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A rapid and physiologically relevant hyperthermia-based induction procedure has been utilized to develop an in vivo model of induced herpes simplex virus (HSV) reactivation in outbred Swiss Webster mice. This procedure was found to efficiently reactivate latent virus from both trigeminal and lumbosacral ganglia. Examination of the time between hyperthermia and virus production demonstrated that detectable levels of infectious virus were present in ganglia as soon as 14 h posttreatment, with peak percent recoveries at 24 h. These data indicated that the switch from latent to active viral gene transcription occurred rapidly following treatment. Immunohistochemical staining for HSV type 1 antigens revealed rare antigen-positive ganglionic neurons 24 h postinduction. HSV antigens were not detected in any other cell type, and lateral spread of the infection was not observed. This is the first report of the detection of HSV antigens in vivo following induced reactivation in the intact nervous system and demonstrates that the neuron is the site of infectious virus production. In addition, our data strongly suggest that at least some neurons in which HSV antigens are detected during reactivation do not survive. Because the temporal and spatial characteristics of HSV reactivation have been clearly defined, this model is uniquely suited for the molecular dissection of the reactivation process.