【作者】 陈宁；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2009， 博士

【摘要】 猪瘟(Classical swine fever, CSF)是严重危害养猪业的传染病之一,具有高传染性和高致病性的特点。其病原为猪瘟病毒(CSFV),属黄病毒科瘟病毒属成员。CSFV是有囊膜的单股正链RNA病毒,基因组长约12.3kb。囊膜糖蛋白E2具有良好的免疫原性,可诱导机体产生中和抗体并对强毒的攻击提供保护。E2在病毒感染过程中也起着重要作用,与病毒吸附,侵入宿主细胞,细胞嗜性和毒力强弱有关。新近研究表明,在CSF流行地区,其症状呈现非典型化,甚至在免疫猪群中也有发病；CSFV流行毒株已经从以前的group 1转向group 2。有迹象表明我国目前使用的group 1兔化弱毒C株疫苗对group 2 CSFV流行毒株难以提供有效保护。本项目的是：(1)建立猪瘟病毒疫苗株和野毒株的鉴别RFLP技术体系；(2)分析我国浙江地区CSFV流行情况；(3)比较CSFV当前流行毒株与早期group 1强毒株和兔化弱毒C株在体外生长特性、囊膜糖蛋白E2的分子变异特征与抗原多样性方面的差异；(4)应用反向遗传学技术,构建基于兔化弱毒C株和携带目前流行毒株E2基因的重组病毒,为开发针对流行毒株的新型标记疫苗奠定基础。1、猪瘟病毒疫苗株和野毒株的鉴别RFLP技术在CSFV-E2基因的上、下游保守区域分别设计两对简并引物用于套式RT-PCR检测。结果表明,该体系具有很好的特异性和灵敏性,检测下限为1400拷贝数的CSFV基因组。根据不同毒株E2基因中MspⅠ酶切位点排布的不同,建立了鉴别检测疫苗株和野毒株的RFLP方法。应用此方法对2003-2008年浙江地区猪场采集的309份组织样品进行CSFV检测,结果发现91份样品能扩增出CSFV特异性条带,阳性PCR产物经MspⅠ酶切,22份样品中含有疫苗株；60份为野毒感染,其余9份能同时检出疫苗株与野毒株。选择4种限制性内切酶BglⅠ, DdeⅠ, DraⅠ和PstⅠ分别对38株流行毒株的E2基因进行酶切,流行毒株可被分为11个不同的RFLP亚型。以上结果表明,浙江地区存在CSFV的流行,且流行毒株在E2基因水平上存在较大差异。2、猪瘟病毒经典强毒株和流行毒株在PK-15细胞和ST细胞中的生长特性比较比较了两个流行毒株(QZ-07和HZ1-08)和石门株在PK-15和ST细胞中的生长特性。强毒石门株在PK-15细胞中的增殖效率明显高于其在ST细胞。在感染PK-15 48h后,滴度可达到107TCID50/ml,而在相同时间,在ST细胞中只能达到105.25TCID50/ml。而两流行毒株在PK-15细胞中的增殖水平明显低于ST细胞,尤其以HZ1-08更为明显。在ST细胞感染的48h后,HZ1-08的滴度可达到105TCID50/ml,而在PK-15细胞中滴度只有102.75TCID50/ml。IFA检测结果同样表明,在感染48小时后,石门株感染的PK-15细胞均为阳性,而HZ1-08只有零星几个荧光斑。虽然三个毒株在感染不同阶段,ST细胞内病毒滴度基本一致,但石门株在增殖过程中释放于上清中的病毒滴度明显高于两个流行毒株。细胞内外感染性病毒粒子的比例在一定程度上可以判断病毒毒力的强弱,根据以上体外生长特性可以初步推断两个流行毒株不属于强毒株。3、猪瘟病毒流行毒株的全基因组比较和基于E2基因的分子变异特征分析对弱毒C株、石门株和分离株QZ-07进行了全基因组测序,结合从GenBank中下载的22个CSFV的全基因组进行遗传进化关系分析。结果表明,25个毒株可以分为两个分支,C株和石门株处于一个分支,而QZ-07位于遗传关系较远的另一个分支。同义突变与非同义突变以及熵值分析结果表明,CSFV多聚蛋白的前1／3编码区(包括病毒的结构蛋白在内的区域)比后2／3编码与病毒RNA复制有关的非结构蛋白区域变异性大,3个结构蛋白和非结构NS5A比较高变,而NS3、NS4B和NS5B相对保守。我们进一步对2004-2008年内流行于浙江地区的34株CSFV囊膜糖蛋白E2的分子变异特征进行深入分析。结果表明,流行毒株属于group 2,除了2004年的一株病毒属于subgroup 2.2以外,其余毒株均属于subgroup 2.1,且都归于genotype 2.1b。而目前使用的疫苗C株属于group 1中的subgroup 1.1。全长E2的核苷酸和氨基酸序列比较结果表明,流行毒株之间核苷酸的同源性在94.6%-99.8%之间,氨基酸的同源性在94.9%-99.7%之间。与C株相比,核苷酸的同源性在81.6%-82.6%之间,氨基酸的同源性在87.4%-89.3%之间。同义突变与非同义突变以及熵值分析结果表明,在E2蛋白中,N端抗原区域的变异程度大于C端,抗原区内鉴定的2个高变区中与抗体相互作用的关键氨基酸位点处于正选择压力,且流行毒株与疫苗株在这些位点上差异很大。4、猪瘟病毒C株E2蛋白单克隆抗体的研制与鉴定为了进一步探索E2蛋白N端高变区域内与抗体识别相关的一些关键氨基酸位点差异对不同毒株抗原结构的影响,我们以C株E2为抗原免疫BALB/c小鼠,应用杂交瘤技术结合IFA筛选,获得了3株持续、稳定分泌小鼠抗E2的单克隆抗体的杂交瘤细胞株1E7、2B6和6B8。免疫印迹和ELISA鉴定结果表明,只有2B6能与变性的E2蛋白反应。E2蛋白N端的6个Cys残基通过相互形成二硫键对维持抗原区域的构象起到关键作用,任何Cys残基的突变将影响相应抗体对该区域的识别。我们通过真核细胞表达不同Cys突变的重组E2以鉴定不同单抗的识别区域,发现1E7和6B8识别位于抗原区域B／C中的构象表位,而2B6能与所有Cys突变的E2蛋白反应,进一步证实该单抗识别的是一线性表位,因此不受E2抗原区域二级结构的影响。应用上述单抗进行流行毒株E2抗原多样性分析,结果表明：单抗2B6识别的抗原表位只存在于group 1毒株中；而单抗1E7和6B8除了不能与LS-05和QZ2-06毒株E2发生反应(因为这两个毒株Cys737突变为Arg,破坏了B／C抗原区的构象),与大部分毒株(8／10)均能发生反应。但是1E7和6B8与流行毒株的反应性比group 1毒株要弱。5、携带流行毒株E2基因的重组猪瘟病毒C株构建与鉴定鉴于流行于我省的CSFV毒株的E2蛋白分子进化特征及抗原多样性,特别是与疫苗毒株C株相比存在较大差异,可能影响疫苗的免疫保护效力。因此,我们建立了CSFV-C株的反向遗传学操作系统,结合分子流行病学研究结果,构建了基于C株、携带流行CSFV毒株E2基因的重组病毒。首先,我们对覆盖CSFV-C株基因组的6个cDNA片段按一定的策略依次克隆于改造的低拷贝质粒,并插入于T7启动子下游,构建了C株的感染性克隆pA-FL22。以线性化的pA-FL22为模板,体外转录的RNA转染细胞后,荧光定量PCR和IFA检测结果表明,转录的RNA在ST细胞中的复制、翻译水平明显高于PK-15。获得的子代病毒FL22注射家兔,产生与亲本毒株C株相同的体温反应,脾脏肿大。作为遗传标记的NcoⅠ位点在子代病毒体内外复制增殖过程中能稳定遗传。在此基础上,构建并拯救了携带石门毒株和流行毒株HZ1-08的E2抗原区域(870bp)的重组病毒FL22-SM-E2和FL22-HZ-E2。两个重组病毒能够通过上述建立的MspⅠ酶切方法与C株相区别,而且重组病毒FL22-HZ-E2还能通过与C株E2单抗反应性的不同与C株相区别。因此,本试验建立的CSFV感染性克隆与重组病毒拯救体系是成功的。总之,本研究深入探索了目前流行于浙江地区CSFV-E2的分子变异特征,初步探明了它们与疫苗株在抗原结构上的差异,建立了CSFV野毒感染和疫苗毒株的鉴别检测和分型技术体系,特别是CSFV感染性克隆的构建与重组病毒拯救体系的成功建立为新型标记疫苗的开发,CSFV的复制机制和致病机理的深入研究奠定了良好基础。更多还原

【Abstract】 Classical swine fever (CSF) is a highly contagious and often fatal disease of swine and wild boar, causing significant economic losses to the swine industry. The causative agent of the disease is classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family. CSFV is an enveloped RNA virus with its genome size of approximately 12.3 kb. The structural glycoprotein E2 is the most immunogenic among the CSFV proteins, inducing neutralizing antibodies and protection against lethal challenge. It also plays multiple roles in viral life cycle, such as virus attachment, entry into target cells, cell tropsim as well as virulence determinant. Currently, the switch of virus populations from historical group 1 to group 2 has been reported and a trend to chronic form of the disease, even in a certain proportion of vaccinated pigs has been reported in the endemic areas. Therefore, the efficacy of the classical group 1 lapinized C-strain vaccine is facing challenge. The present study was aimed (1) to investigate the CSFV infection status in swine population in Zhejiang, (2) to establish an RFLP method for differential identification of the vaccine strain and field isolates, (3) to explore the genetic and antigenetic diversity of the E2 of field isolates, and (4) to develop recombinant C-strain-based vaccine candidate strains containing E2 genes from prevalent field isolates using the reverse genetics technology.1.Differential identification of field CSFV isolates and the vaccine strain by RFLPRT-nested PCR (RT-nPCR) using the consensus-degenerate hybrid oligonucleotide primers targeted on the full-length E2 was used for detection of CFSV. The assay was able to detect as low as 1400 copies of CSFV genomic RNA. Vaccinated and infected CSFV strains could be differentiated by Mspl-based restriction fragment length polymorphism (RFLP) analysis. The assay was applied to identify CSFV isolates from 309 clinical specimens from 2003-2008 in Zhejiang. In 91 CSFV isolates,22 were identified as the C-strain,60 as field strains, and 9 as having both the C-strain and the field ones. RFLP of the RT-nPCR amplicons by BglⅠ, DdeⅠ, DraⅠand PstⅠwas used for subtyping of the field CSFV isolates. Thirty-eight field isolates were divided into 11 subtypes by this RFLP scheme, indicating the genetic diversity of the prevalent isolates in Zhejiang.2. Growth kinetics of prevalent field CSFV isolates and classical virulent isolate Shimen in PK-15 and ST cellsTwo field strains QZ-07 and HZ1-08 were isolated and their replication kinetics in PK-15 and swine testicle ST cell lines were compared with the classical virulent Shimen strain. The Shimen strain replicated more efficiently in PK-15 cells than in ST cells (107 TCID50/ml vs 105.25 TCID50/ml at 48 h post-infection). In contrast, two field strains displayed decreased replication in PK-15 cells (102.75 TCID50/ml for isolate HZ1-08 vs 105 TCID50/ml in ST cells). This was consistent with the protein expression kinetics. Only a few fluorescent foci were detected by IFA in HZ1-08-infected cells, while all cells were positive for Shimen-infected cells at 48h post-infection. Although the cell-associated virus titers were similar among three strains, the ratio of secreted virus versus cell-associated virus was different. The virulent strain Shimen secreted more progeny virus to the culture supernatants than the recent isolates. Considering the increased release of progeny virus particles from the cells as an attribute of high virulence, the prevalent isolates did not seem to have high virulence.3.Characterization of genetic variations of prevalent CSFV isolates in their full genomes and E2 genesThe genome of the strain QZ-07, vaccine C-strain and virulent Shimen were determined. Phylogenetic analysis revealed two major clusters of 25 isolates including those retrieved from the GenBank representing other parts of China and other countries. C-strain and Shimen strain were clustered together, while strain QZ-07 fell into another cluster far away from these historical strains. The variability of the full-length polyprotein of these 25 isolates was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. The first one third of the polyprotein covering all structural proteins was more variable than the last two thirds containing the nonstructural proteins essential for RNA replication. When the individual proteins were compared, three structural proteins and NS5A were more variable as compared with the relative conserved NS3, NS4B and NS5B proteins.Phylogenetic analysis of the E2 gene of 34 CSFV isolates from Zhejiang was further revealed that genotype 2.1b viruses became predominant in Zhejiang with 33 isolates clustered in 2.1b and only 1 isolate belonged to 2.2. Pairwise comparisons demonstrated that isolates in this study had an identity of 94.6%-99.8% at the nucleotide level and 94.9%-99.7% at the amino acid level. The identity ranged from 81.6% to 82.6% for nucleotide and 87.4%-89.3% for amino acids, as compared to the C-strain. Two variable regions in the antigenic domains as well as some positive selected positions located in the identified neutralizing epitopes or related to monoclonal antibodies (mAb) binding were defined. Moreover, these residues in mAb related positions were different between prevalent isolates and vaccine C-strain.4. Characterization of monoclonal antibodies against E2 of the CSFV vaccine strainMonoclonal antibodies to antigenic domains of glycoprotein E2 of C-strain were prepared and used to examine the effect of the variable regions and the specific positive selected positions on antigenic diversity between C-strain and recent field isolates. Three hybridoma cell lines secreting mAb (1E7,2B6 and 6B8) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the purified recombinant E2 protein (rE2). Western blot and ELISA analysis showed that only the mAb 2B6 could react with rE2. Six Cystine residues important for maintenance of the structure of antigenic domains of E2 were mutated in the backbone of nature C-strain E2 and expressed in PK-15 cells. Mutagenic analysis revealed that 1E7 and 6B8 reacted with the conformational epitopes located in antigenic domain B/C, while 2B6 reacted with all Cys mutant E2s independent of the secondary structure. Thus, mAb 2B6 was confirmed to react with a linear epitope. The reactivity of these three mAb with Shimen, QZ-07 and XS-08 indicated that the linear epitope recognized by 2B6 was specific for group 1 strains, while 1E7 and 2B6 reacted weakly with recent group 2 isolates. The reactivity patterns for prevalent isolates were further confirmed by the expression of different field isolates E2 in PK-15 cells. MAbs 1E7 and 2B6 did not react with mutants with substitution of the E2-Cys737 with Arg in the isolates LS-05 and QZ2-06 possibly due to the abolishment of the structure of antigenic domain B/C. This Cys mutant virus may escape from neutralizing antibodies against domain B/C in vivo.5.Genetic rescuing and characterization of recombinant classical swine fever virus vaccine strains containing the E2 gene representing the prevalent field isolatesSince the genetic and antigenic diversity was apparent between recent isolates from Zhejiang and the vaccine C-strain, the vaccine efficacy could be compromised. Thus, we used reverse genetics to engineer recombinant CSF viruses based on the vaccine C-strain and the genetic features of the isolates circulating in Zhejiang. The cDNA fragments covering the genome of the C-strain were assembled and inserted downstream of a T7 promoter to obtain the full-length cDNA clone of pA-FL22. The in vitro synthesis of full-length viral RNA derived from pA-FL22 proven to be replication-competent and infectious after electroporated into ST and PK-15 cells. Real-time PCR and IFA revealed that the RNA replication and protein translation was more efficiently in ST cells than in PK-15 cells. The recombinant virus FL22 recovered from the electroporated cells retained the properties of the parental C-strain in rabbit, fever and enlargement of the spleen. A silent point mutation at position 8036 of the genome which introduced an additional NcoⅠrestriction site as genetic tag was also retained during virus replication in vitro and in vivo. Two infectious recombinant CSF viruses were generated by exchanging the 830-bp region including the antigenic domains in pA-FL22 with the equivalent region of CSFV strains Shimen and HZ 1-08, respectively. The resulting recombinant viruses FL22-SM-E2 and FL22-HZ-E2 could be differentiated from FL22 by MspⅠbased RFLP assay, and virus FL22-HZ-E2 also changed mAb pattern same as the donor strain HZ 1-08. Therefore, the recombinant virus FL22-HZ-E2 may serve as the candidate strain for further marker vaccine development.In summary, our studies revealed the divergence of the prevalent CSFV isolates in Zhejiang and their antigenic variations to the vaccine C-strain. The RT-nPCR-based RFLP could be used for subtyping and differentiation of the field isolates from the vaccine strain. Successful rescuing of the recombinant viruses based on the vaccine strain would be of great use for development of anti-CSFV marker vaccines and for in-depth studies on the replication and pathogenesis of CSFV.更多还原