Organotypic Culture as a Model for EBV Infection of Epithelial Cells

描述（由申请人提供）：EB病毒（EBV）与许多B淋巴细胞和上皮来源的恶性肿瘤相关。其中许多是艾滋病患者特有的或在艾滋病患者中增加，包括头颈部上皮癌。该提案的重点是使用器官型培养物开发 EBV 上皮感染体外模型。器官型培养物与单层培养物的不同之处在于，细胞分化和分层以类似于原位发现的上皮。由于 EBV 可以在体内分化的上皮细胞中复制，因此器官型培养是研究上皮细胞在病毒生命周期中的作用的理想系统。我们的初步数据表明，我们可以使用表达 GFP 作为标记的重组 EBV 有效感染培养物中的原代口腔角质形成细胞，并且我们可以生成有效模仿口腔粘膜上皮的原代口腔角质形成细胞的器官型培养物。基于这些初步数据，我们建议描述器官型培养物中 EBV 感染的特征，识别感染的细胞和表达的病毒基因产物。多项研究表明 EBV 可能在体内建立潜伏感染。我们将确定 EBV 是否可以建立潜伏感染并表征潜伏类型。将检查上皮中表达的病毒基因产物的功能。因此，我们将建立并表征一个用于上皮细胞中EBV研究的模型系统，并利用该系统在R21资助机制的有限范围内尽可能地定义病毒基因产物的功能。这些研究将为未来的实验奠定基础，这些实验将研究有关人类口腔上皮细胞 EBV 感染的几个长期存在的问题。 公共卫生相关性：EB 病毒 (EBV) 与多种人类恶性肿瘤相关，这些恶性肿瘤主要发生在 B 淋巴细胞或上皮细胞中，其中许多恶性肿瘤在艾滋病患者中的发病率有所增加。拟议的研究旨在开发一种 EBV 上皮感染的体外模型，这对于填补我们关于上皮细胞在 EBV 生命周期中的作用的知识空白具有不可估量的价值。由于口腔粘膜是病毒进入和退出的部位，上皮细胞在病毒传播中发挥着重要作用，因此，了解它们在病毒生命周期中的作用不仅将阐明这一重要步骤，而且有可能影响 EBV 感染的所有下游事件。该模型可能不仅对于增加我们对上皮在病毒生命周期中的作用以及病毒对恶性肿瘤发展的贡献的了解很重要，而且还可以用于抗病毒策略的开发和/或测试。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is associated with a number of malignancies of B lymphocyte and epithelial origin. Many of these are specific to or increased in AIDS patients including epithelial cancers of the head and neck. This proposal focuses on the development of an in vitro model of EBV infection of epithelium using organotypic cultures. Organotypic cultures differ from monolayers in that the cells differentiate and stratify to resemble epithelium found in situ. Because EBV can replicate in differentiated epithelial cells in vivo, organotypic culture is an ideal system to study the role of epithelial cells in the viral life cycle. Our preliminary data demonstrate that we can efficiently infect primary oral keratinocytes in culture using a recombinant EBV that expresses GFP as a marker, and that we can generate organotypic cultures of primary oral keratinocytes that effectively mimic oral mucosal epithelium. Based on these preliminary data, we propose to characterize EBV infection in organotypic culture identifying the cells infected and the viral gene products expressed. Several studies have suggested that EBV may establish a latent infection in vivo. We will determine whether EBV can establish a latent infection and characterize the type of latency. The functions of the viral gene products expressed in epithelium will be examined. Thus, we will establish and characterize a model system for the study of EBV in epithelium and use this system to define the functions of the viral gene products as far as possible within the limited scope of the R21 funding mechanism. These studies will set the stage for future experiments that will examine several long standing questions concerning EBV infection of human oral epithelium. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is associated with a variety of human malignancies that occur predominantly in B lymphocytes or epithelial cells, many of which have an increased incidence in AIDS patients. The proposed research is intended to develop an in vitro model of EBV infection of epithelium that will be invaluable in filling in the gaps in our knowledge about the role of epithelial cells in the EBV lifecycle. Because the oral mucosa is the site of entry and exit of the virus, epithelial cells play a major role in viral spread and thus, understanding their role in the viral life cycle will not only shed light on this important step but has the potential to impact all downstream events from EBV infection. This model is likely to be important not only in adding to our knowledge of the role of the epithelium in the viral life cycle and the contribution of the virus to the development of malignancies, but could also be used for the development and/or testing of antiviral strategies.