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。

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.