嵌合动脉炎病毒构建及其病毒学特性
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摘要
1996年成立的动脉炎病毒科(Arteriviridae)其成员包括:马动脉炎病毒(equine arteritis virus,EAV),猪繁殖与呼吸障碍综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)乳酸脱氢酶升高病毒(lactate dehydrogenase-elevating virus,LDV),猴出血热病毒(simian hemorrhagic fever virus,SHFV)。动脉炎病毒感染动物后能引起从无临床症状的持续性感染到致死的急性感染不等的病征。其中PRRSV感染猪引起所谓的“猪蓝耳病”给世界养猪业带来巨大经济损失。目前涉及到动脉炎病毒复制过程的分子机制,尤其是囊膜蛋白结构与功能的关系还不清楚。在本实验室已经拥有的PRRSV和EAV全长感染性克隆的基础上,本研究利用反向遗传操作方法对动脉炎病毒的囊膜蛋白功能、基因转录调控等进行了解析,促进了对动脉炎病毒的进一步了解,为防治PRRSV及其它动脉炎病毒奠定了一定的理论基础。
1.不同基因型PRRSV嵌合体的构建及其病毒学特性
PRRSV分为两个基因型,基因1型与基因2型,两型之间基因相似性仅为60%左右。对本实验室拥有的1型PRRSV株vSHE与2型PRRSV株vAPRRSV进行比对发现其囊膜蛋白GP2a、E、GP3、GP4、GP5之间的相似性在53-76%之间。以2型PRRSV的全长感染性克隆pAPRRS为骨架,先将Asc I酶切位点插入ORF1b与ORF2a之间构建全长克隆pAPRRSasc。再以pAPRRSasc为骨架,将其ORF2-3、ORF2-4、ORF2-5、ORF5分别替换为vSHE的相应区域,构建了全长嵌合克隆pAPRRS-SHE23、pAPRRS-SHE234、pAPRRS-SHE2345、pAPRRS-SHE5。用如上构建的嵌合克隆和pAPRRSasc转染MARC-145细胞,除pAPRRS-SHE23以外均拯救出了相应的感染性子病毒,分别为vAPRRSasc、vAPRRS-SHE234、vAPRRS-SHE2345、vAPRRS-SHE5。间接免疫荧光(IFA)和全长序列测定结果证实了子病毒的嵌合基因组结构。将拯救病毒在体外连续传代至第8代后测序表明嵌合病毒有很好的基因稳定性。用能特异性识别1型PRRSV囊膜蛋白的抗体进行IFA分析表明1型PRRSV基因能在2型骨架PRRSV中有效表达。对拯救出的嵌合病毒进行病毒学特性研究发现:除vAPRRS-SHE5外其它嵌合病毒能在MARC-145上形成与骨架亲本病毒vAPRRS相似的空斑形态;所有嵌合病毒在MARC-145上的复制能力与亲本病毒相似。这表明1型PRRSV的相应囊膜蛋白能在2型PRRSV中发挥相同的功能。采用RT-PCR方法和特异性引物对,从病毒感染细胞的总RNA中扩增出各个亚基因组mRNA(subgenomic mRNA,sg mRNA)。序列测定发现:虽然两型PRRSV位于结构蛋白基因中的转录调控序列(body transcription regulation sequence, TRS-B)不一致,但是1型TRS-B能与2型的位于5’UTR中的转录调控序列(leader transcription regulation sequence, TRS-L)互相作用形成相应的杂合sg mRNA;由于1型TRS-B与2型TRS-L碱基之间的不精确配对导致基因组中一些TRS-B类似序列发挥作用,产生了一些比经典sg mRNA小的亚基因组RNA(sg RNA)。本研究首次获得了不同基因型PRRSV嵌合的感染性子病毒,阐明了1型PRRSV的囊膜蛋白能在2型PRRSV中发挥功能,从理论上表明两型PRRSV之间可能发生重组。这为进一步研究PRRSV囊膜蛋白结构和功能的关系、开发基因标记疫苗奠定了理论基础。
2.不同种动脉炎病毒嵌合体的构建及其病毒学特性
目前对动脉炎病毒细胞受体和病毒结合蛋白的认识还没有统一,大部分人认为病毒结合蛋白是多量囊膜糖蛋白GP5,而有人提出可能是少量囊膜蛋白。这些结论都是基于体外的生物化学实验得到,缺乏遗传学和病毒学上的证据。已知在体外PRRSV只能感染MARC-145细胞,而EAV能感染MARC-145、BHK-21、Vero等多种细胞。在本项研究中,以上一项研究构建的全长感染性克隆pAPRRSasc为骨架,分别将其ORF2?4、ORF5替换为EAV的相应区域构建全长嵌合克隆pAPRRS-EAV234、pAPRRS-EAV5。用构建的嵌合克隆转染MARC-145,发现克隆pAPRRS-EAV234能拯救出感染性的子病毒vAPRRS-EAV234。通过IFA和全长序列测序分析证实了vAPRRS-EAV234的嵌合基因组结构。对第8代病毒测序表明嵌合病毒在体外具有很好的基因稳定性。用特异性识别EAV GP2b的抗体对vAPRRS-EAV234感染细胞进行IFA分析表明嵌入基因能在PRRSV骨架中有效表达。病毒学特性研究发现:嵌合病毒vAPRRS-EAV234获得了供体亲本病毒EAV的细胞感染谱,除了能感染除MARC-145外还能感染BHK-21和Vero细胞,但不能感染骨架亲本病毒PRRSV的体内靶细胞—猪肺泡巨噬细胞(PAM);vAPRRS-EAV234能在MARC-145和BHK-21上实现良好的增殖,其在BHK-21上的最高病毒滴度为在MARC-145上的10倍。对vAPRRS-EAV234的sg mRNA转录谱和sg mRNA序列进行分析发现:虽然EAV和PRRSV的TRS序列差距很大,但是EAV的TRS-B可以与PRRSV的TRS-L结合;由于EAV的TRS-B与PRRSV的TRS-L之间的不精确配对导致sg mRNA2?4的转录量明显下降;产生了一系列具有潜在编码价值的新sg RNA,这些sg RNA的意义还未知。本研究不但在世界上首次成功获得了不同种动脉炎病毒之间完整ORF替换的具有感染性的嵌合病毒,更重要的是首次从遗传学和病毒学的角度阐明少量囊膜蛋白在动脉炎病毒侵入细胞过程中发挥决定性作用。该研究同时提供了一种生产高滴度PRRSV的方法,极有利于疫苗生产。
3.插入嵌合体动脉炎病毒的构建及其病毒学特性
上一项的研究表明EAV的ORF2-4可以在PRRSV中稳定存在并发挥功能,且已知另外一个动脉炎病毒SHFV基因组中拥有2套ORF2-4。该项研究在以上研究基础上以全长感染性克隆pAPRRSasc为骨架,将EAV的ORF2-4插入ORF1b和ORF2a之间,构建了嵌合克隆pAPRRS(EAV234)。用该克隆转染细胞拯救出了具有感染性的插入嵌合病毒vAPRRS(EAV234)。IFA和核酸序列测定结果均证实了子病毒的基因组结构特点且插入基因能有效表达。研究发现vAPRRS(EAV234)在EAV ORF2-4稳定存在的同时PRRSV自身的ORF2-4却发生大范围的缺失。同时还发现插入嵌合病毒vAPRRS(EAV234)能感染MARC-145和BHK-21细胞。本研究表明:PRRSV乃至其它动脉炎病毒可以容纳2套少量囊膜蛋白基因,但是稳定性不强;作为一种潜在外源基因载体,PRRSV可以在ORF1b和ORF2a之间插入较大的外源基因。这为进一步精确解析动脉炎病毒结合蛋白,研究囊膜蛋白之间相互作用、病毒包装,开发PRRSV载体等提供了基础。
The Arteriviridae which was established in 1996 has four members: equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Arterivirus can cause either acute or persistent infections in respective host animals. PRRSV infection (so called porcine reproductive and respiratory syndrome, PRRS), in particular, often leads to high-mortality disease outbreaks and mass economic loss to the swine industry worldwide. Until now, the detail molecular mechanism of how the arterivirus replicate in cell, especially the relationship of structure-function of envelope proteins, is poorly understood. In this study, based on infectious full-length cDNA clones of PRRSV and EAV, the envelope protein functions and properties of transcription regulation are dissected. This will lay foundation to better understand of arterivirus and to develop efficient ways to prevent arterivirus infection.
1. The development and characterization of intra-genotypic chimeric PRRSV
PRRSV is classified into two genotypes, type 1 and type 2, between which share only approximate 60% genetic identity. Bioinformatics analysis showed that the envelope proteins GP2a, E, GP3, GP4, GP5 between vSHE (type 1 strain) and vAPRRS (type 2 strain) share 53?76% identities. Taking the pAPRRS, the full-length cDNA clone of type 2 PRRSV, as backbone, an Asc I restriction enzyme recognition site was introduced immediately upstream of the ORF2 start codon to construct clone pAPRRSasc. Then based on pAPRRSasc, the chimeric full-length clone pAPRRS-SHE23, pAPRRS-SHE234, pAPRRS-SHE2345, pAPRRS-SHE5 were constructed by ORF2?3, ORF2?4, ORF2?5, ORF5 respective substitution with correspond ORFs of vSHE. The viable progeny viruses vAPRRSasc, vAPRRS-SHE234, vAPRRS-SHE2345, vAPRRS-SHE5 were rescued from MARC-145 cells which were transfected with corresponding clones. IFA and complete genome sequencing analysis confirmed the chimeric genomic structures of these rescued viruses. The sequences analysis of passage 8 viruses showed that the chimeric viruses displayed robust genetic stability in vitro. Utilizing antibodies specific for type 1 envelope proteins, further IFA results indicated that these heterologous genes were expressed in type 2 PRRSV backbone. The analysis of virological characteristics showed that all the chimeric viruses displayed similar plaque morphology, except for vAPRRS-SHE5, and similar replication ability in MARC-145 with parental viruses. These results suggested that the envelope proteins of type 1 PRRSV can play full function and work in concert in type 2 virus. Utilizing RT-PCR with specific primers, the subgenomic mRNAs (sg mRNAs) were amplified from intracellular RNA pool and subjected to sequencing. The sequences analysis showed that the TRS-Bs from type 1 can join with TRS-L of type 2 PRRSV to synthesis hybrid sg mRNAs. In addition, some small sg RNAs were generated as the imprecise base paring between the type 2 TRS-L and type 1 TRS-Bs. In this study, the viable intra-genotypic chimeric PRRSVs were fistly successfully rescued in the world, and this study found that the type 1 envelope proteins can play full function in backbone of type 2. This indicated that the recombination between the two genotypic PRRSV is possible, and more importantly, this paved the way to further elucidate the structure-function relationship of PRRSV envelope proteins, and may enable the development of novel gene marker vaccines.
2. The development and characterization of intra-species chimeric arterivirus
The identities of arterivirus cellular receptor and viral attachment proteins are in poor understand. The most well accepted viral attachment protein is GP5, while it is being challenged by the minor envelope proteins. However, both of the two hypotheses are only supported by biochemical evidences, lacking of genetic and virological evidences. In vitro, PRRSV can only infect MARC-145 cell line while EAV display a more broaden cell tropism such as MARC-145, BHK-21, Vero and so on. In this study, taking the previously constructed pAPRRSasc as backbone, the chimeric full-length cDNA clones pAPRRS-EAV234 and pAPRRS-EAV5 were constructed through ORF2?4 and ORF5 substitutions respective with counterparties of EAV. The viable progeny virus vAPRRS-EAV234 was rescued from pAPRRS-EAV234 transfected MARC-145 cells. Trough IFA and complete genome sequencing analysis confirmed that the viable progeny virus has a chimeric genomic structure, and the chimeric virus is genetic stable in vitro. The results of IFA using antibody specific to EAV GP2b indicated that the genes from EAV were translated in PRRSV backbone. The more important was that the chimeric virus vAPRRS-EAV234 obtained the cellular tropism of donor parental virus EAV, it can infect MARC-145, BHK-21 and Vero cell lines, while lost the infectivity to the PRRSV target primary porcine alveolar macrophages (PAM). The vAPRRS-EAV234 can replicate well in MARC-145 and BHK-21, and its’peak titer in BHK-21 was 10 times higher than that in MARC-145. The analysis of transcriptional profiles of APRRS-EAV234 showed that the EAV TRS-Bs join with PRRSV TRS-L, although the base pairs did not match well between them and lead to the down-regulation of sg mRNA2?4. In addition, some novel sg RNAs were generated in chimeric virus which may be translated into unknown proteins. This study first rescued viable chimeric virus in which the intact minor envelope proteins ORFs were substituted by counterparties of EAV, and more importantly, this study provided first genetic and virological evidences to prove that the minor envelope proteins play major role in arterivirus entry into cells. This study also developed a new method to produce high titer PRRSV, and it will be of great significance to vaccine productivity.
3. The development and characterization of chimeric PRRSV containing ORFs from EAV
The above study has shown that the ORF2?4 of EAV can stably exist in PRRSV genome and play function in virus replication. And it is known that another arterivirus member, SHFV, has two panel of minor envelope protein ORFs in genome. In this study, taking the pAPRRSasc as backbone, the ORF2?4 of EAV were inserted between ORF1b and ORF2a of PRRSV backbone to generate chimeric clone pAPRRS(EAV234). Viable progeny virus vAPRRS(EAV234) was rescued from MARC-145 cells transfected with pAPRRS(EAV234). IFA and viral complete genome sequencing analysis showed that the progeny virus shared a chimeric genomic structure and the inserted EAV ORFs were translated in chimera infected cells. The inserted heterologious ORFs stably exist in chimeic virus vAPRRS(EAV234), however, the partial sequences of ORF2?4 of PRRSV were found to be deleted. The vAPRRS(EAV234) can infect both MARC-145 and BHK-21cells. This study indicated that PRRSV, and even other arterivirus, can contain two panel of minor envelope protein ORFs in genome, although its’genetic stability was frustrated. As one potential vector, PRRSV can deliver heterologious genes which were inserted between ORF1b and ORF2a. This paved the way to further identify the arterivirus attachment proteins, dissect the protein-protein interaction and virion assembly mechanism, and develop PRRSV vector.
1.不同基因型PRRSV嵌合体的构建及其病毒学特性
PRRSV分为两个基因型,基因1型与基因2型,两型之间基因相似性仅为60%左右。对本实验室拥有的1型PRRSV株vSHE与2型PRRSV株vAPRRSV进行比对发现其囊膜蛋白GP2a、E、GP3、GP4、GP5之间的相似性在53-76%之间。以2型PRRSV的全长感染性克隆pAPRRS为骨架,先将Asc I酶切位点插入ORF1b与ORF2a之间构建全长克隆pAPRRSasc。再以pAPRRSasc为骨架,将其ORF2-3、ORF2-4、ORF2-5、ORF5分别替换为vSHE的相应区域,构建了全长嵌合克隆pAPRRS-SHE23、pAPRRS-SHE234、pAPRRS-SHE2345、pAPRRS-SHE5。用如上构建的嵌合克隆和pAPRRSasc转染MARC-145细胞,除pAPRRS-SHE23以外均拯救出了相应的感染性子病毒,分别为vAPRRSasc、vAPRRS-SHE234、vAPRRS-SHE2345、vAPRRS-SHE5。间接免疫荧光(IFA)和全长序列测定结果证实了子病毒的嵌合基因组结构。将拯救病毒在体外连续传代至第8代后测序表明嵌合病毒有很好的基因稳定性。用能特异性识别1型PRRSV囊膜蛋白的抗体进行IFA分析表明1型PRRSV基因能在2型骨架PRRSV中有效表达。对拯救出的嵌合病毒进行病毒学特性研究发现:除vAPRRS-SHE5外其它嵌合病毒能在MARC-145上形成与骨架亲本病毒vAPRRS相似的空斑形态;所有嵌合病毒在MARC-145上的复制能力与亲本病毒相似。这表明1型PRRSV的相应囊膜蛋白能在2型PRRSV中发挥相同的功能。采用RT-PCR方法和特异性引物对,从病毒感染细胞的总RNA中扩增出各个亚基因组mRNA(subgenomic mRNA,sg mRNA)。序列测定发现:虽然两型PRRSV位于结构蛋白基因中的转录调控序列(body transcription regulation sequence, TRS-B)不一致,但是1型TRS-B能与2型的位于5’UTR中的转录调控序列(leader transcription regulation sequence, TRS-L)互相作用形成相应的杂合sg mRNA;由于1型TRS-B与2型TRS-L碱基之间的不精确配对导致基因组中一些TRS-B类似序列发挥作用,产生了一些比经典sg mRNA小的亚基因组RNA(sg RNA)。本研究首次获得了不同基因型PRRSV嵌合的感染性子病毒,阐明了1型PRRSV的囊膜蛋白能在2型PRRSV中发挥功能,从理论上表明两型PRRSV之间可能发生重组。这为进一步研究PRRSV囊膜蛋白结构和功能的关系、开发基因标记疫苗奠定了理论基础。
2.不同种动脉炎病毒嵌合体的构建及其病毒学特性
目前对动脉炎病毒细胞受体和病毒结合蛋白的认识还没有统一,大部分人认为病毒结合蛋白是多量囊膜糖蛋白GP5,而有人提出可能是少量囊膜蛋白。这些结论都是基于体外的生物化学实验得到,缺乏遗传学和病毒学上的证据。已知在体外PRRSV只能感染MARC-145细胞,而EAV能感染MARC-145、BHK-21、Vero等多种细胞。在本项研究中,以上一项研究构建的全长感染性克隆pAPRRSasc为骨架,分别将其ORF2?4、ORF5替换为EAV的相应区域构建全长嵌合克隆pAPRRS-EAV234、pAPRRS-EAV5。用构建的嵌合克隆转染MARC-145,发现克隆pAPRRS-EAV234能拯救出感染性的子病毒vAPRRS-EAV234。通过IFA和全长序列测序分析证实了vAPRRS-EAV234的嵌合基因组结构。对第8代病毒测序表明嵌合病毒在体外具有很好的基因稳定性。用特异性识别EAV GP2b的抗体对vAPRRS-EAV234感染细胞进行IFA分析表明嵌入基因能在PRRSV骨架中有效表达。病毒学特性研究发现:嵌合病毒vAPRRS-EAV234获得了供体亲本病毒EAV的细胞感染谱,除了能感染除MARC-145外还能感染BHK-21和Vero细胞,但不能感染骨架亲本病毒PRRSV的体内靶细胞—猪肺泡巨噬细胞(PAM);vAPRRS-EAV234能在MARC-145和BHK-21上实现良好的增殖,其在BHK-21上的最高病毒滴度为在MARC-145上的10倍。对vAPRRS-EAV234的sg mRNA转录谱和sg mRNA序列进行分析发现:虽然EAV和PRRSV的TRS序列差距很大,但是EAV的TRS-B可以与PRRSV的TRS-L结合;由于EAV的TRS-B与PRRSV的TRS-L之间的不精确配对导致sg mRNA2?4的转录量明显下降;产生了一系列具有潜在编码价值的新sg RNA,这些sg RNA的意义还未知。本研究不但在世界上首次成功获得了不同种动脉炎病毒之间完整ORF替换的具有感染性的嵌合病毒,更重要的是首次从遗传学和病毒学的角度阐明少量囊膜蛋白在动脉炎病毒侵入细胞过程中发挥决定性作用。该研究同时提供了一种生产高滴度PRRSV的方法,极有利于疫苗生产。
3.插入嵌合体动脉炎病毒的构建及其病毒学特性
上一项的研究表明EAV的ORF2-4可以在PRRSV中稳定存在并发挥功能,且已知另外一个动脉炎病毒SHFV基因组中拥有2套ORF2-4。该项研究在以上研究基础上以全长感染性克隆pAPRRSasc为骨架,将EAV的ORF2-4插入ORF1b和ORF2a之间,构建了嵌合克隆pAPRRS(EAV234)。用该克隆转染细胞拯救出了具有感染性的插入嵌合病毒vAPRRS(EAV234)。IFA和核酸序列测定结果均证实了子病毒的基因组结构特点且插入基因能有效表达。研究发现vAPRRS(EAV234)在EAV ORF2-4稳定存在的同时PRRSV自身的ORF2-4却发生大范围的缺失。同时还发现插入嵌合病毒vAPRRS(EAV234)能感染MARC-145和BHK-21细胞。本研究表明:PRRSV乃至其它动脉炎病毒可以容纳2套少量囊膜蛋白基因,但是稳定性不强;作为一种潜在外源基因载体,PRRSV可以在ORF1b和ORF2a之间插入较大的外源基因。这为进一步精确解析动脉炎病毒结合蛋白,研究囊膜蛋白之间相互作用、病毒包装,开发PRRSV载体等提供了基础。
The Arteriviridae which was established in 1996 has four members: equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Arterivirus can cause either acute or persistent infections in respective host animals. PRRSV infection (so called porcine reproductive and respiratory syndrome, PRRS), in particular, often leads to high-mortality disease outbreaks and mass economic loss to the swine industry worldwide. Until now, the detail molecular mechanism of how the arterivirus replicate in cell, especially the relationship of structure-function of envelope proteins, is poorly understood. In this study, based on infectious full-length cDNA clones of PRRSV and EAV, the envelope protein functions and properties of transcription regulation are dissected. This will lay foundation to better understand of arterivirus and to develop efficient ways to prevent arterivirus infection.
1. The development and characterization of intra-genotypic chimeric PRRSV
PRRSV is classified into two genotypes, type 1 and type 2, between which share only approximate 60% genetic identity. Bioinformatics analysis showed that the envelope proteins GP2a, E, GP3, GP4, GP5 between vSHE (type 1 strain) and vAPRRS (type 2 strain) share 53?76% identities. Taking the pAPRRS, the full-length cDNA clone of type 2 PRRSV, as backbone, an Asc I restriction enzyme recognition site was introduced immediately upstream of the ORF2 start codon to construct clone pAPRRSasc. Then based on pAPRRSasc, the chimeric full-length clone pAPRRS-SHE23, pAPRRS-SHE234, pAPRRS-SHE2345, pAPRRS-SHE5 were constructed by ORF2?3, ORF2?4, ORF2?5, ORF5 respective substitution with correspond ORFs of vSHE. The viable progeny viruses vAPRRSasc, vAPRRS-SHE234, vAPRRS-SHE2345, vAPRRS-SHE5 were rescued from MARC-145 cells which were transfected with corresponding clones. IFA and complete genome sequencing analysis confirmed the chimeric genomic structures of these rescued viruses. The sequences analysis of passage 8 viruses showed that the chimeric viruses displayed robust genetic stability in vitro. Utilizing antibodies specific for type 1 envelope proteins, further IFA results indicated that these heterologous genes were expressed in type 2 PRRSV backbone. The analysis of virological characteristics showed that all the chimeric viruses displayed similar plaque morphology, except for vAPRRS-SHE5, and similar replication ability in MARC-145 with parental viruses. These results suggested that the envelope proteins of type 1 PRRSV can play full function and work in concert in type 2 virus. Utilizing RT-PCR with specific primers, the subgenomic mRNAs (sg mRNAs) were amplified from intracellular RNA pool and subjected to sequencing. The sequences analysis showed that the TRS-Bs from type 1 can join with TRS-L of type 2 PRRSV to synthesis hybrid sg mRNAs. In addition, some small sg RNAs were generated as the imprecise base paring between the type 2 TRS-L and type 1 TRS-Bs. In this study, the viable intra-genotypic chimeric PRRSVs were fistly successfully rescued in the world, and this study found that the type 1 envelope proteins can play full function in backbone of type 2. This indicated that the recombination between the two genotypic PRRSV is possible, and more importantly, this paved the way to further elucidate the structure-function relationship of PRRSV envelope proteins, and may enable the development of novel gene marker vaccines.
2. The development and characterization of intra-species chimeric arterivirus
The identities of arterivirus cellular receptor and viral attachment proteins are in poor understand. The most well accepted viral attachment protein is GP5, while it is being challenged by the minor envelope proteins. However, both of the two hypotheses are only supported by biochemical evidences, lacking of genetic and virological evidences. In vitro, PRRSV can only infect MARC-145 cell line while EAV display a more broaden cell tropism such as MARC-145, BHK-21, Vero and so on. In this study, taking the previously constructed pAPRRSasc as backbone, the chimeric full-length cDNA clones pAPRRS-EAV234 and pAPRRS-EAV5 were constructed through ORF2?4 and ORF5 substitutions respective with counterparties of EAV. The viable progeny virus vAPRRS-EAV234 was rescued from pAPRRS-EAV234 transfected MARC-145 cells. Trough IFA and complete genome sequencing analysis confirmed that the viable progeny virus has a chimeric genomic structure, and the chimeric virus is genetic stable in vitro. The results of IFA using antibody specific to EAV GP2b indicated that the genes from EAV were translated in PRRSV backbone. The more important was that the chimeric virus vAPRRS-EAV234 obtained the cellular tropism of donor parental virus EAV, it can infect MARC-145, BHK-21 and Vero cell lines, while lost the infectivity to the PRRSV target primary porcine alveolar macrophages (PAM). The vAPRRS-EAV234 can replicate well in MARC-145 and BHK-21, and its’peak titer in BHK-21 was 10 times higher than that in MARC-145. The analysis of transcriptional profiles of APRRS-EAV234 showed that the EAV TRS-Bs join with PRRSV TRS-L, although the base pairs did not match well between them and lead to the down-regulation of sg mRNA2?4. In addition, some novel sg RNAs were generated in chimeric virus which may be translated into unknown proteins. This study first rescued viable chimeric virus in which the intact minor envelope proteins ORFs were substituted by counterparties of EAV, and more importantly, this study provided first genetic and virological evidences to prove that the minor envelope proteins play major role in arterivirus entry into cells. This study also developed a new method to produce high titer PRRSV, and it will be of great significance to vaccine productivity.
3. The development and characterization of chimeric PRRSV containing ORFs from EAV
The above study has shown that the ORF2?4 of EAV can stably exist in PRRSV genome and play function in virus replication. And it is known that another arterivirus member, SHFV, has two panel of minor envelope protein ORFs in genome. In this study, taking the pAPRRSasc as backbone, the ORF2?4 of EAV were inserted between ORF1b and ORF2a of PRRSV backbone to generate chimeric clone pAPRRS(EAV234). Viable progeny virus vAPRRS(EAV234) was rescued from MARC-145 cells transfected with pAPRRS(EAV234). IFA and viral complete genome sequencing analysis showed that the progeny virus shared a chimeric genomic structure and the inserted EAV ORFs were translated in chimera infected cells. The inserted heterologious ORFs stably exist in chimeic virus vAPRRS(EAV234), however, the partial sequences of ORF2?4 of PRRSV were found to be deleted. The vAPRRS(EAV234) can infect both MARC-145 and BHK-21cells. This study indicated that PRRSV, and even other arterivirus, can contain two panel of minor envelope protein ORFs in genome, although its’genetic stability was frustrated. As one potential vector, PRRSV can deliver heterologious genes which were inserted between ORF1b and ORF2a. This paved the way to further identify the arterivirus attachment proteins, dissect the protein-protein interaction and virion assembly mechanism, and develop PRRSV vector.
引文
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