Recrudescence of herpes simplex virus type 1 in latently infected rats after trauma to oral tissues

Tooth extraction in rats was used to trigger a latent HSV-1 infection. HSV-1 was inoculated unilaterally in the rat palates. Eight weeks later two molars were removed bilaterally. The trigeminal ganglia were co-cultivated and HSV-1 was isolated from 63% of the ganglia on the infected sides but from only 11% on control sides. The immune response pattern was analysed by immunoblotting of rat serum, and strong reactivity to HSV-1 specific cell polypeptides and glycoproteins (ICP6, gC, pgC, gD) was seen after reactivation. The extraction sockets were histopathologically evaluated and showed healing on the infected side in 26% compared to 63% in contralateral control sockets. The effect of acyclovir (ACV) treatment was elucidated and was found to influence the subsequent development of antibodies and to promote healing of the sockets. Vesiculation in intra- and subepithelial tissue was present on the infected side in 58% but in only 12% of ACV-treated animals. The present study in rats has shown that a latent HSV-1 infection can be established and reactivated by tooth extraction. Reactivation resulted in delayed healing of sockets on the latently infected side but not on the contralateral control side. HSV-1 reactivation was demonstrated serologically by immunoblotting. Healing was significantly promoted by administration of ACV, which also supports the contention that HSV-1 interferes with the healing process.     

Loss of DNA loop supercoiling and organization in cells infected by herpes simplex virus type 1

In cells infected by herpesviruses, a sequence of nuclear changes during interphase, as well as chromosomal aberrations during mitosis, are commonly observed. These changes suggest the progressive modification of host-cell chromatin. Previous studies have shown that the early chromatin modifications in cells infected by herpes simplex virus type 1 (HSV1) are not due to extensive breakdown of host-cell DNA or disruption of the nucleosomal structure. We have previously shown that infection by HSV1 induces single-stranded breaks in the host-cell DNA early in the course of infection, and that such breaks lead to modifications in the higher-order structure of host-cell chromatin. Here we report that virus-induced DNA breaks produce permanent long-term effects on the state of supercoiling and organization of the nuclear DNA loops, comparable to the DNA loop disorganization produced by high (and irreparable) doses of ultraviolet radiation.     

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-alpha from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-alpha+ and most were also IL-6(+). Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-alpha and/or IL-6. IL-4(+) cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2(+) and/or IFN-gamma+ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-alpha and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

Distribution of herpes simplex virus type 1 and 2 genomes in the human spinal ganglia

Herpes simplex virus (HSV) is well known for its propensity to cause recurrent oral or genital mucosal infections in humans. HSV-1 is involved primarily in oral lesions, whereas HSV-2 is more frequently involved in genital lesions. Based on this, it is thought that HSV-1 may produce latent infections in trigeminal ganglia, and HSV-2 in the sacral ganglia. However the distribution pattern of latent HSV-1 and HSV-2 infections in spinal ganglia remains unknown. Using the polymerase chain reaction we detected latent herpes HSV-1 and HSV-2 in human spinal ganglia obtained from autopsy material. A pair of primers which were specific for a part of the HSV-1 and HSV-2 DNA polymerase domain were employed. HSV-1 and HSV-2 DNAs were detected in 11 of 40 (28%) and 15 of 40 (38%) cervical ganglia, respectively, 52 of 103 (50%) and 47 of 103 (46%) thoracic ganglia, 16 of 53 (30%) and 17 of 53 (32%) lumbar ganglia, and 3 of 20 (15%) and 3 of 20 (15%) sacral ganglia. These findings suggest that latent HSV-1 and HSV-2 infections have a widespread distribution from the cervical ganglia to sacral ganglia. Importantly this study demonstrated latent HSV-1 infection of both the lumbar and sacral ganglia for the first time.     

Induction of reactivation of herpes simplex virus in murine sensory ganglia in vivo by cadmium

Herpes simplex viruses maintained in a latent state in sensory neurons in mice do not reactivate spontaneously, and therefore the factors or procedures which cause the virus to reactivate serve as a clue to the mechanisms by which the virus is maintained in a latent state. We report that cadmium sulfate induces latent virus to reactivate in 75 to 100% of mice tested. The following specific findings are reported. (i) The highest frequency of induction was observed after two to four daily administrations of 100 micrograms of cadmium sulfate. (ii) Zinc, copper, manganese, or nickel sulfate administered in equimolar amounts under the same regimen did not induce viral reactivation; however, zinc sulfate in molar ratios 25-fold greater than those of cadmium induced viral replication in 2 of 16 ganglia tested. (iii) Administration of zinc, nickel, or manganese prior to the cadmium sulfate reduced the incidence of ganglia containing infectious virus. (iv) Administration of cadmium daily during the first week after infection and at 2-day intervals to 13 days after infection resulted in the recovery from ganglia of infectious virus in titers 10- to 100-fold higher than those obtained from animals given saline. Moreover, infectious virus was recovered as late as 11 days after infection compared with 6 days in mice administered saline. (v) Administration of cadmium immediately after infection or repeatedly after establishment of latency did not exhaust the latent virus harbored by sensory neurons, inasmuch as the fraction of ganglia of mice administered cadmium and yielding infectious virus was similar to that observed in mice treated with saline. We conclude that induction of cadmium tolerance precludes reactivation of latent virus. If the induction of metallothionein genes was the sole factor required to cause reactivation of latent virus, it would have been expected that all metals which induce metallothioneins would also induce reactivation, which was not observed. The results therefore raise the possibility that in addition to inducing the metallothionein genes, cadmium inactivates the factors which maintain the virus in latent state.     

Antiviral activity of FA-11H on the infectivity and replication of herpes simplex virus type 1 in cultured cells

A prominent malar complex results in a triangular facial shape. When combined with a prominent mandibular angle, the face appear to be aggressive and masculine. Surgical procedures to correct these features are commonly requested by Asian women. Baek introduced reduction malarplasty by under-positioning of the "osteotomized" zygoma. We modified Baek's procedure by sliding the osteotomized zygoma superoposteriorly. The posterior surface is not separated from the soft tissue to preserve the blood supply to the zygoma. Ten patients with prominent zygomas underwent reduction malarplasty from March 1994 through July 1996. Seven patients were female and 3 patients were male. All patients were satisfied with their results. A symmetrical appearance was achieved in all patients. This method provides for precise malar reduction under good exposure. Symmetry of the zygoma is easily achieved. There is no effect on the survival of the malar bone after the procedure because the osteotomized zygoma has its own blood supply on the posterior surface. The masseteric origin is preserved, which ensures minimal cheek drooping after reduction.     

Dimerization and activation of the herpes simplex virus type 1 protease

The quaternary state of the herpes simplex virus type 1 (HSV-1) protease has been analyzed in relation to its catalytic activity. The dependence of specific activity upon enzyme concentration indicated that association of the 27-kDa subunits strongly increased activity. Size-exclusion chromatography identified the association as a monomer-dimer equilibrium. Isolation of monomeric and dimeric species from a size-exclusion column followed by immediate assay identified the dimer as the active form of the enzyme. Activation of the protease by antichaotropic cosolvents correlated with changes in the monomer-dimer equilibrium. Thus, dimerization of the enzyme was enhanced in solvents containing glycerol or the anions citrate or phosphate. These are substances previously identified as activators of HSV-1 protease (Hall, D. L., and Darke, P. L. (1995) J. Biol. Chem. 270, 22697-22700). The relative potencies of these cosolvents as enzyme activators correlated with their efficiency in promoting dimerization. Under all solvent conditions examined, the dependence of specific activity upon enzyme concentration was consistent with a kinetic model in which only the dimer is active. Dissociation constants for the HSV-1 protease dimer determined with this model at 15 degrees C, pH 7.5, were 964 and 225 nM in 20% glycerol with 0.2 and 0.5 M citrate present, respectively. The activation of the HSV-1 protease by antichaotropic cosolvents was hereby shown to be similar in nature to the activation of the other well characterized herpesvirus protease, that from human cytomegalovirus.     

The normal association between newly replicated DNA and the nuclear matrix is abolished in cells infected by herpes simplex virus type 1

In cells infected by herpes simplex virus type 1 (HSV1), a series of nuclear changes can be observed in a temporal sequence. Such changes are related to important modifications in the higher-order structure of host cell chromatin, such as loss of DNA loop supercoiling and wholesale DNA loop disorganization. It is known that the topological relationship between DNA and the nuclear substructure is a critical factor for proper nuclear physiology. Here we report that the usual association between newly replicated DNA and the nuclear substructure, commonly known as nuclear matrix, is abrogated in cells infected by HSV1, thus establishing a correlation between the virus-induced modifications in chromatin higher-order structure and a major biochemical change within the infected cell nucleus.     

T cell responses to synthetic peptides of herpes simplex virus type 1 glycoprotein D in naturally infected individuals

To locate T cell determinants of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), proliferation assays of lymphocytes obtained from 10 healthy HSV-seropositive individuals were performed using 34 overlapping gD peptides as antigens. Despite large differences between individual responses to the peptides both in number of stimulating peptides and gD regions, three regions (1-54, 110-214, and 290-314) induced a response in 50% or more of the HSV-seropositives. T cells were less frequently stimulated by peptides of region 210-294. No correlation was found between serological data and proliferative responses to the peptides. The diversity in T cell response to the peptides suggests a lack of immunodominance, implying that a single peptide/region of gD, or a combination of peptides, will not be sufficient to serve as a basis for a future HSV-1 vaccine.     

Neuronal sprouting in mouse sensory ganglia infected with herpes simplex virus type 2 (HSV-2): induction of growth-associated protein (GAP-43) and ultrastructural evidence

Herpes simplex virus (HSV) is neurotropic and when inoculated on the mouse footpad is retrogradely transported to the associated dorsal root ganglia (DRG), where infection is established. Previous observations suggest that, after HSV infection, sensory ganglion neurons may mount a sprouting response. In our HSV-infected DRG model, we investigate this issue by (1) examining expression of growth-associated protein (GAP-43), a molecule known to be induced by growing axons, and (2) determining ultrastructurally whether HSV-infected dorsal roots contain neurites. In a time course study, we show that GAP-43 is induced both in HSV-infected DRG and their central processes. The increase in GAP-43 is first seen 2 weeks following unilateral footpad inoculation in both cell bodies and dorsal roots, and is sustained at 1 month post inoculation in roots but not in perikarya. Large bundles of unmyelinated small caliber axons, lacking Schwann cell ensheathment, are observed by electron microscopy in dorsal roots 2 weeks and 1 month following inoculation. These profiles resemble developing or regenerating neurites and are rarely seen in roots of mock-infected or uninfected controls. The increased GAP-43 immunoreactivity and ultrastructural changes shown here, in conjunction with previously documented selective neuropeptide and enzyme alterations, confirm that a sprouting response is mounted in sensory ganglia following acute HSV infection.     

The region of the herpes simplex virus type 1 LAT gene involved in spontaneous reactivation does not encode a functional protein

A family of muscarinic acetylcholine receptor proteins mediates diverse pre- and postsynaptic functions in the hippocampus. However the roles of individual receptors are not understood. The present study identified the pre- and postsynaptic muscarinic acetylcholine receptors at the perforant pathway synapses in rat brain using a combination of lesioning, immunocytochemistry and electron microscopic techniques. Entorhinal cortex lesions resulted in lamina-specific reductions of m2, m3, and m4 immunoreactivity in parallel with the degeneration of the medial and lateral perforant pathway terminals in the middle and outer thirds of the molecular layer, respectively. In contrast, granule cell lesions selectively reduced m1 and m3 receptors consistent with degeneration of postsynaptic dendrites. Direct visualization of m1-m4 by electron microscopic immunocytochemistry confirmed their differential pre- and postsynaptic localizations. Together, these findings provide strong evidence for both redundancy and spatial selectivity of presynaptic (m2, m3 and m4) and postsynaptic (m1 and m3) muscarinic acetylcholine receptors at the perforant pathway synapse.     

Effects of a traditional Chinese herbal medicine, Kanzo-bushi-to, on the resistance of thermally injured mice infected with herpes simplex virus type 1

The protective effect of Kanzo-bushi-to (TJS-038) was investigated on the opportunistic infection of herpes simplex virus type 1 (HSV) in thermally injured mice (TI-Mice). We have previously reported that TI-Mice were approximately 100 times more susceptible to HSV infection than normal mice (N-Mice) and that CD8+ suppressor T (ST)-cells induced by burn injury were involved in causing this increased susceptibility of TI-Mice. Increased susceptibility of TI-Mice to the infection was reversed to the levels observed in N-Mice when TI-Mice were treated intraperitoneally with TJS-038 at a dose of 5 mg/kg 1 and 4 days after thermal injury. The activity of ST-cells was greatly decreased in TI-Mice treated with TJS-038. The generation of Vicia villosa lectin-adherent CD4+ CD28+ TCR-alpha/beta+ contrasuppressor T (Contra-ST)-cells associated with the appearance of ST-cells was expanded and occurred earlier in spleens of TJS-038-treated TI-Mice as compared with that of untreated TI-Mice. The improved resistance of TJS-038-treated TI-Mice to the infection was transferred to untreated TI-Mice by adoptive transfer of Contra-ST-cells prepared from TJS-038-treated TI-Mice. These results suggest that TJS-038 may restore the resistance of TI-Mice to the HSV infection through the expanded generation of Contra-ST-cells.