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A prospective study was conducted that evaluated duration of virus shedding through acute and experimentally-induced recurrent disease episodes in 12 cats, and tissue distribution of latent infections, following intranasal vaccination with a temperature sensitive (ts) mutant strain of feline herpesvirus 1 (FHV1). Six of these cats were challenged with a virulent field strain of the agent to assess the extent to which vaccination affected subsequent shedding of virus and the establishment of latent infections. Virus isolation (VI) tests were done in parallel with a polymerase chain reaction (PCR) assay to compare the performance of each diagnostic method. The PCR confirmed that all 12 cats shed virus throughout the periods of vaccination, challenge or mock-challenge, and a cyclophosphamide-dexamethasone stress protocol to reactivate latent infections. Shedding to the tsFHV1 was documented by VI for up to 25 days following vaccination and for up to 15 days following challenge, but not after experimental stress. Overall, FHV1 was present in 144 of 300 (48%) cat-days of testing by PCR compared to 32 of 300 (11%) by VI. The frequency and distribution of latent FHV1 detected in neurologic, ophthalmic, and other tissues by PCR were identical for vaccine-only and vaccine-challenge groups, thereby disproving previous hypotheses that tsFHV1 mutants administered by this route protect against latency.