Adenovirus Persistence in Human Lymphoid Tissues

描述（由申请人提供）：C亚群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偶发性病毒脱落。虽然腺病毒的持续存在最初被认为是良性的，但最近的研究将腺病毒与哮喘、慢性阻塞性肺病和小细胞肺癌等慢性疾病联系起来，表明这些病毒可能导致更严重的疾病。我们对腺病毒持久性的细胞和分子性质所知甚少。早期的研究人员在没有感染性病毒的淋巴组织中发现了腺病毒DNA，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PCR检测方法，可检测大多数（81%）扁桃体和腺样体标本中单个核细胞制剂中的腺病毒DNA。尽管在一些供体中有大量的病毒基因组，但在这些组织中新分离的传染性病毒很少被恢复。初步实验表明，病毒DNA位于T细胞和第二种细胞类型（可能是吞噬细胞）内。在初步实验中，刺激诱导T细胞活化导致病毒DNA增加和感染性病毒释放。腺病毒持续存在可能导致以前未被发现的长期健康风险，这需要了解病毒持续存在和再激活的细胞和分子机制。为此，特异性目的1将鉴定自然人类扁桃体组织感染中携带C亚群腺病毒的细胞群。将使用多参数FACS分选分离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。将使用限制性稀释法结合实时PCR定量分析分类后的群体，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。特异性目标2将分析淋巴组织中的病毒基因组。需要解决的问题包括：在移除含有病毒DNA的组织时检测到传染性病毒的频率是多少？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mrna的子集？哪些刺激导致病毒转录的诱导？有潜伏期特异性转录吗？特异性目标3将使用人类T细胞系和短期培养来测试一些T细胞能够阻止腺病毒DNA被转录或复制的假设，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。在两种人类T细胞系中观察到的病毒基因表达抑制的性质将被研究，并将这种现象扩展到原代人T细胞。这些研究将揭示持续性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.