Rapid FISH Detection of EBV-infected Cells

描述(由申请人提供)：这项拨款申请的目的是开发一种新的方法，以更好地表征来自老年受试者的EB病毒(EBV)感染细胞。EBV是一种重要的病原体，给卫生保健系统带来了相当大的成本。EB病毒是传染性单核细胞增多症以及鼻咽癌、伯基特淋巴瘤和免疫抑制个体中其他B细胞淋巴瘤的病原体。虽然从体外转化的B淋巴母细胞系的研究中已经了解了很多关于EBV的知识，但关于EBV在体内的致病机制的关键知识空白仍然有待回答，包括EBV是如何在持续的免疫监测下持续存在的。我们有新的数据显示，老年人患有血浆病毒血症(EBV DNA)。此外，分子分析显示，EBV感染的B细胞中存在早期和晚期结构基因的频繁转录。因此，我们迫切需要开发分析方法来更好地表征EBV感染的细胞。我们有令人鼓舞的初步数据，表明我们有能力使用荧光原位杂交(FISH)和流式细胞术相结合的方法在单细胞水平检测EBV感染的B细胞。这项应用本身并不是假设驱动的，但支持一项研究技术的发展，该技术将被用来表征来自经聚合酶链式反应确定的高病毒载量的老年受试者的存档样本中受EBV感染的B细胞。具体目标是：(1)设计和测试针对EBERs的多个反义探针(MAP)；以及(2)应用MAP来表征老年受试者EB病毒感染的B细胞。该项目的成功完成将产生一种新的方法来检测外周血中感染EBV的B细胞，该方法将被纳入更全面的R01应用程序，以研究EBV在衰老过程中的免疫生物学。公共卫生相关性：爱泼斯坦-巴尔病毒(EBV)是一种重要的病原体，给卫生保健系统带来了相当大的成本。EB病毒是传染性单核细胞增多症以及鼻咽癌、伯基特淋巴瘤和免疫抑制个体中其他B细胞淋巴瘤的病原体。虽然从体外转化的B淋巴母细胞系的研究中已经了解了很多关于EBV的知识，但关于EBV在体内的致病机制的关键知识空白仍然有待回答，包括EBV是如何在持续的免疫监测下持续存在的。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，从而更好地了解EBV在衰老过程中的免疫学。

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.