Herpes simplex virus infection of mammalian hosts involves lytic replication at a primary site, such as the cornea, translocation by axonal transport to sensory ganglia and replication, and latent infection at a secondary site, ganglionic neurons. The virus-encoded thymidine kinase, which is a target for antiviral drugs such as acyclovir, is not essential for lytic replication yet evidently is required at the secondary site for replication and some phase of latent infection. To determine the specific stage in viral pathogenesis at which this enzyme is required, we constructed virus deletion mutants that were acyclovir resistant and exhibited no detectable thymidine kinase activity. After corneal inoculation of mice, the mutants replicated to high titers in the eye but were severely impaired for acute replication in trigeminal ganglia and failed to reactivate from ganglia upon cocultivation with permissive cells. Nevertheless, latency-associated transcripts were expressed in neuronal nuclei of ganglia from mutantinfected mice and superinfection of the ganglia with a second virus rescued the latent mutant virus. Thus, contrary to a widely accepted hypothesis, the thymidine kinase-negative mutants established latent infections, implying that neither thymidine kinase activity nor ganglionic replication is necessary for establishment of latency. Rather, thymidine kinase appears to be necessary for reactivation from latency. These results suggest that acyclovir-resistant viruses could establish latent infections in clinical settings and have implications for the use of genetically engineered herpesviruses to deliver foreign genes to neurons.

The purposes of this study were (1) to determine normal values for cervical active range of motion (AROM) obtained with a "cervical-range-of-motion" (CROM) instrument on healthy subjects whose ages spanned 9 decades, (2) to determine whether age and gender affect six cervical AROMs, and (3) to examine the intratester and intertester reliability of measurements obtained. Measurements were made on 337 subjects (171 females and 166 males) whose ages ranged from 11 to 97 years. Measurements were taken by five physical therapists with 7 to 30 years of clinical and teaching experience. Among male and female subjects of the same age, females had a greater AROM than did males for all AROMs except neck flexion. Among both males and females, each of the six cervical AROMs decreased significantly with age. From two pilot studies separate from the acquisition of the normal database, we determined our intratester and intertester reliabilities for making neck AROM measurements with the CROM instrument. We concluded that AROM measurements on the cervical spine with the CROM instrument demonstrated good intratester and intertester reliability, because the intraclass correlation coefficients were generally greater than .80.

We have generated and characterized a deletion mutant of herpes simplex virus type-1, dlLAT1.8, which lacks the putative promoter region, transcriptional start site, and 1,015 base pairs of the DNA sequences specifying the latency-associated transcripts (LATs). When tested in a CD-1 mouse ocular model, dlLATI.8 was replication competent in the eye and in ganglia during acute infection but reactivated from explant cultures of ganglia with reduced efficiency (49%) relative to those of wild-type and marker-rescued viruses (94 and 85%, respectively) despite the fact that levels of mutant viral DNA in ganglia during latent infection were comparable to wild-type levels. The neurovirulence of KOS was not significantly altered by the removal of sequences specifying the LATs, as judged by numbers of animals dying on or before 30 days postinfection. Examination of ganglia latently infected with dlLAT1.8 by in situ hybridization revealed no LAT expression. The genotype of reactivated virus was identical to that of input dlLAT1.8 virus as judged by Southern blot analysis. These studies suggest that although the LATs are not essential for the establishment and reactivation of latency in our model, they may play a role in determining the frequency of reactivation of virus from the latent state.

Using nonsense and deletion mutants of herpes simplex virus type 1, we investigated the roles of three immediate-early proteins (ICP4, ICP27 and ICPO) in the establishment and reactivation of ganglionic latency in a mouse ocular model. DNA hybridization, superinfection-rescue, and cocultivation techniques provided quantitative data that distinguished between the failure of a virus to establish latency in the ganglion and its failure to reactivate. Null mutants with lesions in the genes for ICP4 and ICP27 did not replicate in the eye or in ganglia and failed to establish reactivatable latent infections. Three ICPO deletion mutants which could replicate in the eye and ganglia varied in their ability to establish and reactivate from the latent state, demonstrating that ICPO plays a role both in the establishment and the reactivation of latency. The use of viral mutants and a variety of stage-specffic assays allowed us to better define the stages in the establishment and reactivation of herpes simplex virus type 1 latency.

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