【作者】 冯志新；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪圆环病毒2型（PCV2）为圆环病毒科成员之一，在全球各地已广泛流行，可引起多种临床疾病，包括是引起断奶仔猪多系统衰竭综合征（PMWS），猪皮炎和肾病综合征，已给世界养猪业造成巨大经济损失。该病毒与其它细菌或病毒（如猪繁殖与呼吸障碍综合征病毒）发生混合感染，则能引起更严重的疾病。其致病机理研究尚不十分清楚，在我国也尚无有效控制措施。本研究从我国不同地方PCV2流行毒株的分子遗传特性、PCV2与PRRSV共感染对猪肺泡巨噬细胞的免疫功能两方面探讨其致病机理，并利用腺病毒介导shRNA技术探讨该病防治的新策略，为我国有效防治该病奠定基础。1．中国PCV2流行毒株遗传特性研究。我们收集了2002-2004年全国9个省市具有PMWS临床症状的仔猪病料，分别通过病理学观察、病毒分离，并对不同来源和时间的毒株进行全基因测序及序列分析。结果为，11株PCV2的全序列核苷酸同源性为95.1-99.7％，与GenBank中来源于不同地区、不同时间和不同疾病的49株PCV2序列进行分析比较，全基因核苷酸同源性为93.5-99.70％，ORF2核苷酸同源性为89.9-99.9％。其中与来源于健康猪群的PCV2比较，全基因核苷酸同源性为94.3-99.7％，ORF2核苷酸同源性为89.3-99.7％；与来源于其它疾病（PDNS和流产）的PCV2全基因核苷酸同源性为94.5-99.6％，ORF2同源性为90.2-99.6％。；所有这60株PCV2的系统发生树由两个大分支构成；中国PCV2毒株在两个分支上均有分布。结果证实，PCV2感染及PMWS的发生在我国猪群中已相当普遍；中国PCV2序列与其它国家的毒株均有很近的亲缘关系，但其中大部分与欧洲的毒株同源性较高；中国各地的PCV2存在多种基因型，流行毒株分子遗传特性无时间和地域性差异。基因的差异与临床症状无明显联系。病毒致病特性与病毒分子遗传变异可能无直接相关性。2．PCV2与PRRSV混合感染对猪肺泡巨噬细胞免疫学功能的影响。我们首先成功建立了PCV2、PRRSV以及猪几种细胞因子（INFα，INFγ，TNFα，IL-8和IL-10）的实时定量PCR检测方法，然后制备猪肺泡巨噬细胞（PAM），再将PAM细胞分为6组，分别接种PRRSV和PCV2，即PCV2、PRRSV、PCV2+PRRSV（先接种PCV2,12h后接种PRRSV）、PRRSV+PCV2（先接种PRRSV，12h后接种PCV2）、PRRSV／PCV2（同时接种）和Mock组。接种后培养不同时间观察细胞病变和存活率，并用Real-time PCR和IFA方法检测PAM中PRRSV和PCV2的滴度、以及细胞因子INFα、INFγ、TNFα、IL-8和IL-10的mRNA水平。结果为：①PRRSV能在PAM中增殖，并产生CPE；PCV2在PAM中感染率较低，无CPE。②PRRSV对PCV2增殖没有明显影响，而PCV2先于或同时与PRRSV感染对PRRSV的复制具有抑制作用，但PCV2后于PPRSV感染，可促进PRRSV增殖。③PCV2单独感染PAM后能促进INF-α和INF-γ的大量表达；PRRSV单独感染PAM初期，INF-γ表达量增加，INF-α表达量减少，感染后期INF-α的表达量才有所上升。PCV2与PRRSV混合感染初期则使INF-α和INF-γ表达减少，感染48h后，INF-α的表达量迅速上升，并维持在较高的水平，INF-γ虽然有所上升，但始终维持在较低的水平。④PCV2单独感染对TNF-α的表达量影响不大；而PRRSV感染后，TNF-α表达量持续增加，尤其是与PCV2混合感染后，TNF-α的表达量明显增加。⑤PCV2与PRRSV单独感染后均可以刺激IL-8和IL-10大量表达，感染初期IL-8的表达量高，随后下降，但IL-10的表达量一直维持在很高的水平。PRRSV+PCV2组与PCV2／PRRSV组IL-8和IL-10的平均表达量均明显增加。上述结果表明，PRRSV与PCV2体外共感染对PCV2的增殖没有明显影响，PRRSV感染后如果再感染PCV2，可以明显促进PRRSV病毒增殖，抑制体液免疫和细胞免疫，同时，两种病毒共同刺激了TNF-α、IL-8和IL-10的大量表达，抑制了抗病毒因子INF-γ的表达，从而加重了病理学免疫应答，促进了病理损伤。3．腺病毒介导shRNA在体内外抑制PCV2复制机理研究。（1）靶向PCV2 ORF1和ORF2 shRNA重组腺病毒的构建。首先，我们设计合成分别针对PCV2 ORF1与ORF2基因的发夹小RNA片段（shRNA）S1与S2，分别体外退火形成双链，克隆入pSUPER载体中的H1启动子后的克隆位点，再将H1启动子连同小RNA片段切下后插入重组腺病毒穿梭质粒（pAdTrack-CMV）的多克隆位点，与骨架质粒pAdEasy-1在大肠杆菌BJ5183内进行同源重组，获得的重组腺病毒质粒pAdS1与pAdS2经转染293A细胞生成表达针对PCV2 ORF1与ORF2的shRNA的重组腺病毒，分别命名为：rAdS1与rAdS2。我们用相同的方法将pSUPER载体中的H1启动子插入腺病毒表达载体，构建成功了对照重组腺病毒，rAdH1。（2）重组腺病毒介导的shRNA抑制PCV2在PK15细胞中复制的研究。将rAdS1、rAdS2和对照病毒rAdH1接种PK-15细胞，24h后再接种PCV2病毒，继续培养48h后，用间接免疫荧光试验检测PCV2，结果为，rAdS1和rAdS2均能有效地抑制PCV2病毒的增殖，其中rAdS2抑制效率更强，达96％。分别通过半定量PCR，实时定量PCR和Western blot方法检测上述试验中PCV2的mRNA、DNA和蛋白表达，结果表明，PCV2感染后48h，重组腺病毒rAdS1与rAdS2对PCV2在PK15细胞上的复制有明显的抑制作用，并至少持续120h，且这种抑制作用有重组腺病毒感染剂量依赖性，当表达shRNA重组腺病毒的接种剂量增加至1000个MOI时，rAdS1与rAdS2对PCV2的抑制效率分别升至78％和96％；将这两种重组腺病毒共同感染PK15细胞，发现对病毒复制的抑制作用具有相加性；而且用重组腺病毒接种已经感染PCV2的PK15细胞，病毒的复制也得到部分抑制，其中rAdS1与rAdS2对PCV2的DNA水平的抑制效率分别为48.8％和78.9％。（3）重组腺病毒介导的shRNA抑制PCV2在小鼠体内复制的研究。将重组病毒以10⁸efu／只的剂量通过滴鼻与腹腔注射的方式接种BALB／c小鼠，24h后每只小鼠通过同样的接种方式感染10⁴TCID₅₀ PCV2。感染后观察小鼠的临床症状，并在第7、14，21和28天每组分别宰杀5只小鼠，观察肉眼病变，并通过实时定量PCR方法检测小鼠脾脏及血液中PCV2的病毒含量。结果显示，各组小鼠均未表现出明显的临床症状和肉眼病变。在阳性对照组（PCV2单感染组），小鼠感染PCV2后7-21天，脾脏内PCV2病毒的滴度逐渐增加。在试验组，重组腺病毒rAdS1和rAdS2在感染后前1-2周对小鼠脾脏中PCV2病毒均有轻微的抑制作用，到第3、4周这种抑制作用明显增强，对PCV2病毒的抑制效率可达91.4％-96.8％。结果表明，靶向PCV2基因组不同区域的shRNA可以作为控制该病毒传播的候选策略。更多还原

【Abstract】 Porcine circovirus type 2 （PCV2） has recently prevailed in the world and induced several clinical disease,such as postweaning multisystemic wasting syndrome （PMWS） and porcine dermatitis and nephropathy syndrome （PDNS）. However, the pathogenesis of the virus was not clear. And there is no effective prevention and control method for the PCV2-associated diseases in China. In this study, the pathogenesis of PCV2 was studied by analyzing the genetic characteristic of PCV2 isolates in China and the immunology of porcine alveolar macrophage （PAM） coinfected by PCV2 and PRRSV. Moreover, the inhibition of PCV2 replication were determinded by recombinant adenovirus expressing shRNA targeting to ORF1 and ORF2 regions of PCV2 in PK-15 cells and mouse model.1. Genetic characterization of PCV2 isolates in China.To fully understand the genetic diversity of PCV2 isolates in China, a retrospective study was performed on natural cases of postweaning multisystemic wasting syndrome described from 2002 to 2004 in 9 provinces or municipalities. The data about characterization of prevalence was collected as well as the clinical and histopathological symptoms were observed. To understand the genetic diversity of PCV2, the causative agent of PMWS, complete genomes of 11 PCV2 isolates were sequenced and aligned against the other 49 sequences of PCV2 isolates from Europe, North America, Asia and China. The results indicated that these isolates from 9 provinces or municipalities shared 93.5-99.7% in complete sequence and 89.9-99.9% in ORF2 homology with other 49 PCV2 sequences published in GenBank. In addition, the 11 PCV2 isolates from PMWS cases in this study showed 94.3-99.7% complete sequence identity and 89.3-99.7% homology in ORF2 compared with other strains from healthy cases, and share 94.5-99.6% nucleotide identity in complete sequence with strains from other PCV2-associated diseases （PDNS and abortion）. Phylogenetic analysis of all 60 PCV2 isolates from North America, Europe, Oceania and Asia revealed that these isolates were grouped together in one large tree containing two minor clusters. The Chinese PCV2 isolates were spread in both clusters. It suggested that PCV2 infections and PMWS were common in pig herds in China. The PCV2 isolates in China have many genotypes and closely related to those in other countries in the world. No obvious diversity of geographical origin and time-interval exists. The relation between pathogenesis and genetic diversity of PCV2 was unclear.2. Effect of PCV2 and PRRSV coinfection on immunology of PAM in vitro.At first, we developed real-time PCR assays for PCV2, PRRSV and porcine cytokines （INFα, INFγ, TNFα, IL-8 and IL-10） detection. Then PCV2 and PRRSV were investigated in regard to their effects on monolayer cultures of porcine alveolar macrophages （PAMs）. Two viruses infected PAMs alone or together with the different orders. Cytopathic effect （CPE） and livability of PAMs were observed and calculated. And virus titers and cytokines’ mRNA （INF-α, INF-γ, TNF-α, IL-8 and IL-10） in PAMs were detected by real-time PCR and indirect immunofluorescence assay （IFA）. The results showed as following: （1） PRRSV could replicate in PAMs and induce CPE, but PCV2 could not. （2） PRRSV could not help PCV2 to replicate in PAMs. PCV2 could inhibit the replication of PRRSV in PAMs when infected before or together with PRRSV. But when infected after PRRSV, PCV2 could promote PRRSV replication. （3） Coinfection of PCV2 and PRRSV could produce high level of TNF-α, IL-8 and IL-10 and less INF-γin PAMs than infection of PCV2 or PRRSV alone. It suggested that PRRSV had no effect on replication of PCV2 in vitro. However, the infection of PCV2 after challenge of PRRSV in PAMs could boost up the replication of PRRSV, and suppress the reaction of cellar immunity and humour immunity. The suppression of INF-γand over-expression of TNF-α, IL-8 and IL-10 caused by dual infection of PCV2 and PRRSV might enhance the pathological reaction and induced severer clinical symptom in vivo.3. Adenovirus-mediated RNA interference against PCV2 replication both in vitro and vivo.Firstly, shRNAs （S1 & S2） targeting PCV2 ORF1 and ORF2 were synthesized chemically and cloned into the pSUPER vector following H1 promoter. Then the shRNA expressing cassette was inserted into pAdTrack-CMV vector. The recombinant adenoviral plasmids （pAdS1 and pAdS2） were generated by homologous recombination of recombinant pAdTrack vector and pAdEasy-1 in E. coil BJ5183. Then recombinant adenoviral plasmids were linearized with Pac I and transfected into AD-293 packaging cells to produce recombinant adenovirus, rAdS1 and rAdS2. As the same, rAdH1, the control virus, was constructed by only cloning H1 promoter from pSUPER vector into AdEasy systerm.Secondly, we infected PK-15 cells with recombinant adenovirus rAdS1, rAdS2 and rAdH1. After 24h, the cells were challenged with PCV2. PCV2 antigen was detected by IFA assay at 48h post-infection of PCV2. The result showed that both rAdS1 and rAdS2 could inhibit the replication of PCV2, especially rAdS2 showed stronger inhibition （96%）. Delivery of these shRNAs into cultured PK15 cells caused a significant reduction in PCV2 viral RNA production, viral DNA replication and protein synthesis in infected cells by semi-quantitative RT-PCR, real-time PCR and western blot assay, respectively. The antiviral effect was sequence-specific and dose-dependent and could sustain at least for 120h. Furthermore, by combination of treatment with rAdSl and rAdS2, the viral inhibition cloud be significantly increased. In addition, rAdS1 and rAdS2 displayed partial therapy for PCV2 infection beforehand.Lastly, the BALB/c mice were inoculated intranasally and interperitoneally with rAdSl or rAdS2 （10⁸ efu per mouse）. After 24h, all the mice were challenged with 10⁴ TCID₅₀ of the PCV2 via the same routes. Then the clinical symptom of mice was observed every day. The necropsies of the mice were examined at 7,14, 21 and 28 days post-infection, and the level of PCV2 DNA in Spleens and blood was detected by real-time PCR. The results showed that no obvious clinical signs and macroscopic lesions were detected in each group. In positive group （PCV2 alone-infection）, PCV2 nucleic acid in spleen of mice was increasing and arrived to the tip at 21 days post-infection. However, mice injected with rAdS1 and rAdS2 before PCV2 infection showed substantial and increasing decrease in the level of viral DNA replication in the spleen of the treated mice compared to the controls. This suppression was slight at the first two weeks, while it was significant （91.4%-96.8%） at the 3^rd and 4^th weeks. Meanwhile, no PCV2 was deteced in the blood of nearly all the mice.Taken together, these results indicated that shRNAs mediated by adenovirus could sufficiently and continuously inhibit PCV2 infection both in vitro and in vivo, and might be a potential alternative strategy for controlling PCV2 infection.更多还原