Molecular and Epidemiologic Characterization of a Pathogenic Human Bocavirus

描述(申请人提供)：一种新发现的人类博卡病毒，HBoV-细小病毒家族的成员-似乎是一种在儿童中流行的重要呼吸道病原体。这项申请提出了一项合作努力，由一个拥有广泛的细小病毒研究专业知识的分子病毒学小组和一个在儿科病毒学和流行病学方面有已证明的发现记录的儿科传染病小组共同努力，进一步确定这种病毒的特征。我们已经确定了几个通过诊断性聚合酶链式反应检测到的含有病毒的儿科样本，并从这些样本中克隆了预测的主要病毒衣壳蛋白基因，并将其克隆到杆状病毒载体中。这种杆状病毒感染昆虫细胞，导致博卡病毒多肽的表达，并将其有效地组装成病毒样颗粒(VLP)。将生产足够数量的VLP，以生产针对HboV衣壳的多克隆和单抗，并开发一种用于检测抗衣壳IgG和IgM抗体的酶联免疫吸附试验。在前瞻性和回溯性研究中，将使用聚合酶链式反应和酶联免疫吸附试验相结合的方法，探索HBoV在人类群体中的分布和疾病相关性，特别是在我们的大型儿科研究小组中。我们将尝试培养这种病毒，并在细胞培养中研究其生物学。为此，我们将生产能够检测HBoV感染细胞中表达的假定病毒非结构基因产物的抗体，并建立与之密切相关的牛细小病毒(BPV-1)的感染细胞培养体系，以验证这些试剂的有效性。我们将接种几种潜在合适的人类细胞系，如胚胎肺成纤维细胞、非小细胞肺癌、SV40转化的肺上皮细胞和逐步永生化和转化的呼吸道上皮细胞。将使用如上所述产生的血清学试剂，检查它们支持单轮和多轮感染HoBV的能力。一旦我们确定了一种能够在培养中扩增HBoV的细胞系，我们将从这种细胞中生长的病毒粒子中提纯病毒基因组，通过体外DNA复制构建病毒的全长克隆，并在体外将其转回到合格的宿主细胞中，测试其感染性。这些研究旨在开发新的工具来检测和分离新发现的人类博卡病毒(HboV)，并在人血清中识别该病毒的抗体。这些工具将被用来建立HboV的基本流行病学，并探索其在婴儿呼吸道疾病中的作用。

DESCRIPTION (provided by applicant): A newly discovered human bocavirus, HBoV - a member of the parvovirus family - appears to be a significant respiratory pathogen prevalent in children. This application proposes a collaborative effort to further characterize this virus by a molecular virology group with extensive parvovirus research expertise and a pediatric infectious disease group with a proven track record of discovery in pediatric virology and epidemiology. We have identified several pediatric samples that contain the virus, as detected by diagnostic PCR, and have cloned the gene for the predicted major viral capsid protein from these samples, into a baculovirus vector. Infection of insect cells with this baculovirus results in expression of the bocaviral polypeptide and its efficient assembly into virus-like particles (VLPs). VLPs will be produced in the quantities necessary to produce HboV capsid-specific polyclonal and monoclonal antibodies, and to develop an ELISA for the detection of anti-capsid IgG and IgM antibodies. A combination of PCR and ELISA will be used, in both prospective and retrospective studies, to explore the distribution and disease associations of HBoV in the human population, particularly within our large pediatric study group. We will attempt to grow the virus and study its biology in cell culture. For this we will produce antibodies able to detect putative viral non-structural gene products expressed in HBoV infected cell, and establish an infected-cell culture system for the closely- related bovine parvovirus (BPV-1), in order to validate these reagents. We will inoculate several potentially appropriate human cell lines, such as embryonic lung fibroblasts, non-small cell lung cancer, SV40- transformed lung epithelial cells and stepwise immortalized and transformed airway epithelial cells. These will be examined for their ability to support single- and multiple-round infections with HoBV, using the serological reagents generated as above. Once we have identified a cell line capable of expanding HBoV in culture, we will purify viral genomes from virions grown in such cells, construct a full-length clone of the virus by in vitro DNA replication, and test its infectivity following transfection back into competent host cells in vitro. These studies are designed to develop new tools for the detection and isolation of the newly-discovered human bocavirus (HboV), and to identify antibodies to this virus in human serum. These tools will be used to establish the basic epidemiology of HboV and to explore its role in respiratory illness among infants.