猪圆环病毒2型致病机理和重组腺病毒介导shRNA抑制PCV2在体内外复制的研究
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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10~8efu／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10~4TCID_(50) 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。
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 (10~8 efu per mouse). After 24h, all the mice were challenged with 10~4 TCID_(50) 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.
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10~8efu／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10~4TCID_(50) 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。
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 (10~8 efu per mouse). After 24h, all the mice were challenged with 10~4 TCID_(50) 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.
引文
[1] Allan GM, McNeilly F, Foster JC, Adair BM. Infection of leucocyte cell cultures derived species with pig cirwvirus [J]. Vet Microbiol. 1994,41(3): 267-79.
[2] Allan GM, McNeilly F, Cassidy JP, Reilly GA, Adair B, Ellis WA, McNulty MS. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material [J]. Vet Microbiol.1995,44(1): 49-64.
[3] Allan G Meehan B, Todd D. et al. Novel porcine circovirus from pigs with wasting disease syndrome [J]. Vet Rec, 1998,142(17): 476-468
[4] Allan GM, McNeilly F, Meehan BM, et al. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland [J]. Vet Microbiol, 1999, 66(2): 11523
[5] Allan G, McNeilly F, Meehan B, McNair I, Ellis J, Krakowka S, Fossum C, Wattrang E, Wallgren P, Adair B.Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate [J]. J Vet Diagn Invest. 2003,15(6): 553-60.
[6] Annette Mankertz, Rifat Caliskan, Kim Hattermann,et al. Molecular biology of Porcine circovirus: analyses of gene expression and viral replication [J]. Veterinary Microbiology 2004,98: 81-88
[7] Bassami MR, Berryman D, Wilcox GE, et al. Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circovirus, and chicken anaemia virus [J]. Virology, 1998,249:453-459
[8] Boevink P, Chu PWC, Keese P. Sequence of the geminiviruses subterraneanclover stunt affinities with virus DNA [J]. Virology, 1995,207: 354-361
[9] Bolin, S.R. and Allan, GM. Old virus or new virus-In a world continuous changes [C]. En: PMWS and PCV2 diseases beyond the debate. In: 17th. INTERNATIONAL PIG VETERINARY SOCIETY CONGRESS. Ames. Iowa. USA. 2002: 9-17.
[10] Borel N, Burgi I, Kiupel M, et al. Three cases of postweaning multisystemic wasting syndrome (PMWS) due to porcine circovirus type 2 (PCV2) in Switzerland [J]. Schweiz Arch Tierheilkd, 2001, 143(5): 249-255
[11] Celera VJr, Carasova P. First evidence of porcine circovirus type 2 (PCV-2) infections of pigs in the Czech Republic by semi-nested PCR [J]. J Vet Med B Infect Dis Vet Public Health, 2002, 49(3):155-9.
[12] Cheung AK. Transcriptional analysis of porcine circovirus type 2 [J]. Virology 2002; 305: 168-180.
[13] Cheung AK and Bohn SR. Kinetics of porcine circovirus type 2 replication [J]. Arch Virol 2002b, 147: 43-58.
[14] Choi C, Chae C. ln-situ hybridization for the detection of porcine circovirus in pigs with postweaning multisystemic wasting syndrome [J]. J Comp Pathol, 1999,121(3): 265-70
[15] Clark EG. Postweaning multisystemic wasting syndrome [C]. In:Proceedings of the 28th American Association of Swine Practitioners, Que., Canada, 1997, 28:188-259.
[16] Fenaux M, Halbur PG, Gill M, et al. Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2[J]. J Clin Microbiol, 2000, 38(7):2494-2503.
[17] Gibbs, M.J., Weiller, G.F., Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus [J]. Proc. Natl. Acad. Sci. USA. 1999,14:8022-8027.
[18] Gresham A, Jackson q Giles N, et al. PMWS and porcine dermatitis nephropathy syndrome in Great Britain [J]. Vet Rec, 2000,146(5): 143
[19] Hamel AL, Lin LL, and Nayar GP. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs [J]. J Virol, 1998; 72:5262-5267.
[20] Harding JC. Post-weaning multisystemic wasting syndrome: preliminary epidemiology and clinical findings [C]. In: Proceedings of the Western Canadian Association on Swine Practice, 1996, 21
[21] Harding JC, Clark EG Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS) [J]. Swine Health Prod, 1998,5:201-203
[22] Hattermann K, Roedner C, Schmitt C, Finsterbusch T, Steinfeldt T, Mankertz A. Infection studies on human cell lines with porcine type 1 and porcine circovirus type 2 [J]. Xenotransplantation. 2004a, 11(3): 284-94.
[23] Hattermann K, Maerz A, Slanina H, Schmitt C, Mankertz A. Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-PCR-based search for a human circovirus. Xenotransvlantarion [J]. 2004b, 11(6): 547-50.
[24] Husa P, Chalupa P, Husova L. The SEN virus-will there be another letter in the alphabet of viral hepatitis [J]. Vnitr Lek. 2002, 48(8): 763-6
[25] Jemersic L, Cvetnic Z, Toplak I, et al. Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia [J]. Res Vet Sci, 2004,77(2): 171-5
[26] Josefma Q., Monica B., Joaquim S., Maria C, Gabriela M., RODRIGUEZ ARRIOJA, Juan P., LAMA-D., Mariano D. Experimental inoculation of porcine type 1 (PCV1) and type 2 (PCV2) in rabbits and mice [J]. Vet. Res.2002, 33:229-237.
[27] Katul L, Maiss E, Vetten HJ. Sequence analysis of a faba bean necrotic yellows virus DNA component tative replicase gene [J]. J Gen Virol, 1995,76:475-479
[28] Kenshi S. Tomoyuki S., Yoshiharu L, Hiroshi K., MasanoriK, Koichi K., Evidence of Porcine Infection in Pigs with Wasting Disease Syndrome from 1985 to 1999 in Hokkaido [J], Japan J Yet Med Sci. 2000,62(6): 627-633.
[29] Kiatipattanasakul-Banlunara W, Tantilertcharoen R, Suzuki K, et al. Detection of porcine circovirus 2 (PCV 2) DNA by nested PCR from formalin-fixed tissues of postweaning multi-systemic wasting syndrome (PMWS) pigs in Thailand [J]. J Vet Med Sci, 2002, 64(5): 449-452
[30] Kim, J., Han, D.U., Choi, C, et al. Differentiation of porcine circovirus (PCV)-1 and PCV2 in boar semen using a multiplex nested polymerase chain reaction [J]. Journal of Virological Methods 2001,98: 25-31.
[31] Kim JH, Lyoo YS. Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs [J]. J Vet Sci, 2002, 3(1):31-9
[32] Kiss I, Kecskemeti S, Tuboly T, et al. New pig disease in Hungary: postweaning muitisystemic wasting syndrome caused by circovirus [J]. Acta Vet Hung, 2000, 48(4): 469-475
[33] Kiupel M, Stevenson GW, Choi J, Latimer KS, Kanitz CL, Mittal SK.Viral replication and lesions in BALE/c trice experimentally inoculated with porcine circovirus isolated front a pig with postweaning muitisystemic wasting disease [J]. Vet Pathol. 2001, 38(1): 74-82.
[34] Larochelle R, Magar R, D'Allaire S. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strain from cases presenting various clinical conditions[J].Virus Res, 2002,90(1-2):101-112.
[35] Liu, J., Chen, I., Kwang, J., 2005. Characterization of a previously unidentified viral protein of porcine circovirus type 2-infected cells and its role in virusinduced apoptosis [J]. J. Virol. 79, 8262- 8274.
[36] Liu J, Chen I, Du Q, Chua H, Kwang J. 2006. The ORF3 protein of porcine circovirus type 2 is involved in viral pathogenesis in vitro [J]. J Virol. 80, 5065-73.
[37] Liu Q, Tiskoo S, and Babluk LA. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2 [J]. Virology 2001; 285: 91-99.
[38] Liu Q, Wang L, Willson P, et al. Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica [J]. Can J Vet Res, 2002, 66(4): 225-31
[39] Madec F, Fveno E, Morvan P, et al. Post-weaning muitisystemic wasting syndrome (PMWS) in pigs in France: clinical observations from follow-up studies on affected farms [J]. Livest Prod Sci, 2000, 63(3): 223-233
[40] Magar R, Larochelle R, Thibault S, Lamontagne L.Experimental transmission of porcine type 2 (PCV2) in weaned pigs: a sequential study [J]. J Comp Pathol. 2000,123(4): 258-69.
[41] Maldonado J, Segales J, Calsamiglia M, et al. Postweaning multisystemic wasting syndrome (PMWS) in the Philippines: porcine circovirus type 2 (PCV2) detection and characterization [J]. J Vet Med Sci, 2004,66(5): 533-7
[42] Mankertz A, Mankertz J, Wolf K, et al. Identification of a protein essential for the replication of porcine circovirus [J]. J Gen Virol 1998a; 79:381-384.
[43] Mankertz, J., Bunk, H J., Blaess, G., et al. Transcription analysis of Porcine circovirus (PCV) [J]. Virus Genes. 1998b,16 (3):267-276.
[44] Mankertz A, Domingo M, Folch JM, et al. Characterisation of PCV-2 isolates from Spain, Germany and France [J]. Virus Res, 2000a, 66(1): 65-77
[45] Mankertz A, Hattermann K, Ehlers B, et al. Cloning and sequencing of columbid circovirus (coCV), a new circovirus from pigeons [J]. Arch Virol, 2000b, 145(12): 246979
[46] Mankertz, A., Hillenbrand, B., Replication of Porcine circovirus Type 1 requires two proteins encoded by the viral rep gene [J]. Virology 2001,279 (2), 429-438.
[47] Mankertz A and Hillenbrand B. Analysis of transcription of porcine circovirus type 1 [J]. J Gen Virol 2002; 83: 2743-2751.
[48] Meehan BM, McNeilly F, Todd D, et al. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs [J]. J Gen Virol 1998; 79:2171-2179.
[49] Miyata H, Tsunoda H, Kazi A, et al. Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus [J]. J Virol, 1999,73: 3582-3586
[50] Morozov I, Sirinarumitr T, Sorden SD, et al. Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome [J]. J Clin Microbiol 1998,36(9): 2535-41.
[51] Nawagitgul P, Morozov I, Bolin S, et al. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein [J]. J Gen Virol 2000; 81: 2281-2287.
[52] Niagro FU, Forsthoefel AN, Lawther RP, et al. Beak and feather disease virus and porcine circovirus genomes: intermediates between the germiniviruses and plant circovirus [J]. Arch Virol, 1998.143:1723-1744
[53] Nishizawa T, Okamoto H, Konishi K, et al. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem. Biophys [J]. Res Commun, 1997.241:92-97
[54] Onuki A, Abe K, Togashi K, et al. Tsunemitsu H. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan [J]. J Vet Med Sci, 1999, 61(10): 1119-23
[55] Phenix KV, Weston JH, Ypelaar I, et al. Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae [J]. J Gen Virol, 2001, 82: 2805-9
[56] Pogranichnyy RM, Yoon K-J, Hums PA, et al. Characterization of immune response of young pigs to porcine circovirus type 2 infection [J], Viral Immunol 2000,13:143-153.
[57] Porntippa N, Lgor M, Steven R. Open reading frame 2 porcine circovirus type 2 encodes a major capsid protein [J]. J General Virology, 2000,81: 2281-2287
[58] Renee L, Andrzej B., Peter M., Ronald M. PCR Detection and Evidence of Shedding of Porcine Circovirus Type 2 in Boar Semen [J]. J Clin Micro. 2000,38(12): 4629-4632.
[59] Rohde W, Randies JW, Langridge P, et al. Nucleotide sequence of a circular single-stranded DNAassociated with coconut foliar decay virus [J]. Virology, 1990,176: 648-651
[60] Saoulidis K, Kyriakis SC, Kennedy S, et al. First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy s}mdrome in pigs in Greece [J]. J Vet Med B Infect Dis Vet Public Health, 2002,49(4): 202-5
[61] Sarradell J, Perez AM, Comba E, et al. Pathological findings in pigs affected by the postweaning multisystemic wasting syndrome in Argentina [J]. Rev Argent Microbiol, 2004, 36(3): 118-24
[62] Steinfeldt T, Finsterbusch T, Mankertz A. Rep and Rep' protein of porcine circovirus type 1 bind to the origin of replication in vitro [J]. Virology 2001; 291:152-160
[63] Takahashi K, Iwasa Y, Hijikata M, et al. Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus [J]. Arch Virol 2000, 145(5):979-993
[64] Terregino C, Montesi F, Mutinelli F, et al. Detection of a circovirus-like agent from farmed pheasants in Italy [J]. Vet Rec, 2001,149(11): 340
[65] Thomson J, Smith B, Allan q et al. PDNS, PMWS and porcine circovirus type 2 in Scotland. Porcine dermatitis and nephropathy syndrome. Post-weaning multisystemic wasting syndrome [J].Vet Rec, 2000,146 (22): 651-652
[66] Tischer I., Rasch R, and Tochtermann G Characterization of papovavirus and picornavirus-like particles in permanent pig kidney cell lines [J]. Zentbl Bakteriol Htg A, 1974,226:153-167
[67] Tischer I, Gelderblom H, Vettermann W. A very small porcine virus with circular single-stranded DNA [J]. Nature, 1982, 295: 64-66
[68] Tischer I, Peters D, Pociuli S.Occurrence and role of an early antigen and evidence for transforming ability of porcine circovirus [J]. Arch Virol. 1995; 140(10): 1799-816.
[69] Todd D, Creelan JL, Mackie DP, et al. Purification and biochemical characterization of chicken anaemia agent [J]. J Gen Virol, 1990, 71:819-823
[70] Todd D., Weston J.H., Soike D., et al. Genome sequence determinations and analyses of novel circoviruses from goose and pigeon [J]. Virology 2001.286:354-362
[71] Todd D, Bendinelli M, Biagini P, Hino S, Mankertz A, Mishiro S, Niel C, Okaraoto H, Raidal S, Ritchie BW, Teo GC, 2005. Circociridae [C]. In: Virus Taxonomy, 8th Report of the International Committee for the Taxonomy of Viruses (CM Fauquet, MA Mayo, J Maniloff, U Desselberger and LA Ball, eds), 327-334
[72] Toplak I, Groin J, Hostnik P, et al. Phylogenetic analysis of type 2 porcine circoviruses identified in wild boar in Slovenia [J]. Vet Rec, 2004, 155(6): 178-80
[73] Truong C, Mathe D, Blanchard P, et al. Identification of an immnorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection[J]. Arch Virol, 2001,146(6)1197～1211.
[74] Trujano M, lglesias G, Segales J, et al. PCV-2 from emaciated pigs in Mexico [J]. Vet Rec, 2001 148 (25): 792
[75] Walker IW, Konoby CA, Jewhurst VA, McNair I, McNeilly F, Meehan BM, Cottrell TS, Irllis JA, Allan GM.Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine ckcovirus type 2 [J]. J Vet Diagn Invest. 2000, 12(5): 400-5.
[76] Wallgren P, Hasslung F, Bergstrom G, et. al. Postweaning multisystemic wasting syndrome—PMWS the first year with the disease in Sweden [J]. Vet Q, 2004, 26(4): 170-87
[77] Wang C, Huang TS, Huang CC, et al. Characterization of porcine circovirus type 2 in Taiwan [J]. J Vet Meal Sci, 2004, 66(5): 469-75
[78] Wellenberg GJ, Stockhofe-Zurwieden N, de Jong MF, et al. Excessive porcine circovirus type 2 antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study [J]. Vet Microbiol, 2004, 99(3-4): 203-14
[79] 郎洪武，王力，张广川等．猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断[J]．中国兽医科技，2001，31(3)：3-5．
[80] 林绍荣，黄毓茂，苏丹萍，等，养猪[M]．2003．5：35-36
[81] 郎洪武，张广川，吴发权等．猪断奶多系统衰弱综合征血清抗体检测[J]．中国兽医科技，2000，30(3)：3-5
[82] 周继勇．陈庆新，叶菊秀，等．猪圆环病毒2型感染的血清学分析[J]．中国兽医学报，2004，24，(1)：1-3．
[1] Allan GM, McNeilly F, Foster JC, Adair BM. Infection of leucocyte cell cultures derived species with pig cirwvirus [J]. Vet Microbiol. 1994,41(3): 267-79.
[2] Allan GM, Kennedy S, McNeilly F, Foster JC, Ellis JA, Krakowka SJ, Meehan BM, Adair BM.Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus [J]. J Comp Pathol. 1999,121(1): 1-11.
[3] Allan, G.M., Ellis, J.A., Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation[J], 2000a,12: 3-14.
[4] Allan GM, McNeilly F, Ellis J, et al. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication [J]. Arch Virol, 2000b, 145(11): 2421-2429
[5] Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair I, Walker I, Kennedy S. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication [J]. Arch Virol. 2000c;145(11): 2421-9
[6] Blanchard P, Mahe D, Cariolet R, Keranflec'h A, Baudouard MA, Cordioli P, Albina E, Jestin A. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins [J]. Vaccine. 2003, 21(31): 4565-75.
[7] Chae C. A review of porcine circovirus 2-associated syndromes and diseases [J]. The Veterinary Journal. 2005,169: 326-336
[8] Chang HW, Pang VF, Chen LJ, Chia MY, Tsai YC, Jeng CR. Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages [J]. Vet Microbiol. 2006. 115(4):311-9.
[9] Chianini F, Majo N, Segales J, et al. Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2003, 94(1-2): 63-75
[10] Choi, C, Chae, G, Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephropathy syndrome by double-labeling technique [J]. Veterinary Pathology 2001,38: 436-441.
[11] Chung WB, Chan WH, Chaung HC, et al. Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs [J]. J Virol Methods, 2005,124(1-2): 11-19
[12] Chunmei Ju, Zhengfei Liu, Xin Xi, et al. Immunogenicity of a recombinant pseudorabies vims expressing ORF1 and ORF2 gene of porcine circovirus type2 [J]. Veterinary Microbiology 2005, 109:179-190
[13] Darwich L, Segales J, Domingo M, et al. Changes in CD4(+),CD8(+),CD4(+)CD8(+) and immunoglobulin M-positive peripheral blood mononuclear cells of postweaning multisystemic wasting syndrome-affected pigs and age-matched uninfected wasted and healthy pigs correlate with lesions and porcine circovirus type 2 load in lymphoid tissues [J]. Clin Diagn Lab Immunol, 2002,9(2): 236-242
[14] Darwich L, Balasch M, Plana-Duran J, et al. Cytokine profiles of peripheral blood mononuclear cells of pigs suffering from postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens [J]. J Gen Virol, 2003a, 84: 3453-3457
[15] Darwich L, Pie S, Rovira A, et al. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome [J]. J Gen Virol, 2003b, 84: 2117-2125
[16] Darwich L, Segales J, Mateu E, et al. Pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus 2: an immune riddle [J]. Arch Virol, 2004,149(5): 857-874
[17] Done SH, Bailey M, Gresham ACJ, et al. Postweaning multisystemic wasting syndrome (PMWS) .The current European situation [J]. Pig J, 2002,49: 215-232
[18] Duran, CO., Ramos-Vara, J.A., Render, J.A., Porcine dermatitis and nephropathy syndrome: a new condition to include in the differential diagnosis list for skin discoloration in swine [J]. Swine Health and Production 1997,5: 241-245.
[19] Fenaux M, Opriessnig T, Halbur PCB Elvinger F, Meng XJ.A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs [J].J Virol. 2004a Jun; 78(12): 6297-303.
[20] Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo [J]. J Virol. 2004b, 78(24):13440-6.
[21] Gilpin DF, Stevenson L, McCullough K, et al. Studies on the in vitro and in vivo effect of porcine circovirus type 2 infection of porcine monocytic cells [C]. In: Proc. ssDNA Viruses Plants Birds pigs Primates 97. plants, birds, pigs and primates, Saint-Malo. ZOOPOLE developpement (ISPAIA), France, 2001, 97
[22] Gilpin DF, McCullough K, Meehan BM, et al. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system [J]. Vet Immunol Immunopathol, 2003,94:149-161
[23] Halbur, P.G., Porcine respiratory disease [C]. Proceedings of the International Pig Veterinary Society Congress. 1998,15:1-10.
[24] Hamel, A.L., Lin, L.L., Nayar, G.P., Nucleotide sequence of Porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs [J]. J. Virol. 1998, 72 (6): 5262-5267.
[25] Hamel AL, Lin LL, Sachvie C, et al. PCR detection and characterization of type-2 porcine circovirus [J].Can J Vet Res,2000,64(1):44-52
[26] Harms PA, Sorden SD, Halbur PQ Bolin SR, Lager KM, Morozov I, Paul PS.Experimental reproduction of severe disease in pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus [J]. Vet Pathol. 2001,38(5): 528-39.
[27] Harms, P.A., Halbur, P.G., Sorden, S.D., Three cases of porcine respiratory disease complex associated with porcine circovirus type2 infection [J]. Journal of Swine Health and Production, 2002, 10:27-30.
[28] Hines, R.K., Lukert, P.D., Porcine circovirus as a cause of congenital tremors in newborn pigs [C]. In: Proceedings of the American Association on Swine Practitioners, 1994: 344-345.
[29] Hirai T, Nunoya T, Ihara T, et al. Acute hepatitis in a piglet experimentally inoculated with tissue homogenates from pigs with postweaning multisystemic wasting syndrome [J]. J Vet Med Sci, 2003, 65(9): 1041-5
[30] Josephson, G., Charbonneau, G., Case report of reproductive problems in a new startup operation [J]. Journal of Swine Health and Production 2001, 9: 258-259.
[31] Junghyun K, Chanhee C. A comparison of the lymphocyte subpopulations of pigs experimentally infected with porcine circovirus 2 and/or parvovirus [J].Vet J.2003,165:325-329.
[32] Kennedy S, Moffett D, McNeilly F, et al. Reproduction of lesions of postweaning multisystemic wasting syndrome infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus [J]. J Comp Pathol, 2000,122(1): 9-24
[33] Kennedy, S., Segales, J., Rovira, A., et al. Absence of evidence of porcine circovirus infection in piglets withcongenital tremors [J]. Journal of Veterinary Diagnostic Investigation 2003, 15: 151-156.
[34] Kim, J., Chung, H.-K., Chae, C, Association of porcine circovirus 2 with porcine respiratory disease complex [J]. The Veterinary Journal, 2003,166, 251-256.
[35] Kim J, Ha Y, Jung K, et al. Enteritis associated with porcine circovirus 2 in pigs [J]. Can J Vet Res, 2004a, 68(3): 218-21
[36] Kim, J., Chae, C. Concurrent presence of porcine circovirus type 2 and porcine parvovirus in retrospective cases of exudative epidermitis in pigs [J]. The Veterinary Journal 2004b.167,104-106.
[37] Kim J, Chae C. Necrotising lymphadenitis associated with porcine circovirus type 2 in pigs [J]. Vet Rec, 2005,156(6): 177-8
[38] Krakowka S, Ellis JA, McNeilly F, et al. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV2) [J]. Vet Pathol, 2001, 38(1):31-42
[39] Ladekjaer-Mikkelsen, A.S., Nielsen, J., Storgaard, T., et al. Transplacental infections with PCV2 associated with reproductive failure in a gilt [J]. Veterinary Record. 2001,148: 759-760.
[40] Ladekjaer-Mikkelsen AS, Nielsen J, Stadejek T, et al. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with Porcine circovirus type 2 (PCV2) [J]. Veterinary Microbiology, 2002,89:97-114
[41] Liu J, Chen I, Chua H, Du Q, Kwang J. Inhibition of porcine circovirus type 2 replication in mice by RNA interference [J]. Virology. 2006. 347(2):422-33.
[42] Mandrioli L, Sarli q Panarese S, et al. Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2004,97(l-2):25-37
[43] McNeilly F, McNair I I, O Connor M, et al . Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation , immunohistochemistry,and the polymerase chain reaction [I]. J Vet Diagn Invest, 2002,14(2);106-12
[44] McNulty M., Dale J., Lukert P., et al. Circoviridae. In: van Regenmortel, C.M.F.M.H.V., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Seventh Report of the International Committee on Taxonomy of Viruses,Academic Press, San Diego, 2000: 299-303.
[45] Meerts P, Misinzo G, McNeilly F, Nauwynck HJ.Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages [J]. Arch Virol 2005,150(3):427-410
[46] Nielsen J, Vincent IE, Botner A, et al. Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2003, 92: 97-111
[47] O_Connor, B., Grauvreau, H., West, K., et al. Multiple porcine circovirus 2-associated abortion and repdocutive failure in a multisite swine production unit [J]. Canadian Veterinary Journal 2001,42: 551-553.
[48] Opriessnig T, Yu S, Gallup JM, Evans RB, Fenaux M, Pallares F, Thacker EL, Brockus CW, Ackermann MR, Thomas P, Meng XJ, Halbur PGEffect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus [J]. Vet Pathol. 2003 Sep; 40(5): 521-9
[49] Opriessnig T, Thacker EL, Yu S, et al. Experimental reproduction of postweaning muttisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2 [J]. Vet Pathol, 2004b, 41(6): 624-40
[50] Ouardani M, Wilson L, Jette R, et al. Multiplex PCR for detection and typing of porcine circoviruses [J]. J Clin Microbial, 1999,379120:3917-24
[51] Pallares FJ, Halbur PCB Opriessnig T, et al. Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS) [J]. J Vet Diagn Invest, 2002, 14(6): 515-9
[52] Pesch S, Schmidt U, Ohlinger VF. Proliferative necrotizing pneumonia (PNP) is a result of coinfection with porcine reproductive and respiratory disease virus (PRRSV) and porcine circovirus type 2 (PCV2) [J]. In: Proceedings of the 16th International Congress on Pigs Veterinary Society, 2000,581
[53] Ramos-Vara, J.A., Duran, O., Render, J.A., et al. Porcine dermatitis and nephropathy syndrome in the USA [J]. Veterinary Record 1997,141:479-480.
[54] Romeo E., Sanchez J., Hans J., Nau Francis M.,Gordon M.,Maurice B. Porcine circovirus 2 infection in swine fetuses inoculated at different stages of gestation [J]. Vet Micro.2001,83:169-176.
[55] Rosell C, Segales J, Plana-Duran J, et al. Pathological, immunohistochemical, and in-situ hybridization studies on natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs [J]. J Comp Pathol, 1999,120 (1):59-78
[56] Rosell C, Segales J, Domingo M. Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2 [J]. Vet Pathol, 2000a, 37(6): 687-92
[57] Rosell, C, Segales, J., Ramos-Vara, et al. Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome [J]. Veterinary Record. 2000b,146: 40-43.
[58] Rovira A, Balasch M, Segales J, et al. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2 [J]. J Virol, 2002, 76(7): 3232-3239
[59] Segales J, Alonso F, Rosell C, et al. Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2001, 81(1-2):37-44
[60] Segales J,Pineiro C, Lampreave F, et al. Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Res, 2004,35(3): 275-282
[61] Shibahara T, Sato K, Ishikawa Y, et al. Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome [J]. J Vet Med Sci, 2000,62(11):1125-1131
[62] Sipos W, Duvigneau JC, Willhein m, et al. systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome as determined by semiquantitative RT PCR and flow cytometric intracellular cytokine detection [J]. Vet Immunol Immunopathol, 2004, 99: 63-71
[63] Smith, W.J., Thomson, J.R., Done, S., Dermatitis/nephropathy syndrome of pigs [J]. Veterinary Record. 1993,132, 47.
[64] Stevenson, G., Distribution of porcine circovirus in neonatal pigs with congenital tremors [C]. In: Proceedings of the CRWAD, 1999, p. 217.115(3-4):261-72.
[65] Stevenson GW, Kiupel M, Mittal SK, et al. Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors [J]. J Vet Diagn Invest, 2001, 13: 57-62
[66] Thacker B, Thacker E. The PRDC battle continues [J]. Pig Prog, 2000,16-18
[67] Thacker, E.L., Porcine respiratory disease complex - what is it and why does it remain a problem? [J]. The Pig Journal. 2001,48: 66-70.
[68] Tischer I., Rasch R, and Tochtermann G Characterization of papovavirus and picornavirus-like particles in permanent pig kidney cell lines [J]. Zentbl Bakteriol Htg A, 1974,226:153-167
[69] Todd D., Niagro F.D., Ritchie B.W., et al. Comparison of three animal viruses with circular single-stranded DNA genomes [J]. Arch. Virol. 1991.117:129-135.
[70] Todd D., Weston J.H., Soike D., et al. Genome sequence determinations and analyses of novel circoviruses from goose and pigeon [J].Virology 2001.286:354-362
[71] Wattrang E, McNeilly F, Allan GM, et al. Exudative epidermitis and porcine circovirus-2 infection in a Swedish SPF-herd [J]. Vet Microbiol, 2002,86(4): 281-293
[72] Wellenberg, G.J., Pesch, S., Berndsen, F.W., et al. Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands [J]. Vet. Quart. 2000,22:167-172.
[73] West, K.H., Bystrom, J.M., Wojnarowicz, C, et al. Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2 [J]. Journal of Veterinary Diagnostic Investigation 1999,11: 530-532.
[74] Woods L.W., Latimer K.S., Barr B.C., et al. Circovirus-like infection in a pigeon [J]. J. Vet. Diagn. Invest. 1993,5:609-612.
[75] Yu S, Opriessnig T, Kitikoon P, Nilubol D, Halbur PG, Thacker E. Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs [J]. Vet Immunol Immunopathol. 2007.115(3-4):261-72.
[1] Ait Si Ali S, Guasconi V, Harel Bellan A. RNA interference and its possible use in cancer therapy [J]. Bull Cancer. 2004,91(1):15-18.
[2] Ashrafi K, Chang FY, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory [J], Gene. 2003,421(6920):268～272.
[3] Barton G M, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells [J]. PNAS USA, 2002,99(23):14943-14945.
[4] Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses [J]. BMC Microbiol, 2001,1:34-45.
[5] Boutros M, Kiger AA, Armknecht S, et al. Genome-wide RNAi analysis of growth and viability in Drosophilacells.Science,2004,303(5659):832～835
[6] Brigham PM. Cosuppression comes to the animals [J].Cell. 1997,90(3):385-387.
[7] Brummelkamp TR, Bernards R, Agami R. Asystem for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002,296(5567):550-553.
[8] Castanotto D, Li H, Rossi JJ.Functional siRNA exprssion from transfected PCR products [J]. RNA. 2002.8(11):1454-1460
[9] Chen W.Yan W, Du Q, et al. RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice [J]. J Virol, 2004,78: 6900-6907.
[10] Cocks B G, Theriault T P. Developments in effective application of small inhibitory RNA(siRNA) technology in mammalian cells [J].Targets,2004,3:165-171.
[11] Dykxhoom DM, Novina CD, Sharp PA. Killing the messenger short RNAs that silence gene exprssion [J]. Nat Rev Mol Cell Biol. 2003.4( 6):457-467
[12] Elbashir S M, Harborth J.Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001,411:494-498.
[13] Fedoriw AM, Stein P, Svoboda P, et al.Transgenic RNAi Reveals Essential Function for CTCF in H19 Gene Imprinting [J]. Science. 2004,303(5655):238-240.
[14] Fire A, Xu S, h4ontgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhaditis elegans [J]. Nature, 1998,391:806-811.
[15] Ge Q, Filip L, Bai A, et al.Inhibition of influenza virus production in virus-infected mice by RNA interference [J].Proc Natl Acad Sci USA, 2004,101:8676-8681.
[16] Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells [J]. Nature. 2002.418 (6896): 430-434
[17] Hannon GJ. RNA inference [J]. Nature. 2002,418(6894):244-251
[18] Han S, Mahato R I, Sung Y K, et al. Development of biomaterials for gene therapy [J]. Mol Ther, 2000,2:302-317.
[19] Hugh PC, Tomoyasu S, Chen ES, et al. Comprehensive analysis of heterochromatin and RNAi mediated epigenetic control of the fission yeast genome [J]. Nature Genetics,2005,37(8):809～819.
[20] Kasschau KD, Carrington JC. Acounter defensive strategy of plant viruses: suppression of posttranscriptional gene silencing [J].Cell,1998,95(4):461-470.
[21] Ketting RF, Fischer SE, Bernstein E, et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans [J]. Genes. 2001,15(20):2654-2659.
[22] Khvorova A, Reynolds A, Jayasena S D. Functional siRNAs and miRNAs exhibit strand bias [J].Cell, 2003,115: 209-216.
[23] Kitabwalla M, Ruprecht RM. RNA interference a new weapon a gainst HIV and beyond N Engl [J]. J Med. 2002, 347( 17):1364-1367
[24] Klein C, Bock C T, Wedemeyer H, et al. Inhibition of hepatitis B virus replication in vivo by nucleoside and siRNA [J].Gastroenterology,2003,125:9-18.
[25] Martinoz J, Patkaniowska A, Urlaub H, I.ahrmann R, Tuschl t. Single stranded antisense siRNAs wide Tareet RNA cleavaee in RNAi [J]. Cell. 2002.110(5):563
[26]Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA [J]. Nature. 2004,431(7006):343-349.
[27] Pardridge W M. Intravenous non-viral RNAi gene therapy of brain cance [J]r. Expert Opin Biol Ther. 2002. 41,2435-2438.
[28] Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference [J]. Nat Genet, 2003,33:401-406.
[29] Salvi A, Arici B, DePeLro G, et al. Small interfering RNA urokinase siliencing inhibits a invasion and migraLion of human hepatocellular carcinoma cells [J]. Mol Cancer Ther. 2004,36:671.
[30] Schwarz D S, Hutvagner G, Du T, et al.Asymmetry in the assembly of the RNAi enzyme complex [J]. Cell, 2003,115:199-208.
[31] Svoboda P, Stein P, Hayashi H, et al.RNAi mRNA in ones oocyte by RNA interference [J]. Development, 2000, 127:4147-4156.
[32] Tachibana R, Harashima H, Ide N, et al. Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes [J]. Pharm Res, 2002,377-381.
[33] Timmons L, Court D, Fire A. Ingestion of bacterially expressed dsRNA can produce specific and potent genetic interference in Caenorhabitis elegans [J]. Gene. 2001. 263:103.
[34] Tuschl T, Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double-stranded RNA invitro [J]. Genes. 1999,13(24): 3191-3197.
[35] Tuschl T. Expanding small RNA interference [J]. Nat Bioteclmol, 2002,20: 446-448.
[36] Qin X F, An D S, Chen I S,et al. Inhibiting HIV 1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCRS [J]. PNAS USA,2003,100(1): 183-188.
[37] Ui-Tei K, Naito Y, Takahashi F, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference [J]. Nucleic Acids Res, 2004,32: 936-948.
[38] Van der Krol AR, Mur LA, de Lange P, et al. Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect [J]. Plant Mol Biol, 1990,14(4): 457-466.
[39] Volpe TA, Kidner C, Hall IM, et al. Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi [J]. Science. 2002,297(5588): 1833-1837.
[40] Wang Z, Ren L, Zhao X, et al. Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells [J]. J Virol, 2004,78: 7523-7527.
[41] Wassenegger M. The role of the RNAi machinery in heterochromatin formation [J]. Cell. 2005, 122(1): 13-16.
[42] Yokota T, Sakamoto N, Enomoto N, et al.Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs [J]. EMBO Rep, 2003, 4: 602-608.
[43] Zamore PD, Tuschl T, Sharp PA,et al. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals [J]. Cell,2000,101(1): 25-33.
[44] Zeng Y, Cullen BR. RNA interference in human cells is restricted to the cytoplasm [J]. RNA. 2002, 8 (7): 855-860
[45] Zhao L J, Jian H, Zhu H. Specific gene inhibition by adenovirus mediated expression of small interfering RNA [J]. Gene, 2003,316:137-141.
[46] Zheng ZM, Tang S, Tao MF. Development of Resistancet RNAi in Mammalian Cells [J]. Science,2005,1058: 105-118
[47] 钟苗，雷小勇．RNA沉默：基因组的免疫系统[J]．免疫学杂志，2004，20(2)：83-85．
[1] 郎洪武，王力，张广川等．猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断．中国兽医科技[J]，2001，3：3-5．
[2] Allan G.M., Phenix K.V., Todd D., et al..Some biological and physico-chemical properties of porcine circovirus.J.Vet.Med [J],1994,B41: 17-26
[3] Allan O M, McNeilly F, Cassidy J P, et al. Pathogenesis of porcine curcovirus: experimental infections of colostrums deprived piglets and examination of pig fetal material [J]. Vet Microbio! [J],1995,44: 49-64
[4] Allan G.M., B.Meehan, D.Todd, et.al. Novel porcine circovirus from pigs with wasting disease syndromes. Vet.Rec [J], 1998,142: 467-468
[5] Allan G.M., F.McNeilly, S.Kennedy, et.al. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in USA and Europe. J.Vet. Diagn.Investig [J],1998,10: 3-10
[6] Allan, G.M., Ellis, J.A., Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation [J], 2000,12: 3-14.
[7] Chun WANG, Tien-Shine HUANG, Chin-Cbeng HUANG (2004) Characterization of porcine circovirus type 2 in Taiwan [J]. J Vet Meal Sci 66(5): 469-475
[8] Clark, E.G,.Post-weaning multisystemic syndrome. In: Proceedings of the American Association of Swing Practitioners [J], 1997, p. 499-501
[9] Dulac G.C., Afshar A. Porcine circovirus antigens in PK-15cell line (ATCC CCL-33) and evidence of antibodies to curcovirus in Canadian pigs [J]. Can.J.Vet. Res, 1989, 53: 431-433
[10] F. D. Niagro, A.N.Forsthoefel, R.P.Lawther et al Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circiviruses Arch Virol [J],1998,143: 1723-1744
[11] Fenaux M, Halbur PK, Gill M, et al. Genetic characterization of type 2 porcine circovirus(PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2 [J]. J Clin Microbiol, 2000, 2494-2503.
[12] Gresham, A., Allan, G., McNeilly, F., et al. Links between post-weaning multisystemic wasting syndrome and porcine dermatitis nephropathy syndrome [J]. The Pig Journal, 2001,47:155-159.
[13] Harms, P.A., Halbur, P.G., Sorden, S.D., Three cases of porcine respiratory disease complex associated with porcine circovirus type2 infection [J]. Journal of Swine Health and Production, 2002,10: 27-30.
[14] Jemersic L, Cvetnic Z, Toplak I, Spicic S, Groin J, Barlic-Maganja D, Terzic S, Hostnik P, Lojkic M, Humski A, Habrun B, Krt B. Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia [J]. Res Vet Sci. 2004, 77:171-175
[15] Jin H Kim, Young S Lyoo. Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs [J]. J Vet Sci. 2003, 3(1):31-39
[16] Kim J, Chae C. Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome [J]. Can J Vet Res. 2003, 67(2):133-137
[17] Kim, J., Chung, H. -K., Chae, C., Association of porcine circovirus 2 with porcine respiratory disease complex. The Veterinary Journal [J], 2003b, 166, 251-256.
[18] Mahe D., Blanchard P., Truong C., Arnauld, C., Le Cann P., Cariolet R., Madec F., Albina E., Jestin A. Differential recognition of ORF2 protein from type 1 and 2 porcine circoviruses and identification of immunorelevant epitopes [J]. J. Gen. Virol. 2000, 81:1815-1824.
[19] Maldonado J, Segales J, Calsamiglia M, Llopart D, Sibila M, Lapus Z, Riera P, Artigas C. Postweaning multisystemic wasting syndrome (PMWS) in the Philippines: porcine circovirus type 2 (PCV2) detection and characterization [J]. J Vet Med Sci. 2004, 66(5):533-7.
[20] Meehan, B. M., McNeilly, F., McNair, I., et al. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome. Archives of Virology [J], 2001. 146: 835-842.
[21] Thomson, J., Henderson, L., Meikle, C., MacIntyre, N., Porcine dermatitis and nephropathy syndrome. Veterinary Record [J], 2001a, 148, 282-283.
[22] Tischer I., Mields W, Wolff D, et al. Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol [J], 1986, 91:271-276
[23] Wen L, Guo X, Yang H. Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China [J]. Vet Microbiol. 2005, 110(1-2):141-6
[24] Larochelle R, Magar R, D'Allaire S. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strain from cases presenting various clinical conditions [J]. Virus Res, 2002, 90(1-2):101-112.
[25] Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo [J]. J Virol. 2004, 78(24): 13440-6.
[26] Mankertz A, omingo M, FolchJ M, et al. Characterization of PCV2 isolates from Spain, Germany and France [J]. Virus Res, 2000, 66(1):65-77.
[1] Clark EG. Postweaning Multisystemtic Wasting Syndrome [A]. Proceedings of the American Association of Swine Practitioners [C]. 1997, 499-501.
[2] Allan GM, Mc Neilly F, Meehan BM, et al. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland [J]. Vet Microbiol, 1999, 66(2):115-123.
[3] Choi C, Kim J, Kang IJ, et al. Concurrent outbreak of PMWS and PDNS in a herd of pigs in Korea [J]. Vet Rec, 2002, 151(16):484-485.
[4] Drolet R, Larochelle R, Morin M, et al. Detection rates of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and swine influenza virus in porcine proliferative and necrotizing pneumonia [J]. Vet Pathol, 2003, 40(2): 143-148.
[5] Chun WANG, Tien-Shine HUANG, Chin-Cheng HUANG. Characterization of porcine circovirus type 2 in Taiwan [J]. J Vet Med Sci, 2004, 66(5): 469-475.
[6] Lang H W, Zhang G C, Wu F Q,(郞洪武，张广川，吴发权) et al. Detection of Serum Antibody against Postweaning Multisystemic Wasting Syndrome in Pigs [J]. Chinese Journal of Veterinary Science and Technology(中国兽医科技)，2000，30(3)：3-5．
[7] Cao S B, Cheng H C, Shao S B,(曹胜波，陈焕春，肖少波)et al. The Establishment And Application of PCR to Diagnosis Porcine Circovirus Type 2 [J]. Journal of Huazhong Agricultural(华中农业大学学报)，2001，1：53-56．
[8] Cui S J, Cai Y. Cheng X,(崔尚金，蔡阳，陈欣).Development and appfication of a competitive quantitative assay for monitoring porcine circovirus DNA [J]. Chinese Journal of Preventive Veterinary Medicine(中国预防兽医学报)，2004，26(5)：347-351．
[9] Tischer, I., Rasch, R., Tochtermann, G. Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines[A]. Zentralblatt fur Bakteriologie, Parasitenkund, Infecktionskrankheiten und Hygiene-Erste Abteilung Originale-Reihe A: Medizinische Mikrobiologie und Parasitologie[C]. 1974, 226, 153-167.
[10] Allan GM, McNeilly F, Kennedy S, et al. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA AND Europe [J]. J Vet Diagn Invest, 1998, 10:3-10.
[11] Inger Marit Brunborg, Torfinn Moldal, Christine Monceyron Jonassen. Quantitation of porcine circovirus type 2 isolated from serum/plasma andtissue samples of healthy pigs and pigs with postweaning multisystemicwasting syndrome using a TaqMan-based real-time PCR [J]. J Virol Meth, 2004, 122, 171-178.
[12] Stevenson GW, Kiupe IM, Mittal SK, et al. Ultra structure of porcine circovirus in persistently infected PK-15 cells [J]. Vetpathology, 1999, 36: 368-378.
[13] Wang X W, Jiang P, Li Y F,(王先炜，姜平，李玉峰)et al. Detection and Genetic Characterization of Porcine Circovirus(PCV-2) from Pigs with Postweaning Multisystemic Wasting Syndrome in Mid-east of China [J]. Chinese Journal of Veterinary(中国兽医学报)，2004，24(3)：240-242．
[14] Zhang Zh D, Soren A. Detection of carrier cattle and sheep persistently infected with foot-and-mouth disease virus by a rapid real-time RT-PCR assay [J]. J Virol Meth, 2003, 111: 95-100.
1. Jiang P, Chen P Y, Dong Y Y, et al, Isolation and genome characterization of porcine reproductive and respiratory syndrome virus in PR China[J]. Vet Diagn Invest, 2000, 12: 156-158.
2. Lager K M, Mengeling W L; Brockmeier S L, Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine [J].Vet Microbiol, 1997, 58: 127-133
3. Christoph E,Barbara T,Luzia L, et al. Quantitative TaqMan RT-PCR for the detection and differantiation of European and North American strains of porcine reproductive and respiratory syndrome virus [J]. Jour.Virol Meth, 2001,98:63-75
4. Albina E, Epidemiology of porcine reproductive and respiratory syndrome: an overview [J].Vet Microbiol. 1997, 55: 309-316
5. Will R W, Zimmerman J J, Yoon K J, et al. porcine reproductive and respiratory syndrome vires: routes of excretion [J]. Vet Microbiol, 1997,57:69-81
6. Beyer E M, Fichtner D, Schirrmerier H,et al. porcine reproductive and respiratory syndrome virus: kinetics of infection in lymphatic organs and lung [J].J Vet Med, 2000, 47: 9-25
[1] Abdalla A.O., Kiaii S., Hansson L et al. Kinetics of cytokine gene expression in human CD4+ and CD8+ Tlymphocyte subsets using quantitative real-time PCR [J].Scand. J. Immunol, 2003; 58:601.
[2] Anderson I.E., Reid H.W., Nettleton P.F et al. Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions [J]. Vet. Immunol. Immunopathol, 2001; 83:161.
[3] Audige A., Schlaepfer E., Bonanomi A et al. HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo [J]. J. Immunol, 2004; 172: 2687.
[4] Bailey L.O., Washburn N.R., Simon Jr et al. Quantification of inflammatory cellular responses using real-time polymerase chain reaction [J]. J. Biomed. Mater. Res, 2004; 69A: 305.
[5] Biron C.A. Role of early cytokines, including alpha and beta interferons (IFN-alpha/beta), in innate and adaptive immune responses to viral infections [J]. Semin. Immunol, 1998; 10: 383.
[6] Kvarnstrom M, Jenmalm M.C, Ekerfelt CEffect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4[J]. Cryobiology, 2004; 49:157.
[7] L Darwich, J Segales, E Mateu. Pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus 2: an immune riddle [J]. Arch. Virol, 2004; 149: 857-874
[8] Murtaugh M.P., Baarsch M.J., Zhou Yet al. Inflammatory cytokines in animal health and disease [J]. Vet. Immunol Immunopathol, 1996; 54:45-55
[9] Rottman, J.B. Key role of chemokines and chemokine receptors in inflammation, immunity, neoplasia, and infectious disease[J]. Vet. Pathol, 1999; 36: 357.
[10] Roongroje, Theresa F, Brad J et al. Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model [J]. Vet. Immunol Immunopathol, 2001;79:115-127
[11] Sanipa Suradhat, Roongroje Thanawongnuwech. Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus [J]. J Gen Virol, 2003; 84:2755-2760
[12] W.H. Feng, M.B. Tompkins, J S Xu et al. Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus [J]. Vet. Immunol Immunopathol, 2003; 94: 35-45
[13] Atabak R. Royaee, Robert J. Husmann, Harry D. Dawson et al. Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination [J]. Vet. Immunol Immunopathol, 2004; 102: 199-216
[14] Diaz I, Darwich L, Pappaterra G et al. Immune responses of pigs after experimental infection with a European strain of porcine reproductive and respiratory syndrome virus[J]. J Gen Virol, 2005; 86(Pt7): 1943-51
[1] Allan GM, Ellis JA. Porcine circoviruses: a review [J]. J Vet Diagn Invest, 2000, 12:3-14.
[2] Choi, C., Chae, C. Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in situ hybridization [J]. J. Comp. Pathol, 2000, 123: 302-305.
[3] Collins J.E., Benfield D.A., Christianson W.T., et al. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs [J]. J. Vet. Diag. Invest, 1992, 4:117-126.
[4] D.F. Gilpin, K. McCullough, B.M. Meehan, et al. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system [J]. Veterinary Immunology and Immunopathology, 2003, 94:149-161.
[5] 冯志新，姜平，王先炜，等．猪圆环病毒2型TaqMan实时PCR检测方法的建立[J]．中国病毒学，2006，21(4)：371-374．
[6] Galina L., Pijoan C., Sitjar M., et al. Interaction between Streptococcus suis serotype 2 and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets [J]. Vet. Rec. 1994, 134: 60-64.
[7] Harding JCS, Clark EG. Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS) [J]. J Swine Health Prod. 1997, 5:201-203.
[8] Harding JCS, Clark EG, Strokappe JH, et al. Postweaning multisystemic wasting syndrome: epidemiology and clinical presentation [J].J Swine Health Prod. 1998, 6:249-254.
[9] Harms P.A., Sorden S.D., Halbur P.G., et al. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus [J]. Vet. Pathol. 2001, 38: 528-539.
[10] Hui-Wen Chang, Chian-Ren Jeng, Jiuan J. Liu, et al. Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha [J]. Veterinary Microbiology, 2005, 108:167-177.
[11] Hui-Wen Chang, Victor Fei Pang, Liang-Jiun Chen, et al. Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages [J]. Veterinary Microbiology, 2006, 115(4):311-9.
[12] Laila Darwich, Sandrine Pie', Albert Rovira, et al. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome [J]. Journal of General Virology, 2003, 84:2117-2125.
[13] Lawson, S.R., Rossow, K.D., Collings, J.E., et al. Porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with MAC-387 staining at 21 days post infection [J]. Virus Res. 1997, 51: 105-113.
[14] O'Connor B., Gauvreau H., West K.H., et al. Multiple porcine circovirus2-associated abortions and reproductive failure in a multisystemic swine production unit [J]. Can. Vet. J. 2001, 42: 551-553.
[15] P. A. Harms, S. D. Sorden, P. G. Halbur, et al. Experimental Reproduction of Severe Disease in CD/CD Pigs Concurrently Infected with Type 2 Porcine Circovirus and Porcine Reproductive and Respiratory Syndrome Virus [J]. Vet Pathol, 2001, 38:528-539.
[16] R. Drolet, R. Larochelle, M. Morin, et al. Detection Rates of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, and Swine Influenza Virus in Porcine Proliferative and Necrotizing Pneumonia [J]. Vet Pathol, 2003, 40:143-148.
[17] A. Rovira, M. Balasch, J. Segales, et al. Experimental Inoculation of Conventional Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2 [J]. Journal of Virologs, 2002, 76(7): 3232-3239.
[18] Chiou, M.T., Jeng, C.R., Chueh, L.L., et al. Effects of porcine reproductive and respiratory syndrome virus (isolate tw91) on porcine alveolar macrophages in vitro [J]. Vet. Microbiol, 2000, 71, 9-25.
[19] Oleksiewicz, M.B., Nielsen, J. Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function [J]. Vet. Microbiol, 1999, 66, 15-27.
[20] 冯志新，姜平．猪细胞因子SYBR Green实时PCR检测方法的建立[J]．现代生物医学进展，2007．
[21] 黄娟，姜平，张常印等．猪繁殖与呼吸综合征病毒实时PCR检测方法的建立[J]．中国病毒学，2005，20(5)：530-533．
[22] 彭长凌，，高崧，刘秀梵．PRRSV人工单独感染及其与PCV2人工联合感染的病理学比较[J]．徐州医学院学报，2006，26(1)：49-52
[1] Allan, G. M., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Bother, A., Charreyre, C., 1998. Novel porcine circoviruses from pigs with wasting disease syndromes [J]. Veterinary Record 142, 467-468.
[2] Guo S, Kemphues KJ. 1995. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J].Cell. 81(8): 611-620.
[3] Haibin Xia, Qinwen Mao siRNA-mediated gene silence in vivo and in vitro [J]. Nature biotechnology. 2002.20, 1006-1010
[4] Jana S, Chakraborty C, Nandi S, Deb JK. RNA interference: potential therapeutic targets [J]. Appl Microbiol Biotechnol. 2004. 65(6):649-57.
[5] Liu J, Chen I, Chua H, Du Q, Kwang J. Inhibition of porcine circovirus type 2 replication in mice by RNA interference [J]. Virology. 2006.10; 347(2):422-33.
[6] Meehan, B. M., McNeilly, F., McNair, I., Walker, I., Ellis, J. A., Krakowka, S., Allan, G. M. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome [J]. Archives of Virology. 2001. 146, 835—842.
[7] Chen WZ, Liu MQ, Jiao Y, Yan WY, Wei XF, Chen J, Fei L, Liu Y, Zuo XP, Yang FG, Lu YG, Zheng ZX. Adenovirus-Mediated RNA Interference against Foot-and-Mouth Disease Virus Infection both In Vitro and In Vivo [J]. J Virol. 2006.80(7): 3559-3566.
[8] Han S, Mahato R I, Sung Y K, et al. Development of biomaterials for gene therapy [J]. Mol Ther, 2000, 2:302-317.
[9] Sergio C, Abdullah E, Carol C, Naazneen M, Felix H. S, Fatricia L. M, Nicolas F, Marc S. W, Patrick A. Effective Inhibition of HBV Replication in Vivo by Anti-HBx Short Hairpin RNAs [JJ. Molecular Therapy. 2005. 13(2): 411-421.
[1] Allan, G. M., F. McNeilly, J. P. Cassidy, G. A. Reilly, B. Adair, W. A. Ellis, and M. S. McNulty. Pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material [J]. Vet. Microbiol. 1995.44:49-64.
[2] Allan, G. M., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Botner, A., Charreyre, C. Novel porcine circoviruses from pigs with wasting disease syndromes [J]. Veterinary Record. 1998.142, 467-468.
[3] Brunborg IM, Moldal T, Jonassen CM. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR [J]. J Virol Methods. 2004. 122(2): 171-8.
[4] Chen, W., W. Yah, Q. Du, L. Fei, M. Liu, Z. Ni, Z. Sheng, and Z. Zheng. RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice [J]. J. Virol. 2004. 78:6900—6907.
[5] Cheung, A. K. Transcriptional analysis of porcine circovims type 2 [J]. Virology. 2003.305: 168-180.
[6] Cobum GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference [J]. J Virol. 2002.76(18):9225-31.
[7] Ding, S. W., H. Li, R. Lu, F. Li, and W. X. Li. RNA silencing: a conserved antiviral immunity of plants and animals [J]. Virus Res. 2004.102:109-115.
[8] Dulac GC, Afshar A. Porcine circovirus antigens in pk-15 cell line (ATCCCCL-33) and evidence of antibodies to circovirus in Canadian pigs [J]. Can J Vet Res. 1989. 53: 431-433
[9] Oitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells [J]. Nature. 2002. 418(6896): 430-434
[10] Gitlin, L., and R. Andino. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing [J]. J. Virol. 2003.77:7159-7165.
[11] Haasnoot, P. C., Cupac, D., Berkhout, B., 2003. Inhibition of virus replication by RNA interference. J. Biomed. Sci. 10, 607-616.
[12] Hammond, S. M., Caudy, A. A., Harmon, O. J. Post transcriptional gene silencing by double-stranded RNA [J]. Nat. Rev., Genet. 2001.2, 1110-1119.
[13] Harborth, J., Elbashir, S.M., Vandenburgh, K., Manninga, H., Scaringe, S.A., Weber, K., Tuschi, T. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gone silencing [J]. Antisense Nucleic Acid Drug Dev. 2003.13, 83-105.
[14] Morris, K.V., Chan, S.W.L., Jacobsen, S.E., Looney, D.J. Small interfering RNA-induced transcriptional gone silencing in human cells [J]. Science. 200427, 1289-1292.
[15] Jana S, Chakraborty C, Nandi S, Deb JK. RNA interference: potential therapeutic targets [J]. Appl Microbiol Biotechnol. 2004.65(6):649-57.
[16] Kameoka M, Nukuzuma S, Itaya A, Tanaka Y, Ota K, Ikuta K, Yoshihara K. RNA interference directed against Poly(ADP-Ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells [J]. J Virol. 2004.78(16):8931-4
[17] Li Yao-Chen, Zhao Zhi-Gang, Li Kang-Sheng, RNA interference mediated inhibition of influenza virus NP and PA gone expressions and multiplication in MDCK cell [J]. J Fourth Mil Med Univ. 2006.27(8):681-685.
[18] Liu J,Chen I,Kwang J. Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis [J]. J Virol._2005. 79(13):8262-74.
[19] Liu J, Chen I, Chua H, Du Q, Kwang J. Inhibition of porcine circovirus type 2 replication in mice by RNA interference [J]. Virology. 2006. 10;347(2):422-33.
[20] Liu Q, Wang L, Willson P, Babiuk LA. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome [J]. J Clin Microbiol. 2000.38(9):3474-7.
[21] Mankertz, A., J. Mankertz, K. Wolf, and H. J. Buhk. Identification of a protein essential for replication of porcine circovirus [J]. J. Gen. Virol. 1998.79: 381-383.
[22] McManus, M.T., Sharp, P.A. Gene silencing in mammals by small interfering RNAs [J]. Nat. Rev., Genet. 2002.3, 737-747.
[23] Meehan, B.M., McNeiUy, F., McNair, I., Walker, I., Ellis, J.A.,Krakowka, S., Allan, G.M. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome [J]. Archives of Virology. 2001.146, 835-842.
[24] Moore MD, McGarvey MJ, Russell RA, et al. Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors [J]. J Gene Med. 2005.7(7):918-925.
[25] Nawagitgul, P., I. Morozov, S. R. Bolin, P. A. Harms, and S. D. Sorden. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein [J]. J. Gen. Virol. 2000. 81:2281-2287.
[26] Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HIV-1 infection [J]. Nat Med. 2002.8(7):681-6.
[27] Olvera A, Sibila M, Calsamiglia M, Segales J, Domingo M. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs [J]. J Virol Methods. 2004.117(1):75-80
[28] Persengiev, S.P., Zhu, X., Green, M.R. Nonspecific, concentration dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs) [J]. RNA. 2004.10, 12-18.
[29] Pringle, C. R. Virus taxonomy at the Xlth International Congress of Virology, Sydney, Australia [J]. Arch. Virol. 1999.144:2065-2070.
[30] Randall G, Grakoui A, Rice C M. Clearance ofrplicating hepatitis C virus rplicon RNAs in cell culture by small interfering RNAs [J]. Proc Natl Acad Sci USA, 2003.100(1):235-240.
[31] Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, and Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells [J]. Proc. Natl. Acad. Sci. USA. 2002. 99(8): 5515-5520.
[32] Tischer, I., W. Mields, D. Wolff, M. Vagt, and W. Griem. Studies on epidemiology and pathogenicity of porcine circovirus [J]. Arch. Virol. 1986. 91:271-276.
[33] Zhou XM, Lin JS, Shi Y, Tian DA, Huang HJ, Zhou HJ, Jin YX. Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene [J]. Acta Biochim Biophys Sin (Shanghai). 2005.37(5):310-6.
[34] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal. Biochem. 1976. 72, 248-254
[35] 温立斌．猪圆环病毒2型分子流行病学研究[D]．北京：中国农业大学，2005
[1] Allan, G. M., F. McNeilly, J. P. Cassidy, G. A. Reilly, B. Adair, W. A. Ellis, and M. S. McNulty. Pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material [J]. Vet. Microbiol. 1995.44:49—64.
[2] Allan, G.M., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Botner, A., Charreyre, C. Novel porcine circoviruses from pigs with wasting disease syndromes [J]. Veterinary Record 1998. 142, 467-468.
[3] Brunborg IM, Moldal T, Jonassen CM. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR [J]. J Virol Methods. 2004. 15; 122(2): 171-8.
[4] Chen, W., W. Yan, Q. Du, L. Fei, M. Liu, Z. Ni, Z. Sheng, and Z. Zhong. RNA interference targeting VPI inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice [J]. J. Virol. 2004. 78:6900-6907.
[5] Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference [J]. J Virol. 2002.76(18):9225-31.
[6] Ding, S. W., H. Li, R. Lu, F. Li, and W. X. Li. RNA silencing: a conserved antiviral immunity of plants and animals [J]. Virus Res. 2004. 102:109-115.
[7] Dulac GC, Afshar A. Porcine circovirus antigens in pk-15 cell line (ATCCCCL-33) and evidence of antibodies to circovirus in Canadian pigs [J]. Can J Vet Res. 1989. 53:431—433
[8] Gitlin, L., and R. Andino. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing [J]. J. Virol. 2003.77:7159-7165.
[9] Haasnoot, P.C., Cupac, D., Berkhout, B. Inhibition of virus replication by RNA interference [J]. J. Biomed. Sci. 2003.10, 607—616.
[10] Kiupel, M., Stevenson, G.W., Choi, J., Latimer, K.S., Kanitz, C.L., Mittal, S.K. Viral replication and lesions in BALB/c mice experimentally inoculated with porcine circovirus isolated from a pig with postweaning multisystemic wasting disease [J]. Vet. Pathol. 2001.38, 74-82.
[11] Jana S, Chakraborty C, Nandi S, Deb JK. RNA interference: potential therapeutic targets [J]. Appl Microbiol Biotechnol. 2004.65(6):649-57.
[12] Kameoka M, Nukuzuma S, Itaya A, Tanaka Y, Ota K, Ikuta K, Yoshihara K. RNA interference directed against Poly (ADP-Ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells [J]. J Virol. 2004.78(16):8931-4
[13] Li Yao-Chen, Zhao Zhi-Gang, Li Kang-Sheng. RNA interference mediated inhibition of influenza virus NP and PA gene expressions and multiplication in MDCK cell [J]. J Fourth Mil Med Univ. 2006. 27(8):681-685.
[14] Liu Q, Wang L, Willson P, Babiuk LA. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome [J]. J Clin Microbiol. 2000. 38(9):3474-7.
[15] Lu, S., and B. R. Cullen. Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and microRNA biogenesis [J]. J. Virol. 2004.78:12868-12876.
[16] Morris, K.V., Chan, S.W.L., Jacobsen, S.E., Looney, D.J. Small interfering RNA-induced transcriptional gene silencing in human cells [J]. Science. 2004.27,1289- 1292.
[17] Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HTV-1 infection [J]. Nat Med. 2002. 8(7):681-6.
[18] Olvera A, Sibila M, Calsamiglia M, Segales J, Domingo M. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs [J]. J Virol Methods. 2004.117(1):75-80
[19] Pringle, C. R. Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia [C]. Arch. Virol. 1999. 144:2065-2070.
[20] Quintana, J., Balasch, M., Segale's, J., Calsamiglia, M., Rodn'guez-Arrioja, G.M., Plana-Dura'n, J., Domingo, M. Experimental inoculation of porcine circoviruses type 1 (PCV1) and type 2 (PCV2) in rabbits and mice [J]. Vet. Res. 2002. 33,229-237.
[21] Tischer, I., W. Mields, D. Wolff, M. Vagt, and W. Griem. Studies on epidemiology and pathogenicity of porcine circovirus [J]. Arch. Virol. 1986.91:271-276.
[22] Zhou XM, Lin JS, Shi Y, Tian DA, Huang HJ, Zhou HJ, Jin YX. Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene [J]. Acta Biochim Biophys Sin (Shanghai). 2005. 37(5):310-6.
[23] Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo [J]. J Virol. 2004, 78(24):13440-6.
[2] Allan GM, McNeilly F, Cassidy JP, Reilly GA, Adair B, Ellis WA, McNulty MS. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material [J]. Vet Microbiol.1995,44(1): 49-64.
[3] Allan G Meehan B, Todd D. et al. Novel porcine circovirus from pigs with wasting disease syndrome [J]. Vet Rec, 1998,142(17): 476-468
[4] Allan GM, McNeilly F, Meehan BM, et al. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland [J]. Vet Microbiol, 1999, 66(2): 11523
[5] Allan G, McNeilly F, Meehan B, McNair I, Ellis J, Krakowka S, Fossum C, Wattrang E, Wallgren P, Adair B.Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate [J]. J Vet Diagn Invest. 2003,15(6): 553-60.
[6] Annette Mankertz, Rifat Caliskan, Kim Hattermann,et al. Molecular biology of Porcine circovirus: analyses of gene expression and viral replication [J]. Veterinary Microbiology 2004,98: 81-88
[7] Bassami MR, Berryman D, Wilcox GE, et al. Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circovirus, and chicken anaemia virus [J]. Virology, 1998,249:453-459
[8] Boevink P, Chu PWC, Keese P. Sequence of the geminiviruses subterraneanclover stunt affinities with virus DNA [J]. Virology, 1995,207: 354-361
[9] Bolin, S.R. and Allan, GM. Old virus or new virus-In a world continuous changes [C]. En: PMWS and PCV2 diseases beyond the debate. In: 17th. INTERNATIONAL PIG VETERINARY SOCIETY CONGRESS. Ames. Iowa. USA. 2002: 9-17.
[10] Borel N, Burgi I, Kiupel M, et al. Three cases of postweaning multisystemic wasting syndrome (PMWS) due to porcine circovirus type 2 (PCV2) in Switzerland [J]. Schweiz Arch Tierheilkd, 2001, 143(5): 249-255
[11] Celera VJr, Carasova P. First evidence of porcine circovirus type 2 (PCV-2) infections of pigs in the Czech Republic by semi-nested PCR [J]. J Vet Med B Infect Dis Vet Public Health, 2002, 49(3):155-9.
[12] Cheung AK. Transcriptional analysis of porcine circovirus type 2 [J]. Virology 2002; 305: 168-180.
[13] Cheung AK and Bohn SR. Kinetics of porcine circovirus type 2 replication [J]. Arch Virol 2002b, 147: 43-58.
[14] Choi C, Chae C. ln-situ hybridization for the detection of porcine circovirus in pigs with postweaning multisystemic wasting syndrome [J]. J Comp Pathol, 1999,121(3): 265-70
[15] Clark EG. Postweaning multisystemic wasting syndrome [C]. In:Proceedings of the 28th American Association of Swine Practitioners, Que., Canada, 1997, 28:188-259.
[16] Fenaux M, Halbur PG, Gill M, et al. Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2[J]. J Clin Microbiol, 2000, 38(7):2494-2503.
[17] Gibbs, M.J., Weiller, G.F., Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus [J]. Proc. Natl. Acad. Sci. USA. 1999,14:8022-8027.
[18] Gresham A, Jackson q Giles N, et al. PMWS and porcine dermatitis nephropathy syndrome in Great Britain [J]. Vet Rec, 2000,146(5): 143
[19] Hamel AL, Lin LL, and Nayar GP. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs [J]. J Virol, 1998; 72:5262-5267.
[20] Harding JC. Post-weaning multisystemic wasting syndrome: preliminary epidemiology and clinical findings [C]. In: Proceedings of the Western Canadian Association on Swine Practice, 1996, 21
[21] Harding JC, Clark EG Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS) [J]. Swine Health Prod, 1998,5:201-203
[22] Hattermann K, Roedner C, Schmitt C, Finsterbusch T, Steinfeldt T, Mankertz A. Infection studies on human cell lines with porcine type 1 and porcine circovirus type 2 [J]. Xenotransplantation. 2004a, 11(3): 284-94.
[23] Hattermann K, Maerz A, Slanina H, Schmitt C, Mankertz A. Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-PCR-based search for a human circovirus. Xenotransvlantarion [J]. 2004b, 11(6): 547-50.
[24] Husa P, Chalupa P, Husova L. The SEN virus-will there be another letter in the alphabet of viral hepatitis [J]. Vnitr Lek. 2002, 48(8): 763-6
[25] Jemersic L, Cvetnic Z, Toplak I, et al. Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia [J]. Res Vet Sci, 2004,77(2): 171-5
[26] Josefma Q., Monica B., Joaquim S., Maria C, Gabriela M., RODRIGUEZ ARRIOJA, Juan P., LAMA-D., Mariano D. Experimental inoculation of porcine type 1 (PCV1) and type 2 (PCV2) in rabbits and mice [J]. Vet. Res.2002, 33:229-237.
[27] Katul L, Maiss E, Vetten HJ. Sequence analysis of a faba bean necrotic yellows virus DNA component tative replicase gene [J]. J Gen Virol, 1995,76:475-479
[28] Kenshi S. Tomoyuki S., Yoshiharu L, Hiroshi K., MasanoriK, Koichi K., Evidence of Porcine Infection in Pigs with Wasting Disease Syndrome from 1985 to 1999 in Hokkaido [J], Japan J Yet Med Sci. 2000,62(6): 627-633.
[29] Kiatipattanasakul-Banlunara W, Tantilertcharoen R, Suzuki K, et al. Detection of porcine circovirus 2 (PCV 2) DNA by nested PCR from formalin-fixed tissues of postweaning multi-systemic wasting syndrome (PMWS) pigs in Thailand [J]. J Vet Med Sci, 2002, 64(5): 449-452
[30] Kim, J., Han, D.U., Choi, C, et al. Differentiation of porcine circovirus (PCV)-1 and PCV2 in boar semen using a multiplex nested polymerase chain reaction [J]. Journal of Virological Methods 2001,98: 25-31.
[31] Kim JH, Lyoo YS. Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs [J]. J Vet Sci, 2002, 3(1):31-9
[32] Kiss I, Kecskemeti S, Tuboly T, et al. New pig disease in Hungary: postweaning muitisystemic wasting syndrome caused by circovirus [J]. Acta Vet Hung, 2000, 48(4): 469-475
[33] Kiupel M, Stevenson GW, Choi J, Latimer KS, Kanitz CL, Mittal SK.Viral replication and lesions in BALE/c trice experimentally inoculated with porcine circovirus isolated front a pig with postweaning muitisystemic wasting disease [J]. Vet Pathol. 2001, 38(1): 74-82.
[34] Larochelle R, Magar R, D'Allaire S. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strain from cases presenting various clinical conditions[J].Virus Res, 2002,90(1-2):101-112.
[35] Liu, J., Chen, I., Kwang, J., 2005. Characterization of a previously unidentified viral protein of porcine circovirus type 2-infected cells and its role in virusinduced apoptosis [J]. J. Virol. 79, 8262- 8274.
[36] Liu J, Chen I, Du Q, Chua H, Kwang J. 2006. The ORF3 protein of porcine circovirus type 2 is involved in viral pathogenesis in vitro [J]. J Virol. 80, 5065-73.
[37] Liu Q, Tiskoo S, and Babluk LA. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2 [J]. Virology 2001; 285: 91-99.
[38] Liu Q, Wang L, Willson P, et al. Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica [J]. Can J Vet Res, 2002, 66(4): 225-31
[39] Madec F, Fveno E, Morvan P, et al. Post-weaning muitisystemic wasting syndrome (PMWS) in pigs in France: clinical observations from follow-up studies on affected farms [J]. Livest Prod Sci, 2000, 63(3): 223-233
[40] Magar R, Larochelle R, Thibault S, Lamontagne L.Experimental transmission of porcine type 2 (PCV2) in weaned pigs: a sequential study [J]. J Comp Pathol. 2000,123(4): 258-69.
[41] Maldonado J, Segales J, Calsamiglia M, et al. Postweaning multisystemic wasting syndrome (PMWS) in the Philippines: porcine circovirus type 2 (PCV2) detection and characterization [J]. J Vet Med Sci, 2004,66(5): 533-7
[42] Mankertz A, Mankertz J, Wolf K, et al. Identification of a protein essential for the replication of porcine circovirus [J]. J Gen Virol 1998a; 79:381-384.
[43] Mankertz, J., Bunk, H J., Blaess, G., et al. Transcription analysis of Porcine circovirus (PCV) [J]. Virus Genes. 1998b,16 (3):267-276.
[44] Mankertz A, Domingo M, Folch JM, et al. Characterisation of PCV-2 isolates from Spain, Germany and France [J]. Virus Res, 2000a, 66(1): 65-77
[45] Mankertz A, Hattermann K, Ehlers B, et al. Cloning and sequencing of columbid circovirus (coCV), a new circovirus from pigeons [J]. Arch Virol, 2000b, 145(12): 246979
[46] Mankertz, A., Hillenbrand, B., Replication of Porcine circovirus Type 1 requires two proteins encoded by the viral rep gene [J]. Virology 2001,279 (2), 429-438.
[47] Mankertz A and Hillenbrand B. Analysis of transcription of porcine circovirus type 1 [J]. J Gen Virol 2002; 83: 2743-2751.
[48] Meehan BM, McNeilly F, Todd D, et al. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs [J]. J Gen Virol 1998; 79:2171-2179.
[49] Miyata H, Tsunoda H, Kazi A, et al. Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus [J]. J Virol, 1999,73: 3582-3586
[50] Morozov I, Sirinarumitr T, Sorden SD, et al. Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome [J]. J Clin Microbiol 1998,36(9): 2535-41.
[51] Nawagitgul P, Morozov I, Bolin S, et al. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein [J]. J Gen Virol 2000; 81: 2281-2287.
[52] Niagro FU, Forsthoefel AN, Lawther RP, et al. Beak and feather disease virus and porcine circovirus genomes: intermediates between the germiniviruses and plant circovirus [J]. Arch Virol, 1998.143:1723-1744
[53] Nishizawa T, Okamoto H, Konishi K, et al. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem. Biophys [J]. Res Commun, 1997.241:92-97
[54] Onuki A, Abe K, Togashi K, et al. Tsunemitsu H. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan [J]. J Vet Med Sci, 1999, 61(10): 1119-23
[55] Phenix KV, Weston JH, Ypelaar I, et al. Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae [J]. J Gen Virol, 2001, 82: 2805-9
[56] Pogranichnyy RM, Yoon K-J, Hums PA, et al. Characterization of immune response of young pigs to porcine circovirus type 2 infection [J], Viral Immunol 2000,13:143-153.
[57] Porntippa N, Lgor M, Steven R. Open reading frame 2 porcine circovirus type 2 encodes a major capsid protein [J]. J General Virology, 2000,81: 2281-2287
[58] Renee L, Andrzej B., Peter M., Ronald M. PCR Detection and Evidence of Shedding of Porcine Circovirus Type 2 in Boar Semen [J]. J Clin Micro. 2000,38(12): 4629-4632.
[59] Rohde W, Randies JW, Langridge P, et al. Nucleotide sequence of a circular single-stranded DNAassociated with coconut foliar decay virus [J]. Virology, 1990,176: 648-651
[60] Saoulidis K, Kyriakis SC, Kennedy S, et al. First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy s}mdrome in pigs in Greece [J]. J Vet Med B Infect Dis Vet Public Health, 2002,49(4): 202-5
[61] Sarradell J, Perez AM, Comba E, et al. Pathological findings in pigs affected by the postweaning multisystemic wasting syndrome in Argentina [J]. Rev Argent Microbiol, 2004, 36(3): 118-24
[62] Steinfeldt T, Finsterbusch T, Mankertz A. Rep and Rep' protein of porcine circovirus type 1 bind to the origin of replication in vitro [J]. Virology 2001; 291:152-160
[63] Takahashi K, Iwasa Y, Hijikata M, et al. Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus [J]. Arch Virol 2000, 145(5):979-993
[64] Terregino C, Montesi F, Mutinelli F, et al. Detection of a circovirus-like agent from farmed pheasants in Italy [J]. Vet Rec, 2001,149(11): 340
[65] Thomson J, Smith B, Allan q et al. PDNS, PMWS and porcine circovirus type 2 in Scotland. Porcine dermatitis and nephropathy syndrome. Post-weaning multisystemic wasting syndrome [J].Vet Rec, 2000,146 (22): 651-652
[66] Tischer I., Rasch R, and Tochtermann G Characterization of papovavirus and picornavirus-like particles in permanent pig kidney cell lines [J]. Zentbl Bakteriol Htg A, 1974,226:153-167
[67] Tischer I, Gelderblom H, Vettermann W. A very small porcine virus with circular single-stranded DNA [J]. Nature, 1982, 295: 64-66
[68] Tischer I, Peters D, Pociuli S.Occurrence and role of an early antigen and evidence for transforming ability of porcine circovirus [J]. Arch Virol. 1995; 140(10): 1799-816.
[69] Todd D, Creelan JL, Mackie DP, et al. Purification and biochemical characterization of chicken anaemia agent [J]. J Gen Virol, 1990, 71:819-823
[70] Todd D., Weston J.H., Soike D., et al. Genome sequence determinations and analyses of novel circoviruses from goose and pigeon [J]. Virology 2001.286:354-362
[71] Todd D, Bendinelli M, Biagini P, Hino S, Mankertz A, Mishiro S, Niel C, Okaraoto H, Raidal S, Ritchie BW, Teo GC, 2005. Circociridae [C]. In: Virus Taxonomy, 8th Report of the International Committee for the Taxonomy of Viruses (CM Fauquet, MA Mayo, J Maniloff, U Desselberger and LA Ball, eds), 327-334
[72] Toplak I, Groin J, Hostnik P, et al. Phylogenetic analysis of type 2 porcine circoviruses identified in wild boar in Slovenia [J]. Vet Rec, 2004, 155(6): 178-80
[73] Truong C, Mathe D, Blanchard P, et al. Identification of an immnorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection[J]. Arch Virol, 2001,146(6)1197～1211.
[74] Trujano M, lglesias G, Segales J, et al. PCV-2 from emaciated pigs in Mexico [J]. Vet Rec, 2001 148 (25): 792
[75] Walker IW, Konoby CA, Jewhurst VA, McNair I, McNeilly F, Meehan BM, Cottrell TS, Irllis JA, Allan GM.Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine ckcovirus type 2 [J]. J Vet Diagn Invest. 2000, 12(5): 400-5.
[76] Wallgren P, Hasslung F, Bergstrom G, et. al. Postweaning multisystemic wasting syndrome—PMWS the first year with the disease in Sweden [J]. Vet Q, 2004, 26(4): 170-87
[77] Wang C, Huang TS, Huang CC, et al. Characterization of porcine circovirus type 2 in Taiwan [J]. J Vet Meal Sci, 2004, 66(5): 469-75
[78] Wellenberg GJ, Stockhofe-Zurwieden N, de Jong MF, et al. Excessive porcine circovirus type 2 antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study [J]. Vet Microbiol, 2004, 99(3-4): 203-14
[79] 郎洪武，王力，张广川等．猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断[J]．中国兽医科技，2001，31(3)：3-5．
[80] 林绍荣，黄毓茂，苏丹萍，等，养猪[M]．2003．5：35-36
[81] 郎洪武，张广川，吴发权等．猪断奶多系统衰弱综合征血清抗体检测[J]．中国兽医科技，2000，30(3)：3-5
[82] 周继勇．陈庆新，叶菊秀，等．猪圆环病毒2型感染的血清学分析[J]．中国兽医学报，2004，24，(1)：1-3．
[1] Allan GM, McNeilly F, Foster JC, Adair BM. Infection of leucocyte cell cultures derived species with pig cirwvirus [J]. Vet Microbiol. 1994,41(3): 267-79.
[2] Allan GM, Kennedy S, McNeilly F, Foster JC, Ellis JA, Krakowka SJ, Meehan BM, Adair BM.Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus [J]. J Comp Pathol. 1999,121(1): 1-11.
[3] Allan, G.M., Ellis, J.A., Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation[J], 2000a,12: 3-14.
[4] Allan GM, McNeilly F, Ellis J, et al. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication [J]. Arch Virol, 2000b, 145(11): 2421-2429
[5] Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair I, Walker I, Kennedy S. Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication [J]. Arch Virol. 2000c;145(11): 2421-9
[6] Blanchard P, Mahe D, Cariolet R, Keranflec'h A, Baudouard MA, Cordioli P, Albina E, Jestin A. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins [J]. Vaccine. 2003, 21(31): 4565-75.
[7] Chae C. A review of porcine circovirus 2-associated syndromes and diseases [J]. The Veterinary Journal. 2005,169: 326-336
[8] Chang HW, Pang VF, Chen LJ, Chia MY, Tsai YC, Jeng CR. Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages [J]. Vet Microbiol. 2006. 115(4):311-9.
[9] Chianini F, Majo N, Segales J, et al. Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2003, 94(1-2): 63-75
[10] Choi, C, Chae, G, Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephropathy syndrome by double-labeling technique [J]. Veterinary Pathology 2001,38: 436-441.
[11] Chung WB, Chan WH, Chaung HC, et al. Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs [J]. J Virol Methods, 2005,124(1-2): 11-19
[12] Chunmei Ju, Zhengfei Liu, Xin Xi, et al. Immunogenicity of a recombinant pseudorabies vims expressing ORF1 and ORF2 gene of porcine circovirus type2 [J]. Veterinary Microbiology 2005, 109:179-190
[13] Darwich L, Segales J, Domingo M, et al. Changes in CD4(+),CD8(+),CD4(+)CD8(+) and immunoglobulin M-positive peripheral blood mononuclear cells of postweaning multisystemic wasting syndrome-affected pigs and age-matched uninfected wasted and healthy pigs correlate with lesions and porcine circovirus type 2 load in lymphoid tissues [J]. Clin Diagn Lab Immunol, 2002,9(2): 236-242
[14] Darwich L, Balasch M, Plana-Duran J, et al. Cytokine profiles of peripheral blood mononuclear cells of pigs suffering from postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens [J]. J Gen Virol, 2003a, 84: 3453-3457
[15] Darwich L, Pie S, Rovira A, et al. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome [J]. J Gen Virol, 2003b, 84: 2117-2125
[16] Darwich L, Segales J, Mateu E, et al. Pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus 2: an immune riddle [J]. Arch Virol, 2004,149(5): 857-874
[17] Done SH, Bailey M, Gresham ACJ, et al. Postweaning multisystemic wasting syndrome (PMWS) .The current European situation [J]. Pig J, 2002,49: 215-232
[18] Duran, CO., Ramos-Vara, J.A., Render, J.A., Porcine dermatitis and nephropathy syndrome: a new condition to include in the differential diagnosis list for skin discoloration in swine [J]. Swine Health and Production 1997,5: 241-245.
[19] Fenaux M, Opriessnig T, Halbur PCB Elvinger F, Meng XJ.A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs [J].J Virol. 2004a Jun; 78(12): 6297-303.
[20] Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo [J]. J Virol. 2004b, 78(24):13440-6.
[21] Gilpin DF, Stevenson L, McCullough K, et al. Studies on the in vitro and in vivo effect of porcine circovirus type 2 infection of porcine monocytic cells [C]. In: Proc. ssDNA Viruses Plants Birds pigs Primates 97. plants, birds, pigs and primates, Saint-Malo. ZOOPOLE developpement (ISPAIA), France, 2001, 97
[22] Gilpin DF, McCullough K, Meehan BM, et al. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system [J]. Vet Immunol Immunopathol, 2003,94:149-161
[23] Halbur, P.G., Porcine respiratory disease [C]. Proceedings of the International Pig Veterinary Society Congress. 1998,15:1-10.
[24] Hamel, A.L., Lin, L.L., Nayar, G.P., Nucleotide sequence of Porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs [J]. J. Virol. 1998, 72 (6): 5262-5267.
[25] Hamel AL, Lin LL, Sachvie C, et al. PCR detection and characterization of type-2 porcine circovirus [J].Can J Vet Res,2000,64(1):44-52
[26] Harms PA, Sorden SD, Halbur PQ Bolin SR, Lager KM, Morozov I, Paul PS.Experimental reproduction of severe disease in pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus [J]. Vet Pathol. 2001,38(5): 528-39.
[27] Harms, P.A., Halbur, P.G., Sorden, S.D., Three cases of porcine respiratory disease complex associated with porcine circovirus type2 infection [J]. Journal of Swine Health and Production, 2002, 10:27-30.
[28] Hines, R.K., Lukert, P.D., Porcine circovirus as a cause of congenital tremors in newborn pigs [C]. In: Proceedings of the American Association on Swine Practitioners, 1994: 344-345.
[29] Hirai T, Nunoya T, Ihara T, et al. Acute hepatitis in a piglet experimentally inoculated with tissue homogenates from pigs with postweaning multisystemic wasting syndrome [J]. J Vet Med Sci, 2003, 65(9): 1041-5
[30] Josephson, G., Charbonneau, G., Case report of reproductive problems in a new startup operation [J]. Journal of Swine Health and Production 2001, 9: 258-259.
[31] Junghyun K, Chanhee C. A comparison of the lymphocyte subpopulations of pigs experimentally infected with porcine circovirus 2 and/or parvovirus [J].Vet J.2003,165:325-329.
[32] Kennedy S, Moffett D, McNeilly F, et al. Reproduction of lesions of postweaning multisystemic wasting syndrome infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus [J]. J Comp Pathol, 2000,122(1): 9-24
[33] Kennedy, S., Segales, J., Rovira, A., et al. Absence of evidence of porcine circovirus infection in piglets withcongenital tremors [J]. Journal of Veterinary Diagnostic Investigation 2003, 15: 151-156.
[34] Kim, J., Chung, H.-K., Chae, C, Association of porcine circovirus 2 with porcine respiratory disease complex [J]. The Veterinary Journal, 2003,166, 251-256.
[35] Kim J, Ha Y, Jung K, et al. Enteritis associated with porcine circovirus 2 in pigs [J]. Can J Vet Res, 2004a, 68(3): 218-21
[36] Kim, J., Chae, C. Concurrent presence of porcine circovirus type 2 and porcine parvovirus in retrospective cases of exudative epidermitis in pigs [J]. The Veterinary Journal 2004b.167,104-106.
[37] Kim J, Chae C. Necrotising lymphadenitis associated with porcine circovirus type 2 in pigs [J]. Vet Rec, 2005,156(6): 177-8
[38] Krakowka S, Ellis JA, McNeilly F, et al. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV2) [J]. Vet Pathol, 2001, 38(1):31-42
[39] Ladekjaer-Mikkelsen, A.S., Nielsen, J., Storgaard, T., et al. Transplacental infections with PCV2 associated with reproductive failure in a gilt [J]. Veterinary Record. 2001,148: 759-760.
[40] Ladekjaer-Mikkelsen AS, Nielsen J, Stadejek T, et al. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with Porcine circovirus type 2 (PCV2) [J]. Veterinary Microbiology, 2002,89:97-114
[41] Liu J, Chen I, Chua H, Du Q, Kwang J. Inhibition of porcine circovirus type 2 replication in mice by RNA interference [J]. Virology. 2006. 347(2):422-33.
[42] Mandrioli L, Sarli q Panarese S, et al. Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2004,97(l-2):25-37
[43] McNeilly F, McNair I I, O Connor M, et al . Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation , immunohistochemistry,and the polymerase chain reaction [I]. J Vet Diagn Invest, 2002,14(2);106-12
[44] McNulty M., Dale J., Lukert P., et al. Circoviridae. In: van Regenmortel, C.M.F.M.H.V., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Seventh Report of the International Committee on Taxonomy of Viruses,Academic Press, San Diego, 2000: 299-303.
[45] Meerts P, Misinzo G, McNeilly F, Nauwynck HJ.Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages [J]. Arch Virol 2005,150(3):427-410
[46] Nielsen J, Vincent IE, Botner A, et al. Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2003, 92: 97-111
[47] O_Connor, B., Grauvreau, H., West, K., et al. Multiple porcine circovirus 2-associated abortion and repdocutive failure in a multisite swine production unit [J]. Canadian Veterinary Journal 2001,42: 551-553.
[48] Opriessnig T, Yu S, Gallup JM, Evans RB, Fenaux M, Pallares F, Thacker EL, Brockus CW, Ackermann MR, Thomas P, Meng XJ, Halbur PGEffect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus [J]. Vet Pathol. 2003 Sep; 40(5): 521-9
[49] Opriessnig T, Thacker EL, Yu S, et al. Experimental reproduction of postweaning muttisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2 [J]. Vet Pathol, 2004b, 41(6): 624-40
[50] Ouardani M, Wilson L, Jette R, et al. Multiplex PCR for detection and typing of porcine circoviruses [J]. J Clin Microbial, 1999,379120:3917-24
[51] Pallares FJ, Halbur PCB Opriessnig T, et al. Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS) [J]. J Vet Diagn Invest, 2002, 14(6): 515-9
[52] Pesch S, Schmidt U, Ohlinger VF. Proliferative necrotizing pneumonia (PNP) is a result of coinfection with porcine reproductive and respiratory disease virus (PRRSV) and porcine circovirus type 2 (PCV2) [J]. In: Proceedings of the 16th International Congress on Pigs Veterinary Society, 2000,581
[53] Ramos-Vara, J.A., Duran, O., Render, J.A., et al. Porcine dermatitis and nephropathy syndrome in the USA [J]. Veterinary Record 1997,141:479-480.
[54] Romeo E., Sanchez J., Hans J., Nau Francis M.,Gordon M.,Maurice B. Porcine circovirus 2 infection in swine fetuses inoculated at different stages of gestation [J]. Vet Micro.2001,83:169-176.
[55] Rosell C, Segales J, Plana-Duran J, et al. Pathological, immunohistochemical, and in-situ hybridization studies on natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs [J]. J Comp Pathol, 1999,120 (1):59-78
[56] Rosell C, Segales J, Domingo M. Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2 [J]. Vet Pathol, 2000a, 37(6): 687-92
[57] Rosell, C, Segales, J., Ramos-Vara, et al. Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome [J]. Veterinary Record. 2000b,146: 40-43.
[58] Rovira A, Balasch M, Segales J, et al. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2 [J]. J Virol, 2002, 76(7): 3232-3239
[59] Segales J, Alonso F, Rosell C, et al. Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Immunol Immunopathol, 2001, 81(1-2):37-44
[60] Segales J,Pineiro C, Lampreave F, et al. Haptoglobin and pig-major acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS) [J]. Vet Res, 2004,35(3): 275-282
[61] Shibahara T, Sato K, Ishikawa Y, et al. Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome [J]. J Vet Med Sci, 2000,62(11):1125-1131
[62] Sipos W, Duvigneau JC, Willhein m, et al. systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome as determined by semiquantitative RT PCR and flow cytometric intracellular cytokine detection [J]. Vet Immunol Immunopathol, 2004, 99: 63-71
[63] Smith, W.J., Thomson, J.R., Done, S., Dermatitis/nephropathy syndrome of pigs [J]. Veterinary Record. 1993,132, 47.
[64] Stevenson, G., Distribution of porcine circovirus in neonatal pigs with congenital tremors [C]. In: Proceedings of the CRWAD, 1999, p. 217.115(3-4):261-72.
[65] Stevenson GW, Kiupel M, Mittal SK, et al. Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors [J]. J Vet Diagn Invest, 2001, 13: 57-62
[66] Thacker B, Thacker E. The PRDC battle continues [J]. Pig Prog, 2000,16-18
[67] Thacker, E.L., Porcine respiratory disease complex - what is it and why does it remain a problem? [J]. The Pig Journal. 2001,48: 66-70.
[68] Tischer I., Rasch R, and Tochtermann G Characterization of papovavirus and picornavirus-like particles in permanent pig kidney cell lines [J]. Zentbl Bakteriol Htg A, 1974,226:153-167
[69] Todd D., Niagro F.D., Ritchie B.W., et al. Comparison of three animal viruses with circular single-stranded DNA genomes [J]. Arch. Virol. 1991.117:129-135.
[70] Todd D., Weston J.H., Soike D., et al. Genome sequence determinations and analyses of novel circoviruses from goose and pigeon [J].Virology 2001.286:354-362
[71] Wattrang E, McNeilly F, Allan GM, et al. Exudative epidermitis and porcine circovirus-2 infection in a Swedish SPF-herd [J]. Vet Microbiol, 2002,86(4): 281-293
[72] Wellenberg, G.J., Pesch, S., Berndsen, F.W., et al. Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands [J]. Vet. Quart. 2000,22:167-172.
[73] West, K.H., Bystrom, J.M., Wojnarowicz, C, et al. Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2 [J]. Journal of Veterinary Diagnostic Investigation 1999,11: 530-532.
[74] Woods L.W., Latimer K.S., Barr B.C., et al. Circovirus-like infection in a pigeon [J]. J. Vet. Diagn. Invest. 1993,5:609-612.
[75] Yu S, Opriessnig T, Kitikoon P, Nilubol D, Halbur PG, Thacker E. Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs [J]. Vet Immunol Immunopathol. 2007.115(3-4):261-72.
[1] Ait Si Ali S, Guasconi V, Harel Bellan A. RNA interference and its possible use in cancer therapy [J]. Bull Cancer. 2004,91(1):15-18.
[2] Ashrafi K, Chang FY, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory [J], Gene. 2003,421(6920):268～272.
[3] Barton G M, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells [J]. PNAS USA, 2002,99(23):14943-14945.
[4] Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses [J]. BMC Microbiol, 2001,1:34-45.
[5] Boutros M, Kiger AA, Armknecht S, et al. Genome-wide RNAi analysis of growth and viability in Drosophilacells.Science,2004,303(5659):832～835
[6] Brigham PM. Cosuppression comes to the animals [J].Cell. 1997,90(3):385-387.
[7] Brummelkamp TR, Bernards R, Agami R. Asystem for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002,296(5567):550-553.
[8] Castanotto D, Li H, Rossi JJ.Functional siRNA exprssion from transfected PCR products [J]. RNA. 2002.8(11):1454-1460
[9] Chen W.Yan W, Du Q, et al. RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice [J]. J Virol, 2004,78: 6900-6907.
[10] Cocks B G, Theriault T P. Developments in effective application of small inhibitory RNA(siRNA) technology in mammalian cells [J].Targets,2004,3:165-171.
[11] Dykxhoom DM, Novina CD, Sharp PA. Killing the messenger short RNAs that silence gene exprssion [J]. Nat Rev Mol Cell Biol. 2003.4( 6):457-467
[12] Elbashir S M, Harborth J.Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001,411:494-498.
[13] Fedoriw AM, Stein P, Svoboda P, et al.Transgenic RNAi Reveals Essential Function for CTCF in H19 Gene Imprinting [J]. Science. 2004,303(5655):238-240.
[14] Fire A, Xu S, h4ontgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhaditis elegans [J]. Nature, 1998,391:806-811.
[15] Ge Q, Filip L, Bai A, et al.Inhibition of influenza virus production in virus-infected mice by RNA interference [J].Proc Natl Acad Sci USA, 2004,101:8676-8681.
[16] Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells [J]. Nature. 2002.418 (6896): 430-434
[17] Hannon GJ. RNA inference [J]. Nature. 2002,418(6894):244-251
[18] Han S, Mahato R I, Sung Y K, et al. Development of biomaterials for gene therapy [J]. Mol Ther, 2000,2:302-317.
[19] Hugh PC, Tomoyasu S, Chen ES, et al. Comprehensive analysis of heterochromatin and RNAi mediated epigenetic control of the fission yeast genome [J]. Nature Genetics,2005,37(8):809～819.
[20] Kasschau KD, Carrington JC. Acounter defensive strategy of plant viruses: suppression of posttranscriptional gene silencing [J].Cell,1998,95(4):461-470.
[21] Ketting RF, Fischer SE, Bernstein E, et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans [J]. Genes. 2001,15(20):2654-2659.
[22] Khvorova A, Reynolds A, Jayasena S D. Functional siRNAs and miRNAs exhibit strand bias [J].Cell, 2003,115: 209-216.
[23] Kitabwalla M, Ruprecht RM. RNA interference a new weapon a gainst HIV and beyond N Engl [J]. J Med. 2002, 347( 17):1364-1367
[24] Klein C, Bock C T, Wedemeyer H, et al. Inhibition of hepatitis B virus replication in vivo by nucleoside and siRNA [J].Gastroenterology,2003,125:9-18.
[25] Martinoz J, Patkaniowska A, Urlaub H, I.ahrmann R, Tuschl t. Single stranded antisense siRNAs wide Tareet RNA cleavaee in RNAi [J]. Cell. 2002.110(5):563
[26]Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA [J]. Nature. 2004,431(7006):343-349.
[27] Pardridge W M. Intravenous non-viral RNAi gene therapy of brain cance [J]r. Expert Opin Biol Ther. 2002. 41,2435-2438.
[28] Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference [J]. Nat Genet, 2003,33:401-406.
[29] Salvi A, Arici B, DePeLro G, et al. Small interfering RNA urokinase siliencing inhibits a invasion and migraLion of human hepatocellular carcinoma cells [J]. Mol Cancer Ther. 2004,36:671.
[30] Schwarz D S, Hutvagner G, Du T, et al.Asymmetry in the assembly of the RNAi enzyme complex [J]. Cell, 2003,115:199-208.
[31] Svoboda P, Stein P, Hayashi H, et al.RNAi mRNA in ones oocyte by RNA interference [J]. Development, 2000, 127:4147-4156.
[32] Tachibana R, Harashima H, Ide N, et al. Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes [J]. Pharm Res, 2002,377-381.
[33] Timmons L, Court D, Fire A. Ingestion of bacterially expressed dsRNA can produce specific and potent genetic interference in Caenorhabitis elegans [J]. Gene. 2001. 263:103.
[34] Tuschl T, Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double-stranded RNA invitro [J]. Genes. 1999,13(24): 3191-3197.
[35] Tuschl T. Expanding small RNA interference [J]. Nat Bioteclmol, 2002,20: 446-448.
[36] Qin X F, An D S, Chen I S,et al. Inhibiting HIV 1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCRS [J]. PNAS USA,2003,100(1): 183-188.
[37] Ui-Tei K, Naito Y, Takahashi F, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference [J]. Nucleic Acids Res, 2004,32: 936-948.
[38] Van der Krol AR, Mur LA, de Lange P, et al. Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect [J]. Plant Mol Biol, 1990,14(4): 457-466.
[39] Volpe TA, Kidner C, Hall IM, et al. Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi [J]. Science. 2002,297(5588): 1833-1837.
[40] Wang Z, Ren L, Zhao X, et al. Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells [J]. J Virol, 2004,78: 7523-7527.
[41] Wassenegger M. The role of the RNAi machinery in heterochromatin formation [J]. Cell. 2005, 122(1): 13-16.
[42] Yokota T, Sakamoto N, Enomoto N, et al.Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs [J]. EMBO Rep, 2003, 4: 602-608.
[43] Zamore PD, Tuschl T, Sharp PA,et al. RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals [J]. Cell,2000,101(1): 25-33.
[44] Zeng Y, Cullen BR. RNA interference in human cells is restricted to the cytoplasm [J]. RNA. 2002, 8 (7): 855-860
[45] Zhao L J, Jian H, Zhu H. Specific gene inhibition by adenovirus mediated expression of small interfering RNA [J]. Gene, 2003,316:137-141.
[46] Zheng ZM, Tang S, Tao MF. Development of Resistancet RNAi in Mammalian Cells [J]. Science,2005,1058: 105-118
[47] 钟苗，雷小勇．RNA沉默：基因组的免疫系统[J]．免疫学杂志，2004，20(2)：83-85．
[1] 郎洪武，王力，张广川等．猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断．中国兽医科技[J]，2001，3：3-5．
[2] Allan G.M., Phenix K.V., Todd D., et al..Some biological and physico-chemical properties of porcine circovirus.J.Vet.Med [J],1994,B41: 17-26
[3] Allan O M, McNeilly F, Cassidy J P, et al. Pathogenesis of porcine curcovirus: experimental infections of colostrums deprived piglets and examination of pig fetal material [J]. Vet Microbio! [J],1995,44: 49-64
[4] Allan G.M., B.Meehan, D.Todd, et.al. Novel porcine circovirus from pigs with wasting disease syndromes. Vet.Rec [J], 1998,142: 467-468
[5] Allan G.M., F.McNeilly, S.Kennedy, et.al. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in USA and Europe. J.Vet. Diagn.Investig [J],1998,10: 3-10
[6] Allan, G.M., Ellis, J.A., Porcine circoviruses: a review. Journal of Veterinary Diagnostic Investigation [J], 2000,12: 3-14.
[7] Chun WANG, Tien-Shine HUANG, Chin-Cbeng HUANG (2004) Characterization of porcine circovirus type 2 in Taiwan [J]. J Vet Meal Sci 66(5): 469-475
[8] Clark, E.G,.Post-weaning multisystemic syndrome. In: Proceedings of the American Association of Swing Practitioners [J], 1997, p. 499-501
[9] Dulac G.C., Afshar A. Porcine circovirus antigens in PK-15cell line (ATCC CCL-33) and evidence of antibodies to curcovirus in Canadian pigs [J]. Can.J.Vet. Res, 1989, 53: 431-433
[10] F. D. Niagro, A.N.Forsthoefel, R.P.Lawther et al Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circiviruses Arch Virol [J],1998,143: 1723-1744
[11] Fenaux M, Halbur PK, Gill M, et al. Genetic characterization of type 2 porcine circovirus(PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2 [J]. J Clin Microbiol, 2000, 2494-2503.
[12] Gresham, A., Allan, G., McNeilly, F., et al. Links between post-weaning multisystemic wasting syndrome and porcine dermatitis nephropathy syndrome [J]. The Pig Journal, 2001,47:155-159.
[13] Harms, P.A., Halbur, P.G., Sorden, S.D., Three cases of porcine respiratory disease complex associated with porcine circovirus type2 infection [J]. Journal of Swine Health and Production, 2002,10: 27-30.
[14] Jemersic L, Cvetnic Z, Toplak I, Spicic S, Groin J, Barlic-Maganja D, Terzic S, Hostnik P, Lojkic M, Humski A, Habrun B, Krt B. Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia [J]. Res Vet Sci. 2004, 77:171-175
[15] Jin H Kim, Young S Lyoo. Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs [J]. J Vet Sci. 2003, 3(1):31-39
[16] Kim J, Chae C. Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome [J]. Can J Vet Res. 2003, 67(2):133-137
[17] Kim, J., Chung, H. -K., Chae, C., Association of porcine circovirus 2 with porcine respiratory disease complex. The Veterinary Journal [J], 2003b, 166, 251-256.
[18] Mahe D., Blanchard P., Truong C., Arnauld, C., Le Cann P., Cariolet R., Madec F., Albina E., Jestin A. Differential recognition of ORF2 protein from type 1 and 2 porcine circoviruses and identification of immunorelevant epitopes [J]. J. Gen. Virol. 2000, 81:1815-1824.
[19] Maldonado J, Segales J, Calsamiglia M, Llopart D, Sibila M, Lapus Z, Riera P, Artigas C. Postweaning multisystemic wasting syndrome (PMWS) in the Philippines: porcine circovirus type 2 (PCV2) detection and characterization [J]. J Vet Med Sci. 2004, 66(5):533-7.
[20] Meehan, B. M., McNeilly, F., McNair, I., et al. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome. Archives of Virology [J], 2001. 146: 835-842.
[21] Thomson, J., Henderson, L., Meikle, C., MacIntyre, N., Porcine dermatitis and nephropathy syndrome. Veterinary Record [J], 2001a, 148, 282-283.
[22] Tischer I., Mields W, Wolff D, et al. Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol [J], 1986, 91:271-276
[23] Wen L, Guo X, Yang H. Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China [J]. Vet Microbiol. 2005, 110(1-2):141-6
[24] Larochelle R, Magar R, D'Allaire S. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strain from cases presenting various clinical conditions [J]. Virus Res, 2002, 90(1-2):101-112.
[25] Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo [J]. J Virol. 2004, 78(24): 13440-6.
[26] Mankertz A, omingo M, FolchJ M, et al. Characterization of PCV2 isolates from Spain, Germany and France [J]. Virus Res, 2000, 66(1):65-77.
[1] Clark EG. Postweaning Multisystemtic Wasting Syndrome [A]. Proceedings of the American Association of Swine Practitioners [C]. 1997, 499-501.
[2] Allan GM, Mc Neilly F, Meehan BM, et al. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland [J]. Vet Microbiol, 1999, 66(2):115-123.
[3] Choi C, Kim J, Kang IJ, et al. Concurrent outbreak of PMWS and PDNS in a herd of pigs in Korea [J]. Vet Rec, 2002, 151(16):484-485.
[4] Drolet R, Larochelle R, Morin M, et al. Detection rates of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and swine influenza virus in porcine proliferative and necrotizing pneumonia [J]. Vet Pathol, 2003, 40(2): 143-148.
[5] Chun WANG, Tien-Shine HUANG, Chin-Cheng HUANG. Characterization of porcine circovirus type 2 in Taiwan [J]. J Vet Med Sci, 2004, 66(5): 469-475.
[6] Lang H W, Zhang G C, Wu F Q,(郞洪武，张广川，吴发权) et al. Detection of Serum Antibody against Postweaning Multisystemic Wasting Syndrome in Pigs [J]. Chinese Journal of Veterinary Science and Technology(中国兽医科技)，2000，30(3)：3-5．
[7] Cao S B, Cheng H C, Shao S B,(曹胜波，陈焕春，肖少波)et al. The Establishment And Application of PCR to Diagnosis Porcine Circovirus Type 2 [J]. Journal of Huazhong Agricultural(华中农业大学学报)，2001，1：53-56．
[8] Cui S J, Cai Y. Cheng X,(崔尚金，蔡阳，陈欣).Development and appfication of a competitive quantitative assay for monitoring porcine circovirus DNA [J]. Chinese Journal of Preventive Veterinary Medicine(中国预防兽医学报)，2004，26(5)：347-351．
[9] Tischer, I., Rasch, R., Tochtermann, G. Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines[A]. Zentralblatt fur Bakteriologie, Parasitenkund, Infecktionskrankheiten und Hygiene-Erste Abteilung Originale-Reihe A: Medizinische Mikrobiologie und Parasitologie[C]. 1974, 226, 153-167.
[10] Allan GM, McNeilly F, Kennedy S, et al. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA AND Europe [J]. J Vet Diagn Invest, 1998, 10:3-10.
[11] Inger Marit Brunborg, Torfinn Moldal, Christine Monceyron Jonassen. Quantitation of porcine circovirus type 2 isolated from serum/plasma andtissue samples of healthy pigs and pigs with postweaning multisystemicwasting syndrome using a TaqMan-based real-time PCR [J]. J Virol Meth, 2004, 122, 171-178.
[12] Stevenson GW, Kiupe IM, Mittal SK, et al. Ultra structure of porcine circovirus in persistently infected PK-15 cells [J]. Vetpathology, 1999, 36: 368-378.
[13] Wang X W, Jiang P, Li Y F,(王先炜，姜平，李玉峰)et al. Detection and Genetic Characterization of Porcine Circovirus(PCV-2) from Pigs with Postweaning Multisystemic Wasting Syndrome in Mid-east of China [J]. Chinese Journal of Veterinary(中国兽医学报)，2004，24(3)：240-242．
[14] Zhang Zh D, Soren A. Detection of carrier cattle and sheep persistently infected with foot-and-mouth disease virus by a rapid real-time RT-PCR assay [J]. J Virol Meth, 2003, 111: 95-100.
1. Jiang P, Chen P Y, Dong Y Y, et al, Isolation and genome characterization of porcine reproductive and respiratory syndrome virus in PR China[J]. Vet Diagn Invest, 2000, 12: 156-158.
2. Lager K M, Mengeling W L; Brockmeier S L, Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine [J].Vet Microbiol, 1997, 58: 127-133
3. Christoph E,Barbara T,Luzia L, et al. Quantitative TaqMan RT-PCR for the detection and differantiation of European and North American strains of porcine reproductive and respiratory syndrome virus [J]. Jour.Virol Meth, 2001,98:63-75
4. Albina E, Epidemiology of porcine reproductive and respiratory syndrome: an overview [J].Vet Microbiol. 1997, 55: 309-316
5. Will R W, Zimmerman J J, Yoon K J, et al. porcine reproductive and respiratory syndrome vires: routes of excretion [J]. Vet Microbiol, 1997,57:69-81
6. Beyer E M, Fichtner D, Schirrmerier H,et al. porcine reproductive and respiratory syndrome virus: kinetics of infection in lymphatic organs and lung [J].J Vet Med, 2000, 47: 9-25
[1] Abdalla A.O., Kiaii S., Hansson L et al. Kinetics of cytokine gene expression in human CD4+ and CD8+ Tlymphocyte subsets using quantitative real-time PCR [J].Scand. J. Immunol, 2003; 58:601.
[2] Anderson I.E., Reid H.W., Nettleton P.F et al. Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions [J]. Vet. Immunol. Immunopathol, 2001; 83:161.
[3] Audige A., Schlaepfer E., Bonanomi A et al. HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo [J]. J. Immunol, 2004; 172: 2687.
[4] Bailey L.O., Washburn N.R., Simon Jr et al. Quantification of inflammatory cellular responses using real-time polymerase chain reaction [J]. J. Biomed. Mater. Res, 2004; 69A: 305.
[5] Biron C.A. Role of early cytokines, including alpha and beta interferons (IFN-alpha/beta), in innate and adaptive immune responses to viral infections [J]. Semin. Immunol, 1998; 10: 383.
[6] Kvarnstrom M, Jenmalm M.C, Ekerfelt CEffect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4[J]. Cryobiology, 2004; 49:157.
[7] L Darwich, J Segales, E Mateu. Pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus 2: an immune riddle [J]. Arch. Virol, 2004; 149: 857-874
[8] Murtaugh M.P., Baarsch M.J., Zhou Yet al. Inflammatory cytokines in animal health and disease [J]. Vet. Immunol Immunopathol, 1996; 54:45-55
[9] Rottman, J.B. Key role of chemokines and chemokine receptors in inflammation, immunity, neoplasia, and infectious disease[J]. Vet. Pathol, 1999; 36: 357.
[10] Roongroje, Theresa F, Brad J et al. Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model [J]. Vet. Immunol Immunopathol, 2001;79:115-127
[11] Sanipa Suradhat, Roongroje Thanawongnuwech. Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus [J]. J Gen Virol, 2003; 84:2755-2760
[12] W.H. Feng, M.B. Tompkins, J S Xu et al. Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus [J]. Vet. Immunol Immunopathol, 2003; 94: 35-45
[13] Atabak R. Royaee, Robert J. Husmann, Harry D. Dawson et al. Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination [J]. Vet. Immunol Immunopathol, 2004; 102: 199-216
[14] Diaz I, Darwich L, Pappaterra G et al. Immune responses of pigs after experimental infection with a European strain of porcine reproductive and respiratory syndrome virus[J]. J Gen Virol, 2005; 86(Pt7): 1943-51
[1] Allan GM, Ellis JA. Porcine circoviruses: a review [J]. J Vet Diagn Invest, 2000, 12:3-14.
[2] Choi, C., Chae, C. Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in situ hybridization [J]. J. Comp. Pathol, 2000, 123: 302-305.
[3] Collins J.E., Benfield D.A., Christianson W.T., et al. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs [J]. J. Vet. Diag. Invest, 1992, 4:117-126.
[4] D.F. Gilpin, K. McCullough, B.M. Meehan, et al. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system [J]. Veterinary Immunology and Immunopathology, 2003, 94:149-161.
[5] 冯志新，姜平，王先炜，等．猪圆环病毒2型TaqMan实时PCR检测方法的建立[J]．中国病毒学，2006，21(4)：371-374．
[6] Galina L., Pijoan C., Sitjar M., et al. Interaction between Streptococcus suis serotype 2 and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets [J]. Vet. Rec. 1994, 134: 60-64.
[7] Harding JCS, Clark EG. Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS) [J]. J Swine Health Prod. 1997, 5:201-203.
[8] Harding JCS, Clark EG, Strokappe JH, et al. Postweaning multisystemic wasting syndrome: epidemiology and clinical presentation [J].J Swine Health Prod. 1998, 6:249-254.
[9] Harms P.A., Sorden S.D., Halbur P.G., et al. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus [J]. Vet. Pathol. 2001, 38: 528-539.
[10] Hui-Wen Chang, Chian-Ren Jeng, Jiuan J. Liu, et al. Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha [J]. Veterinary Microbiology, 2005, 108:167-177.
[11] Hui-Wen Chang, Victor Fei Pang, Liang-Jiun Chen, et al. Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages [J]. Veterinary Microbiology, 2006, 115(4):311-9.
[12] Laila Darwich, Sandrine Pie', Albert Rovira, et al. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome [J]. Journal of General Virology, 2003, 84:2117-2125.
[13] Lawson, S.R., Rossow, K.D., Collings, J.E., et al. Porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with MAC-387 staining at 21 days post infection [J]. Virus Res. 1997, 51: 105-113.
[14] O'Connor B., Gauvreau H., West K.H., et al. Multiple porcine circovirus2-associated abortions and reproductive failure in a multisystemic swine production unit [J]. Can. Vet. J. 2001, 42: 551-553.
[15] P. A. Harms, S. D. Sorden, P. G. Halbur, et al. Experimental Reproduction of Severe Disease in CD/CD Pigs Concurrently Infected with Type 2 Porcine Circovirus and Porcine Reproductive and Respiratory Syndrome Virus [J]. Vet Pathol, 2001, 38:528-539.
[16] R. Drolet, R. Larochelle, M. Morin, et al. Detection Rates of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, and Swine Influenza Virus in Porcine Proliferative and Necrotizing Pneumonia [J]. Vet Pathol, 2003, 40:143-148.
[17] A. Rovira, M. Balasch, J. Segales, et al. Experimental Inoculation of Conventional Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2 [J]. Journal of Virologs, 2002, 76(7): 3232-3239.
[18] Chiou, M.T., Jeng, C.R., Chueh, L.L., et al. Effects of porcine reproductive and respiratory syndrome virus (isolate tw91) on porcine alveolar macrophages in vitro [J]. Vet. Microbiol, 2000, 71, 9-25.
[19] Oleksiewicz, M.B., Nielsen, J. Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function [J]. Vet. Microbiol, 1999, 66, 15-27.
[20] 冯志新，姜平．猪细胞因子SYBR Green实时PCR检测方法的建立[J]．现代生物医学进展，2007．
[21] 黄娟，姜平，张常印等．猪繁殖与呼吸综合征病毒实时PCR检测方法的建立[J]．中国病毒学，2005，20(5)：530-533．
[22] 彭长凌，，高崧，刘秀梵．PRRSV人工单独感染及其与PCV2人工联合感染的病理学比较[J]．徐州医学院学报，2006，26(1)：49-52
[1] Allan, G. M., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Bother, A., Charreyre, C., 1998. Novel porcine circoviruses from pigs with wasting disease syndromes [J]. Veterinary Record 142, 467-468.
[2] Guo S, Kemphues KJ. 1995. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed [J].Cell. 81(8): 611-620.
[3] Haibin Xia, Qinwen Mao siRNA-mediated gene silence in vivo and in vitro [J]. Nature biotechnology. 2002.20, 1006-1010
[4] Jana S, Chakraborty C, Nandi S, Deb JK. RNA interference: potential therapeutic targets [J]. Appl Microbiol Biotechnol. 2004. 65(6):649-57.
[5] Liu J, Chen I, Chua H, Du Q, Kwang J. Inhibition of porcine circovirus type 2 replication in mice by RNA interference [J]. Virology. 2006.10; 347(2):422-33.
[6] Meehan, B. M., McNeilly, F., McNair, I., Walker, I., Ellis, J. A., Krakowka, S., Allan, G. M. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome [J]. Archives of Virology. 2001. 146, 835—842.
[7] Chen WZ, Liu MQ, Jiao Y, Yan WY, Wei XF, Chen J, Fei L, Liu Y, Zuo XP, Yang FG, Lu YG, Zheng ZX. Adenovirus-Mediated RNA Interference against Foot-and-Mouth Disease Virus Infection both In Vitro and In Vivo [J]. J Virol. 2006.80(7): 3559-3566.
[8] Han S, Mahato R I, Sung Y K, et al. Development of biomaterials for gene therapy [J]. Mol Ther, 2000, 2:302-317.
[9] Sergio C, Abdullah E, Carol C, Naazneen M, Felix H. S, Fatricia L. M, Nicolas F, Marc S. W, Patrick A. Effective Inhibition of HBV Replication in Vivo by Anti-HBx Short Hairpin RNAs [JJ. Molecular Therapy. 2005. 13(2): 411-421.
[1] Allan, G. M., F. McNeilly, J. P. Cassidy, G. A. Reilly, B. Adair, W. A. Ellis, and M. S. McNulty. Pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material [J]. Vet. Microbiol. 1995.44:49-64.
[2] Allan, G. M., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Botner, A., Charreyre, C. Novel porcine circoviruses from pigs with wasting disease syndromes [J]. Veterinary Record. 1998.142, 467-468.
[3] Brunborg IM, Moldal T, Jonassen CM. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR [J]. J Virol Methods. 2004. 122(2): 171-8.
[4] Chen, W., W. Yah, Q. Du, L. Fei, M. Liu, Z. Ni, Z. Sheng, and Z. Zheng. RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice [J]. J. Virol. 2004. 78:6900—6907.
[5] Cheung, A. K. Transcriptional analysis of porcine circovims type 2 [J]. Virology. 2003.305: 168-180.
[6] Cobum GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference [J]. J Virol. 2002.76(18):9225-31.
[7] Ding, S. W., H. Li, R. Lu, F. Li, and W. X. Li. RNA silencing: a conserved antiviral immunity of plants and animals [J]. Virus Res. 2004.102:109-115.
[8] Dulac GC, Afshar A. Porcine circovirus antigens in pk-15 cell line (ATCCCCL-33) and evidence of antibodies to circovirus in Canadian pigs [J]. Can J Vet Res. 1989. 53: 431-433
[9] Oitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells [J]. Nature. 2002. 418(6896): 430-434
[10] Gitlin, L., and R. Andino. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing [J]. J. Virol. 2003.77:7159-7165.
[11] Haasnoot, P. C., Cupac, D., Berkhout, B., 2003. Inhibition of virus replication by RNA interference. J. Biomed. Sci. 10, 607-616.
[12] Hammond, S. M., Caudy, A. A., Harmon, O. J. Post transcriptional gene silencing by double-stranded RNA [J]. Nat. Rev., Genet. 2001.2, 1110-1119.
[13] Harborth, J., Elbashir, S.M., Vandenburgh, K., Manninga, H., Scaringe, S.A., Weber, K., Tuschi, T. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gone silencing [J]. Antisense Nucleic Acid Drug Dev. 2003.13, 83-105.
[14] Morris, K.V., Chan, S.W.L., Jacobsen, S.E., Looney, D.J. Small interfering RNA-induced transcriptional gone silencing in human cells [J]. Science. 200427, 1289-1292.
[15] Jana S, Chakraborty C, Nandi S, Deb JK. RNA interference: potential therapeutic targets [J]. Appl Microbiol Biotechnol. 2004.65(6):649-57.
[16] Kameoka M, Nukuzuma S, Itaya A, Tanaka Y, Ota K, Ikuta K, Yoshihara K. RNA interference directed against Poly(ADP-Ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells [J]. J Virol. 2004.78(16):8931-4
[17] Li Yao-Chen, Zhao Zhi-Gang, Li Kang-Sheng, RNA interference mediated inhibition of influenza virus NP and PA gone expressions and multiplication in MDCK cell [J]. J Fourth Mil Med Univ. 2006.27(8):681-685.
[18] Liu J,Chen I,Kwang J. Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis [J]. J Virol._2005. 79(13):8262-74.
[19] Liu J, Chen I, Chua H, Du Q, Kwang J. Inhibition of porcine circovirus type 2 replication in mice by RNA interference [J]. Virology. 2006. 10;347(2):422-33.
[20] Liu Q, Wang L, Willson P, Babiuk LA. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome [J]. J Clin Microbiol. 2000.38(9):3474-7.
[21] Mankertz, A., J. Mankertz, K. Wolf, and H. J. Buhk. Identification of a protein essential for replication of porcine circovirus [J]. J. Gen. Virol. 1998.79: 381-383.
[22] McManus, M.T., Sharp, P.A. Gene silencing in mammals by small interfering RNAs [J]. Nat. Rev., Genet. 2002.3, 737-747.
[23] Meehan, B.M., McNeiUy, F., McNair, I., Walker, I., Ellis, J.A.,Krakowka, S., Allan, G.M. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome [J]. Archives of Virology. 2001.146, 835-842.
[24] Moore MD, McGarvey MJ, Russell RA, et al. Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors [J]. J Gene Med. 2005.7(7):918-925.
[25] Nawagitgul, P., I. Morozov, S. R. Bolin, P. A. Harms, and S. D. Sorden. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein [J]. J. Gen. Virol. 2000. 81:2281-2287.
[26] Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HIV-1 infection [J]. Nat Med. 2002.8(7):681-6.
[27] Olvera A, Sibila M, Calsamiglia M, Segales J, Domingo M. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs [J]. J Virol Methods. 2004.117(1):75-80
[28] Persengiev, S.P., Zhu, X., Green, M.R. Nonspecific, concentration dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs) [J]. RNA. 2004.10, 12-18.
[29] Pringle, C. R. Virus taxonomy at the Xlth International Congress of Virology, Sydney, Australia [J]. Arch. Virol. 1999.144:2065-2070.
[30] Randall G, Grakoui A, Rice C M. Clearance ofrplicating hepatitis C virus rplicon RNAs in cell culture by small interfering RNAs [J]. Proc Natl Acad Sci USA, 2003.100(1):235-240.
[31] Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, and Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells [J]. Proc. Natl. Acad. Sci. USA. 2002. 99(8): 5515-5520.
[32] Tischer, I., W. Mields, D. Wolff, M. Vagt, and W. Griem. Studies on epidemiology and pathogenicity of porcine circovirus [J]. Arch. Virol. 1986. 91:271-276.
[33] Zhou XM, Lin JS, Shi Y, Tian DA, Huang HJ, Zhou HJ, Jin YX. Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene [J]. Acta Biochim Biophys Sin (Shanghai). 2005.37(5):310-6.
[34] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal. Biochem. 1976. 72, 248-254
[35] 温立斌．猪圆环病毒2型分子流行病学研究[D]．北京：中国农业大学，2005
[1] Allan, G. M., F. McNeilly, J. P. Cassidy, G. A. Reilly, B. Adair, W. A. Ellis, and M. S. McNulty. Pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material [J]. Vet. Microbiol. 1995.44:49—64.
[2] Allan, G.M., Meehan, B., Todd, D., Kennedy, S., McNeilly, F., Ellis, J., Clark, E.G., Harding, J., Espuna, E., Botner, A., Charreyre, C. Novel porcine circoviruses from pigs with wasting disease syndromes [J]. Veterinary Record 1998. 142, 467-468.
[3] Brunborg IM, Moldal T, Jonassen CM. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR [J]. J Virol Methods. 2004. 15; 122(2): 171-8.
[4] Chen, W., W. Yan, Q. Du, L. Fei, M. Liu, Z. Ni, Z. Sheng, and Z. Zhong. RNA interference targeting VPI inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice [J]. J. Virol. 2004. 78:6900-6907.
[5] Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference [J]. J Virol. 2002.76(18):9225-31.
[6] Ding, S. W., H. Li, R. Lu, F. Li, and W. X. Li. RNA silencing: a conserved antiviral immunity of plants and animals [J]. Virus Res. 2004. 102:109-115.
[7] Dulac GC, Afshar A. Porcine circovirus antigens in pk-15 cell line (ATCCCCL-33) and evidence of antibodies to circovirus in Canadian pigs [J]. Can J Vet Res. 1989. 53:431—433
[8] Gitlin, L., and R. Andino. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing [J]. J. Virol. 2003.77:7159-7165.
[9] Haasnoot, P.C., Cupac, D., Berkhout, B. Inhibition of virus replication by RNA interference [J]. J. Biomed. Sci. 2003.10, 607—616.
[10] Kiupel, M., Stevenson, G.W., Choi, J., Latimer, K.S., Kanitz, C.L., Mittal, S.K. Viral replication and lesions in BALB/c mice experimentally inoculated with porcine circovirus isolated from a pig with postweaning multisystemic wasting disease [J]. Vet. Pathol. 2001.38, 74-82.
[11] Jana S, Chakraborty C, Nandi S, Deb JK. RNA interference: potential therapeutic targets [J]. Appl Microbiol Biotechnol. 2004.65(6):649-57.
[12] Kameoka M, Nukuzuma S, Itaya A, Tanaka Y, Ota K, Ikuta K, Yoshihara K. RNA interference directed against Poly (ADP-Ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells [J]. J Virol. 2004.78(16):8931-4
[13] Li Yao-Chen, Zhao Zhi-Gang, Li Kang-Sheng. RNA interference mediated inhibition of influenza virus NP and PA gene expressions and multiplication in MDCK cell [J]. J Fourth Mil Med Univ. 2006. 27(8):681-685.
[14] Liu Q, Wang L, Willson P, Babiuk LA. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome [J]. J Clin Microbiol. 2000. 38(9):3474-7.
[15] Lu, S., and B. R. Cullen. Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and microRNA biogenesis [J]. J. Virol. 2004.78:12868-12876.
[16] Morris, K.V., Chan, S.W.L., Jacobsen, S.E., Looney, D.J. Small interfering RNA-induced transcriptional gene silencing in human cells [J]. Science. 2004.27,1289- 1292.
[17] Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HTV-1 infection [J]. Nat Med. 2002. 8(7):681-6.
[18] Olvera A, Sibila M, Calsamiglia M, Segales J, Domingo M. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs [J]. J Virol Methods. 2004.117(1):75-80
[19] Pringle, C. R. Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia [C]. Arch. Virol. 1999. 144:2065-2070.
[20] Quintana, J., Balasch, M., Segale's, J., Calsamiglia, M., Rodn'guez-Arrioja, G.M., Plana-Dura'n, J., Domingo, M. Experimental inoculation of porcine circoviruses type 1 (PCV1) and type 2 (PCV2) in rabbits and mice [J]. Vet. Res. 2002. 33,229-237.
[21] Tischer, I., W. Mields, D. Wolff, M. Vagt, and W. Griem. Studies on epidemiology and pathogenicity of porcine circovirus [J]. Arch. Virol. 1986.91:271-276.
[22] Zhou XM, Lin JS, Shi Y, Tian DA, Huang HJ, Zhou HJ, Jin YX. Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene [J]. Acta Biochim Biophys Sin (Shanghai). 2005. 37(5):310-6.
[23] Fenaux M, Opriessnig T, Halbur PG, Elvinger F, Meng XJ. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo [J]. J Virol. 2004, 78(24):13440-6.