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.
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