Zoster (shingles) is a common disorder of the elderly characterized by severe dermatomal pain and rash. Zoster with its attendant neurologic complications of postherpetic neuralgia and encephalitis results from reactivation of varicella zoster virus (VZV), a ubiquitous human herpesvirus that becomes latent in ganglia after childhood chickenpox. Although no animal or in vitro model of VZV latency exists, our application of nucleic acid hybridization to human ganglia and mononuclear cells revealed important differences between VZV, herpes simplex and Epstein-Barr virus, both in the cell targeted for latency and the extent of viral gene expression, supporting our hypothesis that the mechanism by which each herpesvirus is reactivated may be unique. If so, information on the configuration of VZV DNA and the cell type(s) and extent of VZV gene expression n ganglia are crucial to the eventual control of viral reactivation. Using our well-characterized supply of human ganglia, our cloned and mapped VZV DNA fragments, and the complete sequence of the VZV genome, we will identify VZV-positive ganglia by PCR amplification. VZV DNA configuration will be analyzed by hybridization of restriction endonuclease-digested ganglionic DNA separated by standard and pulsed-field electrophoresis to PCR-amplified probes representing terminal and internal parts of the VZV genome. The primary cell type harboring latent VZV will be defined by in situ hybridization and in situ transcription. VZV gene expression will be analyzed by Northern hybridization of RNA from human ganglia to VZV specific probes. The extent and specificity of viral transcription will also be determined by preparing cDNA from ganglionic RNA and using PCR to identify VZV in ganglionic cDNA; VZV-positive cDNA will then be cloned and VZV transcripts will be characterized by sequencing and analysis of VZV gene products encoded by the individual VZV genes. All experiments will be developed in parallel with studies of productive VZV infection in tissue culture. These studies will yield information on the molecular basis of viral latency and reactivation which in turn provides the foundation for future studies designed to eradicate neurologic disease in the elderly produced by VZV.
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