Adenovirus Persistence in Human Lymphoid Tissues

腺病毒在人类淋巴组织中的持续存在

• 批准号: 6611613
• 负责人: Linda R Gooding
• 金额: $ 34.2万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611613
• 关键词: Adenoviridae; T lymphocyte; cell population study; chronic disease /disorder; clinical research; flow cytometry; genetic transcription; human tissue; latent virus infection; leukocyte activation /transformation; lymphatic tissue; respiratory infections; tissue /cell culture; virus DNA; virus replication

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.

描述（由申请方提供）：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内出现偶发性病毒脱落。虽然腺病毒持续存在最初被认为是良性的，但最近的研究将腺病毒与慢性疾病如哮喘、慢性阻塞性肺病和小细胞肺癌联系起来，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。早期的研究者在没有感染性病毒的情况下在淋巴组织中发现了腺病毒DNA，这表明病毒感染的一个阶段，其中病毒在某些细胞类型中处于休眠状态。本实验室开发了一种灵敏的真实的时间PCR检测方法，可检测大多数（81%）扁桃体和腺样体标本单核细胞制剂中的腺病毒DNA。尽管在一些供体中存在大量的病毒基因组，但当新鲜分离时，很少从这些组织中回收感染性病毒。初步实验表明，病毒DNA位于T细胞和第二种细胞类型（可能是吞噬细胞）内。在初步实验中，诱导T细胞活化的刺激导致病毒DNA的增加和感染性病毒的释放。腺病毒持久性引起的以前未发现的长期健康风险的可能性要求理解病毒持久性和再活化的细胞和分子机制。为此，具体目标1将鉴定在扁桃体组织的自然人感染中携带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。还将对吞噬细胞、巨噬细胞和树突细胞进行分选并分析是否存在病毒DNA。将使用有限稀释法结合真实的时间PCR定量分析分选的群体，以评价含病毒细胞的数量和每个细胞的病毒基因组拷贝数。具体目标2将分析淋巴组织中的病毒基因组。需要解决的问题包括：在切除含有病毒DNA的组织时，多久会检测到传染性病毒？什么刺激（T细胞活化或促炎细胞因子）诱导病毒复制？新鲜分离的细胞是否含有病毒mRNA的一个子集，什么样的刺激导致病毒转录的诱导？是否存在潜伏期特异性转录？具体目标3将使用人类T细胞系和短期培养物来测试以下假设：一些T细胞能够阻止腺病毒DNA转录或复制，并且该特性对于在这些细胞中建立潜伏期是必不可少的。将研究在两种人T细胞系中观察到的病毒基因表达抑制的性质，并将该现象扩展到原代人T细胞。这些研究将揭示持续存在的腺病毒导致严重慢性疾病如癌症和自身免疫的潜力。