Reactivation of murine latent HSV infection by epinephrine iontophoresis

Iontophoresis of epinephrine into the cornea of previously infected mice was used in an attempt to induce reactivation of latent herpes simplex virus (HSV) infection of the trigeminal ganglia. BALB/c mice infected with HSV-1 strain McKrae following corneal scarification developed a latent infection of the trigeminal ganglia within 15 days. At 28 days postinfection, mice were subjected to a 3-day cycle of iontophoresis of epinephrine (0.01%) into the cornea. Ocular shedding of HSV occurred in 16/23 (70%) of stimulated mice; these animals did not shed HSV in the 3-day period prior to iontophoresis. Spontaneous shedding of HSV, however, was noted in 3/97 (3%) mice not subjected to epinephrine iontophoresis. "Infectious" virus was isolated only from the trigeminal ganglia of stimulated mice, whereas "latent" virus was isolated from the trigeminal ganglia of both stimulated and nonstimulated mice. All virus isolates were verified to be HSV by neutralization with a known HSV-1 antiserum. This ocular system thus allows for the study of the full spectrum of latent HSV infections, including latency, ganglionic reactivation, and peripheral virus shedding.     

An HSV-1 chimeric containing HSV-2 latency associated transcript (LAT) sequences has significantly reduced adrenergic reactivation in the rabbit eye model

PURPOSE: Both HSV-1 and HSV-2 express LAT during viral latency. Previous studies indicated that deletion of either LAT impairs viral reactivation from latency, but that the HSV-1 LAT confers the ability to reactivate efficiently from trigeminal ganglia onto HSV-2. We sought to determine whether the HSV-2 LAT could function in the context of HSV-1. METHODS: A chimeric HSV-1 that expresses the HSV-2 LAT in place of both copies of its own LAT was constructed. A rescuant virus, in which the wild-type genotype was restored, was also constructed to demonstrate that any phenotypic differences between the mutant virus and wild-type virus were due to the introduced mutations rather than to inadvertent mutations elsewhere in the virus. All viruses were tested in vitro and in vivo (via inoculation of rabbit eyes and induction of reactivation via iontophoresis of epinephrine) to determine the ability of the HSV-2 LAT to substitute for the HSV-1 LAT during acute infection and reactivation from latency. RESULTS: Substitution of the HSV-2 LAT for the HSV-1 LAT had no effect on acute replication or on the ability of the virus to cause disease in the rabbit ocular model. The ability of the mutant virus to reactivate from latency was substantially impaired relative to wild-type HSV-1. CONCLUSIONS: The HSV-2 LAT cannot substitute for the HSV-1 LAT in promoting reactivation from HSV-1 latency from rabbit trigeminal ganglia. This is consistent with the LAT being the major determinant of site-specific HSV reactivation.     

Ganglionic permissivity to HSV-1 replication and acyclovir-induced latency: an in vitro model

HSV-1 is known to establish latent infections in murine trigeminal ganglia. In order to study the genetic influence of the host on permissivity to HSV-1 replication and latency in mouse trigeminal ganglia, an in vitro culture system was developed. Ganglia from HSV sensitive A/J and resistant C57BL/6J mice were harvested and inoculated in vitro with either MP or KOS strains of HSV-1. Infected ganglia were placed in culture for 7 days with or without 150 micrograms/ml acyclovir. Ganglia were then homogenized and assayed for infectious particles or frozen whole for standard immunostaining. A/J ganglia without acyclovir consistently demonstrated a three-fold higher viral replication compared to C57BL/6J ganglia without acyclovir for both MP and KOS. Indirect immunofluorescent staining of actively infected ganglia using rabbit anti-HSV antiserum showed increased staining in A/J ganglionic axons compared to C57BL/6J ganglionic axons. Ganglia from both murine strains showed total suppression of viral replication with acyclovir treatment. Immunostaining for latent viral protein with mouse monoclonal anti-ICP-4 (VP 175) was positive for both A/J and C57BL/6J strains following acyclovir treatment. Desuppression of acyclovir-treated whole ganglia resulted in 54% spontaneous HSV reactivation. These results suggest that: (1) HSV-1 can establish an active infection in whole murine trigeminal ganglia in vitro; (2) HSV-1 replicates to a greater extent in A/J ganglia compared to C57BL/6 ganglia; and (3) in vitro HSV-1 ganglionic infection in both murine strains is equally suppressed to a latent state by acyclovir treatment.     

Herpes simplex virus immediate-early protein ICP4 in murine models of latency

Herpes simplex virus-1 (HSV) infection of trigeminal and dorsal root ganglia was established in mice via corneal scarification and footpad injection, respectively. Trigeminal and dorsal root ganglia were removed during the acute and latent stages of infection and processed for the immediate-early HSV polypeptide ICP4 (VP175) using both a monoclonal reagent and polyclonal antiserum and the avidin biotin complex immunoperoxidase method. ICP4 (VP175) antigen was readily detected during the acute infection of both dorsal and trigeminal ganglia, but not during latency. This antigen could again be detected by reactivation of the latent virus by explantation and organ culture. The detection of ICP4 (VP175) during latency in a rabbit model but not in a murine model may correlate with biologic differences in each system (e.g., rabbits spontaneously reactivate). Alternatively, the discrepancy could reflect viral strain characteristics or may imply that ICP4 (VP175) need not be constitutively expressed (at detectable levels) in all models of latent infection.This work was supported in part by grants from the National Society to Prevent Blindness, Inc. (Dr. Pepose) and from Fight for Sight, Inc. (New York, New York) (Dr. Pepose). Dr. Pepose was the recipient of a Damon Runyon-Walter Winchell Research Fellowship     

Peripheral adrenergic stimulation and indomethacin in experimental ocular shedding of HSV

The application of 6-hydroxydopamine to the cornea by iontophoresis, followed by topical epinephrine, effectively induces herpes simplex virus (HSV) shedding from the external eye of latently infected rabbits. In this study the beta adrenergic blocker, Timolol, reduced virus shedding when applied immediately before the epinephrine, but continued administration resulted in increased viral shedding. While indomethacin, a prostaglandin synthesis inhibitor decreased HSV replication in cell culture, it failed to decrease virus shedding when applied topically to the eye in adrenergically stimulated animals. Timolol may act then by its effect on the peripheral cells of the eye rather than by stimulation of virus production in ganglionic neurons. These same animals were subsequently tested for latent infection of the trigeminal and superior cervical ganglia and corneas 14 months after primary infection. Only 2 of 14 animals had virus in the trigeminal ganglia, a finding which suggests that latent virus may be depleted by repeated reactivations. Virus was recovered from corneas of five rabbits by co-cultivation so it is possible that corneal latency occurs in this rabbit model as it does in humans.     

T lymphocytes in the trigeminal ganglia of rabbits during corneal HSV infection

The results of this investigation reveal, for the first time, the presence of thymus-derived (T) lymphocytes in the trigeminal ganglia of rabbits undergoing primary corneal infection with herpes simplex virus type 1. Infiltration of T cells into the trigeminal ganglion was evident at 15 days after primary ocular infection but these cells were no longer present by 45 days after infection. Corneas and trigeminal ganglia of rabbits sacrificed at 3, 7, 12, 15, 20, 25, 30, 45, 70 and 90 days after infection were assayed for infectious virus and stained for viral antigen and immunoreactive T cells. Infectious virus and cells expressing viral antigens were present in the corneas and trigeminal ganglia during the acute phase (day 0-day 14) of the infection. T cell infiltration of the trigeminal ganglion was present as a perivascular infiltrate along with a sparse scattering of these cells among the nerve fibers. The perivascular infiltration is characteristic of viral infection of a tissue and was not seen in the sections of trigeminal ganglia obtained earlier than 15 days or in ganglia obtained 45 days or more after primary corneal infection. This investigation demonstrates conclusively that the neural ganglia are not completely shielded from the host immune response, as evidenced by the observation that immunocompetent T lymphocytes infiltrate the ganglia subsequent to the infection of a peripheral tissue such as the cornea of the eye.     

Herpes simplex virus: molecular biology and the possibility of corneal latency.

Herpes simplex virus (HSV) is known to be latent in ganglionic neurons. Over the past eight years, a series of reports have described the isolation of HSV after organ culture of human corneas that had been removed in the course of penetrating keratoplasty. None of the corneas showed any clinical signs of active herpetic disease immediately before keratoplasty. Studies in rabbits and mice confirmed that HSV can be recovered from corneas by organ culture long after primary infection has subsided. Recently, sophisticated techniques of molecular biology, such as specific DNA or RNA probes, have been used to detect HSV nucleic acids in the cornea. The crux of the matter is whether the virus recovered from or detected in the cornea is 1) truly latent in cell populations that are nonneuronal; 2) resident in the cornea, replicating at a slow rate; or 3) newly arrived in the cornea following ganglionic reactivation. The evidence suggests that a guarded case can be made for limited HSV latency within corneal cells. HSV corneal latency would allow for reactivation, replication, and the immune response to occur in the absence of ganglionic HSV reactivation. Such a localized phenomenon has not, however, been demonstrated to occur clinically.     

Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultraviolet B radiation.

The authors characterized a murine model of herpes simplex virus (HSV) reactivation in which recurrent herpetic keratitis was obtained in up to 80% of animals. Five weeks after ganglionic latency was established in National Institutes of Health inbred mice after corneal inoculation, HSV type 1 (HSV-1) was reactivated by irradiating the previously inoculated eye with ultraviolet (UV) light. Comparison of different UV wavelengths showed UVB to be optimal for reactivation, with peak viral recurrence being induced by a total exposure of approximately 250 mJ/cm2. Reactivated infectious virus generally began to appear in trigeminal ganglia 2 days postirradiation and was subsequently detectable in the cornea by both corneal swabbing and immunostaining for viral antigens. Two consecutive outbreaks of viral recurrence at the ocular surface were induced in selected animals by serial exposure to UVB. Advantages of this model over other models of recurrent keratitis are discussed.     

Immunomodulation of experimental murine herpes simplex keratitis: II. Glycoprotein D protection

Subcutaneous immunization with purified HSV-1 glycoprotein D (gD) protects susceptible A/J mice against keratitis and encephalitis following corneal HSV-1 challenge. Mice were immunized with gD, in complete Freund's adjuvant, 3.0 micrograms/mouse followed by two booster doses of 1.5 micrograms/mouse at weeks 2 and 4. Control groups of A/J mice were injected with either complete Freund's adjuvant (unimmunized controls) or live HSV-1 MP strain (immunized controls). All mice were challenged ocularly at week 5 with HSV-1, F strain (6.5 x 10(3) PFU) after corneal scarification. None of the 16 animals immunized with gD developed stromal keratitis; only 3 out of 12 animals immunized with live HSV-1 developed a stromal keratitis; 13 out of 16 CFA primed unimmunized mice developed severe stromal keratitis within 14 days post corneal challenge, and 3 out of 16 control CFA primed animals died within 16 days post corneal challenge. At the time of sacrifice (3 weeks post corneal challenge), the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these experiments indicate that immunization with gD produces protection against latent ganglionic infection in 56% of the immunized animals, and provides protection against keratitis and death following HSV corneal challenge.     

Herpetic imprint on privileged areas of its target organs: local latency and reactivation in herpetic keratitis

Herpetic ocular disease is still one of the major causes of corneal blindness. Due to its unique morphological structure, the eye is one of the most studied organs--in both clinical and experimental models of herpetic infections. Herpes simplex virus (HSV) can react with human host cells to produce cytocidal infection, persistent infection, or latent infection. Whilst the establishment of viral latency in the sensory ganglia was demonstrated and extensively studied, recent evidences based on: (a) demonstration of viral particles by electron microscopy and of HSV positive antigen cells in human corneae with inactive stromal keratitis; (b) experimental animal and in vitro studies with the use of organ cultures, co-cultivation methods and molecular biology techniques; suggest the possibility of local latency in non-neural tissues as an additional source of dormant viruses that could reactivate and lead to local reinfection of the affected target organ. Reactivation of a herpetic infection may therefore require both the existence of a dormant herpetic reservoir in the ganglia, and a predilected target organ with possible independent mechanisms of local latency and reactivation. Possible mechanisms of triggers for reactivation of herpetic ocular disease are discussed.     

The isolation of herpes simplex virus from rabbit corneas during latency

Herpes simplex virus type 1 (HSV-1) latency, as operationally defined, is a state in which cell-free infectious virus cannot be demonstrated in tissue at the time of sacrifice, but infectious virus can be isolated from the same tissue after prolonged cultivation. Latent HSV has been routinely detected in sensory ganglia of the infected dermatome. We have isolated HSV-1 (RE) from the corneas of 11% of infected rabbits which harbored virus in a latent state in trigeminal ganglia. HSV-1 (RE) was isolated from 10 of 88 cultures of corneal cells established following collagenase digestion of individual corneas taken from asymptomatic animals 118 days after infection. Virus was recovered only after prolonged primary culture and in some cases serial passage of corneal cells (range 5 to 26 days to initial cytopathic effect, n = 10). Virus was isolated from 68 of 68 trigeminal ganglia from the same rabbits by cocultivation of ganglion pieces with Vero cells (range 9 to 20 days to initial cytopathic effect, n = 68) while no cell-free virus was isolated from ganglia at the time of sacrifice. Virus isolation from corneas during the latent period occurred in a manner independent of prior antiviral or antiviral plus immunosuppressive therapy. Clinical evaluation of the corneas throughout the course of acute disease, stromal disease, and at the time of sacrifice provided no evidence that could be used to predict which corneas would yield virus. These data suggest that HSV-1 can remain in a nonreplicative state characteristic of latency in cells of rabbit corneas for long periods after infection and therapy of herpetic eye disease.     

单纯疱疹病毒性角膜炎的发病机制

单纯疱疹病毒性角膜炎(herpes simplex keratitis,HSK)是一种常见的眼部疾病,由单纯疱疹病毒(herpes simplex virus,HSV)感染引起。人群中超过90%的人曾经感染过HSV。HSV可以在神经组织及角膜组织长期潜伏。在适宜的刺激下,如紫外线照射、发热、精神压力、高温、低温、手术等,病毒活化增殖导致HSK。HSV感染引起的免疫反应是造成角膜组织损害的主要机制。HSK的免疫反应主要是由CD4+细胞介导的,而CD8+细胞对病毒感染具有保护作用。     

Effect of acyclovir on thermal stress-induced herpesvirus reactivation

PURPOSE: Acyclovir has been shown to be effective in preventing recurrent herpes simplex virus lesions of the genitalia and oral labia. The purpose of the current study was to determine the effect of acyclovir on the appearance of infectious virus in the peripheral nervous system and in an end organ, the eye. MATERIALS AND METHODS: Mice latent for the McKrae strain of herpes simplex virus type 1 were given 3.5 mg/ml acyclovir in their drinking water. Control animals received water without drug. Acyclovir treatment was continued for 4 successive days. On the third day, the mice were subjected to a brief period of hyperthermic stress to induce viral reactivation. Twenty-four hours after stress induction, swabs of the ocular surface and homogenates of the cornea and trigeminal ganglia were analyzed for the presence of infectious herpes simplex virus type 1 and viral DNA. RESULTS: Acyclovir treatment significantly decreased the frequency of infectious virus in the ocular tear film and the cornea but not in the trigeminal ganglion. The corneal homogenates of acyclovir-treated animals contained smaller amounts of viral DNA compared with untreated controls, whereas the amounts of viral DNA in the trigeminal ganglia of acyclovir-treated and untreated animals were similar. CONCLUSIONS: These results suggest that oral administration of acyclovir, at least at the dose used in this study, is effective in modestly reducing viral replication in peripheral tissues such as the eye but is not effective in inhibiting viral reactivation and viral DNA synthesis in the peripheral nervous system in mice subjected to induction of reactivation by hyperthermic stress.     

Acetylsalicylic acid reduces viral shedding induced by thermal stress

PURPOSE: To investigate the effect of acetylsalicylic acid on ocular shedding of herpes simplex virus type 1 (HSV-1). MATERIALS AND METHODS: Mice that were latent for the McKrae strain of HSV-1 were treated with acetylsalicylic acid, a nonspecific inhibitor of cyclooxygenases, either prophylactically or at the time of heat stress-induced viral reactivation. The effect of the drug on viral shedding in the tear film, infectious virus in the cornea and trigeminal ganglion, and viral DNA in the cornea and trigeminal ganglion was determined. RESULTS: Acetylsalicylic acid inhibited heat stress-induced shedding of virus in the tears and reduced the numbers of corneal and trigeminal ganglion homogenates containing virus. Intraperitoneal therapeutic and oral prophylactic plus therapeutic treatments were similar in their ability to inhibit reactivation. CONCLUSIONS: The results indicate that a cyclooxygenase inhibitor such as acetylsalicylic acid can reduce recurrent viral infection in mice. These findings may implicate prostaglandins as agents in the viral reactivation process and suggest that therapy to suppress viral reactivation using nontoxic inhibitors of prostaglandin synthesis may be effective in humans.     

Protective immunity against herpetic ocular disease in an outbred mouse model

Immunization of outbred mice by subdermal (footpad) inoculation with the F strain of herpes simplex virus type 1 (HSV-1) induces an immune response which protects the animals against herpetic ocular disease and encephalitis, and reduces the incidence of latent trigeminal ganglion infections following corneal challenge with the RE strain of HSV type 1. The protective effects are proportional to the dose of virus used for immunization. Heat-killed virus preparations also protected the mice against encephalitis and stromal keratitis, but failed to prevent epithelial keratitis and establishment of latency.     

Recovery of a latent HSV-1 thymidine kinase negative strain following iontophoresis and co-cultivation in the ocularly-infected rabbit model

Previous studies in the mouse and guinea pig have reported little or no colonization of sensory ganglia by strains of herpes simplex type 1 failing to express the enzyme, thymidine kinase (TK). The current study in the rabbit demonstrated trigeminal ganglionic colonization and reactivation of a latent thymidine kinase negative strain of HSV-1 by two independent methods: iontophoresis-induced ocular shedding and co-cultivation. Treatment with topical steroids during the acute infection did not enhance the latency rate. Following reactivation, back mutation with phenotypic reversion to thymidine kinase positive was demonstrated in a few recovered isolates. The current study also emphasized the importance of species differences to explain differing experimental results in studies of HSV-1 TK negative latency.     

Study of HSV-1 DNA species from trigeminal ganglia of rabbits during acute and latent infections

Recurrent herpetic keratitis remains a major cause of corneal blindness in developed countries. A fundamental unanswered question regarding herpes simplex virus infection concerns the relationship between the virus and host cell DNA during latency. In the present study DNA was extracted from trigeminal ganglia during both acute and latent infection following ocular inoculation. Extracted, purified DNA was utilized for transfection and for hybridization studies using a 32P-labeled HSV-1 DNA probe. DNA extracted during acute infection was complete, linear and non-integrated. Autoradiographic patterns of DNA isolated during latent infection were suggestive of two separate DNA species.     

Recovery of herpes simplex virus from ocular tissues of latently infected inbred mice

Evidence for latent infection of ocular tissues following topical corneal inoculation with herpes simplex virus type 1 (HSV) was sought in three strains of inbred mice that differ in susceptibility to HSV stromal keratitis. Corneas of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV. At 6-8 weeks after inoculation, when no active ocular infection was present, minced whole eyes and trigeminal ganglia were assayed for latent virus. Virus was recovered by explantation from minced eyes of all three strains (DBA/2 = 20%; BALB/c = 17%; C57BL/6 = 7%). In order to determine which ocular structures harbored virus, corneas, retinas and choroid-sclera were cultivated separately. Virus was activated from corneas of DBA/2 and BALB/c mice, but not from corneas of C57BL/6 mice. These findings suggest that HSV is capable of establishing latent infection in ocular tissue of inbred mice and that the rate of establishment of latency is under host genetic control. Since neural cell bodies are not present in the cornea, the data suggest that latency is established in cells other than neurons.     

Variation in the number of sites of latency of herpes simplex virus in trigeminal ganglia of inbred mice

Following uniocular topical corneal inoculation with herpes simplex virus type 1 (HSV), 176-fold more virus was recovered by 14-day cultivation in vitro from latently infected ipsilateral trigeminal ganglia (TG) of BALB/c mice than from TG of C57BL/6 mice (p = 0.002). Since these quantitative differences may reflect a difference in the number of latently infected cells or a difference in the ability of virus to replicate in secondarily infected cells during cultivation in vitro, experiments were designed to estimate the actual number of sites of latency. Two to four months after topical corneal inoculation, when no active ocular disease was present, minced TG were digested with 2% collagenase. The dissociated cells were placed on monolayers of vero cells, incubated at 31 degrees C, and observed for cytopathic effect (CPE) for 14 days. Ipsilateral TG from BALB/c mice produced five-fold more foci of infection than TG from C57BL/6 mice (p = 0.007). The number of foci of infection was also dependent upon the dose of virus used to infect the eye. Following infection with high doses of HSV, virus was reactivated from contralateral TG, but in lower numbers than from ipsilateral TG. In vitro studies showed that the replication of virus in ganglia from BALB/c mice was 3-8-fold greater than that in ganglia from C57BL/6 mice. These data support the hypothesis that host genetic factors and the number of infectious particles inoculated influence the number of latently infected cells in the trigeminal ganglion after corneal infection with HSV.(ABSTRACT TRUNCATED AT 250 WORDS)