Rapid FISH Detection of EBV-infected Cells

描述（由申请人提供）：该资助申请的目的是开发一种新方法，以更好地表征老年受试者的EB病毒（EBV）感染细胞。EB病毒是一种重要的病原体，给卫生保健系统带来相当大的成本。EBV是免疫抑制个体中传染性单核细胞增多症以及鼻咽癌、伯基特淋巴瘤和其它B细胞淋巴瘤的病原体。虽然已经从体外转化的B淋巴母细胞系的研究中了解了很多关于EBV的知识，但关于EBV体内发病机制的知识的关键空白仍有待回答，包括EBV如何在持续的免疫监视下持续存在。我们有新的数据表明，老年受试者有血浆病毒血症（EBV DNA）。此外，分子分析揭示了EBV感染的B细胞中早期和晚期结构基因的频繁转录。因此，我们迫切需要开发更好地表征EBV感染细胞的检测方法。我们有令人鼓舞的初步数据，证明我们有能力检测EB病毒感染的B细胞在单细胞水平上使用荧光原位杂交（FISH）结合流式细胞术。本申请本身不是假设驱动的，但支持开发一种研究技术，该技术将用于表征通过PCR测定具有高病毒载量的老年受试者存档标本中EBV感染的B细胞。具体目标是：（1）设计和测试靶向EBER的多种反义探针（MAP）;和（2）MAP用于表征来自老年受试者的EBV感染的B细胞的应用。该项目的成功完成将产生一种检测外周血中EBV感染的B细胞的新方法，该方法将被纳入更全面的R 01应用程序中，以研究EBV在衰老中的免疫生物学。与公共卫生的相关性：EB病毒（EBV）是一种重要的病原体，给卫生保健系统带来相当大的成本。EBV是免疫抑制个体中传染性单核细胞增多症以及鼻咽癌、伯基特淋巴瘤和其它B细胞淋巴瘤的病原体。虽然已经从体外转化的B淋巴母细胞系的研究中了解了很多关于EBV的知识，但关于EBV体内发病机制的知识的关键空白仍有待回答，包括EBV如何在持续的免疫监视下持续存在。我们将开发一种新的基于FISH的检测方法，在单细胞水平上检测外周血中EBV感染的细胞，这可以用来更好地了解EBV在衰老中的免疫学。

DESCRIPTION (provided by applicant): The objective of this grant application is to develop a new method to better characterize Epstein-Barr virus (EBV)-infected cells from aged subjects. EBV is an important pathogen and results in considerable cost to the health care system. EBV is the causative agent of infectious mononucleosis as well as nasopharyngeal carcinoma, Burkitt's lymphoma, and other B-cell lymphomas in immunosuppressed individuals. Although much has been learned about EBV from studies of in vitro transformed B-lymphoblastoid cell lines, critical gaps in knowledge regarding EBV pathogenesis in vivo remain to be answered, including how EBV persists in spite of the continuous immune surveillance. We have new data which shows that aged subjects have plasma viremia (EBV DNA). In addition, molecular analysis revealed frequent transcription of early and late structural genes in EBV-infected B-cells. Thus, we have an immediate need to develop assays to better characterize EBV-infected cells. We have encouraging preliminary data that demonstrates our ability to detect EBV-infected B cells at the single-cell level using fluorescent in situ hybridization (FISH) combined with flow cytometry. This application is not hypothesis driven per se but supports the development of a research technology that will be used to characterize EBV-infected B-cells in archived specimens from aged subjects that have high viral load as determined by PCR. The specific aims are: (1) Design and test multiple antisense probes (MAPs) that target EBERs; and (2) Application of MAPs to characterize EBV-infected B-cells from aged subjects. The successful completion of this project will yield a novel method to detect EBV-infected B cells in peripheral blood, and this methodology will be incorporated into a more comprehensive R01 application to investigate the immunobiology of EBV in aging. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is an important pathogen and results in considerable cost to the health care system. EBV is the causative agent of infectious mononucleosis as well as nasopharyngeal carcinoma, Burkitt's lymphoma, and other B-cell lymphomas in immunosuppressed individuals. Although much has been learned about EBV from studies of in vitro transformed B-lymphoblastoid cell lines, critical gaps in knowledge regarding EBV pathogenesis in vivo remain to be answered, including how EBV persists in spite of the continuous immune surveillance. We will develop a novel FISH-based assay to detect EBV-infected cells in peripheral blood at the single-cell level which can be used to better understand the immunology of EBV in aging.