【作者】 宫晓炜；

【导师】 才学鹏；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2014， 博士

【摘要】 牛病毒性腹泻病（Bovine viral diarrhea, BVD）是由牛病毒性腹泻病毒（Bovine viral diarrheavirus, BVDV）感染引起的，以急慢性粘膜病、持续性感染和免疫抑制为主要特征的传染性疾病；该病被世界动物卫生组织（OIE）列入OIE疾病目录。目前BVDV感染宿主的范围不断扩大，流行趋势呈明显上升状态，给BVD防控计划的实施带来了严重的挑战。病毒诱导的细胞自噬不仅介导细胞先天性抗病毒免疫反应，也参与病毒抗原的提呈；但很多病毒可演化出不同的机制来破坏、逃避和抑制自噬，甚至利用自噬来增强自身的繁殖。BVDV和宿主蛋白相互作用所导致的细胞损伤是BVDV致病的关键。目前有关BVDV与自噬及宿主蛋白相互作用的报道极为有限。本论文旨在从宿主角度探索BVDV感染复制的相关机制，并取得了如下成果：1）近几年，我国西北地区流行的BVDV亚型主要是BVDV-1b、BVDV-1d及BVDV-1q。通过RT-PCR方法检测宁夏和青海奶牛血样及牦牛血样中BVDV的感染情况，结果显示BVDV阳性率分别为26.8%和24%。对5′UTR和Npro进行测序和遗传进化分析，证实流行的毒株主要为BVDV-1b、BVDV-1d及BVDV-1q三个亚型。此外，对BVDV抗原阳性的血样进行病毒分离，均为ncp型毒株；电镜和直接免疫荧光检测可观察到细胞中病毒粒子的存在。2）制备高亲和力的兔抗-Core和兔抗Npro多克隆抗体。构建原核表达载体pET-30a-Core和pET-30a-Npro，转化BL21（DE3），经IPTG诱导、Ni-NTA亲和层析纯化和电洗脱包涵体，获得浓度和纯度较高的Core和Npro重组蛋白，Western-blot检测具有很好的反应原性；利用纯化的蛋白制备兔抗血清，Western-blot和IFA检测表明，两种多克隆抗体均能识别BVDV病毒粒子和细胞中天然表达的Core和Npro蛋白，可用于后续的试验。3）BVDV感染诱导的细胞自噬促进自身在细胞中的复制。通过透射电镜、Western-blot及荧光显微镜检测，发现BVDV oregon C24V感染MDBK细胞后24h，细胞质中会出现大量的自噬空泡；BVDV感染可引起LC3-Ⅰ向LC3-Ⅱ的转化以及p62的降解；在GFP-LC3质粒转染的细胞，能看到增多的聚集颗粒。用药物抑制细胞自噬或干扰Beclin-1的表达，会降低病毒滴度和病毒蛋白的表达。4）BVDV Core蛋白与PIAS4蛋白相互作用有利于BVDV的生长和复制。本研究以Core蛋白为诱饵，利用酵母双杂交系统从牛单核细胞文库中筛选出与其相结合的关键宿主蛋白（PIAS4）；通过酵母回交试验和免疫共沉淀试验，证实Core蛋白和PIAS4发生相互作用；激光共聚焦显微镜观察二者在细胞质共定位；下调和上调细胞内PIAS4的表达，病毒滴度和Core蛋白的表达量会发生相应的下降和增加。本论文研究和发现了宿主细胞内参与BVDV复制的核心元件，为进一步研究自噬和BVDV的互作及BVDV抗宿主免疫应答的机制提供了线索。更多还原

【Abstract】 Bovine viral diarrhea (BVD) is an important viral disease and listed as a reportable disease of cattleby the World Organization for Animal Health (OIE). BVDV is the causative agent of Bovine viraldiarrhea which causes a series of clinical symptoms of acute or chronic mucosal disease, persistentinfection and immunosuppression. At present, the BVDV prevalence rate is rising in a worldwide andthe host range has expanded, which brings more challenges to prevent and control BVDV infection.Autophagy is induced by virus infection. It may not only contribute as an intrinsic host defensemechanism against invading viruses but may also mediate antigen presentation, which plays crucial rolein the control of intracellular pathogen. However, many viruses have a number of mechanisms to blockautophagy or even manipulate autophagy for their own benefit. In addition, the interactions betweenBVDV and cellular protein can destroy the normal function of cell and lead to cell damage, which is akey element of causing disease. However, the studies on BVDV-autophagy and BVDV-host interactionsare limited. In order to elucidate the BVDV replication mechanism, the following results were obtained:1. In recent years, three subgentypes: BVDV-1b, BVDV-1d and BVDV-1q are circulating inWestern China. In order to investigate possible infection in dariy cattle of Ningxia and yaks of Qinghai,all samples were tested for viral nucleic acids by RT-PCR. The results showed the prevalence was26.8%and24%in dariy cattle and yak, respectively. The phylogenetic reconstructions demonstratedthat these samples are BVDV-1, specifically belonging to subgenotypes BVDV-1b, BVDV-1d andBVDV-1q, through the nucleotide analysis of5′UTR and Nproregions. Thirteen viruses were isolatedfrom the antigen-positive samples, and all of them were ncp biotype. Virus particles can be observedunder electron microscopy and direct immunofluorescence.2. Preparation of high affinity antibodies with Core and Nproproteins. The prokaryotic expressionplasmids pET-30a-Core and pET-30a-Nprowere constructed and transformed into E.coli BL21. Highlypurified Core and Nproproteins were obtained by an affinity chromatography and electroelution afterinduction by IPTG. The western-blot assay indicated that recombinant proteins had good antigenicity.Then, polyclonal antibodies against Core and Nproproteins were prepared. IFA and western-blotexperiments demonstrated that polyclonal antibodies can identify the natural proteins and can be used infollow-up studies.3. Induction of autophagy can enhance BVDV replication. To determine the role of autophagy inthe infection of BVDV, we performed on MDBK cell infected by BVDV strain Oregon C24V. A largenumber of autophagosome-like double-memberane vesicles in the cytoplasm were observed undertransmission electron microscopy. BVDV infection resulted in conversion of LC3-Ⅰ/Ⅱ, degradation ofp62and the accumulation of GFP-LC3dot. Inhibition of autophagy using wortmannin and smallinterfering RNAs targeting Beclin-1can reduce BVDV replication and expression of Npro.4. Interaction of cellular PIAS4with BVDV core protein is beneficial to BVDV replication. Usinga yeast two-hybrid screen, the interacting partners of the Core protein were screened in PBMC cDNAlibrary and PIAS4as a novel interacting partner was selected. The interaction between Core and PIAS4 was confirmed by co-immunoprecipitaion and confocal assays. Moreover, Knockdown of PIAS4bysmall interfering RNA resulted in reduction of the virus titer as well as Core protein, whileoverexpression of PIAS4promoted BVDV growth and expression of Core protein.According to the research, the key host factors that regulate the BVDV replication have beendiscovered, which provide clues for further study on virus-autophagy interaction and mechanism inresistance to host immunity.更多还原