调节Ⅱ型鲤疱疹病毒复制的病毒MicroRNA分子的鉴定与功能分析

MicroRNAs(miRNAs) are increasingly being recognized as major regulators of gene expression in many organisms, including viruses. Among viruses, members of the family Herpesviridae account for the majority of the well-known miRNA-encoding virus. The discovery of miRNAs in herpesvirus indicates that viruses have evolved to exploit RNA silencing mechanism for regulation of both viral and host genes. The miRNAs involved in regulating the viral gene expression and regulatory immune factor of host cell are not identified in Cyprinid herpesvirus 2 (CyHV-2). To answer the question, bioinformatics analysis and high-throughput sequencing are used at first in this project to screen miRNAs with significantly differential expression following CyHV-2 infection in three different ambient temperatures. Together with the sequencing results of cDNA library, target genes silencing by CyHV-2 miRNAs will be predicted and the differential expression of target genes will be verified. Secondly，their spatial and temporal expression and regulation of viral genes’ expression and the host immune regulatory factor will be real-time quantitative analysis. Finally, overexpression and inhibition of miRNAs will used to clarify the key miRNAs’ function for virus replication in vivo and vitro. The completion of this project will help to understand the regulatory mechanism of CyHV-2 miRNAs during the process Of viral replication. Meanwhile, it will provide a theoretical basis for revealing the mechanism of viral infection with host, and provide a theoretical reference for the prevention and control strategy of cyprinid herpesvirus diseases.

MicroRNA（miRNA）是一类重要的非编码小RNA，通过对靶基因的转录后抑制来调节基因的翻译。疱疹病毒编码的miRNA利用沉默机制可同时调控病毒和宿主的基因。II型鲤疱疹病毒（CyHV-2）编码的miRNA对其病毒基因的表达和宿主免疫调控因子的调节作用机制尚不清楚。为此，本项目利用生物信息学和高通量测序技术，选择在三个不同环境温度下，筛选CyHV-2病毒感染过程中显著差异表达的miRNAs；联合cDNA文库测序结果预测并验证miRNAs的靶基因（病毒或宿主）；定量分析miRNAs的时空表达，鉴定miRNA在体内、体外与靶标基因的作用，阐明其调节病毒基因表达和调控宿主靶基因的功能；本项目的顺利完成，有助于了解CyHV-2编码的miRNAs对病毒复制的分子调控机制，为揭示鲤疱疹病毒和宿主间的作用机制提供理论基础，为鱼类鲤科疱疹病毒性疾病的防控策略提供理论参考。

MicroRNA是一类重要的非编码小RNA，通过对靶基因的转录后抑制来调节基因的翻译。疱疹病毒编码的miRNA利用其沉默机制可同时调控病毒和宿主的基因。II型鲤疱疹病毒（CyHV-2）编码的miRNA对其病毒基因的表达和宿主免疫调控因子的调节作用机制尚不清楚。为此，本项目利用生物信息学和高通量测序技术，筛选出CyHV-2病毒感染异育银鲫脾脏组织过程中显著差异表达的miRNAs；联合cDNA文库测序结果预测并验证miRNAs的靶基因（病毒或宿主）。验证miRNA有多个靶基因，这些靶基因参与多种信号途径，如Jak-STAT信号通路、B细胞受体信号通路、凋亡和MAPK信号通路等。研究证实CyHV-2编码miRNA的病毒基因组序列上主要在两个区域成簇状分布，其中一个位于ORF42附近，包括miR-C3，miR-C4，和miR-C5，另外一个区域位于ORF114附近，包括miR-C1，miR-C15，和miR-C16。研究证明miR-C4能与ORF4、IRF3、RBMX和PIN1基因的3’ UTR 区域直接结合，而IRF3、RBMX和PIN1是RIG-I信号通路上的重要基因，因此推测CyHV-2通过编码miRNA参与调控RIG-I信号通路，从而逃避宿主的免疫反应。为进一步探miR-C12在病毒侵染过程中的调控机制，将miR-C12 mimics，miR-C12 inhibitor，CASP-siRNA-1和相应的对照物转染细胞，研究结果证实转染miR-C12 inhibitor抑制CyHV-2的复制。由此证实CyHV-2通过编码miR-C12抑制病毒诱导的细胞凋亡，进而促进病毒在细胞内复制。本项目的顺利完成，解析了CyHV-2编码的miRNAs对病毒复制的分子调控机制，为揭示鲤疱疹病毒和宿主间的作用机制提供理论基础，为鱼类鲤科疱疹病毒性疾病的防控策略提供理论参考。