【作者】 徐绍建；

【导师】 柴同杰； 王金宝；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2013， 博士

【摘要】 猪圆环病毒（porcine circovirus type, PCV）是一种环状、无囊膜的单股DNA病毒，它包括猪圆环病毒1型（PCV1）和猪圆环病毒2型（PCV2）两个基因型。其中圆环病毒2型具有致病性，能够引起断奶仔猪多系统衰竭综合征等多种疾病，并可以引起猪的免疫抑制。2000年我国第一次报道猪群检测出PCV2，随后多个省市陆续报道了PCV2的感染，流行呈现逐渐蔓延的趋势。该病毒往往与其他病原混合或继发感染，给我国养猪业造成了巨大损失，严重影响了我国养猪业的发展。近年来，各国学者对PCV2的病原学研究和防控技术研究逐渐深入，但目前PCV2病原学及流行病学许多问题仍未解决，该病毒山东省等地的流行规律、危害，尤其是在2010年以来，经过疫苗大面积使用之后的流行规律还未见报道。PCV2的致病机理仍然处于探索阶段，PCV2感染引发细胞信号转导的研究有助于阐明PCV2的致病机理。PCV2感染PK-15细胞中JAK-STAT信号通路的激活机制，JAK-STAT信号通路是如何抑制病毒蛋白表达和病毒增殖，与病毒的复制相关性尚未见报道；因此，本研究对山东省PCV2的分子流行病学情况进行了调查；对山东分离株和其他PCV2分离株全基因组序列和主要保护性抗原蛋白序列进行了分析，应用本实验室建立的PK-15细胞α-IFN效应因子实时荧光定量RT-PCR检测方法和PCV2病毒含量实时荧光定量RT-PCR检测方法，并进一步研究了JAK-STAT信号通路与PCV2感染的相互关系。以下分三部分论述：1.山东省猪圆环病毒2型流行病学调查与PCV2毒株分离及其感染增殖特性研究为了掌握PCV2在山东省猪群中的感染和流行情况，为猪圆环病毒病的防制提供参考，2010年~2012年对山东100余家大中型猪场进行猪圆环病毒流行病学情况调查，采用PCR技术和ELISA方法分别对采集的猪抗凝血和疑似猪圆环病毒感染病猪的淋巴结、脾脏、肺脏等脏器样品进行PCV2抗原及抗体检测。结果显示，2010年、2011年、2012年抗体阳性率分别为72.95%、61.85%和59.89%；核酸阳性率分别为34.66%、29.59%、21.38%。在流行病学调查的基础上，用PCV2抗原检测阳性的病料感染PK-15细胞，获得2株PCV2病毒的分离株。将其盲传20代后病毒滴度为分别为106.0TCID50/ml和106.2TCID50/ml，通过对其中一株PCV2SD-JN株进行病毒的生长曲线测定发现，PCV2在细胞中生长至第72小时后，达到TCID50的最高峰106.2TCID50/ml。PCV2分离株的获得，对进行病毒体外增殖特性研究，阐明PCV2与细胞间的相互作用机制，进一步研制高效优质的PCV2疫苗奠定了基础。2.猪圆环病毒2型分离株遗传演化、时空界定及PCV2b山东株全基因序列分析为了解山东省PCV2流行株的遗传变异情况，2010～2012年，本研究从患病猪组织、血样中扩增克隆出PCV2的全基因组序列，获得8株PCV2山东株全基因序列，其中PCV2b型为7株。同时对2005～2012间本实验室获得的26株山东分离株进行了核苷酸和主要蛋白氨基酸序列分析。结果发现，26株山东PCV2分离株核苷酸同源性为69.9%-100%，其中2006年分离的DQ478947与2010年分离的HM142894同源性最低。应用DNAStar分析软件对26株山东分离株Rep蛋白氨基酸序列和Cap蛋白氨基酸序列进行了分析，结果表明，根据Cap蛋白第86-89位氨基酸序列为SNPR(L)或者TNKI判断，26株PCV2山东分离株中，HM142897、HM142900和SD8-1三株序列第86-89位氨基酸序列为TNKI，为PCV2a亚型，其余的23株均为SNPR(L)，为PCV2b亚型。26株PCV2ORF1编码的314或315个的氨基酸，ORF2编码233、234或235个的氨基酸，Rep蛋白的氨基酸同源性为98.3%-100%，Cap蛋白的氨基酸同源性为83.7%-100%。Rep蛋白上的三个潜在的糖基化位点24aa-26aa(NPS),256aa-258aa(NQT),286aa-288aa(NAT)均没有变化。Cap蛋白变异较大，主要变异区位于51aa-78aa,121aa-134aa,169aa-215aa。将2005～2012间本实验室获得的26株山东分离株与GenBank登录的国内外其他PCV2全基因序列共90株，进行核苷酸进化树和ORF2编码氨基酸进行分析，结果表明，PCV2的流行经历了1998年至2002年以PCV2a亚型为主要流行毒株阶段，2003年为PCV2a和PCV2b亚型并存阶段，2003年至今以PCV2b为主要流行毒株阶段的三个阶段演变过程。PCV2b亚型中国分离毒株在遗传进化上，以秦岭淮河为界限，按照地域分为了中国南部和中国北部两个大的集群。从ORF2进化树分析看出，26株山东分离株中，24株的ORF2基因集中分布于进化树两端的两个大分支上，其中9株位于进化树的上部A区分支上，与法国、匈牙利分离株遗传距离较近，15株位于进化树的下部B区分支上，与英国分离株遗传距离较近。3.JAK-STAT信号通路与PCV2感染的相互作用由于α-IFN激活JAK-STAT信号通路后，激活α-IFN效应因子Mx1、OAS基因转录表达，因此本实验室根据GenBank中收录的猪β-actin、Mx1、OAS基因序列，选择其中的保守区，利用Premier5.0软件设计相应的引物，建立了针对对PK-15细胞的β-actin、Mx1、OAS基因的荧光定量PCR方法。参照该方法进行α-IFN的最佳作用时间和最佳作用浓度筛选。发现使用浓度为750U/ml的α-IFN，处理PK-15细胞12小时后，α-IFN的效应因子Mx1、OAS相对表达量最高，因此确定α-IFN处理的最佳作用时间为12h，最佳浓度为750U/ml。分别对JAK-STAT信号通路特异性抑制剂AG490最佳作用浓度以及最佳作用时间进行优化。结果发现，使用10μM的AG490作用于α-IFN处理的PK-15细胞6小时后，OAS的相对表达量达到最低点接近空白对照细胞，而12小时左右Mx1的表达量才达到相对表达量最低，接近空白对照细胞，基本完全阻断JAK-STAT信号通路，从而确定将AG490的最佳作用时间定为12小时，最佳作用浓度为10μM。以PK-15细胞作为空白对照，通过检测分组检测不同时间点OAS和Mx1基因的相对表达量发现，当PK-15细胞接种PCV2后，OAS和Mx1的相对表达量高于空白对照细胞低于α-IFN激活细胞JAK-STAT信号通路组。以PCV2单独感染组作为对照感染组，利用本实验室建立的PCV2特异性荧光定量PCR方法，对PCV2的最佳收获时间进行确定并检测不同分组PCV2的绝对表达量。结果发现，随着培养时间的增长，病毒液中PCV2的含量逐渐增加，于接毒后72h病毒含量到达顶峰（3.109×108copies），随后病毒含量开始逐渐降低。应用AG490抑制JAK-STAT信号通路后，PCV2的病毒绝对表达量量高于对照感染组，而通过α-IFN激活JAK-STAT信号通路组，PCV2病毒绝对表达量大大低于与PCV2单独感染组。结果表明，PCV2的感染能够激活PK-15细胞中JAK-STAT信号通路，但信号通路的激活又会反过来抑制PCV2在细胞中的增值。本研究明确了PCV2感染PK-15细胞与JAK-STAT信号通路的相互作用，丰富了PCV2的感染和发病机制理论，为阐明PCV2组织细胞感染致病机理奠定了基础。更为预防和控制PCV2的感染，研制PCV2型的新型疫苗提供新的靶向分子。更多还原

【Abstract】 Porcine circovirus (PCV) is a nonenveloped, icosahedral virus containing a circularsingle-stranded DNA genome, which is one of the minimum DNA animal viruses. Porcinecircovirus is divided into two serotypes: PCV1and PCV2, PCV1is not pathogenic whilePCV2is pathogenic. The spread of PCV2causing huge economic losses to the pig industry.Ithas seriously hampered the development of the pig industry.More than a dozen provinces reported porcine circovirus virus type2(PCV2) infectionssince porcine circovirus virus type (PCV) antigen has been detected by Lang Hongwu firstlyin2000. It spread gradually and seriously hampered the healthy development of pig industryof China. Currently, the pathogenesis of PCV2is still at the exploratory stage.The study ofcellular signal transduction caused by PCV2infection will help to elucidate the pathogenesisof PCV2.There are no reports about the way JAK-STAT signaling pathway in PK-15cellsinfected with PCV2curbing viral protein expression and virus proliferation.To understand thegenetic variation of PCV2epidemic strains in Shandong Province the molecular prevalence ofPCV2in Shandong Province from2010to2012was investigated. The whole genomesequence of PCV2isolates of Shandong have been compared to the submitted sequence of theGenBank with the whole genome sequence.PK15cells were infected with PCV2isolatedfrom diseased or serum which antigen test is positive. The relationship between JAK-STATsignaling pathway and PCV2infection was studied by PCV2-specific fluorescencequantitative PCR and PK-15cells α-IFN real-time quantitative RT-PCR.There are four parts of this study.1. Epidemiological investigation proliferation properties of porcine circovirus virus type2in Shandong provinceIn order to understand PCV2infection situation of swinery in Shandong province inrecent years, we collected lung, lymph nodes, spleen and other organs of the pathologicalchanges, anticoagulant, serum from2010to2012December from300big farms in Shandongprovince, and detected the antigen and antibody of PCV2using PCR and ELISA method. Theresult of survey showed that the antibody positive rate in2010,2011,2012was72.95%,61.85%,59.89%, while antigen positive rate was34.66%,29.59%,21.38%, respectively. Inaddition, the PCV2antibody levels of serum in pig farms which did not immune PCV2 vaccine were as high as88.72%. This study was aimed at analyzing systematically PCV2infection and popular rule in pigs in our province, which provide reference for prevention andcontrol of PCV2. PK15cells were infected by tissues or serum which was positive in PCV2antigen detection.The virus isolates were obtained, and the titer of virus generation in vitro was assayed.The results of tests showed that two isolates were obtained from tissues or serum. In theindirect immunofluorescent assay (IFA) test, the nucleus sent dense green fluorescents andcytoplasm appeared sparse green fluorescent, while there were no fluorescents in control cells.The growth curve of PCV2isolated strain (SD-JN) was detected. The result showed that theTCID50of PCV2achieved the peak when PK15growth to72hours, and then began todecline.The result of animal regression test showed that Kunming mouse infected PCV2andcontrol mouse did not appear obvious clinic symptoms, but the result of PCR was positive intest group. The obtaining of PCV2isolates laid a solid foundation for the research of theproliferation characteristics of viruses in vitro, to clarifying the mechanism of interactionbetween PCV2and PK15cells, to develop further high quality vaccine.2.Spatial and temporal distribution and genetic evolution of porcine circovirus type2andthe genome sequence of PCV2b virus isolates from Shandong provinceThe study of amplification cloned whole genome sequence of PCV2from sick pig in thetissue, blood, and26strains of isolates in Shandong from our laboratory were sequenced andcompared with the sequence of PCV2isolates submitted in the GenBank, the results showedthat26strains of Shandong PCV2isolates nucleotide homology of69.9%-69.9%, amongisolated strains of DQ478947in2006and separate strain of HM142894in2010of homologylowest.The whole genome of90strains PCV2from the isolated gene sequences downloadedfrom Shandong Province26PCV2gene sequences and in GenBank (26strains of otherprovinces in China’s mainland, Taiwan7strains and30strains in foreign) were analyzed towhole sequence of nucleotides and ORF2amino acids encoded by gene phylogenetic tree.The results show that53PCV2of the separation from China’s mainland, PCV2b subtypes of45strains, subtype PCV2a of8strains, full description PCV2b subtypes. At present, the domesticmainland major PCV2major pandemic strain. From looking at the year of separation, since1998,the ring virus epidemic strain went from PCV2a as the main epidemic strains,thecoexistence of PCV2a and PCV2b subtypes, with PCV2b as the main evolution of theepidemic strains.On the genetic tree, currently the isolation and identification of PCV2bsubtype of HIV strains, with Qinling huaihe River as the boundary, in accordance with the region divided into southern China and Northern China two large clusters.From ORF2evolution tree analysis, isolates from Shandong24PCV2ORF2gene concentrated on the twobranches of both ends of the evolutionary tree, which9Article Shandong separation strains ofORF2gene is located in evolution tree of the upper part of Zone A branch and France, andHungary separation strains genetic distance more near,15article Shandong separation strainsof ORF2gene is located in evolution tree of the lowe part of zone B branch, and UnitedKingdom separation strains genetic distance more near. The purpose of this test is tounderstand the genetic variation of PCV2epidemic strains of Shandong Province, to provide atheoretical basis for the diagnosis of porcine circovirus disease and vaccine research anddevelopment.3.The interaction of JAK-STAT signaling pathways with infection of PCV2Specificity primers were designed according the gene sequences of β-actin、Mx1、OASfrom pig in the GenBank for β-actin、Mx1、OAS from PK-15cell and a real-time RT-PCRmethod was established in our lab. The best action time and the optimal concentration aboutα-IFN in PK-cell was defined that were750U/ml and12hours.The best time to gather thePCV2from PK-cell is72hours post infection. The best action time and the optimalconcentration about AG490(special inhibitor to JAK-STAT pathway) in PK-cell wereconfirmed by real-time RT-PCR method of β-actin、Mx1、OAS and PCV2. The results showedthat the optimal condition is10μM and12hours. The expression level of Mx1、OAS geneswere tested in different time. The results are that the genetic expressions of Mx1、OASincreased after PCV2infection in PK-cell than the control PK-cell. But it is lower than thegenetic expressions of Mx1、OAS in PK-cell treated with the α-IFN. We can confirm that theinfection of PK-cell can activate the JAK-STAT singaling Pathway. There are high level ofPCV2in the PK-cell in which the JAK-STAT pathway was inhibitted completely by AG490than the PK-cell as control. And there are low level of PCV2in the PK-cell in which theJAK-STAT pathway was activated completely by α-IFN than the PK-cell as control.Then,wecan determine that JAK-STAT pathway activation can inhibit the proliferation of PCV2.This conclusion can help us to study the biological mechanism of PCV2infection.更多还原