人博卡病毒 1(human bocaparvovirus 1,HBoV1)为感染人并引起疾病的两种细小病毒之一.其感染 2-5 岁婴幼儿,能引起轻度或重度急性呼吸道疾病,严重时可危及生命.HBoV1 基因组末端含末端反向重复序列(repeat the sequence in reverse,ITR),为病毒基因组复制所必需,但是难以进行PCR扩增合成.本研究通过分步合成末端ITR及分子克隆方法成功构建HBoV1 的全长感染性克隆pSKHBoV1.经转染HEK293 细胞后,分别从重要非结构蛋白的表达、病毒RNA转录后修饰与加工、病毒基因组复制水平以及子代病毒粒子基因组鉴定等方面,证实构建的感染性克隆在转染 HEK293 细胞后能够进入正常的复制周期并具有拯救出病毒粒子的潜力,这为后续研究HBoV1 的复制增殖、病毒与宿主互作关系以及病毒疫苗的研发奠定了基础.
Construction and characterization of an infectious clone for human bocaparvovirus HBoV1
Human bocaparvovirus 1(HBoV1)is one of the two parvoviruses that infect humans and cause diseases.Infection with HBoV1 in infants and young children aged 2-5 years can lead to mild or severe acute respiratory diseases,with the most severe cases posing a life-threatening risk.Similar to other parvoviruses,the HBoV1 DNA genome consists of two terminal reverse repeats(ITRs)at its ends,which are necessary for viral genome replication.However,up to now,it has remained a technical challenge to clone the entire ITRs through PCR amplification.In this study,we successfully constructed a full-length infectious clone of HBoV1,termed as pSKHBoV1,by synthesizing and cloning the terminal ITRs in a stepwise manner.After transfecting HEK293 cells with the infectious clone pSKHBoV1,we were able to reconstitute the viral replication cycle.This included the expression of key non-structural proteins,post-transcriptional modification and processing of viral RNA,viral genome replication,and potentially the production of progeny virions containing the defined DNA genome.The successful construction of the infectious clone pSKHBoV1 lays the foundation for future studies on HBoV1 replication and propagation,virus-host interaction,and the development of viral vaccines.
human bocaparvovirusinfectious clonesynthetic constructionviral replication
万方数据
维普
