【作者】 郭雪坤；

【导师】 封文海；

【作者基本信息】 中国农业大学 ， 微生物学， 2014， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS),俗称蓝耳病,是由猪繁殖与呼吸综合征病毒(PRRS virus, PRRSV)引起的猪传染性疾病,近年来给我国乃至全世界养猪业造成了巨大的经济损失。最近研究表明,在病毒感染过程中宿主microRNAs (miRNAs)可以靶向病毒基因组RNA而导致病毒的复制受到抑制,这种作用方式被人们视为一种潜在的宿主抗病毒防御机制。然而宿主miRNAs是否可以靶向PRRSV的基因组RNA并抑制PRRSV复制的问题并不是很清楚,本论文针对这一问题进行了一定的研究。基于已经鉴定的猪miRNAs数目很少,本文首先通过分子克隆和高通量测序技术鉴定了猪肺泡巨噬细胞(Porcine alveolar macrophages, PAMs)中miRNA表达谱,并分析了PAMs在感染PRRSV前后miRNAs表达的差异。初步结果显示PAMs可以表达800多种miRNAs,且PRRSV感染后几乎所有miRNAs的表达均呈现下降趋势。通过生物信息学软件(RegRNA和ViTa)对PRRSV的基因组RNA进行miRNA靶位点预测,结果显示microRNA-181(miR-181)可能通过种子区(seed region)碱基配对的方式靶向PRRSV JXwn06基因组RNA上的13684-13692位点,而且这个位点在美洲株(属于type2)中的保守性非常高,可达96%以上。体外过表达miR-181的相关实验结果表明,在PAMs和Marc-145细胞中miR-181的确可以抑制PRRSV病毒基因的表达和病毒粒子的产生。有意思的是,miR-181家族的四个成员(a、b、c和d)都可以抑制PRRSV复制。通过分析病毒在PAMs中的生长曲线后发现,过表达miR-181家族可以持续地抑制PRRSV在PAMs中生长复制,且miR-181c抑制PRRSV复制的能力最强。荧光素酶报告基因系统分析miR-181靶位点的实验结果表明,miR-181的确可以直接靶向PRRSV基因组RNA上的特定序列来抑制病毒复制。实验结果还表明,病毒RNA可以与荧光素酶报告载体上miR-181靶位点竞争结合miR-181,导致miR-181对荧光素酶活性的抑制受到削弱,间接证明了PRRSV的RNA可以与miR-181相互结合。RNA免疫共沉淀(RNA-IP)的实验结果显示,miR-181可以特异性地与PRRSV基因组RNA结合在miRISC复合体上,为证明miR-181可以直接靶向PRRSV基因组RNA提供了更有力的证据。检测和比较不同原代细胞(包括PAMs、外周血单核细胞、小胶质细胞和腹腔巨噬细胞)和不同组织中的miRNAs后还发现,不易感染PRRSV的细胞或组织中的miR-181和其他潜在靶向PRRSV miRNAs(如miR-206)的表达量显著高于易感染PRRSV的细胞或组织。最后,动物体内实验的结果表明,在感染高致病性PRRSV的猪中滴鼻给药miR-181c可以降低动物血液中的病毒载量并缓解PRRSV引起的发烧症状,证明了miR-181也可以在动物体内抑制PRRSV的复制。以上结果表明,宿主miR-181的确可以通过直接靶向病毒基因组RNA来抑制PRRSV复制,并且在不同细胞中miR-181的表达与PRRSV感染能力或复制能力呈负相关性。这些发现暗示了宿主miRNAs在调控PRRSV的感染复制和致病机理中具有重要作用,同时也为宿主miRNAs作为抗病毒治疗工具提供了理论依据。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) also known as blue-ear pig disease is caused by PRRS virus (PRRSV) which continuously caused huge economic losses in our country or even throughout the world. Emerging evidence indicates that host microRNAs (miRNAs) can target viral RNAs during infections, resulting in inhibition of virus replication, which has been regarded as a new antiviral defense. Whether host miRNAs can target PRRSV RNA and be used to inhibit virus infection has not been well characterized. This dissertation work was focused on this question.As only a few of pig miRNAs have been identified, the miRNA expression profilings of porcine alveolar macrophages (PAMs) when infected with or without PRRSV were identified first by molecular cloning and deep sequencing. The results showed that PAMs could express over800distinct miRNAs, and most of them were down-regulated by PRRSV. The analysis of miRNA target prediction in PRRSV genomic RNA by two bioinformatic softwares (RegRNA and ViTa) showed that microRNA-181(miR-181) could target the site (nt13684to13692) in PRRSV JXwn06genomic RNA through seed base-pairing, and the target site was highly conserved (over96%) in type2PRRSV. Then our results showed that overexpressed miR-181indeed inhibited viral gene expression and PRRSV production in PAMs or Marc-145cells. Interestingly, all the four members of miR-181family (a, b, c and d) could inhibit PRRSV replication. Analysis of dynamics of PRRSV growth indicated that overexpressed miR-181could steadily inhibit PRRSV replication in PAMs and miR-181c was the strongest inhibitor of PRRSV. Direct targeting of the PRRSV genomic RNA by miR-181was then identified by luciferase reporter assays. Meanwhile, the viral RNA could compete with miR-181target site in luciferase vector to bind to miR-181, resulting in less inhibition of luciferase activity, which further demonstrated the specific interaction between miR-181and PRRSV RNA. Moreover, physical interaction of miR-181with viral genomic RNA in the miRNA-induced silencing complex (miRISC) was then tested by RNA immunoprecipitation (RNA-IP) assay, which provided stronger evidence for this model. Detection of miRNAs from different primary cells (including PAMs, peripheral blood mononuclear cells, microglia and peritoneal macrophages) and different tissues showed that miR-181and other potential PRRSV-targeting miRNAs (such as miR-206) were expressed much more abundantly in minimally permissive cells or tissues than in highly permissive cells or tissues. Finally, highly pathogenic PRRSV-infected pigs treated with miR-181c showed substantially decreased viral loads in blood and relief from PRRSV-induced fever, supporting that miR-181could inhibit PRRSV replication in vivo.Taken together, these results indicate that miR-181can inhibit PRRSV replication by directly targeting viral genomic RNA and the expression of miR-181is negatively correlated with viral replication capacity. These findings suggest an important role of host miRNAs in modulating PRRSV infection and replication, and viral pathogenesis. They also support the idea that host miRNAs could be useful for antiviral therapeutic strategies.更多还原