鱼淋巴囊肿病毒中国株目的miRNAs的鉴定及其调控作用

Viral miRNAs play important regulation roles in the process of virus infection. Functional resolution of miRNAs of lymphocystis disease virus (LCDV) could contribute to the further illumination of the pathogenic mechanism of LCDV. Furthermore, candidate pre-miRNAs encoded by lymphocystis disease virus China (LCDV-C) were predicted in bioinformatics methods by the applicant. In this project, LCDV-C was as a model virus to screen mature miRNAs encoded by LCDV-C in bioinformatics prediction, solexa sequencing and RT-PCR methods. The expression characteristics and timing of viral miRNAs were analyzed in Northern blot and RT-PCR methods. Then virus-specific miRNAs were identified. Target genes of virus-specific miRNAs were identified in bioinformatics prediction and the dual-luciferase reporter gene system. The regulation of virus-specific miRNAs to the expression of target genes after the up and down regulation of virus-specific miRNAs were revealed in qRT-PCR and Western blot methods. The regulation of virus-specific miRNAs to the viral replication and the growth of host cells after the silence and overexpression of virus-specific miRNAs were interpreted in qRT-PCR, Western blot and cell proliferation assay, etc. Regulation roles of virus-specific miRNAs in the pathogenesis of LCDV-C were illustrated. This study would lay the foundation for the elucidation of the pathogenic mechanism of LCDV in a new dimension.

病毒miRNAs在病毒感染过程中起着重要的调控作用。鱼淋巴囊肿病毒(LCDV) miRNAs的功能解析有助于LCDV致病机制的深入阐明，且申请人预测到鱼淋巴囊肿病毒中国株(LCDV-C)候选pre-miRNAs。本项目拟以LCDV-C为模式病毒，通过生物信息学分析、深度测序及RT-PCR等筛选LCDV-C成熟miRNAs，采用Northern blot和qRT-PCR等分析miRNAs的表达特性和时序性，进而鉴定目的miRNAs；应用生物信息学和双荧光素酶报告基因系统鉴定目的miRNAs的靶基因；利用qRT-PCR和Western blot揭示上调和下调目的miRNAs对其靶基因表达的调控，通过qRT-PCR、Western blot和细胞增殖检测等诠释沉默和过表达目的miRNAs对病毒复制和宿主细胞生长的调控，解析目的miRNAs的调控作用，为在新的层面阐明LCDV的致病机制奠定基础。

鱼淋巴囊肿病毒可以引起全球140种以上的鱼类发生淋巴囊肿病，对鱼类养殖造成了严重的危害。但淋巴囊肿病的防治及鱼淋巴囊肿病毒的致病机制仍未得以解析。且目前全球已发现鱼类淋巴囊肿病毒的多个基因型，病毒的遗传多样性更是加重了疾病防治的难度。有大量研究发现同一物种的不同分离株间，病毒miRNAs具有较高的保守性，且病毒miRNAs在病毒感染过程中起着重要的调控作用。因此本研究以鱼淋巴囊肿病毒中国株为模式病毒，应用生物信息学方法预测到鱼淋巴囊肿病毒中国株编码107个miRNA前体，编码428个成熟miRNAs。对鱼淋巴囊肿病毒中国株在细胞系GCO和FG中的感染特性进行了研究，鱼淋巴囊肿病毒中国株在GCO中可以产生明显的CPE现象，并通过PCR检测和电镜切片验证了鱼淋巴囊肿病毒中国株在GCO中成功增殖。然后，通过GCO细胞富集了大量病毒液，制备了8个时间点的24个样品，进行深度测序。通过深度测序成功获得474个已知的miRNAs，195个未知的miRNAs，且应用TPM方法筛选出差异表达的已知miRNAs 514个和未知miRNAs 99个，发现已知的差异表达的514个miRNAs参与了肌动蛋白细胞骨架调节、PI3K-Akt信号通路、RAP1信号通路、RAS信号通路、病毒致癌、神经活性配体-受体相互作用、甲型流感致病等调控。通过对差异表达的未知miRNAs进行靶基因预测、GO和pathway显著性富集分析，进一步筛选目标miRNAs；然后将筛选出的miRNAs与生物信息学预测到的miRNA进行比较，筛选出的其中2个目的miRNA正在进行功能验证实验，其它筛选出的目的miRNA也将在后续工作中进行实验验证，还有一部分目的miRNAs还在进一步筛选鉴定中。分析了2个目的miRNAs的表达特性和表达时序性，靶基因和调控作用的验证还未彻底做完。因疫情而停止的研究工作，等学校通知返校后，将继续开展，致力于构建鱼淋巴囊肿病毒中国株编码miRNAs的调控网络。为鱼类淋巴囊肿病的诊断及致病机制的解析做出贡献。