Reactivation Of HSV-1 Research Articles

The persistent elevated cytokine mRNA levels in trigeminal ganglia of mice latently infected with HSV-1 are not due to the presence of latency associated transcript (LAT) RNAs

Trigeminal ganglia (TG) from mice latently infected with wild type HSV-1 contain detectable levels of cytokine transcripts that are not present in TG from uninfected mice. This suggests that during HSV-1 neuronal latency, the immune system is stimulated by the production of one or more viral proteins. Since the LAT (latency associated transcript) gene is essential for wild type levels of spontaneous reactivation and is the only highly active viral gene during latency, the stimulation of cytokines may indicate the presence of a LAT encoded latency protein. We therefore compared the cytokine transcript profiles in the TG of mice latently infected with wild type and LAT negative viruses. Mice were latently infected with either: (1) the LAT null mutant dLAT2903; (2) its marker rescued virus dLAT2903R; or (3) the parental wild type HSV-1 strain McKrae. As expected, reactivation following explant cultivation of TG from latently infected mice was significantly decreased with dLAT2903 ( P<0.05)(40±8%, n=24) compared with dLAT2903R (85±7.6%, n=36) or the parental virus (70±10.0%, n=36). The relative levels of various cytokines was determined by RT-PCR analysis of TG extracts. None of the cytokine transcripts detected in mice latently infected with the wild type or marker rescued viruses were missing or decreased in mice latently infected with the LAT null mutant 30 or 60 days post infection. There were also no differences in the HSV-1 antibody titers induced by the LAT negative virus compared to the LAT positive viruses. Thus, although LAT facilitated reactivation of HSV-1 from explanted mouse TG, expression of LAT during latency did not appear to be involved in persistent cytokine expression in TG. This suggests that during latency, HSV-1 does not produce a highly antigenic abundant LAT encoded protein.

Assessment of a selective inhibitor of herpes simplex virus thymidine kinase (L-653,180) as therapy for experimental recurrent genital herpes.

Herpes simplex virus (HSV)-coded thymidine kinase (TK) is important in efficient reactivation of latent infection. These studies were designed to investigate whether treatment of latently infected animals with a TK inhibitor altered the natural history of recurrent HSV disease. 9-([(Z)-2-(hydroxymethyl)cyclohexyl]methyl) guanine (L-653,180) is a potent and selective nonsubstrate inhibitor of HSV TK which can suppress or delay reactivation of HSV-1 from latently infected cells in vitro without affecting viral replication. In an initial study, six female Hartley guinea pigs were treated with L-653,180 in their diet (25 mg/30 g of food) and water (300 mg/liter) for 7 days. Blood, urine, kidney, liver, spinal cord, and cerebral cortex specimens were collected. L-653,180 was detected in all specimens at concentrations which, although low, were higher than the in vitro 50% inhibitory concentration of the drug against HSV TK. In the second study, 20 female Hartley guinea pigs were randomized into two groups following recovery from primary genital HSV-2 infection. One group received L-653,180 in diet and water for 4 weeks beginning 21 days postinoculation. Animals were examined daily for recurrent lesions for 10 weeks. Treated animals experienced fewer recurrences during the treatment period but the results were not significantly different from results with controls. During the first 2-week posttreatment period, L-653,180-treated animals had significantly fewer recurrences than control animals (P = 0.02). Over the entire 10-week observation period, treated animals experienced fewer recurrences (P = 0.06). These results suggest that inhibitors of viral TK may be useful in limiting reactivation of latent virus and thus recurrent infections. In these experiments, the amount of drug that could be administered to the animals was limited by its poor solubility. Further studies with more potent and soluble inhibitors of HSV TK appear to be warranted.

Effect of ibuprofen on the in vitro and in vivo reactivation of latent HSV-1

Prostaglandins have been suggested to play an important role in the reactivation of latent herpes simplex virus. To further understand the role of prostaglandins in the reactivation process, we investigated the effects of ibuprofen, a nonsteroidal anti-inflammatory drug with prostaglandin synthesis inhibitory activity, on the in vitro and in vivo reactivation of latent type 1 herpes simplex virus in mouse ganglia and rabbits, respectively. Ibuprofen, at a concentration of 50 or 100 μM, did not alter the titer of reactivated virus from explanted ganglia with latent virus, but, at a concentration of 200 or 500 μM, it significantly reduced the reactivated viral titer from the ganglia. Ibuprofen also directly inhibited the replication of herpes simplex virus in trigeminal ganglia and Vero cell monolayers, which indicates that the drug reduced the recovery of reactivated viral titers from explanted ganglia with latent virus by acting on the replication process rather than on the reactivation mechanism in vitro. The systemic administration of ibuprofen failed to demonstrate any significant effect on the ocular shedding of virus after attempted reactivation by 6-hydroxydopamine iontophoresis in rabbits with latent herpes simplex virus infection. This failure in vivo could be due to the short half-life and low concentration of ibuprofen at the site of reactivation and replication of latent virus. Alternatively, in the clinical setting, it is conceivable that ibuprofen may not have an effect on in vivo reactivation of latent herpes.

Herpes simplex virus latency in the nervous system--a new model.

Permissive herpes simplex virus (HSV) infection in tissue culture results in host cell destruction. Latent HSV infection in vivo occurs in neurons of peripheral sensory ganglia (PSG) and it therefore can not take place in neurons in which the virus has completed a lytic replication cycle similar to that present in vitro. Our hypothesis, based on experimental data and observations in humans, suggests that establishment of latent infection and reactivation of HSV-1 does not involve neuronal cell loss. Latency is established in neurons in which the virus does not replicate and is determined, in part, by the tissue levels of a herpes transactivating protein (Vmw65) that is a component of the viral tegument. We also suggest that reactivation of latent infection does not involve destruction of neurons and is due to replication of virus at the peripheral mucocutaneous tissues to where virus or viral DNA have been transported from the nervous tissue. Alternatively, reactivation is initiated in the PSG using a replication cycle which does not involve irreversible damage to neurons. This model explains the lack of damage to neurons which continue to serve as permanent reservoirs of latent virus for the entire life of the host.

The role of cyclic nucleotide mediators in latency and reactivation of HSV-1 infected neuroblastoma cells.

The mechanisms that control herpes simplex virus type 1 latency and reactivation are still poorly understood. We developed an in vitro murine neuroblastoma cell HSV-infected, acyclovir suppressed model to study the influence of different cyclic nucleotide mediators on the latency and reactivation of HSV-1. A positive cDNA 'in situ' hybridisation for HSV genome was used to prove the establishment of a viral-host cell nuclear relationship. An ABC-immunoperoxidase reaction to cell surface HSV mature glycoproteins was also performed to determine the time of viral reactivation with formation of mature virions. Supernates of cultured cells were placed on Vero cells for confirmation of reactivation by classic cytopathic effect. Theophylline (50 micrograms/ml) and dibutyryl-cAMP (0.1, 0.5, 1 mg/ml) produced the most pronounced response, accelerating HSV reactivation time by 150%. Epinephrine (10, 20 micrograms/ml) had an intermediate effect on accelerating viral reactivation; and verapamil (20, 50 micrograms/ml), theophylline and epinephrine at lower doses had a smaller effect. Carbamylcholine (10 micrograms/ml) prolonged the time to viral reactivation by 100%, 36 hours compared to control time of 18 hours. Insulin (0.1, 0.5, 1 mg/ml) also prolonged HSV 'latency' by six hours. Exogenous dibutyryl-cGMP and carbamylcholine at lower concentrations did not have an effect on viral reactivation. These findings suggest that there is a relationship between changes of intracellular concentration of cyclic nucleotides and HSV latency and reactivation.

Host species and strain differences affect the ability of an HSV-1 ICP0 deletion mutant to establish latency and spontaneously reactivate in vivo

HSV-1 latency and reactivation were studied in vivo by spontaneous and iontophoresis-induced ocular viral shedding in New Zealand rabbits, Balb/c and A/J mice latently infected with wild-type KOS, and dl × 3.1, a progeny ICP0 deletion mutant. The presence of trigeminal ganglionic latency was demonstrated by the in vitro methods of cocultivation and in situ hybridization. Although the efficiency of ganglionic latency was significantly less ( P < .0001) for dl × 3.1 than for KOS in both mice and rabbits, only dl × 3.1 shed spontaneously in the NZ rabbit. lontophoresis of adrenergic agents failed to induce reactivation and ocular viral shedding of KOS or dl × 3.1 in mice or rabbits. The establishment of latency and reactivation of KOS and dl × 3.1 was dependent on the host animal. We conclude that host factors as exemplified by host species and host strain differences significantly affected the ability of KOS and dl × 3.1 to establish latency, to reactivate, and to shed spontaneously. ICP0 expression was not required for the establishment or maintenance of latency, nor was it required for the reactivation of latent HSV-1. Furthermore, the biological activity of KOS and dl × 3.1 during latency in vivo did not correlate with latency studies based on in vitro methods.

Vanadate promotes reactivation and ionotophoresis-induced ocular shedding of latent HSV-1 W in different host animals

Vanadate is a potent inhibitor of calcium stimulated ATPase, Na, K-ATPase, and may have adrenergic activity. Using the iontophoresis method, we compared vanadate to a BSS control and the standard iontophoresis model (6-hydroxydopamine/epinephrine) by measuring induced ocular shedding of latent HSV-1 in different host animals. Latent trigeminal ganglionic infections were established in Balb/c mice and New Zealand rabbits following corneal inoculation with HSV-1 [W] strain, and later confirmed by cocultivation. Latently-infected animals (greater than 1 month post-infection) were divided into three treatment groups. Each group was iontophoresed with BSS, vanadate 1% or 6-HD 1%, and then treated topically for 10 days with BSS, vanadate or epinephrine respectively. Reactivation and recovery of latent HSV-1 was detected by daily ocular swabbing, plating, and observing progressive viral growth in Vero cells. The vanadate group had more virus-positive eyes than the BSS control group in mice, (8/32 vs. 1/32 P less than .01), and also in rabbits (14/20 vs 6/22 P less than .01). Virus-positive animals and total positive swabs were also higher for vanadate than BSS in both mice and rabbits. Furthermore, while vanadate was associated with fewer virus-positive eyes than 6-HD & EPI (8/32 vs. 17/32 P less than .02) in mice, there were no significant differences in rabbits. We conclude that vanadate promotes ocular shedding of latent HSV-1, and may act through an adrenergic mechanism.

The effect of alpha blockade on iontophoresis-induced ocular shedding of latent HSV-1 W in different host animals

Previous studies demonstrated that non-specific beta blockade promoted ocular shedding of latent HSV-1 in the mouse and rabbit iontophoresis models. The present study examined the effect of topical alpha blockers, thymoxamine and corynanthine, on reactivation and induced ocular shedding of latent HSV-1 W in different host animals. Latent trigeminal ganglionic infection was established in Balb/c mice and New Zealand rabbits following corneal inoculation with HSV-1 W strain, and later confirmed by co-cultivation. Treatment with coded eye drops (thymoxamine, corynanthine or BSS was begun one day prior to iontophoresis induction and continued BID OU for 5 days. Reactivation and recovery of latent HSV-1 was determined by daily ocular swabs, and characteristic HSV-1 cytopathic effect in Vero cells. In Balb/c mice, topical administration of thymoxamine 0.5% or corynanthine 5% significantly reduced the number of virus-positive eyes, virus-positive mice, and total virus-positive swabs per experiment, whereas the inhibitory effect was minimal in NZ rabbits. We conclude that alpha blockade may alter the reactivation signal that is mediated via the adrenergic system, and that different host factors (as expressed in different species) may play an important role in this process.

Effects of acyclovir therapy during simultaneous reactivation of latent HSV-1 in rabbits

Acyclovir (ACV) therapy with a simultaneous reactivation of latent HSV-1 was evaluated in HSV-1 infected rabbit eyes. When the latently infected rabbits received epinephrine iontophoresis into corneas without ACV therapy 100% of eyes shed virus into tear film. The shedding was initiated on the second day of the epinephrine iontophoresis and lasted for an average of 4.6 days. When the rabbits received ACV (60 mg/kg body weight) intravenously once daily and topically (5% ACV ointment) twice daily for 6 consecutive days while a 0.01% epinephrine solution was iontophoresed into cornea for the first 3 consecutive days, 33% ( 2 6 ) of eyes showed detectable HSV-1 in the tear film only after terminating the ACV therapy, and the duration of shedding was for only one day. The average quantity of virus detectable in the tear film was decreased 14-fold in the latter group compared to the epinephrine iontophoresis group without ACV therapy. Four days after the last ACV therapy the titer of HSV-1 in the cell-free homogenates of the trigeminal and superior cervical ganglia was determined. For the epinephrine iontophoresed group, 67% of ganglia ( 8 12 ) were HSV-1 positive, while only 33% ( 4 12 ) of the ganglia from the combined treatment group were HSV-1 positive. The difference was statistically significant ( p = 0.034). Furthermore, the titer of virus detectable in the cell-free homogenates of the virus-positive ganglia from the combined treatment group was less than that from the ganglia of the epinephrine iontophoresed group or untreated group. This suggests a reduction in the total number of latent foci for the combined treatment group.

HSV-1 latency: thymidine kinase requirement and the round-trip theory.

The present study used the rabbit iontophoresis model to examine the thymidine kinase (TK) requirement for HSV-1 latency, and test the round trip theory of latency with emergent phenotypic mutations of the HSV-1 TK negative (TK-) inoculating strain. The results demonstrated repeated induced ocular shedding of latent HSV-1 in 14-100% of rabbits. The TK phenotype of recovered tear film following spontaneous shedding and repeated iontophoresis induction was thymidine kinase negative in 90% of eyes. Sequential shedding of TK- virus from the same eye over time was demonstrated in 21% of eyes in 33% of rabbits. We conclude that TK is not an absolute requirement for the establishment or reactivation of latent HSV-1 in the rabbit model. The round trip theory of latency was not supported as TK+ isolates and syncytial variants of the TK- inoculating strain which were recovered at the ocular surface after the initial iontophoresis could not be demonstrated following subsequent trials of iontophoresis.

Enhanced survival of ultraviolet-irradiated herpes simplex virus in cells exposed to antiviral agents

Enhanced survival of UV-irradiated HSV-1 is demonstrated in monkey cells exposed to inhibitors of viral DNA synthesis. Phosphonoacetic acid (PAA), adenine arabinoside (ara-A), and cytosine arabinoside (ara-C) pretreatment of infected cells is associated with concentration-dependent reactivation of UV-HSV-1. At concentrations that result in enhanced virus survival, inhibition of cell DNA synthesis is observed by either ara-A or ara-C, but not by PAA. Pretreatment of uninfected cells with acycloguanosine (ACG) is not associated with reactivation of irradiated HSV-1, and this is probably due to insufficient generation of ACG-triphosphate, the active inhibitor of viral and cell DNA synthesis.