【作者】 韦祖樟；

【导师】 袁世山；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2008， 博士

【摘要】 除马动脉炎病毒(equine arteritis virus, EAV)外,动脉炎病毒科还包括猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、乳酸脱氢酶升高症病毒(lactate dehydrogenase elevating virus, LDV)以及猴出血热病毒(simian hemmorrhagic fever virus, SHFV)。动脉炎病毒感染的宿主主要为马和驴(EAV)、猪(PRRSV)、鼠(LDV)和非洲、亚洲一些种属的猴(SHFV)。感染动物主要表现为无明显临床症状的持续性带毒;另外,也可造成流产或致死性的出血性高热。关于动脉炎病毒基因组RNA的复制、亚基因组mRNA的转录和翻译、病毒颗粒组装以及病毒致病机理等许多问题尚待研究。本研究旨在通过建立EAV全长cDNA克隆,进而通过构建不同EAV或其它动脉炎病毒之间的嵌合cDNA克隆,进而研究不同基因组区域及其编码产物在病毒复制过程中的作用,为进一步剖析病毒遗传组份的结构与功能及其在致病机理奠定基础。1、马动脉炎病毒基因组全长cDNA克隆的构建及其序列分析根据马动脉炎病毒Bucyrus株全基因组序列(GenBank NO NC 002532),设计并合成EAV特异引物,进而应用RT-PCR技术分6段扩增了EAV M9544株的全基因组cDNA。将扩增的各个cDNA重叠片断PE124、PE631、PE1854、PE5191、PE61107、PE97Q分别克隆到载体pCR-BluntⅡ-TOPO中,建立了EAV各扩增片段的cDNA克隆。在扩增5′末端时,引入NotI酶切位点和T7启动子序列;在基因组3′末段Poly(A)尾后引入XhoI酶切位点,后者供cDNA模板的线性化之用。将上述片断在pCR BluntⅡ-TOPO中依次连接,最后克隆到质粒pBluescriptⅡSK(+)中,获得了EAV全长基因组cDNA克隆pWEAV。核酸序列分析表明:该毒株基因组全长为12704个核苷酸(不包括poly(A)尾),与EAV北美谱系代表株Bucyrus株的同源性为99.1%;与欧洲谱系分离株同源性为85.5%。与Bucyrus株全长序列相比,M9544株EAV全长基因组中共有106个核苷酸变异,有两个核苷酸位于5‵UTR中,在编码区中有68个核苷酸为沉默突变,另外的36个核苷酸相应地导致编码区内36个氨基酸发生变异。本研究构建了EAV M9544株基因组全长cDNA克隆pWEAV,为进一步获取EAV感染性克隆并研究动脉炎病毒基因组结构和功能奠定了基础。2、动脉炎病毒复制过程结构蛋白调控因子的初步研究动脉炎病毒的基因组RNA复制、亚基因组mRNA转录和翻译、病毒颗粒包装等病毒生命周期的调控机制,尤其是病毒编码蛋白在其中所起的作用尚待进一步研究。将所构建的M9544株全长cDNA克隆pWEAV体外合成RNA后转染BHK-21细胞,通过免疫荧光和RT-PCR方法检测转染细胞及培养上清,结果均不能检测到病毒的复制、转录和细胞病变(CPE),说明pWEAV含有致死性变异位点。为了查明pWEAV中关乎病毒复制调控的致死性变异位点,本研究将细胞适应株感染性克隆pEAV030(Snijder博士提供)为骨架,将pWEAV基因组不同区域cDNA片段置换嵌合到pEAV030中,构建了一系列pEAV030/pWEAV的嵌合cDNA克隆。将后者体外转录RNA后转染BHK-21,病毒拯救试验表明,上文所提及106个变异核苷酸中,位于5‵UTR中的两个核苷酸变异不影响病毒的感染性;在ORF1编码的非结构蛋白中共有10个氨基酸的变异和ORF2-4编码的结构蛋白GP2-4中11个氨基酸变异也不影响病毒的感染性。另一方面,发现在pWEAV克隆基因组片段1651nt-4264nt间(nsp2的N端和nsp3 C端)的6个氨基酸中存在致死性变异位点。nsp2和nsp3中这些氨基酸变异影响了病毒基因组RNA复制和亚基因组RNA转录。而下游嵌合克隆pEAVexb (以pWEAV相应序列替换pEAV030的11488nt-11901nt)能够在BHK-21中进行复制转录,但是在转染细胞以及多次传代细胞中未出现CPE。说明GP5中位点170 A→S氨基酸突变影响病毒复制的转录后(post-transcriptional)阶段,可能阻断了病毒颗粒组装以及出芽等病毒复制步骤。在pEAVxx(以pWEAV相应序列替换pEAV030的11948nt-12704nt)N蛋白位点56 H→Y位点为病毒感染性致死性变异位点;通过对pEAVxx进行回复突变(Y→H)获得了拯救病毒,说明该氨基酸对病毒转录后的翻译、病毒颗粒包装或出芽等步骤可能起调控作用。总之,通过利用无感染性的M9544株与感染性克隆pEAV030进行嵌合,我们发现了28个氨基酸变异不影响EAV的复制过程;而另一方面, nsp2的N端和nsp3C端中6个氨基酸变异影响病毒基因组RNA复制和亚基因组RNA转录过程。而GP5和N蛋白中两个位点对病毒基因转录后阶段起着重要的调控作用。3、Nsp6在动脉炎病毒复制中的作用动脉炎病毒的非结构蛋白( nsp1-13 )装配成所谓的“复制转录酶复合体”(Replicase/Transcriptiase Complex, RTC),后者是动脉炎病毒基因组RNA复制及亚基因组mRNA转录等过程的引擎。目前对各个nsp在RTC中所起的作用及机制并不清楚,其中,nsp6作为已知最小的nsp(13-22aa),其结构和功能有待研究。为了研究nsp6在动脉炎病毒复制中的作用,本研究通过重叠延伸PCR (SOE-PCR)法对PRRSV全长感染性克隆pAPRRS的nsp6(16aa)进行了系列缺失,同时与其它动脉炎病毒的nsp6进行替换,构建了一系列以pAPPRS为骨架的全长cDNA克隆突变体。病毒拯救试验表明, nsp6全部(16)或部分(3、6个)氨基酸缺失的突变体不能产生病毒,但RT-PCR结果显示突变体能够进行亚基因组的转录,说明nsp6对病毒的转录是非必需的。欧洲型PRRSV nsp6和pAPRRS的nsp6之间存在3个氨基酸差异,用前者替换的嵌合克隆能够被拯救出相应的嵌合病毒,说明上述三个氨基酸序列不具有型间特异性;将LDV nsp6替换pAPRRS nsp6,改变了nsp6中的6个氨基酸,RT-PCR和IFA能够检测到嵌合克隆基因组RNA和亚基因组RNA的复制和转录,但是不能产生CPE。EAV nsp6(22aa)替换pAPRRS nsp6后改变整个APRRS nsp6的结构,嵌合克隆未被检测到复制和转录。综上所述,本研究表明,nsp6对动脉炎病毒的感染性是必需的,其作用机制尚待研究;另一方面,我们发现其中三个氨基酸变异不影响了病毒复制过程,而更大幅度的突变在更大程度上影响了病毒的复制过程,RNA合成翻译甚或病毒颗粒成熟过程。4、强弱毒株PRRSV嵌合感染性克隆的构建及鉴定PRRSV是近两年来流行于我国大部分省份的“猪高热综合征”的主要病原体。该病毒在增殖过程中极易发生遗传及抗原变异。查明PRRSV致病性大幅增高的机制,进而研制用于防治易变的流行PRRSV变异株的高效疫苗无疑是兽医工作者的当务之急。在弱毒株APRRS的全长感染性克隆pAPRRS及其含有多克隆位点(PacI,SwaI,AscI)的突变感染性克隆pCSA以及我室构建的高致病(high pathogencity, HP) PRRSV感染性克隆pJX143的基础上,构建了一系列包括PRRSV ORF1a、ORF1b以及ORF2-7等的强弱毒PRRSV嵌合感染性克隆。将构建的嵌合克隆转染Marc-145,4天后观察到典型的CPE。通过RT-PCR和免疫荧光证明获得了一系列强弱毒株之间的嵌合病毒。这些嵌合病毒的构建成功和相应反向遗传操作平台的建立及应用为研发预防HP PRRSV的高效嵌合疫苗来奠定了基础。更重要的是,该类嵌合感染性cDNA克隆也为解析目前流行的HP PRRSV毒力因子和高致病力机制奠定了物质基础。更多还原

【Abstract】 Equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevating virus (LDV) and simian haemorrhagic fever virus (SHFV) were united in the family of enveloped positive-stranded RNA viruses, the Arteriviridae. The host range of the currently known arteriviruses is restricted to horses and donkeys (EAV), pigs (PRRSV), mice (LDV), and several genera of African and Asian monkeys (SHFV).The outcome of arterivirus infection can range from an asymptomatic, persistent carrier state to abortion or lethal haemorrhagic fever. There are still lots of mysteries about arterivirus genome replication, transcription and protein translation. The development of infectious cDNA clones made it possible to construct chimeric arteriviruses to investigate the functional complementation of a protein in two inter- or intra- species viruse strains. Chimeric viruses can also provide a powerful tool for molecular dissection of the pathogenesis of the increaseing virulence of arteriviruses.1: Construction of full-length cDNA clone of equine arteritis virus and analysis of genome sequenceTo develop a reverse genetics system of EAV, six pairs of oligonucleotides were designed based on the full-length genomic sequence of EAV Bucyrus strain. By RT-PCR, six overlapping cDNA fragments, designated as PE124,PE631,PE1854, PE5191, PE61107 and PE97Q respectively, were amplified, followed by being cloned into pCR BluntⅡ-TOPO vector. A NotI enzyme site and a T7 promoter sequence were introduced immediately upstream of 5′-end, with a XhoI cutting site in downstream of poly(A) tail in 3′-end. Full-length cDNA clone pWEAV was obtained by connecting the six cDNA fragments utilizing single restriction endonuclease site into the pBluescriptⅡKS(+) vector. The construction of a full-length genomic cDNA clone of EAV is a crucial step to obtain the infectious clone,which may facilitate further dissecting of structure and function relationship of EAV genome.2: Investation of the regulator of arterivirus replicationIn arterivirus, the regulating mechanisms of the virus life circle, such as genomic RNA replication, mRNA transcription, translation and virus particle packaging were mysterious. Furthermore, the deeper investigations need to be done to determine the roles virus coding proteins taking in these regulating mechanisms.The in vitro transcript of full length cDNA clone pWEAV was transfected in BHK-21 cell, and then IFA and RT-PCR were used to detect the virus protein expression and RNA replication. No RNA synthesis and protein expression were observed during the passages, so lethal mutations existed in the cDNA clone pWEAV. In order to identify the lethal mutations, based on the backbone of infectious clone pEAV030, we constructed a series of chimeric pEAV030/pWEAV cDNA clones. The in vitro transcript of the chimeric cDNA clones were transfected in the BHK-21.The results showed that two nucleotides variants in 5‵UTR and 10 amino acids variants in nsp1-nsp12 and 11 amino acids varants in GP2-GP4 also did not effect t the infectity of chimeric clone. On the other hand, we found that 6 animo acids variants in nsp2-nsp3 of chimeric clone are lethal. There animo acids effect genomic RNA replication and subgenomic RNA transcription. We also found one single amino acids mutation (A→S ) in GP5 and H→Y mutation in N protein, respectively, did not effect the genomic RNA replication and subgenomic RNA transcription. But both amino acids were responsible for the lethal of the pWEAV. It indicated that the both two amino acids may have a function in virus particle packaging and budding. Roles of these lethal mutations involving in the EAV life cycle will be studied in the future.3: The function of nsp6 in the arteriviruses replicationArterivirus non-structural protein (nsp1-nsp13) can assemble a Replicase/Transcriptiase Complex (RTC) for genomic RNA replication and subgenomic RNA transcription. The function and mechanism of each nsp assosiated for RTC is unclear. Nsp6, only 13-22 amino acids,.is the smallest protein in the arterivirus. From now on, the structure and function of nsp6 is unknown. To study the role of the nsp6 in replication of arterivirus, in this study, we construct a series of clones with nsp6 deletion and substitution with other arterivirus nsp6 by SOE-PCR. RT–PCR and IFA were used to detect the genomic RNA replication and subgenomic RNA transcription of the nsp6 mutants. The results showed that the mutants with nsp6 completely (16aa) or partially (3aa, 6aa) deletions can not be rescued, but all mutants were detected subgenomic RNA synthesis. The pAPRRS mutant whose nsp6 was substituted with LV, 3 amino acids changed, was viable. When pAPRRS nsp6 was substituted with LDV nsp6, ,the chimeric clone presented replication, transcription but not CPE, although 6 amino acids changed. Then pAPRRS nsp6 was replaced with the EAV nsp6, resulting in the lethal chimeric clone which can not tolerate the 22aa substitution. In conclusion, nsp6 is essential for virus infectivity, but the mechanism of nsp6 action will be studied in next step.We also found that 3 amino acid mutations were nonessential for virus replication and virus infectivity; however more mutations of nsp6 effected the process of virus replication in a way. 4:The construction of chimeric clone of high pathogencity PRRSV and attenuated PRRSV strain and identification of chimeric viruses rescuedPRRSV, the causative agent of the ongoing“porcine high fever syndrome”in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. In this study, based on an infectious cDNA clone of an attenuated TypeⅡPRRSV strain pCSA, which was further manipulated by inserting polylinker (PacI,SwaI,AscI)between ORF1 and ORF2, and the high pathogencity PRRSV cDNA clone pJX143, we replaced the coding sequence of pCSA structural protein(s) and/or non-structual protein with those of the HP PRRSV to develop a series of chimeric clones. Upon transfection of chimeric clones cDNAs into Marc-145 cells, typical PRRSV cytopathic effects were observed. This study provided a valuable tool to develop the chimeric PRRSV as vaccine candidate offering cross-protection to HP PRRSV strains. Furthermore the infectious chimeric cDNA clone provides a powerful tool to molecular dissection of the mechanism of pathogenesis of the increasing- virulence of the on-going prevalent PRRSV in China.更多还原