Huang Qinfeng, Deng Xuefeng, Yan Ziying, Cheng Fang, Luo Yong, Shen Weiran, Lei-Butters Diana C M, Chen Aaron Yun, Li Yi, Tang Liang, Söderlund-Venermo Maria, Engelhardt John F, Qiu Jianming
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
PLoS Pathog. 2012;8(8):e1002899. doi: 10.1371/journal.ppat.1002899. Epub 2012 Aug 30.
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.
人博卡病毒1型（HBoV1）已被确定为急性呼吸道感染幼儿喘息的病原体之一。在本研究中，我们使用从一名感染患者的鼻咽抽吸物中提取的病毒DNA获得了全长HBoV1基因组序列（包括两个末端），克隆了全长HBoV1基因组，并证明了DNA复制、单链DNA基因组的衣壳化以及HBoV1病毒粒子从人胚肾293细胞中的释放。从基于该细胞系的生产系统产生的HBoV1病毒粒子呈现出典型的二十面体结构，直径约为26nm，并且能够从顶端表面有效感染极化的原代人气道上皮细胞（HAE）。受感染的HAE表现出肺气道损伤的特征，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，从永生化气道上皮细胞系CuFi-8（最初来源于一名囊性纤维化患者）培养的极化HAE也支持HBoV1的有效感染。因此，我们建立了一个反向遗传学系统，并生成了第一个基于细胞系的HBoV1感染研究培养系统，这将显著推进HBoV1复制和发病机制的研究。
