The antiviral resistance and replication of cidofovir-resistant adenovirus variants in the New Zealand White rabbit ocular model.

PURPOSE: To determine the antiviral resistance of three cidofovir (CDV)-resistant variants of adenovirus type 5 (Ad5) and their ability to replicate in the New Zealand White rabbit ocular model. METHODS: Rabbits were inoculated topically in both eyes with the CDV-resistant variants R1, R2, and R3, and the Ad5 parental strain. On day 1, rabbits from each virus inoculation were divided into two topical treatment groups: 0.5% CDV and PBS control. Treatment was administered twice daily in both eyes for 7 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Using viral outcome parameters, CDV resistance was determined for each virus by comparing each CDV-treated virus group to its respective PBS control, and altered pathogenesis was assessed by comparing viral replication in the PBS control groups of the Ad5 parent and the three resistant variants. RESULTS: Topical 0.5% CDV treatment demonstrated significant antiviral inhibitory activity in the Ad5 parental group (e.g., reduced total Ad5-positive cultures, reduced daily Ad5-positive cultures on days 5, 9, 11, and 14, and duration of ocular shedding), but had no effect on the three CDV-resistant variants. There were no significant differences in pathogenicity between the Ad5 parent and the CDV-resistant variants. CONCLUSIONS: The Ad5 variants R1, R2, and R3 were resistant to topical treatment with 0.5% cidofovir in the rabbit ocular model. However, the acquisition of CDV resistance did not alter the replication of the three Ad5 CDV variants on the rabbit eye.

The effects of topical nonsteroidal anti-inflammatory drugs on adenoviral replication.

OBJECTIVE: To evaluate the antiviral activity of topical diclofenac sodium (Voltaren Ophthalmic) and ketorolac tromethamine (Acular) (2 nonsteroidal anti-inflammatory drugs [NSAIDs]) on adenoviral replication in vitro and in the adenovirus (Ad) 5 McEwen-New Zealand rabbit ocular model. METHODS: The 50% inhibitory concentration of ketorolac and diclofenac and their respective preservative components were determined for common ocular adenoviral serotypes (Ad8, Ad19, Ad1, and Ad5). In a series of experiments, Ad5 McEwen-inoculated New Zealand rabbit eyes were treated topically 4 times daily for 18 days with either ketorolac, diclofenac, prednisolone acetate (Pred Forte), or control vehicle (Comfort Tears). MAIN OUTCOME MEASURES: Outcome measures included serial ocular tear film titers and the formation of subepithelial immune corneal infiltrates. RESULTS: In vitro, neither ketorolac nor diclofenac demonstrated significant inhibitory activity against Ad1, Ad5, Ad8, or Ad19. In the rabbit model, there were no statistically significant differences among ketorolac, diclofenac, and the control vehicle with respect to viral replication or the formation of subepithelial immune infiltrates. In contrast, 1% prednisolone prolonged viral shedding and inhibited immune infiltrates (P < .001 for both). CONCLUSIONS: Our experimental study suggests that treatment of epidemic keratoconjunctivitis with topical NSAIDs may be a safer alternative than topical steroids. Only controlled clinical trials can determine whether topical NSAIDs can provide symptomatic relief and not interfere with normal viral clearance.

Multiple adenoviral serotypes demonstrate host range extension in the New Zealand rabbit ocular model.

PURPOSE: Although several human adenoviral serotypes demonstrated the genetic capability of replicating in New Zealand rabbit corneas in organ culture, only a single adenovirus (Ad) serotype, Ad5, has been reported to replicate in vivo in New Zealand rabbit eyes. The purpose of this study was to determine whether additional adenoviral serotypes could extend their host range to the New Zealand rabbit ocular model. METHODS: Six rabbits per viral isolate were inoculated in each eye after corneal scarification with 1.5 x 10(6) plaque-forming units per eye with one of the following reference or clinical adenovirus isolates: Ad1 ATCC, Ad1 Kmetz, Ad2 ATCC, Ad2 Wolf, Ad5 ATCC, Ad5 McEwen, Ad6 ATCC, Ad 19 ATCC, and Ad8 Cray (five rabbits). Eyes were cultured on days 0, 1, 3, 4, 5, 7, 9, 11, 14, 16, 18, and 21 after inoculation, and their tear film viral titers were determined on A549 cells. RESULTS: Ad19 ATCC and Ad8 Cray demonstrated no apparent viral replication. The mean duration of shedding was 1.5 and 0.3 days, respectively, and the total percentage of Ad-positive eyes was 13% and 3%, respectively. In contrast to Ad19 ATCC and Ad8 Cray, all other isolates demonstrated productive infection. The mean duration of shedding was 8 to 16 days (P < 0.0001), and the total percentage of Ad-positive eyes was 33% to 79% (P < 0.0002). The durations of shedding for Ad1 ATCC, Ad1 Kmetz, Ad2 ATCC, Ad2 Wolf, and Ad6 ATCC did not differ statistically from Ad5 McEwen, whereas Ad5 ATCC demonstrated a duration of shedding longer than all isolates (P < 0.0001). CONCLUSIONS: This was the first demonstration of host range extension by additional clinical and reference isolates of adenovirus types 1, 2, 5, and 6 in the New Zealand rabbit ocular model. These results suggested that host specificity was less stringent than previously thought.

Topical corticosteroids reverse the antiviral effect of topical cidofovir in the Ad5-inoculated New Zealand rabbit ocular model.

PURPOSE: To determine how the addition of topical corticosteroids would affect the anti-adenoviral inhibitory effect of topical cidofovir (S-HPMPC) in the Ad5 New Zealand (Ad5/NZ) rabbit ocular model. METHODS: In a series of experiments (two-eye design), Ad5-inoculated/NZ rabbits (10(6) pfu/eye) were treated with 1 of 3 treatment regimens. Group 1 was administered 1% cidofovir (CDV) twice a day for 3 days plus comfort tears four times a day for 14 days. Group 2 was administered 1% CDV twice a day for 3 days plus 1% Pred Forte four times a day for 14 days. Group 3 was administered vehicle twice a day for 3 days plus comfort tears four times a day for 14 days and served as the control. All eyes were evaluated for 21 days for serial eye titers, Ad5 positive eyes, and duration of Ad5 shedding. RESULTS: Compared to control eyes in the Ad5/NZ rabbit ocular model, CDV alone demonstrated a significant antiviral inhibitory effect: reduced mean Ad5 eye titer during the early phase of infection (days 3 to 7), fewer Ad5-positive eyes during the early and late (days 9 to 21) phases of infection, and shortened duration of shedding. However, concomitant treatment with both Pred Forte and CDV significantly reversed the antiviral inhibitory activity of CDV: increased mean Ad5 eye titer, increased Ad5-positive eyes (early and late phases) and prolonged duration of shedding. CONCLUSIONS: These experimental data further support the clinical development of cidofovoir as a topical antiviral agent, but they do not support a treatment regimen that includes a combination of topical corticosteroids and topical cidofovir as a desirable strategy for the treatment of symptomatic adenoviral ocular infection.

Isolation of human adenovirus type 5 variants resistant to the antiviral cidofovir.

PURPOSE: Cidofovir (S-HPMPC) is a potent broad-spectrum antiviral drug with potential clinical application against infections caused by human cytomegalovirus, herpes simplex virus, and adenovirus (AD). This study sought to determine whether variants of AD5 could be isolated in vitro that demonstrated increased resistance to this new antiviral drug. METHODS: Homogenous stocks of wild-type AD5 (ATCC strain VR-5) were generated from isolated plaques grown in A549 cells. The stocks subsequently were serially passaged in cells containing increasing levels (from 5 to 75 micrograms/ml) of cidofovir. The recovered virus either was passaged, titrated, or assayed for 50% inhibitory concentration (IC50) of cidofovir. RESULTS: Three independently isolated variants were obtained that demonstrated increased resistance to cidofovir. Viral resistance to the drug increased on stepwise passage in higher concentrations. Compared to the ATCC AD5 reference (IC50 = 6.2 micrograms/ml), stable cidofovir-resistant variants showed fivefold to eightfold resistance (AD5 RI IC50 = 36.5 micrograms/ml; AD5 R2 IC50 = 36.7 micrograms/ml; and AD5 R3 IC50 = 32.6 micrograms/ml; analysis of variance, P = 0.000001). However, a variable number of passages (1 to 13) at each concentration of cidofovir was performed to obtain robust infectious virus suitable for testing at the next higher concentration. All resistant virus isolates grew to levels of virus titer comparable to the parental virus and showed no apparent phenotypic changes in growth rates, plaque size, or efficiency of plaque formation. CONCLUSIONS: The successful isolation of AD5 variants in tissue culture resistant to cidofovir has important clinical implications with respect to the anticipated use of this antiviral drug in treating adenoviral ocular infections.

The effects of corticosteroids of adenoviral replication.

OBJECTIVE: To evaluate the effects of Pred Forte (prednisolone acetate; Allergan Pharmaceutical, Irvine, Calif) on the replication of different adenoviral serotypes in vitro and in the adenovirus type 5/New Zealand rabbit ocular model. METHODS: The 50% inhibitory doses of Pred Forte and its components were determined for common ocular serotypes. The effects of continuous topical treatment with Pred Forte for 18 days were evaluated (eg, conjunctivitis, subepithelial immune infiltrates, and serial ocular viral titers) in the adenovirus 5/New Zealand rabbit ocular model. RESULTS: Pred Forte and prednisolone acetate inhibited adenoviruses 1, 5, 8, and 19 in vitro. In vivo, 1% Pred Forte significantly reduced conjunctivitis (P = .04) and subepithelial infiltrates (P = .02), but enhanced viral replication (P = .01) on days 9 to 21 and increased the duration of viral shedding (P < .001). CONCLUSIONS: Despite demonstrated anti-inflammatory and anti-immune effects, prolonged treatment of acute adenoviral infections with topical Pred Forte is not recommended because of the enhanced risks of viral transmission and community epidemics.

HPMPC, a broad-spectrum topical antiviral agent, inhibits herpes simplex virus type 1 replication and promotes healing of dendritic keratitis in the New Zealand rabbit ocular model.

Previously, we demonstrated that HPMPC, a new, broad-spectrum antiviral agent, inhibited adenovirus type 5 in the New Zealand (NZ) rabbit ocular model (Cornea 1992; 11:529-33). Historically, no antiviral agent has been demonstrated to be effective against both herpes simplex virus type 1 (HSV-1) and adenovirus eye infections in an experimental animal model. In this study, we compared topical 0.2% HPMPC to 1% trifluridine and vehicle control in the NZ rabbit HSV-1 keratitis model. Using a double-masked, two-eye design, NZ rabbits were inoculated in both eyes with HSV-1 W strain (10(5) pfu/eye), and dendritic keratitis and HSV-1 ocular titers were measured serially. Compared with the control group, both topical 0.2% HPMPC and 1% trifluridine significantly reduced healing time of HSV-1 dendritic keratitis, lowered HSV-1 ocular titers on days 3 through 11, and shortened duration of HSV-1 shedding in the tear film. For all outcome parameters measured, topical 0.2% HPMPC was as effective as 1% trifluridine. A new concept of a broad-spectrum topical antiviral agent was shown to be effective against HSV-1 in an NZ rabbit keratitis model, and further development toward clinical application appears desirable.

Topical HPMPC inhibits adenovirus type 5 in the New Zealand rabbit ocular replication model.

PURPOSE: To evaluate the antiviral inhibitory activity of HPMPC against ocular adenoviral serotypes in vitro and to determine the therapeutic efficacy and ocular toxicity of treatment with topical HPMPC on established adenovirus type 5 (AD5) McEwen infection in the New Zealand (NZ) rabbit ocular replication model. METHODS: The 50% inhibitory dose (ID50) of HPMPC was determined for various clinical isolates of AD5 and AD8 by plaque assay in A549 cells. In vivo inhibitory effects were measured by serial ocular titers and duration of viral shedding in the AD5-NZ rabbit ocular model. Local ocular toxicity was evaluated by external and slit lamp examination for blepharitis, conjunctivitis, keratitis, and iritis. RESULTS: The mean ID50 for seven isolates of AD8 was 0.47 (range, 0.02 microgram/ml to 0.82 microgram/ml), and the mean ID50 for seven isolates of AD5 was 1.03 (range, 0.15 microgram/ml to 2.80 micrograms/ml). In a series of in vivo experiments, topical administration of HPMPC for as long as 10 days (total dose, > 2.8 mg) significantly reduced both AD5 ocular titers and the number of days of viral shedding compared to that for vehicle-treated control eyes. Local ocular toxicity was not clinically significant at a total dose of < 10 mg administered for as long as 10 days. CONCLUSIONS: HPMPC, a broad-spectrum, long-acting nucleoside monophosphate analog, is a promising candidate for the treatment of epidemic keratoconjunctivitis infections. Further studies to ensure safety and efficacy in humans are warranted.

Prolonged recovery of desiccated adenoviral serotypes 5, 8, and 19 from plastic and metal surfaces in vitro.

BACKGROUND: Prevention of the spread of epidemic keratoconjunctivitis (EKC) at eye care facilities (doctors' offices, clinics, hospitals) has been a major public health goal for ophthalmology for more than 50 years. The authors explored a potentially contributing attribute of the adenovirus serotypes that cause EKC. Specifically, they investigated the capacity of different clinical and laboratory ocular serotypes (AD8, 19, and 5) to survive for extended periods of time in a desiccated state. METHODS: Twenty microliters containing 2000 plaque-forming units of different ATCC laboratory adenoviral ocular serotypes (AD8, 19, and 5) and clinical isolates (AD8 Cray, AD19 Kowalski, and AD5 McEwen) were inoculated onto 7-mm plastic disks and 6-mm aluminum foil disks and were allowed to completely desiccate. At weekly intervals up to 7 weeks, eight desiccated virus-inoculated plastic or metal disks per serotype were added to tissue culture medium, and the amount of recoverable virus was determined by plaque assay on A549 cells. RESULTS: Ocular adenoviral serotypes AD8, 19, and 5 could be recovered up to 49 days from plastic, and 35 to 49 days from metal. Sufficient virus concentrations (> 100 plaque-forming units/disk) to be clinically infectious were recovered up to 28 days. Differences in recovery among serotypes (AD19 > AD5, AD8) were demonstrated, but laboratory and clinical isolates of the same serotype were usually comparable. CONCLUSIONS: Ocular isolates of adenovirus that cause EKC are much harder than previously suspected, and the capacity to survive in a desiccated state may possibly play some role in office-based mini-epidemics of EKC.

A comparison of enzyme immunoassay and polymerase chain reaction with the clinical examination for diagnosing ocular herpetic disease.

PURPOSE: The results of two laboratory diagnostic herpes simplex virus (HSV) tests, an enzyme immunoassay (improved Herpchek [iHC]) and the polymerase chain reaction (PCR), were compared with the clinical examination in the diagnosis of HSV. We determined when diagnostic laboratory tests provided the initial diagnosis of HSV ocular disease and when they were only confirmatory. METHODS: The sensitivity and specificity of iHC and PCR were determined using 22 HSV culture-positive clinical samples, 10 adenovirus culture-positive clinical samples, 5 samples from normal conjunctivas, 4 bacterial samples, and 1 sample containing Varicella zoster virus. The medical history of the 22 patients with positive HSV cultures were reviewed to determine the initial diagnosis by clinical examination and the initial therapy. RESULTS: For typical presentations of ocular HSV disease, the clinical examination is as accurate as iHC (P = 0.99) and PCR (P = 0.24). However, for atypical presentations of ocular HSV disease, iHC (P = 0.000005) or PCR (P = 0.00006) were more accurate in detecting HSV infection than the clinical examination. CONCLUSION: Laboratory diagnosis of HSV from ocular samples was most useful to the clinician in atypical presentations of herpetic ocular disease.

Pretreatment with topical 0.1% (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine inhibits adenovirus type 5 replication in the New Zealand rabbit ocular model.

Currently there is no clinically effective antiviral agent for the prevention or treatment of ocular adenoviral infections. Using a paired-eye, masked design, we tested the antiviral efficacy of topical 0.1% (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine in the New Zealand rabbit ocular model after topical and intrastromal inoculation with 100 microliters (4 x 10(5) plaque-forming units per eye) of adenovirus type 5 McEwen, a clinical isolate. Prevention studies involved pretreatment (six times a day) 1 day before inoculation and continuing for 4 additional days. Compared with the control eyes, the pretreated eyes showed a significant reduction in the peak viral eye titers on days 3, 4, 5, and 7 after treatment (p < 0.03-0.005), and a reduction in the duration of viral sheeding (p < 0.02). Rebound increase in adenoviral titers was detected in five of 20 eyes (25%) after cessation of treatment, suggesting a therapeutic effect and a need for further studies to optimize the treatment regimen.

An ocular model of adenovirus type 5 infection in the NZ rabbit.

Ocular adenoviral infections occur worldwide, and currently, there is no ocular animal model for evaluating new antivirals or studying pathogenesis. With a paired-eye design, an ocular model was developed in 32 New Zealand rabbits following topical and intrastromal inoculation with a clinical isolate of adenovirus type 5 (Ad5 McEwen). Clinical signs of infection--conjunctivitis, corneal edema, subepithelial infiltrates, and iritis--and seroconversion were evaluated. Replicating virus on the ocular surface was determined by serial ocular titers. Reproducible acute ocular infection was demonstrated in 32 of 32 infected eyes (100%), with mean viral replication lasting for 8.3 days. Peak ocular viral titers (10(3) plaque forming units/ml) were achieved on day three after inoculation and represented a 2 log increase (100 times) over day one. Ocular viral replication was associated with acute conjunctivitis (24/34 eyes, 75%), and delayed-onset presumed immune-mediated clinical disease was associated with: blepharoconjunctivitis (21/32 eyes, 66%), iritis (29/32 eyes, 91%), corneal edema (32/32 eyes, 100%), and subepithelial corneal infiltrates (30/32 eyes, 94%). Seroconversion was demonstrated in 26 of 31 rabbits (84%). The study concludes that a potentially useful animal model of adenoviral ocular infection can be attained.

Inhibitory effect of (S)-HPMPC, (S)-HPMPA, and 2'-nor-cyclic GMP on clinical ocular adenoviral isolates is serotype-dependent in vitro.

Currently, there is no effective treatment for ocular adenoviral infections that occur in epidemics worldwide, produce significant patient morbidity, and cause substantial economic losses. We tested several new antivirals in vitro, and found that (S)-HPMPC, (S)-HPMPA, and 2'-nor-cyclic GMP demonstrated significant serotype-dependent inhibitory activity by plaque reduction assay (ID50 = 0.017-17.0 micrograms/ml) against common clinical ocular isolates and standard adenoviral serotypes (Ad 1, Ad 5, Ad 8, and Ad 19). (S)-HPMPC was the least toxic (CD50 in A549 cells = 306 micrograms/ml), and (S)-HPMPC and (S)-HPMPA had high selectivity indices.

Replication of ocular isolates of human adenovirus is serotype-dependent in rabbit corneal organ culture.

The goal of the present in vitro study was to determine the ability of unadapted human adenoviral ocular isolates to replicate in the rabbit cornea. Rabbit corneas grown in organ culture (24 well plate) were inoculated topically with 50 microliters (5 x 10(5) pfu) of different ocular adenoviral serotypes (ATCC and clinical isolates). Control wells (no cornea present) were inoculated in a similar fashion. Viral replication was determined by serial aliquots titrated on A549 cells. We demonstrated sustained viral replication over time of all isolates (100%) of Ad1, 2, 5, 6, 8, 9, 11 and 37 tested. No isolates (0%) of Ad3, 7A, 19, and 4 demonstrated replication in our model. Peak titers varied among successful serotypes from 10(2) pfu/ml (Ad11) to 10(5) PFU/ml (Ad5), and among different isolates of a given serotype. We conclude that the ability of unadapted human Ad serotypes to replicate in rabbit corneas was serotype-dependent, and that subgroup C (Ad1, 2, 5, and 6) appeared to be the most successful subgroup.

Prolonged recoverability of desiccated adenovirus type 19 from various surfaces.

Epidemic keratoconjunctivitis is a highly contagious disease whose transmission has been linked to the ophthalmologist's office. The authors studied the ability of adenovirus 19 (ADV 19) to survive on surfaces commonly found in the office setting. An initial in vitro laboratory experiment demonstrated that ADV 19 in a desiccated state could be recovered up to 8 days from paper, and up to 10 days from cloth, metal, and plastic. The amount of recovered ADV 19 was significantly greater (analysis of variance, P less than 0.0001) from nonporous surfaces (plastic, metal) compared with porous surfaces (cloth, paper). A second experiment demonstrated that 35 days was the maximum length of time that desiccated ADV 19 could be recovered from a nonporous surface (plastic). The authors conclude that despite drying, ADV 19 is a hearty virus that remains potentially infectious for a long time on various surfaces that may be found in an ophthalmologist's office.

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 x 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 less than .0001) for dl x 3.1 than for KOS in both mice and rabbits, only dl x 3.1 shed spontaneously in the NZ rabbit. Iontophoresis of adrenergic agents failed to induce reactivation and ocular viral shedding of KOS or dl x 3.1 in mice or rabbits. The establishment of latency and reactivation of KOS and dl x 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 x 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 x 3.1 during latency in vivo did not correlate with latency studies based on in vitro methods.

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.

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.