RESUMO
Herpes simplex virus (HSV) latent infection of ganglion neurons follows axoplasmic transport of HSV, probably in the form of nucleocapsid from peripheral sites of infection (e.g. footpad). This raises the possibility that latency is dependent on this particular means of presenting HSV to ganglion neurons. To investigate this, we directly infected ganglia of mice with HSV and evaluated latency. Initially, ganglia were surgically exposed in intact mice, infected with HSV and after 4 weeks evaluated for HSV latency-associated transcript (LAT) expression. LAT expression suggested latency. To more fully evaluate latency after direct ganglion inoculation, a transplant model was developed. In this model, ganglia were removed from mice, inoculated with HSV, transplanted into syngeneic recipients and evaluated for latency after several weeks. Latency was evident in transplanted ganglia by (1) the presence of LAT in neurons; (2) the lack of HSV ICP4 RNA or viral antigen, and (3) the isolation of HSV from explants of transplants but not from direct homogenates. The transplant model was then used to evaluate the effect of inhibition of HSV replication on latency. Antivirals which inhibited HSV replication markedly decreased the number of LAT-positive neurons in transplants, suggesting a role for HSV replication mechanisms and latency. It is thought that direct ganglion inoculation and ganglion transplant methods will permit unique investigations of mechanisms of latency.
Assuntos
Gânglios Espinais/virologia , Herpes Simples/virologia , Simplexvirus/fisiologia , Latência Viral , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Aciclovir/farmacologia , Animais , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Famciclovir , Gânglios Espinais/citologia , Gânglios Espinais/transplante , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Herpes Simples/metabolismo , Herpes Simples/patologia , Proteínas Imediatamente Precoces/análise , Proteínas Imediatamente Precoces/genética , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/citologia , Neurônios/virologia , RNA Viral/análise , Simplexvirus/isolamento & purificação , Células Vero , Carga Viral , Latência Viral/efeitos dos fármacos , Latência Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
Sensory ganglia latently infected with herpes simplex virus (HSV) were transplanted beneath the renal capsule of syngeneic recipients, and the latent infection remaining was investigated. HSV latency-associated transcript (LAT) expression and reactivation of HSV after explant of transplanted dorsal root ganglia were monitored as markers of latency. Two to four weeks after transplantation, both indicated evidence of HSV latency in transplants. At those times, infectious virus was not detected in direct ganglion homogenates. In addition, viral antigen and infected cell polypeptide 4 RNA were not detected. Taken together, the results suggested that HSV latent infection rather than persistent infection was present in transplants. From these results, two explanations seemed possible: latency was maintained in transplanted neurons, or alternatively, latency developed after transplantation, in neurons not previously latently infected. The latter was considered putative secondary latency and was investigated in three ways. First, evidence of reactivation which might serve as a source for secondary latency was evaluated. Reactivation of HSV in transplants was evident from HSV antigen expression (52% of transplants) and the presence of cell-free virus (38% of transplants) 3 to 5 days after transplantation. Second, putative secondary latency was investigated in recipients immunized with HSV prior to receiving latently infected ganglia. Reactivation was not detected 3 to 5 days after transplantation in immunized recipients, and LAT expression was rare in these recipients after 3 to 4 weeks. Lastly, the possibility of secondary latency was investigated by comparing results obtained with standard HSV and with reactivation-defective thymidine kinase-negative (TK-) HSV. Defective reactivation of TK- HSV was demonstrated by immunohistochemistry and by the inability to isolate infectious virus. Donor dorsal root ganglia latently infected with TK+ HSV showed many LAT-positive neurons 2 or more weeks after transplantation (average, 26 per transplant). However, LAT expression was undetectable or minimal > 2 weeks after transplantation in donor ganglia latently infected with TK- HSV (average, 0.2 per transplant). Impaired reactivation of TK- HSV-infected donor ganglia after transplantation, therefore, was correlated with subsequent limited LAT expression. From these results, the occurrence of secondary latency was concluded for ganglia latently infected with TK+ HSV and transplanted beneath the kidney capsule. In vivo reactivation in this transplant model may provide a more useful means to investigate HSV reactivation than in usual in vitro explant models and may complement other in vivo reactivation models. The occurrence of secondary latency was unique. The inhibition of secondary latency by the immune system may provide an avenue to evaluate immunological control of HSV latency.
Assuntos
Gânglios Espinais/transplante , Gânglios Espinais/virologia , Simplexvirus/fisiologia , Latência Viral , Animais , Antígenos Virais/análise , Feminino , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Simplexvirus/genética , Simplexvirus/imunologia , Timidina Quinase/análise , Ativação ViralRESUMO
Herpes simplex virus (HSV) is a common neurotropic virus, and latent infection of sensory ganglion neurons readily occurs in humans and in experimentally infected animals. During HSV latency, infectious virus and viral antigen are not detected, and HSV transcription is limited to specific RNA termed latency-associated transcript (LAT). In the present study, the effect of altered nervous system function on HSV latent infection was investigated in dorsal root ganglia (drg) of experimentally infected mice. Latent infection of lumbar drg was established by footpad inoculation of HSV. During latency, sciatic neurectomy was performed in order to modify the in vivo function of latently infected neurons, and HSV LAT and HSV DNA in drg were investigated. Neurectomy has been used in many neurobiological studies to alter neuronal RNA and protein expression. After neurectomy there was a marked decrease in the number of LAT-positive neurons and in the amount of ganglion LAT. This was determined by in situ and RNA (Northern) blot hybridization. The neurectomy-related decrease of HSV LAT was apparent 9-10 days after neurectomy and was more marked after 21 days. The decrease was noted both in drg latently infected with standard thymidine kinase-positive (TK+) HSV and in ganglia infected with mutant TK- HSV. Since TK- HSV is largely reactivation defective, it is concluded that the neurectomy-induced decrease of LAT was probably not the result of in vivo HSV reactivation. It is acknowledged, however, that abortive reactivation by TK- HSV may occur, and decrease of latency may have resulted from neuronal or other host mechanisms subsequent to this. In order to investigate residual HSV latency, in addition to viral transcription, HSV DNA in drg was evaluated by polymerase chain reaction techniques. Decrease of HSV DNA was noted after neurectomy in drg latently infected with either TK+ or TK- HSV. It is suggested that the decrease in LAT expression detected was due to the change in neuronal transcription which is part of the neurectomy-induced axon reaction. Decreased HSV LAT may have led to decreased HSV DNA and latency. The decrease in the molecular markers of HSV latency following neurectomy emphasized the importance of neuronal control mechanisms in the pathogenesis of HSV latent infection.
Assuntos
Herpes Simples/microbiologia , Neurônios/microbiologia , Simplexvirus/fisiologia , Animais , Sequência de Bases , Contagem de Células , DNA Viral/metabolismo , Feminino , Gânglios Espinais/microbiologia , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/fisiologia , RNA Viral/metabolismo , Nervo Isquiático/microbiologia , Nervo Isquiático/cirurgia , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/metabolismo , Transcrição Gênica , Replicação ViralRESUMO
The presence of herpes simplex virus type 2 (HSV-2) transcription during in vivo latent infection was investigated by in situ hybridization. Latent infection of mouse dorsal root ganglion was investigated with the BamHI p fragment of HSV-2, which resulted in evidence of ganglion hybridization, and other fragments representing approximately 40% of the genome, which did not result in hybridization. Strand specificity of hybridization was investigated in studies with synthetic oligonucleotides, which supported the conclusion that a latency-associated transcript(s) had been detected. Hybridization was detected with oligonucleotides complementary to the infected-cell polypeptide 0 (ICP0) template strand but not with oligonucleotides synthesized from the ICP0 template strand. Although most hybridization occurred over neurons, in some instances hybridization appeared to occur over nonneuronal ganglion cells, and this was more evident when tissue sections were examined by phase contrast microscopy. Although these results supported the usual neuronal site of HSV-2 latency, latency in nonneuronal cells may be important in considering the pathobiology of HSV-2 infections.
Assuntos
Gânglios Espinais/microbiologia , Herpes Simples/microbiologia , RNA Viral/biossíntese , Simplexvirus/genética , Animais , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Masculino , Camundongos , Neurônios/microbiologia , Hibridização de Ácido Nucleico , RNA Viral/análise , Coelhos , Mapeamento por RestriçãoRESUMO
The presence of herpes simplex virus (HSV) latency-associated transcript (LAT) was investigated in sensory ganglion neurons of mice after inoculation with thymidine kinase (TK) mutants of HSV. Ganglion serial sections were examined in order to quantitate numbers of LAT-positive neurons. After inoculation with TK-positive HSV, virus was isolated during latency from explants of most ganglia, and LAT was detected by in situ hybridization in 96% of ganglia. After inoculation with HSV TK mutants, virus was isolated from 0% of ganglia, but LAT was detected in 95 to 100% of ganglia. After inoculation of TK mutants of HSV, therefore, although latent infection as indicated by the isolation of virus from ganglion explants was not detected, the presence of LAT was common. These results suggest that the lack of reactivatable virus after inoculation of HSV TK mutants may be related to a role for HSV TK expression in the reactivation process.
Assuntos
Gânglios/microbiologia , Neurônios Aferentes/microbiologia , Simplexvirus/genética , Timidina Quinase/genética , Transcrição Gênica , Animais , Sondas de DNA , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Mutação , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Simplexvirus/enzimologiaRESUMO
Dorsal root ganglion neurons, which express herpes simplex virus (HSV) latency-associated transcript (LAT) during experimental latent infection, were investigated by in situ hybridization. The number of LAT-positive neurons was determined by examination of ganglion serial sections. In other latently infected mice, the number of ganglion neurons that reactivated HSV antigen after explant culture was determined in serial sections. LAT was detected in 100 percent of ganglia, with an average of 19.5 LAT-positive neurons per ganglion. After explant culture of latently infected ganglia (in the presence of colchicine to decrease spread of reactivated virus), HSV antigen was detected in 94 percent of ganglia, with an average of 13.1 positive neurons in the antigen-positive ganglia. The similar quantities of LAT- and antigen-positive neurons within ganglia support the hypothesis that LAT-positive neurons were the neurons from which HSV was reactivated.
Assuntos
Gânglios Espinais/microbiologia , Herpes Simples/microbiologia , Neurônios/microbiologia , Animais , Sondas de DNA , Camundongos , Hibridização de Ácido Nucleico , Fatores de TempoRESUMO
Section of the sciatic nerve during the period of herpes simplex virus (HSV) latent infection was performed to evaluate residual latency in mouse dorsal root ganglion. In control mice without sciatic neurectomy, latency was present in 90-100%, while in those which underwent a neurectomy procedure, latent infection was surprisingly decreased to 28-50%. To investigate the hypothesis that the decrease of latency resulted from HSV reactivation and replication (with subsequent neuron destruction), groups of mice were treated with acyclovir to inhibit HSV reactivation, after having undergone a neurectomy procedure. Acyclovir treatment largely prevented the neurectomy-related elimination of latency and supported the hypothesized mechanism.
Assuntos
Gânglios Espinais/microbiologia , Herpes Simples/microbiologia , Simplexvirus/fisiologia , Aciclovir/uso terapêutico , Animais , Denervação , Feminino , Herpes Simples/tratamento farmacológico , Masculino , Camundongos , Recidiva , Nervo Isquiático/cirurgiaRESUMO
Infection of trigeminal ganglion by herpes simplex virus (HSV) thymidine kinase-negative (TK-) mutants was investigated in mixed infection studies in mice. Mice were corneally inoculated with TK- HSV alone or with mixtures of TK- HSV-TK+ HSV. When inoculated alone, an arabinosylthymine-selected HSV type 1 TK- mutant and a HSV type 2 TK- deletion mutant infected mouse ocular tissues but rarely infected ganglion tissues. However, both TK- mutants readily infected ganglion tissues when they were inoculated in mixtures with TK+ HSV. By means of mixed infection studies, it was demonstrated that TK- HSV could readily establish acute and latent ganglion infections. It was thought that the frequent infection of trigeminal ganglion tissue by both TK- mutants after mixed TK(-)-TK+ HSV infection was the result of in vivo complementation. After mixed TK(-)-TK+ HSV infection and subsequent cultivation of ganglion explants in arabinosylthymine, results supported the conclusion that when TK- was present in ganglia it was in the same neurons that contained TK+ HSV.
Assuntos
Ceratite Dendrítica/microbiologia , Simplexvirus/genética , Timidina Quinase/genética , Gânglio Trigeminal/microbiologia , Nervo Trigêmeo/microbiologia , Proteínas Virais/genética , Animais , Feminino , Teste de Complementação Genética , Masculino , Camundongos , Simplexvirus/enzimologia , Simplexvirus/fisiologia , Cultura de VírusRESUMO
Determinations of the possible importance of herpes simplex virus type 1 (HSV) thymidine kinase (TK) expression in the pathogenesis of viral latency depend in part on the use of defined mutants. In a recent study by A. E. Sears, B. Meignier, and B. Roizman (J. Virol. 55, 410-416 (1985], in which they utilized genetically engineered viral recombinants considered to be TK-, the role of HSV TK expression in latency was reported to be minimal. To further investigate this conclusion we intensively studied the TK phenotypes of their M316-2 and M316-10 HSV-1 mutants. TK activity was investigated by phosphorylation of thymidine, by arabinosylthymine (ara-T) inhibition and by virus plaque autoradiography. TK activity of the M316-2 and M316-10 HSV mutants was not detected in 5-min assays (as performed by Sears et al.), but in longer assays substantial activity was apparent. In contrast, in assays of control TK- viruses, activity was minimal or absent at all time points. In ara-T inhibition assays the M316-2 and M316-10 viruses were inhibited more than 10-fold, consistent with viruses of intermediate TK activity. By plaque autoradiography both of these viruses produced plaques which incorporated significant amounts of thymidine. Based on these results we conclude that the M316-2 and M316-10 viruses should likely be considered to express intermediate levels of TK activity. HSV latency results using these mutants may need to be interpreted with this in mind.