and bhv5-miR-B10 was most abundently expressed in infected epithelial cells, but the role of this miRNA in the neuropathogenecity of BoHV-5 is not known. In this project we aim to investigate the function of this miRNA by construction of anti-miRNA reporter system and a mutant virus expressing miRNA decoy by homologous recombination. The expression of miRNA and target genes will be detected by quantitative PCR, northern blot and western blot assays. Then the virulence of the mutant virus will be tested by animal experiments in rabbits and calves inoculated through nostrils, and clinical symptoms will be compared. Rabbits infected with mutant and wild type BoHV-5 will be subject to magnetic resonance imaging after anaesthesia. The amount of viral antigen and infection loci in brain will be compared by virus isolation and immunohistochemistry. And finally the in vivo expression of bhv5-miR-B10 and its targets will be analysed. The purpose of this study is to reveal the neuropathogenecity of BoHV-5 based on noncoding RNA, and the result of this study might provide clues to the development of novo vaccine as well as therapeutic drugs for alphaherpesvirus-associated diseases.

牛疱疹病毒5型（BoHV-5）引起犊牛脑炎和急性死亡，对大脑的侵袭性是BoHV-5区别于其它牛源α疱疹病毒的主要特征。在前期研究中我们证实了BoHV-5具有编码microRNA（miRNA）的能力，其中仅bhv5-miR-B10呈高丰度表达，但该miRNA在BoHV-5神经致病性中的作用不清楚。本课题拟构建表达anti-miRNA的报告系统，用同源重组方法构建突变病毒，以"miRNA诱饵"策略抑制bhv5-miR-B10的表达，通过定量PCR、northern blot、western blot等检测miRNA的表达水平，及其对靶基因的调控作用；进而通过鼻窦腔接种比较试验动物的临床症状，用免疫组织化学、核磁共振、病毒分离试验等研究miRNA对病毒沿嗅神经通路侵入大脑的作用。本课题研究结果为从非编码基因角度研究BoHV-5的神经致病性提供实验证据，对新型疫苗或药物的研制也具有重要的指导意义。

牛疱疹病毒5型（Bovine herpesvirus 5，BoHV-5）具有嗜神经性特征。BoHV-5能够编码16条成熟的miRNA，其中miR-10 是表达量最大的。本课题通过体内外试验研究其功能，具体如下。.1.miR-10靶基因的预测及验证:首先经过生物信息学预测软件（miRanda）分析，经分析比较挑出UL23、BICP22、UL28、UL37、UL39和Us3。继而用双荧光素酶报告系统进行验证。结果表明，UL39和Us3是miR-10的靶基因。.2.过量表达miR-10-5p对病毒增殖的影响：我们将miR-10-5p转染MDBK细胞，接种BoHV-5。结果表明：过表达miR-10-5p后病毒的滴度变化不大，说明miR-10-5p的过量表达对病毒在上皮细胞中的复制与增殖没有影响。.3.过量表达miR -10-5p后病毒对上皮细胞凋亡的影响：由于Us3在病毒感染细胞诱导凋亡的过程中有重要作用，我们将miR-10-5p转染MDBK细胞，western blot和流式细胞仪检测结果显示过量表达miR-10-5p后，能够上调感染细胞中caspase-3的表达量。表明miR-10-5p能够促进病毒感染诱导的上皮细胞凋亡。.4.过量表达miR -10-5p后病毒对神经细胞凋亡的影响：由于BoHV-5具有嗜神经性，我们进一步将miR-10-5p转染N2a细胞，同时接种BoHV-5野毒，western blot和流式细胞仪检测结果显示miR-10-5p能够促进BoHV-5感染神经细胞的凋亡。.5.含miRNA诱饵系统的质粒的构建：我们把Tet-on调控系统与能够吸附特异miRNA的诱饵元件（TuD）串联起来，用Tet-on调控系统调控EGFP基因表达，从而实现对TuD元件的可控表达。.6.含miRNA诱饵系统的重组病毒的构建：将含有上下游同源臂的诱饵系统中间转移质粒和BoHV-5基因组以及辅助转移质粒共转染，挑取含有荧光的重组病毒。.7.重组病毒miRNA诱饵系统功能的验证：我们一方面通过生物学特性的变化直接反映诱饵系统的效果；另一方面是通过荧光定量PCR和western blot对knock down相应miRNA后的效果进行定量检测。最后将重组病毒接种家兔，比较了多西环素诱导后家兔神经症状的变化。.8. 发表SCI文章2篇，获授权专利1项，培养研究生4名。