嵌合动脉炎病毒全长cDNA克隆的构建及应用
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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。
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 sequence
To 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 replication
In 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 replication
Arterivirus 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 rescued
PRRSV, 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.
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。
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 sequence
To 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 replication
In 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 replication
Arterivirus 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 rescued
PRRSV, 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.
引文
1. 高志强,郭鑫,杨汉春,等.猪繁殖与呼吸综合征病毒缺失变异株的基因组特征[J].畜牧兽医学报,2005,36(6):578—584.
2. 郭宝清,陈章水,刘文兴,等. 从疑似PRRS 流产胎儿分离猪生殖和呼吸综合征病毒(PRRSV)的研究[J].中国畜禽传染病1996,87(2):1-5
3. 郭宝清.从疑似PRRS流产胎儿分离猪生殖和呼吸综合症病毒(PRRSV)的研究[J].中国畜禽传染病,1996, 2: 1～3.
4. 梁成珠,陈国忠,孟广校,等马病毒性动脉验血清中和试验的研究及应用[J].中国兽医科技,1996,26 (9):13-141.
5. 吕健,张建武,孙志,刘维全,袁世山]. 高致病性猪蓝耳病病毒感染性克隆的构建及应用[J]. 微生物与感染,万遂如.猪无名高热综合征[J]. 养殖和饲料,2006, 10: 14-17.
6. 袁世山,韦祖樟. PRRSV全长感染性cDNA克隆的构建:非结构蛋白和结构蛋白编码区之间的分离[J].中国科学C辑.2008.38(1):1-8.
7. 张建武,庄金山,袁世山. 中国部分地区“猪高热综合征”的病原学调查及高致病性猪繁殖与呼吸综合征病毒的分子流行病学研究[J]. 中国农业科学,2008 录用。.
8. 朱来华,梁成珠,李保家,等.马动脉炎病毒血清抑制试验研究及其应用[J].中国兽医科技,2001,31(2):32-341.
9. Alexander E. Gorbalenya, and Eric J. Snijder. Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle [J]. J Virol, 2006,1653–1661.
10. Alexander E. Gorbalenya, Luis Enjuanes, John Ziebuhr, et al. Nidovirales: Evolving the largest RNA virus genome[J]. Virus Research, 2006, 117(1): 17–37.
11. Allende, R., T. L. Lewis, Z. Lu,et al. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions[J]. J Gen Virol, 1999. 80.307–315.
12. An TQ, Zhou Y, Tong G. Genetic diversity and phylogenetic analysis of glycoprotein 5 of PRRSV isolates in mainland China from 1996 to 2006: Coexistence of two NA-subgenotypes with great diversity, Vet. Microbiol.. 2007.02.025
13. Anja Seybert, Clara C. Posthuma, Leonie C. van Dinten, Eric J. Snijder, et al. A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases [J]. Journal of Virology, 2005, 79(2), 696–704.
14. Bautista, E. M., K.S. Faaberg, D. Mickelson and E. D. McGruder. Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus [J]. Virology, 2002,298, 258–270.
15. Beeren N and Snijder. RNA signals in the 3' terminus of the genome of Equine arteritis virus are required for viral RNA synthesis [J].J Gen Virol, 2006,87(7), 1977-1983.
16. Beerens N, Selisko B, Ricagno S, Imbert I, De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase. J Virol. 2007 Aug; 81 (16):8384-95.
17. Bryans JT, Crown ME, Doll ER. Isolation of a filterable agent causing arteritis of of horses and abortion by mares: its differentiation from the equine abortion (influenza) virus [J]. Cornell Vet, 1957, 47(1):3-41.
18. Buck, K. W. Comparison of the replication of positive-stranded RNA viruses of plants and animals[J]. av. Virus Res. 47:159–251.
19. Byungjoon Kwon, Israrul H. Ansari, Fernando A. Osorio. Infectious clone-derived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model [J].Vaccine ,2006,24 7071–7080.
20. Changhee Lee , Dongwan Yoo. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties [J]. Virology, 2006,355, 30–43.
21. Changhee Lee, Douglas Hodgins , Jay G. Calvert, et al. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication .Virology, 2006, 346(1): 238-250.
22. Clara C. Posthuma, Danny D. Nedialkova, Jessika C. Zevenhoven-Dobbe, et al. Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle [J]. Journal of Virology, 2006, 80(4): 1653–1661.
23. Collins J, Benfield D A, Nelson Eet al. Isolation of swine infertibility and respratory syndrome (SIRS) virus (isolate VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J Vet Diagn Invest, 1992, 4:117-127.
24. Danny van Aken , Willemien E. Benckhuijsen et al. Expression, purification, and in vitro activity of an arterivirus main proteinase [J]. Virus Res, 2006, 120(1-2): 97–106.
25. Danny van Aken, Eric J. Snijder, Alexander E. Gorbalenya. Mutagenesis Analysis of the nsp4 Main Proteinase Reveals Determinants of Arterivirus Replicase Polyprotein Autoprocessing [J]. J Virol, 2006, 80(7):3428–3437.
26. Danny van Aken, Willemien E. Benckhuijsen, Jan W. et al .Expression, purification, and in vitro activity of an arterivirus main proteinase [J]. Virus Research, 2006, 120(1-2): 97–106.
27. De Lima M, Pattnaik AK, Flores EF et al. Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus. Virology, 2006, 353(2): 410-21.
28. De VAAF, Glaser A L , et al. Genetic manipulation of equine arteritis virus using full length cDNA clones: separation of overlapping genes and expression of a foreign epitope [J]. Virology, 2000, 270: 84～97.
29. De Vries, A. A. F., M. J. B. Raamsman, H. A. van Dijk, M. C. et al. The small envelope glycoprotein (GS) of equine arteritis virus folds into three distinct monomers and a disulfide-linked dimmer [J].Journal of Virology, 1995, 69(6): 3441–3448.
30. DeVAAF, Glaser A L , et al. Recombinant equine arteritis virus as an exp ression vector [J]. Virology, 2001, 284: 259～276.
31. Dobbe J C, V an D M , Y, Spaan, et al. Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus trop ismin cell culture [J]. Virology, 2001, 288: 283～294.
32. Dongwan Yoo, Siao-Kun W. Welch, Changhee Lee, et al.Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors[J].Veterinary Immunology and Immunopathology, 2004, 102(3): 143–154.
33. E.Gorbalenya,and John Ziebuhr. A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases [J]. Journal of Virology, 2005, 79(2): 696–704.
34. Elida M. Bautista, Kay S. Faaberg, Dan Mickelson et a . Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome .Virus Virology, 2002, 298(2): 258–270.
35. Ellis, J.A., Krakowa, S., Allan, G., Clark, E., Kennedy, S. The clinical scope of porcine reproductive and respiratory syndrome virus infection has expanded since 1987: an alternative perspective[J]. Vet Pathol. 1999,36:262-265.
36. Eric J Snijder, Janneke J M Meulenberg. The molecular biology of arteriviruses [J]. J Gen Virol, 1998,79 (pt5): 961-9791.
37. Eric J. Snijder, Hans van Tol, Norbert Roos and Ketil W. Pedersen Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex [J]. Journal of General Virology,2001,82,985–994.
38. Eric J. Snijder, Hans van Tol, Norbert Roos et al. Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex [J]. Journal of General Virology, 2001, 82(pt5): 985–994.
39. Eric J. Snijder, Jessika C. Dobbe, Willy J. M. Spaan. Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity [J]. Journal of Virology, Jan. 2003, 77(1):97–104.
40. Fang Y, Rowland RR, Roof M et al. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region. J Virol. 2006. 80(23):11447-55.
41. Fang, Y., Kimb, D., Ropp, S.,.Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States[J].Virus Research,2004,100: 229–235
42. Groot Bramel-Verheije, M.H., Rottier, P.J.M., Meulenberg, J.J.M.,. Expression of a foreign epitope by porcine reproductive and respiratory syndrome virus [J]. Virology, 2000,278, 380–389.
43. Guo B Q, Chen Z S, Liu W X et al. Isolation of PRRSV from aborted fetus with typical clinical PRRS manifestations. Chinese J Preventive Vet Med, 1996. 2:1-5.
44. Han J, Liu G, Wang Y, Faaberg KS. Identification of Nonessential Regions of the nsp2 Replicase Protein of Porcine Reproductive and Respiratory Syndrome Virus Strain VR-2332 for Replication in Cell Culture. J Virol, 2007, 81(18): 9878-90.
45. Israrul H. Ansari, Byungjoon Kwon, Fernando A. Osorio. Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability to Induce Neutralizing Antibodies.Journal of Virology, 2006, 80(8): 3994–4004.
46. J. Zhang1;_, F. Miszczak2;_, S. Pronost2, C.Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus[J],Arch Virol, 2007.
47. Johan A. Den Boon, Kats. Faaberg et al. Processing and Evolution of the N-Terminal Region of the Arterivirus Replicase ORF1a Protein: Identification of Two Papainlike Cysteine Proteases. Journal of Virology, 1995, 69(7): 4500–4505.
48. John Ziebuhr, Eric J. Snijder , Alexander E. Gorbalenya .Virus-encoded proteinases and proteolytic processing inthe Nidovirales[J].Journal of General Virology ,2000,81(pt4): 853–879.
49. Keffaber K. K. Reproductive failure of unknown etiology [J]. Am.Assoc. SwinePrac News,1989, 1: 1-9.
50. Kegong Tian, Xiuling Yu, Tiezhu Zhao et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark [J]. LoS one. 2007, 13(2):e526.
51. Lee C, Calvert JG, Welch SK, Yoo D. A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity [J]. Virology. 2005. 331(1):47-62.
52. Lee, C., Calvert, J.G., Welch, S.K.W., Yoo, D. A DNA launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity [J]. Virology, 2004.,74 (11): 5213–5223.
53. Leoneie C. Van Dinden, Hans Van tol, et al. The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis [J]. Journal of Virology, June 2000, 74 (11): 5213–5223.
54. M. B. Oleksiewicz, A. B?tner, J. Nielsen .Determination of 50-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs [J] .Arch Virol, 1999, 144: 981–987.
55. M. Ostrowski, J. A. Galeota, A. M. Jar, K. B. Platt et al.Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain[J]. Journal of Virology, 2002, 76(13): 4241–4250.
56. M.A., Schild, G.C., et al., Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine [J]. J.Virol, 989, 63, 1338–1344.
57. M.H. Verheije, M.V. Kroese, I.F.A. van der Linden, Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs [J]. Vaccine, 2003, 21, 2556–2563.
58. MA, Nedialkova DD, Zevenhoven-Dobbe JC, Gorbalenya AE, et al. Arterivirus Subgenomic mRNA Synthesis and Virion Biogenesis Depend on the Multifunctional nsp1 Autoprotease[J]. J Virol, 2007, 81(19):10496-505.
59. MacLachlan NJ, Balasuriya UB. Equine viral arteritis [J].Adv Exp Med Biol, 2006, 581:429-33.
60. Maines TR, Young M, Dinh MN. Two cellular proteins that interact with a stem loop in the simian hemorrhagic fever virus 3’(+)NCR RNA[J]. Virus Res, 2005, 109(2):109-24.
61. Mardassi, H., P. Gonin, C.A.Gagnon, B.Massie. A subset of porcine reproductive and respiratory syndrome virus GP3 glycoprotein is released into the culture medium of cells as a non-virion-associated and membrance-free (soluble) form. Journal of Virology, 1998, 72(8): 6298-6306.
62. Marieke A. Tijms and Eric J. Snijder. Equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mRNA synthesis, interacts with the cellular transcription co-factor p100[J]. J Gen Virol. 2003, 84(9):2317-22.
63. Mateu E, Martin M, Vidal D. Genetic diversity and phylogenetic analysis of glycoprotein 5 of European-type porcine reproductive and respiratory virus strains in Spain. J Gen Virol. 2003. 84(Pt 3):529-34.
64. Meng XJ. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol. 2000. 74(4):309-29.
65. Mengeling WL, Lager KM, Vorwald AC. The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance [J]. Anim Reprod Sci. 2000. 60-61:199-210.
66. Mengeling, W.L., Lager, K.M., Vorwald, A.C. Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of“atypical” PRRS [J]. Am. J. Vet. Res, 1998, 59: 1540-1544.
67. Meulenberg JJ, Bos-de Ruijter JN, van de Graaf R et al. Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. J Virol. 1998. 72(1):380-7.
68. Meulenberg, J. J. M., A. P. van Nieuwstadt, A, van Essen-Zandbergen, et al. Localization and fine mapping of antigenic sites on the nucleocapsid protein of porcine reproductive and respiratory syndrome virus with monoclonal antibodies. Virology, 1998, 252(1): 106-114.
69. Meulenberg, J.J.M., Bos-de Ruijter, J.N.A., van de Graaf, R., Wensvoort, G., Moormann, R.J.M.,. Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus[J]. J. Virol. 1998, 72, 380–387.
70. Nancy Beerens and Eric J. Snijder. An RNA Pseudoknot in the 3’ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis [J]. J Virol, 2007, 81(17), 9426–9436.
71. Nancy Beerens and Eric J. Snijder. RNA signals in the 39 terminus of the genome of Equine arteritis virus are required for viral RNA synthesis [J]. Journal of General Virology ,2006, 87, 1977–1983
72. Nelsen CJ, Murtaugh MP, Faaberg KS. Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents [J]. J Virol. 1999. 73(1):270-80.
73. Nielsen HS, Liu G, Nielsen J. et al. Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus [J]. J Virol. 2003, 77(6):3702-11.
74. Pastermal A O,Van den Born E, Spaan W J M et al. The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor In arterivirus subgenomic mRNA synthesis[J]. J Virol, 2003,7(2):1175-83.
75. Pasternak A O, Alexander P, et al. Genetic manipulation of arterivirus alternative mRNA leader-body junction sites reveals tight regulation of structural protein expression [J]. J Virol, 2000, 74:11642-11653.
76. Pasternak A O, Spaan W J M, Snijder E J. Regulation of relative abundance of arterivirus subgenomic mRNAs[J]. J Virol, 2004, 78:8102–8113.
77. Pasternak A O, van den Born E, Spaan W J M, et al. Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis [J]. EMBO J, 2001, 20: 7220-7228.
78. Peter L. Delputte and Hans J. Nauwynck. Porcine Arterivirus Infection of Alveolar Macrophages Is Mediated by Sialic Acid on the Virus [J]. Journal of Virology, 2004, 78(15): 8094–8101.
79. Ran ZG, Chen XY, Yang HC et al. Recovery of an infectious virus from the full-length cDNA of PRRSV BJ-4. Wei Sheng Wu Xue Bao. 2007 Jun 4; 47(3):423-9.
80. Raymond R.R. Rowland, Dongwan Yoo. Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences.Virus Research, 2003, 95(1-2): 23-33.
81. Roeland Wieringa, Antoine A. F. de Vries, Jannes van der Meulen, et al. Structural Protein Requirements in EquineArteritis Virus Assembly[J]. Journal of Virology, Dec. 2004, 78(23): 13019–13027.
82. Rossow KD, Shivers JL, Yeske PE, et al. Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence. Vet Rec. 1999. 144 (16):444-8.
83. Rowland R. R.,R. Kervin,C. Kuckcleburg, et al. The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence [J]. Virus Research, 1999, 64:1-12.
84. Sarah K. Wootton, Raymond R. R. Rowland, Dongwan Yoo. Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein. Journal of Virology, 2002, 76(20): 10569–10576.
85. Shen S, Kwang J, Liu W et al. Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and dentification of the Nsp2 gene with a unique insertion [J]. Arch. Virol. 2000.145: 871–883.
86. Shen S,Kwang J,LiuW ,et a1.Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion.Arch Virol,2000,145(5) :871—883.
87. Shishan Yuan, Daniel Mickelson, Michael P .Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains [J]. Virus Research, 7(4 ), 2001;99–110
88. Snijder, E. J., Brinton, M. A., Faaberg, K. S.et al. Family Arteriviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomyof Viruses. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press. 2004
89. Snijder, E. J., Wassenaar, A. L., van Dinten, L.C.,.The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases[J].Journal of Biological Chemistry,1996,271, 4864±4871.
90. Snijder, E.J, Wassenaar,A.L Spaan,W.J. Proteolytic processing of the replicase ORF1a protein of equine arteritis virus[J]. J Virol, 1994, 8(9): 5755±5764.
91. Snijder, E.J, Wassenaar, A.L, et al.The arterivirus Nsp2 protease An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsinlike proteases [J]. Journal of Biological Chemistry, 1995, 270(28):
92. Snijder, E.J Wassenaar, A.L.& Spaan,W.J..Proteolytic processing of the replicase ORF1a protein of equine arteritis virus [J]. Journal of Virology, 1994, 68, 755±5764.
93. T.V. Grebennikova,a,* D.F. Clouser,b A.C. Vorwald,b M.I .Genomic characterization of virulent, attenuated, and revertant passages of a North American porcine reproductive and respiratory syndrome virus strain Virology[J]. 2004, 321;383– 390.
94. Tian K, Yu Y, Zhao T et al. Emergence of Fatal PRRSV Variants: Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the Unique Hallmark [J]. 2007. PLoS ONE 2007. 6:e526
95. Tong G-Z, Zhou Y-J, Hao X-F, Tian Z-J, An T-Q, Qiu H-J. Highly pathogenic porcine reproductive and respiratory syndrome, China [letter]. Emerg Infect Dis. 2007 Sep; Emerg Infect Dis Letter, 2007, 9(13): 1434—1436
96. Truong, H.M., Lu, Z., Kutish, G.F., Galeota, J., Osorio, F.A., Pattnaik, A.K.,. A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus [J]. Virology 2004,325, 308–319.
97. Udeni B.R. Balasuriya, N. James MacLachlan. The immune response to equine arteritis virus:potential lessons for other arteriviruses. Veterinary Immunology and Immunopathology, 2004, 102(3): 107–129.
98. Udeni B.R. Balasuriya,, Jessika C, Hans W et al, Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus[J]. Journal of General Virology, 2002, 83(4): 795–800.
99. Udeni B.R. Balasuriya,, Jessika C, Hans W et al. Expression of the Two Major Envelope Proteins of Equine Arteritis Virus as a Heterodimer Is Necessary for Induction of Neutralizing Antibodies in Mice Immunized with Recombinant Venezuelan Equine Encephalitis Virus Replicon Particles [J]. Journal of Virology, Nov.2000, 10623–10630.
100. Udeni B.R. Balasuriya,, Jessika C. Dobbe, Hans W. Heidner et al. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone [J].Virology , 2004, 321(2): 235– 246.
101. Udeni B.R. Balasuriya,, Jessika C. Dobbe, Hans W. Heidner, Victoria L. Smalley,Andrea Navarrette, Eric J. Snijder, and N. James MacLachlan Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone[J]. Virology, 2004,321, 235– 246.
102. V erheijeM H, W elting T J M , Jansen H T, et al. Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting [J]. Virology, 2002,303: 364～373.
103. Van Dinten, L. C., Rensen, S., Gorbalenya, A. E. & Snijder, E. J.. Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication [J].Journal of Virology, 1999, 73, 2027±20.
104. Van Dinten, L.C, Wassenaar, A. L, Gorbalenya, A. E., Spaan, et al. Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains [J].Journal of Virology. 1996, 70(10):6625-6633.
105. Van Dinten, L.C., den Boon, J.A., Wassenaar, A.L.M., Spaan, W.J.M., Snijder, E.J. An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription[J]. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 991–996.
106. VerheijeM H, KroeseM V , V an D L , et al. Safety and protective efficacy of porcine reproductive and respiratory syndrome virus vaccines in young pigs[J]. Vaccine, 2003, 21: 2556～2563.
107. Wai-Hong Wu, Ying Fang, Raymond R.R. Rowland. Nelson The 2b protein as a minor structural component of PRRSV [J]. Virus Research, 2005, 114(22): 177–181.
108. Wang Y, Liang Y, Han J.Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence [J]. Virology, 2008,371(2):418-29
109. Wassenaar, A.L., W.J. Spaan, A.E. Gorbalenya. Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease[J]. J.Virol. 1997,71: 9313-9322.
110. Welch S KW, Jo lie R, PearceD S, et al. Construction and evaluation of replication incompetent porcine reproductive and respiratory syndrome virus vaccines[J]. Vet Immunol Immunopathol, 2004, 102: 279～292.
111. Wensvoort, G., Terpstra, C., Pol, J. M., Mystery swine disease in the Netherlands: the isolation of Lelystad virus[J].Veterinary Quarterly, 1991, 13: 121-130.
112. Wieringa R, De V A A F, et al. Intra- and inter-molecular disulfide bonds of the GP2b glycop rotein of equine arteritis virus: relevance for virus assembly and infectivity [J]. Journal of Virology, 2003, 77:12996～13004.
113. Wieringa, R., A. A. F. de Vries, P. J. M. Rottier. Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus [J]. Journal of Virology, 2003, 77(11): 6216–6226.
114. Wieringa, R.,A.A. F. de Vries, M. J. B. Raamsman, et al. Characterization of two new structural glycoproteins, GP3 and GP4, of equine arteritis virus[J]. Journal of Virology, 2002, 76(21): 10829–10840.
115. Wootton, S, K., D. Yoo. Homo-oligomerization of the procine reproductive and respiratory syndrome virus nucleid protein and the rold of disulfide linkages [J]. Journal of Virology, 2003, 77, 4546-4557.
116. Wootton, S.,G. Koljesar, L.Yoon,D. Yoo. Antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein [J]. Clin.Diagn. Lab. Immunol. 2001, 8(3): 598-603.
117. Yan Y,Guo X,Chen Y,Cha Z et al. Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcine reproductive and respiratory syndrome virus [J]. Virus res.2007, 126(1-2): 207-15.
118. Yoo D, Welch SK, Lee C, Calvert JG. Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors [J]. Vet Immunol Immunopathol. 2004. 102(3):143-54.
119. Yufeng Li, Xinglong Wang, Ping Jiang. Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China[J].The Veterinary Journal, 2007, 174(3):577-84
120. Yu-Jeong Choi, Sang-Im Yun, Shien-Young Kang, and Young-Min Lee. Identification of 5’ and 3’ cis-Acting Elements of the Porcine Reproductive and Respiratory Syndrome Virus: Acquisition of Novel 5’ AU-Rich Sequences Restored Replication of a 5’ Proximal 7-Nucleotide Deletion Mutan[J]. Journal of Virology, 2006, 723–736.
121. Ziebuhr, J., E. J. Snijder, and A. E. Gorbalenya. Virus-encoded proteinases and proteolytic rocessing in the Nidovirales. J. Gen. Virol. 2000,81 Pt 4: 853–879.
122. Zimmerman, J.J., Yoon, K.J., Wills, R.W., Swenson, S.L. General over view of PRRSV: a perspective rom the United States. Vet. Microbiol. 1997,55:187-96.
2. 郭宝清,陈章水,刘文兴,等. 从疑似PRRS 流产胎儿分离猪生殖和呼吸综合征病毒(PRRSV)的研究[J].中国畜禽传染病1996,87(2):1-5
3. 郭宝清.从疑似PRRS流产胎儿分离猪生殖和呼吸综合症病毒(PRRSV)的研究[J].中国畜禽传染病,1996, 2: 1～3.
4. 梁成珠,陈国忠,孟广校,等马病毒性动脉验血清中和试验的研究及应用[J].中国兽医科技,1996,26 (9):13-141.
5. 吕健,张建武,孙志,刘维全,袁世山]. 高致病性猪蓝耳病病毒感染性克隆的构建及应用[J]. 微生物与感染,万遂如.猪无名高热综合征[J]. 养殖和饲料,2006, 10: 14-17.
6. 袁世山,韦祖樟. PRRSV全长感染性cDNA克隆的构建:非结构蛋白和结构蛋白编码区之间的分离[J].中国科学C辑.2008.38(1):1-8.
7. 张建武,庄金山,袁世山. 中国部分地区“猪高热综合征”的病原学调查及高致病性猪繁殖与呼吸综合征病毒的分子流行病学研究[J]. 中国农业科学,2008 录用。.
8. 朱来华,梁成珠,李保家,等.马动脉炎病毒血清抑制试验研究及其应用[J].中国兽医科技,2001,31(2):32-341.
9. Alexander E. Gorbalenya, and Eric J. Snijder. Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle [J]. J Virol, 2006,1653–1661.
10. Alexander E. Gorbalenya, Luis Enjuanes, John Ziebuhr, et al. Nidovirales: Evolving the largest RNA virus genome[J]. Virus Research, 2006, 117(1): 17–37.
11. Allende, R., T. L. Lewis, Z. Lu,et al. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions[J]. J Gen Virol, 1999. 80.307–315.
12. An TQ, Zhou Y, Tong G. Genetic diversity and phylogenetic analysis of glycoprotein 5 of PRRSV isolates in mainland China from 1996 to 2006: Coexistence of two NA-subgenotypes with great diversity, Vet. Microbiol.. 2007.02.025
13. Anja Seybert, Clara C. Posthuma, Leonie C. van Dinten, Eric J. Snijder, et al. A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases [J]. Journal of Virology, 2005, 79(2), 696–704.
14. Bautista, E. M., K.S. Faaberg, D. Mickelson and E. D. McGruder. Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus [J]. Virology, 2002,298, 258–270.
15. Beeren N and Snijder. RNA signals in the 3' terminus of the genome of Equine arteritis virus are required for viral RNA synthesis [J].J Gen Virol, 2006,87(7), 1977-1983.
16. Beerens N, Selisko B, Ricagno S, Imbert I, De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase. J Virol. 2007 Aug; 81 (16):8384-95.
17. Bryans JT, Crown ME, Doll ER. Isolation of a filterable agent causing arteritis of of horses and abortion by mares: its differentiation from the equine abortion (influenza) virus [J]. Cornell Vet, 1957, 47(1):3-41.
18. Buck, K. W. Comparison of the replication of positive-stranded RNA viruses of plants and animals[J]. av. Virus Res. 47:159–251.
19. Byungjoon Kwon, Israrul H. Ansari, Fernando A. Osorio. Infectious clone-derived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model [J].Vaccine ,2006,24 7071–7080.
20. Changhee Lee , Dongwan Yoo. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties [J]. Virology, 2006,355, 30–43.
21. Changhee Lee, Douglas Hodgins , Jay G. Calvert, et al. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication .Virology, 2006, 346(1): 238-250.
22. Clara C. Posthuma, Danny D. Nedialkova, Jessika C. Zevenhoven-Dobbe, et al. Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle [J]. Journal of Virology, 2006, 80(4): 1653–1661.
23. Collins J, Benfield D A, Nelson Eet al. Isolation of swine infertibility and respratory syndrome (SIRS) virus (isolate VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J Vet Diagn Invest, 1992, 4:117-127.
24. Danny van Aken , Willemien E. Benckhuijsen et al. Expression, purification, and in vitro activity of an arterivirus main proteinase [J]. Virus Res, 2006, 120(1-2): 97–106.
25. Danny van Aken, Eric J. Snijder, Alexander E. Gorbalenya. Mutagenesis Analysis of the nsp4 Main Proteinase Reveals Determinants of Arterivirus Replicase Polyprotein Autoprocessing [J]. J Virol, 2006, 80(7):3428–3437.
26. Danny van Aken, Willemien E. Benckhuijsen, Jan W. et al .Expression, purification, and in vitro activity of an arterivirus main proteinase [J]. Virus Research, 2006, 120(1-2): 97–106.
27. De Lima M, Pattnaik AK, Flores EF et al. Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus. Virology, 2006, 353(2): 410-21.
28. De VAAF, Glaser A L , et al. Genetic manipulation of equine arteritis virus using full length cDNA clones: separation of overlapping genes and expression of a foreign epitope [J]. Virology, 2000, 270: 84～97.
29. De Vries, A. A. F., M. J. B. Raamsman, H. A. van Dijk, M. C. et al. The small envelope glycoprotein (GS) of equine arteritis virus folds into three distinct monomers and a disulfide-linked dimmer [J].Journal of Virology, 1995, 69(6): 3441–3448.
30. DeVAAF, Glaser A L , et al. Recombinant equine arteritis virus as an exp ression vector [J]. Virology, 2001, 284: 259～276.
31. Dobbe J C, V an D M , Y, Spaan, et al. Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus trop ismin cell culture [J]. Virology, 2001, 288: 283～294.
32. Dongwan Yoo, Siao-Kun W. Welch, Changhee Lee, et al.Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors[J].Veterinary Immunology and Immunopathology, 2004, 102(3): 143–154.
33. E.Gorbalenya,and John Ziebuhr. A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases [J]. Journal of Virology, 2005, 79(2): 696–704.
34. Elida M. Bautista, Kay S. Faaberg, Dan Mickelson et a . Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome .Virus Virology, 2002, 298(2): 258–270.
35. Ellis, J.A., Krakowa, S., Allan, G., Clark, E., Kennedy, S. The clinical scope of porcine reproductive and respiratory syndrome virus infection has expanded since 1987: an alternative perspective[J]. Vet Pathol. 1999,36:262-265.
36. Eric J Snijder, Janneke J M Meulenberg. The molecular biology of arteriviruses [J]. J Gen Virol, 1998,79 (pt5): 961-9791.
37. Eric J. Snijder, Hans van Tol, Norbert Roos and Ketil W. Pedersen Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex [J]. Journal of General Virology,2001,82,985–994.
38. Eric J. Snijder, Hans van Tol, Norbert Roos et al. Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex [J]. Journal of General Virology, 2001, 82(pt5): 985–994.
39. Eric J. Snijder, Jessika C. Dobbe, Willy J. M. Spaan. Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity [J]. Journal of Virology, Jan. 2003, 77(1):97–104.
40. Fang Y, Rowland RR, Roof M et al. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region. J Virol. 2006. 80(23):11447-55.
41. Fang, Y., Kimb, D., Ropp, S.,.Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States[J].Virus Research,2004,100: 229–235
42. Groot Bramel-Verheije, M.H., Rottier, P.J.M., Meulenberg, J.J.M.,. Expression of a foreign epitope by porcine reproductive and respiratory syndrome virus [J]. Virology, 2000,278, 380–389.
43. Guo B Q, Chen Z S, Liu W X et al. Isolation of PRRSV from aborted fetus with typical clinical PRRS manifestations. Chinese J Preventive Vet Med, 1996. 2:1-5.
44. Han J, Liu G, Wang Y, Faaberg KS. Identification of Nonessential Regions of the nsp2 Replicase Protein of Porcine Reproductive and Respiratory Syndrome Virus Strain VR-2332 for Replication in Cell Culture. J Virol, 2007, 81(18): 9878-90.
45. Israrul H. Ansari, Byungjoon Kwon, Fernando A. Osorio. Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability to Induce Neutralizing Antibodies.Journal of Virology, 2006, 80(8): 3994–4004.
46. J. Zhang1;_, F. Miszczak2;_, S. Pronost2, C.Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus[J],Arch Virol, 2007.
47. Johan A. Den Boon, Kats. Faaberg et al. Processing and Evolution of the N-Terminal Region of the Arterivirus Replicase ORF1a Protein: Identification of Two Papainlike Cysteine Proteases. Journal of Virology, 1995, 69(7): 4500–4505.
48. John Ziebuhr, Eric J. Snijder , Alexander E. Gorbalenya .Virus-encoded proteinases and proteolytic processing inthe Nidovirales[J].Journal of General Virology ,2000,81(pt4): 853–879.
49. Keffaber K. K. Reproductive failure of unknown etiology [J]. Am.Assoc. SwinePrac News,1989, 1: 1-9.
50. Kegong Tian, Xiuling Yu, Tiezhu Zhao et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark [J]. LoS one. 2007, 13(2):e526.
51. Lee C, Calvert JG, Welch SK, Yoo D. A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity [J]. Virology. 2005. 331(1):47-62.
52. Lee, C., Calvert, J.G., Welch, S.K.W., Yoo, D. A DNA launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity [J]. Virology, 2004.,74 (11): 5213–5223.
53. Leoneie C. Van Dinden, Hans Van tol, et al. The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis [J]. Journal of Virology, June 2000, 74 (11): 5213–5223.
54. M. B. Oleksiewicz, A. B?tner, J. Nielsen .Determination of 50-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs [J] .Arch Virol, 1999, 144: 981–987.
55. M. Ostrowski, J. A. Galeota, A. M. Jar, K. B. Platt et al.Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain[J]. Journal of Virology, 2002, 76(13): 4241–4250.
56. M.A., Schild, G.C., et al., Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine [J]. J.Virol, 989, 63, 1338–1344.
57. M.H. Verheije, M.V. Kroese, I.F.A. van der Linden, Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs [J]. Vaccine, 2003, 21, 2556–2563.
58. MA, Nedialkova DD, Zevenhoven-Dobbe JC, Gorbalenya AE, et al. Arterivirus Subgenomic mRNA Synthesis and Virion Biogenesis Depend on the Multifunctional nsp1 Autoprotease[J]. J Virol, 2007, 81(19):10496-505.
59. MacLachlan NJ, Balasuriya UB. Equine viral arteritis [J].Adv Exp Med Biol, 2006, 581:429-33.
60. Maines TR, Young M, Dinh MN. Two cellular proteins that interact with a stem loop in the simian hemorrhagic fever virus 3’(+)NCR RNA[J]. Virus Res, 2005, 109(2):109-24.
61. Mardassi, H., P. Gonin, C.A.Gagnon, B.Massie. A subset of porcine reproductive and respiratory syndrome virus GP3 glycoprotein is released into the culture medium of cells as a non-virion-associated and membrance-free (soluble) form. Journal of Virology, 1998, 72(8): 6298-6306.
62. Marieke A. Tijms and Eric J. Snijder. Equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mRNA synthesis, interacts with the cellular transcription co-factor p100[J]. J Gen Virol. 2003, 84(9):2317-22.
63. Mateu E, Martin M, Vidal D. Genetic diversity and phylogenetic analysis of glycoprotein 5 of European-type porcine reproductive and respiratory virus strains in Spain. J Gen Virol. 2003. 84(Pt 3):529-34.
64. Meng XJ. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol. 2000. 74(4):309-29.
65. Mengeling WL, Lager KM, Vorwald AC. The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance [J]. Anim Reprod Sci. 2000. 60-61:199-210.
66. Mengeling, W.L., Lager, K.M., Vorwald, A.C. Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of“atypical” PRRS [J]. Am. J. Vet. Res, 1998, 59: 1540-1544.
67. Meulenberg JJ, Bos-de Ruijter JN, van de Graaf R et al. Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. J Virol. 1998. 72(1):380-7.
68. Meulenberg, J. J. M., A. P. van Nieuwstadt, A, van Essen-Zandbergen, et al. Localization and fine mapping of antigenic sites on the nucleocapsid protein of porcine reproductive and respiratory syndrome virus with monoclonal antibodies. Virology, 1998, 252(1): 106-114.
69. Meulenberg, J.J.M., Bos-de Ruijter, J.N.A., van de Graaf, R., Wensvoort, G., Moormann, R.J.M.,. Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus[J]. J. Virol. 1998, 72, 380–387.
70. Nancy Beerens and Eric J. Snijder. An RNA Pseudoknot in the 3’ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis [J]. J Virol, 2007, 81(17), 9426–9436.
71. Nancy Beerens and Eric J. Snijder. RNA signals in the 39 terminus of the genome of Equine arteritis virus are required for viral RNA synthesis [J]. Journal of General Virology ,2006, 87, 1977–1983
72. Nelsen CJ, Murtaugh MP, Faaberg KS. Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents [J]. J Virol. 1999. 73(1):270-80.
73. Nielsen HS, Liu G, Nielsen J. et al. Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus [J]. J Virol. 2003, 77(6):3702-11.
74. Pastermal A O,Van den Born E, Spaan W J M et al. The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor In arterivirus subgenomic mRNA synthesis[J]. J Virol, 2003,7(2):1175-83.
75. Pasternak A O, Alexander P, et al. Genetic manipulation of arterivirus alternative mRNA leader-body junction sites reveals tight regulation of structural protein expression [J]. J Virol, 2000, 74:11642-11653.
76. Pasternak A O, Spaan W J M, Snijder E J. Regulation of relative abundance of arterivirus subgenomic mRNAs[J]. J Virol, 2004, 78:8102–8113.
77. Pasternak A O, van den Born E, Spaan W J M, et al. Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis [J]. EMBO J, 2001, 20: 7220-7228.
78. Peter L. Delputte and Hans J. Nauwynck. Porcine Arterivirus Infection of Alveolar Macrophages Is Mediated by Sialic Acid on the Virus [J]. Journal of Virology, 2004, 78(15): 8094–8101.
79. Ran ZG, Chen XY, Yang HC et al. Recovery of an infectious virus from the full-length cDNA of PRRSV BJ-4. Wei Sheng Wu Xue Bao. 2007 Jun 4; 47(3):423-9.
80. Raymond R.R. Rowland, Dongwan Yoo. Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences.Virus Research, 2003, 95(1-2): 23-33.
81. Roeland Wieringa, Antoine A. F. de Vries, Jannes van der Meulen, et al. Structural Protein Requirements in EquineArteritis Virus Assembly[J]. Journal of Virology, Dec. 2004, 78(23): 13019–13027.
82. Rossow KD, Shivers JL, Yeske PE, et al. Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence. Vet Rec. 1999. 144 (16):444-8.
83. Rowland R. R.,R. Kervin,C. Kuckcleburg, et al. The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence [J]. Virus Research, 1999, 64:1-12.
84. Sarah K. Wootton, Raymond R. R. Rowland, Dongwan Yoo. Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein. Journal of Virology, 2002, 76(20): 10569–10576.
85. Shen S, Kwang J, Liu W et al. Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and dentification of the Nsp2 gene with a unique insertion [J]. Arch. Virol. 2000.145: 871–883.
86. Shen S,Kwang J,LiuW ,et a1.Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion.Arch Virol,2000,145(5) :871—883.
87. Shishan Yuan, Daniel Mickelson, Michael P .Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains [J]. Virus Research, 7(4 ), 2001;99–110
88. Snijder, E. J., Brinton, M. A., Faaberg, K. S.et al. Family Arteriviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomyof Viruses. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press. 2004
89. Snijder, E. J., Wassenaar, A. L., van Dinten, L.C.,.The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases[J].Journal of Biological Chemistry,1996,271, 4864±4871.
90. Snijder, E.J, Wassenaar,A.L Spaan,W.J. Proteolytic processing of the replicase ORF1a protein of equine arteritis virus[J]. J Virol, 1994, 8(9): 5755±5764.
91. Snijder, E.J, Wassenaar, A.L, et al.The arterivirus Nsp2 protease An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsinlike proteases [J]. Journal of Biological Chemistry, 1995, 270(28):
92. Snijder, E.J Wassenaar, A.L.& Spaan,W.J..Proteolytic processing of the replicase ORF1a protein of equine arteritis virus [J]. Journal of Virology, 1994, 68, 755±5764.
93. T.V. Grebennikova,a,* D.F. Clouser,b A.C. Vorwald,b M.I .Genomic characterization of virulent, attenuated, and revertant passages of a North American porcine reproductive and respiratory syndrome virus strain Virology[J]. 2004, 321;383– 390.
94. Tian K, Yu Y, Zhao T et al. Emergence of Fatal PRRSV Variants: Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the Unique Hallmark [J]. 2007. PLoS ONE 2007. 6:e526
95. Tong G-Z, Zhou Y-J, Hao X-F, Tian Z-J, An T-Q, Qiu H-J. Highly pathogenic porcine reproductive and respiratory syndrome, China [letter]. Emerg Infect Dis. 2007 Sep; Emerg Infect Dis Letter, 2007, 9(13): 1434—1436
96. Truong, H.M., Lu, Z., Kutish, G.F., Galeota, J., Osorio, F.A., Pattnaik, A.K.,. A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus [J]. Virology 2004,325, 308–319.
97. Udeni B.R. Balasuriya, N. James MacLachlan. The immune response to equine arteritis virus:potential lessons for other arteriviruses. Veterinary Immunology and Immunopathology, 2004, 102(3): 107–129.
98. Udeni B.R. Balasuriya,, Jessika C, Hans W et al, Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus[J]. Journal of General Virology, 2002, 83(4): 795–800.
99. Udeni B.R. Balasuriya,, Jessika C, Hans W et al. Expression of the Two Major Envelope Proteins of Equine Arteritis Virus as a Heterodimer Is Necessary for Induction of Neutralizing Antibodies in Mice Immunized with Recombinant Venezuelan Equine Encephalitis Virus Replicon Particles [J]. Journal of Virology, Nov.2000, 10623–10630.
100. Udeni B.R. Balasuriya,, Jessika C. Dobbe, Hans W. Heidner et al. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone [J].Virology , 2004, 321(2): 235– 246.
101. Udeni B.R. Balasuriya,, Jessika C. Dobbe, Hans W. Heidner, Victoria L. Smalley,Andrea Navarrette, Eric J. Snijder, and N. James MacLachlan Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone[J]. Virology, 2004,321, 235– 246.
102. V erheijeM H, W elting T J M , Jansen H T, et al. Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting [J]. Virology, 2002,303: 364～373.
103. Van Dinten, L. C., Rensen, S., Gorbalenya, A. E. & Snijder, E. J.. Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication [J].Journal of Virology, 1999, 73, 2027±20.
104. Van Dinten, L.C, Wassenaar, A. L, Gorbalenya, A. E., Spaan, et al. Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains [J].Journal of Virology. 1996, 70(10):6625-6633.
105. Van Dinten, L.C., den Boon, J.A., Wassenaar, A.L.M., Spaan, W.J.M., Snijder, E.J. An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription[J]. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 991–996.
106. VerheijeM H, KroeseM V , V an D L , et al. Safety and protective efficacy of porcine reproductive and respiratory syndrome virus vaccines in young pigs[J]. Vaccine, 2003, 21: 2556～2563.
107. Wai-Hong Wu, Ying Fang, Raymond R.R. Rowland. Nelson The 2b protein as a minor structural component of PRRSV [J]. Virus Research, 2005, 114(22): 177–181.
108. Wang Y, Liang Y, Han J.Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence [J]. Virology, 2008,371(2):418-29
109. Wassenaar, A.L., W.J. Spaan, A.E. Gorbalenya. Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease[J]. J.Virol. 1997,71: 9313-9322.
110. Welch S KW, Jo lie R, PearceD S, et al. Construction and evaluation of replication incompetent porcine reproductive and respiratory syndrome virus vaccines[J]. Vet Immunol Immunopathol, 2004, 102: 279～292.
111. Wensvoort, G., Terpstra, C., Pol, J. M., Mystery swine disease in the Netherlands: the isolation of Lelystad virus[J].Veterinary Quarterly, 1991, 13: 121-130.
112. Wieringa R, De V A A F, et al. Intra- and inter-molecular disulfide bonds of the GP2b glycop rotein of equine arteritis virus: relevance for virus assembly and infectivity [J]. Journal of Virology, 2003, 77:12996～13004.
113. Wieringa, R., A. A. F. de Vries, P. J. M. Rottier. Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus [J]. Journal of Virology, 2003, 77(11): 6216–6226.
114. Wieringa, R.,A.A. F. de Vries, M. J. B. Raamsman, et al. Characterization of two new structural glycoproteins, GP3 and GP4, of equine arteritis virus[J]. Journal of Virology, 2002, 76(21): 10829–10840.
115. Wootton, S, K., D. Yoo. Homo-oligomerization of the procine reproductive and respiratory syndrome virus nucleid protein and the rold of disulfide linkages [J]. Journal of Virology, 2003, 77, 4546-4557.
116. Wootton, S.,G. Koljesar, L.Yoon,D. Yoo. Antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein [J]. Clin.Diagn. Lab. Immunol. 2001, 8(3): 598-603.
117. Yan Y,Guo X,Chen Y,Cha Z et al. Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcine reproductive and respiratory syndrome virus [J]. Virus res.2007, 126(1-2): 207-15.
118. Yoo D, Welch SK, Lee C, Calvert JG. Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors [J]. Vet Immunol Immunopathol. 2004. 102(3):143-54.
119. Yufeng Li, Xinglong Wang, Ping Jiang. Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China[J].The Veterinary Journal, 2007, 174(3):577-84
120. Yu-Jeong Choi, Sang-Im Yun, Shien-Young Kang, and Young-Min Lee. Identification of 5’ and 3’ cis-Acting Elements of the Porcine Reproductive and Respiratory Syndrome Virus: Acquisition of Novel 5’ AU-Rich Sequences Restored Replication of a 5’ Proximal 7-Nucleotide Deletion Mutan[J]. Journal of Virology, 2006, 723–736.
121. Ziebuhr, J., E. J. Snijder, and A. E. Gorbalenya. Virus-encoded proteinases and proteolytic rocessing in the Nidovirales. J. Gen. Virol. 2000,81 Pt 4: 853–879.
122. Zimmerman, J.J., Yoon, K.J., Wills, R.W., Swenson, S.L. General over view of PRRSV: a perspective rom the United States. Vet. Microbiol. 1997,55:187-96.