Molecular and Epidemiologic Characterization of a Pathogenic Human Bocavirus

致病性人博卡病毒的分子和流行病学特征

• 批准号: 7315319
• 负责人: Peter J. Tattersall
• 金额: $ 20.63万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7315319
• 关键词: Age Distribution; Antibodies; Back; Baculoviruses; Biological Assay; Biology; Bovine Papillomavirus-1; Capsid; Capsid Proteins; Cell Culture System; Cell Line; Cells; Child; Childhood; Clinical; Cohort Studies; Communicable Diseases; Cultured Cells; DNA biosynthesis; Detection; Diagnostic; Disease Association; Embryo; Enzymes; Epidemiologic Studies; Epidemiology; Epithelial Cells; Family; Fibroblasts; Genome; Human; Human Cell Line; Immunoglobulin G; Immunoglobulin M; In Vitro; Infant; Infection; Insecta; Length; Link; Lung; Molecular; Molecular Genetics; Molecular Virology; Monoclonal Antibodies; Mus; Non-Small-Cell Lung Carcinoma; Oryctolagus cuniculus; Parvovirus; Patients; Polymerase Chain Reaction; Population; Principal Investigator; Proteins; Rate; Reagent; Recombinants; Research; Research Design; Respiratory Tract Diseases; Retrospective Studies; Role; Sampling; Serological; Serum; Simian virus 40; Symptoms; Testing; Transfection; Viral; Viral Genome; Virion; Virus; Virus-like particle; base; bovine parvovirus; gene cloning; member; pathogen; polyclonal antibody; polypeptide; programs; prospective; respiratory; tool; vector; virology

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.

描述（由申请人提供）：一种新发现的人类博卡病毒，HBoV（细小病毒家族的成员）似乎是儿童中流行的重要呼吸道病原体。该申请提议由具有广泛细小病毒研究专业知识的分子病毒学小组和在儿科病毒学和流行病学方面拥有良好发现记录的儿科传染病小组合作，进一步表征该病毒。我们已经通过诊断性 PCR 检测到了几个含有该病毒的儿科样本，并将这些样本中预测的主要病毒衣壳蛋白的基因克隆到杆状病毒载体中。用这种杆状病毒感染昆虫细胞会导致博卡病毒多肽表达并有效组装成病毒样颗粒（VLP）。 VLP 的生产量将用于生产 HboV 衣壳特异性多克隆和单克隆抗体，并开发用于检测抗衣壳 IgG 和 IgM 抗体的 ELISA。将在前瞻性和回顾性研究中结合使用 PCR 和 ELISA，以探索 HBoV 在人群中的分布和疾病关联，特别是在我们的大型儿科研究组中。我们将尝试在细胞培养中培养病毒并研究其生物学。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，并建立密切相关的牛细小病毒（BPV-1）的感染细胞培养系统，以验证这些试剂。我们将接种几种可能合适的人类细胞系，例如胚胎肺成纤维细胞、非小细胞肺癌、SV40转化的肺上皮细胞以及逐步永生化和转化的气道上皮细胞。将使用上述生成的血清学试剂检查它们支持 HoBV 单轮和多轮感染的能力。一旦我们确定了能够在培养物中扩增 HBoV 的细胞系，我们将从在此类细胞中生长的病毒体中纯化病毒基因组，通过体外 DNA 复制构建病毒的全长克隆，并在体外转染回感受态宿主细胞后测试其感染性。 这些研究旨在开发新工具来检测和分离新发现的人类博卡病毒（HboV），并鉴定人血清中针对该病毒的抗体。这些工具将用于建立 HboV 的基本流行病学并探讨其在婴儿呼吸道疾病中的作用。