马传染性贫血病毒弱毒疫苗株与强毒株感染对靶细胞作用差异的研究

Equine infectious anemia virus (EIAV) and HIV-1 are both members of the Lentiviruses Genus and are similar in major virologycal characters. The live attenuated EIAV vaccine developed by Chinese scientists in the 1970s is the first lentiviral vaccine that was approved safe and effective after being applied country-widely. The decoding of the functional mechanism of the live attenuated EIAV vaccine should provide a valuable reference for the development of other lentiviral vaccines. Our preliminary studies revealed that there were significant differences between the vaccine strain and its parental pathogenic strain in their genomic sequences and compositions, immunogenicities and effects on target cells. These results strongly suggest that the effect variation of these viruses on target cells may provide significant impact on the viral pathogenicity and immunogenicity. This proposal intends to analyze the differences of the protein and microRNA expression levels of target cells in vitro infected respectively by the vaccine strain and the pathogenic strain by using the techniques of iTRAQ (isobaric tags for relative and absolute quantitation) and microRNA high-throughput sequencing. The differentially expressed cluster analysis will be performed using bioinformatics, and the impact of differently expressed proteins and microRNAs on the virus-host interactions will be tested by approaches of the over-expression or knockdown of target genes and the application of specific chemical inhibitors to identify the roles of these proteins and microRNAs in the "anti-virus network" of cells infected with the vaccine and pathogenic strain. Combined with results of our preliminary studies, this proposal is expected to provide the essential molecular biology information for better understanding the mechanism, under which the Chinese attenuated EIAV vaccine induces protective immunity to the infection of homologous and heterologous pathogenic strains.

同属慢病毒的马传染性贫血病毒（EIAV）与艾滋病毒相似。我国研制的EIAV 弱毒疫苗，是首例经大规模应用证实安全、有效的慢病毒疫苗。阐明该疫苗的作用机理，将为其他慢病毒疫苗研究提供借鉴。前期研究表明，EIAV疫苗株与强毒株在基因组构成和免疫原性方面存在显著差异，并对靶细胞的影响明显不同，强烈提示EIAV与靶细胞相互作用的差异对病毒毒力和免疫诱导能力的影响。本项目拟利用同位素标记相对和绝对定量的质谱技术及微小RNA 高通量测序方法，分析疫苗株与强毒株体外感染对靶细胞蛋白质和微小RNA表达的影响及其差异。通过生物信息学方法进行差异表达聚类分析，应用过表达、基因抑制以及特异性抑制剂等方法，进行差异蛋白质和微小RNA对病毒和细胞相互作用影响的研究，以确定其在疫苗株和强毒株感染后靶细胞"对抗网络"中的位点。结合前期研究结果，本项目将为揭示EIAV弱毒疫苗的作用机理提供不可缺少的分子生物学信息。

同属慢病毒的马传染性贫血病毒（EIAV）与艾滋病毒相似。我国研制的EIAV 弱毒疫苗，是首例经大规模应用证实安全、有效的慢病毒疫苗。阐明该疫苗的作用机理，将为其他慢病毒疫苗研究提供借鉴。前期研究表明，EIAV 疫苗株与强毒株在基因组构成和免疫原性方面存在显著差异，并对靶细胞的影响明显不同，强烈提示EIAV 与靶细胞相互作用的差异对病毒毒力和免疫诱导能力的影响。.本项目拟通过比较蛋白质组学及microRNA差异表达分析，发现EIAV弱毒疫苗和强毒株与靶细胞相互作用的关键差异点，以期发掘出EIAV 弱毒疫苗作用机理重要的分子生物学信息。本项目研究主要内容包括2部分: 1）感染EIAV弱毒疫苗株与致病性强毒株靶细胞的差异表达蛋白质筛选及功能分析；2）感染EIAV弱毒疫苗株与致病性强毒株靶细胞的差异表达microRNA筛选及功能分析。.通过比较蛋白质组学发现，EIAV强弱毒感染靶细胞后有82个差异表达蛋白质与线粒体组分及氧化磷酸化生物功能功能相关，占差异蛋白质总数的18.1%，是发生蛋白质差异表达最多的细胞器，结合透射电镜结果表明EIAV强毒感染靶细胞后可诱导线粒体发生更多的分裂，而弱毒感染靶细胞后可诱导线粒体肿胀和膜通透性增加，表明靶细胞线粒体对EIAV弱毒疫苗株和强毒株有不同的应激反应，这可能与弱毒疫苗的作用机制有着非常重要的联系。.通过microRNA深度测序发现，EIAV强弱毒感染靶细胞后的差异表达microRNA共28个，其所涉及的生物学功能包括TLR和RIG信号通路、氧化磷酸化途径、蛋白酶体途径、内质网应激途径以及线粒体应激和先天免疫反应等。利用聚类分析发现多个microRNA表达簇，除此之外，通过与EIAV基因组比对，发现1个病毒所编码的microRNA，经预测可以与病毒本身及宿主基因组作用。该部分结果表明，EIAV强弱毒感染，可诱导靶细胞不同的microRNA表达谱，而这种生物学现象也与EIAV弱毒疫苗的作用机理有着相关联系。.总之，在原有研究基础上，通过该项目的执行，发现了不同的线粒体应激反应和microRNA表达谱均与EIAV弱毒疫苗作用机理有着重要联系，发现了“对抗网络”的关键点，促进了对EIAV弱毒疫苗作用机理深层次的理解，除此之外，还开辟了关于EIAV与宿主线粒体和非编码RNA相互作用的新研究领域。