Adenovirus Persistence in Human Lymphoid Tissues

描述(申请人提供)：C亚组腺病毒引起幼儿呼吸道感染，导致初次感染后数月至数年的间歇性病毒脱落。虽然腺病毒的持久性最初被认为是良性的，但最近将腺病毒与哮喘、慢性阻塞性肺疾病和小细胞肺癌等慢性疾病联系起来的研究表明，这些病毒可能导致更严重的疾病。值得注意的是，人们对腺病毒持续存在的细胞和分子性质知之甚少。早期的研究人员在淋巴组织中发现了腺病毒DNA，在没有传染性病毒的情况下，这表明病毒感染的一个阶段，病毒在某些细胞类型中处于休眠状态。该实验室开发了一种灵敏的实时聚合酶链式反应方法，可以从大多数(81%)扁桃体和腺样体标本中检测单个核细胞标本中的腺病毒DNA。尽管在一些捐赠者中存在大量的病毒基因组，但在新鲜分离的这些组织中，很少能从这些组织中提取到传染性病毒。初步实验表明，病毒DNA位于T细胞和第二种细胞类型中，可能是吞噬细胞。在初步实验中，诱导T细胞激活的刺激会导致病毒DNA的增加和传染性病毒的释放。腺病毒持续存在可能导致以前未被检测到的长期健康风险，因此需要了解病毒持续存在和重新激活的细胞和分子机制。为此，特定目标1将鉴定自然感染人类扁桃体组织中携带C亚群腺病毒的细胞群。将使用多参数FACS分类来分离T淋巴细胞亚群，以确定哪些细胞携带病毒。吞噬细胞、巨噬细胞和树突状细胞也将被分类和分析是否存在病毒DNA。将使用有限稀释法和实时聚合酶链式反应定量相结合的方法对分类的种群进行分析，以评估含病毒细胞的数量和每个细胞的病毒基因组拷贝数。特定目标2将分析淋巴组织中的病毒基因组。要解决的问题包括：在切除含有病毒DNA的组织时，多久检测到一次传染性病毒？是什么刺激(T细胞激活或促炎细胞因子)诱导病毒复制？新分离的细胞是否含有病毒mRNAs的一个子集？什么刺激可以诱导病毒转录？有潜伏期特定的转录吗？特殊目的3将使用人类T细胞系和短期培养来测试假设，即一些T细胞能够阻止腺病毒DNA被转录或复制，并且这种特性对于在这些细胞中建立潜伏期是必不可少的。将研究在两个人类T细胞系中观察到的病毒基因表达抑制的本质，并将这一现象扩展到原代人类T细胞。这些研究将揭示持续腺病毒可能导致严重的慢性疾病，如癌症和自身免疫。

DESCRIPTION (provided by applicant): The subgroup C adenoviruses cause respiratory tract infections in young children leading to episodic virus shedding for months to years after primary infection. Although adenovirus persistence was originally believed to be benign, recent studies linking adenovirus to chronic ailments such as asthma, chronic obstructive pulmonary disease, and small cell lung cancer suggest that these viruses may contribute to more serious diseases. Remarkably little is known about the cellular and molecular nature of adenovirus persistence. Early investigators found adenoviral DNA in lymphoid tissues in the absence of infectious virus, suggesting a stage of virus infection in which the virus is dormant in some cell type. This laboratory has developed a sensitive real time PCR assay that detects adenoviral DNA in mononuclear cell preparations from the majority (81%) of tonsil and adenoid specimens tested. Despite large numbers of viral genomes in some donors, infectious virus is infrequently recovered from these tissues when freshly isolated. Preliminary experiments suggest that viral DNA is located within T cells and a second cell type, perhaps a phagocyte. Stimuli that induce T cell activation lead to increases in viral DNA and release of infectious virus in preliminary experiments. The potential for previously undetected long-term health risks arising from adenovirus persistence requires that the cellular and molecular mechanisms underlying virus persistence and reactivation be understood. To this end, Specific aim 1 will identify the cell populations harboring subgroup C adenoviruses in natural human infection of tonsil tissue. T lymphocyte subpopulations will be isolated using multi parameter FACS sorting to determine which cells harbor the virus. Phagocytic cells, macrophages and dendritic cells will also be sorted and analyzed for the presence of viral DNA. Sorted populations will be analyzed using a limiting dilution approach coupled with quantitation by real time PCR to evaluate the number of virus-containing cells and the number of copies of the viral genome per cell. Specific aim 2 will analyze the viral genome in lymphoid tissues. Questions to be addressed include: How often is infectious virus detected at the time of removal of tissues containing viral DNA? What stimuli (T cell activation or pro-inflammatory cytokines) induce viral replication? Do freshly isolated cells contain a subset of viral mRNAs and what stimuli lead to induction of viral transcription? Is there latency-specific transcription? Specific aim 3 will use human T cell lines and short term cultures to test the hypothesis that some T cells are able to prevent adenoviral DNA from being transcribed or replicated, and that this property is essential for the establishment of latency in these cells. The nature of the inhibition of viral gene expression observed in two human T cell lines will be investigated and the phenomenon extended to primary human T cells. These studies will reveal the potential for persistent adenovirus to contribute to serious chronic diseases such as cancer and autoimmunity.