摘要
呼吸道病毒是引发婴幼儿急性呼吸道感染疾病的主要病原体,目前仍有60%的小儿呼吸道疾病病理未阐明。2005年瑞典科学家Allander从患呼吸道疾病的婴幼儿感染样本中分离到一种新病毒,命名为人类博卡病毒(Human Bocavirus, HBoV)。HBoV为单链、无包膜DNA病毒,属于细小病毒科,博卡病毒属。最近,在胃肠道疾病患者的粪便中又发现了三种不同基因型的人类博卡病毒HBoV2、HBoV3和HBoV4。目前,由于未分离得到HBoV病毒粒子,感染性克隆的构建又受阻于难以获得病毒基因组末端回文序列(ITR),因此,对人类博卡病毒的研究多局限于流行病学方面,而有关病毒致病机制的研究报道甚少。本实验采集患有呼吸道疾病婴幼儿痰液样本,从中鉴定出人类博卡病毒(HBoV1),构建病毒基因组中间大片段克隆(缺少ITR),并获得包含HBoV1基因组的重组杆状病毒。以构建的克隆及重组杆状病毒为工具,对该病毒的转录机制、蛋白表达、启动子活性及病毒非结构蛋白对该启动子的调控作用进行分析。研究结果为深入揭示人类博卡病毒感染途径和致病机理提供理论基础,将促进病毒性呼吸道感染的治疗和预防。本论文的研究主要取得了以下几个方面的进展：
采用PCR扩增方法,以人类博卡病毒的NP1基因作为目标基因,对2007年10月-2009年3月收集的941例下呼吸道感染患者的痰液标本进行检测。941份标本中共检测到33份HBoV阳性扩增产物,阳性率为3.51%(33/941),且在所检出的阳性样品中,1岁以下婴幼儿样品共有24份,占阳性样72.7%,表明人类博卡病毒的敏感人群为1周岁以下婴幼儿。以检测的阳性样本DNA为模板,通过分子生物学方法,构建了含有HBoV中间大片段基因组克隆,命名为WHL-1,基因组序列比对为HBoV1型,序列全长5299bp (GenBank Acession NO. GU139423)。运用BLAST分析该病毒株与GenBank中HBoV1基因的同源性,结果显示WHL-1病毒株与目前报道的其他几株人类博卡病毒有高度同源的基因组序列。目前为止还未得到病毒基因组的末端回文序列。
PCR扩增人类博卡病毒左端唯一启动子区,分别构建病毒启动子增强型绿色荧光蛋白(EGFP)/荧光素酶报告基因重组载体：pGL3-pBoV-EGFP/pGL3-Basic-pBoV,转染哺乳动物细胞,通过检测绿色荧光蛋白表达和荧光素酶活性定性并定量研究人类博卡病毒启动子在哺乳动物细胞中的活性。结果显示,启动子在试验细胞中都具有活性,且比强启动子巨细胞病毒启动子(Cytomegalovirus,CMV)活性更高,在293T细胞中HBoV1启动子活性是CMV的4-5倍。将252bp启动子序列截短成7段,克隆到pGL3-Basic载体中,通过测定荧光素酶报告基因发光值检测各片段的活性,我们发现1-95nt序列对启动子活性无影响,而96-145nt区域对启动子活性有很大影响,推测该区段为转录调控区域。通过共转染试验,发现在被转染的293T和HeLa细胞中,病毒非结构蛋白NS1对HBoV1启动子具有反式激活作用,荧光素酶活性检测实验证实启动子活性增强2-3倍,推测NS1蛋白在病毒转录过程中发挥作用。
通过RT-PCR及RACE实验,我们在pWHL-1质粒转染的293T细胞中检测到NP1基因的转录产物。两个转录子的起始位点都位于186nt处,在mRNA1中,只发生一次剪接(供体位点在241nt处,受体位点在2236nt处),在mRNA2中,发生两次剪接(第一次位于241nt、2044nt处,第二次位于2164nt、2236nt处)：发现两个不同的NP1mRNA多聚腺苷酸位点,分别位于3233nt和3389nt。Southern blot试验显示,pWHL-1质粒转染的细胞中未发生病毒DNA复制,进一步证明ITR结构对病毒DNA复制是必需的,且HBoVl启动子为早期强启动子。启动子在试验细胞中启动病毒NP1基因转录并翻译,产生的非结构蛋白NP1定位在细胞核中。
利用Bac-to-Bac杆状病毒表达系统构建了包含HBoVl中间大片段的重组杆状病毒Bac-HBoVl及包含启动子-EGFP片段的重组杆状病毒Bac-Bov-EGFP。通过流式细胞仪统计发绿色荧光的细胞,我们发现在试验的多数哺乳动物细胞中,重组杆状病毒Bac-Bov-EGFP的转导效率比重组质粒pGL-pboca-EGFP的转染效率高,如相对转染效率较低的A-549细胞,重组杆状病毒的转导效率达到50%-60%。由于操作过程中不需要使用昂贵且对细胞有毒性的转染试剂,又可通过昆虫细胞扩增得到大量重组杆状病毒粒子,该系统为转染效率较低细胞接纳外源基因提供更为优化的平台。同时,构建的重组杆状病毒Bac-HBoV1转导293T、HeLa和A549细胞,都可检测到HBoVl-NP1基因的转录及表达,与pWHL-1质粒转染细胞的结果一致,进一步证明该系统的可行性及优越性。
Respiratory viruses are the major pathogens of acute lower respiratory tract infection (LRTI) in infants and young children. To date, pathogenesis of60%respiratory diseases has not been identified yet. In2005, Allander et al. first isolated a novel parvovirus isolated from hospitalized children with pneumonia and named this virus the human bocavirus (HBoV). HBoV is a linear single-stranded DNA virus without envelop and classified in the Bocaviurs genus of the Parvovirinae subfamily within the Parvoviridae family. Three other new species of human Bocavirus, HBoV2,3and4, have recently been isolated from fecal specimens. No infectious clone or efficient cell culture system has been described so far to support the replication of the virus. Most studies of HBoV were focused on epidemiology but not pathogenesis of the virus. In this study, HBoV1was detected from nasopharyngeal aspirates collected from children with lower respiratory tract infections. The recombinant plasmid and baculovirus containing the nearly full length genome of HBoV1were successfully constructed. Using these constructs, we studied the mechanism of transcription and expression strategy of the viral genome, activity of the HBoV1promoter and the regulation of the nonstructural proteins on the promoter activity. The results provided us with pro-fundamental basis for further studies on the HBoV1infection pathway and viral pathogenesis. The main results of this study are summarized as follows:
We screened941nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections in the Children's Hospital of Hubei province from October9,2007to March20,2009. The conserved region of HBoV NP1gene was used as the detection target by PCR. Our results showed that33of941samples (3.51%) were detected positive for HBoV;24(72.7%) HBoV-positive samples were from children under12months. Furthermore, to obtain a full-length HBoV clone, three segments which covered the nearly full-length genome were amplified by PCR from HBoV positive samples separately and cloned into pBluescript SKⅡ vector to generate the plasmid WHL-1(GenBank Acession NO. GU139423). The whole genome of WHL-1was sequenced and compared with those in GenBank. Phylogenetic analyses suggested that WHL-1shares high homology with other bocavirus isolates whose genotype belongs to was HBoV1. However, the both termini of HBoV1have not been obtained.
We constructed the both EGFP and luciferase reporter gene vectors under the control of the HBoV unique promoter, respectively. The data of fluorescent microscopy observation of the EGFP and luciferase reporter assays demonstrated that the promoter of HBoV1was highly active in most mammalian cell lines transfected by recombinant vectors. Moreover, the activity of HBoV1promoter was4-5folds higher than that of the Cytomegalovirus (CMV) promoter in293T cells. The region from1-95nt made no difference to the basal activity of the promoter and the putative transcription factor binding region located from96to145nt. In addition, we found that the transcriptional activity of this promoter could be transactivated by the viral nonstructural protein NS1in293T and HeLa cells, suggesting that the NS1protein may be play an important role in virus genome transcription.
We designed two specific NP1primers to analyze mRNAs of NP1gene by RT-PCR. All mRNAs were transcribed from a single promoter located at the left side of the HBoV1genome and NP1RT-PCR product displayed two fragments:one about980bp and another about1100bp. Sequencing of the fragments revealed that the two transcripts which presumably encode the NP1protein were alternative spliced. In mRNA1, only one splicing occurred (donor site241nt and acceptor site2236nt), whereas in mRNA2, splicing occurred twice (donor site241nt and acceptor site2044nt; donor site2164nt and acceptor site2236nt). The sequence between positions2044nt and2164nt is retained in the mRNA2. We next determined the3'terminal sequence of the NP1mRNA transcripts by3'-RACE. Sequence results showed that these two NP1PCR fragments of approximately790bp and940bp were properly annealed to the polyadenosine tails and the mRNAs were polyadenylated at two different positions proximal to the3'terminus of the NP1gene (position3268nt and3403nt), which corresponded to two polyadenylation signals situated at position3233nt and3389nt, respectively. Southern Blot analysis showed that no viral DNA replication was detected in293T cells transfected with pWHL-1plasmid, suggesting that ITRs were essential for viral DNA replication during infection cycle. The HBoV1promoter was an early promoter and the NP1gene was transcriped under the control of this promoter. The NP1protein was detected in pWHL-1transfected cells and localized in the nucleus.
We developed two recombinant baculoviruses:Bac-Bov-EGFP containing the reporter enhanced green fluorescent protein (EGFP) gene under the HBoVl promoter control, and Bac-HBoV1containing the whole HBoV1genome without both termini. A number of mammalian cells transduced by Bac-Bov-EGFP expressed the transgene with high efficiency, suggesting that baculovirus-mediated gene transfer was more efficient than the plasmid-based vector pGL-pboca-EGFP which contains the HBoV promoter and EGFP reporter gene tranfected by Lipofectamine into the cell lines such as human lung epithelial A549cells and HeLa cells. A HBoV1recombinant baculovirus has been shown to efficiently deliver the HBoV1genome into A549cells resulting in HBoV1transcription and translation. In summary, this system provides a useful tool for the analysis of the transcription and translation not only for HBoV1but also for other viruses without permissive cell culture system.
引文
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