应用RNAi技术抑制猪圆环病毒2型的复制和表达猪圆环病毒2型衣壳蛋白的杆状病毒的免疫保护作用
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摘要
猪圆环病毒(porcine circovirus,PCV)属于圆环病毒科,为单股环状DNA病毒。分为1型(porcine circovirus type 1,PCV1)和2型(porcinecireovirus type 2,PCV2),其中PCV1不致病,PCV2具有致病性,能引起猪的多种病症,如断奶仔猪多系统衰竭综合征(post-weaning multisystemicwasting syndrome,PMWS)、猪皮炎与肾病综合征(porcine dermatitis andnephropathy syndrome,PDNS)及繁殖障碍等。自加拿大1991年首次报道发生PMWS以来,现在全世界大多数养猪国家均有该病发生。PCV2感染能造成免疫抑制,易继发或并发其它病原体感染,对养猪业造成很大的经济损失。因此,寻找一些新的方法防制该病已成为一项紧迫任务。对该病的防制,一方面在于研制新型疫苗,如DNA疫苗和亚单位苗;另一方面,一些新技术的应用,如RNA干扰(RNA interference,RNAi)技术。RNA干扰是近年来发展起来的一种新技术,可通过对特定基因转录出的mRNA进行降解,对病原体的复制产生抑制,从而防止疾病的发生。本研究首先从临床上疑为PMWS的病例中分离到了10株PCV2,测定其全基因组序列,并对基因序列进行了遗传分析,获得了基因组的变异信息。对一株PCV2参考毒株的开放阅读框2进行了克隆,鉴定后构建了表达PCV2衣壳蛋白(capsid,Cap)的重组杆状病毒,对其表达的Cap蛋白进行了免疫生物学特性研究,同时进行了杆状病毒表达的Cap蛋白对小鼠的免疫保护试验。对测定的和网上登录的PCV基因组序列进行了同源性分析,应用相关软件和网上资源设计了6个PCV特异性小干扰RNA(small interference RNA,siRNA)片段,构建了siRNA表达质粒,鉴定出了相应的插入序列正确的克隆,对PCV1和PCV2毒株在Dulac细胞及PCV2毒株在小鼠体内复制的抑制作用进行了初步研究。
1.江苏部分地区10株猪圆环病毒2型分离株的分子流行病学
从PMWS病例中分离到10株PCV2,对其全基因组序列进行了测定和分析,并将10株PCV2毒株基因组序列提交到GenBank。分析结果表明,分离到的10株PCV2全长均为1767个核苷酸,分属于3个不同的基因亚群,其中苏州0511毒株在一个较远的分支上,扬州0705与其它8个毒株分布在不同分支上,遗传上有一定差异,但总体上遗传关系较近,基因组同源性在98.3-99.5%之间,同处于PCV2基因1群中。其中一株分离自家养野猪的PCV2和其它7株遗传关系非常近,可能是同一个起源。这些分离株的cap基因变异程度不大,可作为亚单位苗或DNA疫苗的良好候选基因。
2.表达猪圆环病毒2型衣壳蛋白的重组杆状病毒对小鼠的免疫保护作用
通过PCR方法扩增出PCV2 XSC株cap基因,克隆到pGEM-T~(?)easy载体,测序验证后亚克隆到转座载体pFastBac~(TM)1,经再次测序验证后将含有PCV2衣壳蛋白基因的表达盒转座到穿梭载体Bacmid中。将获得的重组质粒Bacmid-Cap转染Sf9细胞,获得了重组杆状病毒,病毒扩增后重组杆状病毒的滴度达到5×10~8pfu/ml,以10~5pfu的reBac-Cap感染Sf9细胞,经SDS-PAGE和Western blot证实Cap蛋白已表达,分子量约28kDa,与天然衣壳蛋白分子量大小一致。SDS-PAGE证实,PCV2 Cap蛋白非分泌性、可溶性地表达于Sf9细胞胞浆内。取感染reBac-Cap的Sf9细胞进行免疫荧光试验,能与猪抗PCV2高免血清发生特异性反应,显示重组核衣壳蛋白具有良好的抗原性。重组杆状病毒免疫小鼠,应用间接免疫荧光方法对接种后7d、14d、21d、28d、35d、42d、49d、56d的小鼠进行血清抗体检测。结果显示从接种后21d开始小鼠血清产生了针对PCV2 Cap蛋白的特异性抗体,攻毒后不同时间应用建立的TaqMan探针定量PCR方法检测血清中PCV2病毒拷贝数,结果发现表达PCV2 Cap的重组杆状病毒免疫后对病毒在小鼠体内的复制具有一定的抑制作用。
3.质粒表达的siRNA片段对猪圆环病毒在Dulac细胞中复制的抑制作用
在对PCV2基因组进行分析的基础上设计了siRNA片段,合成后构建了表达载体。对GenBank中登录和实验室测定的PCV基因组序列进行分析,将PCV毒株基因保守区序列提交到Ambion公司提供的siRNA查找工具,产生候选siRNA片段,将候选siRNA片段在internet上进行同源比对,将与其它物种同源性高的siRNA片段剔除。结合已发表文献中的siRNA片段序列,最终确定了6个siRNA片段,其中4个是针对猪圆环病毒rep基因的,命名为SH1(S50-68)、SH2(S554-572)、SH3(S653-671)、SH4(S624-642);2个针对PCV2 cap基因,命名为SH5(R1429-1411)、SH6(R1307-1289)。根据所用表达载体RNAi-Ready pSIREN-RetroQ ZsGreen的要求设计了6对67个碱基的DNA片段,该序列可在转染细胞中产生发夹结构RNA(shorthairpin RNA,shRNA)分子。人工合成相应DNA片段,退火后形成双链,与载体试剂盒中的针对萤光素酶(Luciferase,Luc)的67个碱基的siRNA相应DNA片段及阴性对照片段(46个碱基)分别连接到载体鼠源U6启动子下游,转化大肠杆菌DH5α,挑取阳性克隆,测序鉴定,得到了正确的克隆,分别命名为Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc和Retro-NC。
首先对siRNA表达质粒Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6的抑制作用进行检测,筛选出抑制作用较强的siRNA。重悬后的对数生长期Dulac细胞0.5ml/孔(2.0×10~5个细胞)置于24孔细胞板中进行培养,6h后加入梭华-Sofast~(TM)基因转染试剂及0.6μg的siRNA表达质粒Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc和对照质粒复合物,12h、24h后分别进行荧光观察。24h后每孔加入0.1ml含有10~5 TCID_(50)的PCV2 XSC株病毒液感染上述细胞,感染后72h收集上清和细胞,应用TaqMan探针Real-time PCR法对PCV2病毒拷贝数进行检测。结果发现,细胞在转染上述质粒后12h开始出现绿色荧光,24h后荧光较强。观察到荧光表明转染成功,siRNA片段可能已经表达。siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6转染细胞后病毒基因拷贝数与未转染质粒细胞组和转染其它质粒对照组差异显著,说明Retro-SH1,Retro-SH4,Retro-SH6表达的siRNA片段作用较强。用筛选出的3个对病毒复制抑制作用较强的siRNA表达质粒进行以下试验。
转染质粒后不同时间对后续接种的病毒复制的干扰作用进行了比较,转染不同质粒及三种质粒的混合体24h后加0.1ml含10~5TCID_(50)的PCV2 XSC参考株感染细胞,分别于感染后24h、36h、48h、60h、72h、84h、96h、120h收集上清和细胞,应用TaqMan探针real-time PCR法对PCV2病毒拷贝数进行检测。结果发现,感染60h后载体表达的特异性siRNA片段对PCV2病毒的复制表现出很强的抑制作用,可以持续到96h以后,不同的siRNA片段抑制作用有一定差异,其中表达质粒Retro-SH4的干扰作用最强,而3个siRNA表达质粒混合体对PCV2病毒的干扰作用与单独作用最强的处理组的干扰作用没有明显差异,并未显示出协同作用。
对不同浓度的质粒产生的干扰作用进行了比较,转染50ng、100ng,500ng、1000ng、1500ng、2500ng表达siRNA质粒的Dulac细胞在感染PCV2后60h收集上清和细胞,检测其病毒在细胞内的拷贝数,发现不同浓度的表达质粒对PCV2 XSC株的复制均有抑制作用,且500ng-2500ng浓度的质粒其干扰作用无明显差别。综合考虑质粒对细胞的干扰强度相当且节省质粒等原则,随后在细胞中的抑制试验均选用500ng的质粒。
应用流式细胞术对500ng的Retro-SH1,Retro-SH4,Retro-SH6表达的siRNA片段对病毒复制的抑制作用进行了分析,结果表明三个质粒表达的siRNA对PCV2的复制均有一定的抑制作用,其中Retro-SH4作用最强。
Dulae细胞感染PCV2 XSC株后12h、24h、36h、48h和60h分别转染表达siRNAs的质粒,运用定量PCR方法进行检测,发现细胞感染12h后转染表达PCV2特异的siRNAs质粒对PCV2毒株的复制有很强的抑制作用,细胞感染PCV2 36h后再转染质粒其抑制作用降低,而混合质粒对PCV2病毒的干扰与单独抑制能力最强的质粒的作用没有明显差异,并未显示协同作用,Retro-Luc和对照质粒组则没有抑制作用。以上结果说明,细胞感染PCV2后,再转染表达的特异性siRNA的质粒能有效抑制病毒的繁殖,但以细胞感染PCV2 12h内即转染干扰质粒其抑制效果最好。
siRNA对临床分离毒株在Dulac细胞上复制的抑制作用进行了比较试验,500ng siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6分别转染Dulac细胞,置于24孔细胞板上培养,24h后加入10~5个TCID_(50)的不同PCV2分离株的细胞培养物,结果表明,500ng的质粒Retro-SH1,Retro-SH4,Retro-SH6对不同PCV2分离株在细胞中的复制均有一定抑制作用,不同的siRNA分子作用有差异,但不同毒株之间差异不大。
500ng siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6分别转染Dulac细胞,培养24h后加入10~5个TCID_(50)的PCV1毒株的细胞培养物,结果表明,Retro-SH1,Retro-SH4对PCV1复制有很强的抑制作用,而Retro-SH6对PCV1在细胞中的复制没有明显抑制作用。
4.表达siRNA的质粒对PCV2在小鼠体内复制的抑制作用
将72只8周龄BALB/c小鼠随机分为12组,每组6只,第1-4组每只分别肌肉注射10μg表达的siRNA质粒Retro-SH1,Retro-SH4,Retro-SH6,Retro-NC,第5组肌肉注射10μg Retro-SH1,Retro-SH4和Retro-SH6混合质粒。24h后每只按10~5TCID_(50)的量口服及腹腔注射0.1ml的PCV2病毒。第6-10组先用10~5TCID_(50) PCV2攻毒,24h后每只分别肌肉注射10μg表达的siRNA质粒Retro-SH1,Retro-SH4,Retro-SH6或它们的混合体及Retro-NC。第11组为攻毒对照组,第12组为健康对照组。分别于感染后第5天及第11天进行扑杀采样,每次采集3只小鼠血清和脾脏,应用real-timePCR方法检测血清中PCV2病毒拷贝数,通过免疫组化和组织学方法检测组织中病毒抗原与病理损伤。结果表明10μg siRNA表达质粒对PCV2攻毒的小鼠有很好的保护力,但不同的siRNA分子保护力有差异。3个siRNA表达质粒混合体与单独作用最强的质粒组的保护力没有明显差异,并未显示协同作用。而对已感染PCV2的BALB/c小鼠进行治疗时发现,载体表达的PCV2特异性的siRNA分子对小鼠也有一定的保护作用。
综上所述,本研究从临床上出现PMWS症状的病猪中分离出10株PCV2,测定了它们的基因组序列,遗传发生分析结果表明,10个毒株分属于3个遗传分支,但总体上遗传距离很近,核苷酸的同源性在98.9%以上,尤其是cap基因很保守。通过PCR扩增出了cap基因,构建了表达该基因的重组杆状病毒,动物实验证明,重组病毒免疫后能够抑制PCV2在小鼠体内的复制。设计出了6个针对猪圆环病毒的siRNA片段,构建了shRNA分子的质粒表达载体,筛选出了3个作用较强的siRNA片段(Retro-SH1,Retro-SH4,Retro-SH6),证实了这3个表达的siRNA片段在Dulac细胞和小鼠体内都能够对PCV2的复制产生抑制作用,其中2个在Dulac细胞中对PCV1也有抑制作用,以上siRNA表达质粒可能成为控制猪圆环病毒2型感染的新工具之一。
Porcine circovirus type 2(PCV2) infection has been associated with post-weaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex and reproductive disorders.Since the first case was reported in Canada in 1991, PCV2 infections have been confirmed in many countries of the world.The pigs infected with PCV2 were in an immunosuppressive status which could trigger co-infection with other pathogens.It is not an easy task to get the epidemics under control or eradication because of wide spread of the virus.It is necessary to explore some novel approaches for better prevention and control of the disease. Within the scope of the development of new vaccines against PCV2 infection, subunit vaccines based on the baculovirus expression system should be a putative prophylactic tool.RNA interference(RNAi) is a recently developed technology in which double-stranded RNA(dsRNA) directs sequence-specific degradation of messenger RNA(mRNA) and serves as a potential therapeutic strategy for the inhibition of virulence-associated genes of some pathogens.
In this study,10 isolates of PCV2 were obtained from the clinical cases infected with PWMS in Jiangsu Province.Complete genomes of these PCV2 strains were sequenced and analyzed.The cap gene of PCV2 strain XSC was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice.Several small interference RNAs(siRNAs) specific to porcine circovirus type 2 were designed,synthesized and expressed by the plasmid vector. Their inhibition effect on the replication of PCV1 and PCV2 by these siRNAs was evaluated in both Dulac cells and mice.
1.Molecular characteristics of ten isolates of PCV2 from Jiangsu Province
Ten isolates of PCV2 were obtained from the clinical cases infected with PMWS by passaging in Dulac cells.Complete genomes of these PCV2 strains were amplified by over-lapping PCR and sequenced directly.The whole genomic sequences of all these isolates were spliced successfully and all of them were 1767 nucleotides long,which have been deposited in GenBank.Sequence analysis showed that these PCV2 isolates exhibited high level of nucleotide similarity and were classified into group 1 of PCV2 based on their genomic sequences.The phylogenetic tree analysis and multiple alignments of nucleotide sequences present that the isolate Suzhou 0511 and Yangzhou 0705 were in other cluster departed from other 8 isolates.All genomics of these isolates exhibited 98.3-99.5%nucleotide sequence identities and their cap genes were also in higher similarity.
2.The immunogenicity of the PCV2 capsid protein expressed by the recombinant baculovirus
The capsid protein gene of PCV2 strain XSC was amplified by PCR and cloned into pGEM-T(?) easy vector,and subcloned into pFastBac~(TM)1 donor plasmid,then inserted into baculovirus shuttle vector Bacmid by site-specific transposition.The recombinant baculovirus,designated as reBac-Cap,was constructed when the recombinant shuttle vector Bacmid-Cap was transfected into Sf9 cells.To evaluate the expression level of capsid protein gene,a viral stock with a high-titer up to 5×10~8 pfu/ml was obtained and determined by the plaque assay.Infected Sf9 cells with 10~5 pfu reBac-Cap were harvested at 72 h post infection,the expressed capsid protein with molecular weight of 28 kDa,the same size as the natural capsid protein,was identified to be expressed in cytoplasm in soluble,non-secreted form.Furthermore,the expression product was observed to react with the pig antisera specific to PCV2 virus.Sera samples collected from all control and vaccinated animals at 0,7,14,21,28,35,42 49,56 day post-innoculation(dpi) were assayed for anti-PCV2 capsid antibodies by IFA.The results indicated that the recombinant baculovirus reBac-Cap induced specific antibodies against the pathogenic PCV2 capsid antigen from 21dpi.The sera were detected by real-time PCR.The results indicated that the recombinant baculovirus reBac-Cap was experienced the highest replication inhibition rate of PCV2.
3.Inhibition of the replication of the porcine circovirus type 2 in the Dulac cells by the siRNAs expressed by the plasmids
Sequences of PCV capsid protein genes and replicase genes determined in this study or from the GenBank were submitted to the website of Ambion company for target sequences searching,and some candidate siRNA sequences were generated.The candidate siRNA sequences were blasted,those shared any identity with other known organism genes were exclued.Six siRNAs were chosen to be evaluated for their selected inhibition effect on the replication of PCVs,four of these were specific to the rep gene of PCVs,designated as SH1(S50-68)、SH2(S554-572)、SH3(S653-671)、SH4(S624-642),and the others were specific to the cap gene of PCV2,designated as SH5(R1429-1411) and SH6(R1307-1289). Six DNA fragments with 67 nucleotides producing short hairpin RNA(shRNA) were designed based on the plasmid vector.The corresponding DNA fragments were synthesized,annealed and ligated to the downstream of the mouse originated U6 promoter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector.As controls, the siRNAs specific to Luciferase gene within the vector and negative fragments for any organisms were also included.Recombinant plasmids were transformed into the host bacteria DH5αand positive clones were selected.Positive clones carried the expected insertions were identified by sequencing and designated as Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc and Retro-NC,respectively.
To test whether siRNAs expressed by these six plasmids inhibit the PCV2 replication,we first examined their effect on the replication of the PCV2 virus in the Dulac cells.The plamids were introduced into the Dulac cells using Sofast~(TM) transfection reagent.Each samples had triplication.As controls,Retro-Luc and Retro-NC were similarly introduced into the same cells,followed by PCV2 virus infection.The cells were collected at 72 h after infection.Virus loads was detected by real-time PCR assay established in our lab.The results showed that virus loads strongly reduced in cells transfected by Retro-SH1,Retro-SH4 and Retro-SH6,while did not reduce in the cells transfected by other plasmids and the controls.These three siRNAs expressed plasmids were further evaluated in the following experiments.
The kinetic effect of siRNAs expressed by plasmids on PCV2 replication in Dulac cells was determined according to the method mentioned above.Three kinds of siRNA expressing plasmids and their mixture were transfected into the cells,and then infected by PCV2 at 24 h after transfection.Collected cell cultures at 24 h,36 h,48 h,60 h,72 h,96 h,120 h after infection were determined for the copies of genomic DNA of PCV2 by real-time PCR.The results showed that there was strong inhibition effect on PCV2 replication from 60 h post its infection,and the effect lasted at least 48 h.As the dose of plasmids transfected,a positive dose-dependent effect was confirmed.500 ng of each plasmid was optimal.The inhibition effect of the Retro-SH4 was the strongest among three plasmids,and no synergism was observed in the cells transfected by the mixture of the three plasmids.The results were also confirmed by the FACS technique.When Dulac cells were infected with PCV2 XSC strain,then each of three plasmids and their mixture were transfected into above cells at different intervals.The results indicated that the siRNAs expressing plasmids should be tranfected as early as possible in order to get strong inhibitions.Transfection of the plasmids at 36 h after virus infection could not inhibit the ongoing replication of the PCV2 effectively.For ten of PCV2 field isolates,the siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could inhibit the replication of all isolates in Dulae cells. For PCV1,the siRNAs expressed by Retro-SH1,Retro-SH4 specific to the rep gene of PCV exhibited inhibition effect on the replication of PCV1 in Dulac cells while Retro-SH6 did not.
4.Inhibition of the replication of the porcine circovirus type 2 in mice by the siRNAs expressed by the plasmids
Seventy-two of 8-week-old BALB/c mice were divided randomly into 12 groups,6 mice for each.Mice in four groups received 10μg of Retro-SH1, Retro-SH4,Retro-SH6 and Retro-NC,respectively,another group were intramuscularly injected with the mixture of Retro-SH1,Retro-SH4 and Retro-SH6,mice in these five groups were challenged by oral administration and intraperitoneal injection with 10~5 TCID_(50) of PCV2.Mice in other five groups were oral administration and intraperitoneal injected with 10~5 TCID_(50) of PCV2 and then intramuscularly introduced by Retro-SH1,Retro-SH4,Retro-SH6 or their mixture and Retro-NC at 24 h after PCV2 infection.One group was treated as challenge control and the other as a blank control group.All of the mice were slaughted for sera and tissues collection at 5 d and 11 d post challenge.The sera were detected by real-time PCR and the tissues were examined by histopathological and immunohistochemical techniques.Both of the prophylactic and therapy experiments showed that siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could offer some protection against PCV2 challenge, Retro-SH4 inoculation group experienced the highest replication inhibition rate up to 99.8%.No synergism was observed in the group transfected by the mixture of the three plasmids.
In summary,10 isolates of PCV2 were obtained from the clinical cases infected with PMWS in Jiangsu Province.Genomic sequences of these isolates were in high level of homology.The cap gene of PCV2 was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice. The mice immunized with the recombinant baculovirus could elicit specific antibody against PCV2.Six of siRNAs specific for PCV2 genomic fragments were designed and the plasmid-based system for generating unimolecular,hairpin siRNAs were constructed successfully and three of them(Retro-SH1,Retro-SH4, Retro-SH6) were selected.These siRNAs expressed by the plasmids could inhibit the replication of the PCVs in the Dulac cells and provide some protection against PCV2 infection in mice.These data indicated that siRNA technique should be a potential useful tool for developing novel strategy to prevent and control PCV2 infection.
1.江苏部分地区10株猪圆环病毒2型分离株的分子流行病学
从PMWS病例中分离到10株PCV2,对其全基因组序列进行了测定和分析,并将10株PCV2毒株基因组序列提交到GenBank。分析结果表明,分离到的10株PCV2全长均为1767个核苷酸,分属于3个不同的基因亚群,其中苏州0511毒株在一个较远的分支上,扬州0705与其它8个毒株分布在不同分支上,遗传上有一定差异,但总体上遗传关系较近,基因组同源性在98.3-99.5%之间,同处于PCV2基因1群中。其中一株分离自家养野猪的PCV2和其它7株遗传关系非常近,可能是同一个起源。这些分离株的cap基因变异程度不大,可作为亚单位苗或DNA疫苗的良好候选基因。
2.表达猪圆环病毒2型衣壳蛋白的重组杆状病毒对小鼠的免疫保护作用
通过PCR方法扩增出PCV2 XSC株cap基因,克隆到pGEM-T~(?)easy载体,测序验证后亚克隆到转座载体pFastBac~(TM)1,经再次测序验证后将含有PCV2衣壳蛋白基因的表达盒转座到穿梭载体Bacmid中。将获得的重组质粒Bacmid-Cap转染Sf9细胞,获得了重组杆状病毒,病毒扩增后重组杆状病毒的滴度达到5×10~8pfu/ml,以10~5pfu的reBac-Cap感染Sf9细胞,经SDS-PAGE和Western blot证实Cap蛋白已表达,分子量约28kDa,与天然衣壳蛋白分子量大小一致。SDS-PAGE证实,PCV2 Cap蛋白非分泌性、可溶性地表达于Sf9细胞胞浆内。取感染reBac-Cap的Sf9细胞进行免疫荧光试验,能与猪抗PCV2高免血清发生特异性反应,显示重组核衣壳蛋白具有良好的抗原性。重组杆状病毒免疫小鼠,应用间接免疫荧光方法对接种后7d、14d、21d、28d、35d、42d、49d、56d的小鼠进行血清抗体检测。结果显示从接种后21d开始小鼠血清产生了针对PCV2 Cap蛋白的特异性抗体,攻毒后不同时间应用建立的TaqMan探针定量PCR方法检测血清中PCV2病毒拷贝数,结果发现表达PCV2 Cap的重组杆状病毒免疫后对病毒在小鼠体内的复制具有一定的抑制作用。
3.质粒表达的siRNA片段对猪圆环病毒在Dulac细胞中复制的抑制作用
在对PCV2基因组进行分析的基础上设计了siRNA片段,合成后构建了表达载体。对GenBank中登录和实验室测定的PCV基因组序列进行分析,将PCV毒株基因保守区序列提交到Ambion公司提供的siRNA查找工具,产生候选siRNA片段,将候选siRNA片段在internet上进行同源比对,将与其它物种同源性高的siRNA片段剔除。结合已发表文献中的siRNA片段序列,最终确定了6个siRNA片段,其中4个是针对猪圆环病毒rep基因的,命名为SH1(S50-68)、SH2(S554-572)、SH3(S653-671)、SH4(S624-642);2个针对PCV2 cap基因,命名为SH5(R1429-1411)、SH6(R1307-1289)。根据所用表达载体RNAi-Ready pSIREN-RetroQ ZsGreen的要求设计了6对67个碱基的DNA片段,该序列可在转染细胞中产生发夹结构RNA(shorthairpin RNA,shRNA)分子。人工合成相应DNA片段,退火后形成双链,与载体试剂盒中的针对萤光素酶(Luciferase,Luc)的67个碱基的siRNA相应DNA片段及阴性对照片段(46个碱基)分别连接到载体鼠源U6启动子下游,转化大肠杆菌DH5α,挑取阳性克隆,测序鉴定,得到了正确的克隆,分别命名为Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc和Retro-NC。
首先对siRNA表达质粒Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6的抑制作用进行检测,筛选出抑制作用较强的siRNA。重悬后的对数生长期Dulac细胞0.5ml/孔(2.0×10~5个细胞)置于24孔细胞板中进行培养,6h后加入梭华-Sofast~(TM)基因转染试剂及0.6μg的siRNA表达质粒Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc和对照质粒复合物,12h、24h后分别进行荧光观察。24h后每孔加入0.1ml含有10~5 TCID_(50)的PCV2 XSC株病毒液感染上述细胞,感染后72h收集上清和细胞,应用TaqMan探针Real-time PCR法对PCV2病毒拷贝数进行检测。结果发现,细胞在转染上述质粒后12h开始出现绿色荧光,24h后荧光较强。观察到荧光表明转染成功,siRNA片段可能已经表达。siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6转染细胞后病毒基因拷贝数与未转染质粒细胞组和转染其它质粒对照组差异显著,说明Retro-SH1,Retro-SH4,Retro-SH6表达的siRNA片段作用较强。用筛选出的3个对病毒复制抑制作用较强的siRNA表达质粒进行以下试验。
转染质粒后不同时间对后续接种的病毒复制的干扰作用进行了比较,转染不同质粒及三种质粒的混合体24h后加0.1ml含10~5TCID_(50)的PCV2 XSC参考株感染细胞,分别于感染后24h、36h、48h、60h、72h、84h、96h、120h收集上清和细胞,应用TaqMan探针real-time PCR法对PCV2病毒拷贝数进行检测。结果发现,感染60h后载体表达的特异性siRNA片段对PCV2病毒的复制表现出很强的抑制作用,可以持续到96h以后,不同的siRNA片段抑制作用有一定差异,其中表达质粒Retro-SH4的干扰作用最强,而3个siRNA表达质粒混合体对PCV2病毒的干扰作用与单独作用最强的处理组的干扰作用没有明显差异,并未显示出协同作用。
对不同浓度的质粒产生的干扰作用进行了比较,转染50ng、100ng,500ng、1000ng、1500ng、2500ng表达siRNA质粒的Dulac细胞在感染PCV2后60h收集上清和细胞,检测其病毒在细胞内的拷贝数,发现不同浓度的表达质粒对PCV2 XSC株的复制均有抑制作用,且500ng-2500ng浓度的质粒其干扰作用无明显差别。综合考虑质粒对细胞的干扰强度相当且节省质粒等原则,随后在细胞中的抑制试验均选用500ng的质粒。
应用流式细胞术对500ng的Retro-SH1,Retro-SH4,Retro-SH6表达的siRNA片段对病毒复制的抑制作用进行了分析,结果表明三个质粒表达的siRNA对PCV2的复制均有一定的抑制作用,其中Retro-SH4作用最强。
Dulae细胞感染PCV2 XSC株后12h、24h、36h、48h和60h分别转染表达siRNAs的质粒,运用定量PCR方法进行检测,发现细胞感染12h后转染表达PCV2特异的siRNAs质粒对PCV2毒株的复制有很强的抑制作用,细胞感染PCV2 36h后再转染质粒其抑制作用降低,而混合质粒对PCV2病毒的干扰与单独抑制能力最强的质粒的作用没有明显差异,并未显示协同作用,Retro-Luc和对照质粒组则没有抑制作用。以上结果说明,细胞感染PCV2后,再转染表达的特异性siRNA的质粒能有效抑制病毒的繁殖,但以细胞感染PCV2 12h内即转染干扰质粒其抑制效果最好。
siRNA对临床分离毒株在Dulac细胞上复制的抑制作用进行了比较试验,500ng siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6分别转染Dulac细胞,置于24孔细胞板上培养,24h后加入10~5个TCID_(50)的不同PCV2分离株的细胞培养物,结果表明,500ng的质粒Retro-SH1,Retro-SH4,Retro-SH6对不同PCV2分离株在细胞中的复制均有一定抑制作用,不同的siRNA分子作用有差异,但不同毒株之间差异不大。
500ng siRNA表达质粒Retro-SH1,Retro-SH4,Retro-SH6分别转染Dulac细胞,培养24h后加入10~5个TCID_(50)的PCV1毒株的细胞培养物,结果表明,Retro-SH1,Retro-SH4对PCV1复制有很强的抑制作用,而Retro-SH6对PCV1在细胞中的复制没有明显抑制作用。
4.表达siRNA的质粒对PCV2在小鼠体内复制的抑制作用
将72只8周龄BALB/c小鼠随机分为12组,每组6只,第1-4组每只分别肌肉注射10μg表达的siRNA质粒Retro-SH1,Retro-SH4,Retro-SH6,Retro-NC,第5组肌肉注射10μg Retro-SH1,Retro-SH4和Retro-SH6混合质粒。24h后每只按10~5TCID_(50)的量口服及腹腔注射0.1ml的PCV2病毒。第6-10组先用10~5TCID_(50) PCV2攻毒,24h后每只分别肌肉注射10μg表达的siRNA质粒Retro-SH1,Retro-SH4,Retro-SH6或它们的混合体及Retro-NC。第11组为攻毒对照组,第12组为健康对照组。分别于感染后第5天及第11天进行扑杀采样,每次采集3只小鼠血清和脾脏,应用real-timePCR方法检测血清中PCV2病毒拷贝数,通过免疫组化和组织学方法检测组织中病毒抗原与病理损伤。结果表明10μg siRNA表达质粒对PCV2攻毒的小鼠有很好的保护力,但不同的siRNA分子保护力有差异。3个siRNA表达质粒混合体与单独作用最强的质粒组的保护力没有明显差异,并未显示协同作用。而对已感染PCV2的BALB/c小鼠进行治疗时发现,载体表达的PCV2特异性的siRNA分子对小鼠也有一定的保护作用。
综上所述,本研究从临床上出现PMWS症状的病猪中分离出10株PCV2,测定了它们的基因组序列,遗传发生分析结果表明,10个毒株分属于3个遗传分支,但总体上遗传距离很近,核苷酸的同源性在98.9%以上,尤其是cap基因很保守。通过PCR扩增出了cap基因,构建了表达该基因的重组杆状病毒,动物实验证明,重组病毒免疫后能够抑制PCV2在小鼠体内的复制。设计出了6个针对猪圆环病毒的siRNA片段,构建了shRNA分子的质粒表达载体,筛选出了3个作用较强的siRNA片段(Retro-SH1,Retro-SH4,Retro-SH6),证实了这3个表达的siRNA片段在Dulac细胞和小鼠体内都能够对PCV2的复制产生抑制作用,其中2个在Dulac细胞中对PCV1也有抑制作用,以上siRNA表达质粒可能成为控制猪圆环病毒2型感染的新工具之一。
Porcine circovirus type 2(PCV2) infection has been associated with post-weaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex and reproductive disorders.Since the first case was reported in Canada in 1991, PCV2 infections have been confirmed in many countries of the world.The pigs infected with PCV2 were in an immunosuppressive status which could trigger co-infection with other pathogens.It is not an easy task to get the epidemics under control or eradication because of wide spread of the virus.It is necessary to explore some novel approaches for better prevention and control of the disease. Within the scope of the development of new vaccines against PCV2 infection, subunit vaccines based on the baculovirus expression system should be a putative prophylactic tool.RNA interference(RNAi) is a recently developed technology in which double-stranded RNA(dsRNA) directs sequence-specific degradation of messenger RNA(mRNA) and serves as a potential therapeutic strategy for the inhibition of virulence-associated genes of some pathogens.
In this study,10 isolates of PCV2 were obtained from the clinical cases infected with PWMS in Jiangsu Province.Complete genomes of these PCV2 strains were sequenced and analyzed.The cap gene of PCV2 strain XSC was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice.Several small interference RNAs(siRNAs) specific to porcine circovirus type 2 were designed,synthesized and expressed by the plasmid vector. Their inhibition effect on the replication of PCV1 and PCV2 by these siRNAs was evaluated in both Dulac cells and mice.
1.Molecular characteristics of ten isolates of PCV2 from Jiangsu Province
Ten isolates of PCV2 were obtained from the clinical cases infected with PMWS by passaging in Dulac cells.Complete genomes of these PCV2 strains were amplified by over-lapping PCR and sequenced directly.The whole genomic sequences of all these isolates were spliced successfully and all of them were 1767 nucleotides long,which have been deposited in GenBank.Sequence analysis showed that these PCV2 isolates exhibited high level of nucleotide similarity and were classified into group 1 of PCV2 based on their genomic sequences.The phylogenetic tree analysis and multiple alignments of nucleotide sequences present that the isolate Suzhou 0511 and Yangzhou 0705 were in other cluster departed from other 8 isolates.All genomics of these isolates exhibited 98.3-99.5%nucleotide sequence identities and their cap genes were also in higher similarity.
2.The immunogenicity of the PCV2 capsid protein expressed by the recombinant baculovirus
The capsid protein gene of PCV2 strain XSC was amplified by PCR and cloned into pGEM-T(?) easy vector,and subcloned into pFastBac~(TM)1 donor plasmid,then inserted into baculovirus shuttle vector Bacmid by site-specific transposition.The recombinant baculovirus,designated as reBac-Cap,was constructed when the recombinant shuttle vector Bacmid-Cap was transfected into Sf9 cells.To evaluate the expression level of capsid protein gene,a viral stock with a high-titer up to 5×10~8 pfu/ml was obtained and determined by the plaque assay.Infected Sf9 cells with 10~5 pfu reBac-Cap were harvested at 72 h post infection,the expressed capsid protein with molecular weight of 28 kDa,the same size as the natural capsid protein,was identified to be expressed in cytoplasm in soluble,non-secreted form.Furthermore,the expression product was observed to react with the pig antisera specific to PCV2 virus.Sera samples collected from all control and vaccinated animals at 0,7,14,21,28,35,42 49,56 day post-innoculation(dpi) were assayed for anti-PCV2 capsid antibodies by IFA.The results indicated that the recombinant baculovirus reBac-Cap induced specific antibodies against the pathogenic PCV2 capsid antigen from 21dpi.The sera were detected by real-time PCR.The results indicated that the recombinant baculovirus reBac-Cap was experienced the highest replication inhibition rate of PCV2.
3.Inhibition of the replication of the porcine circovirus type 2 in the Dulac cells by the siRNAs expressed by the plasmids
Sequences of PCV capsid protein genes and replicase genes determined in this study or from the GenBank were submitted to the website of Ambion company for target sequences searching,and some candidate siRNA sequences were generated.The candidate siRNA sequences were blasted,those shared any identity with other known organism genes were exclued.Six siRNAs were chosen to be evaluated for their selected inhibition effect on the replication of PCVs,four of these were specific to the rep gene of PCVs,designated as SH1(S50-68)、SH2(S554-572)、SH3(S653-671)、SH4(S624-642),and the others were specific to the cap gene of PCV2,designated as SH5(R1429-1411) and SH6(R1307-1289). Six DNA fragments with 67 nucleotides producing short hairpin RNA(shRNA) were designed based on the plasmid vector.The corresponding DNA fragments were synthesized,annealed and ligated to the downstream of the mouse originated U6 promoter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector.As controls, the siRNAs specific to Luciferase gene within the vector and negative fragments for any organisms were also included.Recombinant plasmids were transformed into the host bacteria DH5αand positive clones were selected.Positive clones carried the expected insertions were identified by sequencing and designated as Retro-SH1、Retro-SH2、Retro-SH3、Retro-SH4、Retro-SH5、Retro-SH6、Retro-Luc and Retro-NC,respectively.
To test whether siRNAs expressed by these six plasmids inhibit the PCV2 replication,we first examined their effect on the replication of the PCV2 virus in the Dulac cells.The plamids were introduced into the Dulac cells using Sofast~(TM) transfection reagent.Each samples had triplication.As controls,Retro-Luc and Retro-NC were similarly introduced into the same cells,followed by PCV2 virus infection.The cells were collected at 72 h after infection.Virus loads was detected by real-time PCR assay established in our lab.The results showed that virus loads strongly reduced in cells transfected by Retro-SH1,Retro-SH4 and Retro-SH6,while did not reduce in the cells transfected by other plasmids and the controls.These three siRNAs expressed plasmids were further evaluated in the following experiments.
The kinetic effect of siRNAs expressed by plasmids on PCV2 replication in Dulac cells was determined according to the method mentioned above.Three kinds of siRNA expressing plasmids and their mixture were transfected into the cells,and then infected by PCV2 at 24 h after transfection.Collected cell cultures at 24 h,36 h,48 h,60 h,72 h,96 h,120 h after infection were determined for the copies of genomic DNA of PCV2 by real-time PCR.The results showed that there was strong inhibition effect on PCV2 replication from 60 h post its infection,and the effect lasted at least 48 h.As the dose of plasmids transfected,a positive dose-dependent effect was confirmed.500 ng of each plasmid was optimal.The inhibition effect of the Retro-SH4 was the strongest among three plasmids,and no synergism was observed in the cells transfected by the mixture of the three plasmids.The results were also confirmed by the FACS technique.When Dulac cells were infected with PCV2 XSC strain,then each of three plasmids and their mixture were transfected into above cells at different intervals.The results indicated that the siRNAs expressing plasmids should be tranfected as early as possible in order to get strong inhibitions.Transfection of the plasmids at 36 h after virus infection could not inhibit the ongoing replication of the PCV2 effectively.For ten of PCV2 field isolates,the siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could inhibit the replication of all isolates in Dulae cells. For PCV1,the siRNAs expressed by Retro-SH1,Retro-SH4 specific to the rep gene of PCV exhibited inhibition effect on the replication of PCV1 in Dulac cells while Retro-SH6 did not.
4.Inhibition of the replication of the porcine circovirus type 2 in mice by the siRNAs expressed by the plasmids
Seventy-two of 8-week-old BALB/c mice were divided randomly into 12 groups,6 mice for each.Mice in four groups received 10μg of Retro-SH1, Retro-SH4,Retro-SH6 and Retro-NC,respectively,another group were intramuscularly injected with the mixture of Retro-SH1,Retro-SH4 and Retro-SH6,mice in these five groups were challenged by oral administration and intraperitoneal injection with 10~5 TCID_(50) of PCV2.Mice in other five groups were oral administration and intraperitoneal injected with 10~5 TCID_(50) of PCV2 and then intramuscularly introduced by Retro-SH1,Retro-SH4,Retro-SH6 or their mixture and Retro-NC at 24 h after PCV2 infection.One group was treated as challenge control and the other as a blank control group.All of the mice were slaughted for sera and tissues collection at 5 d and 11 d post challenge.The sera were detected by real-time PCR and the tissues were examined by histopathological and immunohistochemical techniques.Both of the prophylactic and therapy experiments showed that siRNAs expressed by Retro-SH1, Retro-SH4,Retro-SH6 could offer some protection against PCV2 challenge, Retro-SH4 inoculation group experienced the highest replication inhibition rate up to 99.8%.No synergism was observed in the group transfected by the mixture of the three plasmids.
In summary,10 isolates of PCV2 were obtained from the clinical cases infected with PMWS in Jiangsu Province.Genomic sequences of these isolates were in high level of homology.The cap gene of PCV2 was expressed by the recombinant baculovirus reBac-Cap and its immunogenicity was evaluated in mice. The mice immunized with the recombinant baculovirus could elicit specific antibody against PCV2.Six of siRNAs specific for PCV2 genomic fragments were designed and the plasmid-based system for generating unimolecular,hairpin siRNAs were constructed successfully and three of them(Retro-SH1,Retro-SH4, Retro-SH6) were selected.These siRNAs expressed by the plasmids could inhibit the replication of the PCVs in the Dulac cells and provide some protection against PCV2 infection in mice.These data indicated that siRNA technique should be a potential useful tool for developing novel strategy to prevent and control PCV2 infection.
引文
1.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Biackwell publishing,2006:299-308.
2.Meehan B M,McNeilly F,Todd D,et al.Characterization of novel circovirus DNAs associated with wasting syndromes in pig[J].Journal of General Virology,1998,79(9):2171-2179.
3.Allan G M,Meehan B,Todd D,et al.Novel porcine circovirus from pigs with wasting disease syndromes[J].Vet Rec,1998,142(17):467-468.
4.Allan G M,Mcneilly E,Kennedy S,et al.PCV2 associated PDNS in Northern Ireland in 1990.Porcine dermatitis and nephropathy syndrome[J].Vet Rec,2000,146(24):711-712.
5.Stevenson G W,Kiupel M,Mittal S K,et al.Tissue distribution and genetic typing of porcine circovirus in pigs with naturally occurring congenital tremors[.I].J Vet Diagn Invest,2001,13(1):57-62.
6.Segales J,Rosell C and Domingo M.Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[J].Vet Microbiol,2004,98(2):137-149.
7.Harding J C.Postweaning multisystemie wasting syndrome:preliminary epidemiology and clinical findings.In:Proceedings of the Western Canadian Association on Swine Practice,1996,22-25.
8.Ellis J A,Wiseman B M,Allan G,et al.Analysis of seroconversion to porcine circovirus 2 among veterinarians from the United States and Canada[J].J Am Vet Med Assoc, 2000,217(11):1645-1646.
9.Choi C and Chae C.In-situ hybridization for the detection of porcine circovirus in pigs with post-weaning multisystemic wasting syndrome[J].Comp Patho,1999,121(3):265-270.
10.Onuki A,Abe K,Togashi K,et al.Detection of porcine circovirns from lesions of a pig with wasting disease in Japan[J].Vet Meal Sci,1999,61(10):1119-1123.
11.Plana-Duran J,Balasch M,Segales J,et al.Post-weaning multisystemic wasting syndrome in Spain[J].Vet Rec,1999,1450):87-88.
12.Allan G M,McNeilly F,Meehan B M,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.
13.Spillane P,Kennedy S,Meehan B,et al.Porcine circovirus infection in the Republic of Ireland[J].Vet Rec,1998,143(18):511-512.
14.Mankertz A,Domingo M,Folch J M,et al.Characterisation of PCV-2 isolates from Spain,Germany and France[J].Virus Res,2000,66(1):65-77.
15.Madec F,Eveno E,Morvan P,et al.Post-weaning multisystemic 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.
16.Kiss I,Kecskemeti S,Tuboly T,et al.New pig disease in Hungary:post-weaning multisystemic wasting syndrome caused by circovirus(short communication)[J].Acta Vet Hung,2000,48(4):469-475.
17.Trujano M,Iglesias G,Segales J,et al.PCV-2 from emaciated pigs in Mexico[J].Vet Rec,2001,148(25):792.
18.Borel N,Burgi E,Kiupei M,et al.Three cases of post-weaning multiaystemic wasting syndrome(PMWS) due to porcine circovirus type 2(PCV 2) in Switzerland[J].Schweiz Arch Tierheilkd,2001,143(5):249-255.
19.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.
20.Celera V Jr and Carasova P.First evidence of porcine circovirus type 2(PCV2) infection of pigs in the Czech Republic by semi-nested PCR[J].Vet Meal B Infect Dis Vet Public Health,2002,49(3):155-159.
21.Liu Q,Wang L,Willson P,et al.Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica.[J].Vet Res,2002,66(4):225-231.
22.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 Q,2000,22(3):167-172.
23.Saoulidis K,Kyriakis S C,Kennedy S,et al.First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome in pigs in Greece [J].Vet Med B Infect Dis Vet Public Health,2002,49(4):202-205.
24.Sarradell J,Perez A M,Andrada M,et al.PMWS in Argentina[J].Vet Rec,2002,150(10):323.
25.Cano J P,Rodriguez-Carino C,Sogbe E,et al.Post-weaning multisystemic wasting syndrome in pigs in Venezuela[J].Vet Rec,2005,156:620.
26.Kiatipattanasakul-Banlunara W,Tantilertcharoen R.SuzukiK,et al.Detection of porcine circovirus 2(PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome(PMWS) pigs in Thailand[J].J Vet Med Sci,2002,64(5):559-452.
27.Maldonado J,Segales J,Calsamiglia M,et al.Post-waning multisystemic wasting syndrome(PMWS) in the Philippines:porcine circovirus type 2(PCV2) detection and characterization[J].Vet Med Sci,2004,66(5):533-537.
28.Wallgren P,Hasslung F,Bergsrom G,et al.Post-weaning multisystemic wasting syndrome--PMWS.The first year with the disease in Sweden[J].Vet Q,2004,26(4):170-187.
29.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):171-175.
30.Toplak I,Grom J,Hostnik P,et al.Phylogenetic analysis of type 2 porcine circoviruses identified in wild boar in Slovenia[J].Vet Rec,2004,155(6):178-180.
31.Wang C,Huang T S,Huang C C,et al.Characterization of porcine circovirus type 2 in Taiwan[J].Vet Med Sci,2004,66(5):469-475.
32.郎洪武,张广川,吴发权,等.断奶猪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。
33.潘饶霖,单松华,刘洪云,等.猪圆环病毒2型的分离与鉴定[J]。上海农业学报,2006。22(3):4-7。
34。王忠田,杨汉春,郭鑫.规模化猪场猪圆环病毒2型感染的流行病学调查[J]。中国兽医杂志,2002,10(38):3-6.
35.宋建国,高生智,魏润生.规模化猪场猪圆环病毒2型感染的血清学调查[J].中国兽医科技,2004,34(12):26-28.
36.吕艳丽,杨汉春,郭鑫,等.猪圆环病毒2型的分离与鉴定[J].中国兽医杂志,2004,40(2):14-18.
37.杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医,2004 31(5):41-43。
38.Buchen-Osmond,C.(Ed).ICTVdB Management(2006).00.016.0.01.Circovirus.In:ICTVdB - The Universal Virus Database,version 4,Columbia University,New York,USA.
39.Tischer I,Rasch R and Tochtermann G.Characterization of papovavirus-and picomavirus-like particles in permanent pig kidney cell lines[J].Zentralbl Bakteriol,1974,226(2):153-167.
40.Tischer I,Gelderblom H,Vettermann W,et al.A very small porcine virus with a circular single-stranded DNA[J].Nature,1982,295(5844):64-66.
41.Allan G M,Phenix K V,Todd D,et al.Some biological and physico-chemical properties of porcine circovirus[J].Zentralbl Veterinarmed,1994,41(1):17-26.
42.O'Dea M A,Hughes A P,Davies L J,et al.Thermal stability of porcine circovirus type 2 in cell culture[J].J Virol Methods,2008,147(1):61-66.
43.Tischer I,Peters D,Rash R,et al.Replication of porcine circovirus:induction by glucose mine and cell cycle dependence[J].Arch Virol,1987,96(1-2):39-57.
44.Stevenson G W,Kiupel M,Mittal S K,et al.Ultrastructure of porcine circovirus in persistently infected PK-15 cells[J].Vet Pathol,1999,36(5):368-378.
45.Allan G M,Kennedy S and McNeilly F.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.
46.Misinzo G,Delputte P L and Nauwynck H J.Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells[J].J Virol,2008,82(3):1128-1135.
47.Misinzo G,Delputte P L,Lefebvre D J,et al.Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification[J].Arch Virol,2008,153(2):337-342.
48.Mankertz A,Persson F,Mankertz J,et al.Mapping and characterization of the origin of DNA replication of porcine circovirus[J].Journal of Virology,1997,71(3):2562-2566.
49.Todd D,Niagro F,Richie BW,et al.Genome sequence determinations and analyses of novel circovirus from goose and pigeon[J].Virology,2001,286(2):354-362.
50.Cheung A K.Transcriptional analysis of porcine circovirus type 2[J].Virology,2003,305(1):168-180.
51.Cheung A K.A stem-loop structure,sequence non-specific,at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication[J].Virology,2007,363(1):229-235.
52.Cheung A K.Identification of an octanucleotide motif sequence essential for viral protein,DNA,and progeny virus biosynthesis at the origin of DNA replication of porcine circovirus type 2[J].Vitology,2004,324(1):28-36.
53.Steinfeldt T,Finsterbusch T and Mankertz A.Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1[J].Virol,2007,81(11):5 696-5704.
54.Hamel A L,Lin L L and Nayar G P.Nucleotide sequence of porcine circovirus with postweaning multisystemic wasting syndrome in pigs[J].Journal of Virology,1998,72(6):5262-5267.
55.Mankertz A,Mankertz J,Wolf K,et al.Identification of a protein essential for the replication of porcine circovirus[J].J Gen Virol,1998,79(2):381-384.
56.Mahe D,Bianchard P,Ttuong C,et al.Diferential recognition of ORF2 protein f rom type 1 and type 2 porcine cicovir2uses and identification of immunorelevant epitopes[J]. Journal of General Virology, 2000, 81(pt 7):1815-1824.
57. 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.
58. Steinfeldt T, Finsterbusch T and Mankertz A. Rep and Rep 'protein of porcine circovirus type 1 bind to the origin of replication in vitro [J]. Virology, 2001, 291(1): 152-160.
59. Vega-Rocha S, Gronenborn B, Gronenborn AM, et al. Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures. Biochemistry, 2007,46 (21): 6201-6212.
60. Nawagitgul P, Morozov I, Bolin S, et al. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J]. Journal of General Virology,2000, 81: 2281-2287.
61. Truong C, Mahe D, Blanchard P, et al. Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection [J]. Arch Virol, 2001,146 (5): 1197-1211.
62. Blanchard P, Mahe D, Cariolet R, et al. An ORF2 protein-based E1ISA for porcine circovirus type 2 antibodies in multisystemic wasting syndrome[J]. Vet Microbiol, 2003, 94(3): 183-94.
63. Liu Q, T ikoo S K, Babiuk L A. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2[J]. Virology, 2001,285(1): 91-99.
64. Lefebvre D J, Costers S, Van Doorsselaere J, et al. Antigenic differences among porcine circovirus type 2 strains, as demonstrated by the use of monoclonal antibodies[J]. J Gen Virol, 2008, 89(pt 1):177-187.
65. Tischer I, Mields W, Wolff D, et al. Studies on epidemiology and pathogeniciry of porcine circovirus[J]. Arch Virol, 1986, 91(3-4):271-276.
66. Edwards S, Sands J J. Evidence of circovirus infection in British pigs [J]. Vet Rec, 1994, 134(26): 680-681.
67. Dulac G C and Afshar A. Porcine circovirus antigens in PK15 cell line (ATCC CC1-33) and evidence of antibodies to circovirus in Canadian pigs [J]. Can J Vet Res, 1989,53(4): 431-433.
68. Clark E G Post-weaning multisystemic wasting syndrome[J]. Proceeding of the Americns assocination of swine practitioners, 1997,28:499-501.
69. Grau-Roma L and Segales J. Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain[J]. Vet Microbiol, 2007,119 (2-4): 44-51.
70. Ramirez-Mendoza H, Martinez C, Mercado C, et al. Porcine circovirus type 2 antibody detection in backyard pigs from Mexico City[J].Res Vet Sci,2007,83(1):130-132.
71.Cheung A K,Lager K M,Kohutyuk O I,et al.Detection of two porcine circovirus type 2 genotypic groups in United States swine herds[J].Arch Virol,2007,152(5):1035-1044.
72.Dorr P M,Baker R B,Almond G W,et al.Epidemiologic assessment of porcine circovirus type 2 coinfection with other pathogens in swine[J].J Am Vet Med Assoc,2007,230(2):244-250.
73.Gagnon C A,Tremblay D,Tijssen P,et al.The emergence of porcine circovirus 2b genotype(PCV-2b) in swine in Canada[J].Can Vet J,2007,48(8):811-819.
74.Finlaison D,Kirldand P,Luong R,et al.Survey of porcine circovirus 2 and postweaning multisystemic wasting syndrome in New South Wales piggeries[J].Aust Vet J,2007,85(8):304-310.
75.An D J,Rob I S,Song D S,et al.Phylogenetic characterization of porcine circovirus type 2 in PMWS and PDNS Korean pigs between 1999 and 2006[J].Vires Res,2007,129(2):115-122.
76.Allan G M,McNeilly F,McMenamy M,et al.Temporal distribution of porcine circovirus 2 genogroups recovered from postweaning multisystemic wasting syndrome affected and nonaffected farms in Ireland and Northern Ireland[J].J Vet Diagn Invest,2007,19(6):668-673.
77.Yang Z Z,Shual J B,Dai X J,et al.A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou,Zhejiang Province,China[J].J Zhejiang Univ Sci B,2008,9(2):148-153.
78.Yang J S,Song D S,Kim S Y,et al.Detection of porcine circovirus type 2 in feces of pigs with or without enteric disease by polymerase chain reaction[J].J Vet Diagn Invest,2003,15(4):369-73.
79.Bolin S R,Stoffregen W C,Nayar G P,et al.Post-weaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived,colostrums -deprived piglets with type 2 porcine circovirus[J].J Vet Diagn Invest,2001,13(3):185-194.
80.Allan G M,McNeilly F,Cassidy J P,et al.Pathogenesis of porcine circovirus:experimental infection of colostrums deprived and examination of pig foetal material[J].Veterinary Microbiology,1995,44(1):49-64.
81.Olvera A,Cortey M and Segales J.Molecular evolution of porcine circovirus type 2genomes:phylogeny and clonality[J].Virology,2007,357(2):175-85.
82.Larochelle R,Magar R and D' Allaire S.Genetic characterization and phylogenetic analysis of porcine circovirus type 2(PCV2) strains from cases presenting various clinical condition[J].Virus Res,2002,90(1-2):101-112.
83.Famham M W,Choi Y K,Goyai S M,et al.Isolation and characterization of porcine circovirus type 2 from sera of stillborn fetuses[J].Can J Vet Res,2003.67(2):108-113.
84.Fenaux M,Opriessnig T,Halbur P G,et al.Detection and in vitro and in vivo characterization of porcine circovirus DNA from a porcine-derived commercial pepsin product[J].Gen Virol,2004,85(11):3377-3382.
85.Niagro F D,Forsthoefel A N,Lawther R P,et al.Beak and feather disease virus and porcine circovirus genomes:intermediates between the geminiviruses and plant circoviruses[J].Arch Virol,1998,143(9):1723-1744.
86.Gibbs M J and 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,1999,96(14):8022-8027.
87。马相如,陈焕春,肖少波,等.猪圆环病毒(PCV)序列分析与进化[J].中国兽医学报,2004(2):105-108.
88.Bolin S R,Stonffregen W C,Nayar G P,et al.Postweaning multisystemic wasting syndrome induced aRer experimental inoculation of eesarean-deiived,colostrums-deprived piglets with type 2 porcine eirovirus[J].J Vet Diagn Invest,2001,13(3):185-194.
89.Krakowka S,Ellis J A,Meehan B,et al.Viral wasting syndrome of swine:experi-mental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcineparvovirus[J].Vet Pathol,2000,37(3):254-263.
90.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 eircovirus and porcine repreductive and respiratory syndrome virus[J].Vet Pathol,2001,38(5):528-539.
91.Rosell Q,Segales J,Piana-Duran J,et al.Pathological,immunohistochemieal and in-situ hybridization studies of natural cases of postweaning multisystemie wasting syndrome in pigs[J].J Comp Pathol,1999,120(1):59-78.
92.McNeilly F,Allan G M,Foster C,et al.Effect of porcine circovirus infection on porcine alveolar macrophage function[J].Vet Immunol Immunopathol,1996,49(4):295-306.
93.Shibahara T,Sato K,Ishilcawa Y,et al.Porcine cireovirus induces B lymphocyte depletion in pigs with wasting disease[J].J Vet Med Sci,2000,62(11):1125-1131.
94.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.
95.Darwich I,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 cireovirus type 2 load in lymphoid tissues[J].Clin Diagn lab Immunol,2002,9(2):236-242.
96.Allan G M,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 PCV 2 replication[J].Arch Virol,2000,145(11):2421-2429.
97.Krakowka S,Ellis J A and McNeilly R.Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus2(PCV2)[J].Vet Pathol,2001,38(1):31-42.
98.Allan G M,McNeilly.F,Foster C.et al.Infection of leucocyte cell cultures from different species with porcine circovirus[J].Vet Microbiol,1994,41:267-279.
99.Sanchez R E J,Meerts P,Nauvsynck H,et al.Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life[J].Vet Microbiol,2003,95(1-2):15-25.
100.Gilpin D F,McCullough K,Meehan B M,et al.In vitro studies on the and replication of porcine circovirus type 2 in ceils of the porcine immune system[J].Vet Immunol Immunopathol,2003,94(3-4):149-161.
101.Kekarainen T,Montoya M,Mateu E,et al.Porcine circovirns type 2-induced interleukin-10 modulates recall antigen responses[J].J Gen Virol,2008,89(pt 3):760-765.
102.Kekarainen T,Montoya M,Dominguez J,et al.Porcine circovirus type 2(PCV2) viral components immunomodulate recall antigen responses[J].Vet Immunol Immunopathol,2008(in press).
103.Vincent I E,Balmelli C,Meehan B,et al.Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA[J].Immunology,2007,120(1):47-56.
104.Wikstrom F H,Meehan B M,Berg M,et al.Structure-dependent modulation of alpha interferon production by porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells[J].J Virol,2007,81(10):4919-4927.
105.Trundova M and Celer V.Expression of porcine circovirus 2ORF2 gene requires codon optimized E.coli cells[J].Virus Genes,2007,34(2):199-204.
106.Yu S,Opriessnig T,Kitikoon P,et al.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):261-272.
107.Chang H W,Jeng C R,Lin C M,et al.The involvement of Fas/FasL interaction in porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus co-inoculation-associated lymphocyte apoptosis in vitro[J].Vet Microbiol,2007,122(1-2):72-82.
108.Meehan B M,McNeilly F,McNair I,et al.Isolation and characterization of porcine circovirus2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome[J].Arch Virol,2001,146(4):835-842.
109.Choi C and Chae C.Insitu hybridization for the detection of porcine circovirus in pigs with postweaning multisystemic wasting syndrome[J].J Comp Pathol,1999,121(3):265-270.
110. Choi C and Chae C. Distribution of porcine parvovirus in porcine circovirus-2 infected pigs with postweaning multisystemic wasting syndrome as shown by insit u hybridization[J]. J Comp Pathol, 2000,123 (4): 302-305.
111. Nawagitgul P, Morozov I, Sirinarumitr T, et al. Development of probes to differentiate porcine circovirus types 1 and 2 i n vitro by in situ hybridization[J]. Vet Microbiol, 2000,75(1):83-89.
112. Sirinarumitr T, Morozov I, Nawagitgul P, et al. Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome[J]. J Vet Diagn Invest, 2000, (6): 562-565.
113. Kim J and Chae C. Simultaneous detection of porcine circovirus 2 and porcine parvovirus in naturally and experimentally coinfected pigs by double in situ hybridization[J]. J Vet Diagn Invest, 2002, 14 (3):236-240.
114. larochelle R, Antaya M, Morin M, et al. Typing of porcine circovirus in clinical specimens by multiplex PCR [J]. J Virol Meth, 1999, 80(1): 69-75.
115. Kim J and Chae C. Optimized protocols for the detection of porcine circovirus 2 DNA from formalin-fixed paraffinembedded tissues using nested polymerase chain reaction and comparison of nested PCR with in situ hybridization [J]. J Virol Methods, 2001, 92(2): 105-111.
116. Kiatipattanasakul-Banlunara W, Tantilertcharoen R, Suzuki K, et al. Detection of porcine circovirus 2 ( PCV-2) DNA by nested PCR from formalin2fixed tissues of postweaning multisystemic wasting syndrome ( PMWS) pigs in Thailand[J]. J Vet Med Sci, 2002, 64 (5): 449-452.
117. Kim J , Han D U , Choi C , et al. Differentiation of porcine circovirus PCV1 and PCV2 in boar semen using a multiplex nested polymerase chain reaction[J]. J Virol Methods, 2001,98(1): 25-31.
118. Fenaux M, Halbur P G, Gill M, et al. Genetic characterization of type 2 porcine circovirus PCV2 from pigs with postweaningmultisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR2restriction fragment length polymorphism assay to detect and differentiate between infections with PCV1 and PCV2[J]. J Clin Microbiol, 2000,38(7): 2494-2503.
119. Hamel A L, lin L L, Sachvie C, et al. PCR detection and characterization of type-2 porcine circovirus[J]. Can J Vet Res, 2000,64(1): 44-52.
120. Giammarioli M, Pellegrini C, Casciari C, et al. Development of a novel hot-start multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and porcine parvovirus[J]. Vet Res Commun, 2008,32(3): 255-262.
121. Lee C S, Moon H J, Yang J S, et al. Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs[J]. J Virol Methods,2007,139(1):39-43.
122.Allan G M,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(1):3-10.
123.Walker I W,Konoby C A,Jewhurst V A,et al.Development and application of a compe titive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2[J].J Vet Diagn Invest.2000,12(5):400-405.
124.Nawagitgul P,Harms P A,Morozov I,et al.Modified indirect porcine circovirus(PCV)type2 based and recombinant capsid protein(ORF2) based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clin Diagn lab Immunol,2002,9(1):33-40.
125.Bianchard P,Mahe D,Cariolet R.et al.An ORF2 protein-based ElISA for porcine circovirus type 2 antibodies in multisystemic wasting syndrome[J].Vet Microbiol,2003,94(3):1831-94.
126.McNeilly F,McNair I,Mackie D P,et al.Production,characterization and applications of monoclonal antibodies to porcine circovirus 2[J].Arch Virol,2001,146(5):909-922.
127.Wu P C,Chien M S,Tseng Y Y,et al.Expression of the porcine circovirus type 2capsid protein subunits and application to an indirect ELISA[J].J Biotechnol,2008,133(1):58-64.
128.Blanchard P,Mabe D,Cariolet R,et al.Protection of swine against postweaning multisystemic wasting syndrom(PMWS) by porcine circovirus type 2(PCV2)proteins[J].Vaccine,2003,21(31):4565-4575.
129.Kamstrup S,Barfoed A M,Frimann T H,et al.Immunization against PCV2 structural protein by DNA vaccination of mice[J].Vaccine,2004,22(11-12):1358-1361.
130.Fenaux M,Opriessnig T,Halbur P C B,et al.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,2004 Jun;78(12):6297-6303.
131.Fenaux M,Opriessnig T,Halbur P G,et al.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):13440-13446.
132.Wang X,Jiang P and Li Y.Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2[J].Vet Microbiol,2007,121(3-4):215-224.
133.Song Y,Jin M,Zhang S,et al.Generation and immunogenicity of a recombinant pseudorabies virus expressing cap protein of porcine circovirus type 2[J].Vet Microbiol,2007,119(2-4):97-104.
134.Fachinger V,Bischoff R.Jedidia S B,et al.The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex[J].Vaccine, 2008,26(11):1488-1499.
135.Fort M,Sibila M,Allepuz A,et al.Porcine circovirus type 2(PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins[J].Vaccine,26(8):1063-1071.
136.Fan H,Xiao S,Tong T,et al.Immunogenicity of porcine circovirus type 2 capsid protein targeting to different subcellular compartments[J].Mol Immunol,2008,45(3):653-660.
137.Opfiessnig T,Patterson A R,Elsener J,et al.Influence of maternal antibodies on efficacy of porcine circovirus type 2(PCV2) vaccination to protect pigs from experimental infection with PCV2[J].Clin Vaccine Immunol.2008,15(3):397-401.
138.Feng Z,Jiang P,Wang X,et al.Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo[J].Antiviral Res,2008,77(3):186-194.
139.Liu J,Chen I,Chua H,et al.Inhibition of porcine circovirus type 2 replication in mice by RNA interference[J].Virology,2006,347(2):422-433.
1.Fire A,Xu S,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
2.Bosher J M,and Labouesse M.RNA interference:genetic wand and genetic watchdog[J].Nat Cell Biol,2000,2(2):E31-36.
3.Wiebusch L,Truss M and Hagemeier C.Inhibition of human cytomegalovirus replication by small interfering RNAs[J].J Gen Virol,2004,85(1):179-184
4.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(14):7523-7527.
5.Lu W W,Hsu Y Y,Yang J Y,et al.Selective inhibition of enterovirus 71 replication by short hairpin RNAs[J].Biochem Biophys Res Commun,2004,325(2):494-499.
6.Kahana R,Kuznetzova L,Rogel A,et al.Inhibition of foot-and-mouth disease virus replication by small interfering RNA[J].J Gen Virol.2004,85(pt 11):3213-3217.
7.Ni B,Shi X,Li Y,et al.Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA[J].Antivir Ther,2005,10(4):527-533.
8.Lee N S,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-1 revtranscdpts human cells[J].Nat Biotechnol,2002,20(5):500-505.
9.Baulcombe D.RNA silencing[J].Curr Biol,2002,12(3):R82-84.
10.Baulcombe D.RNA silencing in plants[J].Nature,2004,431(7006):356-363.
11.Hamilton A J and Baulcombe D C.A species of small antisense RNA in posttranscriptional gene silencing in plants[J].Science,1999,286(5441):950-952.
12.Tuschl T,Zamore P D,Lehmann R,et al.Targeted mRNA degradation by double-stranded RNA in vitro[J].Genes Dev,1999,13(24):3191-3197.
13.Zamore P D,Tuschl T,Sharp P A,et al.RNAi:double-stranded RNA directs the ATP-dependent cleavage ofmRNA at 21 to 23 nucleotide intervals[J].Ceil,2000,101(l):25-33.
14.Ingelbrecht I,Van Houdt H,Van Montagu M,et al.Post-transcriptional silencing of reporter transgenes in tobacco correlates with DNA methylation[J].Proc.Natl.Acad.Sci.USA,1994.91(22):10502-10506.
15.Bemstein E,Candy A A,Hammond S M,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409(6818):363-366.
16.Nykanen A,Haley B and Zamore PD.ATP requirement and small interfering RNA structure in the RNA interference pathway[J].Cell,2001,I07(3):309-321.
17.Bass B L.RNA interfference the short answer[J].Nature,422(6836):494-498.
18.Pal-Bhadra M,Bhadra U and Birchler J A.RNAi-related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila[J].Mol Cell,2002,9(2):315-327.
19.Elbashir S M,Lendeckel W and Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2001,15(2):188-200.
20.Bemstein E,Caudy A A,Hammond S M,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409(6818):363-366.
21.Ketting R F,Fischer S E,Bemstein E,et al.Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C.elegans[J].Genes Dev,2001,15(20):2654-2659.
22.Lipardi C,Wei Q and Paterrson B M.RNAi as randomdegradation PCR:siRNA palmers convert mRNA into dsRNA that are degraded to generate new siRNAs[J].Cell,2001,107(3):297-307.
23.Sijen T,Fleenor J,Simmer F,et al.On the role of RNA amplification in dsRNA-triggered gone silencing[J].Cell,2001,107(4):465-476.
24.Dykxhoom D M,Novina C D and Sharp P A..Killing the messenger:short RNAs that silence gene expression[J].Nat Rcv Mo Cell Biol,,2003,4(6):457-467.
25.Harmon G J.RNA Interference[J].Nature,2002,418(6894):244-251.
26.Zamore P D.Ancient pathways programmed by small RNAs[J].Science,2002,296(5571):1265-1259.
27.宋尔卫,主编,RNA干扰的生物学原理与应用[M].北京,高等教育出版社.2005,47-48.
28.孙建国,廖荣霞,陈正堂.RNA干涉分子机制研究进展[J].生物化学与生物物理学进展,2002,29(5):678-681.
29.Kumiko U,Yuki N,Fumitaka T,et al.Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference[J].Nue Aei Res,2004,32(3):936-948.
30.Reynolds A,Anderson E M,Vermeulen A,et al.Induction of the interferon response by siRNA is cell type- and duplex length-dependent[J].Rna,2006,12(6):988-993.
31.Brummelkamp T R and Bernards R.New tools for functional mammalian cancer genetics[J].Nat Rev Cancer,2003,3(10):781-789.
32.Matthew M,Michael S D and Andrew P.The utility of siRNA transcripts produced by RNA polymerase I in down regulating viral gene expression and replication of negativeand positive-strand RNA viruses[J].Virology,2003,313:514-524.
33.An D S,Xin Y,Mao S H,et al.Efficient lentiviral vectors for short haipin RNA delivery into human cells[J].Hum Gene Ther,2003,14:1207-1212.
34.Eric K H,Ee M Y,Dong S A,et al.Inhibition of influenza virus matrx(M1) protein expression and vires replication by U6 promoter-driven and lentivirus-mediated delivery of siRNA[J].Journal of General Virology,2004,85(pt 7):1877-1884.
35.Bantounas I,Glover C P,Kelly S,et aL Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons:inhibition of endogenous caspase-3 activity and protection from apoptotic cell death[J].J Neurosci Res,2005,79(5):661-669.
36.Castanotto D.and Scherer L.Targeting cellular genes with PCR cassettes expressing short interfering RNAs[J].Methods Enzymol,2005,392:173-185.
37.Derroe E and Silver P A.Retrovirus-delivered siRNA[J].13MC Biotechnol,2002,2:15-19.
38.Yang G,Thompson J A,Fang B,et al.Silencing of H-ras gene expression by tetrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer[J].Oncogene,2003,22(36):5694-5701.
39.Abbas-Terki,Blanco-Bose T and Deglon W N.Lentiviral-mediated RNA interference[J]. Hum Gene Ther,2002,13(18):2197-2201.
40.Matta H,Hozayev B,Tomar R,et al.Use of lentiviral vectors for delivery of small interfering RNA[J].Cancer Biol Ther,2003,2(2):206-210.
41.Tiscomia G,Singer O,Ikawa M,et al.A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA[J].Proc Natl Acad Sci USA,2003,100:1844-1848.
42.Xia H,Mao Q,Paulson H L,et al.siRNA-mediated gene silencing in vitro and in vivo[J].Nat Biontechnol,2002,20(10):1006-1010.
43.Shen C,Buck A K,Liu X,et al.Gene silencing by adenovirus-delivered siRNA[J].FEBS Lett,2003,539(1-3):111-114.
44.黄海,黄健.RNA干扰在基因功能和病毒防治中的应用[J].现代临床医学生物工程学杂志,2003,9(4):237-370.
45.Li W X,Li H,Lu R,et al.Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[J].Proc Natl Acad Sci USA,2004,101(5):1350-1355.
46.Joost Haasnoot PC,Daniel C and Ben B.Inhibition of virus replication by RNA interference[J].J Biomedical sci,2003,(10):607-616.
47.Ge Q,Michael T M.,Nguyen T,et al.RNA interfence of influenza viurs production for directly targenting mRNA for degradation and indirectly ingibiting all viral RNA transcription[J].Proc Natl Acad Sci USA,2003,100(23):2718-2723.
48.Stepen M T,Chia-Yun L,Terrence M,et al.Protection against lethal influenza virus challenge by RNA interference in vivo[J].Proc Natl Acad Sci USA,2004,101(23):8682-8686.
49.Zhou Y,Chan J H,Chan A Y,et al.Transgenic plant-dedved siRNAs can suppress propagation of influenza virus in mammalian cells[J].FEBS Lett,2004,577(3):345-350.
50.Hu W Y,Bushman F D and Siva A C.RNA interference against retroviruses[J].Virus Res,2004,102(1):59-64.
51.Jacque J M,Trique L,Stevenson M.Modulation of HIV-1 replication by RNA interference[J].Nature,2002,418:435-438.
52.Lee N S,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-I rev transcripts in human cells[J].Nat Biotechnol,2002,20(5):500-505.
53.Novina C D,Murray M F,Dykxhoorn D M,et al.SiRNA-directed inhibition of HIV-1infection[J].Nature Med,2002,8(7):681-686.
54.Qin X F,An D S,Chen I S,et al.Inhibiting HIV-linfection in human T cells by lentiviral-medinted delivery of small interfering RNA against CCRS[J].Proc Nail Acad Sci USA,2003,100(1):183-188.
55.Randall G,Grakoui A,Rice C M.Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs[J].Proc Natl Acad Sci USA,2003, 100(1):235-240.
56.Kapadia S B,Bridean-Andersen A and Chisari F V.Interference of hepatitis C virus RNA replication by short interfering RNAs[J].Proc Natl Acad Sci USA,2003,100(4):2014-2018.
57.Wilson J A,Jayasena S,Khvonrova A,et al.RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cell[J].Proc Natl Acad Sci USA,2003,100(5):2783-2788.
58.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(6):1-7.
59.MeCaffrey A P,Meuse L,Pham T T,et al.RNA interference in adult mice[J].Nature,2002,418(6893):38-39.
60.Saleh M C,Van RIJ R P and Anding R.RNA silencing in viral infections:insights from poliovirus[J].Virus Res,2004,102(1):11-17.
61.Citlin L,Karelsky S and AndinoR.Short interfering RNA confers intracellular antiviral immunity in human ceils[J].Nature,2002,418(6896):430-434.
62.Adelman Z,Sanchez-Vargas I,Travanty E,et al.RNA Silencing of Dengue Virus Type 2 R.eplieation in Transformed C6/36 Mosquito Cells Transcribing an Inverted Repeat RNA Derived from the Virus Genome[J].Journal of virology,2002,76(24):12925-12933.
63.Zhang W,Singam R,Hellermann G,et al.Attenuation of dengue virus infection by adeno2associated virus2mediated siRNA delivery[J].Genetic Vaccines Ther,2004,2(1):8.
64.朱旭东,党颖,冯怡,等。用RNaseⅢ制备的小干涉RNA降解SARS冠状病毒基因的细胞内转录物[J].生物工程学报,2004,24(4):485-489。
65.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(23):8676-8681.
66.刘文博,张小荣,曹永忠,等.表达禽流感病毒特异性siRNA分子减毒鼠伤寒沙门氏菌的构建[J].病毒学报,2007,23(3):125-129.
67.Chen Y,Du D,Wu J,et al.Inhibition of hepatitis B virus replication by stably expressed shRNA[J].Biochem Biophys Res Commun,2003,311(2):398- 404.
68.Mcmanusmt M T and Sharp P A.Gene silencing in mammals by small interfering RNAs[J].Nat Rev Genet,2002,3(10):737- 747.
69.Ying C,De Clereq E and Neyts J.Selective inhibition of hepatitis B virus replication by RNA interference[J].Biochem Biophys Res Commun,2003,309(2):482-484.
70.MeCaffrey A P,Nakai H,Pandey K,et al.Inhibition of hepatitis B virus in mice by RNA interference[J].Nat Biotechnol,2003,21(6):639- 644.
71.Shlomai A and Shaul Y.Inhibition of hepatitis B virus expression and replication by RNA interference[J].Hepatology,2003,37(4):764- 770.
1.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Blackwell publishing,2006:299-308.
2.Vicente J,Segales J,Here,U et al.Epidemiological study on porcine circovirus type 2(PCV2) infection in the European wild boar(Sus scrofa),Vet Res,2004,35(2):243-253.
3.Olvera A,Cortey M and Segales J.Molecular evolution of porcine circovirus type 2genomes:phylogeny and clonality[J].Virology,2007,357(2):175-85.
4.Laura B.Postweaning multisystemic wasting syndrome(PMWS) in Quebec,is it an emerging disease? American Association of swine veterinarians,2006:383-385.
5.Quintana J,Segales J,.Rosell C,et al.Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome[J].Vet Rec,2001,149(12):357-361.
6.An D J,Roh I S,Song D S,et al.Phylogenetic characterization of porcine circovirus type 2 in PMWS and PDNS Korean pigs between 1999 and 2006[J].Virus Res,2007,129(2):115-122.
7.Grau-Roma L,Crisci E,Sibila M,et al.A proposal on porcine circovirus type 2(PCV2)genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence.Vet Microbiol,2008,128(1-2):23-25.
8.Meehan B M,McNeilly F,Todd D,et al.Characterization of novel circovirus DNAs associated with wasting syndromes in pig[J].Journal of General Virology,1998,79(9):2171-2179.
9.Wang C,Huang T S,Huang C C,et al.Characterization of porcine circovirus type 2 in Taiwan[J].Vet Med Sci,2004,66(5):469-475.
10.Celera V Jr and Carasova P.First evidence of porcine circovirus type 2(PCV2) infection of pigs in the Czech Republic by semi-nested PCR[J].Vet Med B Infect Dis Vet Public Health,2002,49(3):155-159.
11.Larochelle R,Magar R and D' Allaire S.Genetic characterization and phylogenetic analysis of porcine circovirus type 2(PCV2) strains from cases presenting various clinical condition[J].Virus Res,2002,90(1-2):101-112.
12.Rodriguez-Arrioja G M,Segales J,Resell C,et al.Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain[J].J Vet Med B Infect Dis Vet Public Health,2003,50(2):99-101.
13.Lipej Z,Segales J,Jemersic L,et al.First description of postweaning multisystemic wasting syndrome(PMWS) in wild boar(Sus scrofa) in Croatia and phyiogenetic analysis of partial PCV2 sequences[J].Acta Vet Hung,2007,55(3):389-404.
1.Meehan B M,McNeilly F,Todd D,et al.Characterization of novel circovirus DNAs associated with wasting syndromes in pig[J].Journal of General Virology,1998,79(9):2171-2179.
2.Harding J C.Postweaning multisystemic wasting syndrome:preliminary epidemiology and clinical findings.In:Proceedings of the Western Canadian Association on Swine Practice,1996,22-25.
3.Ellis J A,Wiseman B M,Allan G,et al.Analysis of seroconversion to porcine circovirus 2 among veterinarians from the United States and Canada[J].J Am Vet Med Assoc,2000, 217(11):1645-1646.
4.Choi C and Chae C.In-situ hybridization for the detection of porcine circovirus in pigs with post-weaning multisystemic wasting syndrome[J].Comp Patho,1999,121(3):265-270.
5.Onuki A,Abe K,Togashi K,et al.Detection of porcine circovirus from lesions of a pig with wasting disease in Japan[J].Vet Med Sci,1999,61(10):1119-1123.
6.Plana-Duran J,Baiasch M,Segaies J,et al.Post-weaning multisystemic wasting syndrome in Spain[J].Vet Rec,1999,145(3):87-88.
7.Allan G M,McNeilly F,Meehan B M,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.
8.Spillane P,Kennedy S,Meehan B,et al.Porcine circovirus infection in the Republic of Ireland[J].Vet Rec,1998,143(18):511-512.
9.Mankertz A,Domingo M,Foich J M,et al.Charactefisation of PCV-2 isolates from Spain,Germany and France[J].Virus Res,2000,66(1):65-77.
10.Madec F,Eveno E,Morvan P,et al.Post-weaning multisystemic 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.
11.Kiss I,Kecskemeti S,Tuboly T,et al.New pig disease in Hungary:post-weaning multisystemic wasting syndrome caused by circovirus(short communication)[J].Acta Vet Hung,2000,48(4):469-475.
12.Trujano M,Iglesias G,Segales J,et al.PCV-2 from emaciated pigs in Mexico[J].Vet Rec,2001,148(25):792.
13.Borel N,Burgi E,Kiupel M,et al.Three cases of post-weaning multisystemic wasting syndrome(PMWS) due to porcine circovirus type 2(PCV 2) in Switzerland[J].Schweiz Arch Tierheilkd,2001,143(5):249-255.
14.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.
15.Celera V Jr and Carasova P.First evidence of porcine circovirns type 2(PCV2) infection of pigs in the Czech Republic by semi-nested PCR[J].Vet Med B Infect Dis Vet Public Health,2002,49(3):155-159.
16.Liu Q,Wang L,Willson P,et al.Seroprevaience of porcine circovirus type 2 in swine populations in Canada and Costa Pdca.[J].Vet Res,2002,66(4):225-231.
17.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 Q,2000,22(3):167-172.
18.Saoulidis K,Kyriakis S C,Kennedy S,et al.First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome in pigs in Greece [J].Vet Med B Infect Dis Vet Public Health,2002,49(4):202-205.
19.Sarradell J,Perez A M,Andrada M,et al.PMWS in Argentina[J].Vet Rec,2002,150(10):323.
20.Cano J P,Rodriguez-Carino C,Sogbe E,et al.Post-weaning multisystemic wasting syndrome in pigs in Venezuela[J].Vet Rec,2005,156:620.
21.Kiatipattanasakul-Banlunara W,Tantilertcharoen R,SuzukiK,et al.Detection of porcine circovirus 2(PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome(PMWS) pigs in Thailand[J].J Vet Med Sci,2002,64(5):559-452.
22.Maldonado J,Segales J,Calsamiglia M,et al.Post-waning multisystemic wasting syndrome(PMWS) in the Philippines:porcine circovirus type 2(PCV2) detection and characterization[J].Vet Med Sci,2004,66(5):533-537.
23.Wallgren P,Hasslung F,Bergsrom G,et al.Post-weaning multisystemic wasting syndrome--PMWS.The first year with the disease in Sweden[J].Vet Q,2004,26(4):170-187.
24.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):171-175.
25.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):178-180.
26.Wang C,Huang T S,Huang C C,et al.Characterization of porcine circovirus type 2 in Taiwan[J].Vet Med Sci,2004,66(5):469-475.
27.Allan G M,Kennedy S and McNeilly F.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.
28.Misinzo G,Delputte P L and Nauwynck H J.Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells[J].J Virol,2008,82(3):1128-1135.
29.郎洪武,张广川,吴发权,等。断奶猪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.
30.杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医,2004 31(5):41-43.
31.Condreay J P and Kost T A.Baculovirus expression vectors for insect and mammalian cells[J].Curr Drug Targets,2007,8(10):1126-31.
32.Nawagitgul P,Morozov I,Bolin S,et al.Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J].Journal of General Virology,2000,81:2281-2287.
33.萨姆布鲁克等主编.高福主译.分子克隆指南[M].第二版.北京:科学出版社.1998,19-68.
34.Brunborg I M,Moldal T,Christine M J,et al.Quantitation of porcine circovirus type 2isolated 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,122(2):171-178.
35.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Blackwell publishing,2006:299-308.
36.Mahe D,Blanchard P,Ttuong C,et al.Diferential recognition of ORF2 protein from type 1 and type 2 porcine cicovir2uses and identification of immunorelevant epitopes[J].Journal of General Virology,2000,81(pt 7):1815-1824.
37.Blanchard P,Mahe D,Cariolet R,et al.Protection of swine against postweaning multisystemic wasting syndrom(PMWS)by porcine circovirus type 2(PCV2) proteins[J].Vaccine,2003,21(31):4565-4575.
38.Kamstrup S,Barfoed A M,Frimann T H,et al.Immunization against PCV2 structural protein by DNA vaccination of mice[J].Vaccine,2004,22(11-12):1358-1361.
39.Fenaux M,Opriessnig T,Halbur P C B,et al.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,2004 Jun;78(12):6297-6303.
40.Fenaux M,Opriessnig T,Halbur P G,et al.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):13440-13446.
1.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Blackwell publishing,2006:299-308.
2.Steinfeldt T,Finsterbusch T and Mankertz A.Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1[J].Viral,2007,81(11):5 696-5704.
3.Nawagitgul P,Morozov I,Bolin S,et al.Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J].Journal of General Virology,2000,81:2281-2287.
4.Fire A,Xu S,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
5.Bosher J M,and Labouesse M.RNA interference:genetic wand and genetic watchdog[J].Nat Cell Biol,2000,2(2):E31-36.
6.宋尔卫。主编,RNA干扰的生物学原理与应用[M].北京,高等教育出版社.2005,47-48.
7.Li W X,Li H,Lu R,et al.Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[J].Proc Natl Acad Sci USA,2004,101(5):1350-1355.
8.Joost Haasnoot PC,Daniel C and Ben B.Inhibition of virus replication by RNA interference[J].J Biomedical sci,2003,(10):607-616.
9.Ge Q,Michael T M.,Nguyen T,et al.RNA interfence of influenza viurs production for directly targenting mRNA for degradation and indirectly ingibiting all viral RNA transcription[J].Proc Natl Acad Sci USA,2003,100(23):2718-2723.
10.Stepen M T,Chia-Yun L,Terrence M,et al.Protection against lethal influenza virus challenge by RNA interference in vivo[J].Proc Natl Acad Sci USA,2004,101(23):8682-8686.
11.Zhou Y,Chan J H,Chan A Y,et al.Transgenic plant-derived siRNAs can suppress propagation of influenza virus in mammalian cells[J].FEBS Lett,2004,577(3):345-350.
12.Hu W Y,Bushman F D and Siva A C.RNA interference against retroviruses[J].Virus Res,2004,102(1):59-64.
13.Jacque J M,Trique L,Stevenson M.Modulation of HIV-1 replication by RNA interference[J].Nature,2002,418:435-438.
14.Lee N S,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells[J].Nat Bioteehnol,2002,20(5):500-505.
15.Novina C D,Murray M F,Dykxhoom D M,et al.SiRNA-directed inhibition of HIV-1infection[J].Nature Med,2002,8(7):681-686.
16.Qin X F,An D S,Chen I S,et al.Inhibiting HIV-linfection in human T cells by lentiviral-medinted delivery of small interfering RNA against CCR5[J].Proe Natl Aead Sei USA,2003,100(1):183-188.
17.Randall G,Grakoui A,Rice C M.Clearance of replicating hepatitis C virus replieon RNAs in cell culture by small interfering RNAs[J].Proe Natl Aead Sci USA,2003,100(1):235-240.
18.Kapadia S B,Bridean-Andersen A and Chisari F V.Interference of hepatitis C virus RNA replication by short interfering RNAs[J].Proc Natl Acad Sci USA,2003,100(4):2014-2018.
19.Wilson J A,Jayasena S,Khvonrova A,et al.RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cell[J].Proc Natl Acad Sci USA,2003,100(5):2783-2788.
20.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(6):1-7.
21.McCaffrey A P,Meuse L,Pham T T,et al.RNA interference in adult mice[J].Nature,2002,418(6893):38-39.
22.Saleh M C,Van RIJ R P and Anding R.RNA silencing in viral infections:insights from poliovirus[J].Virus Res,2004,102(1):11-17.
23.Citlin L,Karelsky S and AndinoR.Short interfering RNA confers intracellular antiviral immunity in human cells[J].Nature,2002,418(6896):430-434.
24.Adelman Z,Sanchez-Vargas I,Travanty E,et al.RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted Repeat RNA Derived from the Virus Genome[J].Journal of virology,2002,76(24):12925-12933.
25.Zhang W,Singam R,Hellermann G,et al.Attenuation of dengue virus infection by adeno2associated virus2mediated siRNA delivery[J].Genetic Vaccines Ther,2004,2(1):8.
26.朱旭东,党颖,冯怡,等.用RNaseⅢ制备的小干涉RNA降解SARS冠状病毒基因的细胞内转录物[J].生物工程学报,2004,24(4):485-489.
27.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(23):8676-8681.
28.刘文博,张小荣,曹永忠,等.表达禽流感病毒特异性siRNA分子减毒鼠伤寒沙门氏菌的构建[J].病毒学报,2007,23(3):125-129.
29.Chen Y,Du D,Wu J,et al.Inhibition of hepatitis B virus replication by stably expressed shRNA[J].Biochem Biophys Res Commun,2003,311(2):398- 404.
30.Mcmanusmt M T and Sharp P A.Gene silencing in mammals by small interfering RNAs[J].Nat Rev Genet,2002,3(10):737- 747.
31.Ying C,De Clereq E and Neyts J.Selective inhibition of hepatitis B virus replication by RNA interference[J].Biochem Biophys Res Commun,2003,309(2):482- 484.
32.McCaffrey A P,Nakai H,Pandey K,et al.Inhibition of hepatitis B virus in mice by RNA interference[J].Nat Biotechnol,2003,21(6):639- 644.
33.Shlomai A and Shaul Y.Inhibition of hepatitis B virus expression and replication by RNA interference[J].Hepatology,2003,37(4):764- 770.
34.Liu J,Chen I,Chua H,et al.Inhibition of porcine cireovirus type 2 replication in mice by RNA interference[J].Virology,2006,347(2):422-433.
35.Feng Z,Jiang P,Wang X,et al.Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo[J].Antiviral Res,2008,77(3):186-194.
2.Meehan B M,McNeilly F,Todd D,et al.Characterization of novel circovirus DNAs associated with wasting syndromes in pig[J].Journal of General Virology,1998,79(9):2171-2179.
3.Allan G M,Meehan B,Todd D,et al.Novel porcine circovirus from pigs with wasting disease syndromes[J].Vet Rec,1998,142(17):467-468.
4.Allan G M,Mcneilly E,Kennedy S,et al.PCV2 associated PDNS in Northern Ireland in 1990.Porcine dermatitis and nephropathy syndrome[J].Vet Rec,2000,146(24):711-712.
5.Stevenson G W,Kiupel M,Mittal S K,et al.Tissue distribution and genetic typing of porcine circovirus in pigs with naturally occurring congenital tremors[.I].J Vet Diagn Invest,2001,13(1):57-62.
6.Segales J,Rosell C and Domingo M.Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease[J].Vet Microbiol,2004,98(2):137-149.
7.Harding J C.Postweaning multisystemie wasting syndrome:preliminary epidemiology and clinical findings.In:Proceedings of the Western Canadian Association on Swine Practice,1996,22-25.
8.Ellis J A,Wiseman B M,Allan G,et al.Analysis of seroconversion to porcine circovirus 2 among veterinarians from the United States and Canada[J].J Am Vet Med Assoc, 2000,217(11):1645-1646.
9.Choi C and Chae C.In-situ hybridization for the detection of porcine circovirus in pigs with post-weaning multisystemic wasting syndrome[J].Comp Patho,1999,121(3):265-270.
10.Onuki A,Abe K,Togashi K,et al.Detection of porcine circovirns from lesions of a pig with wasting disease in Japan[J].Vet Meal Sci,1999,61(10):1119-1123.
11.Plana-Duran J,Balasch M,Segales J,et al.Post-weaning multisystemic wasting syndrome in Spain[J].Vet Rec,1999,1450):87-88.
12.Allan G M,McNeilly F,Meehan B M,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.
13.Spillane P,Kennedy S,Meehan B,et al.Porcine circovirus infection in the Republic of Ireland[J].Vet Rec,1998,143(18):511-512.
14.Mankertz A,Domingo M,Folch J M,et al.Characterisation of PCV-2 isolates from Spain,Germany and France[J].Virus Res,2000,66(1):65-77.
15.Madec F,Eveno E,Morvan P,et al.Post-weaning multisystemic 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.
16.Kiss I,Kecskemeti S,Tuboly T,et al.New pig disease in Hungary:post-weaning multisystemic wasting syndrome caused by circovirus(short communication)[J].Acta Vet Hung,2000,48(4):469-475.
17.Trujano M,Iglesias G,Segales J,et al.PCV-2 from emaciated pigs in Mexico[J].Vet Rec,2001,148(25):792.
18.Borel N,Burgi E,Kiupei M,et al.Three cases of post-weaning multiaystemic wasting syndrome(PMWS) due to porcine circovirus type 2(PCV 2) in Switzerland[J].Schweiz Arch Tierheilkd,2001,143(5):249-255.
19.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.
20.Celera V Jr and Carasova P.First evidence of porcine circovirus type 2(PCV2) infection of pigs in the Czech Republic by semi-nested PCR[J].Vet Meal B Infect Dis Vet Public Health,2002,49(3):155-159.
21.Liu Q,Wang L,Willson P,et al.Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica.[J].Vet Res,2002,66(4):225-231.
22.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 Q,2000,22(3):167-172.
23.Saoulidis K,Kyriakis S C,Kennedy S,et al.First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome in pigs in Greece [J].Vet Med B Infect Dis Vet Public Health,2002,49(4):202-205.
24.Sarradell J,Perez A M,Andrada M,et al.PMWS in Argentina[J].Vet Rec,2002,150(10):323.
25.Cano J P,Rodriguez-Carino C,Sogbe E,et al.Post-weaning multisystemic wasting syndrome in pigs in Venezuela[J].Vet Rec,2005,156:620.
26.Kiatipattanasakul-Banlunara W,Tantilertcharoen R.SuzukiK,et al.Detection of porcine circovirus 2(PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome(PMWS) pigs in Thailand[J].J Vet Med Sci,2002,64(5):559-452.
27.Maldonado J,Segales J,Calsamiglia M,et al.Post-waning multisystemic wasting syndrome(PMWS) in the Philippines:porcine circovirus type 2(PCV2) detection and characterization[J].Vet Med Sci,2004,66(5):533-537.
28.Wallgren P,Hasslung F,Bergsrom G,et al.Post-weaning multisystemic wasting syndrome--PMWS.The first year with the disease in Sweden[J].Vet Q,2004,26(4):170-187.
29.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):171-175.
30.Toplak I,Grom J,Hostnik P,et al.Phylogenetic analysis of type 2 porcine circoviruses identified in wild boar in Slovenia[J].Vet Rec,2004,155(6):178-180.
31.Wang C,Huang T S,Huang C C,et al.Characterization of porcine circovirus type 2 in Taiwan[J].Vet Med Sci,2004,66(5):469-475.
32.郎洪武,张广川,吴发权,等.断奶猪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。
33.潘饶霖,单松华,刘洪云,等.猪圆环病毒2型的分离与鉴定[J]。上海农业学报,2006。22(3):4-7。
34。王忠田,杨汉春,郭鑫.规模化猪场猪圆环病毒2型感染的流行病学调查[J]。中国兽医杂志,2002,10(38):3-6.
35.宋建国,高生智,魏润生.规模化猪场猪圆环病毒2型感染的血清学调查[J].中国兽医科技,2004,34(12):26-28.
36.吕艳丽,杨汉春,郭鑫,等.猪圆环病毒2型的分离与鉴定[J].中国兽医杂志,2004,40(2):14-18.
37.杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医,2004 31(5):41-43。
38.Buchen-Osmond,C.(Ed).ICTVdB Management(2006).00.016.0.01.Circovirus.In:ICTVdB - The Universal Virus Database,version 4,Columbia University,New York,USA.
39.Tischer I,Rasch R and Tochtermann G.Characterization of papovavirus-and picomavirus-like particles in permanent pig kidney cell lines[J].Zentralbl Bakteriol,1974,226(2):153-167.
40.Tischer I,Gelderblom H,Vettermann W,et al.A very small porcine virus with a circular single-stranded DNA[J].Nature,1982,295(5844):64-66.
41.Allan G M,Phenix K V,Todd D,et al.Some biological and physico-chemical properties of porcine circovirus[J].Zentralbl Veterinarmed,1994,41(1):17-26.
42.O'Dea M A,Hughes A P,Davies L J,et al.Thermal stability of porcine circovirus type 2 in cell culture[J].J Virol Methods,2008,147(1):61-66.
43.Tischer I,Peters D,Rash R,et al.Replication of porcine circovirus:induction by glucose mine and cell cycle dependence[J].Arch Virol,1987,96(1-2):39-57.
44.Stevenson G W,Kiupel M,Mittal S K,et al.Ultrastructure of porcine circovirus in persistently infected PK-15 cells[J].Vet Pathol,1999,36(5):368-378.
45.Allan G M,Kennedy S and McNeilly F.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.
46.Misinzo G,Delputte P L and Nauwynck H J.Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells[J].J Virol,2008,82(3):1128-1135.
47.Misinzo G,Delputte P L,Lefebvre D J,et al.Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification[J].Arch Virol,2008,153(2):337-342.
48.Mankertz A,Persson F,Mankertz J,et al.Mapping and characterization of the origin of DNA replication of porcine circovirus[J].Journal of Virology,1997,71(3):2562-2566.
49.Todd D,Niagro F,Richie BW,et al.Genome sequence determinations and analyses of novel circovirus from goose and pigeon[J].Virology,2001,286(2):354-362.
50.Cheung A K.Transcriptional analysis of porcine circovirus type 2[J].Virology,2003,305(1):168-180.
51.Cheung A K.A stem-loop structure,sequence non-specific,at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication[J].Virology,2007,363(1):229-235.
52.Cheung A K.Identification of an octanucleotide motif sequence essential for viral protein,DNA,and progeny virus biosynthesis at the origin of DNA replication of porcine circovirus type 2[J].Vitology,2004,324(1):28-36.
53.Steinfeldt T,Finsterbusch T and Mankertz A.Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1[J].Virol,2007,81(11):5 696-5704.
54.Hamel A L,Lin L L and Nayar G P.Nucleotide sequence of porcine circovirus with postweaning multisystemic wasting syndrome in pigs[J].Journal of Virology,1998,72(6):5262-5267.
55.Mankertz A,Mankertz J,Wolf K,et al.Identification of a protein essential for the replication of porcine circovirus[J].J Gen Virol,1998,79(2):381-384.
56.Mahe D,Bianchard P,Ttuong C,et al.Diferential recognition of ORF2 protein f rom type 1 and type 2 porcine cicovir2uses and identification of immunorelevant epitopes[J]. Journal of General Virology, 2000, 81(pt 7):1815-1824.
57. 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.
58. Steinfeldt T, Finsterbusch T and Mankertz A. Rep and Rep 'protein of porcine circovirus type 1 bind to the origin of replication in vitro [J]. Virology, 2001, 291(1): 152-160.
59. Vega-Rocha S, Gronenborn B, Gronenborn AM, et al. Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures. Biochemistry, 2007,46 (21): 6201-6212.
60. Nawagitgul P, Morozov I, Bolin S, et al. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J]. Journal of General Virology,2000, 81: 2281-2287.
61. Truong C, Mahe D, Blanchard P, et al. Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection [J]. Arch Virol, 2001,146 (5): 1197-1211.
62. Blanchard P, Mahe D, Cariolet R, et al. An ORF2 protein-based E1ISA for porcine circovirus type 2 antibodies in multisystemic wasting syndrome[J]. Vet Microbiol, 2003, 94(3): 183-94.
63. Liu Q, T ikoo S K, Babiuk L A. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2[J]. Virology, 2001,285(1): 91-99.
64. Lefebvre D J, Costers S, Van Doorsselaere J, et al. Antigenic differences among porcine circovirus type 2 strains, as demonstrated by the use of monoclonal antibodies[J]. J Gen Virol, 2008, 89(pt 1):177-187.
65. Tischer I, Mields W, Wolff D, et al. Studies on epidemiology and pathogeniciry of porcine circovirus[J]. Arch Virol, 1986, 91(3-4):271-276.
66. Edwards S, Sands J J. Evidence of circovirus infection in British pigs [J]. Vet Rec, 1994, 134(26): 680-681.
67. Dulac G C and Afshar A. Porcine circovirus antigens in PK15 cell line (ATCC CC1-33) and evidence of antibodies to circovirus in Canadian pigs [J]. Can J Vet Res, 1989,53(4): 431-433.
68. Clark E G Post-weaning multisystemic wasting syndrome[J]. Proceeding of the Americns assocination of swine practitioners, 1997,28:499-501.
69. Grau-Roma L and Segales J. Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain[J]. Vet Microbiol, 2007,119 (2-4): 44-51.
70. Ramirez-Mendoza H, Martinez C, Mercado C, et al. Porcine circovirus type 2 antibody detection in backyard pigs from Mexico City[J].Res Vet Sci,2007,83(1):130-132.
71.Cheung A K,Lager K M,Kohutyuk O I,et al.Detection of two porcine circovirus type 2 genotypic groups in United States swine herds[J].Arch Virol,2007,152(5):1035-1044.
72.Dorr P M,Baker R B,Almond G W,et al.Epidemiologic assessment of porcine circovirus type 2 coinfection with other pathogens in swine[J].J Am Vet Med Assoc,2007,230(2):244-250.
73.Gagnon C A,Tremblay D,Tijssen P,et al.The emergence of porcine circovirus 2b genotype(PCV-2b) in swine in Canada[J].Can Vet J,2007,48(8):811-819.
74.Finlaison D,Kirldand P,Luong R,et al.Survey of porcine circovirus 2 and postweaning multisystemic wasting syndrome in New South Wales piggeries[J].Aust Vet J,2007,85(8):304-310.
75.An D J,Rob I S,Song D S,et al.Phylogenetic characterization of porcine circovirus type 2 in PMWS and PDNS Korean pigs between 1999 and 2006[J].Vires Res,2007,129(2):115-122.
76.Allan G M,McNeilly F,McMenamy M,et al.Temporal distribution of porcine circovirus 2 genogroups recovered from postweaning multisystemic wasting syndrome affected and nonaffected farms in Ireland and Northern Ireland[J].J Vet Diagn Invest,2007,19(6):668-673.
77.Yang Z Z,Shual J B,Dai X J,et al.A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou,Zhejiang Province,China[J].J Zhejiang Univ Sci B,2008,9(2):148-153.
78.Yang J S,Song D S,Kim S Y,et al.Detection of porcine circovirus type 2 in feces of pigs with or without enteric disease by polymerase chain reaction[J].J Vet Diagn Invest,2003,15(4):369-73.
79.Bolin S R,Stoffregen W C,Nayar G P,et al.Post-weaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived,colostrums -deprived piglets with type 2 porcine circovirus[J].J Vet Diagn Invest,2001,13(3):185-194.
80.Allan G M,McNeilly F,Cassidy J P,et al.Pathogenesis of porcine circovirus:experimental infection of colostrums deprived and examination of pig foetal material[J].Veterinary Microbiology,1995,44(1):49-64.
81.Olvera A,Cortey M and Segales J.Molecular evolution of porcine circovirus type 2genomes:phylogeny and clonality[J].Virology,2007,357(2):175-85.
82.Larochelle R,Magar R and D' Allaire S.Genetic characterization and phylogenetic analysis of porcine circovirus type 2(PCV2) strains from cases presenting various clinical condition[J].Virus Res,2002,90(1-2):101-112.
83.Famham M W,Choi Y K,Goyai S M,et al.Isolation and characterization of porcine circovirus type 2 from sera of stillborn fetuses[J].Can J Vet Res,2003.67(2):108-113.
84.Fenaux M,Opriessnig T,Halbur P G,et al.Detection and in vitro and in vivo characterization of porcine circovirus DNA from a porcine-derived commercial pepsin product[J].Gen Virol,2004,85(11):3377-3382.
85.Niagro F D,Forsthoefel A N,Lawther R P,et al.Beak and feather disease virus and porcine circovirus genomes:intermediates between the geminiviruses and plant circoviruses[J].Arch Virol,1998,143(9):1723-1744.
86.Gibbs M J and 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,1999,96(14):8022-8027.
87。马相如,陈焕春,肖少波,等.猪圆环病毒(PCV)序列分析与进化[J].中国兽医学报,2004(2):105-108.
88.Bolin S R,Stonffregen W C,Nayar G P,et al.Postweaning multisystemic wasting syndrome induced aRer experimental inoculation of eesarean-deiived,colostrums-deprived piglets with type 2 porcine eirovirus[J].J Vet Diagn Invest,2001,13(3):185-194.
89.Krakowka S,Ellis J A,Meehan B,et al.Viral wasting syndrome of swine:experi-mental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcineparvovirus[J].Vet Pathol,2000,37(3):254-263.
90.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 eircovirus and porcine repreductive and respiratory syndrome virus[J].Vet Pathol,2001,38(5):528-539.
91.Rosell Q,Segales J,Piana-Duran J,et al.Pathological,immunohistochemieal and in-situ hybridization studies of natural cases of postweaning multisystemie wasting syndrome in pigs[J].J Comp Pathol,1999,120(1):59-78.
92.McNeilly F,Allan G M,Foster C,et al.Effect of porcine circovirus infection on porcine alveolar macrophage function[J].Vet Immunol Immunopathol,1996,49(4):295-306.
93.Shibahara T,Sato K,Ishilcawa Y,et al.Porcine cireovirus induces B lymphocyte depletion in pigs with wasting disease[J].J Vet Med Sci,2000,62(11):1125-1131.
94.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.
95.Darwich I,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 cireovirus type 2 load in lymphoid tissues[J].Clin Diagn lab Immunol,2002,9(2):236-242.
96.Allan G M,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 PCV 2 replication[J].Arch Virol,2000,145(11):2421-2429.
97.Krakowka S,Ellis J A and McNeilly R.Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus2(PCV2)[J].Vet Pathol,2001,38(1):31-42.
98.Allan G M,McNeilly.F,Foster C.et al.Infection of leucocyte cell cultures from different species with porcine circovirus[J].Vet Microbiol,1994,41:267-279.
99.Sanchez R E J,Meerts P,Nauvsynck H,et al.Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life[J].Vet Microbiol,2003,95(1-2):15-25.
100.Gilpin D F,McCullough K,Meehan B M,et al.In vitro studies on the and replication of porcine circovirus type 2 in ceils of the porcine immune system[J].Vet Immunol Immunopathol,2003,94(3-4):149-161.
101.Kekarainen T,Montoya M,Mateu E,et al.Porcine circovirns type 2-induced interleukin-10 modulates recall antigen responses[J].J Gen Virol,2008,89(pt 3):760-765.
102.Kekarainen T,Montoya M,Dominguez J,et al.Porcine circovirus type 2(PCV2) viral components immunomodulate recall antigen responses[J].Vet Immunol Immunopathol,2008(in press).
103.Vincent I E,Balmelli C,Meehan B,et al.Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA[J].Immunology,2007,120(1):47-56.
104.Wikstrom F H,Meehan B M,Berg M,et al.Structure-dependent modulation of alpha interferon production by porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells[J].J Virol,2007,81(10):4919-4927.
105.Trundova M and Celer V.Expression of porcine circovirus 2ORF2 gene requires codon optimized E.coli cells[J].Virus Genes,2007,34(2):199-204.
106.Yu S,Opriessnig T,Kitikoon P,et al.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):261-272.
107.Chang H W,Jeng C R,Lin C M,et al.The involvement of Fas/FasL interaction in porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus co-inoculation-associated lymphocyte apoptosis in vitro[J].Vet Microbiol,2007,122(1-2):72-82.
108.Meehan B M,McNeilly F,McNair I,et al.Isolation and characterization of porcine circovirus2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome[J].Arch Virol,2001,146(4):835-842.
109.Choi C and Chae C.Insitu hybridization for the detection of porcine circovirus in pigs with postweaning multisystemic wasting syndrome[J].J Comp Pathol,1999,121(3):265-270.
110. Choi C and Chae C. Distribution of porcine parvovirus in porcine circovirus-2 infected pigs with postweaning multisystemic wasting syndrome as shown by insit u hybridization[J]. J Comp Pathol, 2000,123 (4): 302-305.
111. Nawagitgul P, Morozov I, Sirinarumitr T, et al. Development of probes to differentiate porcine circovirus types 1 and 2 i n vitro by in situ hybridization[J]. Vet Microbiol, 2000,75(1):83-89.
112. Sirinarumitr T, Morozov I, Nawagitgul P, et al. Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome[J]. J Vet Diagn Invest, 2000, (6): 562-565.
113. Kim J and Chae C. Simultaneous detection of porcine circovirus 2 and porcine parvovirus in naturally and experimentally coinfected pigs by double in situ hybridization[J]. J Vet Diagn Invest, 2002, 14 (3):236-240.
114. larochelle R, Antaya M, Morin M, et al. Typing of porcine circovirus in clinical specimens by multiplex PCR [J]. J Virol Meth, 1999, 80(1): 69-75.
115. Kim J and Chae C. Optimized protocols for the detection of porcine circovirus 2 DNA from formalin-fixed paraffinembedded tissues using nested polymerase chain reaction and comparison of nested PCR with in situ hybridization [J]. J Virol Methods, 2001, 92(2): 105-111.
116. Kiatipattanasakul-Banlunara W, Tantilertcharoen R, Suzuki K, et al. Detection of porcine circovirus 2 ( PCV-2) DNA by nested PCR from formalin2fixed tissues of postweaning multisystemic wasting syndrome ( PMWS) pigs in Thailand[J]. J Vet Med Sci, 2002, 64 (5): 449-452.
117. Kim J , Han D U , Choi C , et al. Differentiation of porcine circovirus PCV1 and PCV2 in boar semen using a multiplex nested polymerase chain reaction[J]. J Virol Methods, 2001,98(1): 25-31.
118. Fenaux M, Halbur P G, Gill M, et al. Genetic characterization of type 2 porcine circovirus PCV2 from pigs with postweaningmultisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR2restriction fragment length polymorphism assay to detect and differentiate between infections with PCV1 and PCV2[J]. J Clin Microbiol, 2000,38(7): 2494-2503.
119. Hamel A L, lin L L, Sachvie C, et al. PCR detection and characterization of type-2 porcine circovirus[J]. Can J Vet Res, 2000,64(1): 44-52.
120. Giammarioli M, Pellegrini C, Casciari C, et al. Development of a novel hot-start multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and porcine parvovirus[J]. Vet Res Commun, 2008,32(3): 255-262.
121. Lee C S, Moon H J, Yang J S, et al. Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs[J]. J Virol Methods,2007,139(1):39-43.
122.Allan G M,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(1):3-10.
123.Walker I W,Konoby C A,Jewhurst V A,et al.Development and application of a compe titive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2[J].J Vet Diagn Invest.2000,12(5):400-405.
124.Nawagitgul P,Harms P A,Morozov I,et al.Modified indirect porcine circovirus(PCV)type2 based and recombinant capsid protein(ORF2) based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clin Diagn lab Immunol,2002,9(1):33-40.
125.Bianchard P,Mahe D,Cariolet R.et al.An ORF2 protein-based ElISA for porcine circovirus type 2 antibodies in multisystemic wasting syndrome[J].Vet Microbiol,2003,94(3):1831-94.
126.McNeilly F,McNair I,Mackie D P,et al.Production,characterization and applications of monoclonal antibodies to porcine circovirus 2[J].Arch Virol,2001,146(5):909-922.
127.Wu P C,Chien M S,Tseng Y Y,et al.Expression of the porcine circovirus type 2capsid protein subunits and application to an indirect ELISA[J].J Biotechnol,2008,133(1):58-64.
128.Blanchard P,Mabe D,Cariolet R,et al.Protection of swine against postweaning multisystemic wasting syndrom(PMWS) by porcine circovirus type 2(PCV2)proteins[J].Vaccine,2003,21(31):4565-4575.
129.Kamstrup S,Barfoed A M,Frimann T H,et al.Immunization against PCV2 structural protein by DNA vaccination of mice[J].Vaccine,2004,22(11-12):1358-1361.
130.Fenaux M,Opriessnig T,Halbur P C B,et al.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,2004 Jun;78(12):6297-6303.
131.Fenaux M,Opriessnig T,Halbur P G,et al.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):13440-13446.
132.Wang X,Jiang P and Li Y.Protection of pigs against post-weaning multisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2[J].Vet Microbiol,2007,121(3-4):215-224.
133.Song Y,Jin M,Zhang S,et al.Generation and immunogenicity of a recombinant pseudorabies virus expressing cap protein of porcine circovirus type 2[J].Vet Microbiol,2007,119(2-4):97-104.
134.Fachinger V,Bischoff R.Jedidia S B,et al.The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex[J].Vaccine, 2008,26(11):1488-1499.
135.Fort M,Sibila M,Allepuz A,et al.Porcine circovirus type 2(PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins[J].Vaccine,26(8):1063-1071.
136.Fan H,Xiao S,Tong T,et al.Immunogenicity of porcine circovirus type 2 capsid protein targeting to different subcellular compartments[J].Mol Immunol,2008,45(3):653-660.
137.Opfiessnig T,Patterson A R,Elsener J,et al.Influence of maternal antibodies on efficacy of porcine circovirus type 2(PCV2) vaccination to protect pigs from experimental infection with PCV2[J].Clin Vaccine Immunol.2008,15(3):397-401.
138.Feng Z,Jiang P,Wang X,et al.Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo[J].Antiviral Res,2008,77(3):186-194.
139.Liu J,Chen I,Chua H,et al.Inhibition of porcine circovirus type 2 replication in mice by RNA interference[J].Virology,2006,347(2):422-433.
1.Fire A,Xu S,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
2.Bosher J M,and Labouesse M.RNA interference:genetic wand and genetic watchdog[J].Nat Cell Biol,2000,2(2):E31-36.
3.Wiebusch L,Truss M and Hagemeier C.Inhibition of human cytomegalovirus replication by small interfering RNAs[J].J Gen Virol,2004,85(1):179-184
4.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(14):7523-7527.
5.Lu W W,Hsu Y Y,Yang J Y,et al.Selective inhibition of enterovirus 71 replication by short hairpin RNAs[J].Biochem Biophys Res Commun,2004,325(2):494-499.
6.Kahana R,Kuznetzova L,Rogel A,et al.Inhibition of foot-and-mouth disease virus replication by small interfering RNA[J].J Gen Virol.2004,85(pt 11):3213-3217.
7.Ni B,Shi X,Li Y,et al.Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA[J].Antivir Ther,2005,10(4):527-533.
8.Lee N S,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-1 revtranscdpts human cells[J].Nat Biotechnol,2002,20(5):500-505.
9.Baulcombe D.RNA silencing[J].Curr Biol,2002,12(3):R82-84.
10.Baulcombe D.RNA silencing in plants[J].Nature,2004,431(7006):356-363.
11.Hamilton A J and Baulcombe D C.A species of small antisense RNA in posttranscriptional gene silencing in plants[J].Science,1999,286(5441):950-952.
12.Tuschl T,Zamore P D,Lehmann R,et al.Targeted mRNA degradation by double-stranded RNA in vitro[J].Genes Dev,1999,13(24):3191-3197.
13.Zamore P D,Tuschl T,Sharp P A,et al.RNAi:double-stranded RNA directs the ATP-dependent cleavage ofmRNA at 21 to 23 nucleotide intervals[J].Ceil,2000,101(l):25-33.
14.Ingelbrecht I,Van Houdt H,Van Montagu M,et al.Post-transcriptional silencing of reporter transgenes in tobacco correlates with DNA methylation[J].Proc.Natl.Acad.Sci.USA,1994.91(22):10502-10506.
15.Bemstein E,Candy A A,Hammond S M,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409(6818):363-366.
16.Nykanen A,Haley B and Zamore PD.ATP requirement and small interfering RNA structure in the RNA interference pathway[J].Cell,2001,I07(3):309-321.
17.Bass B L.RNA interfference the short answer[J].Nature,422(6836):494-498.
18.Pal-Bhadra M,Bhadra U and Birchler J A.RNAi-related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila[J].Mol Cell,2002,9(2):315-327.
19.Elbashir S M,Lendeckel W and Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2001,15(2):188-200.
20.Bemstein E,Caudy A A,Hammond S M,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409(6818):363-366.
21.Ketting R F,Fischer S E,Bemstein E,et al.Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C.elegans[J].Genes Dev,2001,15(20):2654-2659.
22.Lipardi C,Wei Q and Paterrson B M.RNAi as randomdegradation PCR:siRNA palmers convert mRNA into dsRNA that are degraded to generate new siRNAs[J].Cell,2001,107(3):297-307.
23.Sijen T,Fleenor J,Simmer F,et al.On the role of RNA amplification in dsRNA-triggered gone silencing[J].Cell,2001,107(4):465-476.
24.Dykxhoom D M,Novina C D and Sharp P A..Killing the messenger:short RNAs that silence gene expression[J].Nat Rcv Mo Cell Biol,,2003,4(6):457-467.
25.Harmon G J.RNA Interference[J].Nature,2002,418(6894):244-251.
26.Zamore P D.Ancient pathways programmed by small RNAs[J].Science,2002,296(5571):1265-1259.
27.宋尔卫,主编,RNA干扰的生物学原理与应用[M].北京,高等教育出版社.2005,47-48.
28.孙建国,廖荣霞,陈正堂.RNA干涉分子机制研究进展[J].生物化学与生物物理学进展,2002,29(5):678-681.
29.Kumiko U,Yuki N,Fumitaka T,et al.Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference[J].Nue Aei Res,2004,32(3):936-948.
30.Reynolds A,Anderson E M,Vermeulen A,et al.Induction of the interferon response by siRNA is cell type- and duplex length-dependent[J].Rna,2006,12(6):988-993.
31.Brummelkamp T R and Bernards R.New tools for functional mammalian cancer genetics[J].Nat Rev Cancer,2003,3(10):781-789.
32.Matthew M,Michael S D and Andrew P.The utility of siRNA transcripts produced by RNA polymerase I in down regulating viral gene expression and replication of negativeand positive-strand RNA viruses[J].Virology,2003,313:514-524.
33.An D S,Xin Y,Mao S H,et al.Efficient lentiviral vectors for short haipin RNA delivery into human cells[J].Hum Gene Ther,2003,14:1207-1212.
34.Eric K H,Ee M Y,Dong S A,et al.Inhibition of influenza virus matrx(M1) protein expression and vires replication by U6 promoter-driven and lentivirus-mediated delivery of siRNA[J].Journal of General Virology,2004,85(pt 7):1877-1884.
35.Bantounas I,Glover C P,Kelly S,et aL Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons:inhibition of endogenous caspase-3 activity and protection from apoptotic cell death[J].J Neurosci Res,2005,79(5):661-669.
36.Castanotto D.and Scherer L.Targeting cellular genes with PCR cassettes expressing short interfering RNAs[J].Methods Enzymol,2005,392:173-185.
37.Derroe E and Silver P A.Retrovirus-delivered siRNA[J].13MC Biotechnol,2002,2:15-19.
38.Yang G,Thompson J A,Fang B,et al.Silencing of H-ras gene expression by tetrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer[J].Oncogene,2003,22(36):5694-5701.
39.Abbas-Terki,Blanco-Bose T and Deglon W N.Lentiviral-mediated RNA interference[J]. Hum Gene Ther,2002,13(18):2197-2201.
40.Matta H,Hozayev B,Tomar R,et al.Use of lentiviral vectors for delivery of small interfering RNA[J].Cancer Biol Ther,2003,2(2):206-210.
41.Tiscomia G,Singer O,Ikawa M,et al.A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA[J].Proc Natl Acad Sci USA,2003,100:1844-1848.
42.Xia H,Mao Q,Paulson H L,et al.siRNA-mediated gene silencing in vitro and in vivo[J].Nat Biontechnol,2002,20(10):1006-1010.
43.Shen C,Buck A K,Liu X,et al.Gene silencing by adenovirus-delivered siRNA[J].FEBS Lett,2003,539(1-3):111-114.
44.黄海,黄健.RNA干扰在基因功能和病毒防治中的应用[J].现代临床医学生物工程学杂志,2003,9(4):237-370.
45.Li W X,Li H,Lu R,et al.Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[J].Proc Natl Acad Sci USA,2004,101(5):1350-1355.
46.Joost Haasnoot PC,Daniel C and Ben B.Inhibition of virus replication by RNA interference[J].J Biomedical sci,2003,(10):607-616.
47.Ge Q,Michael T M.,Nguyen T,et al.RNA interfence of influenza viurs production for directly targenting mRNA for degradation and indirectly ingibiting all viral RNA transcription[J].Proc Natl Acad Sci USA,2003,100(23):2718-2723.
48.Stepen M T,Chia-Yun L,Terrence M,et al.Protection against lethal influenza virus challenge by RNA interference in vivo[J].Proc Natl Acad Sci USA,2004,101(23):8682-8686.
49.Zhou Y,Chan J H,Chan A Y,et al.Transgenic plant-dedved siRNAs can suppress propagation of influenza virus in mammalian cells[J].FEBS Lett,2004,577(3):345-350.
50.Hu W Y,Bushman F D and Siva A C.RNA interference against retroviruses[J].Virus Res,2004,102(1):59-64.
51.Jacque J M,Trique L,Stevenson M.Modulation of HIV-1 replication by RNA interference[J].Nature,2002,418:435-438.
52.Lee N S,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-I rev transcripts in human cells[J].Nat Biotechnol,2002,20(5):500-505.
53.Novina C D,Murray M F,Dykxhoorn D M,et al.SiRNA-directed inhibition of HIV-1infection[J].Nature Med,2002,8(7):681-686.
54.Qin X F,An D S,Chen I S,et al.Inhibiting HIV-linfection in human T cells by lentiviral-medinted delivery of small interfering RNA against CCRS[J].Proc Nail Acad Sci USA,2003,100(1):183-188.
55.Randall G,Grakoui A,Rice C M.Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs[J].Proc Natl Acad Sci USA,2003, 100(1):235-240.
56.Kapadia S B,Bridean-Andersen A and Chisari F V.Interference of hepatitis C virus RNA replication by short interfering RNAs[J].Proc Natl Acad Sci USA,2003,100(4):2014-2018.
57.Wilson J A,Jayasena S,Khvonrova A,et al.RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cell[J].Proc Natl Acad Sci USA,2003,100(5):2783-2788.
58.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(6):1-7.
59.MeCaffrey A P,Meuse L,Pham T T,et al.RNA interference in adult mice[J].Nature,2002,418(6893):38-39.
60.Saleh M C,Van RIJ R P and Anding R.RNA silencing in viral infections:insights from poliovirus[J].Virus Res,2004,102(1):11-17.
61.Citlin L,Karelsky S and AndinoR.Short interfering RNA confers intracellular antiviral immunity in human ceils[J].Nature,2002,418(6896):430-434.
62.Adelman Z,Sanchez-Vargas I,Travanty E,et al.RNA Silencing of Dengue Virus Type 2 R.eplieation in Transformed C6/36 Mosquito Cells Transcribing an Inverted Repeat RNA Derived from the Virus Genome[J].Journal of virology,2002,76(24):12925-12933.
63.Zhang W,Singam R,Hellermann G,et al.Attenuation of dengue virus infection by adeno2associated virus2mediated siRNA delivery[J].Genetic Vaccines Ther,2004,2(1):8.
64.朱旭东,党颖,冯怡,等。用RNaseⅢ制备的小干涉RNA降解SARS冠状病毒基因的细胞内转录物[J].生物工程学报,2004,24(4):485-489。
65.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(23):8676-8681.
66.刘文博,张小荣,曹永忠,等.表达禽流感病毒特异性siRNA分子减毒鼠伤寒沙门氏菌的构建[J].病毒学报,2007,23(3):125-129.
67.Chen Y,Du D,Wu J,et al.Inhibition of hepatitis B virus replication by stably expressed shRNA[J].Biochem Biophys Res Commun,2003,311(2):398- 404.
68.Mcmanusmt M T and Sharp P A.Gene silencing in mammals by small interfering RNAs[J].Nat Rev Genet,2002,3(10):737- 747.
69.Ying C,De Clereq E and Neyts J.Selective inhibition of hepatitis B virus replication by RNA interference[J].Biochem Biophys Res Commun,2003,309(2):482-484.
70.MeCaffrey A P,Nakai H,Pandey K,et al.Inhibition of hepatitis B virus in mice by RNA interference[J].Nat Biotechnol,2003,21(6):639- 644.
71.Shlomai A and Shaul Y.Inhibition of hepatitis B virus expression and replication by RNA interference[J].Hepatology,2003,37(4):764- 770.
1.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Blackwell publishing,2006:299-308.
2.Vicente J,Segales J,Here,U et al.Epidemiological study on porcine circovirus type 2(PCV2) infection in the European wild boar(Sus scrofa),Vet Res,2004,35(2):243-253.
3.Olvera A,Cortey M and Segales J.Molecular evolution of porcine circovirus type 2genomes:phylogeny and clonality[J].Virology,2007,357(2):175-85.
4.Laura B.Postweaning multisystemic wasting syndrome(PMWS) in Quebec,is it an emerging disease? American Association of swine veterinarians,2006:383-385.
5.Quintana J,Segales J,.Rosell C,et al.Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome[J].Vet Rec,2001,149(12):357-361.
6.An D J,Roh I S,Song D S,et al.Phylogenetic characterization of porcine circovirus type 2 in PMWS and PDNS Korean pigs between 1999 and 2006[J].Virus Res,2007,129(2):115-122.
7.Grau-Roma L,Crisci E,Sibila M,et al.A proposal on porcine circovirus type 2(PCV2)genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence.Vet Microbiol,2008,128(1-2):23-25.
8.Meehan B M,McNeilly F,Todd D,et al.Characterization of novel circovirus DNAs associated with wasting syndromes in pig[J].Journal of General Virology,1998,79(9):2171-2179.
9.Wang C,Huang T S,Huang C C,et al.Characterization of porcine circovirus type 2 in Taiwan[J].Vet Med Sci,2004,66(5):469-475.
10.Celera V Jr and Carasova P.First evidence of porcine circovirus type 2(PCV2) infection of pigs in the Czech Republic by semi-nested PCR[J].Vet Med B Infect Dis Vet Public Health,2002,49(3):155-159.
11.Larochelle R,Magar R and D' Allaire S.Genetic characterization and phylogenetic analysis of porcine circovirus type 2(PCV2) strains from cases presenting various clinical condition[J].Virus Res,2002,90(1-2):101-112.
12.Rodriguez-Arrioja G M,Segales J,Resell C,et al.Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain[J].J Vet Med B Infect Dis Vet Public Health,2003,50(2):99-101.
13.Lipej Z,Segales J,Jemersic L,et al.First description of postweaning multisystemic wasting syndrome(PMWS) in wild boar(Sus scrofa) in Croatia and phyiogenetic analysis of partial PCV2 sequences[J].Acta Vet Hung,2007,55(3):389-404.
1.Meehan B M,McNeilly F,Todd D,et al.Characterization of novel circovirus DNAs associated with wasting syndromes in pig[J].Journal of General Virology,1998,79(9):2171-2179.
2.Harding J C.Postweaning multisystemic wasting syndrome:preliminary epidemiology and clinical findings.In:Proceedings of the Western Canadian Association on Swine Practice,1996,22-25.
3.Ellis J A,Wiseman B M,Allan G,et al.Analysis of seroconversion to porcine circovirus 2 among veterinarians from the United States and Canada[J].J Am Vet Med Assoc,2000, 217(11):1645-1646.
4.Choi C and Chae C.In-situ hybridization for the detection of porcine circovirus in pigs with post-weaning multisystemic wasting syndrome[J].Comp Patho,1999,121(3):265-270.
5.Onuki A,Abe K,Togashi K,et al.Detection of porcine circovirus from lesions of a pig with wasting disease in Japan[J].Vet Med Sci,1999,61(10):1119-1123.
6.Plana-Duran J,Baiasch M,Segaies J,et al.Post-weaning multisystemic wasting syndrome in Spain[J].Vet Rec,1999,145(3):87-88.
7.Allan G M,McNeilly F,Meehan B M,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.
8.Spillane P,Kennedy S,Meehan B,et al.Porcine circovirus infection in the Republic of Ireland[J].Vet Rec,1998,143(18):511-512.
9.Mankertz A,Domingo M,Foich J M,et al.Charactefisation of PCV-2 isolates from Spain,Germany and France[J].Virus Res,2000,66(1):65-77.
10.Madec F,Eveno E,Morvan P,et al.Post-weaning multisystemic 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.
11.Kiss I,Kecskemeti S,Tuboly T,et al.New pig disease in Hungary:post-weaning multisystemic wasting syndrome caused by circovirus(short communication)[J].Acta Vet Hung,2000,48(4):469-475.
12.Trujano M,Iglesias G,Segales J,et al.PCV-2 from emaciated pigs in Mexico[J].Vet Rec,2001,148(25):792.
13.Borel N,Burgi E,Kiupel M,et al.Three cases of post-weaning multisystemic wasting syndrome(PMWS) due to porcine circovirus type 2(PCV 2) in Switzerland[J].Schweiz Arch Tierheilkd,2001,143(5):249-255.
14.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.
15.Celera V Jr and Carasova P.First evidence of porcine circovirns type 2(PCV2) infection of pigs in the Czech Republic by semi-nested PCR[J].Vet Med B Infect Dis Vet Public Health,2002,49(3):155-159.
16.Liu Q,Wang L,Willson P,et al.Seroprevaience of porcine circovirus type 2 in swine populations in Canada and Costa Pdca.[J].Vet Res,2002,66(4):225-231.
17.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 Q,2000,22(3):167-172.
18.Saoulidis K,Kyriakis S C,Kennedy S,et al.First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome in pigs in Greece [J].Vet Med B Infect Dis Vet Public Health,2002,49(4):202-205.
19.Sarradell J,Perez A M,Andrada M,et al.PMWS in Argentina[J].Vet Rec,2002,150(10):323.
20.Cano J P,Rodriguez-Carino C,Sogbe E,et al.Post-weaning multisystemic wasting syndrome in pigs in Venezuela[J].Vet Rec,2005,156:620.
21.Kiatipattanasakul-Banlunara W,Tantilertcharoen R,SuzukiK,et al.Detection of porcine circovirus 2(PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome(PMWS) pigs in Thailand[J].J Vet Med Sci,2002,64(5):559-452.
22.Maldonado J,Segales J,Calsamiglia M,et al.Post-waning multisystemic wasting syndrome(PMWS) in the Philippines:porcine circovirus type 2(PCV2) detection and characterization[J].Vet Med Sci,2004,66(5):533-537.
23.Wallgren P,Hasslung F,Bergsrom G,et al.Post-weaning multisystemic wasting syndrome--PMWS.The first year with the disease in Sweden[J].Vet Q,2004,26(4):170-187.
24.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):171-175.
25.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):178-180.
26.Wang C,Huang T S,Huang C C,et al.Characterization of porcine circovirus type 2 in Taiwan[J].Vet Med Sci,2004,66(5):469-475.
27.Allan G M,Kennedy S and McNeilly F.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.
28.Misinzo G,Delputte P L and Nauwynck H J.Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells[J].J Virol,2008,82(3):1128-1135.
29.郎洪武,张广川,吴发权,等。断奶猪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.
30.杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医,2004 31(5):41-43.
31.Condreay J P and Kost T A.Baculovirus expression vectors for insect and mammalian cells[J].Curr Drug Targets,2007,8(10):1126-31.
32.Nawagitgul P,Morozov I,Bolin S,et al.Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J].Journal of General Virology,2000,81:2281-2287.
33.萨姆布鲁克等主编.高福主译.分子克隆指南[M].第二版.北京:科学出版社.1998,19-68.
34.Brunborg I M,Moldal T,Christine M J,et al.Quantitation of porcine circovirus type 2isolated 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,122(2):171-178.
35.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Blackwell publishing,2006:299-308.
36.Mahe D,Blanchard P,Ttuong C,et al.Diferential recognition of ORF2 protein from type 1 and type 2 porcine cicovir2uses and identification of immunorelevant epitopes[J].Journal of General Virology,2000,81(pt 7):1815-1824.
37.Blanchard P,Mahe D,Cariolet R,et al.Protection of swine against postweaning multisystemic wasting syndrom(PMWS)by porcine circovirus type 2(PCV2) proteins[J].Vaccine,2003,21(31):4565-4575.
38.Kamstrup S,Barfoed A M,Frimann T H,et al.Immunization against PCV2 structural protein by DNA vaccination of mice[J].Vaccine,2004,22(11-12):1358-1361.
39.Fenaux M,Opriessnig T,Halbur P C B,et al.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,2004 Jun;78(12):6297-6303.
40.Fenaux M,Opriessnig T,Halbur P G,et al.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):13440-13446.
1.Straw B E,Zimmerman J J,D'Allaire S,et al.Diseases of swine[M].9th edition,Iowa:Blackwell publishing,2006:299-308.
2.Steinfeldt T,Finsterbusch T and Mankertz A.Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1[J].Viral,2007,81(11):5 696-5704.
3.Nawagitgul P,Morozov I,Bolin S,et al.Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J].Journal of General Virology,2000,81:2281-2287.
4.Fire A,Xu S,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
5.Bosher J M,and Labouesse M.RNA interference:genetic wand and genetic watchdog[J].Nat Cell Biol,2000,2(2):E31-36.
6.宋尔卫。主编,RNA干扰的生物学原理与应用[M].北京,高等教育出版社.2005,47-48.
7.Li W X,Li H,Lu R,et al.Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[J].Proc Natl Acad Sci USA,2004,101(5):1350-1355.
8.Joost Haasnoot PC,Daniel C and Ben B.Inhibition of virus replication by RNA interference[J].J Biomedical sci,2003,(10):607-616.
9.Ge Q,Michael T M.,Nguyen T,et al.RNA interfence of influenza viurs production for directly targenting mRNA for degradation and indirectly ingibiting all viral RNA transcription[J].Proc Natl Acad Sci USA,2003,100(23):2718-2723.
10.Stepen M T,Chia-Yun L,Terrence M,et al.Protection against lethal influenza virus challenge by RNA interference in vivo[J].Proc Natl Acad Sci USA,2004,101(23):8682-8686.
11.Zhou Y,Chan J H,Chan A Y,et al.Transgenic plant-derived siRNAs can suppress propagation of influenza virus in mammalian cells[J].FEBS Lett,2004,577(3):345-350.
12.Hu W Y,Bushman F D and Siva A C.RNA interference against retroviruses[J].Virus Res,2004,102(1):59-64.
13.Jacque J M,Trique L,Stevenson M.Modulation of HIV-1 replication by RNA interference[J].Nature,2002,418:435-438.
14.Lee N S,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells[J].Nat Bioteehnol,2002,20(5):500-505.
15.Novina C D,Murray M F,Dykxhoom D M,et al.SiRNA-directed inhibition of HIV-1infection[J].Nature Med,2002,8(7):681-686.
16.Qin X F,An D S,Chen I S,et al.Inhibiting HIV-linfection in human T cells by lentiviral-medinted delivery of small interfering RNA against CCR5[J].Proe Natl Aead Sei USA,2003,100(1):183-188.
17.Randall G,Grakoui A,Rice C M.Clearance of replicating hepatitis C virus replieon RNAs in cell culture by small interfering RNAs[J].Proe Natl Aead Sci USA,2003,100(1):235-240.
18.Kapadia S B,Bridean-Andersen A and Chisari F V.Interference of hepatitis C virus RNA replication by short interfering RNAs[J].Proc Natl Acad Sci USA,2003,100(4):2014-2018.
19.Wilson J A,Jayasena S,Khvonrova A,et al.RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cell[J].Proc Natl Acad Sci USA,2003,100(5):2783-2788.
20.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(6):1-7.
21.McCaffrey A P,Meuse L,Pham T T,et al.RNA interference in adult mice[J].Nature,2002,418(6893):38-39.
22.Saleh M C,Van RIJ R P and Anding R.RNA silencing in viral infections:insights from poliovirus[J].Virus Res,2004,102(1):11-17.
23.Citlin L,Karelsky S and AndinoR.Short interfering RNA confers intracellular antiviral immunity in human cells[J].Nature,2002,418(6896):430-434.
24.Adelman Z,Sanchez-Vargas I,Travanty E,et al.RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted Repeat RNA Derived from the Virus Genome[J].Journal of virology,2002,76(24):12925-12933.
25.Zhang W,Singam R,Hellermann G,et al.Attenuation of dengue virus infection by adeno2associated virus2mediated siRNA delivery[J].Genetic Vaccines Ther,2004,2(1):8.
26.朱旭东,党颖,冯怡,等.用RNaseⅢ制备的小干涉RNA降解SARS冠状病毒基因的细胞内转录物[J].生物工程学报,2004,24(4):485-489.
27.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(23):8676-8681.
28.刘文博,张小荣,曹永忠,等.表达禽流感病毒特异性siRNA分子减毒鼠伤寒沙门氏菌的构建[J].病毒学报,2007,23(3):125-129.
29.Chen Y,Du D,Wu J,et al.Inhibition of hepatitis B virus replication by stably expressed shRNA[J].Biochem Biophys Res Commun,2003,311(2):398- 404.
30.Mcmanusmt M T and Sharp P A.Gene silencing in mammals by small interfering RNAs[J].Nat Rev Genet,2002,3(10):737- 747.
31.Ying C,De Clereq E and Neyts J.Selective inhibition of hepatitis B virus replication by RNA interference[J].Biochem Biophys Res Commun,2003,309(2):482- 484.
32.McCaffrey A P,Nakai H,Pandey K,et al.Inhibition of hepatitis B virus in mice by RNA interference[J].Nat Biotechnol,2003,21(6):639- 644.
33.Shlomai A and Shaul Y.Inhibition of hepatitis B virus expression and replication by RNA interference[J].Hepatology,2003,37(4):764- 770.
34.Liu J,Chen I,Chua H,et al.Inhibition of porcine cireovirus type 2 replication in mice by RNA interference[J].Virology,2006,347(2):422-433.
35.Feng Z,Jiang P,Wang X,et al.Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo[J].Antiviral Res,2008,77(3):186-194.