伪狂犬病毒变异株感染细胞的microRNA表达谱及病毒抗原性分析

发布时间：2018-06-10 15:42

本文选题：伪狂犬病毒 + miRNA表达谱　；参考：《中国农业科学院》2015年博士论文

【摘要】：猪伪狂犬病(Pseudorabies,PR)是由伪狂犬病毒(Pseudorabies virus,PRV)引起的以造成新生仔猪神经系统疾病和呼吸系统紊乱及怀孕母猪的繁殖障碍为主要症状的传染性疾病。疫苗免疫曾经有效地控制了此病的流行和危害,但近年来,许多免疫猪群暴发伪狂犬病,并造成了严重的经济损失。本实验室于2012年在江苏某免疫猪场发病死亡的仔猪脑组织中分离到一株PRV JS-2012,经全基因组序列分析和感染试验证明该PRV发生了较大变异(Ye et al.,2015),对各年龄的猪致病力明显增强(Tong et al.,2015),而且现有的疫苗不能提供完全保护。本试验试图通过分析PRV JS-2012感染PK-15细胞后引起的细胞和病毒microRNA(mi RNA)表达谱变化,为进一步研究miRNA转录后对PRV复制、免疫及持续感染的调控作用奠定基础。通过sRNA文库构建、高通量测序,我们在PRV感染和未感染PK-15细胞的样品中分别得到109,595,539和112,037,904条高质量的小RNAs(sRNA)。利用生物信息学软件分析,在PRV JS-2012感染的PK-15细胞样品中初步检测到了36条mi RNA,其中11条为数据库中得到验证的高表达的保守的miRNA,其余25条可能是新的病毒miRNA(vmiRNA);除prv-miR-1、6、16、21和25外,其余20条vmiRNA均通过stem-loop RT-qPCR方法得到证实。与α-疱疹病毒miRNA通常编码在潜伏转录相关因子(LLT)区不同的是,PRV JS-2012编码的vmiRNA中只有3条(prv-miR-11-5p、prv-miR-12a-3p和prv-miR-17a-5p)分布在LLT区,其余至少17条则像β-疱疹病毒一样散在分布于PRV整个基因组,这在α-疱疹病毒中是首次发现。这些可能的新的mi RNA中,prv-mi R-LLT10a/b-3p、prv-miR-LLT11a/b-5p、prv-miR-12a/b-3p、prv-miR-13a/b-5p、prv-miR-14a/b-5p和prv-miR-17a/b-5p位于内部重复序列(IRS)和末端重复序列(TRS),而prv-miR-18至25、prv-miR-12 b、prv-miR-13 b、prv-miR-14 b和prv-miR-17由PRV基因组的负链编码。生物信息学预测,这些vmiRNA能调控包括参与病毒裂解复制和潜伏感染的相关基因。双荧光素酶表达系统验证结果表明分布在LLT区的prv-miR-LLT1、prv-miR-LLT7和prv-miR-LLT9对PRV转录极早期基因IE180表达有抑制作用,prv-mi R-LLT7对参与病毒复制调控的UL48基因(VP16蛋白)表达有抑制作用,因此这些vmiRNA可能在病毒感染过程中调节病毒的复制,而进一步功能试验表明体外过表达这些vmiRNA的模拟物,进而感染PRV后并未明显抑制IE180和VP16的蛋白表达水平。病毒感染细胞后还能通过影响宿主miRNA表达谱进而有利于病毒的复制。通过对PRV JS-2012感染组和未感染组的sRNA测序结果进行比较,分别在感染和未感染的sRNA文库中确认了303条和295条已知的猪源细胞miRNA,283条已知的和239条新的宿主miRNA在两个sRNA文库中是共有。感染的样品中有8条宿主miRNA显著上调,5条宿主miRNA显著下调。通过GO富集分析,这些异常表达的宿主miRNA的靶基因主要参与新陈代谢通路调控、生物过程的调节和先天性免疫等过程。通过合成这些宿主差异miRNA的模拟物或抑制剂,并将其在PK-15细胞中过表达,感染病毒后并没有发现这些宿主差异的miRNA对病毒的复制有显著影响。新出现的伪狂犬病毒显然能突破现有疫苗的免疫防线,使得免疫的猪群发病。为研究PRV JS-2012抗原性变异情况,本研究制备了JS-2012、SC(经典伪狂犬病强毒)和Bartha-K61(基因缺失弱毒疫苗株)的全病毒兔源和鼠源多克隆抗体。交叉中和试验显示Bartha-K61的全病毒多抗不能很好的中和JS-2012病毒,初步推断JS-2012变异株的抗原性可能发生了改变。此外,我们利用Bartha-K61和Bucharest疫苗免疫仔猪。利用制备的疫苗多抗与JS-2012、SC、Bartha-K61和Bucharest病毒进行交叉中和试验。两种疫苗均在免疫14 d后出现中和抗体,并且两个疫苗均是针对自身疫苗毒株中和效价最高,针对JS-2012的中和效价最低。该结果进一步说明了JS-2012的抗原性发生了变化。本研究为进一步鉴定PRV流行毒株的抗原变异奠定基础。综上所述,本研究首次分析了变异PRV JS-2012感染PK-15细胞后miRNA表达谱情况,分析了JS-2012的vmiRNA的保守性并鉴定了多条新的vmi RNA,并对其功能做了初步的验证。此外,本文鉴定了变异PRV感染PK-15细胞后对的宿主miRNA产生的影响,加深了对病毒感染细胞后的miRNA调控的理解和认识。同时为研究流行的变异PRV毒株的抗原性差异奠定基础。
[Abstract]:Pseudorabies (PR) is a contagious disease caused by the Pseudorabies virus (PRV), which causes the nervous system diseases and respiratory disorders of newborn piglets and the reproductive disorders of pregnant sows. Vaccine immunity has effectively controlled the epidemic and harm of this disease, but in recent years, many immune pigs have been immunized. In 2012, a strain of PRV JS-2012 was isolated from the brain tissue of a piggery that died in an immune pig farm in Jiangsu. The whole genome sequence analysis and infection test showed that the PRV had a large variation (Ye et al., 2015), and significantly increased the pathogenicity of pigs of all ages (Tong). Et al., 2015), and the existing vaccines do not provide complete protection. This trial attempts to analyze the changes in the expression profiles of microRNA (MI RNA) induced by PRV JS-2012 infected PK-15 cells, laying the foundation for further research on the regulation of PRV replication, immune and persistent infection after miRNA transcription. Sequencing, 109595539 and 112037904 high quality small RNAs (sRNA) were obtained in the samples of PRV infected and uninfected PK-15 cells. Using bioinformatics software, 36 mi RNA were preliminarily detected in the PK-15 cell samples infected with PRV JS-2012, of which 11 were the highly expressed conservative miRN in the database. A, the other 25 may be new virus miRNA (vmiRNA); except for prv-miR-1,6,16,21 and 25, the remaining 20 vmiRNA are confirmed by the stem-loop RT-qPCR method. Only 3 of the PRV JS-2012 encoded vmiRNA are encoded in the latent transcription related factor (LLT) region. R-17a-5p) is distributed in the LLT region, and at least 17 others are distributed in the whole PRV genome like beta herpes virus. This is the first discovery in the alpha herpes virus. In these possible new mi RNA, prv-mi R-LLT10a/b-3p, prv-miR-LLT11a/b-5p, prv-miR-12a/b-3p, prv-miR-13a/b-5p, prv-miR-14a/b-5p, and prv-miR-17a/b-5p are within the internal weight. The complex sequence (IRS) and the end repeat sequence (TRS), and prv-miR-18 to 25, prv-miR-12 B, prv-miR-13 B, prv-miR-14 B and prv-miR-17 are encoded by the negative chain of the PRV genome. Bioinformatics predicts that these vmiRNA can regulate the related genes involved in viral lysis replication and latent infection. The results of the dual luciferase expression system verification show distribution Prv-miR-LLT1, prv-miR-LLT7 and prv-miR-LLT9 have inhibitory effect on the early gene IE180 expression of PRV transcriptional pole in LLT region, prv-mi R-LLT7 has a inhibitory effect on the expression of UL48 gene (VP16 protein) involved in the regulation of viral replication, so these vmiRNA may be replicating the virus in the process of virus infection, and the further functional test indicates in vitro Overexpression of these vmiRNA mimics, and then PRV infection did not significantly inhibit the protein expression level of IE180 and VP16. The virus infected cells can also contribute to the replication of the virus by affecting the host miRNA expression profile. By comparing the sRNA sequencing results of the PRV JS-2012 infection group and the uninfected group, the infection and non infection of the infected and uninfected groups are respectively infected and uninfected. In the sRNA library, 303 and 295 known pig source cells were identified, 283 known and 239 new host miRNA were shared in two sRNA libraries. 8 host miRNA was significantly up-regulated in the infected samples and 5 host miRNA significantly decreased. The target genes for these abnormal expressed host miRNA were mainly involved in the new target genes of the host miRNA. The processes of regulation of metabolic pathways, biological processes, and innate immunity. By synthesizing these miRNA mimics or inhibitors, and overexpressing them in PK-15 cells, after infection, the miRNA has no significant impact on the replication of the virus. The new pseudorabies virus is clearly able to break through In order to study the antigenic variation of PRV JS-2012, the whole virus rabbit source and mouse source polyclonal antibody of JS-2012, SC (classical pseudorabies strong poison) and Bartha-K61 (gene deletion attenuated vaccine strain) were prepared. The cross neutralization test showed that the total virus polyclonal antibody of Bartha-K61 could not be very strong. Good neutralization of the JS-2012 virus, preliminarily infer that the antigenicity of the JS-2012 variant may have changed. In addition, we use Bartha-K61 and Bucharest vaccines to immunization piglets. Using the prepared vaccine, we cross neutralization tests with JS-2012, SC, Bartha-K61 and Bucharest viruses. The two vaccines are neutralized after 14 d. The neutralization titers of the two vaccines were the highest and the neutralization titer against JS-2012 was the lowest. The results further indicated that the antigenicity of JS-2012 was changed. This study was the basis for further identification of the antigen variation of the PRV epidemic strains. To sum up, this study was the first to analyze the variant PRV JS-2012 infection of PK-15 cells. After miRNA expression profiles, the conservatism of JS-2012 vmiRNA was analyzed and a number of new VMI RNA were identified, and their function was preliminarily verified. In addition, the effects of PRV infected PK-15 cells on the host miRNA production were identified, and the understanding and understanding of miRNA regulation after the virus infected cells were deepened. The variation of the antigenicity of the variant PRV strain laid the foundation.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

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