Molecular characterization of the bocaviruses

描述（由申请人提供）：人博卡病毒（HBoV）与急性呼气性喘息和肺炎有关。在世界范围内，在患有急性呼吸道疾病的幼儿呼吸道标本中经常检测到HBoV基因组，范围从2%-19%不等。分化的人气道上皮细胞最近被证明支持HBoV复制；但是，需要高病毒载量。HBoV的感染性DNA克隆尚未建立，这影响了对该病毒发病机制的研究。犬科微小病毒（MVC）是犬科bocavavirus的一种，在幼犬中引起呼吸困难的呼吸道疾病和严重腹泻的肠炎。准许型WRD细胞的MVC感染在感染早期的s期扰乱细胞进程，有利于病毒复制，在感染后期的G2/ m期扰乱细胞进程，最终导致细胞凋亡。此外，在MVC感染期间检测到DNA损伤反应。因此，独立于非结构和结构病毒蛋白的MVC感染诱导的细胞死亡一定是一种宿主防御机制，可能是DNA损伤反应，旨在通过消除感染细胞来阻止病毒复制，并且必须受到调节。此外，在细小病毒中，bocavavirus的独特之处在于第三个ORF，即中间ORF，编码一种新的非结构蛋白NP1。由于HBoV和MVC在基因组的组织和序列以及引起疾病的症状上都有相似之处，对犬bocavavirus MVC的研究将最终解答NP1在bocavvirus DNA复制中的功能以及bocavvirus诱导的细胞周期阻滞和细胞死亡的分子基础。最终，从MVC研究中获得的知识将有助于我们了解HBoV的分子发病机制。在本应用中，我们拟发现NP1蛋白在bocavavirus DNA复制中的功能，特别是鉴定与NP1相互作用的细胞因子；以及博卡病毒感染如何阻滞细胞周期并导致细胞凋亡；并尝试从临床标本中构建具有传染性的HBoV克隆。此外，bocavavirus感染用于诱导DNA损伤反应，导致细胞周期阻滞和细胞死亡的策略是不寻常的，因为病毒蛋白不参与其中。在易处理的bocavavirus生物系统中检查这一策略为我们提供了一个有吸引力的机会，以深入了解涉及防御病毒感染的基本细胞机制。

DESCRIPTION (provided by applicant): Human bocavirus (HBoV) is associated with acute expiratory wheezing and pneumonia. The HBoV genome has been frequently detected worldwide, ranging from 2%-19%, in respiratory specimens from young children with acute respiratory illnesses. Differentiated human airway epithelial cells have been recently shown to support HBoV replication; however, a high virus load is required. An infectious DNA clone of HBoV has not been constructed, which impairs study of the pathogenesis of this virus. Minute virus of canines (MVC), a canine bocavirus, causes respiratory diseases with breathing difficulty and enteritis with severe diarrhea in pups. MVC infection of permissive WRD cells perturbs cell progression at the S-phase in favor of viral replication in early infection and at the G2/M-phase in later infection, and ultimately leads to an apoptotic cell death. In addition, a DNA damage response is detected during MVC infection. Therefore, cell death induced by MVC infection, which is independent of both nonstructural and structural viral proteins, must be a host defense mechanism, presumably the DNA damage response, designed to arrest viral replication by eliminating infected cells, and must be regulated. In addition, bocaviruses are unique among parvoviruses in that a third ORF, the mid-ORF, encodes a novel non-structural protein, NP1. Because of the similarities between HBoV and MVC, both in the organization and sequence of the genomes and in the symptoms of caused diseases, study of the canine Bocavirus MVC will eventually answer questions about the functions of NP1 in Bocavirus DNA replication and the molecular basis of Bocavirus-induced cell cycle arrest and cell death. Ultimately, knowledge obtained from study of MVC will help us to understand the molecular pathogenesis of HBoV. In this application, we propose to discover the function of the NP1 protein in Bocavirus DNA replication, especially, to identify cellular factors that interact with the NP1; and how Bocavirus infection arrests the cell cycle and causes an apoptotic cell death; and attempt to construct an infectious clone of HBoV from clinical specimens. In addition, the strategy that Bocavirus infection uses to induce a DNA damage response, leading to cell cycle arrest and cell death, is unusual in that viral proteins are not involved. Examining this strategy within the tractable biological system of Bocavirus provides us an attractive opportunity to gain insight into the basic cellular mechanism involved in defending against viral infection. PUBLIC HEALTH RELEVANCE: Human bocavirus is pathogenic to humans, causes lower respiratory tract infections in children. Study of the animal bocavirus will help us to understand the replication and pathogenesis of the bocaviruses in general. Our experiments will thus answer critical questions in the pathogenesis of human bocavirus infection, and have the potential to provide prophylactic and therapeutic interventions to diseases caused by human bocavirus.