Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.
Establishment of a reverse genetics system for studying human bocavirus in human airway epithelia.
复制标题
DOI：
10.1371/journal.ppat.1002899
复制
发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Qiu J
中科院分区：
文献类型：
--
作者：
Huang Q;Deng X;Yan Z;Cheng F;Luo Y;Shen W;Lei-Butters DC;Chen AY;Li Y;Tang L;Söderlund-Venermo M;Engelhardt JF;Qiu J
人类1型博卡病毒(HBoV1)是引起婴幼儿急性呼吸道感染喘息的病原体之一。在这项研究中，我们从感染患者的鼻咽抽提物中提取病毒DNA，获得了全长HBoV1基因组(包括两端)的序列，克隆了全长HBoV1基因组，并证明了DNA复制、单链DNA基因组的封装以及从人胚胎肾脏293细胞中释放HBoV1病毒粒子。这种基于细胞系的生产系统产生的HBoV1病毒粒子呈现出直径约26 nm的典型二十面体结构，并能够从根尖表面高效地感染极化的原代人呼吸道上皮(HAE)。感染的HAE表现出肺部呼吸道损伤的特征，包括紧密连接屏障的破坏，纤毛丧失和上皮细胞肥大。值得注意的是，从永生化的呼吸道上皮细胞系CUFI-8(最初来自囊性纤维化患者)培养的极化HAE也支持HBoV1的生产性感染。因此，我们建立了用于研究HBoV1感染的反向遗传学体系，并建立了第一个基于细胞系的培养体系，这将大大推进HBoV1复制和致病机制的研究。人类1型博卡病毒(HBoV1)是引起婴幼儿急性呼吸道感染喘息的病原体之一。HBoV1可有效感染极化的原代人呼吸道上皮细胞。然而，目前还没有建立起允许感染HBoV1的细胞系。更重要的是，HBoV1基因组两端的序列仍然未知。我们在这项研究中解决了这两个问题。我们已经对HBoV1的全长基因组进行了测序，并将其克隆到了一个质粒中。我们进一步证明了该HBoV1质粒在人胚胎肾293细胞中复制并产生病毒。这些HBoV1子代病毒粒子的感染在极化的人呼吸道上皮细胞中产生了明显的细胞病变效应，表现为上皮屏障的破坏。此外，当HBoV1被极化时，我们鉴定出支持HBoV1感染的呼吸道上皮细胞系。这是首次获得全长HBoV1基因组，阐明HBoV1感染在人呼吸道上皮细胞中的发病机制，并鉴定第一个支持生产性HBoV1感染的细胞系。
Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis. Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. HBoV1 productively infects polarized primary human airway epithelia. However, no cell lines permissive to HBoV1 infection have yet been established. More importantly, the sequences at both ends of the HBoV1 genome have remained unknown. We have resolved both of these issues in this study. We have sequenced a full-length HBoV1 genome and cloned it into a plasmid. We further demonstrated that this HBoV1 plasmid replicated and produced viruses in human embryonic kidney 293 cells. Infection of these HBoV1 progeny virions produced obvious cytopathogenic effects in polarized human airway epithelia, which were represented by disruption of the epithelial barrier. Moreover, we identified an airway epithelial cell line supporting HBoV1 infection, when it was polarized. This is the first study to obtain the full-length HBoV1 genome, to demonstrate pathogenesis of HBoV1 infection in human airway epithelia, and to identify the first cell line to support productive HBoV1 infection.
登录
查看更多内容
影响因子：
3.1
作者：
GIORGI, PL;OGGIANO, N;KANTAR, A
通讯作者：
KANTAR, A
DOI：
10.1073/pnas.0504666102
发表时间：
2005-09-06
影响因子：
11.1
作者：
Allander, T;Tammi, MT;Andersson, B
通讯作者：
Andersson, B
影响因子：
9.4
作者：
Edner, Niklas;Castillo-Rodas, Paul;Allander, Tobias
通讯作者：
Allander, Tobias
影响因子：
5.4
作者：
Chen, Aaron Yun;Luo, Yong;Qiu, Jianming
通讯作者：
Qiu, Jianming
影响因子：
3.1
作者：
Don, Massimiliano;Soderlund-Venermo, Maria;Korppi, Matti
通讯作者：
Korppi, Matti