Timolol induces HSV-1 ocular shedding in the latently infected rabbit

Timolol iontophoresis into the eye can induce herpes simplex virus type 1 (HSV-1) shedding in rabbits latently infected with HSV-1 strain McKrae. Anodal iontophoresis of 0.01% timolol was done at 0.8 mAmp for 8 min once a day for 3 consecutive days. Viral shedding was determined by the presence of HSV-1 in the preocular tear film obtained by eye swabs. In two experiments, iontophoresis of 0.01% timolol resulted in all eyes (18/18) shedding HSV-1 for an average duration of 4.3 days. When 5.0% timolol was applied topically to rabbit eyes supersensitized by iontophoresis of 6-hydroxydopamine (6-HD), all eyes (10/10) shed virus for an average duration of 2.9 days. All eyes (12/12) receiving iontophoresis of 6-HD, pre- and posttreatment with topical application of 5.0% timolol, and posttreatment with topical application of 1.0% epinephrine shed virus for an average duration of 3.6 days. Eyes treated with topical application of 5.0% timolol alone showed no difference in HSV-1 ocular shedding, compared with untreated eyes. We concluded that both iontophoresis of 0.01% timolol and topical application of 5.0% timolol to adrenergically supersensitized eyes induced HSV-1 shedding reliably and with a high frequency, and that topically applied timolol does not block the HSV-1 ocular shedding induced by epinephrine in adrenergically supersensitized eyes.     

Reactivation of HSV-1 in primates by transcorneal iontophoresis of adrenergic agents

Transcorneal iontophoresis of adrenergic agents has been shown to reactivate latent herpes simplex virus type 1 (HSV-1) and to produce viral shedding in the tear film in rabbits and mice, but not, to date, in nonhuman primates. In this study, we demonstrated induced reactivation of latent HSV-1, viral shedding, and production of ocular lesions in nine squirrel monkeys (Saimiri sciureus) by iontophoresis of 6-hydroxydopamine (6-HD) with topical instillation of epinephrine or with iontophoresis of timolol. Monkey corneas were scarified and inoculated with McKrae strain HSV-1 on three separate occasions. All monkeys received daily intramuscular prednisolone for 39 or 46 days prior to ionotophoresis. Once latency was established, a single ionotphoresis of 6-HD was performed on both eyes in five of the monkeys, followed by 1% epinephrine given topically four times daily for 5 days. Iontophoresis of timolol was performed on both eyes in the other four monkeys once per day on 3 consecutive days. Eight of the ten eyes receiving 6-HD and epinephrine shed HSV-1; seven eyes developed deep punctate, dendritic, or geographic corneal lesions. Seven of the eight eyes receiving timolol shed HSV-1; six of the eyes developed lesions suggestive of HSV-1 specific corneal lesions. The methods used in this report were slightly different from those used to reactivate HSV-1 in rabbits, in that repeated inoculations with HSV-1 and repeated intramuscular injections with prednisolone were required; however, these results demonstrate that iontophoresis of adrenergic agents can produce shedding and recurrent epithelial lesions in the nonhuman primate.     

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.     

The development of an improved murine iontophoresis reactivation model for the study of HSV-1 latency

The present study reviews the development of an effective murine iontophoresis reactivation model for the study of HSV-1 latency. In a series of experiments, Balb C mice latently infected with HSV-1 McKrae strain were iontophoresed with epinephrine X 3 days (EPI X 3/ION) or 6-hydroxydopamine X 1 day followed by topical epinephrine (6-HD ION/EPI). Reactivation and recovery of latent HSV-1 was determined by daily ocular swabs, titration, and neutralization. Additional studies determined the effect of topical ocular steroids on viral recovery rate. The results demonstrated no recovery of McKrae strain in Balb C (0%) with EPI X 3/ION, and no enhancement with topical steroids. 6-HD ION/EPI demonstrated a low recovery rate in mice (8%). However, the recovery rate was significantly increased to 50% by the addition of topical steroids to form the 6-HD ION/EPI/STEROID model, a useful experimental tool. The substitution of a clinical isolate, W strain, for McKrae strain further improved the model. The results demonstrated that, following the acute infection in mice, W strain was associated with a significantly higher (P = .001) survival rate than McKrae strain (81% vs. 52%). There was no statistically significant difference between the two strains, W vs McKrae, in Balb C mice comparing keratitis, establishment of latency (by co-cultivation), spontaneous shedding rate, or induced ocular shedding following iontophoresis. The development of an effective murine iontophoresis model offers an economical method which is uniquely suited for immunological and genetic studies of HSV-1 latency.     

Corneal nerves are necessary for adrenergic reactivation of ocular herpes

Herpes simplex virus type 1 (HSV-1) can infect the cornea and achieve ganglionic latency. HSV-1 can later be activated by a variety of effectors although the exact mechanism of reactivation is unknown. Rabbits harboring latent HSV-1 strain McKrae can be induced to shed virus by ocular iontophoresis of epinephrine to the cornea. No studies have been done to investigate if corneal nerves are necessary for epinephrine induction of HSV-1 ocular shedding. We did penetrating keratoplasty (PKP) in one eye each of 23 rabbits; the other eye served as an unoperated control. The surgery effectively denervates the area of the transplant for up to 90 days. Eighteen rabbits carrying latent HSV-1 strain McKrae received corneas from uninfected rabbits. Five uninfected rabbits with no latent virus received corneas from rabbits harboring latent HSV-1. On days 10-14 after penetrating keratoplasty, 24 eyes in the HSV-1 latent group and all ten uninfected eyes received iontophoresis of 0.01% epinephrine (0.8 mAmps for 8 min or 0.6 mAmps for 6 min) once daily for 3 days by means of an eye cup whose diameter was less than the diameter of the transplant. Six rabbits in the HSV-1 latent group received intravenous injections of cyclophosphamide (75 mg/kg) and dexamethasone (4 mg/kg). Following iontophoretic or immunosuppressive induction, the eyes were swabbed daily for 9 days. Of the 12 rabbits with latent virus which were treated by iontophoresis, one of the transplanted eyes and eight of the nontransplanted eyes were induced to shed virus. The mean duration of shedding in the nontransplanted eyes was 3.25 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iontophoresis of epinephrine isomers to rabbit eyes induced HSV-1 ocular shedding

Iontophoresis of 0.01% levo(-) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days induces ocular shedding of herpes simplex virus type 1 (HSV-1) in latently infected rabbits. In the present experiment, we tested dextro(+) and levo(-) epinephrine for their comparative effects on induced HSV-1 ocular shedding. One hundred percent of the eyes shed virus after either 0.01% dextro(+) or levo(-) epinephrine iontophoresis (8 min, 0.8 mAmp). However, the shedding frequency caused by 0.005% levo(-) epinephrine was significantly higher (P less than 0.05) than that by 0.005% dextro(+) epinephrine when the iontophoresis was conducted at 0.4 mAmp for 4 min. Iontophoresis of 0.001% levo(-) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days and iontophoresis of 0.001% dextro(+) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days did not induce HSV-1 ocular shedding in latently infected rabbits. The data suggest that the mechanism of induction of HSV-1 ocular shedding by epinephrine is correlated to the receptor potency of levo(-) epinephrine.     

Induction of ocular herpes simplex virus shedding by iontophoresis of epinephrine into rabbit cornea

Ocular herpes simplex virus type 1 (HSV-1) shedding from the latently infected rabbit was induced by iontophoresis of 0.01% epinephrine into the eye. The iontophoresis of epinephrine was at 0.8 mAmp for 8 min once a day for 3 consecutive days. Shedding was determined by the presence of HSV-1 in the tear film obtained with eye swabs. Unilateral epinephrine iontophoresis performed 60 days after inoculation of the virus resulted in ipsilateral HSV-1 shedding in all cases (7/7). Bilateral epinephrine iontophoresis performed on selected days during 170 to 365 days after inoculation resulted in HSV-1 shedding in 75% of the eyes (21/28) and 100% of the rabbits (14/14). All shedding was initiated within 3 days after the third treatment with epinephrine iontophoresis. The shedding frequency induced by epinephrine iontophoresis was significantly higher (pb less than 0.05) than that induced by the other methods employed. HSV-1 was detected in one or both cocultivated explants of trigeminal and superior cervical ganglia for every eye in all experimental groups, indicating that all eyes had the potential to shed. In conclusion, epinephrine iontophoresis induced ocular HSV-1 shedding reliably and with a high frequency in the latently infected rabbits. Furthermore, we suggest that this easily reproducible model of viral shedding offers a system for studying the factors involved in recurrent HSV-1 ocular infections.     

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.     

Vanadate promotes reactivation and iontophoresis-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.     

Quantitation and kinetics of induced HSV-1 ocular shedding

Iontophoresis of 6-hydroxydopamine (6-HD) to the rabbit eye, followed by topical instillation of 2% epinephrine, induces ocular shedding of herpes simplex virus type 1 (HSV-1) reliably and with a high frequency in latently infected rabbits. Rabbit eyes inoculated with HSV-1 (McKrae strain) showed dendritic lesions indicative of acute HSV infection and subsequently shed virus spontaneously at least once during days 20 to 39 postinoculation (P.I.). Two iontophoretic conditions were employed. Group A (3 rabbits, 60.3 days P.I.) received iontophoresis of 1.0% 6-HD at 0.75 mAmp for 3 min. Group B (three rabbits, 67.3 days P.I.) received iontophoresis of 0.1% 6-HD at 0.5 mAmp for 8 min. Following iontophoresis, 2% epinephrine was instilled topically once on the day of iontophoresis and twice daily for four consecutive days. Tear film was collected on Dacron swabs and titered on African green monkey kidney cells by a plaque assay procedure. In group A, 100% (6/6) of the eyes shed virus, and the average duration of shedding was 4.0 days. The titers ranged from 2.0 to 7.7 X 10(4) plaque-forming units (PFU) per eye. The highest daily average titer, 9.89 X 10(3) PFU/eye, occurred on day 5 following iontophoresis. In Group B, 100% (6/6) of the eyes also shed virus, and the average duration of shedding was 5.3 days. The viral titer of the tear film ranged from 5.0 to 1.4 X 10(5) PFU/eye. The highest daily average titer, 4.68 X 10(3) PFU/eye, also occurred on day 5 following iontophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

HSV-1 quantitation from rabbit neural tissues after epinephrine-induced reactivation

Epinephrine iontophoresis into the eye can induce ocular herpes simplex virus type-1 (HSV-1) shedding with a high frequency from latently infected rabbits. The present study was designed to qualify and quantify infectious HSV-1 from neural tissues of latently infected rabbits after ocular epinephrine iontophoresis. Epinephrine iontophoresis was performed daily for 3 consecutive days on selected days during 220-227 days postinoculation. The induced ocular shedding was detected in the tear film with a frequency of 83% (10/12) within 72 hr after the initial iontophoresis. The rabbits were killed 24 hr after the last iontophoretic treatment, and the corneas and neural tissues were homogenized immediately. The cell-free supernatants were inoculated on primary rabbit kidney cell monolayers for qualitative assays of infectious virus and later titrated on CV-1 monolayers. The frequencies of the recovery of infectious HSV-1 from the cell-free homogenates were 0% of the corneas (0/12), 83% (10/12) from the superior cervical ganglion (SCG), 100% (12/12) from the trigeminal ganglion (TG), 42% (5/12) from the ophthalmic branch of the trigeminal nerve (TN), 8% (1/12) from the root entry zone of the trigeminal nerve into the brain-stem (REZ), and 0% (0/12) from the cerebellum. The authors conclude that epinephrine iontophoresis can reactivate the latent HSV-1 in neural tissues and infectious virus can be quantified from the cell-free homogenates. To the best of our knowledge, this is the first report to quantify HSV-1 with a high frequency from neural tissues following induced reactivation.     

Adrenergically induced recurrent HSV-1 corneal epithelial lesions

Herpes simplex virus type 1 (HSV-1) ocular shedding and recurrent HSV-1 corneal epithelial lesions were assessed after ocular iontophoresis of 0.1% 6-hydroxydopamine followed by topical ocular instillation of 0.1% Propine in ten rabbits latently infected with HSV-1 strain McKrae. Iontophoresis was performed once at 0.5 mAmp for five minutes and 0.1% Propine drops were instilled four times a day beginning three days after iontophoresis and continuing for five consecutive days. Over an eight day period beginning three days after iontophoresis, ocular tear film samples were collected on Dacron swabs with care taken to avoid contact with the corneal epithelium. The corneas were examined daily for the presence of epithelial lesions using a slit-lamp biomicroscope. Three types of lesions were observed: deep punctate lesions, dendritic lesions, and geographic epithelial defects. The ratio of positive HSV-1 eye swabs to total eye swabs was 36/157 (23%). The ratio of total positive days of corneal lesions to total days was 40/160 (25%). There were 23 deep punctate lesions, 13 dendritic lesions, and four geographic epithelial defects. There were 24/36 (67%) positive HSV-1 eye swabs associated with concurrent HSV-1 corneal epithelial lesions. There were 105/121 (87%) negative eye swabs with concurrent negative slit-lamp examinations. Chi square analysis showed significant (p less than 0.001) association of HSV-1 positive eye swabs and HSV-1 corneal lesions. These results suggest that adrenergic ocular treatment may induce both HSV-1 ocular shedding (reactivation) and HSV-1 corneal epithelial lesions (recurrence) in rabbits latently infected with HSV-1 strain McKrae.     

Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits

Spontaneous ocular shedding and adrenergic induction of ocular shedding were examined in rabbits infected with ten strains of herpes simplex virus type 1 (HSV-1): McKrae, KOS, F, Rodanus, 17 Syn+, RE, E-43, SC-16, MacIntyre, and CGA-3. All ocular inoculations were with 50 microliter of HSV-1 with titers between 1-10 X 10(6) PFU/ml. All corneas, except those that received the McKrae strain, were scarified. Acute ocular infection was determined by slit-lamp biomicroscopy. Dendritic keratitis or geographic ulcers developed in all eyes of all rabbits within 10 days after ocular inoculation. All eyes of all surviving rabbits were swabbed for 20 consecutive days during days 20-39 postinoculation (PI). On PI day 19, no active lesions were present as judged by slit-lamp biomicroscopy. Ocular tear film was collected on a Dacron-tipped swab and placed on primary rabbit kidney cell monolayers. The cell monolayers were monitored for cytopathic effects consistent with HSV-1 infection. Spontaneous HSV-1 shedding was detected in some eyes from all groups of latently infected rabbits, except those infected with CGA-3. Spontaneous shedding (positive swabs/total swabs) of the other nine strains ranged from 0.7% to 15.7%. After PI day 42, the rabbit eyes received 6-hydroxydopamine by iontophoresis, followed for 5 days by topical application of 2% epinephrine. This procedure results in induced HSV-1 ocular shedding for a duration of 3-5 days in rabbits infected with the McKrae strain. In rabbits latently infected with KOS, F, RE, MacIntyre, and CGA-3, no induced HSV-1 shedding was detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trifluridine decreases ocular HSV-1 recovery, but not herpetic lesions after timolol iontophoresis

To determine the effect of a topically applied antiviral agent on shedding of herpes simplex virus type 1 (HSV-1) into the tear film and corneal epithelial lesions, ten rabbits latently infected with HSV-1 were subjected to transcorneal iontophoresis of 0.01% timolol once a day for 3 consecutive days to induce viral shedding and lesions. Iontophoretic induction was performed similarly in five uninfected rabbits as controls. Half of the infected rabbits and all of the uninfected controls received topical 1.0% trifluridine five time a day for 9 days, beginning the day after the first iontophoresis. All eyes were examined daily for 10 days by slit-lamp biomicroscopy and tear film samples collected on swabs were analyzed for virus. In the infected rabbits, the eyes treated with trifluridine had significantly fewer swabs positive for HSV-1 than the untreated eyes (P less than 0.001); however, there was no significant difference in the numbers of lesions in the treated and untreated eyes. The uninfected controls had no positive swabs and developed no lesions. These results suggest that topical treatment with trifluridine may reduce recovery of HSV-1 from the tear film, but does not affect the incidence of iontophoretically induced corneal epithelial lesions.     

Herpes simplex virus recovery in neural tissues after ocular HSV shedding induced by epinephrine iontophoresis to the rabbit cornea

Ocular HSV-1 shedding from latently infected rabbits was induced by iontophoresis of 0.01% epinephrine into the eye. Anodal Iontophoresis of epinephrine was performed at 0.8 mAmps for 8 min once a day for 3 consecutive days. Shedding was determined by the presence of HSV-1 in the preocular tear film obtained via eye swabs. Bilateral epinephrine iontophoresis performed on selected days during 220-280 days after inoculation resulted in HSV-1 shedding in 75% of the eyes (30/40) and 100% of the rabbits (20/20). Following the induction of ocular HSV-1 shedding, rabbits were killed and selected neural tissues were homogenized. Cell-free preparations were assayed for the presence of infectious virions using primary rabbit kidney cell monolayers. When the tissues were homogenized immediately after death, virus was detected in only one neural tissue, the trigeminal ganglia. However, when the tissues were incubated in vitro for 18-24 hours prior to the homogenization, infectious HSV-1 was recovered from homogenates of the trigeminal ganglion, superior cervical ganglion, the ophthalmic branch of the trigeminal nerve, and the root entry zone of the trigeminal nerve. A relationship was noted between the time of the last ocular shedding and recovery of infectious HSV from the tissue homogenates. Furthermore, a positive correlation in 11 eyes between the recovery of HSV-1 from the perocular tear film and HSV-1 recovery from one or more corresponding neural tissues was found. These results suggested that epinephrine iontophoresis to the cornea triggered an "alteration" in the state of the virus in the neural tissues of the latently infected rabbits and that the change can be related to the induced ocular shedding.     

Corneal nerves contain intra-axonal HSV-1 after virus reactivation by epinephrine iontophoresis

Experimental ocular models of herpes simplex virus type 1 (HSV-1) reactivation have been used to monitor viral shedding in the tear film and the appearance of corneal epithelial lesions, but the temporal correlation between reactivation and the presence of viral particles in the corneal nerves has not been made. Two New Zealand white rabbits were inoculated with 20 microliters of HSV-1 McKrae strain (5.0 x 10(6) PFU/ml) in each eye. Beginning on postinfection day 82, ocular iontophoresis (0.8 mAmps for 8 min) of 0.01% epinephrine was done once a day for 3 consecutive days to induce reactivation. Ten limbal nerves from four corneas processed for transmission electron microscopy contained 883 unmyelinated and 40 myelinated axons. Seven nerves were positive for virus. Viral particles were found only in unmyelinated axons, and in low frequency (24/883). Virus was not found in Schwann cells, perineurium, or adjacent stroma nor were virus particles seen exiting axons. No enveloped virions were found. Axons from six nerves of four control corneas from rabbits with latent, but not reactivated, HSV-1 did not contain virus particles. Induction by corneal iontophoresis of epinephrine suggests that HSV-1 is translocated from the ganglion to the cornea through axonal transport mechanisms. For the first time, evidence of anterograde, intra-axonal transport of HSV-1 particles in response to epinephrine reactivation is demonstrated.     

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.     

The effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection

Recurrent herpes simplex virus-1 (HSV-1) ocular infection is a frequent cause of morbidity and blindness. Factors that trigger viral reactivation are poorly understood and the role of topical ocular corticosteroid administration in the development of recurrent HSV-1 ocular disease is not clear. Clinical reports and epidemiological studies suggested topical corticosteroids may reactivate latent HSV-1 and result in recrudescent ocular disease; however, experimental studies to establish this causal relationship produced inconsistent results. The previous experimental studies were performed by infecting unnatural host species with HSV-1 and aspects of viral behavior and reactivation within these animals may differ from the host for which the virus is adapted. The purpose of the study reported here was to determine if topical ocular corticosteroid administration results in viral reactivation and recrudescent ocular disease in a host-adapted pathogen animal model of HSV-1 recurrent ocular disease. Canine herpesvirus-1 (CHV-1) is a corticosteroid-sensitive alphaherpesvirus that is biologically related to HSV-1 and induces similar ocular lesions in canids during recurrent infection. A randomized, masked, placebo-controlled, crossover study was performed. Primary ocular CHV-1 infection was experimentally induced in mature specific pathogen-free beagles by topical ocular inoculation and the presence of reactivatable latency was later confirmed by administration of an immunosuppressive dosage of systemic corticosteroid to the dogs. Twelve months following experimental CHV-1 reactivation, dogs were administered either topical ocular prednisolone acetate (1.0% ophthalmic suspension, one drop in both eyes, four times daily) or placebo (artificial tear solution, one drop in both eyes, four times daily) for 28 days. After a 14 day washout period, the treatment groups were reversed and study agents administered for an additional 28 days. Ophthalmic examinations, in vivo ocular confocal microscopy, real-time quantitative CHV-1 polymerase chain reaction assays, and CHV-1 serum neutralization antibody titers were performed at regular intervals throughout the study. Viral reactivation was not detected in dogs administered topical ocular prednisolone or placebo as determined by clinical ocular disease recrudescence, in vivo ocular confocal microscopic findings, ocular viral shedding, and serologic response. Similar to other animal models of recurrent HSV-1 ocular infection, the behavior of latent CHV-1 in dogs may differ from HSV-1 in humans; however, results of the present study suggest administration of topical ocular prednisolone at the evaluated drug concentration, dosing frequency, and treatment duration is not likely to result in detectable reactivation of latent CHV-1 in experimentally infected dogs. This may be attributed to insufficient systemic absorption of locally administered corticosteroid to reactivate latent virus and produce recurrent disease. Crystalline corneal opacities that were apparently not associated with viral reactivation were detected by clinical examination and in vivo confocal microscopy in two dogs during topical ocular prednisolone administration. The crystalline keratopathy may have resulted from corneal degeneration associated with metaherpetic disease, corticosteroid administration, or a combination of both factors.     

Intentional reactivation of latent ocular herpes infection during BVDU therapy

Sixteen adult New Zealand white rabbits with previously confirmed herpes simplex virus type 1 (HSV-1) infections were stimulated by iontophoresis of 6-hydroxydopamine into the cornea and were followed-up by topical epinephrine treatment to confirm latency. A total of 224 ocular cultures were obtained, of which 73 were positive for HSV during the seven day cycle. Twenty-seven of the 32 eyes (84%) had at least one positive culture. Animals were randomly divided into two treatment groups. Upon repeat stimulation (Cycle 2), concurrent with oral and topical bromovinyl-deoxyuridine (BVDU) therapy, only 3/104 ocular cultures were HSV positive, while 24/112 ocular cultures from placebo-treated animals were positive. Anti-HSV serum titers were comparable before and after BVDU therapy and the HSV isolates from BVDU treated animals did not develop drug resistance (i.e., ED-50 values were approximately 0.1-0.2 ug/ml both before and after therapy). It was concluded that BVDU had a demonstrable therapeutic effect on the recovery of HSV-1 from ocular cultures during intentional reactivation, but the latent ganglionic infection was not eliminated.     

Human alpha and gamma interferon analogs in rabbits with herpetic keratitis

Complete gene synthesis methods have been used to construct analogs of human interferons (IFNs): these include a consensus of the known human IFN-alpha S, designated IFN-alpha Con1 and a variant of human IFN-gamma, designated IFN-gamma 4A. These interferons, in purified form, were used topically against herpes simplex virus type 1 (HSV-1) induced ocular keratitis in rabbits. Eyes pretreated with IFN-alpha Con1 had decreased signs of infection and a lower incidence of HSV-1 positive trigeminal ganglia (3 of 14 positive) compared to the placebo treated (10 of 14 positive). IFN-alpha Con1 was as effective as natural IFN-alpha subtypes on a units basis, despite the very high specific activity of this analog. IFN-gamma 4A used under similar conditions do not result in beneficial effects with treatments beginning 24 or 48 hr before or 4 hr after virus inoculation. Rabbits with confirmed latent HSV infection were treated topically with IFN-alpha Con1 (10(6) units per eye each day) either before or before and after attempts to intentionally reactivate the infection by bilateral iontophoresis of 6-hydroxydopamine plus topical epinephrine treatment of the corneas. These IFN-alpha Con1 treatment regimens along with intentional reactivation during latency did not: (1) lessen the frequency of inducible ocular shedding episodes; (2) alter the mean time of 3-5 days between attempts to reactive latent infection and the appearance of HSV in tears; or (3) significantly change the incidence of HSV-positive trigeminal ganglia (83-100% HSV positive).