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）：新发现的人类博卡病毒HBOV（细小病毒家族的成员）似乎是儿童普遍存在的重要呼吸病原体。该应用程序提出了一项协作努力，以进一步描述具有广泛细小病毒研究专业知识的分子病毒学组和一个儿科传染病群体，其儿科病毒学和流行病学的发现记录有了可靠的记录。我们已经确定了几个小儿样本，这些样本含有诊断性PCR检测到的病毒，并将预测的主要病毒衣壳蛋白的基因从这些样品中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Bocaviral多肽的表达及其有效组装到病毒样颗粒（VLP）中。 VLP将以产生HBOV帽特异性多克隆和单克隆抗体的必要量产生，并开发ELISA以检测抗Capsid IgG和IgM抗体。在前瞻性和回顾性研究中，将使用PCR和ELISA的结合，以探索HBOV人口中HBOV的分布和疾病关联，尤其是在我们大型儿科研究小组中。我们将尝试种植病毒并研究其在细胞培养中的生物学。为此，我们将生产能够检测在HBOV感染细胞中表达的推定病毒非结构基因产物，并为密切相关的牛小扇联病毒（BPV-1）建立感染细胞培养系统，以验证这些试剂。我们将接种几种可能适当的人类细胞系，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和转化的气道上皮细胞。这些将使用如上所述产生的血清学试剂支持HOBV支持单轮和多轮感染的能力。一旦我们确定了能够在培养中扩展HBOV的细胞系，我们将通过体外DNA复制来纯化从这种细胞中生长的病毒体中的病毒基因组，构建病毒的全长克隆，并在体外转染后将其感染回到体外。 这些研究旨在开发新的工具，用于检测和隔离新发现的人类博卡病毒（HBOV），并鉴定人类血清中该病毒的抗体。这些工具将用于建立HBOV的基本流行病学，并探索其在婴儿中呼吸道疾病中的作用。