Molecular characterization of the bocaviruses

描述（由申请方提供）：人博卡病毒（HBoV）与急性呼气性喘鸣和肺炎相关。HBoV基因组在世界范围内经常被检测到，范围从2%-19%，在患有急性呼吸道疾病的幼儿的呼吸道标本中。分化的人气道上皮细胞最近已显示支持HBoV复制;然而，需要高病毒载量。目前尚未构建HBoV的感染性DNA克隆，这影响了对该病毒致病性的研究。犬细小病毒（MVC）是一种犬博卡病毒，可引起幼犬呼吸困难的呼吸道疾病和严重腹泻的肠炎。MVC感染允许的WRD细胞在S期扰乱细胞进程，有利于早期感染中的病毒复制，在后期感染中在G2/M期扰乱细胞进程，并最终导致凋亡性细胞死亡。此外，在MVC感染期间检测到DNA损伤反应。因此，由MVC感染诱导的细胞死亡，其独立于非结构和结构病毒蛋白，必须是宿主防御机制，推测是DNA损伤反应，旨在通过消除感染的细胞来阻止病毒复制，并且必须受到调节。此外，博卡病毒在细小病毒中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NP 1。由于HBoV和MVC之间的相似性，无论是在组织和序列的基因组和引起的疾病的症状，犬博卡病毒MVC的研究将最终回答有关NP 1在博卡病毒DNA复制和博卡病毒诱导的细胞周期停滞和细胞死亡的分子基础的功能的问题。最终，从MVC的研究中获得的知识将有助于我们了解HBoV的分子发病机制。在这个应用中，我们建议发现NP 1蛋白在博卡病毒DNA复制中的功能，特别是，以确定与NP 1相互作用的细胞因子;以及博卡病毒感染如何阻止细胞周期并导致凋亡性细胞死亡;并尝试从临床标本中构建HBoV的感染性克隆。此外，博卡病毒感染用于诱导DNA损伤反应，导致细胞周期停滞和细胞死亡的策略是不寻常的，因为病毒蛋白不参与。在博卡病毒的易处理的生物系统中检查这种策略为我们提供了一个有吸引力的机会，以深入了解防御病毒感染的基本细胞机制。 公共卫生相关性：人类博卡病毒对人类具有致病性，可引起儿童下呼吸道感染。对动物博卡病毒的研究将有助于我们从总体上了解博卡病毒的复制和致病机制。因此，我们的实验将回答人类博卡病毒感染的发病机制中的关键问题，并有可能为人类博卡病毒引起的疾病提供预防和治疗干预措施。

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.